This is Volume 27 in the FISH PHYSIOLOGY series Edited by Anthony P. Farrell and Colin J. Brauner Honorary Editor: William S. Hoar and David J. Randall A complete list of books in this series appears at the end of the volume Academic Press is an imprint of Elsevier 84 Theobald’s Road, London WC1X 8RR, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA First edition 2009 Copyright # 2009 Elsevier Inc. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: permissions@elsevier.com. 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Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made British Library Cataloging in Publication Data A catalog record for this book is available from the British Library Library of Congress Cataloging in Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-374632-0 For information on all Academic Press publications visit our website at www.elsevierdirect.com Printed and bound in 09 10 11 12 10 9 8 7 6 5 4 3 2 1 CONTRIBUTORS The numbers in parentheses indicate the pages on which the authors’ contributions begin. MARK BAYLEY (361), Institute of Biological Sciences Zoophysiology, University of Aarhus, Aarhus C Denmark DENISE L. BREITBURG (1), Senior Scientist, Smithsonian Environmental, Research Center, Edgewater, Maryland LAUREN J. CHAPMAN (25), Department of Biology, McGill University, Montreal, Quebec, Canada NGUYEN VAN CONG (361), College of Environment and Natural Resources, Cantho University, Cantho City, Vietnam ROBERT J. DIAZ (1), School of Marine Science, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, USA W. R. DRIEDZIC (301), Ocean Sciences Centre, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada ANTHONY P. FARRELL (487), Faculty of Land and Food Systems & Department of Zoology, The University of British Columbia, Vancouver, British Columbia, Canada A. KURT GAMPERL (301), Ocean Sciences Centre, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada K. M. GILMOUR (193), Department Biology, University of Ottawa, Ottawa, Ontario, Canada DO THI THANH HUONG (361), College of Aquaculture and Fisheries, Cantho University, Cantho City, Vietnam M. G. JONZ (193), Department Biology, University of Ottawa, Ottawa, Ontario, Canada ix x CONTRIBUTORS GISELA LANNIG (143), Alfred Wegener Institute, Am Handelshafen 12, Bremerhaven, Germany SJANNIE LEFEVRE (361), Institute of Biological Sciences Zoophysiology, University of Aarhus, Aarhus C Denmark DAVID J. MCKENZIE (25), Institut des Sciences de l’Evolution de Montpellier (UMR 5554 CNRS-Université de Montpellier 2), Station Méditerranéenne de l’Environnement Littoral, 1, quai de la Daurade, France GÖRAN E. NILSSON (397), Division of General Physiology, Department of Biology, University of Oslo, Oslo, Norway S. F. PERRY (193), Department Biology, University of Ottawa, Ottawa, Ontario, Canada HANS O. PÖRTNER (143), Laboratory of Ecophysiology and Ecotoxicology, Alfred Wegener Institute, Am Handelshafen 12, Bremerhaven, Germany JEFFREY G. RICHARDS (443), Department of Zoology, The University of British Columbia, Vancouver, British Columbia, Canada JONATHAN A. W. STEYCK (397), Department of Molecular Biosciences, University of Oslo, Blindern, Oslo, Norway MATTI VORNANEN (397), Professor of Animal Physiology, University of Joensuu, Faculty of Biosciences, Joensuu, Finland TOBIAS WANG (361), Institute of Biological Sciences Zoophysiology, University of Aarhus, Aarhus C Denmark RUFUS M. G. WELLS (255), School of Biological Sciences, The University of Auckland, Auckland, New Zealand RUDOLF WU (79), Department of Biology and Chemistry, City University of Hong Kong, Kowloon Tong, Hong Kong PREFACE Periods of environmental hypoxia are extremely common in aquatic systems due to both natural causes such as diurnal oscillations in algal respiration, seasonal flooding, stratification, ice cover in lakes, and isolation of densely vegetated water bodies, as well as more recent anthropogenic causes (e.g., eutrophication). In view of this, it is perhaps not surprising that among all vertebrates, half of which are fish, fish boast the largest number of hypoxiatolerant species; hypoxia has clearly played an important role in shaping the evolution of many unique adaptive strategies. These unique adaptive strategies either allow fish to maintain function at low environmental oxygen levels, thus extending hypoxia tolerance limits, or permit them to defend against the metabolic consequences of oxygen levels that fall below a threshold where metabolic functions cannot be maintained. The past several decades have seen an explosion of research on the responses of fish to hypoxia. The breadth of advances include the evolutionary and ecological consequences of hypoxia exposure in fish in addition to the morphological, behavioral, physiological, biochemical, cellular, and molecular responses that occur in some fish in response to hypoxia exposure. However, with an ever-expanding area of research, the breadth of information available on the responses and adaptations of fish to hypoxia has grown beyond the capacity of a single review article. Fish respond to and survive hypoxia exposure through the integration of numerous adaptive traits, thus a review of the current literature that integrates and synthesizes across levels of biological organization is needed. With this need in mind, we conceived the idea of devoting a single volume of Fish Physiology to the responses and adaptations of fish to hypoxia. As a result, the aim of this volume is two-fold. First, this book will review the behavioral, morphological, physiological, biochemical, and molecular strategies used by fish to survive hypoxia exposure and place them within an environmental and ecological context. Second, through the development of a synthesis chapter this book attempts to provide an integrative overview of the responses of fish to hypoxia. The production of this volume would not have been possible without the contributions of our colleagues. We are truly grateful to all of our colleagues xi xii PREFACE for their thoughtful, knowledgeable, and enthusiastic contributions to this volume. Also, we are grateful to the many reviewers for their constructive comments. Finally, we thank Kristi Gomez and the staV of Elsevier for their support. Jeffrey G. Richards Anthony P. Farrell Colin J. Brauner 1 THE HYPOXIC ENVIRONMENT ROBERT J. DIAZ DENISE L. BREITBURG 1. Importance of Oxygen and Hypoxia 2. Hypoxia Distribution and Causes 2.1. Where Hypoxia Occurs 2.2. Rise of Anthropogenic Influence on Oxygen Budgets 2.3. Oxygen Budgets and Global Climate Change 3. Hypoxia and Fish 3.1. Consequences for Fish 3.2. Consequences for Fish Habitat 4. Conclusions Low dissolved oxygen environments occur in a wide range of aquatic systems, and vary in temporal frequency, seasonality, and persistence. While there have always been naturally occurring low dissolved oxygen habitats, anthropogenic activities related primarily to organic and nutrient enrichment have led to increases in hypoxia and anoxia in both freshwater and marine systems. Lakes and coastal areas with seasonal stratification tend to be highly sensitive to the consequences of anthropogenic nutrient enrichment. Many systems that are currently hypoxic were not reported to have low dissolved oxygen concentrations when first studied. The rapid rise in the number of coastal hypoxic systems lagged about 20 years behind the increased use of industrial fertilizer. The future status of hypoxia and its consequences for fishes will depend on a combination of climate change (primarily from warming, and altered patterns for wind, currents, and precipitation) and land use change (primarily from expanded agriculture and nutrient loadings). If in the next 50 years humans continue to modify and degrade coastal systems as in previous years, human population pressure will likely be the main driving factor in spreading of coastal dead zones and climate change 1 Hypoxia: Volume 27 FISH PHYSIOLOGY Copyright # 2009 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(08)00001-0 2 ROBERT J. DIAZ AND DENISE L. BREITBURG factors will be secondary. Climate forcing, however, will tend to make systems more susceptible to development of hypoxia through direct eVects on stratification, solubility of oxygen, metabolism, and mineralization rates, particularly in lakes and semienclosed coastal areas. 1. IMPORTANCE OF OXYGEN AND HYPOXIA Oxygen is necessary to sustain the life of fishes and invertebrates dependent on aerobic respiration. When the supply of oxygen is cut oV or consumption exceeds resupply, dissolved oxygen (DO) concentrations can decline below levels required by most animal life. This condition of low DO is known as hypoxia; water devoid of oxygen is referred to as anoxic and can contain lethal concentrations of metabolic products of microbial anaerobic respiration. Thus hypoxia and anoxia diVer quantitatively in the availability of oxygen, as well as qualitatively in the presence of toxic compounds such as hydrogen sulfide. While many authors and water quality regulations focus on concentrations of DO below 2–3 mg O2/L as a threshold value for marine and estuarine environments, and 5–6 mg O2/L in some freshwater habitats, such arbitrary limits may be unsuitable when examining potential impacts of hypoxia on any one given species or on the way that oxygen concentrations aVect interactions among species. Species and life stages diVer in their basic oxygen requirements, and oxygen requirements increase as energy‐demanding metabolic processes are mobilized. Depending on temperature and salinity, water contains 20–40 times less oxygen by volume and diVuses about ten thousand times more slowly through water than air (Graham, 1990). This relatively low solubility and diVusion of oxygen in water combined with two principal factors lead to the development of hypoxia and anoxia. These factors are density stratification of the water column that isolates the bottom water from exchange with oxygen‐rich surface water and the atmosphere, and decomposition of organic matter in the isolated bottom water that consumes dissolved oxygen. The combination of these factors can allow hypoxia to develop and persist in deeper waters by causing oxygen consumption to exceed resupply. For lakes, factors aVecting vertical water mixing such as wind and temperature aVect seasonal changes in the DO depth profile and can lower DO in bottom waters (Green et al., 1973). Ice and snow cover on lakes and streams can also block photosynthesis and reaeration, and may lead to hypoxia and ‘‘winterkills’’ (Greenbank, 1945; Magnuson et al., 1985; Graham, 2006). In tropical freshwaters oxygenation is often greater in rainy seasons with more water flow than during dry stagnant water seasons (Val and Almeida‐Val, 1995; Graham, 2006). 1. 3 THE HYPOXIC ENVIRONMENT Because of the low solubility of oxygen in water small changes in the absolute amount of oxygen dissolved in water (resulting from microbial or macrofaunal respiration) lead to large diVerences in per cent air saturation. Thus what appear to be small changes in DO can have major consequences to animals living in an oxygen‐limited milieu. For example, 9.1 mg O2 will dissolve in a liter of freshwater at 20oC; at this temperature a 1 mg O2/L drop in oxygen is equivalent to an 11% decline in air saturation (Figure 1.1). Going from freshwater to seawater (35 psu) at the same temperature reduces air saturation to 7.2 mg O2/L (Benson and Krause, 1984). Some species are particularly sensitive to even small changes in oxygen concentrations. For example, for some salmonids, the limiting factor of DO becomes operative at relatively high values and even air saturation can be limiting at higher temperatures (Fry, 1971). Hypoxia has been a potent force in evolution. Air breathing and the ultimate evolution of terrestrial vertebrates is thought to have been an evolutionary response to low atmospheric and dissolved oxygen concentrations during the Devonian (Clack, 2007). Within aquatic environments, fishes have developed a wide range of mechanisms to secure more oxygen 12 8 350 10 300 6 4 SW 10⬚C 8 mg O2/L 150 mL O2/L µM 250 200 FW 10⬚C FW 30⬚C SW 30⬚C 6 4 100 2 2 50 0 0 0 0 20 0 40 60 80 % air saturation 50 100 100 150 120 200 PO2 (torr or mmHg) 0 5 10 15 20 25 PO2 (kPa) Fig. 1.1. Nomogram for dissolved oxygen in freshwater (FW) and seawater (SW) at 10 C and 30 C. (J. G. Richards, Unpublished data). 4 ROBERT J. DIAZ AND DENISE L. BREITBURG from their environment in situations where oxygen availability is critically low (Hoar and Randall, 1984; Brauner et al., 1995; Gonzales et al., 2006). The large number of hypoxia‐tolerant aquatic species, and wide variety of anatomical, physiological, and behavioral adaptations to hypoxia, indicate that after the great atmospheric oxygenation event some 2.3 billion years ago (Catling et al., 2001), low DO environments have played an important role in the evolution of many adaptive strategies (Guppy and Withers, 1999; Val, 2000; Bickler and Buck, 2007; see Chapters 2 to 9). 2. HYPOXIA DISTRIBUTION AND CAUSES 2.1. Where Hypoxia Occurs Oceanic oxygen minimum zones (OMZs) are the largest low DO areas on earth. OMZs form under areas of high surface productivity, which sinks and in the process of microbial metabolism oxygen is consumed (Figure 1.2). They are widespread and stable oceanic features occurring at intermediate depths (typically 400–1000 m), are particularly severe in regions of sluggish circulation, persist for long periods of time (at greater than decadal scales), and are controlled by natural processes and cycles (Wyrtki, 1966; Kamykowski and Zentara, 1990; Helly and Levin, 2004). Where OMZs contact the bottom, globally about a million square kilometers along the continental margins, specialized communities have evolved to survive at DO concentrations as low as 0.1 mg O2/L (Graham, 1990; Childress and Seibel, 1998; Levin, 2002; Helly and Levin, 2004). Upwelling areas can also develop extensive hypoxia as deep‐water nutrients are added to surface waters increasing production that eventually sinks and decomposes. Hypoxia associated with upwelling is not as long‐lived and stable as that associated with OMZs. Hypoxia is a natural component of many freshwater habitats such as swamps and backwaters that circulate poorly, stratify, and have large loads of terrestrial organic matter. Primary productivity, depth, and temperature are the main determinants of the degree of hypolimnetic oxygen depletion in lakes, with both naturally and culturally eutrophic lakes experiencing summer oxygen depletion (Cornett and Rigler, 1980; Wetzel, 2001). In addition, some deep, amictic oligotrophic lakes, like Lake Tanganyika (Coulter, 1967), develop year‐round hypoxia and anoxia gradually over time through sinking and decomposition of organic matter. Hypoxia is also common in reservoirs, and lateral variability of hypoxia tends to be greater in these systems than in lakes because of spatial variability in inflow, withdrawal, and loads of particulate organic matter (Thornton et al., 1990). Reservoirs are also more prone to metalimnetic oxygen minima, which are rare in lakes. 5 Concern Documented Improved OMZ areas Fig. 1.2. Global distribution of major OMZ areas and coastal hypoxic systems. Systems with documented hypoxia are red circles, areas of concern for being hypoxic are blue circles, and areas that have recovered from hypoxic conditions are yellow circles. Shading indicates the tropical regions (20 north and south of the equator) most likely to experience naturally low dissolved oxygen conditions. [Based on Diaz and Rosenberg (2008), Helly and Levin (2004), and Selman et al. (2008).] 6 ROBERT J. DIAZ AND DENISE L. BREITBURG Areas of naturally low DO in coastal marine systems are limited to fjord‐ like systems prone to water column stratification and deep depositional basins, such as Oslofjord, Norway (Karlson et al., 2002) or the central basin of the Black Sea, currently the largest pool of naturally occurring anoxic water on earth (Kideys, 2002). In shallow water, depending on the balance between production and respiration, a natural diel cycling of DO from supersaturation during the day to hypoxic or near anoxic during the night can occur. In highly productive systems, calm weather conditions and extended periods of cloud cover often exacerbate the problem. Water also becomes hypoxic on floodplains (Townsend and Edwards, 2003; Val et al., 2006), wetlands, and shallow embayments or margins of smaller systems with high productivity and restricted circulation. Among tropical habitats, coral reef crevices can become severely hypoxic at night owing to respiration of coral and associated organisms (Gonzales et al., 2006; Nilsson et al., 2007). During intertidal exposure organisms without adaptations for air‐breathing experience hypoxia along with hypercapnia (elevated CO2) (Warren, 1984; Burnett, 1997). 2.2. Rise of Anthropogenic Influence on Oxygen Budgets Eutrophication can be defined simply as the production of organic matter in excess of what an ecosystem is normally adapted to processing (Nixon, 1995), however, it is only part of a complex web of stressors that interact to shape and direct ecosystem level processes (Breitburg et al., 1998; Cloern, 2001). The primary driver of eutrophication in both freshwater and marine systems is excess nutrient enrichment, but physical conditions that limit reaeration are also necessary for the development of hypoxia. Thienemann (1926, in Cornett and Rigler, 1980) was one of the first to note that production and morphometry influence oxygen depletion. Phosphorus is generally the limiting nutrient in freshwater (Schindler, 1978) and increases in anthropogenic phosphorus have caused increased algal production and eutrophication in freshwater ecosystems worldwide even where human waste is treated or only a minor contributor to declining water quality (Carpenter et al., 1999; Smith, 2003, 2006). For marine systems the limiting nutrient tends to be nitrogen (Boesch, 2002). This basic diVerence is related to the physical properties of phosphorus and nitrogen compounds, and their biogeochemical cycling through the freshwater and marine environments. Eutrophication and associated hypoxia in freshwater systems became widespread in the mid–late 20th century, but eVective nutrient management has reversed this trend where it has been rigorously implemented (Jeppesen et al., 2005). In tidal portions of rivers and other water bodies near dense population centers, severe hypoxia and anoxia has been caused by discharge 1. 7 THE HYPOXIC ENVIRONMENT of raw sewage, which is high in both nutrients and organic matter. Areas devoid of fishes were reported at least as early as the late 1800s and persisted until improvements in sewage treatment were implemented (Jones, 2006). Much of the hypoxia and anoxia in shallow coastal marine and estuarine areas is recent in origin (Diaz and Rosenberg, 1995). These areas of hypoxia, commonly called dead zones (Rabalais et al., 2002), tend to be related to a combination of agriculture, human waste, and atmospheric deposition of nitrogen, which has led to a general eutrophication. Within the last 50 years, dissolved oxygen conditions of many shallow coastal ecosystems around the world have been adversely aVected by eutrophication (see Figure 1.2). As more organic matter was produced more oxygen was needed to remineralize the organics, primarily through the microbial loop, and as ecosystems became overloaded DO declined. The declining trend in dissolved oxygen lagged about 20 years behind increased use of chemical fertilizer after World War II (Figure 1.3). For European systems that have historical data from the early 1900s, declines in DO started in the 1950s and 1960s. However, declining dissolved oxygen levels were noted as early as the 1930s in the deep central basin of the Baltic Sea (Fonselius, 1969). Among marine systems with long‐term DO data, benthic hypoxia became a problem in the 1950s in the Baltic Sea proper (Fonselius, 1969), the 1960s in the northern Adriatic (Justić et al., 1987), the 1970s in the Kattegat (Baden et al., 1990), and the 1980s on the Northwest continental shelf of the Black Sea (Mee, 1992). Annual hypoxia does not appear to be a natural condition for marine waters except for those systems previously described. Even in Teragrams of nitrogen 150 Period of explosive increase of coastal eutrophication 400 Total reactive N 300 Industrially fixed (mainly fertilizer) 100 200 100 50 N-fixing crops Fossil fuel combustion 0 1900 1920 1940 1960 1980 2000 Number of hypoxic systems Cumulative number of hypoxic sites 200 0 Fig. 1.3. Relationship between fertilizer use and rise of dead zones. [Modified from Boesch (2002) and Diaz and Rosenberg (2008).] 8 ROBERT J. DIAZ AND DENISE L. BREITBURG Chesapeake Bay, which had hypoxia when DO measurements were first made in the 1910s in the Potomac River (Sale and Skinner, 1917) and 1930s in the mainstem channel (OYcer et al., 1984), the geological record suggests that low DO was not an annual, seasonally persistent feature of the system prior to European colonization (Cooper and Brush, 1991; Zimmerman and Canuel, 2000; Cronin and Vann, 2003). Geochronologies from the hypoxic area on the continental shelf of the northern Gulf of Mexico also indicate that the current seasonal hypoxia, which can cover over 20 000 km2, did not form annually prior to the 1950s (Sen Gupta et al., 1996). Hypoxia was recorded with the first DO measurement made in the area in the summer of 1973 on the central Louisiana continental shelf (Harper et al., 1981) and has been an annual event ever since. Geochronologies from both of these systems that go back over a 1000 years are at times punctuated by low DO markers that appeared aperiodically and likely marked major discharge events that led to low DO (Osterman et al., 2007). Recent research and monitoring suggests that once a system develops hypoxia, it can quickly become an annual event and a prominent feature aVecting energy flow (Elmgren, 1989; Pearson and Rosenberg, 1992; Baird et al., 2004). From the 1980s to the present, the number of systems reporting hypoxia has increased from <50 in 1960 to about 400 at present (Diaz and Rosenberg, 2008). Only in systems that have experienced intensive regulation of nutrient or carbon inputs have oxygen conditions improved, primarily from initiation of sewage treatment that at first removed organic matter and later from substantial upgrades in treatment level reduced nutrients. Examples include the Hudson River, New York, Delaware River, Pennsylvania‐ New Jersey, and the Mersey Estuary in England (Patrick, 1988; Brosnan and O’Shea, 1996; Jones, 2006). The northwest shelf of the Black Sea once experienced annual hypoxic events, but is now in a state of recovery largely due to the economic collapse of Eastern Europe in the early 1990s, which greatly reduced the use of fertilizer and subsequent nutrient loading in runoV. Within 3 years, the hypoxic area in the northwest shelf of the Black Sea went from a maximum area of about 40 000 km2 to none. While no hypoxic events were recorded on the shelf between 1993 and 2001, a full recovery of the Black Sea is far from certain. Climatic conditions caused a large hypoxic area to form during a warmer than average 2001 and expected recovery of farming in Eastern Europe will likely lead to increased nutrient loadings (Mee, 2006). Temporary improvements have also been seen in systems with changes in hydrology or nutrient inputs. In the northern Gulf of Mexico the size of the hypoxic area responds annually to Mississippi River discharge with low flow years having less hypoxia and high flow years more (Rabalais et al., 2007). Large‐scale meteorological events that disrupt stratification are also capable 1. 9 THE HYPOXIC ENVIRONMENT of reducing the area of hypoxia, as in the Gulf of Mexico (Rabalais et al., 2007) and the Gulf of Finland (Karlson et al., 2002). In most coastal marine systems and in many freshwater habitats, hypoxia appears to be a consequence of general ecosystem eutrophication. As a result, it is diYcult to separate the eVects of hypoxia from eVects of other symptoms of nutrient enrichment or other co‐occurring stressors (overfishing, habitat loss, contaminants) on ecosystem functioning (Cloern, 2001; Breitburg, 2002; Breitburg et al., in press). Nutrients are closely linked to a system’s secondary productivity and to a point enhance biomass and fisheries yield (Caddy, 1993, 2000; Nixon and Buckley, 2002). The general eVect of eutrophication to favor benthic species with opportunistic life histories and eliminate sensitive species leads to higher production of benthic invertebrates (prey resources) during normoxic periods, which can either become available or be lost to higher‐level predators depending on the severity and extent of hypoxia (Baird et al., 2004). Another critical point in a system’s trajectory of decline is the appearance of anoxia and associated H2S, which have the potential to produce mass mortality of both benthic and pelagic species. The positive eVect of nutrient enrichment on fisheries (i.e., total fisheries’ landings, not individual species) may last until as much as 40% of the bottom is aVected by hypoxia (Breitburg et al., 2009). The frequency and duration of hypoxic events vary among systems, over time, and with varying nutrient loads or organic accumulation. Hypoxia ranges from aperiodic events with years to decades between reoccurrences to a persistent year‐round feature that can last for years or centuries at a time. Dominant faunal responses diVer by type of hypoxia (Figure 1.4). Aperiodic Periodic >1 event per year Little mortality Diel 1 event per day Stressed Seasonal 1 event per year Mortality Persistent Event lasts most or all of year None to little macrofauna Increasing growth and reproductive impairment opportunistic feeding Fishes Sessile fauna Increasing avoidance Description <1 event per year, Mass mortality sometimes years between events Increasing mortality Hypoxia type Recovery Multi-year Hours to days Hours Annual Multi-year or none Fig. 1.4. Types of hypoxia and generalized faunal response. Sessile fauna is primarily macrobenthos. Arrows indicate direction of increased impact on fishes. Mortality in fishes is more likely from aperiodic hypoxia, with complete avoidance of persistent hypoxia. Physiological impairment and opportunistic feeding are greatest for periodic and diel hypoxia. 10 ROBERT J. DIAZ AND DENISE L. BREITBURG Aperiodic hypoxia, resulting from unusual or uncommon climate conditions, elicits the most dramatic response of mass mortality in sessile and, at times, mobile species. For benthic invertebrates, this dramatic response is due to the large numbers of sensitive species usually present prior to the hypoxic event. For example, the onetime hypoxic event in the New York Bight in 1976 caused mass mortality of many commercial and noncommercial species (Boesch and Rabalais, 1991). Many systems that now experience seasonal hypoxia started out with reports of aperiodic hypoxic events. Wind‐ or tidal‐ mixing periodically disrupts stratification and hypoxia in many systems lessening the eVects on sessile fauna and at times allowing mobile fanua to return. This form of periodic hypoxia, with several to many events per year, is also common on the edges of seasonal and persistent dead zones as currents or wind‐driven upwelling cause hypoxic bottom waters to move. The form of periodic hypoxia with the most frequent recurrences is diel cycling hypoxia, which appears to be common in shallow systems, and is driven by the balance between oxygen production during daylight and respiration at night. Seasonal hypoxia, typically occurring during summer or early autumn, is common and often causes mortality of benthos followed by benthic recolonization, as well as avoidance by mobile species with their return as oxygen concentrations increase. Persistent hypoxia that develops anoxia has the greatest eVect on benthic fauna by removing all habitat value of the bottom for extended periods of time. 2.3. Oxygen Budgets and Global Climate Change If in the next 50 years humans continue to modify and degrade coastal systems as in previous years (Halpern et al., 2008), human population pressure will likely continue to be the main driving factor in the persistence and spreading of coastal dead zones (Figure 1.5). Expanding agriculture for production of crops to be used for food and biofuels will result in increased nutrient loading and expand eutrophication eVects (EPA Science Advisory Board, 2007; Rabalais et al., 2007). Climate change, however, may make systems more susceptible to development of hypoxia through direct eVects on stratification, solubility of oxygen, metabolism, and mineralization rates. This will likely occur primarily though warming, which will lead to increased water temperatures, decreased oxygen solubility, increased organism metabolism and remineralization rates, and enhanced stratification. Changing temperatures will lead to spatial shifts in habitat suitability for fishes and will favor some species over others in a wide range of habitats. Warming may be particularly important to the development of hypoxia in lakes by leading to expanded periods of thermal stratification and deepening of thermoclines, which can lead to an increase in oxygen demand for aerobic decomposition, 1. 11 THE HYPOXIC ENVIRONMENT Land use Climate change More hypoxia Increasing Higher metabolism Increased stratification Lower solubility of water column of oxygen in water Higher temperature Nutrient inputs Increased storm intensity/frequency Decreasing Higher nutrient loading Higher runoff lower Decreased stratification of water column Lower nutrient loading Less hypoxia Fig. 1.5. Relative contribution of global climate change and land use to future hypoxia. Thickness of the arrows indicates relative magnitude of eVect. promote an upward flux of phosphorus from sediments, and thereby increase the concentration and amount of phosphorus in the hypolimnion (Magnuson and Destasio, 1996; Komatsu et al., 2007). Climate warming is, however, projected to lessen or eliminate winterkills in some lakes by reducing the period of ice cover in higher latitude lakes (Fang and Stefan, 2000; Fang et al., 2004). The future pervasiveness of hypoxia in all ecosystems will depend upon a combination of climate change and land management. Climate change will aVect water column stratification, organic matter production, nutrient discharges, and rates of oxygen consumption. Land management will also aVect the concentrations of nutrients through agriculture. General circulation models predict a decrease in the global oceanic oxygen inventory through increased stratification and warming (Keeling and Garcia, 2002), which may lead to expanding OMZs. Large changes in rainfall patterns are also predicted (IPCC, 2007). If these changes in rainfall lead to increased runoV to estuarine and coastal ecosystems, stratification and nutrient loads are likely to increase and worsen oxygen depletion (Justić et al. 2007). Conversely, if stratification decreases due to lower runoV or is disrupted by increased storm activity or intensity, the chances for oxygen depletion should decrease. For the Mississippi River basin associated with the northern Gulf of Mexico 12 ROBERT J. DIAZ AND DENISE L. BREITBURG seasonal dead zone, climate predictions suggest a 20% increase in river discharge (Miller and Russell, 1992) that would lead to elevated nutrient loading, a 50% increase in primary production, and expansion of the oxygen‐ depleted area (Justić et al., 1996). 3. HYPOXIA AND FISH Fishes respond to hypoxia through a wide range of physiological, anatomical, and behavioral adaptations that vary among species, life stages, and habitats (see Chapter 2). At the physiological level, critical adaptations include mechanisms to reduce metabolic rates and increase tolerance of ionic and pH disturbances during exposure to hypoxia, and mechanisms to reduce free‐radical damage during reaeration (reviewed in Bickler and Buck, 2007). Anatomical adaptations ranging from highly vascularized buccal cavities to lungs permit the use of atmospheric oxygen by fish residing in waters with low dissolved oxygen concentrations (Brauner et al., 1995; Randall et al., 2004; Soares et al., 2006). Changes in behavior can allow fishes to access more highly oxygenated environments (Kramer, 1987) and reduce the eVects of respiration by nearby individuals on local oxygen concentrations (Domenici et al., 2007). The stress of hypoxia can lead to a dramatic decrease in preferred temperature to gain physiological advantages associated with lower temperatures (Crawshaw and O’Connor, 1997). Fish species vary widely in their tolerance to low oxygen, from highly sensitive species to carp (Carassius spp.), which can survive months of hypoxia and 2 days of anoxia at low temperatures (reviewed in Nilsson and Renshaw, 2004; see Chapter 9). 3.1. Consequences for Fish EVects of hypoxia on fishes and adaptations of fishes to low oxygen environments have been longstanding areas of interest in research and management. By 1913, experiments established behavioral avoidance of low dissolved oxygen concentrations (Shelford and Allee, 1913) as well as variation among species and eVects of body size in tolerance to low oxygen (Wells, 1913). Studies of adaptations to low oxygen in swamps were already well underway in the 1930s (Carter and Beadle, 1931). Research in the 1940s examined eVects of winter hypoxia on fishes in north‐temperate lakes (e.g., Greenbank, 1945; Cooper and Washburn, 1949). Jones (1952) recognized that a lack of DO was a major hazard to fishes and that by the 1920s the literature on the eVects of oxygen deficiency on fishes was extensive (Gardner, 1926; Black, 1951). Davis (1975) reviewed oxygen requirements 1. THE HYPOXIC ENVIRONMENT 13 of Canadian freshwater and marine species with the intention of determining water quality criteria relative to minimum dissolved oxygen concentrations. These are some of the first works to point to the critical importance of DO, but it was not obvious that DO would become critical in shallow coastal marine systems until the 1970s and 1980s when large areas of low dissolved oxygen started to appear with associated mass mortalities of invertebrates and fishes (Diaz and Rosenberg, 1995). Before the 1950s, there were few reports of mass mortalities of marine animals related to lack of oxygen (Brongersma‐Sanders, 1957). However, in the 1940s, hypoxia‐driven migrations of mobile organisms to the edge of the water (Jubilee) were reported in Mobile Bay, Alabama (Loesch, 1960). In addition, there were a number of highly urbanized rivers and estuaries that were devoid of fishes by the late 1800s (Araújo et al., 2000; Jones, 2006). Behavioral responses of fishes to low DO concentrations have been well studied (Duque et al., 1988; Pihl et al., 1991; Plante et al., 1998; see Chapter 2). In general freshwater and marine fishes are capable of actively avoiding low DO water (e.g., Jones, 1952; Schurmann and SteVensen, 1992; Schurmann et al., 1998; Breitburg, 2002). However, the point at which various fishes initiate behavioral response to declining DO and eventually suVocate varies widely among species and habitats. For North American freshwater fishes 5 mg O2/L appeared to be a lower limit for maintaining a desirable riverine fish fauna (Jones, 1952). But for coldwater salmonids behavioral responses are initiated at 8 mg O2/L. Marine fishes avoid DO concentrations similar to those that reduce growth (Breitburg, 2002), but growth reductions related to hypoxia occur in the field as a result of imperfect avoidance, the energetic costs of avoiding hypoxia, and density‐dependent processes in normoxic parts of systems (Breitburg, 1992; Taylor and Miller, 2001; Perez‐Dominguez et al., 2006; StierhoV et al., 2006). Consequences of low DO are often sublethal and aVect growth (Stewart et al., 1967; Andrews et al., 1973; Pedersen, 1987), immune responses (Thomas et al., 2007), and reproduction (Wu et al., 2003; see Chapter 3). For example, cod from the northern Gulf of St. Lawrence may be less productive than other stocks not only because they live in cold water (Brander, 1995; Dutil et al., 1999), but also because deep waters in the northern Gulf of St. Lawrence are hypoxic and some segments of the cod stock are found in deep waters (D’Amours, 1993; Gilbert et al., 2005). Growth is a determinant of cod surplus production in the northern Gulf of St. Lawrence and factors that aVect growth such as DO need to be considered to better forecast stock status (Dutil et al., 1999). While the global distribution of coastal hypoxic zones is centered on major population centers or closely associated with developed watersheds that deliver large quantities of nutrients (Howarth et al., 1996), these same 14 ROBERT J. DIAZ AND DENISE L. BREITBURG areas have been major fishing grounds. The direct connection of hypoxia to fisheries’ landings at large regional scales is weak because of a number of factors that include confounding eVects of overfishing and compensatory mechanisms that alter or mask eVects of hypoxia on landings (Breitburg et al., 2009, in press). Both mobile species and fishers can distribute themselves to avoid low DO and utilize prey‐enriched areas (Breitburg, 2002; Eby and Crowder, 2004; Craig and Crowder, 2005). 3.2. Consequences for Fish Habitat When a system becomes hypoxic, fishes have to contend with loss of habitat and, for demersal feeders, loss of prey resources. The interaction of hypoxia with habitat requirements varies by species and life stage. Habitat compression or habitat ‘‘squeeze’’ can occur where hypoxia overlaps with nursery habitat or makes deeper, cooler water unavailable during the summer (Coutant, 1990). Spawning success of cod in the central Baltic is hindered by hypoxic and anoxic water below the halocline (70–80 m) where salinity is high enough to provide buoyancy for cod eggs (Nissling and Vallin, 1996; Cardinale and Modin, 1999). The elimination of benthic prey and compression of habitat by hypoxia also have profound eVects on ecosystem energetics as organisms die and are decomposed by microbes. Under certain circumstances demersal feeding fishes are able to utilize low DO‐stressed benthic prey. Over a narrow range of conditions, hypoxia can therefore facilitate the upward trophic transfer as physiologically stressed benthic fauna forced to the sediment surface during hypoxia may be exploited by predators (Pihl et al., 1992; Nestlerode and Diaz, 1998). Aggregation of demersal predators on the edge of dead zones may be a combination of responses that include flight from physiological stress and trophic advantage (Craig and Crowder, 2005). Thus, short‐lived and mild hypoxia may not have a net negative eVect on trophic transfer as does severe seasonal hypoxia. This low DO‐associated facilitation is most prevalent under diel cycling hypoxic conditions (see Figure 1.4). Diel cycling hypoxia may be a common phenomenon to which fishes respond in an opportunistic manner (D’Avanzo and Kremer, 1994; Layman et al., 2000; Smith and Able, 2003; Tyler and Targett, 2007). In Delaware coastal creek systems, fish emigrated only when DO dropped to very low values and returned quickly with improving DO (Ross, 2003; Tyler and Targett, 2007). Acoustic tagging of juvenile weakfish in Pepper Creek, Delaware, demonstrated that these fish track the dynamics of DO, moving down the creek as DO falls and returning as DO rises (Brady and Targett, unpublished data). While it may be physiologically stressful for juvenile fishes to remain in or near low DO water, the gains in trophic 1. THE HYPOXIC ENVIRONMENT 15 resources and added protection from predation may be greater and diel hypoxic areas can still serve as important nursery habitats. In areas where hypoxia is intermittent and does not cause substantial mortality of the benthos, the behavior of benthos may facilitate energy transfer to oxygen‐ tolerant bottom feeding fish, which are physiologically capable of withstanding short‐term exposure to DO levels to take advantage of weakened benthic prey and receive an energetic gain (Diaz et al., 1992; Pihl et al., 1992; Nestlerode and Diaz, 1998; Seitz et al., 2003). Upward energy transfer is inhibited in areas where hypoxia is severe as either benthic resources are killed directly and/or predators capable of detecting low DO would avoid the area. In all cases, the increase in the proportion of production transferred to predators is temporary and as mortality of benthos occurs, microbial pathways quickly dominate energy flows (Baird et al., 2004). This energy diversion tends to occur in ecologically important places and at the most inopportune time for predator energy demands, causes overall reduction in ecosystems’ functional ability to transfer energy to higher trophic levels, and renders ecosystems less resilient to other stressors. 4. CONCLUSIONS Hypoxia occurs in a wide range of aquatic systems and varies in temporal frequency, seasonality, and persistence. The oxygen minimum zones of the world’s oceans as well as deep basins of permanently stratified lakes and semienclosed seas represent large expanses of severely hypoxic or anoxic environments that exclude all but the most highly specialized fish species and their prey. In these systems, low oxygen is primarily a physically driven process. Persistent stratification prevents reaeration of the lower water column and allows microbial respiration to deplete dissolved oxygen even where anthropogenic enhancement of nutrient loads is low. In temperate latitudes, bottom waters can remain hypoxic or anoxic for hours to months during summer and autumn, with oxygen concentrations fluctuating with tides, winds, and depth during that period. Hypoxia also develops in shallow waters of organically enriched systems, with oxygen concentrations fluctuating in a diel cycle dependent on the balance between respiration and photosynthesis, and minimum concentrations varying among days depending on cloud cover, wind mixing, and temporal variability in phytoplankton and macrophyte biomass. A diVerent seasonality characterizes the development of hypoxia in ‘‘winterkill’’ lakes where ice cover prevents reaeration and snow pack reduces light available for photosynthesis and oxygen generation during the coldest 16 ROBERT J. DIAZ AND DENISE L. BREITBURG parts of the year. Floodplains that develop on a seasonal basis or as a result of storm‐related flooding can create large expanses of habitat that physically expand the boundaries of the aquatic realm, but are often characterized by low oxygen concentrations, particularly where they greatly increase loadings of nutrients from crop and livestock agriculture. There has been a growing appreciation of the importance of low oxygen in microhabitats such as coral reef crevices and burrows in selection for hypoxia tolerance. In addition, fish that utilize intertidal habitats may experience environmental hypoxia in tide pools, and like fishes inhabiting other shallow, chronically hypoxic habitats, many possess adaptations for aerial respiration, or physiological mechanisms to deal with hypoxia and hypercapnia if they remain above the tide line. Finally, under some circumstances fish can deplete oxygen concentrations in embayments and similar habitats with limited circulation. In general, freshwater systems are more prone to hypoxia and anoxia, with a long history of occurrence. Lakes and coastal areas with seasonal stratification tend to be highly sensitive to anthropogenic nutrient enrichment. Many of the systems that are currently hypoxic were not when first studied. The rapid rise in the number of coastal hypoxic systems lagged about 20 years behind the increase in the use of industrial fertilizer (see Figure 1.3), which led to a general eutrophication of many freshwater and marine systems. Recovery of a system from a hypoxic event involves two components for fishes: physical habitat value and trophic value. Unless the physical structure of a habitat is biological and perished during hypoxia, the habitat value of an area returns once hypoxia and other toxic compounds, such as H2S, dissipate. The issue of trophic value is more complex. Generally, hypoxia favors enhanced diversion of energy flows into microbial pathways to the detriment of higher trophic levels. But under certain circumstances hypoxia‐enhanced trophic transfer to fishes may occur with periodic hypoxia, diel hypoxia, and along the edges of seasonal hypoxia. For juvenile fishes there may be a benefit to occupying physiologically stressful habitats that are subjected to diel hypoxia as the gains in trophic resources and added protection from predation may be greater and diel hypoxic areas can still serve as important nursery habitats. The future status of hypoxia and its consequences for fishes will depend on a combination of climate change (primarily from warming, and altered patterns for wind, currents, and precipitation) and land use change (primarily from expanded agriculture and nutrient loadings). 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Physiological performance of juvenile southern flounder, Paralichthys lethostigma (Jordan and Gilbert, 1884), in chronic and episodic hypoxia. J. Exp. Mar. Biol. Ecol. 258, 195–214. Thienemann, A. (1926). Der Nahrungskreislauf im Wasscr. Vcrh. Dtsch. Zool. Ges. 2, 29–79. Thomas, P., Rahman, M. S., Khan, I. A., and Kummer, J. A. (2007). Widespread endocrine disruption and reproductive impairment in an estuarine fish population exposed to seasonal hypoxia. Proc. R. Soc. Biol. Sci. 274, 2693–2701. Thornton, K. W., Kimmel, B. L., and Payne, F. E. (1990). ‘‘Reservoir Limnology: Ecological Perspectives.’’ Wiley, New York. Townsend, S. A., and Edwards, C. A. (2003). A fish kill event, hypoxia and other limnological impacts associated with early wet season flow into a lake on the Mary River floodplain, tropical northern Australia. Lakes Reservoirs: Res. Manag. 8, 169–176. Tyler, R. M., and Targett, T. E. (2007). Juvenile weakfish Cynoscion regalis distribution in relation to diel‐cycling dissolved oxygen in an estuarine tributary. Mar. Ecol. Prog. Ser. 333, 257–269. Val, A. L., Paula da Silva, M. N., and Almeida-Val, V. M. F. (2006). Environmental eutrophication and its effects on fish of the Amazon. In ‘‘Proceedings of the Ninth International Symposium on Fish Physiology, Toxicology and Water Quality’’, April 24–28, 2006, Hotel San Michele, Capri, Italy (Brauner C. J., Suvajdzic, K., Nilsson, G., and Randall, D. J., Eds.), pp 65-72, EPS/600/R-7/010. United States Environmental Protection Agency, Environmental Research Laboratory, Athens, Georgia. Val, A. L. and Almeida-Val, V. M. F. (1995). ‘‘Fishes of the Amazon and their Environment: Physiological and Biochemical Aspects.’’ Springer-Verlag, Berlin. Val, V. (2000). Evolutionary features of hypoxia tolerance in fish of the Amazon: from molecular to behavioral aspects. In ‘‘Evolution of Physiological and Biochemical Traits in Fish. 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A geochemical record of eutrophication and anoxia in Chesapeake Bay sediments: Anthropogenic influence on organic matter composition. Mar. Chem. 69, 117–137. 2 BEHAVIORAL RESPONSES AND ECOLOGICAL CONSEQUENCES LAUREN J. CHAPMAN DAVID J. MCKENZIE 1. Introduction 2. Aquatic Surface Respiration and Air-Breathing 2.1. Aquatic Surface Respiration 2.2. Air-breathing 3. Effects of Hypoxia on Activity 3.1. Spontaneous Swimming Activity 3.2. Other Locomotor Responses to Hypoxia 4. Hypoxia and Parental Care Behavior 5. Hypoxia and Ecological Interactions 5.1. Hypoxic Refugia from Piscine Predators 5.2. A Shift in the Beneficiary – Increased Prey Vulnerability under Hypoxia Stress 5.3. When Prey Becomes Predator: Hypoxia and Fish–Invertebrate Interactions 5.4. Hypoxia and Social Interactions 6. Summary Fishes employ many and diverse strategies to increase oxygen transfer from the environment to their tissues and/or avoid problems associated with hypoxia. Some of these responses can be activated quickly (e.g., hours, days), whereas others are developmentally plastic and/or genetically fixed. Shortterm physiological and biochemical responses provide regulatory mechanisms to deal with variable oxygen in habitats. Behavioral responses can provide additional flexibility to mitigate exposure to hypoxic stress, and many occur at levels of aquatic oxygen availability far higher than lethal levels. Fish may avoid hypoxic areas through movement or they may compensate for hypoxia through air-breathing or aquatic surface respiration (ASR), ventilating their gills with water from the air–water interface. As a result, behavioral responses to hypoxia can influence other critical 25 Hypoxia: Volume 27 FISH PHYSIOLOGY Copyright # 2009 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(08)00002-2 26 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE components of the behavior of fishes in their environment, including habitat use and selection, predator–prey interactions, competitive interactions, and patterns of aggregation. In this chapter, we review behavioral responses to hypoxia, including aquatic surface respiration, air-breathing, changes in spontaneous activity, and parental care. We then consider the role of hypoxia in modulating ecological interactions, in particular the interaction between predator and prey. Hypoxic alterations to predator–prey interactions can influence other components of the food web and assemblage structure; so predicting whether predator or prey is the beneficiary of hypoxic stress is fundamental to understanding community level impacts of hypoxia, whether natural or anthropogenically induced. 1. INTRODUCTION The abiotic environment has had a major influence on the ecology and evolution of organisms. For fishes, the availability of dissolved oxygen (DO) is one physicochemical factor that can limit habitat quality, distribution, growth, reproduction, and survival. All fishes require oxygen for long-term survival; however, the physical properties of water (high viscosity, low oxygen content at saturation) make oxygen uptake challenging for fishes even at high DO levels. In addition to these constraints imposed by the physical properties of water, there are many habitats in which dissolved oxygen is depressed below saturation periodically or chronically. Hypoxia occurs naturally in habitats characterized by low mixing or light limitation, such as heavily vegetated swamps, flooded forests, floodplain lakes and ponds, ephemeral pools, spring boils, and the profundal waters of deep lakes; it is particularly widespread in tropical waters where high temperatures elevate rates of organic decomposition and reduce oxygen tensions and contents (Kramer, 1983; Chapman and Liem, 1995; Chapman et al., 1999; see Chapter 1). Hypoxic (and anoxic) environments have existed through geological time, but human environmental degradation is increasing the occurrence of hypoxia, as influxes of municipal wastes and fertilizer runoVs accelerate eutrophication and pollution of water bodies (Diaz, 2001). Increasing hypoxia is now recognized as an environmental issue of global importance to fresh and coastal waters, which can result in changes in species composition, population decline, mass mortalities, and production of extensive ‘‘dead zones,’’ such as that in the Gulf of Mexico and Lake Erie that can aVect important fisheries (Diaz, 2001; Dybas, 2005; Pollock et al., 2007). Thus, it has become increasingly important to understand the consequences of hypoxic stress on the behavior and ecology of aquatic organisms, and so to predict cascading eVects of hypoxia on community function. 2. BEHAVIORAL RESPONSES TO HYPOXIA 27 Fishes have evolved a variety of solutions to hypoxic stress, including morphological adaptations, physiological adjustments, and biochemical and molecular defenses (see Chapters 5, 6, 7, 9, and 10). Behavioral responses provide fishes with additional flexibility in responding to temporal and spatial variation in DO, through avoidance of hypoxic zones, changes in activity level, use of aquatic surface respiration, and, when possible, changes in air-breathing frequency (Kramer, 1987; Timmerman and Chapman, 2004). These behavioral responses to hypoxia can interact with other physiological and biochemical adjustments to alter crucial components of the interactions between a fish and its environment, in particular predator–prey interactions, schooling behavior, dominance, aggression, and parental care. Given the growing incidence of anthropogenically induced hypoxia in aquatic environments, there is accelerating interest in predicting levels at which hypoxia becomes ecologically active and its role in modifying ecological interactions. In this chapter, we address behavioral responses to hypoxia and associated changes in spatial and temporal distributions, density, and ecological interactions. We begin by reviewing two major behavioral mechanisms for increasing oxygen uptake (aquatic surface respiration and air-breathing) and the ecological consequences of these behaviors that reflect costs and benefits of surfacing. We then consider eVects of hypoxia on a major component of the energy budget of active fishes—swimming. Many fishes exhibit changes in spontaneous swimming activity when exposed to hypoxia; the literature indicates these responses can comprise either reductions or increases in activity, as a function of species and context. Another significant metabolic cost for some fish species is the energy directed to parental care. Costs of parental care may be particularly severe under hypoxia, although high levels of parental care may actually be necessary under hypoxic conditions, to ensure survival of the eggs and young. We explore the degree to which DO is a driver of parental care behavior across a range of strategies, from guarding to viviparity, and the ability of fishes to modify their care behaviors in response to variations in oxygen availability Finally, we integrate behavioral responses to hypoxia and the relative tolerance of species, to develop a picture of how hypoxia influences species interactions, in particular predator–prey relationships, clearly an area of growing concern given the global increase in hypoxia. Whether hypoxia favors the predator or the prey depends, at least in part, on the relative tolerance of the interactants and can ultimately influence other components of the food web and assemblage. Thus predicting whether the prey or the predator is the beneficiary of hypoxic stress is critical for understanding community level impacts of hypoxia, for predicting response to anthropogenically induced hypoxia, and for setting habitat conservation or restoration objectives in the context of rehabilitation or management policy. 28 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE 2. AQUATIC SURFACE RESPIRATION AND AIR-BREATHING Aquatic surface respiration (ASR) and air-breathing are two of the most pronounced behavioral responses by bony fishes to aquatic hypoxia. Neither behavior has been described in more plesiomorphic fish groups, the agnathans, elasmobranches, or chondrichthyans; but air-breathing is found in a number of primitive bony fishes and in the ancient crossopterygians (Randall et al., 1981a; Graham, 1997; Reid et al., 2006; McKenzie et al., 2007a). 2.1. Aquatic Surface Respiration As the name implies, aquatic surface respiration (ASR) involves rising to the surface and ventilating the layer of water in contact with the atmosphere, which is richer in DO than the underlying bulk water (Kramer and McClure, 1982). Many teleost species have evolved this behavioral response, in freshwater and marine environments, both temperate and tropical (Lewis, 1970; Gee et al., 1978; Kramer and McClure, 1982; Kramer, 1983; Gee and Gee, 1995; Nordlie 2006; McNeil and Closs, 2007). A number of species have morphological features, such as upturned mouths and flattened heads, which appear to improve the eYciency of ASR (Lewis, 1970; Cech et al., 1985). In some species, the morphological adaptations are very pronounced such as the dermal lip protuberances in various tropical teleosts (Saint-Paul 1984; Winemiller 1989; Reid et al., 2006). For example, in the Neotropical tambaqui Colossoma macropomum, hypoxia causes the lower lip to swell extensively, to form a funnel that skims the surface layer of water into the mouth (Val and Almeida-Val, 1995; Sundin et al., 2000; Figure 2.1). Chapman et al. (1994) found evidence to suggest that inverted swimming in the upside-down catfish Synodontis nigriventris may also increase the eYciency of ASR. Many species also hold an air bubble (or bubbles) in their mouth when they perform ASR, which may have a dual role of increasing oxygen levels in the bucco-opercular cavity, and maintaining the fish buoyant at the water surface (Burggren, 1982; Gee and Gee, 1991, 1995; Chapman et al., 1995). Table 2.1 provides a review of species for which ASR responses have been observed and for which hypoxic oxygen partial pressure (PO2) thresholds for the behavior have been determined. There is a great deal of variability in the thresholds. A higher threshold can be indicative of a lower specific tolerance of hypoxia, because it has been directly associated with a higher threshold for regulation of basal aerobic metabolic rate in hypoxia (the critical oxygen tension PO2, Pcrit) in comparative studies over a fairly wide range of species (Chapman et al., 1995, 2002; Rosenberger and Chapman, 2000; Melnychuk and Chapman, 2002; Schofield et al., 2007; Table 2.1). Figure 2.2 shows the 2. BEHAVIORAL RESPONSES TO HYPOXIA 29 Fig. 2.1. Morphological adaptations for aquatic surface respiration in the tambaqui, Colossoma macropomum. (A) Lateral view of the head of a tambaqui approaching the water surface illustrating the initial swelling of the lower lip following exposure to an hypoxic water PO2 of 2.0 kPa; (B) a dorsal view of the same fish following 3.5 h exposure to the same level of hypoxia, illustrating the forward expansion of the lip near full development. From Sundin et al. (2000) with permission from the Company of Biologists. profile of the ASR response as a function of water PO2 in three tropical teleosts as described by Kramer and McClure (1982). This response profile, where there is no ASR until quite a discrete threshold at a relatively low PO2, beyond which it rapidly becomes the dominant behavior, has since been described in the vast majority of species that exhibit the ASR response. Gee et al. (1978) demonstrated that the PO2 threshold at which 50% of fathead minnows, Pimephales promelas, performed ASR was directly related to acclimation temperature, rising from approximately 1 kPa (7 mmHg) at 6 C to approximately 6 kPa (28 mmHg) at 31 C. Kramer and Mehegan (1981) also demonstrated that ASR activity increased with increasing water temperature in the guppy Poecilia reticulata. Sloman et al. (2006; 2008) found that the threshold for ASR increased with body mass in the oscar Astronotus ocellatus and the tidepool sculpin Oligocottus maculosus (Table 2.1). Prior acclimation to hypoxia (lab-induced or field conditions) has been shown to lower the ASR threshold in a wide variety of species (Kramer and Mehegan, 1981; Olowo and Chapman, 1996; Melnychuk and Chapman, 2002; Chapman et al., 2002; Timmerman and Chapman, 2004; Table 2.1). This is associated with changes to other physiological traits indicative of improved hypoxia tolerance, including increased gill surface area, increased haematocrit, and lower Pcrit (Olowo and Chapman, 1996; Melnychuk and Chapman, 2002; Chapman et al., 2002; Martinez et al., 2004). ASR thresholds have also been demonstrated to vary with season. Love and Rees (2002) found seasonal diVerences in ASR thresholds and tolerance of hypoxia in Fundulis grandis, whereby thresholds were lower and tolerance greater in summer. Although ASR is clearly a behavioral response to hypoxia, it is in fact a reflex that is driven by oxygen-sensitive chemoreceptors (Shingles et al., 2005; Table 2.1 Oxygen partial pressure thresholds (kPa) at which various species of fish perform aquatic surface respiration (ASR), reported as the percentage proportion of individuals performing the behavior, and thresholds for the regulation of routine metabolic rate (Pcrit, in kPa), as reported by various authors Forklength (mm) Temp ( C) 63–114 16.5  1 26–88 63  11 73  5 16.5  1 22 22 2.74 1.41 2.57 2.08 0.95 1.65 0.58 4–28 23 3.33 2.26 1.47 2.78–3.39 25  1 2.24 Hyphessobrycon pulchripinnis Metynnis sp. 0.53–0.64 25  1 3.55 2.50 1.69 25  1 3.68 2.76 Paracheirodon axelrodi 0.51 25  1 2.50 1.97 Paracheirodon innesi 0.15–0.22 25  1 1.97 1.18 Roeboides guatemalensis 0.26–0.49 25  1 2.89 2.24 Cichlidae Aequidens coeruleopunctatus 2.37–5.02 25  1 1.97 Species Catostomidae Catostomus commersoni Centrarchidae Ambloplites rupestris Lepomis gulosus Lepomis marginatus Centropomidae Lates niloticus Characidae Gymnocorymbus sp. Mass (g) 4.0  2.6 6.1  1.1 10% ASR 50% ASR 90% ASR Pcrit Source Gee et al. (1978)a 1.79 Gee et al. (1978)a Schofield et al. (2007) Schofield et al. (2007) 3.51 Chapman et al. (2002) 2.24 Kramer and McClure (1982)b Kramer and McClure (1982)b Kramer and McClure (1982)b Kramer and McClure (1982)b Kramer and McClure (1982)b Kramer and McClure (1982)b 1.71 Kramer and McClure (1982)b Astatoreochromis alluaudi Astatotilapia ‘‘wrought-iron’’ Astatotilapia aeneocolor 3.9  1.0 64  6 23–26 2.64 1.74 1.30 1.9–10.1 18–20 1.97 1.30 0.62 1.78 3.6–7.1 18–20 2.02 1.50 0.98 1.97 23 3.62 2.55 1.53 1.71 0.79 42  10 Astatotilapia velifer Astronotus ocellatus 2.05–4.08 25  1 0.92 Astronotus ocellatus Astronotus ocellatus Cichlasoma biocellatum 16.2  9 230  11 2.92–4.60 28  1 28  1 25  1 2.93  0.49e 6.52  1.29e 1.71 1.45 Cyprichromis leptosoma Haplochromis ‘‘rock kribensis’’ Hemichromis bimaculatus Hemichromis letourneuxi Labrochromis ishmaeli Neochromis nigricans Neolamprologis tretocephalus Oreochromis esculentis Oreochromis niloticus Prognathochromis perrieri 8.8  2.0 3.4  1.4 23–26 23–26 1.59 1.92 1.05 1.38 25  1 2.23 1.71 96  5 58  9 3.59–4.06 Chapman et al. (1995) 9.00 6.66 1.10 4.6  2.6 3.9  1.0 5.4  1.5 4.1  0.6 63  11 68  6 72  6 60  2 25 23–26 23–26 23–26 0.80 2.24 1.32 3.53 0.49 1.18 1.00 2.26 0.21 0.20 0.39 21.2  5.1 40.1  9.1 5.8  2.0 111  9 137  11 79  8 23–26 23–26 23–26 0.97 4.64 2.00 0.80 0.91 1.45 0.67 0.59 0.92 Melnychuk and Chapman (2002) Melnychuk and Chapman (2002) Rosenberger and Chapman (2000) Kramer and McClure (1982)b Sloman et al. (2006) Sloman et al. (2006) Kramer and McClure (1982)b Chapman et al. (1995) Chapman et al. (1995) Kramer and McClure (1982)b Schofield et al. (2007) Chapman et al. (1995) Chapman et al. (1995) Chapman et al. (1995) Chapman et al. (1995) Chapman et al. (1995) Chapman et al. (1995) (continued) Table 2.1 (continued ) Species Mass (g) Forklength (mm) Temp ( C) 10% ASR 50% ASR 90% ASR Pcrit 75  11 23 2.57 1.83 1.14 1.58 59  8 23 1.96 1.11 0.72 1.05 53  6 60  2 62  9 23–26 23–26 23–26 2.24 3.88 2.72 1.74 2.50 2.29 0.95 1.97 2.17 0.34–0.37 25  1 3.15 3.15 Kramer and McClure (1982)b 0.50–0.70 25  1 1.97 1.18 Kramer and McClure (1982)b Olowo and Chapman (1996) Olowo and Chapman (1996) Kramer and McClure (1982)b Kramer and McClure (1982)b Kramer and McClure (1982)b McNeil and Closs (2007) Gee et al. (1978)a McNeil and Closs (2007) Prognathochromis venator Pseudocrenilabrus multicolor Pyxichromis orthostoma Tropheus moorii Yssichromis argens 1.9  4.4 4.1  0.6 2.7  1.6 Cobitidae Botia sidthimunki Cyprinidae Barbus everetti Barbus neumayeri c 68 – 91 20  1 2.94 1.53 0.97 Barbus neumayeri d 68–91 20  1 1.58 0.68 0.26 Barbus nigrofasciatus 0.21–0.40 25  1 3.42 2.89 Barbus schwanenfeldi 18.66 25  1 2.24 1.97 Brachydanio albolineatus 0.65–1.04 25  1 3.16 2.50 25 1.99 16.5  1 25 1.99 Carassius auratus Chrosomus eos Cyprinus carpio Source 44–51 0.50 1.25 0.50 Rosenberger and Chapman (2000) Rosenberger and Chapman (2000) Chapman et al. (1995) Chapman et al. (1995) Chapman et al. (1995) Hybognathus hankinsoni Labeo bicolor Nocomis biguttatus Notropis atherinoides Notropis cornutus Notropis hudsonius Pimephales promelas Rasboro taeniata 54–61 0.21–0.29 64–112 51–76 38–51 63–112 40–63 0.28–0.38 16.5  1 25  1 16.5  1 16.5  1 16.5  1 16.5  1 16.5  1 25  1 2.76 1.49 2.24 3.95 1.80 2.05 2.11 2.26 1.80 2.89 2.89 1.99 1.05 1.84 1.04 2.24 Rhinichthys atratulus Rhinichthys cataractae Semotilus atromaculatus Semotilus margarita Roeboides guatemalensis 0.26–0.49 16.5  1 16.5  1 16.5  1 16.5  1 25  1 Cyprinodontidae Epiplatys dageti 0.70–1.31 25  1 5.39 1.97 Rivulus hartii 2.91–4.19 25  1 2.89 1.97 25 2.74 63–76 45–78 63–89 88–115 Eleotridae Hypseleotris sp. Esocidae Esox lucius 89–115 16.5  1 Gee et al. (1978)a Kramer and McClure (1982)b Gee et al. (1978)a Gee et al. (1978)a Gee et al. (1978)a Gee et al. (1978)a Gee et al. (1978)a Kramer and McClure (1982)b Gee et al. (1978)a Gee et al. (1978)a Gee et al. (1978)a Gee et al. (1978)a Kramer and McClure (1982)b Kramer and McClure (1982)b Kramer and McClure (1982)b 1.25 0.8 McNeil and Closs (2007) Gee et al. (1978)a (continued) Table 2.1 (continued ) Species Mass (g) Forklength (mm) Galaxidae Galaxias rostratus 25 38–53 31–38 Culaea inconstans Pungitus pungitus Hemiodidae Hemiodopis sp. Mormyridae Gnathonemus victoriae Petrocephalus catostoma 76–115 38–64 Percidae Etheostoma exile Etheostoma nigrum 50% ASR 16.5  1 16.5  1 Pcrit <2.50 5.49 2.89 90% ASR Source McNeil and Closs (2007) 2.46 6.57 Gee et al. (1978)a Gee et al. (1978)a 2.76 Kramer and McClure (1982)b 1.35 0.45 Gee et al. (1978)a Gee et al. (1978)a 2.09–2.63 25  1 0.79 0.79 Kramer and McClure (1982)b 1.31 25  1 1.97 1.97 Kramer and McClure (1982)b 1–12 1–4 23 23 1.94 1.71 1.43 0.40 20 3.00e Mugilidae Liza aurata Mugil cephalus Mugil cephalusf 10% ASR 16.5  1 16.5  1 25  1 8.90 Ictaluridae Ictalurus melas Noturus gyrinus Mastacembelidae Mastacembelus circumcinctus Mochokidae Synodontis nigrita Temp ( C) 212  33 565  38 565  38 31–63 37–50 0.46 1.65 1.51 Chapman et al. (2002) Chapman et al. (2002) 24 24 2.53 1.04 Lefrançois and Domenici (2005) Shingles et al. (2005) Shingles et al. (2005) 16.5  1 16.5  1 1.61 2.21 Gee et al. (1978)a Gee et al. (1978)a Perca flavescens Perca fluviatilis 50–127 Percina maculata 51–58 Percichthyidae Nannoperca australis Pimelodidae Pimelodella picta 1.38–1.41 Poeciliidae Gambusia holbrooki 16.5  1 25 1.54 <2.50 5.49 16.5  1 1.09 25 2.3 25  1 2.24 25 5.49 0.60 0.96–2.05 25  1 4.47 2.76 Xiphophorus helleri 0.72–1.36 25  1 2.89 2.50 25 6.23 25  1 4.21 Retropinnidae Retropinna semoni Siluridae Krytopterus bicirrhus a 0.61–0.62 3.68 McNeil and Closs (2007) Kramer and McClure (1982)b 1.97 Poecilia sphenops Gee et al. (1978)a McNeil and Closs (2007) Gee et al. (1978)a 1.05 McNeil and Closs (2007) Kramer and McClure (1982)b Kramer and McClure (1982)b 3.49 McNeil and Closs (2007) Kramer and McClure (1982)b Gee et al. (1978) found that Oncorhynchus mykiss, Salvelinus alpinus, Coregonus clupeaformis (Salmonidae), and Stizostedion vitreum (Percidae) did not perform ASR at any water PO2. b These values are not thresholds but refer to the PO2 at which between 1 and 3 fishes of each species performed ASR 10% or 50% of the time (Kramer and McClure, 1982). c Fish collected from relatively normoxic riverine ecotones (Olowo and Chapman, 1996). d Fish collected from hypoxic swamp ecotones (Olowo and Chapman, 1996). e Mean ( SEM) hypoxic PO2 at which fish broke the surface for the first time. f Same fish as the line above, but in the presence of a model avian predator. 36 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE 100 80 Pterophyllum 60 40 20 Percent time 80 Copella 60 40 20 80 Osteoglossum 60 40 20 0 0 3 6 9 12 15 18 21 PO2 (kPa) Fig. 2.2. The relationship between bulk water PO2 and percentage time spent performing aquatic surface respiration (ASR) in three species of tropical freshwater fish. Dotted lines were fitted by eye and drawings show posture adopted by the fish during ASR. Figure redrafted from Kramer and McClure (1982) with permission from Elsevier. Florindo et al., 2006). That is, when a fish senses that oxygen is low or limiting, they perform the behavior. Shingles et al. (2005) used the oxygenchemoreceptor stimulant sodium cyanide (NaCN) to demonstrate that the ASR response is elicited by chemoreceptors sensitive to oxygen levels in the 2. BEHAVIORAL RESPONSES TO HYPOXIA 37 ventilatory water stream and the blood stream of flathead grey mullet (Mugil cephalus). Florindo et al. (2006) demonstrated that the chemoreceptors that stimulate ASR in the tambaqui are innervated by cranial nerves that serve the bucco-opercular cavity and gills. These chemoreceptor sensory modalities and innervations would appear to be homologous, therefore, to those that drive reflex gill hyperventilation in all fish groups studied to date (Burleson et al., 1992; Taylor et al., 1999; see Chapter 5), including species that appeared in the fossil record prior to teleosts (McKenzie et al., 1995a; McKendry et al., 2001). Thus, ASR may use the pre-existing sensory arm of such hypoxic ventilatory reflexes, integrating a new motor output that involves rising to the water surface to ventilate the surface layer. Presumably, cessation of this behavior is also driven by information from the same chemoreceptors (Shingles et al., 2005). Gill hyperventilation chemoreflexes in fish are mediated by the medulla, or hindbrain, which contains the central respiratory pattern generator (Taylor et al., 1999). Clearly, ASR is a much more complex chemoreflex with a very large behavioral component, which must involve significant inputs from higher brain centres (Shingles et al., 2005; Sloman et al., 2006, 2008). Teleost fish also exhibit behavioral modulation of gill ventilation patterns (Johnsson et al., 2001), and such higher-order inputs to the respiratory medulla must, presumably, have been a prerequisite for the evolution of the complex ASR motor responses. The widespread prevalence of the ASR response, and the evolution of specific morphological adaptations for increasing the eYciency of this behavior, must be considered an indicator that ASR provides a physiological advantage to fishes that inhabit periodically or chronically hypoxic habitats (Kramer and McClure, 1982; Kramer, 1987). Nonetheless, there exists no direct quantitative demonstration that ASR increases the ability of fish to regulate their aerobic metabolic rate independent of bulk water oxygen availability in hypoxia. As stated above, the behavior typically shows a sudden increase in prevalence below a relatively discrete threshold water PO2 (Figure 2.2 and Table 2.1), and this threshold is often quite close to the Pcrit, which indicates that the behavior only begins when oxygen becomes physiologically limiting in the bulk water (Table 2.1). Rantin et al. (1998) found that the threshold for initiation of ASR in the pacu, Piaractus mesopotamicus, was slightly below their Pcrit (Table 2.1). Rosenberger and Chapman (2000), Chapman et al. (2002), and Melnychuk and Chapman (2002) have all reported that the threshold for initiation of ASR coincided fairly closely with the Pcrit in various tropical freshwater species (Table 2.1). Sloman et al. (2006) found that the threshold for ASR coincided with the Pcrit of large oscars, Astronotus ocellatus, but was significantly below the Pcrit of small oscars (Table 2.1), a diVerence in response that they attributed to greater fear of predation in smaller fish (see Section 5.1 below). On the other hand, Sloman et al. (2008) found that the hypoxic threshold for ASR behavior 38 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE in the tidepool sculpin (Table 2.1) was well above the hypoxic PO2 at which biochemical disturbances were measurable in fish that were denied access to the response. However, as described in Section 3.2 below, the appearance of metabolic disturbances coincided with the threshold for spontaneous emergence from the water in this species (Sloman et al., 2008). Burggren (1982) demonstrated that permitting goldfish (Carassius auratus) to perform ASR and bubble-holding allowed them to maintain arterial oxygen levels higher in deep hypoxia. There are also various demonstrations that ASR improves tolerance of deep hypoxia, measured as survival time (Kramer and Mehegan, 1981; Kramer and McClure, 1982) or as time to loss of equilibrium (Chapman et al., 1995). Rutledge and Beitinger (1989) found that access to ASR in deep hypoxia increased tolerance of acute increases in water temperature (critical thermal maximum) in three species of North American freshwater fish. Lefrançois et al. (2005) found that denying access to ASR in deep hypoxia impaired performance of the fast-start escape response in golden grey mullet (Liza aurata). StierhoV et al. (2003) found that Fundulus heteroclitus exhibited less impairment of growth in deep hypoxia if they were allowed access to the surface to perform ASR. As described in Section 5.1 below, a major ecological benefit to the performance of ASR can be that it allows fish to colonize hypoxic refugia that less tolerant predatory species cannot occupy successfully (Chapman et al., 1995; Rosenberger and Chapman, 1999; Chapman et al., 2002). Kramer (1987) argues that one major physiological cost to ASR is the increased locomotor activity required for repeated surfacing and skimming. However, as described in Section 5 below, another major ecological cost to ASR relates to predation, in that the behavior places fish at significantly greater risk from aerial predation by birds (Kramer et al., 1983). Perhaps not surprisingly, if fish perceive a risk of predation they can modulate the behavioral component of the ASR chemoreflex (Shingles et al., 2005; Sloman et al., 2006, 2008). Shingles et al. (2005) found that exposure of flathead grey mullet to a model avian predator delayed the onset of ASR in hypoxia or in response to direct chemoreceptor stimulation with NaCN. Furthermore, the fish surfaced preferentially under a sheltered area in their experimental chamber or close to the walls (Figure 2.3A). In turbid water, the fish could not see the model predator and it had no eVect on the onset of ASR but, in turbidity, all the mullet preferentially surfaced around the walls of their chamber (Figure 2.3B). The tidepool sculpin will release alarm substance from epithelial cells when damaged during a predation event (Hugie et al., 1991). When Sloman et al. (2008) added this alarm substance into their water, tidepool sculpins showed a significantly lower threshold for initiation of ASR in response to progressive hypoxia. Thus, the behavioral component of the ASR reflex is plastic; it can be modulated by inputs from higher centers, in particular as a function of perceived risk of predation. 2. BEHAVIORAL RESPONSES TO HYPOXIA 39 A B Fig. 2.3. Aerial view of locations of aquatic surface respiration (ASR) events performed by Mugil cephalus in response to an external application of 300 mg NaCN into the ventilatory stream via a buccal cannula (A) or when exposed to aquatic hypoxia (B) in clear water (left panel) or in turbid water containing 300 NTU Polsperse 10 kaolin (right panel) at a temperature of 25 C. Open circles represent ASR events in the absence of a model avian predator; solid circles represent events in the presence of the model. The outer lines at the top of the diagrams represent a sheltered area in the aquarium. From Shingles et al. (2005) with permission from the University of Chicago Press. 2.2. Air-breathing A number of bony fishes have evolved bimodal respiration, meaning that they retain functional gills but can also gulp air at the water surface and store this in an air-breathing organ (ABO). The comparative physiology and evolution of air-breathing in fishes have been the subject of extensive reviews and dedicated books (Johansen, 1970; Randall et al., 1981a; Graham, 1997; 40 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE Graham and Lee, 2004; Brauner and Val, 2006; Lam et al., 2006; Reid et al., 2006); the reader is referred to these and only a synthetic overview is provided here, with the emphasis upon responses to hypoxia. In freshwater species, the ABO is typically a highly vascularized diverticulum of the buccal cavity, pharynx, or gut; the primitive air-breathing fishes typically use modified swimbladders whereas the more modern teleosts use the branchial chambers or the gut itself (Graham, 1997). Air-breathing appears to have evolved independently multiple times in freshwater fishes (Randall et al., 1981a; Graham, 1997; Brauner and Val, 2006). The prevailing opinion is that hypoxia was the essential driving force for its evolution, and that it evolved from pre-existing ASR and bubble-holding behaviors (Gee and Gee, 1995; Graham, 1997; Graham and Lee, 2004). There are a large number of highly derived marine teleosts that occupy the intertidal zone and which are believed to have evolved air-breathing abilities and an amphibious lifestyle independently of the freshwater air-breathers. The selection pressures may have been an ability to tolerate emersion during low tide and to escape extremes of salinity and hypoxia in tidepools (Martin, 1995; Graham, 1997; Graham and Lee, 2004; Sayer, 2005; Lam et al., 2006), although the amphibious lifestyle may also have provided a means of protecting their eggs and young from aquatic predators (Graham, 1997; Shimizu et al., 2006). These species typically use the skin, gills, and branchial chambers as air-breathing organs (Graham, 1997; Graham and Lee, 2004; Sayer, 2005; Lam et al., 2006). It has been suggested that terrestrial vertebrates have evolved from freshwater air-breathing ancestors, rather than from an amphibious marine ancestor (Graham, 1997; Graham and Lee, 2004). Fishes with bimodal respiration have been classified into two functional groups, either facultative or obligate air-breathers (Johansen, 1970; Graham, 1997). Facultative air-breathers, by definition, supplement oxygen uptake at the surface but can survive by gill ventilation alone if denied access to air in normoxic water. Obligate air-breathers drown if denied access to air-breathing, even in normoxic water, and this is typically because they have very reduced gill surface areas. The reduction in gill surface area is because, in the all bony fishes, oxygenated blood leaving the ABO must traverse the gills to enter the systemic circulation; hence reduced gills will reduce potential loss of oxygen across gills to hypoxic water (reviewed by Graham, 1997). The gills cannot be lost completely because they retain an essential role in excretion of carbon dioxide and ammonia, and for ionic, osmotic, and acid–base balance (Randall et al., 1981a; Graham, 1997; Brauner and Val, 2006). It should be noted that the facultative/obligate classification is not absolute, there are many species that utilize both strategies as a function of their developmental stage or environmental conditions (Graham, 1997; Reid et al., 2006), and animals that have been considered to be obligate air-breathers in laboratory 2. BEHAVIORAL RESPONSES TO HYPOXIA 41 studies, such as species of African lungfish Protopterus (Johansen, 1970), have proven to be less so when studied by telemetry in their natural environment (Mlewa et al., 2005). What is irrefutable is that hypoxia stimulates air-breathing behavior in all fish with bimodal respiration. Figure 2.4 shows the proportion of oxygen uptake that is met by air-breathing as a function of water PO2 in a number of freshwater species (figure after Graham, 1997). Although the various species show diVering degrees of reliance on air-breathing in normoxia, all of them show an increased reliance in hypoxia (Figure 2.4). Much less is known about how hypoxia influences air-breathing behavior in amphibious marine species (Reid et al., 2006; Lam et al., 2006). As described in Section 3.2 intertidal blennies (Blennioidiae) and sculpins (Cottidae) that use their skin as an ABO will spontaneously emerge from hypoxic water into air (Martin, 1995; Yoshiyama et al., 1995; Sloman et al., 2008). The giant mudskipper, Periophthalmodon schlosseri, is an obligate air-breathing fish that uses a modified bucco-opercular cavity as an ABO, and it alternates brief periods of gill ventilation using water with longer periods when it holds air in the gill pouch ABO. Aguilar et al. (2000) exposed the giant mudskipper to either aquatic or aerial hypoxia, and found that only aerial hypoxia elicited an increase in air-breathing activity, water oxygen levels being without eVect. Mudskippers live in j-shaped burrows and create an air-pocket in the end of the burrow by transporting in air in their mouth (Lee et al., 2005). Lee et al. (2005) demonstrated that, if oxygen levels in the air are experimentally reduced below a certain PO2 threshold (about 50% of air saturation) in the burrow of Scartelaos histophorus, it will actively expel the air and replace it with fresh normoxic air from the surface. Gonzales et al. (2006) found that aquatic hypoxia caused a significant stimulation of air-breathing frequency in the burrow-dwelling eel goby, Odontamblyopus lacepedii. This species is sympatric with the mudskippers but is not truly amphibious and remains in its burrow, full of hypoxic water, during low tide (Gonzales et al., 2006). As was the case for ASR, although air-breathing can be considered a behavioral response to hypoxia, it is a chemoreflex driven by oxygensensitive receptors (see Smatresk, 1990; Taylor et al., 1996; Reid et al., 2006, for reviews). The surfacing and gulping response in freshwater airbreathing species can be stimulated by chemoreceptors that sense oxygen levels in either the ventilatory water or the blood stream (Smatresk, 1986; Smatresk et al., 1986; McKenzie et al., 1991), and the gulping element of the motor output is believed to be a modification of pre-existing suction feeding movements (Liem, 1987, reviewed in Taylor et al., 1996; Graham, 1997; Reid et al., 2006). Once again, much less is known about how air-breathing is controlled in marine amphibious species. 42 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE Amia Ancistrus Channa Clarias Erythrinus Gymnotus Hoplerythrinus Lepisosteus ⊕ Piabucina Polypterus Protopterus Synbranchus % oxygen uptake from air 100 80 60 40 20 0 0 5 10 15 Water PO2 (kPa) 20 25 Fig. 2.4. EVects of aquatic hypoxia on the percentage of oxygen uptake that is met by airbreathing in 12 genera of air-breathing fish. The figure is reproduced from Graham (1997), with the addition of new data for two species, Hoplerythrinus unitaeniatus and Synbranchus marmoratus, taken from McKenzie et al. (2007b) and McKenzie et al. (submitted). Amia calva at 18 C, data estimated from Johansen et al. (1970); Ancistrus chagresi at 25 C, data from Graham (1983); Channa argus at 25 C, data from Itazawa and Ishimatsu (1981); Clarias lazera at 28 to 32 C, data from Babiker 1979; Erythrinus erythrinus at 27 to 30 C, data from Stevens and Holeton (1978); Gymnotus carapo at 29 to 33 C, data from Liem et al. (1984); H. unitaeniatus at 25 C, data from McKenzie et al. (2007b); Lepisosteus oculatus at 20 C, data from Smatresk and Cameron (1982); Piabucina festae at 25 C, data from Graham et al. (1977); Polypterus senegalus at 28 C, data from Babiker (1984), Protopterus annectens at 28 C, data from Babiker (1984); S. marmoratus at 25 C, data from McKenzie et al. (submitted), note that the greater than 100% oxygen uptake from air in this species reflects loss of oxygen to the water across the gills in deep hypoxia. The primary benefit of air-breathing is that it makes oxygen uptake and aerobic metabolism entirely independent of the prevailing water oxygen availability (reviewed by Johansen, 1970; Graham, 1997, see also Perry et al., 2004; Seifert and Chapman, 2006; McKenzie et al., 2007b) such that fish, can, in theory, colonize hypoxic areas successfully. There is some evidence that oxygen taken up from the ABO may be lost to the surrounding water across the gills in very deep hypoxia in some species, but this eVect is minor (Randall et al., 1978, 1981b; Smatresk and Cameron, 1982). It has been suggested that the persistence of a few relict species of phylogenetically 2. BEHAVIORAL RESPONSES TO HYPOXIA 43 ancient primitive bony and lobe-finned fishes, such as the polypterids, Amia, the gars, and the lungfishes, is due in part to their ability to breathe air (Ilves and Randall, 2007). Air-breathing should, in theory, be energetically more eYcient than water-breathing for fishes, because air is so much richer in oxygen and requires much less eVort to ventilate (Kramer, 1983, 1987; Graham, 1997). Nonetheless, there are less than 400 known species of airbreathing fish (Graham, 1997), amongst some 25 000 species of bony fish. With the possible exception of the labyrinthine fishes of south-east Asia (Rüber et al., 2006), there is little evidence that the acquisition of a capacity for air-breathing was followed by the extensive adaptive radiation that has occurred in groups of purely water-breathing fish, for example, following the appearance of the hinged jaw apparatus that provides flexibility in feeding strategies (Mabuchi et al., 2007). Indeed, as reviewed in detail in Section 5 below, there must be significant physiological and ecological costs to air-breathing, which presumably include costs of surfacing and increased risk of predation (Kramer, 1983, 1987; Kramer et al., 1983; Bevan and Kramer, 1986; Randle and Chapman, 2004). As was the case for fish performing ASR, there is evidence that air-breathing patterns and behavior are significantly influenced by perceived risk of predation. Smith and Kramer (1986) reported that exposure of an obligate airbreather, the Florida gar Lepisosteus platyrhincus, to a model avian predator resulted in a decrease in air-breathing frequency and an increase in gill ventilation eVort. Herbert and Wells (2001) found that fear of predation reduced air-breathing frequency by the blue gourami, Trichogaster trichopterus, an obligate air-breather, which compensated by reducing overall rates of activity. Thus, higher processing can influence reflexive air-breathing behaviors, with adaptive responses that would allow the fish to conserve the O2 stored in their air-breathing organs. There is also evidence that some air-breathing fishes perform the behavior most frequently at night, when the risk of predation might be less (Grigg, 1965; Horn and Riggs, 1973; Babiker, 1979). It is also possible to speculate about other potential physiological and behavioral costs to air-breathing. The vast majority of air-breathing fishes have reduced relative gill areas compared with closely related and/or sympatric water-breathing species (Graham, 1997). The aerobic metabolic scope necessary for all activities such as sustained swimming, recovery from intense exercise, or digestion of food would, presumably, have to be met to some extent by increased air-breathing activity (Farmer and Jackson, 1998; McKenzie et al., 2007c; Wells et al., 2007). This is an interesting area for future research. It might, presumably, constrain their options in terms of habitat choice (requiring cover from predation) and in terms of their diel rhythms in activity. Air-breathing could also interfere with social interactions if fish were dependent upon constant visits to the surface. 44 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE 3. EFFECTS OF HYPOXIA ON ACTIVITY 3.1. Spontaneous Swimming Activity Changes in spontaneous swimming activity have been described in a wide variety of fish groups and species when exposed to hypoxia, including elasmobranchs, chondrosteans, and teleosts. As reviewed in this section, these behavioral responses can comprise either a reduction in activity or an increase in activity, depending upon the species and the context. Table 2.2 lists the species that have been reported to change spontaneous swimming activity in hypoxia and the nature of the response including, when relevant, as a function of the context. Figure 2.5 shows species for which such responses have been quantified. It has been suggested that species that reduce their activity in hypoxia tend to be demersal or bentho/pelagic, with a relatively sedentary lifestyle during which they may often encounter hypoxia in their habitat; whereas species that increase activity tend to be active pelagic schooling fishes (Domenici et al., 2000; Herbert and SteVensen, 2005, 2006). Swimming is typically considered to represent a major component of the energy budget of active fishes, and high-intensity aerobic swimming can utilize a very significant proportion of a fish’s aerobic metabolic scope (Fry, 1971; Claireaux and Lefrançois, 2007). Thus, for those species that reduce levels of spontaneous swimming activity in hypoxia, this has been interpreted as an energy-saving response (Metcalfe and Butler, 1984; Fischer et al., 1992). The Crucian carp, Carassius carassius, can tolerate complete anoxia for many days. Nilsson et al. (1993) used video-tracking to show that one of the energy-saving strategies used by this species was to reduce their spontaneous activity by 50% (Figure 2.5). Nilsson et al. (1993) calculated that the reduced activity would provide a saving of approximately 35% of overall energy requirements in anoxia. Schurmann and SteVensen (1994) studied eVects of gradual stepwise progressive hypoxia on spontaneous swimming activity of Atlantic cod acclimated to three temperatures, 5 C, 10 C, and 15 C. As shown in Figure 2.6, at all temperatures routine normoxic activity was maintained at levels until a threshold degree of hypoxia beyond which it declined. This response pattern is in fact very similar to the pattern for regulation of aerobic metabolic rate by cod (and, indeed, most fish) in hypoxia (Schurmann and SteVensen, 1997). Interestingly, Schurmann and SteVensen observed that the water oxygen threshold at which the decline in activity of the cod occurred was not directly related to temperature but was highest at 10 C and similar at 5 C and 15 C (Figure 2.6). This is somewhat unexpected because the critical PO2 threshold for regulation of standard metabolic rate (critical PO2, Pcrit) is highly temperature-dependent in cod, with cod at 15 C being significantly Table 2.2 Fish species that have been reported to change their spontaneous locomotor activity in response to hypoxia Species (Family) Elasmobranchs Carcharhinus acronotus (Carcharhinidae) Mustelus norrisi (Triakidae) Response Context Source Increased swimming speed Progressive moderate hypoxia Carlson and Parsons (2001) Decreased swimming speed Progressive moderate hypoxia Carlson and Parsons (2001) Decreased activity Rapid exposure to moderate hypoxia Progressive moderate hypoxia Metcalfe and Butler (1984) McKenzie et al. (1995b) Scyliorhinus canicula (Scyliorhinidae) Sphyrna tiburo (Carcharhinidae) Chondrosteans Acipenser naccarii (Acipenseridae)a Increased swimming speed Acipenser naccarii (Acipenseridae)a Reduced swimming speed Water PO2 declining to mild hypoxia Stable mild hypoxia Reduced swimming speed Deep hypoxia Behrens and SteVensen (2007) Reduced swimming speed Increased swimming speed Anoxia Progressive moderate hypoxia Clupea harengus (Clupeidae) Clupea harengus (Clupeidae) Decreased swimming speed Increased swimming speed Gadus morhua (Gadidae) Increased swimming speed Deep hypoxia Water PO2 declining to moderate and deep hypoxia Water PO2 declining to mild hypoxia Nilsson et al. (1993) Domenici et al. (2000); Herbert and SteVensen (2006) Domenici et al. (2000) Herbert and SteVensen (2006) Gadus morhua (Gadidae) Decreased swimming speed Teleosteans Ammondytes tobianus (Ammobdytidae) Carassius carassius (Cyprinidae) Clupea harengus (Clupeidae) Increased swimming speed Stable moderate to deep hypoxia Carlson and Parsons (2001) McKenzie et al. (1995b) Schurmann and SteVensen (1994); Herbert and SteVensen (2005); Johansen et al. (2006) Schurmann and SteVensen (1994); Herbert and SteVensen (2005); Johansen et al. (2006); Skjæraasen et al. (2008) (continued) Table 2.2 (continued ) Species (Family) Response Katsuwonus pelamis (Scombridae) Increased swimming speed Menidia beryllina (Atherinidae) Pomatoschistus minutus (Gobiidae) Solea solea (Soleidae)a Thunnus albacares (Scombridae) Urophycis chuss (Phycidae) Zoarces viviparous (Zoarcidae) Increased swimming speed Increased ‘‘restless’’ activity Decreased activity level Increased swimming speed Increased swimming activity Progressive decline in activity a Context Declining water PO2 in moderate hypoxia Moderate hypoxia; larval fishes Deep hypoxia Moderate hypoxia Moderate hypoxia Moderate to deep hypoxia Progressive moderate to deep hypoxia Source Dizon (1977) Weltzien et al. (1999) Petersen and Petersen (1990) Dalla Via et al. (1998) Bushnell and Brill (1991) Bejda et al. (1987) Fischer et al. (1992) Also shows intense agitation in deep hypoxia (Randall et al., 1992; Dalla Via et al., 1998; McKenzie et al., 2008). 2. 47 BEHAVIORAL RESPONSES TO HYPOXIA 80 Acipenser (0.35) Ammobdytes (0.93) Carcharhinus (0.45) Carassius (0.31) Clupea (2.52) Clupea (0.65) Gadus (0.32) Katsuwonus (1.95) Mustelus (0.44) Sphyrna (0.35) 60 % change in swimmingspeed 40 20 0 −20 −40 −60 −80 −100 100 80 60 40 20 Water O2 saturation (%) 0 Fig. 2.5. EVects of hypoxia on swimming speed in fishes. Quantitative data are available for nine species, this figure presents mean percentage changes in speed relative to the normoxic control, note also the inverted abscissa. For each species, the number in brackets is the mean normoxic swimming speed in bodylengths sec 1. Acipenser naccarii exposed for 3 h to either normoxia or mild hypoxia at 23 C (McKenzie et al. 1995b). Ammobdytes tobianus exposed to progressive reductions in water PO2 each 10 min and then maintained for 1 h at a low PO2 below 20% at 10 C. There was a profound decline in swimming speed when water PO2 had stabilized below 20% (Behrens and SteVensen, 2007). Carcarhinus acronotus exposed to progressive hypoxia in a sealed respirometer at 26 C and speed measured at four PO2 intervals (Carlson and Parsons, 2001). Carassius carassius exposed to anoxia for 1 h at 8 C (Nilsson et al., 1993); Clupea harengus dotted line is mean response to progressive reductions in water PO2 each 10 min at 15 C (Domenici et al., 2000). Clupea harengus diamonds show eVects of exposure for 30 min to a stepwise series of progressively more hypoxic PO2s at 10 C (white diamonds) and the response to declining PO2 between each step (intervening black diamonds). The declining PO2 caused a significant increase in swimming speed by the herring, which disappeared when PO2 stabilized (Herbert and SteVensen, 2006). Gadus morhua exposed to progressive reductions in water PO2 each 30 min at 10 C (Schurmann and SteVensen, 1994). Note that Herbert and SteVensen (2005) found that this species increased swimming speed when exposed to declining PO2, but speed then stabilized at a lower level when the hypoxic PO2 stabilized (data not shown). Katsuwonus pelamis exposed to progressive reduction in PO2 each 10 min at 24 C (Dizon, 1977). Mustelus norrisi and Sphyrna tiburo exposed to progressive hypoxia in a sealed respirometer at 26 C and speed measured at four PO2 intervals (Carlson and Parsons, 2001). 48 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE 200 Swimming distance (m) 160 120 80 5°C 40 0 10°C 15°C 0 20 40 60 80 100 Oxygen saturation (%) Fig. 2.6. Swimming distance of Atlantic cod Gadus morhua at diVerent oxygen saturations, at three temperatures of 5 C, 10 C, and 15 C. Each point represents the mean distance (m) covered by eight cod during 30 min. At all oxygen saturations, the activity levels (swimming distance) at 5 C and 15 C are similar and there was a tendency for cod at 10 C to be more active at higher oxygen saturations. At oxygen saturations below 30%, the activity levels were similar at all temperatures and decreased with decreasing oxygen saturation. From Schurmann and SteVensen (1994) with permission from the Company of Biologists. less tolerant of hypoxia than those at 5 C (Schurmann and SteVensen, 1997). Skjæraasen et al. (2008)) have subsequently, however, demonstrated that cod show greater hypoxia depression of spontaneous activity at 15 C than they do at 5 C, which is consistent with their relative overall hypoxia tolerance at these temperatures (Schurmann and SteVensen, 1997). The thresholds for reduced activity by cod in hypoxia have consistently been higher than the corresponding Pcrit for cod at that same temperature (Schurmann and SteVensen, 1994, 1997; Herbert and SteVensen, 2005; Skjæraasen et al., 2008), indicating that the reduced swimming activity may have reflected reduced aerobic metabolic scope as hypoxia progressed toward the Pcrit (Claireaux and Lagardère, 1999). Behrens and SteVensen (2007) studied eVects of progressive hypoxia on spontaneous swimming activity in schools of lesser sandeels, Ammobdytes tobianus. These small marine fish bury in the sand for much of the time, but emerge to feed in large schools. Their spontaneous swimming speed was 2. BEHAVIORAL RESPONSES TO HYPOXIA 49 0.9 BL sec-1 in normoxia, and this speed was maintained until a threshold PO2 of about 15% saturation, beyond which the animals reduced their activity by over 90% and came to rest on the substrate (Figure 2.5). This threshold for reduced activity is below their Pcrit at the same temperature (20% saturation), indicating that the reduced activity may have reflected an inescapable metabolic depression, although all of the animals recovered activity when returned to normoxia (Behrens and SteVensen, 2007). Dalla Via et al. (1998) studied the responses of Dover sole, Solea solea, to progressive hypoxia. Although swimming activity was not quantified, the authors report that spontaneous activity was reduced at oxygen tensions between 80% and 20% air saturation. Sole of the size used by Dalla Via et al. (1998) have a Pcrit below 20% saturation (van den Thillart et al., 1994; Couturier et al., 2007). Dalla Via et al. (1998) found that, as this threshold approached, the fish tended to remain immobile except for a tendency to raise portions of their body oV the substrate, possibly to ventilate the blind side (Nonnotte and Kirsch, 1978; McKenzie et al., 2008). At 5% air saturation or lower, the fish exhibited intense agitation followed by a loss of equilibrium and cessation of movements. McKenzie et al. (2008) found qualitatively similar patterns of behavioral and metabolic responses to hypoxia in very early life stages of this flatfish species (including in pre-settlement larvae at 9–13 days old), which suggests that the responses arise early in ontogeny (Weltzien et al., 1999). Both Dalla Via et al. (1998) and McKenzie et al. (2008) found that the agitation response occurred below the corresponding Pcrit for their life-stages, but the Pcrit and thresholds for the behaviors were very much higher in the early life stages, being 60% and 56%, respectively, in larvae, and 48% and 29%, respectively, in post-settlement juveniles (15–20 days old). The intense agitation of fish in deep hypoxia below the Pcrit has been interpreted as an acute escape response (Bejda et al., 1987; Randall et al., 1992; Van Raaij et al., 1996). For those species that increase their level of spontaneous activity in hypoxia, this has been interpreted as a response to escape the hypoxic area (Dizon, 1977; Bejda et al., 1987; Petersen and Petersen, 1990; Weltzien et al., 1999; Domenici et al., 2000; Herbert and SteVensen, 2005, 2006). Petersen and Petersen (1990) found that oxygen saturations below 40% caused the sand goby Pomatoschistus minutus to become restless and perform random activity. Young red hake Urophycis chuss moved upwards in the water column if the oxygen concentration fell below about 50% saturation (Bejda et al., 1987). These responses arise very early in life; Weltzien et al. (1999) found that larvae of the inland silverside, Menidia beryllina, exhibited an avoidance response to hypoxic water within hours of hatching. Larvae were placed in a water column with two salinity layers, when the larvae drifted into a layer that was maintained hypoxic at less than 55% oxygen saturation, this 50 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE stimulated bursts of swimming activity at up to 6.4 BL sec-1 that were directed toward the normoxic layer (Figure 2.5). Dizon (1977) studied two species of tuna, the skipjack, Katsuwonus pelamis, and the yellowfin, Thunnus albacares, which swim spontaneously at speeds of about 1.6 and 1.2 BL sec-1, respectively. When these tunas were exposed to a gradual decline in oxygen tension, the skipjack showed an abrupt doubling of swimming speed, up to 2.2 BL sec-1, as oxygen fell below 55% saturation (Figure 2.5), although there was no eVect of hypoxia on activity in yellowfin. Dizon (1977) attributed this species diVerence in behavior to a greater sensitivity to hypoxia in the skipjack, which therefore engendered an escape response. Bushnell and Brill (1991) subsequently reported that the yellowfin did increase swimming speed by about 20% when exposed to hypoxia at 60% of air saturation, and also attributed this to an escape response. Herring school spontaneously at speeds of between 0.8 and 3 BL sec-1 and show progressive increases in speed when exposed to stepwise progressive hypoxia (Domenici et al., 2000; Herbert and SteVensen, 2006). Domenici et al. (2000) found that the lower the spontaneous speed in normoxia, the greater the increase in speed in hypoxia. Both Domenici et al. (2000) and Herbert and SteVensen (2006) found that the most marked increase in activity occurred when water PO2 fell below about 40% of air saturation; Herbert and SteVensen (2006) found that the most marked increases in swimming speed occurred when water PO2 was declining rather than at a stable degree of hypoxia (Figure 2.5). These same authors could not detect evidence of a transition to anaerobic metabolism (no increase in plasma lactate) at the hypoxic PO2s at which the changes in swimming speed occurred, indicating that the herring were above their Pcrit. This response may help herring to avoid hypoxic areas in their natural environment (Neuenfeldt, 2002; Herbert and SteVensen, 2006). Herring also form vast schools, and the fish at the middle/back of the school may experience hypoxia due to oxygen uptake by those preceding and around them (McFarland and Moss, 1967; Domenici et al., 2002, see Section 5.2). The increased swimming speed would allow the fish to reshuZe and change position, toward better-oxygenated areas in the school (Domenici et al., 2000; Herbert and SteVensen, 2006). As might be expected, many species can exhibit both types of response (increased or decreased activity), depending on the degree of hypoxia, the rate of change in water oxygen tension, and their degree of activity in normoxia. Thus, as already discussed, Dover sole will reduce activity as they approach their Pcrit but then exhibit intense agitation prior to loss of equilibrium in deep hypoxia (Dalla Via et al., 1998; McKenzie et al., 2008). Schools of herring exhibit exactly the opposite response, showing increased activity as oxygen levels drop down to about 25% air saturation, followed by a decline in activity and disruption of the school at lower PO2 (Domenici 2. BEHAVIORAL RESPONSES TO HYPOXIA 51 et al., 2000; Figure 2.5). Schurmann and SteVensen (1994) and Herbert and SteVensen (2005) demonstrated that when oxygen tensions are declining there is a transient increase in activity in Atlantic cod, and it is when hypoxic conditions are stable that activity is down-regulated (Figure 2.5). These authors postulate that the two contrasting responses would be adaptive if the increased activity allowed the fish to escape hypoxia as they encountered it (or as it developed in their surroundings), whereas the reduced activity would allow them to ‘‘sit out’’ hypoxia from which escape was impossible. McKenzie et al. (1995b) found that the responses of Adriatic sturgeon, Acipenser naccarii, to hypoxia diVered as a function of their degree of activity in normoxia. This species shows constant sustained activity at a speed between 0.2 and 0.5 BL sec-1. Animals that were most active in normoxia reduced their activity when exposed to hypoxia, as oxygen levels declined and then stabilized (at 50% of air saturation, Figure 2.5). Animals that were less active in normoxia showed a transient increase in activity as oxygen levels declined toward hypoxia, but then returned to their previous normoxic swimming speed (McKenzie et al., 1995b). This species must swim in order to regulate metabolism in hypoxia, which would argue for a role of ram ventilation (McKenzie et al., 1995b, 2007d) although when animals swam at incremental sustained speeds (McKenzie et al., 2001) there was no evidence of the cessation of gill ventilation that accompanies a transition to ram ventilation in many teleosts (Freadman, 1979; SteVensen, 1985). Some large open-ocean pelagic species swim constantly and have evolved a dependence upon ram ventilation to generate a flow of water across the gills (Brown and Muir, 1970; Roberts, 1978; Parsons and Carlson, 1998; Carlson and Parsons, 2001). Although tunas rely on ram ventilation (Roberts, 1978), their increased swimming speeds in hypoxia have been attributed to an escape response (Dizon, 1977; Bushnell and Brill, 1991) because a model relating swimming speed and mouth gape to rates of oxygen uptake revealed that observed increases in swimming and gape in hypoxia would not be suYcient to maintain the highly elevated routine normoxic rates of oxygen uptake in these species (Bushnell and Brill, 1991). In a number of large sharks with ram ventilation, however, a spontaneous increase in swimming speed in hypoxia does appear to be an adaptive response to increase rates of gill ventilation (Parsons and Carlson, 1998; Carlson and Parsons, 2001). Carlson and Parsons (2001) compared locomotor and metabolic responses to hypoxia in three species of shark, two large species with ram ventilation (bonnethead, Sphyrna tiburo, and blacknose, Carcharhinus acronotus) and a dogfish with active gill ventilation (Florida smoothhound shark, Mustelus norrisi). All of the species swam spontaneously at a speed of about 0.4 BL sec-1 in normoxia; but as the fish were exposed to progressive hypoxia, the bonnethead and blacknose (ram ventilators) increased their swimming speed by up to 50% 52 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE and 25%, respectively (Figure 2.5). The dogfish, on the other hand, reduced swimming speed by up to 36% at the lowest degree of hypoxia tested (approximately 40% of air saturation, Figure 2.5). In all species, these changes started at a threshold of about 70% of air saturation; in the ram ventilators they were associated with an increase in oxygen uptake relative to normoxia whereas oxygen uptake was regulated unchanged from normoxic rates in the dogfish (Carlson and Parsons, 2001; Figure 2.5). In many of these studies, modulation of routine rates of spontaneous activity in hypoxia occurs above the Pcrit of the fish, hence at levels of hypoxia that are not, presumably, associated with respiratory distress and a transition to anaerobic metabolism (Herbert and SteVensen, 2005, 2006). These changes in routine activity are also, presumably, stimulated by information from the chemoreceptors, which have been demonstrated to monitor oxygen levels in the water and/or the blood of fishes (Burleson et al., 1992; Sundin et al., 2007), although this remains to be confirmed. The routine swimming speeds that have been measured in normoxia all fall within the range of sustainable aerobic activities that are dependent upon the performance of red muscle (Webb, 1993, 1998), hence reducing these activities may relieve hypoxic constraints on aerobic metabolic scope (Claireaux and Lagardère, 1999; Claireaux et al., 2000; Lefrançois and Claireaux, 2003). The locomotor responses to hypoxia arise very early in ontogeny, in particular the avoidance and agitation responses (Weltzien et al., 1999; McKenzie et al., 2008). The agitation responses seen in some species in deep hypoxia below the Pcrit would seem indicative of the recruitment of white muscle for a transient burst of anaerobic swimming activity (Webb, 1993, 1998). 3.2. Other Locomotor Responses to Hypoxia There are other locomotor responses to hypoxia that are characteristic of species with particular life histories. Sand eels (Ammodytidae) occupy burrows on sandy sea beds, lying with their snout a few millimeters below the surface. Behrens et al. (2007) used novel planar optodes to quantify oxygen levels around buried lesser sandeels, and demonstrated that they breathe by advective transport through the permeable interstice, forming an inverted cone of oxygenated porewater in front of their mouth. Oxygen levels around the buried eel tended, however, to be very low and, every once in a while, the sandeels wriggled in the sand and re-oxygenated their surroundings. It is not known why they do this, because the skin does not appear to play any role in gas exchange (Behrens et al., 2007). If the overlying water is made hypoxic, the sandeel moved toward the surface and its head emerged when PO2 fell below its Pcrit (20% saturation, Behrens and SteVensen, 2007), eventually emerging in extreme hypoxia (5%) to lay on the surface (Behrens et al., 2007). 2. BEHAVIORAL RESPONSES TO HYPOXIA 53 Another response to aquatic hypoxia is spontaneous emersion, whereby amphibious and semi-amphibious species leave water to go onto land. A number of freshwater and marine species have been reported to venture onto land, a role for aquatic hypoxia in this response has only been studied in a few species (see Graham, 1997; Sayer, 2005, for reviews). The fishes for which the hypoxic emersion response is most well-described are the marine species that inhabit rockpools in the intertidal zone and will emerge from water to maintain gas exchange in air via their skin (Martin, 1995; Graham, 1997; Sayer, 2005). A number of species of blenny (Blennioids) exhibit this response. Graham (1970) found that a tropical blenny, Mnierpes macrocephalus, could be stimulated to emerge from hypoxic water in the laboratory. Field observations have reported that other blennies (Helcogramma medium, Blennius pholis) crawl from hypoxic tidepools (Davenport and Woolmington, 1981; Innes and Wells, 1985). Intertidal sculpins of the family Cottidae inhabit tidepools that can become isolated and extremely hypoxic during low tide, and many of these species will spontaneously emerge from hypoxic water (Davenport and Woolmington, 1981; Yoshiyama et al., 1995; Graham, 1997; Sayer, 2005). Davenport and Woolmington found that Taurulus (Cottus) bubalis emerged from hypoxic tidepools when PO2 fell below 5% saturation. Yoshiyama et al. (1995) found that four diVerent species of intertidal sculpin that inhabit rockpools in the intertidal zone of the temperate Pacific (Oligocottus snyderi, O. maculosus, Clinocottus globiceps, and Ascelichthys rhodorus) also emerged spontaneously in laboratory experiments. The propensity to perform this behavior was directly related to the capacity of the species to breathe air, being greatest in C. globiceps and least in A. rhodorus (Yoshiyama et al., 1995); however, the PO2 thresholds for emergence were not reported. Sloman et al. (2008) studied emersion responses by the tidepool sculpin, O. maculosus, comparing two size-classes of fish in the laboratory, in artificial tidepool mesocosms, and in their natural habitat. These authors found that both size-classes of fish tended to emerge from the water when oxygen fell below 20% of air saturation, and this was similar in laboratory, mesocosm, and field. This hypoxic threshold coincided with the threshold for the appearance of anaerobic end-products in the tissues of fish that were denied access to emergence. This indicates that in this species, emergence is a last-ditch attempt to avoid hypoxic depression of aerobic metabolism; in air the animals were able to maintain their aerobic metabolism at routine normoxic levels but may suVer increased risk of predation by birds (Yoshiyama et al., 1995; Sloman et al., 2008). Liem (1987) investigated whether hypoxia stimulated an emersion response in a number of tropical labyrinthine fishes (Anabantoids), airbreathing freshwater species that are known to venture onto land. Hypoxia did not stimulate emersion in the climbing perch (Anabas testudineus), the 54 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE Siamese fighting fish (Beta splendens), or various gourami species (Macropodus opercularis and Trichogaster trichopterus). In species such as the mangrove killifish Krytpolebias (Rivulus) marmoratus, an emergence response can be stimulated by poor water quality (increased water H2S content, Abel et al., 1987). Similar emersion responses to escape ‘‘poor water quality’’ have been anecdotally reported for many tropical air-breathing fish species such as erythrinids, walking catfish (family Claridae), snakeheads (Channidae), swamp eels (Synbranchidae), and eels of the genus Anguilla (Tesch, 1972; Graham, 1997). A potential role of hypoxia in the emergence responses of these species remains, however, to be described. 4. HYPOXIA AND PARENTAL CARE BEHAVIOR In addition to the large energetic costs associated with swimming and other locomotor responses, another significant metabolic cost for some fish species is the energy directed to parental care. Among fishes that exhibit post-fertilization care, there is an amazing diversity of strategies ranging from simple nest guarding, to mouth brooding, to live bearing. The energetic cost of reproduction increases as more energy is invested into parental care (Fryer and Iles, 1972; Jones and Reynolds, 1999a), and one would predict that alternative oxygen environments would aVect the costs and benefits of parental care (Hale et al., 2003). Some oviparous fishes protect their developing young after spawning by selecting suitable nesting sites, as well as nest building and guarding the young, fanning to aerate eggs, and mouthing to clean and remove dead and diseased eggs (Wootton, 1990). In mouth brooders (where the eggs and larvae are held in the mouth of a parent) and other forms of live-bearing fishes (e.g., seahorses or other pouch bearers) the parent can protect the young from predators and other environmental stressors by moving to more suitable environments (Wourms and Lombardi, 1992; Goodwin et al., 2002; Wen-Chi Corrie et al., 2007). Ovoviviparous fish take parental care a step further retaining the embryos in either the ovary or uterus until they reach a more advanced and less vulnerable stage of development. Some live-bearing species are viviparous and have evolved specialized tissues to provide nutrients to developing young (Thibault and Schultz, 1978; Blackburn et al., 1985). The costs to the parent associated with carrying the young either internally or externally include high energetic costs of bearing the young and increased predation risk due to reduced mobility (Thibault and Schultz, 1978; Blackburn et al., 1985; Goodwin et al., 2002; Timmerman and Chapman, 2003). Mouth brooders bear an additional cost as most are prohibited from feeding while brooding. Costs of parental care in fishes may be particularly severe under hypoxia due to the challenge of providing oxygen to the eggs, the need for 2. BEHAVIORAL RESPONSES TO HYPOXIA 55 physiological and biochemical mechanisms to facilitate oxygen uptake for the parent, and increased predation risk associated with surfacing behavior. However, high levels of parental care may be necessary under hypoxic conditions to ensure survival of the eggs and young. Roberts (1973) noted an interesting link between hypoxic habitats and parental care in his review of the ecology of fishes of the Amazon and Congo basins. He observed that in these large tropical rivers, parental care occurs primarily in fishes in which the adults breed in swamps and other oxygen-deficient habitats. For example, in the African lungfish (Protopterus spp.) a nest is constructed and guarded by the male (Johnels and Svensson, 1954; Bouillon, 1961; Greenwood, 1987). In the central Congo basin, Protopterus dolloi excavates a burrow that receives air through a chimney-like structure, without which the eggs would be deprived of oxygen. The small anabantid fish Ctenopoma damasi and the characoid Hepsetus odoe construct floating nests of foam in which the eggs are supported (Berns and Peters, 1969; Roberts, 1973). The most abundant cichlids in the dense interior of East African swamps are mouth-brooding haplochromines and tilapiines (Chapman et al., 2002, 2006a,b) that can move the young to microhabitats with better levels of oxygen. The eVects of low-oxygen stress on parental care of fishes is a topic of growing concern, given the widespread and increasing occurrence of aquatic hypoxia. Literature in this area is still depauperate but, nonetheless, demonstrates that DO is an important driver of parental care behavior across a range of strategies from guarding to viviparity. Many nest-guarding fishes use fanning or other behaviors that increase ambient oxygen levels (Mertz and Barlow, 1966; Wootton, 1976; Zoran and Ward, 1983; Coleman, 1992; Jones and Reynolds, 1999a,b; Takegaki and Nakazono, 1999). Some species have been shown to alter their ventilation behaviors in response to changing levels of DO, including three-spined stickleback (Reebs et al., 1984), the anemonefish, Amphiprion melanopus (Green and McCormick, 2004), the sand goby, Pomatoschistus minutus (Lissaker et al., 2003), the land-locked goby, Rhinogobius sp. (Maruyama et al., 2008), and the common goby, Pomatoschistus microps (Jones and Reynolds, 1999a), which supports a role for parental care in oxygen replenishment in fish nests. The importance of male parental care is evident in the sand goby, P. minutus, where females were shown consistently to prefer males with elevated levels of parental care under hypoxia (Lindström et al., 2006). However, the response of the Florida flagfish, Jordanella floridae, is inconsistent with these earlier studies. Hale et al. (2003) found that male flagfish devoted less time to parental care (including fanning) as DO declined. Hale and colleagues hypothesized that the increasing costs of care as DO declined outweighed the benefits for this species, and they noted that earlier studies may not have exposed parental fish to DO levels suYciently low enough to preclude a benefit of fanning. 56 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE More complex parental care has been described for the amphibious mudskippers (Oxudercinae), fish that are well adapted for life on intertidal mudflats, highly productive systems that are characterized by severe environmental challenges. Mudskippers lay their eggs in mud burrows that contain extremely hypoxic water. In their study of the Japanese mudskipper, Periophthalmus modestus, Ishimatsu et al. (2007) discovered that eggs are deposited on the walls of an air-filled chamber where oxygen is maintained via activity of the guarding male that deposits mouthfuls of fresh air into the chamber at each low tide. After completion of egg development, the male was shown to remove the air from the chamber, flooding the eggs to induce hatching (Ishimatsu et al., 2007). For mouth-brooding fishes, costs of parental care may be severe under hypoxia because of the increased requirements of oxygenating eggs when the parent cannot eat, and the elevated predation risks associated with any surfacing behavior. Wen-Chi Corrie et al. (2007) quantified the behavioral response to progressive hypoxia of the widespread mouth-brooding African cichlid, Pseudocrenilabrus multicolor victoriae. This species responded to progressive hypoxia by performing ASR; however, brooding females showed higher ASR thresholds than males, and initiated ASR at a much higher threshold. Non-brooding females did not diVer from males for any ASR threshold. A high ASR threshold in brooding females may reflect various costs such as churning behavior, which is used to move the brood inside the mouth, potentially enhancing ventilation and cleaning of the eggs and young (Oppenheimer and Barlow, 1968; Keenleyside, 1991). This may add to the energy expenditure of the female, particularly under hypoxic conditions. Evidence that brooding aVects responses to hypoxia has been reported in other species. In their study of cardinal fishes, paternal mouth brooders, Östlund-Nilsson and Nilsson (2004) found that the critical oxygen tension of brooders was almost twice as high as non-brooders. Similarly, costs of carrying young that aVect responses to hypoxia are evident in livebearers. Timmerman and Chapman (2003) found that gestating female sailfin mollies (Poecilia latipinna) spent 27% more time at the surface using ASR than non-gestating females. They attributed this to a high mass-specific oxygen requirement in the young, which increased the total oxygen requirement of females (Boehlert and Yoklavich, 1984; Boehlert et al., 1986; Dygert and Gunderson, 1991). The increased time allocated to ASR may directly aVect maternal predation risk of mollies in hypoxic waters, as risk of aerial predation has been demonstrated to increase with time spent conducting ASR (Kramer et al., 1983). In the field, one might anticipate gestating females to exhibit behaviors that reduce the risk, such as selection of microhabitats with elevated DO or areas that reduce aerial predation risk, such as vegetated cover. 2. BEHAVIORAL RESPONSES TO HYPOXIA 57 5. HYPOXIA AND ECOLOGICAL INTERACTIONS There is a growing body of empirical evidence that hypoxia can influence species interactions, in particular predator–prey relationships, by altering the success rate of the predator and/or the vulnerability of the prey. Low-oxygen conditions may shift the balance of the interaction to favor predator or prey depending, at least in part, on the relative tolerance of the interactants (Domenici et al., 2007). Hypoxia can elicit behaviors such as ASR or air breathing that increase the risk of predation; it can negatively impact faststart performance of prey, and it can alter the dynamics of schooling behavior. For aquatic water-breathing predators, hypoxia can decrease predation through metabolic depression, lowered appetite, or decreased performance. The outcome of altered predator–prey interactions can ultimately influence other components of the food web and assemblage; therefore, predicting whether the prey or the predator is the beneficiary of hypoxic stress is critical for understanding community level impacts of hypoxia, whether natural or anthropogenically induced. 5.1. Hypoxic Refugia from Piscine Predators Studies of predator–prey interactions in fishes suggest that hypoxia may be an important modulator; the prey may benefit if high tolerance to hypoxia permits access to refugia from less tolerant predators. For example, for some potential prey in the Lake Victoria basin of East Africa, hypoxic refugia have mitigated impacts of a large introduced piscivore. The explosive speciation of haplochromine cichlid fishes in Lake Victoria is unrivaled among vertebrates; however, over 40% of its endemic fishes disappeared between 1980 and 1986 associated with various anthropogenic perturbations including the upsurge of the invasive predatory Nile perch (Lates niloticus) (Kaufman, 1992; Kaufman et al., 1997; Seehausen et al., 1997a,b; Balirwa et al., 2003). A similar pattern of faunal collapse was observed in other lakes in the basin where Nile perch was introduced (Kyoga, Nabugabo). However, some indigenous species persisted with the Nile perch and were resilient to increasing eutrophication and other stressors. Over the years, interest in conservation of this residual fauna has sparked studies directed toward identification of faunal refugia; habitats where native fishes are protected from Nile perch predation. Wetlands in the Lake Victoria basin serve as both structural and low-oxygen refugia for fishes that can tolerate wetland conditions, and function as barriers to dispersal of Nile perch (Chapman et al., 1996a,b; Balirwa, 1998; Schofield and Chapman, 1999; Chapman et al., 2002; Mnaya et al., 2006). Based on a suite of ecophysiological studies on the fishes of the 58 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE Lake Nabugabo system (critical oxygen tension, ASR thresholds), Chapman and colleagues found that some cichlids and some native non-cichlids that are relatively tolerant of hypoxia are able to persist in the dense interior of hypoxic swamps (Chapman et al., 1995; Chapman and Chapman, 1998; Rosenberger and Chapman, 2000; Chapman et al., 2002; Rutjes, 2006), while the Nile perch cannot, as indicated by its high threshold for ASR, its high critical oxygen tension, and its distribution (Schofield and Chapman, 2000; Chapman et al., 2002). This permits some fishes to persist in wetlands under reduced predator pressure from Nile perch (Chapman et al., 2002). The ecotone of the wetland/open water is a particularly important refugium because interaction with the main lake waters elevates DO, but structural complexity is still high. Nile perch are present but rare in ecotonal wetlands, and species richness is higher than in the interior swamp (Chapman et al., 1996a,b; Balirwa, 1998; Schofield and Chapman, 1999; Chapman et al., 2002). However, even areas deep within the fringing swamp are important in the maintenance of a subset of the basin fauna (Chapman et al., 1996b, 2002). There is now a growing body of empirical support, beyond the Lake Victoria basin, for the use of hypoxic refugia by fishes. Nilsson and ÖstlundNilsson (2004) quantified the critical oxygen tension of 31 coral reef fishes in the Great Barrier Reef, Australia and reported a surprisingly high level of tolerance to hypoxia. They suggested that widespread tolerance to aquatic hypoxia in coral reef teleosts may reflect use of hypoxic spaces in the coral as nocturnal refugia from predators or use of isolated tide pools that experience hypoxia (Nilsson and Östlund Nilsson, 2004). In their review of respiratory ecophysiology of coral reef fishes, Nilsson et al. (2007) noted that air breathing allows some coral reef gobies to stay in their coral refugia during air exposure at low tides, thereby minimizing predator risk. The crucian carp is another example of a species that may use physiological exclusion to minimize predator risk. This carp is well known for its extreme anoxia tolerance, surviving in shallow ponds in Northern Europe that can become anoxic in the winter and are therefore free of piscine predators (Nilsson and Renshaw, 2004; see Chapter 9). Robb and Abrahams (2003) evaluated hypoxic tolerance of small yellow perch (Perca flavescens) and fathead minnows (Pimephales promelas), both potential prey of large yellow perch. They found that both within and between species, smaller individuals were the most tolerant of hypoxic environments, and suggested that prey may intentionally seek out low-oxygen habitats under risk of predation. However, Abrahams et al. (2007) argued that diVerences in hypoxia tolerance that fall within the range of physiological acclimation of the predator may only benefit the prey if interactions are emphemeral. Hypoxia has been implicated as a determinant of fish assemblage structure in other systems where piscine predators are prevalent. For example, 2. BEHAVIORAL RESPONSES TO HYPOXIA 59 Tonn and Magnuson (1982) demonstrated that in Wisconsin lakes, a centrarchid-Esox predator assemblage was dominant if winter oxygen levels were high, while Umbra-cyprinid prey assemblages dominated in lakes with low winter oxygen levels. McNeil and Closs (2007) found a generally high level of tolerance to periodic hypoxia in the fishes of the Ovens River floodplain in south-east Australia with the exception of three species, one of which was the predatory introduced redfin perch (Perca fluviatilis), again supporting the role of hypoxic habitats as refuge for tolerant prey and emphasizing the implications of species-specific variation in hypoxia-tolerance for community structure. Finally, in their study of the fish community along a DO gradient in a Florida spring, McKinsey and Chapman (1998) found that the mosquitofish, Gambusia holbrooki, was the most abundant species at the boil of the spring where DO averaged 0.20 mg L-1. They suggested that the boil area may serve as refugium from predation for G. holbrooki; only one large predatory species was observed in the boil region (Amia calva, an air breather), whereas other piscivores are found in the main river. Avian predation could compromise the value of the boil refugium; however, G. holbrooki resides primarily in heavy vegetation along the boil margins (McKinsey and Chapman, 1998). In the context of predator–prey interactions under hypoxia, Robb and Abrahams (2003) suggested that there may be an ecological advantage of being small based on studies showing smaller fish to be more hypoxia tolerant than larger fish. Robb and Abrahams reviewed two plausible mechanisms to explain this size sensitivity: the negative allometric relationship for mass-specific gill-surface area (Muir, 1969; Hughes, 1984), and a fractal scaling relationship whereby larger fish may be limited by the fixed size of the red blood cells for gas exchange (West et al., 1997). Recently, Nilsson and Östlund-Nilsson (2008) reviewed the literature on eVects of body size on hypoxia tolerance in fishes. They argued that body size per se does not influence oxygen uptake ability because the gill respiratory surface has a similar scaling relationship as metabolic rate. In addition, where anaerobic ATP production is required for survival, large fish seem to have an advantage because of their lower mass-specific metabolic rate (Nilsson and Östlund-Nilsson, 2008). Indeed a physiological advantage of small size under hypoxic stress is not always evident, yet hypoxic refugia may still occur. For example, Sloman and colleagues (2006) reported that small Amazonian oscars (Astronotus ocellatus) seek out hypoxic habitats as refuge, but found evidence to suggest that the juveniles are not more tolerant than larger conspecifics, but rather, accept a greater physiological compromise to access hypoxic shelter. Although relationships between fish size and hypoxia tolerance are not consistent across studies, there is a growing body of empirical support for the role of hypoxic habitats in modulating piscine predator eVects by serving as refugia. 60 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE Hypoxia may also decrease vulnerability of fish prey to piscine predators if predator activity is reduced. There are a number of studies demonstrating decreased spontaneous activity (see Section 3 above), feeding rate, metabolism, and/or predator activity under hypoxic stress (e.g., Bejda et al., 1987; Breitburg et al., 1994; Shoji et al., 2005; Ripley and Foran, 2006). In this situation, risk of predation by piscivores may be reduced for fish prey; however, other aquatic predators may still take advantage of the negative eVects of hypoxia on the ability of fish to escape (Domenici et al., 2007), thus shifting the balance toward a new player in the game. For example, in a study of the eVects of hypoxia on the predation of larval red sea bream (Pagrus major) by the jellyfish, Aurelia aurit, and the juvenile Spanish mackerel, Scomberomorous niphonius, Shoji et al. (2005) found that lower DO induced higher predation rates by the tolerant jellyfish and lower predation rates by the juvenile mackerel, which had become physiologically stressed. The authors suggested that the increase in the number of jellyfish and its predation on juvenile sea bream have been driven by high nutrient loading in the Seto Inland Sea, which has accelerated eutrophication and exacerbated oxygen depletion. A similar shift in predator eVects was reported by Breitburg et al. (1997), who showed that under hypoxia, predation on naked gobies (Gobiosoma bosc) by adult piscivores decreased, while predation by the sea nettle (the jellyfish Chrysaora quniquecirrha) increased, reflecting both the high tolerance of the sea nettle and impaired fast-start performance (see Section 5.3 below) of the fish prey. 5.2. A Shift in the Beneficiary – Increased Prey Vulnerability under Hypoxia Stress 5.2.1. Risk of Surfacing Hypoxia may increase vulnerability of fish to predation by aVecting their vertical distribution, thereby increasing their potential encounter rate with air-breathing predators. As reviewed in Section 2.1, many fishes respond to hypoxia by using ASR. However, there are clearly costs associated with use of ASR. As DO levels approach zero, many fishes spend most of their time at the surface (Gee et al., 1978; Kramer and McClure, 1982; Chapman et al., 1995; Olowo and Chapman, 1996; Figure 2.2). ASR not only takes time away from other activities, but could place fish at higher risk of predation when they leave shelter and move to the surface where visibility is increased to both avian and other air-breathing or highly tolerant taxa (Kramer et al., 1983; Kramer, 1987; Domenici et al., 2007). Theoretically, air breathing is energetically more eYcient than water breathing for fishes because of air’s superior properties as a respiratory medium (Kramer, 1983, 1987). However, as discussed in Section 2.2, the 2. BEHAVIORAL RESPONSES TO HYPOXIA 61 rarity of air-breathing fishes and the richness and diversity of water breathers, even in habitats with low DO, suggest that there must be significant costs to air breathing (Kramer, 1983, 1987). These costs may include increased vulnerability to aerial predation and increased energetic costs of travel to the surface (Kramer, 1983; Kramer et al., 1983; Bevan and Kramer, 1986; Randle and Chapman, 2004). Kramer et al. (1983) used a trained heron to evaluate the risk of aerial predation for air-breathing fishes and non-airbreathing fishes that use ASR in response to hypoxic stress. They found that fish using ASR tended to surface at lower DO thresholds than air breathers, though surfacing time was longer. Kramer and colleagues suggested that fish using ASR may incur less risk of avian predation at moderate DO levels, but air breathers seem to have an advantage under extreme hypoxia. Given the potential costs of surfacing, it is not surprising that fishes show many behaviors to minimize the risk. For example, several air-breathing fishes use some form of synchronous air breathing, where individuals in a group breathe together or in rapid succession. Examples include: Lepisosteus osseus, L. oculatus (Hill, 1972), Hoplosternum thoracatum, Piabucina festae, Trichogaster leeri, Ancistrus chagresi (Kramer and Graham, 1976), and Clarias liocephalus (Chapman and Chapman, 1994). It is argued that the selective factor underlying synchrony is predator pressure, with clumped breathing reducing potential for encounter with predators in a manner analogous to schooling (Kramer and Graham, 1976; Gee, 1980; Chapman and Chapman, 1994). Fish have also been shown to reduce the risk of surfacing by selecting less risky habitats (Wolf and Kramer, 1987; Shingles et al., 2005) or at less risky times of the diel cycle (Saint-Paul and Soares, 1987). 5.2.2. Fast-start Recent studies have demonstrated negative eVects of hypoxia on the escape response in fishes (Domenici et al., 2007). In response to predator attack, fast-starts are a critical evasion strategy in fishes that consist of a sudden acceleration in a direction away from the stimulus. In teleost, this is often characterized by bending the body into a C-shape. The fast-start escape response in fishes is driven anaerobically (Webb, 1993, 1998; Wakeling and Johnston, 1998); however, recent work indicates that hypoxia can still have detrimental eVects on the response. In the golden grey mullet (Liza aurata), Lefrançois and colleagues (2005) reported that hypoxia aVected escape performance by impairing both responsiveness and directionality, suggesting reduced sensitivity of fishes to mechanico-acoustic stimuli. In the golden grey mullet, additional negative eVects on locomotor performance were observed when surface access was denied. The golden grey mullet uses ASR in response to hypoxic stress, and by doing so can reduce the negative eVects of hypoxia on fast-start performance, but this can increase exposure to avian predation 62 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE (Lefrançois et al., 2005). In contrast, the European sea bass, Dicentrarchus labrax, does not respond to hypoxia by using ASR. Lefrançois and Domenici (2005) concluded that locomotor variables associated with the fast-start response in the sea bass were not aVected by hypoxia exposure; however, similar to the mullet, the main eVect was decreased responsiveness and directionality. At this stage there are an insuYcient number of studies to draw generalities with respect to the impact of hypoxia on escape behavior. But, there are clearly species-specific features, and it will be important to broaden the geographical, ecological, and phylogenetic scope of the species studied. In addition, the ecological implications of impaired fast-start performance on vulnerability need to be both studied and interpreted in light of eVects on water-breathing predators. 5.2.3. Schooling Schooling in fishes has been related primarily to predator risk and prey detection (Godin, 1986; Magurran, 1990; Pitcher and Parrish, 1993), and hypoxia can potentially aVect the benefits of schooling by influencing spatial structure and velocity of the group. Schooling may actually induce hypoxia along the axis of motion (toward the rear) because of the oxygen consumption of the fish in the front of the school (McFarland and Moss, 1967; Domenici et al., 2007). The response to hypoxia observed in the Atlantic herring, Clupea harengus (Domenici et al., 2002), is an increase in the school volume and width (Moss and McFarland, 1970; Domenici et al., 2000, 2002), which may counteract hypoxic stress by increasing the spacing among individuals. However, this could negatively aVect the sensory modalities used in school coordination and/or decrease energetic advantages (Domenici et al., 2007). Moss and McFarland (1970) reported an increase in speed under hypoxia in the anchovy, Engraulis mordax, but not until near lethal levels were reached. In the Atlantic herring, Herbert and SteVensen (2006) also reported increased swimming speed in response to stepwise progressive hypoxia, which they argued was an adaptive avoidance response. 5.3. When Prey Becomes Predator: Hypoxia and Fish–Invertebrate Interactions Hypoxia has also been implicated as a factor underlying shifts in trophic interactions between fish and their invertebrate prey. Again, the beneficiary of the interaction often depends on the relative tolerance to hypoxic stress. Invertebrate species that are not tolerant of hypoxia have been observed to avoid seasonal hypolimnetic oxygen depletion by migration up into the water column or to the ice–water interface in the case of winterkill lakes (Nagell, 1977; Magnuson et al., 1985). However, in lakes with fish this can 2. BEHAVIORAL RESPONSES TO HYPOXIA 63 result in very high invertebrate prey mortality (Rahel and Kolar, 1990). Invertebrate species capable of tolerating low DO may use hypoxic benthic areas in stratified lakes as refugia to avoid fish predation (Rahel and Kolar, 1990; Stirling et al., 1990; Kolar and Rahel, 1993). In a series of behavioral experiments, Kolar and Rahel (1993) found that highly mobile taxa with low tolerance to hypoxia (e.g., mayflies and amphipods) moved upward from their benthic refuge in response to oxygen depletion at the substrate–water interface and were preyed upon by fish. Other taxa were less mobile and therefore less vulnerable to the fish. Species vulnerable to both hypoxia and predation altered their behavior to remain longer in the benthic zone in the presence of fish, thus demonstrating a tradeoV between costs of hypoxia and predation risk. Tolerance to hypoxia by fish predators can limit the eVectiveness of benthic refugia. When fish predators are capable of tolerating lethal levels of low oxygen for short periods, they can potentially forage in hypoxic waters. Rahel and Nutzman (1994) examined the foraging behavior of the central mudminnow (Umbra limi) in a stratified lake in Wisconsin. Although DO in the benthic zone of the lake was lethal to the mudminnows, they routinely ventured into the environment for short-term foraging bouts. They proposed the conditions promoting foraging in lethal environments represented a tradeoV between food availability in non-lethal waters and the cost of short-term exposure to abiotic stress. McParland and Paszkowski (2006) provided experimental evidence to suggest that small fish that colonize eutrophic, hypoxia-prone prairie potholes in Alberta can reduce aquatic invertebrate densities. By adding brook stickleback (Culaea inconstans) and fathead minnow (Pimephales promelas) to fishless potholes, they were able to show that these small-bodied hypoxia-tolerant fish reduced invertebrate prey, which altered the foraging behavior of blue-winged teal and other waterbirds. 5.4. Hypoxia and Social Interactions Given the potential eVects of hypoxia exposure on metabolic rate, activity, motivation, and habitat use, it is not surprising to find evidence for changes in social behavior in response to aquatic oxygen availability. EVects of hypoxia on the dynamics and structure of fish schools has been discussed in the context of increased school volume as a mechanism to elevate DO within the school. It has been argued that spacing within schools allows fish to keep track of one another without colliding. Domenici and colleagues (2002) suggested that the fast sound pulses emitted by some fishes, which may assist in synchronous response to predators (Gray and Denton, 1991) may be less eVective when school volume is increased under hypoxic stress. Hypoxia may also aVect sensory channels involved in fish maneuverability, and thus impair fast antipredator manoeuvres (Domenici et al., 2007). 64 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE The use of ASR in response to extreme hypoxia has been demonstrated to aVect social behaviors within groups of conspecifics. For example, in their study of ASR in swamp-dwelling and open-water populations of the haplochromine cichlid Astatotilapia ‘‘wrought-iron,’’ Melnychuk and Chapman (2002) found that the pre-ASR aggression rate was higher in swamp-dwelling ‘‘wrought-iron’’ than in the open-water populations, but the aggression rate dropped in both open-water and swamp-dwelling fish between the pre-ASR and post-ASR periods. The use of ASR may impose both time and energetic constraints that reduce aggression. This could aVect the development and maintenance of dominance hierarchies in cichlids and other species with complex social systems. For example, in three-spined stickleback from lotic and lentic sites, Sneddon and Yerbury (2004) found that dominance hierarchies were less stable when fish from the river site were exposed to hypoxia (20% saturation). In addition, fish under hypoxic conditions from both sites showed a decreased frequency of aggressive acts with the exception of the most dominant fish, and the most dominant fish lost mass under hypoxia conditions. Sneddon and Yerbury (2004) hypothesized that the maintenance of aggression under hypoxia in the dominant fish had a significant energetic cost. Hypoxia may also aVect aggressive contests among fishes by altering display signals. Abrahams et al. (2005) explored the eVect of hypoxia on the opercular displays of Siamese fighting fish, Betta splendens. The fish reduced their opercular displays under hypoxic conditions, which was interpreted as an honest signal that indicated the physiological condition of the contestant. Although many behavioral studies of physiological stressors employ an acclimation protocol, Marks et al. (2005) examined the influence of development under hypoxia in altering aggression in the zebrafish (Danio rerio). They found evidence for an eVect of the developmental environment and the adult behavioral environment (acclimation). Aggression levels of fish reared in hypoxia were lower than in those reared in normoxia. However, when zebrafish were acclimated to either hypoxia or normoxia, their aggression level was highest in the environment in which they had been reared, providing evidence that hypoxic stress during development aVects behavioral responses of this species and emphasizing the importance of understanding both developmental and acclimation responses. 6. SUMMARY From this review of behavioral responses to hypoxia, it is clear that low DO can become ecologically active at levels far above those that are lethal, and can induce behaviors that alter fish distributions and their inter- and intra-specific interactions. Variation in oxygen availability can influence 2. BEHAVIORAL RESPONSES TO HYPOXIA 65 spatial and temporal patterns of distribution at various scales, from the micro- to the macrohabitat, and alter crucial components of the interactions between a fish and its environment including predator–prey interactions, schooling behavior, dominance, aggression, and parental care. Aquatic surface respiration and air breathing are two of the most pronounced behavioral responses by bony fishes to aquatic hypoxia. Both behaviors are reflexes driven by oxygen-sensitive chemoreceptors, but can be modulated by inputs from higher centers, in particular as a function of perceived risk of predation and other costs. Many fishes also exhibit changes in spontaneous swimming activity when exposed to hypoxia that can comprise either a reduction in activity or an increase in activity, depending upon the species and the context. A reduction in spontaneous swimming activity has been interpreted as an energy-saving response, while an increase has been interpreted as an avoidance response. As might be expected, many species can exhibit both types of response, depending on the degree of hypoxia, the rate of change in DO, and their degree of activity in normoxia. Another locomotor response that can reduce exposure to hypoxia is spontaneous emersion, whereby amphibious and semi-amphibious species leave water to go onto land; however, the role of hypoxia as a driver has only been studied in a few species, and this remains an area ripe for investigation. In addition to the large energetic costs associated with locomotor activity, another significant metabolic cost for some fish species is the energy directed to parental care. Costs of parental care in fishes may be particularly severe under hypoxia, due to the challenge of providing oxygen to the eggs. High levels of parental care may, however, be essential under hypoxic conditions, to ensure survival of the eggs and young. Although the literature in this area is still depauperate, DO seems to be an important driver of parental care behavior across a range of strategies from guarding to viviparity. Given the potential eVects of hypoxia exposure on metabolic rate, activity, and motivation, it is not surprising to find evidence for changes in social behavior in response to oxygen availability, including shifts in school volume and synchronous response with schools, as well as levels of aggression that could aVect the development and maintenance of dominance hierarchies in cichlids and other species with complex social systems. An integration of knowledge on behavioral responses to hypoxia and the relative tolerance of species supports the role of hypoxia as a modulator of species interactions, in particular predator–prey relationships where it can alter the success rate of the predator and/or the vulnerability of the prey. Whether hypoxia favors the predator or prey depends, at least in part, on the relative tolerance of the interactants. Hypoxia can elicit behaviors that increase predation risk such as ASR or an increase in the frequency of air breathing; it can negatively impact fast-start performance of prey or alter the 66 LAUREN J. CHAPMAN AND DAVID J. MCKENZIE dynamics of schooling behavior. For aquatic water-breathing predators, hypoxia can decrease predation through metabolic depression, lowered appetite, or decreased performance. Hypoxia has also been implicated as a factor underlying shifts in trophic interactions between fish and their invertebrate prey. Again, the beneficiary of the interaction often depends on the relative tolerance of the species to hypoxic stress. Tolerance to hypoxia by fish predators can limit the eVectiveness of benthic refugia for macroinvertebrates. 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DiVerential propensities for aerial emergence in intertidal sculpins (Teleostei; Cottidae). J. Exp. Mar. Biol. Ecol. 191, 195–207. Zoran, M. J., and Ward, J. A. (1983). Parental egg care behavior and fanning activitiy for the orange chromide, Etroplus maculatus. Environ. Biol. Fish. 8, 301–310. 3 EFFECTS OF HYPOXIA ON FISH REPRODUCTION AND DEVELOPMENT RUDOLF S. S. WU 1. Introduction 1.2. Occurrence of Hypoxia in the Aquatic Environment 1.2. Global Changes in Fish Populations and Communities 2. Hypoxia and Fish Reproduction 2.1. Control of Reproductive Processes in Fish 2.2. EVects of Hypoxia on the HPG Axis, Steroidogenesis, and Sex Hormones 2.3. Hypoxia Impairs Fish Reproduction 3. Hypoxia and Fish Development 3.1. Regulation of Sex DiVerentiation, Sex Development, and Sex Determination 3.2. Hypoxia Impairs Fish Development 4. Supporting Evidence for the Effects of Hypoxia on Reproduction and Development in other Vertebrates 4.1. In Vitro Evidence 4.2. In Vivo Evidence 5. The Role of Hypoxia-Inducible Factors 6. Biological and Ecological Implications 7. Conclusions Hypoxia has a profound eVect on fish reproduction and development. Behavioral studies revealed that hypoxia can aVect courtship behaviors, mate choice, and reproductive eVorts in fish. Both laboratory and field evidence showed that hypoxia can cause major reproductive impairments by inhibiting testicular and ovarian development, aVecting production and quality of sperm and egg, reducing fertilization and hatching success, and aVecting larval survivorship as well as the quality and fitness of juveniles. Emerging evidence further showed that hypoxia does not impair these key reproductive processes through a general down-regulation of metabolism and reproductive functions, but does so by aVecting specific hormones, neurotransmitters, and receptors along the 79 Hypoxia: Volume 27 FISH PHYSIOLOGY Copyright # 2009 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(08)00003-4 80 RUDOLF S. S. WU hypothalamus–pituitary–gonad axis as well as certain enzymes controlling steroidogenesis and vitellogenesis. In zebrafish, hypoxia has been shown to down-regulate CYP19 and alter the ratio of testosterone to estradiol during early sex development, leading to a male-biased F1 generation. Hypoxia has been shown to delay embryonic development and hatching in many fish species, and embryos in some species may undergo complete developmental arrest under anoxia. In zebrafish embryos, blastomeres were arrested during the S and G2 phases of the cell cycle under anoxia. Fish embryos developed under hypoxia lost their normal synchronization, and abnormalities in spinal and vascular development are commonly observed. Results of both laboratory and field studies showed a higher percentage of malformation in fish developed under hypoxic conditions, possibly through altering their normal apoptosis. Both in vitro and in vivo studies demonstrated that expression levels of certain genes directly or indirectly related to cell cycle, cell proliferation, and apoptosis, which underpin some of the fundamental processes related to development, are aVected by hypoxia. Whether hypoxic inducible factor is involved in mediating the changes in gene expression and the observed reproductive and development impairments remains unclear. 1. INTRODUCTION 1.2. Occurrence of Hypoxia in the Aquatic Environment Hypoxia is generally defined as dissolved oxygen less than 2.8 mg O2/L (equivalent to 2 mL O2/L or 91.4 mM) (Diaz and Rosenberg, 1995) and anoxia means no oxygen. Hypoxia/anoxia occurs in a variety of marine, estuarine, and freshwater habitats, and can be a natural phenomenon caused by vertical stratification such as formation of haloclines and thermoclines (Rosenberg et al., 1991; Pihl et al., 1992; Hoback and Barnhart, 1996). Globally, the total area of permanently hypoxic continental shelf and bathyal sea floor with dissolved oxygen <0.5 ml O2/L (minimal oxygen zones) is estimated at more than one million square kilometers (Helly and Levin, 2004). More often, however, the occurrence of hypoxia is due to excessive anthropogenic input of nutrients and organic matters into water bodies with poor circulation (Pihl et al., 1992; Dalla Via et al., 1994; Peckol and Rivers, 1995; Gamenick et al., 1996; Sandberg, 1997; Wu and Lam, 1997; Aarnio et al., 1998; Mason, 1998). Nowadays, hypoxia or anoxia aVecting thousands of square kilometers of marine waters has been commonly reported for waters around North and South America, Africa, Europe, India, Southeast Asia, Australia, Japan, and China (Nixon, 1990; Diaz and Rosenberg, 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 81 1995; Wu, 1999). Likewise, hypoxia also commonly occurs in freshwater systems in many countries (Sabo et al., 1999; Keister et al., 2000; Fontenot et al., 2001; Breitburg et al., 2003). In China, for example, over 77% of the freshwater ecosystems are now considered under serious threat by hypoxia (Ma and Li, 2002). Indeed, hypoxia caused by eutrophication is now considered to be one of the most serious threats to aquatic ecosystems worldwide. Hypoxia has not only increased in terms of frequency, severity, and areas aVected in the last two decades, but is likely to be further exacerbated in the coming years (Diaz and Rosenberg, 1995; Goldberg, 1995; Gray et al., 2002; Wu, 2002). The eminence of the problem is clearly exemplified by the global increase in the number of ‘‘dead zones’’ from 150 in 2004 to 200 in 2006 (UNEP, 2006). 1.2. Global Changes in Fish Populations and Communities Unlike mammals, which can only tolerate a narrow range of oxygen regimes, fish often have to contend with large fluctuations of oxygen in their natural environment, which sometimes can occur very rapidly (e.g., within a day) or within minutes if they swim through a hypoxic region. Indeed, there is no other environmental parameter, except perhaps temperature, in the aquatic ecosystem that can change so drastically, within such a short time, as dissolved oxygen. Thus, it is not surprising that many fish species have evolved a variety of molecular, biochemical, and physiological adaptations to cope with hypoxia in the course of evolution (see Hochachka and Somero, 2002). Hypoxia has already led to major changes in fish species composition, alteration of food webs and community structure, decrease in species richness and diversity, population declines and extinction of sensitive species in both marine and freshwater systems in many parts of the world (Wu, 1982; Dauer, 1993; Pihl, 1994; Diaz and Rosenberg, 1995; Alexander et al., 2000; Diaz, 2001; Wanink et al., 2001; Wu, 2002). Massive fish kills over large areas due to hypoxia have been reported in coastal areas all over the world, and sensitive species have been permanently or periodically removed in many places (Wu, 1982; Diaz and Rosenberg, 1995). Massive fish kills in aquaculture due to hypoxia are equally common (Townsend et al., 1992; Grantham et al., 2004; Azanza et al., 2005; Parvez et al., 2006; Bouchet et al., 2007). Besides causing direct death, hypoxia may also reduce growth, alter behaviors of fishes, and change their food items, thereby reducing their abundance and diversity (Breitburg, 2002). Reductions in the biomass and landing of fish have been reported in many hypoxic areas (Dyer et al., 1983; Rosenberg and Loo, 1988; Baden et al., 1990; Pihl et al., 1991; Breitburg, 1992; Lekve et al., 1999). Petersen and Pihl (1995) demonstrated a significant 82 RUDOLF S. S. WU 30% 50% 100% Biomass (kg wet wt. h−1) 80 60 40 20 0 0 2 4 6 8 10 Oxygen concentration (mg L−1) Fig. 3.1. The relationship between fish biomass (kg wet wt. h-1 trawling) and oxygen concentration in the bottom water of SE Kattegat. Plaice (<, p < 0.02), Dab (, p < 0.01) in the years 1984 to 1990 (After Petersen and Pihl, 1995.) relationship between biomass (catch per unit eVort) of plaice and dab and oxygen concentration in the bottom water of Kattegat, Sweden (Figure 3.1). Hypoxia might also favor the selection of small benthic species with a shorter life cycle, and such long-term changes in prey species, coupled with a lower level of oxygen in bottom waters, have been related to a shift in dominance from demersal to pelagic fish in the Kattegat, Sweden (Pihl, 1994). The observed decline in natural fish populations may also be caused by reproductive impairments resulting from chronic hypoxia, although it would be diYcult to decipher the exact cause, or to attribute the observed population decline and community changes to hypoxia per se, since hypoxia in the natural environment is often associated with other confounding factors such as pollution and overfishing. Arguably, reproductive output, quality of gametes, and survival of larvae and juveniles are the most important factors in determining reproductive success and hence fitness and survival of any species. At the same time, both reproduction and development involve a myriad of intricate processes, making these life stages particularly vulnerable to environmental stresses (Connell et al., 1999), especially since these intricate processes are tightly controlled by hormones that are very sensitive to environmental changes (Bhattacharya, 1999; Seale et al., 2002; Okuzawa et al., 2003). Notably, many 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 83 coastal areas, which serve as important spawning and nursery grounds, are located in areas where occurrence of hypoxia is common. Surprisingly, the eVects of hypoxia on reproduction and development of fish, especially on natural populations, remain poorly understood (Wu, 2002). A field study in the Atchafalaya River of Louisiana (Fontenot et al., 2001) demonstrated a strong, positive relationship between dissolved oxygen level and the abundance of larval sunfish (Lepomis spp.) and shad (Dorosoma spp.). The field study of Ingendahl (2001) reported that sea trout (Salmo trutta) alevins only emerged from covered redds in tributaries of the Rhine where the mean dissolved oxygen level was above 6.9 mg O2/L (56% saturation). More recently, Dumas et al. (2007) reported that low oxygen delayed brown trout alevin development and growth in a tributary of the Adour river in south-west France. The above field evidence oVers indirect evidence supporting the postulation that hypoxia can aVect reproduction and/or larval development in their natural habitats, contributing to the population decline and community changes observed in hypoxic areas worldwide. 2. HYPOXIA AND FISH REPRODUCTION Hypoxia impairs reproductive success by aVecting a number of key reproductive processes, including gametogenesis, the number and quality of sperm and egg, reproductive behaviors, fertilization success, hatching, and, subsequently, larval survivorship and the quality and fitness of juveniles. These impairments may be mediated through disrupting the various hormones and enzymes regulating these key reproductive processes, or by reducing food intake and, hence, the energy available for reproductive investment. 2.1. Control of Reproductive Processes in Fish 2.1.1. The Hypothalamus^Pituitary^Gonad Axis Despite the fact that reproductive processes in fish are highly diverse and vary among species, the intricate process is very conservative and tightly regulated by the hypothalamus–pituitary–gonad (HPG) axis. The HPG axis controls gametogenesis, reproductive behavior, and reproduction, including the release of gametes and fertilization via positive and/or negative feedback loops (for a detailed review, please see Ankley and Johnson, 2004; Thomas, 2008). The control of reproductive hormone synthesis and secretion along the HPG axis is schematically shown in Figure 3.2. The basic features and control of the HPG axis in fish closely resembles those in higher vertebrates. 84 RUDOLF S. S. WU External stimuli Internal stimuli (e.g. temperature, photoperiod) (e.g. biological clock, nutrient status) Hypothalamus +/− +/− Dopamine GnRH Serotonin Pituitary +/− +/− + LH FSH Testis FSH-R StAR CYP17 StAR CYP11A +/− Other pituitary hormones 17 β-HSD 3 β-HSD CYP11A 17 β-HSD CYP17 Ovary FSH-R CYP11 β 20 β-HSD 3 β-HSD CYP19A 20 β-HSD Thecal cells Granulosa cells Leydig cells 11-KT Testicular development spermatogenesis T E2 Ovarian development oogenesis Progestin Gamete maturation spawning success Fig. 3.2. The HPG axis and control of reproductive hormone synthesis and secretion in male and female teleosts. Key regulatory hormones for steroidogenesis are highlighted in yellow, hypoxiaresponsive genes are highlighted in green, and steroid hormones involved in gametogenesis are highlighted in purple. (Modified from Weltzien et al., 2004.) Environmental cues (e.g., temperature, photoperiod, and nutritional changes as well as hypoxia) detected by various sensory systems are relayed to the hypothalamus. The hypothalamus releases various neurotransmitters and neuropeptides, leading to the secretion of gonadotropin-releasing hormone (GnRH) into the intercellular space of the hypophysis of the pituitary through the hypothalamic neurons. The decapeptide GnRH then binds to the specific receptors on the plasma membrane of the gonadotropes from the anterior pituitary and stimulates the production and/or release of two types of glycoprotein gonadotropic hormones (GtHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Both FSH and LH consist of an a subunit (which is common to both GtHs and thyroid-stimulating hormone) and a b subunit, which is hormone specific. These two gonadotropins are then transported to the gonads through blood circulation where they bind to specific G-protein-coupled membrane GtH receptors (GtH-Rs) and activate G-proteins, adenyl cyclase, and Ca2+-dependent second messenger signaling pathways, which subsequently lead to the production and 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 85 secretion of steroid hormones. FSH primarily induces oogenesis and spermatogenesis, while LH directs maturation and release of gametes. FSH and LH stimulate the thecal and granulosa cells of the ovary to produce the female steroid hormone 17b-estradiol, and FSH induces the enzyme aromatase in the granulosa cells, which converts testosterone to estradiol. 17bestradiol stimulates oocyte development in the ovary and the synthesis of vitellogenin (the egg yolk precursor protein) in the liver for release into the bloodstream. In the male fish, gonadotropin stimulates Leydig cells to produce the androgens (testosterone and 11-ketotestosterone), which, in turn, activates Sertoli cells to stimulate premitotic spermatogonia to complete spermatogenesis (Nagahama et al., 1994; Thomas, 2008). Breakdown products of sex hormones may act as pheromones directing behavior (Sorensen et al., 2004). A number of neurotransmitters also play an important role in modulating reproductive processes in fish. For example, the monoamine neurotransmitter serotonin (5-HT) acts on GnRH and potentiates LH secretion. Another neurotransmitter, dopamine (DA), inhibits LH secretion in some species (e.g., carp and catfish) but not in others (e.g., Atlantic croakers). Other neurotransmitters and neuropeptides such as neuropeptide Y, gamma aminobutyric acid (GABA), glutamate, and taurine are also implicated in the neuroendocrine control of GTH release in teleosts (Kah et al., 1993). 2.1.2. Steroidogenesis Similar to other vertebrates, gametogenesis and sex behaviors in fish are directly controlled by sex steroid hormones. The synthesis of sex steroid hormones (steroidogenesis) mainly takes place in adrenal tissues (zona glomerulosa, zona fasciculate, and zona reticularis) and gonadal tissues (male testes and female ovaries) (Young et al., 2004). A schematic representation of the key steps involved in steroidogenesis in teleosts is shown in Figure 3.3. Cholesterol is the common precursor for all sex steroid hormones. The first rate-determining step involves the importation of cholesterol into the inner mitochondrial membrane. This step, which initiates steroidogenesis, is regulated by the steroidogenic acute regulatory protein (StAR), and the production of StAR is upregulated by GtH in fish (Bauer et al., 2000; Stocco, 2001; Kusakabe et al., 2002). Subsequent steps of the steroidogenic pathway are controlled by a number of steroidogenic enzymes including cytochrome P450 enzymes and hydroxysteroid dehydrogenases (HSDs) (Miller, 1988; Senthilkumaran et al., 2004; Weltzien et al., 2004; Miller, 2005). Cholesterol is converted to pregnenolone by the P450 enzyme cholesterol side chain cleavage (P450scc or CYP 11A) on the inner membrane of the mitochondria (Takahashi et al., 1993). Pregnenolone is then converted through a series of steps to androgens by 3b-hydrosteroid dehydrogenase (3bHSD), 86 RUDOLF S. S. WU LH, FSH Tropic hormone receptor Lipid droplet ATP cAMP Cholesterol ester Cholesterol ester hydrolase StAR PKA ? PKC Transfer of cholesterol to inner mitochondrial membrane HO P450scc Free cholesterol Mitochondrion CH3 CH3 CO CO HO O Progesterone Pregnenolone P450c17 CH3 CO OH 3b-HSD CH3 CO CH3 20b-HSD H O OH OH OH O HO 17α-hydroxypregnenolone O 17α-hydroxyprogesterone 17α,20β-dihydroxy4-pregnen-3-one P450c17 O HO 3b-HSD O O Dehydroepiandrosterone Androstenedione 17b-HSD OH HO Androstenediol OH P450arom O OH HO Testosterone Estradiol-17β P45011b OH 11b-HSD OH O HO O 11β-hydroxytestosterone O 11-ketotestosterone Fig. 3.3. A schematic pathway of steroidogenesis in the gonads of teleost fish. White arrows indicate the proposed androgen synthesis pathway. Gray arrows indicate the proposed progestogen synthesis pathway. Genes known to be inducible by hypoxia are circled in red; hormones known to be aVected by hypoxia are framed in blue. (Modified from Young et al., 2004.) 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 87 17a-hydroxylase (P450c17), 21-hydroxylase (P450c21), 11b-hydrolase (P450c11), and 20b-dehydroxysteroid dehydrogenase (20b-HSD) to form progestins (Nagahama, 2000) and then 11-ketotestosterone (11-KT). Finally, testosterone is converted to estrogen by aromatase (encoded by P450arom or CYP19). There is good evidence to show that reproductive processes in fish are regulated by the expression levels of the various steroidogenic enzymes. In the channel catfish (Ictalurus punctatus), for example, P450c17, P450scc, and P450arom were up-regulated at the onset of ovarian recrudescence and during early vitellogenic growth of the oocytes, but subsided upon completion of vitellogenesis (Kumar et al., 2000). In salmonids, FSH stimulates the expression and activity of P450arom, and regulates the production of E2 in the ovary (Montserrat et al., 2004). The above evidence shows that GtH may regulate sex steroid levels through regulating various steroidogenic enzymes. It also appears that the genes encoding these three steroidogenic cytochrome P450s have a similar regulatory mechanism (Kumar et al., 2000). 2.1.3. Sex Differentiation and Sex Determination Unlike mammals, fish exhibit considerable plasticity in sex determination irrespective of their genotypic sex. Many environmental factors (e.g., temperature, photoperiod, and social behavior), chemicals, and sex hormones may influence sex diVerentiation and determination (Jobling, 1995). In some gonochoristic species such as zebrafish, the gonads will first pass through a juvenile ‘‘ovary’’ phase before diVerentiating into testes or ovary. A similar sexual developmental pattern is generally found in the protogynous species. The genetic and molecular mechanisms underlying sex determination and diVerentiation in fish remain unclear. In particular, the reason why genetic makeup in fish is relatively easy to override by environmental factors as compared to mammals remains unknown, both from a mechanistic and evolutionary points of view. Unlike mammals, sex chromosomes have only been identified in only about 10% of fish (Devlin and Nagahama, 2002). Recently, the DMY (Y-specific DM domain) gene has been identified as the sex-determining gene in the Y chromosome of freshwater medaka (Oryzias latipes) (Matsuda et al., 2002). Gonadotropin, thyroid hormones, growth hormone, insulin, and insulin-like growth factors have been shown to aVect ovarian growth in brown trout (Salmo trutta), rainbow trout (Oncorhynchus mykiss), and Chinook salmon (Oncorhynchus tshawytscha) (Tyler and Sumpter, 1996), and hence sex development. Since the regulation of the HPG axis is a complex and highly intricate process, disruption of the HPG axis is likely to alter sex diVerentiation and gametogenesis. The early work of Yamamoto (1961) revealed that complete sex reversal can occur in medaka when steroid hormones (estrogens, androgens, or 88 RUDOLF S. S. WU progestins) were administered during their early developmental stages, resulting in a phenotypic male or female irrespective of genetic sex. Similarly, administration of androgens can alter sex diVerentiation in Chinook salmon (Oncorrhychus tshawytcha), turning genotypic females into males (Piferrer et al., 1993). Subsequent investigations further revealed that inhibition of the cytochrome P450 aromatase complex (by androgens or aromatase inhibitors) during sex diVerentiation in fish can turn genotypic females into phenotypic males. For example, sex change was reported in the Japanese flounder (Paralichthys olivaceus) when treated with aromatase inhibitor and 17a-methyl-testosterone (Kitano et al., 2000); sex change in the goby (Gobiodon histrio) was attributable to aromatase activities and levels of 11-KT (Kroon et al., 2003). In the Nile tilapia (Oreochromis niloticus), genotypic female fry treated with dietary Fadrozole (an aromatase inhibitor) during sexual diVerentiation led to an increase in the percentage of males (Kwon et al., 2000). Fenske and Segner (2004) showed that aromatase modulation alters gonad diVerentiation in zebrafish. The above evidence clearly demonstrated that sex diVerentiation and sex determination in many fish species are modulated by sex hormones, and factors aVecting key enzymes regulating steroidogenesis may alter the balance of sex hormones and hence sex determination. In particular, P450arom, which converts testosterone into estradiol and aVects the ratio of androgens to estrogens, could be expected to play a critical role in fish reproduction and sex diVerentiation. Since the cytochrome P450 enzymes demand oxygen (Nishimura et al., 2006), hypoxia may potentially disrupt normal steriodogenesis and interfere with sex diVerentiation and sex determination via these enzymes. This, however, may be only one of the many ways in which hypoxia modulates sex diVerentiation and determination in fish. 2.2. EVects of Hypoxia on the HPG Axis, Steroidogenesis, and Sex Hormones 2.2.1. Gene Expression Profile A detailed review of the eVects of hypoxia on gene expression profile is given in Chapter 10, and only those hypoxia-responsive genes relating to fish reproduction and development will be reviewed here. Several attempts have been made to map out the global responses of genes to hypoxia, using cDNA microarray technology. Some of the genes responsive to hypoxia, as revealed in these studies, are indirectly or remotely related to neurotransmitters, hormones, cell cycle, cell proliferation, and apoptosis, which underpin some of the fundamental processes related to reproduction and development. 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 89 Gracey et al. (2001) examined gene expression in liver, brain, skeletal muscle, and heart from adult gobies (Gillichthys mirabilis and G. seta) exposed to hypoxia (0.8 mg O2/L, 10% saturation) for 6 days. Up-regulation of various genes involved in glycolysis, iron metabolism, amino acid metabolism, and growth suppression were found in the liver, and down-regulation of genes involved in protein translation and muscle contraction were found in both skeletal muscle and heart. Induction of MAP kinase phosphatase 1 (KP-1), which stimulates cell growth, was found in all tissues, whereas the anti-proliferation genes transducer, Erb-B2 (Tob) and B-cell translocation gene-1 (BTG-1), were induced in the liver. Using the same approach, Ton et al. (2003) studied the expression patterns of 4512 genes in whole embryos of zebrafish [24 hours post-fertilization (hpf )] exposed to extreme hypoxia (5% saturation, levels at which zebrafish embryos are unable to survive for more than 24 h) for 24 h followed by 5 h of recovery. Hypoxia increased the expression of HIF-1 and certain glycolytic genes, but down-regulated genes involved in oxidative carbohydrate metabolism, muscle contraction, translation, and cell cycle progression. Hypoxia also repressed high-motility group proteins HMG-Y and HMG2a, histone H3, proliferating cell nuclear antigen (PCNA), and cyclin G1 and G2/mitotic-specific cyclin involved in cell division. This is consistent with the observations of Padilla and Roth (2001) that the S and G2 phases of the cell cycle in zebrafish embryonic development (in 4-cell stage embryos) was arrested under hypoxia and anoxia. Down-regulation of intracellular transducers e.g., small GTP-binding protein Rab, which may be related to suppression of cell growth and proliferation under hypoxia, and induction of HSP70, which is known to protect cells against apoptosis, were also found. Using a microarray containing 8046 medaka (Oryzias latipes) genes, Ju et al. (2007) reported that 501 genes in the brain, 442 in the gill, and 715 in the liver were diVerentially expressed in medaka exposed to hypoxia. Among these, two genes relating to neurotransmitter transport in the brain were down-regulated, while two genes in the brain related to response to hormones were up-regulated during hypoxia. The above three microarray screening studies provided evidence that certain genes relating directly or indirectly to reproductive hormones and fundamental processes in development (e.g., apoptosis, cell proliferation, and cell growth) in fish may be aVected by hypoxia. Despite the fact that neither apoptosis nor necrosis was found in the gills of zebrafish subjected to hypoxia (10% saturation for 21 days), a number of genes relating to apoptosis and growth regulation were responsive to hypoxia (Figure 3.4), and the majority of the former are anti-apoptotic genes (van der Meer et al., 2005). HSP70, which is known to protect cells against apoptosis (Höhfeld, 1998), was also up-regulated. 90 RUDOLF S. S. WU A B Beta-1 tubulin Simple type ii keratin kBa s1:s1 Swap-70 arp2/3 complex 16kda subunit Map 1 light chain 3-like protein 1 Type ii cytokeratin; ckii Alpha-tubulin p115 p115 Beta-2-tubulin aa 1– 443 Vacuolar atpase sub unit f: vatf ma binding motif protein 5 Zinc finger protein 216 Human emapii dap-3 rbap46 Cell death-regulatory protein grim 19 dad-1 d4-gdp-dissociation inhibitor 8¥ 4¥ 2¥ 1:1 2¥ 4¥ 8¥ Repressed Induced Fig. 3.4. Genes related to (A) apoptosis and (B) growth regulation found to be diVerentially expressed under hypoxic conditions in the gill of zebrafish. Quantitative changes in gene expression are induced genes (red) and repressed genes (green). (After van de Meer et al., 2005.) However, many genes specifically related to HPG, steroidogenic enzymes, and neurotransmitters that were found to be aVected by hypoxia in zebrafish, medaka, Gulf killifish, and Atlantic croakers in several independent studies (see Sections 2.2.2 and 2.2.4 below) were not revealed in any of these microarray screening studies, suggesting that some of these microarray data should be viewed with great caution, in particular the study by Ton et al. (2003) where fish were exposed to an extremely low oxygen level at which death would begin to occur. 2.2.2. GnRH and GtH It is well known that fish reproduction is regulated by the hypothalamus– pituitary–gonad (HPG) axis; despite this knowledge, studies on the eVects of hypoxia on fish reproduction, thus far, have almost exclusively focused on sex steroid hormones in gonads. Only very limited information is available on the eVects of hypoxia at the hypothalamus and pituitary levels. Whether hypoxia does aVect GnRH, GtHs, and their receptors and the manner in which it does so remain unknown. Lu et al. (2007) reported a significant reduction in mRNA of pituitary FSHb in female zebrafish after exposure to hypoxia (0.6 mg O2/L, 8% saturation) for 3 weeks. Thomas et al. (2007) reported that levels of plasma LH were below detection limit in Atlantic croakers exposed to hypoxia (in the saline-injected group), but levels of LH became detectable and showed an inverse relationship to oxygen concentration after GnRH injection. In contrast, Wang et al. (2008) found a significant reduction in serum LH level when carp was exposed to long-term hypoxia (1 mg O2/L, 11% saturation) for more than 2 months. 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 91 2.2.3. Neurotransmitters Lu et al. (2006) showed that 3–4-week-old marine medaka (Oryzias melastigma) exposed to hypoxia (1.8 mg O2/L, 28% saturation) for 3 months until sexual maturity was reached showed a significant reduction in mRNA of both tryptophan hydroxylase (TPH, the rate limiting enzyme of serotonin synthesis) in the brains and FSH receptor in the ovaries of female fish, while no significant changes could be found in GnRH, GnRH receptors, or FSH and LH in the brain of hypoxic males, suggesting that the responses of neurotransmitters to hypoxia may be sex dependent. A recent attempt has been made to investigate whether the observed disruption of sex steroid hormones by hypoxia may also aVect the neuroendocrine function at the brain and pituitary levels (Thomas et al., 2007). Atlantic croakers (Micropogonias undulatus) were injected with GnRHa or saline after exposure to normoxia or hypoxia. LH secretion in response to GnRHa injection was significantly attenuated in croakers exposed to hypoxia, showing a decrease in the responsiveness of the pituitary to GnRHa. The expression of GnRH mRNA was also significantly decreased in the preoptic-anterior hypothalamus. Exposure to hypoxia also caused a decrease in serotonin (5-HT) concentration as well as the activity of tryptophan hydroxylase (the enzyme responsible for synthesis of 5-HT) in the hypothalamus. Artificial restoration of hypothalamic 5-HT levels restored neuroendocrine function, indicating that the stimulatory serotonergic neuroendocrine pathway is a major site of hypoxia-induced inhibition. It was further suggested that inhibition of tryptophan hydroxylase activity could be an adaptive mechanism to down-regulate reproductive activity and survive hypoxia (Thomas et al., 2007). In vitro mammalian studies showed that hypoxia induced the release of catecholamines, acetylcholine, dopamine, and tyrosine hydroxylase (the enzyme regulating the synthesis of dopamine) in pheochromocytoma 12 cells (Kumar et al., 1998; Kumar et al., 2003; Kim et al., 2004), and oVered further evidence to support the notion that neurotransmitters are responsive to hypoxia. 2.2.4. Steroidogenic Enzymes Steroidogenic enzymes are primarily regulated at the transcriptional level under the control of the pituitary gland (Omura and Morohashi, 1995). Thus, any interference with their transcription may alter the production of sex hormones. Increasing evidence shows that genes regulating steroidogenesis are important target sites for various endocrine disrupting chemicals (Thibaut and Porte, 2004; Sanderson, 2006). The synthesis of sex steroid hormones requires molecular oxygen (RaV & Bruder, 2006). As such, hypoxia may be expected to aVect steroidogenesis, and hence the production of sex hormones. 92 RUDOLF S. S. WU Shang et al. (2006) showed that 3b-HSD, CYP11A, and CYP19B in 10 dpf zebrafish were significantly down-regulated by hypoxia (at which time expression of CYP19A was still under the detection limit in both normoxic and hypoxic fish). At 40 dpf, all genes investigated were down-regulated in the hypoxia treatment (Figure 3.5). Expression of b actin (the housekeeping gene for normalization) was not aVected by hypoxia, indicating that hypoxic eVects on these steroidogenic enzymes were specific, but not due to a general down-regulation of metabolism. Ex vivo studies on fish ovarian follicles further showed that FSH can stimulate the expression of CYP19 in brown trout (Montserrat et al., 2004), indicating that the suppression of CYP19A expression in the ovary is under the control of FSHb in the pituitary. Both in vitro and in vivo studies in mammals and mammalian cell lines lend support to the postulation that hypoxia can aVect the expression level of steroidogenic enzymes. In rat, CYP11A1 was stimulated while steroidogenic acute regulator (StAR) was inhibited under hypoxia (Bruder et al., 2002, Fold change A 10 dpf 1.5 1 *** 0.5 *** *** 0 3b-HSD CYP11A CYP19A CYP19B Fold change B 40 dpf 1.5 1 * 0.5 *** 0 3b-HSD CYP11A *** CYP19A *** CYP19B Fig. 3.5. Expression levels of the various sex hormones that control genes in zebrafish at (A) 10 dpf and (B) 40 dpf upon exposure to normoxia (5.8 mg of O2/L, 74% saturation) and hypoxia (0.8 mg O2/L, 10% saturation) (n = 4 replicates, each replicate was pooled from 10 individuals, mean  SD). Values significantly diVerent from the normoxic control are indicated by asterisks (t-test, * p < 0.05, *** p < 0.001). (Reproduced from Shang et al., 2006.) 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 93 2004, 2005). In sheep, chronic hypoxia represses the expression of CYP11A1 and CYP17 (Myers et al., 2005). In vitro studies also showed that hypoxia also reduces the level of CYParom, aldosterone, cortisol, and progesterone receptor in adrenal tissues (RaV et al., 2004). 2.2.5. Sex Hormones Several studies provided evidence to suggest that hypoxia can disrupt levels of sex hormones, vitellogenin, and triiodothyronine in fish (including carp, zebrafish, Gulf killifish, and Atlantic croakers), and shed light on the underlying mechanisms for the observed reproductive impairments such as retarded gonadal development and a reduction in spawning success, sperm motility, fertilization success, hatching rate, and larval survival. Serum levels of testosterone (T), estradiol (E2), and triiodothyronine (T3) were clearly disrupted in carp (Cyprinus carpio) upon chronic exposure to hypoxia. A significant increase in T and E2, and a significant decrease in T3, were clearly evident in male carps exposed to hypoxia for 4 weeks. After 8 weeks of exposure to hypoxia, T and T3 levels were significantly reduced, but E2 levels increased significantly in male carp. Female carp exposed to hypoxia for 8 weeks showed a significant reduction in serum T, E2, and T3 levels (Table 3.1). These hormonal changes were associated with retarded gonadal development in both male and female carp, reduced spawning success, sperm motility, fertilization success, hatching rate, and larval survival (see Figure 3.12), indicating that the adverse eVects of hypoxia on reproductive performance resulted from endocrine disruption (Wu et al., 2003). Chronic exposure of Atlantic croaker (Micropogonias undulatus) to hypoxia (1.7 and 2.7 mg O2/L, 24 and 38% saturation, respectively) showed dramatic suppression of sex steroid hormones (E2, T, 11-KT), as well as hepatic estrogen receptor and plasma vitellogenin. These hormonal disruptions were clearly related to a decrease in gonadal somatic index, ovarian and testicular development, sperm and egg production, and fecundity (Thomas et al., 2006, 2007). A recent study by Landry et al. (2007) also showed similar hormonal disruption and reproductive impairments in the Gulf killifish Fundulus grandis exposed to hypoxia (1.34 mg O2/L, 19% saturation), in which a 50% reduction in E2 and 11-KT was found in hypoxic females and hypoxic males, respectively. T and VTG, however, remained unchanged in either sex after hypoxic exposure. Female Gulf killifish exposed to hypoxia also produced significantly fewer eggs, and spawning occurred later than in their normoxic counterpart. A diVerent pattern was observed in the Pacu, Piaractus brachypomus, in which plasma T and 11-KT in males, as well as T and E2 in females, were significantly reduced, while 17, 20b-dihydroxy-4-pregnen-3-one (17,20bP) in both sexes remain unchanged when exposed to hypoxia (2.0–4.5 mg O2/L, 25–56% saturation) for 3 days. The concentration of spermatozoa, however, was not aVected (Dabrowski et al., 2003). Table 3.1 Hormonal levels (mean  SEM) in diVerent fish species exposed to hypoxia Fish species Common carp Zebrafish (embryo at blastula stage) Zebrafish (embryo at blastula stage) Pacu Pacu Gulf killifish Gulf killifish Atlantic croaker Sex Immature male Male Duration of exposure (days) 28 60 120 Female 60 120 Mature male 3 Mature female Mature male 3 Mature female Adult female (1-year-old) 30 30 70 Mode of study DO level (mg O2/L) Laboratory 7.0 1.0 Laboratory 5.8 0.8 5.8 0.8 Laboratory 5.8 0.8 5.8 0.8 Laboratory 5.5–7.5 2.0–4.5 5.5–7.5 2.0–4.5 Laboratory 6.68  2.1 1.34  0.45 6.68  2.1 1.34  0.45 Laboratory 5.7 2.7 1.7 % saturation Temperature ( C) 81 12 75 10 75 10 75 10 75 10 69–94 25–56 69–94 25–56 93 19 93 19 80 38 24 22.5  0.5 28.5 28.5 26–27.5 27.1  0.3 23–24 11-KT (ng/mL) T (ng/mL) E2 (ng/mL) T/E2 ratio N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. 37.68  5.48 5.98  1.61** N.A. N.A. 0.34  0.076 0.15  0.03* N.A. N.A. N.A. N.A. N.A. 4.68  1.44 13.46  2.78 * 13.4  2.44 3.53  0.3 ** 15.69  5.32 10.75  4.1 2.02  0.08 2.37  0.36 4.34  0.54 6.83  0.68 * 6.76  0.98 0.53  0.1** 6.1  1.15 1.58  0.19** 0.6  0.12 0.37  0.03 0.43  0.07 0.36  0.01 N.A. N.A. N.A. 0.04  0.005 0.24  0.035 ** 10.87  0.79 3.27  0.21 *** 19.42  8.34 13.08  8.09 9.4  1.23 5.89  1.1 9.97  3.33 4.33  0.34 N.A. N.A. 7.46  0.32 3.92  0.23** N.A. N.A. 2.99  0.65 1.27  0.26* 5.78  0.77 3.45  0.79* 0.821  0.22*** 117  0.33 56.08  0.25 *** 1.2  0.14 1.07 0.03 1.02  0.17 1.17  0.25 0.22  0.02 0.42  0.03 ** 0.57  0.15 1.57  0.05 *** N.A. N.A. 0.82  0.19 0.4  0.13 N.A. N.A. 0.14  0.27 0.28  0.21 N.A. N.A. N.A. References Wu et al. (2003) Shang et al. (2006) Dabrowski and Richard (2003) Landry et al. (2007) Thomas et al. (2006) Atlantic croaker Adult male (1-year-old) 70 Atlantic croaker Adult male (1-year-old) N.A. Atlantic croaker Adult female (1-year-old) N.A. Laboratory 5.33  0.02 2.7  0.01 1.72  0.01 Field 4.62–5.52 1.2–4.8 1.32–3.2 6.65–7.03 6.68 2.22–4.72 Field 4.62–5.52 1.2–4.8 1.32–3.2 80 38 24 67–80 18–71 19–45 97–102 97 31–66 67–80 18–71 19–45 23 24.56–25.23 (Oct 2003) 23.59–24.12 (Nov 2003) 24.56–25.23 (Oct 2003) 5.67  0.33 2.67  0.33* 2.0  0.27* 1.38–1.63 0.88* 0.63–1.19* 1.25–1.5 1.25 0.38–0.63*** N.A. N.A. N.A. 7.5  0.83 7.92  0.42 8.33  0.83 1–1.03 0.69 0.5–0.875*** 0.48–0.53 0.59 0.28–0.45** 0.87–1.19 0.49* 0.43–0.76*** N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. 1.63–2.69 0.98* 0.73–1.47*** N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. 0.43–0.52 0.5 0.31–1.31 Thomas et al. (2007) Thomas et al. (2007) Asterisks indicate values in the hypoxic treatments are significantly diVerent from their counterparts in the normoxic control:*, p < 0.05; **, p < 0.01; ***, p < 0.001. DO, dissolved oxygen; E2, estradiol; 11-KT, 11-ketotestosterone; T, testosterone. 96 RUDOLF S. S. WU Since hormones in fish also regulate functions other than those involved in reproductive processes, their disruption does more than just impair reproduction and decrease reproductive output and success. It is well known that maternal hormones also play an important role in the development of fish larvae. For example, levels of cortisol (a stress hormone) in female fish can be transferred to the egg yolk and aVect larval developmental rates. A field manipulating experiment on damselfish (Pomacentrus amboinensis) showed that cortisol levels strongly influenced the yolk size of larvae at hatching, and elevated cortisol levels in the egg reduced larval length. Elevated testosterone also appears to influence yolk utilization rates and increase yolk sac size. Maternally derived cortisol and testosterone have been shown to be important in regulating growth, development, and nutritional reserves of fish embryo and larvae, which may, in turn, aVect larval survival and fitness (McCormick, 1998, 1999). Table 3.1 summarizes the changes in levels of sex steroid hormones (T, E2, and 11-KT), in five species of fish upon exposure to various levels of hypoxia. With a few exceptions, decreases in T, E2, and 11-KT were generally observed in hypoxic fishes, regardless of species and sex, indicating that hypoxia reduces the production of sex steroid hormones, presumably by down-regulating some of the steroidogenic enzymes. A significant increase in the T/E2 ratio was clearly evident in female zebrafish upon exposure to hypoxia (Shang et al., 2006) but not in the other species. A dose–response relationship between hormonal changes and level of hypoxia was also found in the Atlantic croakers (Thomas et al., 2007). Importantly, decreases in the level of sex hormones were associated with reproductive impairments in all these studies. One of the problems in deciphering the eVects of hypoxia on reproduction and development is that hypoxia aVects a wide range of physiological and biochemical systems and pathways, and it would be diYcult to distinguish between direct and indirect eVects of hypoxia. For example, a reduction in metabolism associated with hypoxia will cause many changes as the cell machinery is making adjustments to this new state. These changes are diYcult to separate from those hypoxia-induced changes directed at regulating a specific pathway. 2.3. Hypoxia Impairs Fish Reproduction 2.3.1. Reproductive Behaviors Various laboratory experiments and field studies, to date, have established that many fish species can actively avoid hypoxia (Gray, 1990; Pihl et al., 1991; Wannamaker and Rice, 2000), showing that hypoxia can aVect fish behavior. However, only limited studies have shown that hypoxia can 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 97 also aVect reproductive behavior (e.g., mate choice, courtship, reproductive eVorts, and investment), which may, in turn, aVect reproductive output. No courtship behavior was observed when male and female carps were reared separately under hypoxia and mixed at the time of spawning, whereas in the normoxic control, male fish followed the females and pushed them with their nose close to the anal papilla about an hour after mixing (Wang et al., 2008), suggesting that normal courtship behavior in fish was aVected by chronic hypoxic exposure to hypoxia. Behavioral studies showed that hypoxia can aVect mate choice and reproductive eVorts in fish. Female marine gobies (Pomatoschistus microps) preferred to spawn with males in nests that already contained eggs that had been spawned earlier by other females. However, this preference was reversed under hypoxia (30% saturation). Under normoxia, females preferred to mate with males with the smallest nest entrance, whereas males exhibiting the signal of willingness to provide parental care would be preferred under hypoxia (Jones and Reynolds, 1999a; Reynolds and Jones, 1999), clearly indicating the adjustment of mate choice in response to selection pressure (in this case, hypoxia) prevailing at diVerent times and in diVerent environments. Hypoxia can also change reproductive eVorts in fish. Under hypoxia (35% saturation), male marine gobies would increase their time and eVort in ventilating the eggs, and correspondingly reduced their time in selecting females (Jones and Reynolds, 1999b) (Figure 3.6). During hypoxia, male sand gobies (Pomatoschistus minutus) built nests with larger entrances and increased fanning activities to increase oxygen supply to their eggs (Lissåker et al., 2003) (Figure 3.7). In a noncompetitive environment, male mosquito fish (Gambusia holbrooki) spent more time following females and increased copulations under hypoxia (15–20% saturation). However, hypoxia had no eVect when males were competing for copulations (Carter and Wilson, 2006) (Figure 3.8). The fact that hypoxia can aVect synthesis of sex hormones, while the breakdown products of sex hormones may act as pheromones (Sorensen et al., 2004), suggests the possibility that hypoxia may aVect the production of pheromones and hence reproductive behaviors in fish. In goldfish, responses to pheromone is mediated through microvillous olfactory receptor cells (Zippel et al., 1997). In rabbits, it has been shown that the number of olfactory neurons is significantly reduced under hypoxia (Drobyshevsky et al., 2006). Conceivably, hypoxia may also reduce olfactory sensitivity thereby aVecting the ability of fish to detect pheromones in the environment. Whether hypoxia can aVect fish pheromones or the sensing of pheromone remains unknown, and studies addressing this important topic are required, since even a small change in sex pheromones may lead to reproductive failure in natural fish populations. 98 RUDOLF S. S. WU C 50 12 60 40 13 20 13 12 0 B 80 Percent time near female 13 40 13 30 20 10 Control Control Low O2 Low O2 eggs no eggs eggs no eggs 60 Courtship intensity Percent time fanning A 80 12 13 12 12 0 Control Control Low O2 Low O2 eggs no eggs eggs no eggs 13 40 20 12 0 Control Control Low O2 Low O2 eggs no eggs eggs no eggs Fig. 3.6. (A) Percentage of time spent fanning by male marine gobies in the presence of a restrained female. (B) Percentage of time spent by males near a restrained female. (C) Intensity of courtship by males during a 20-min observation sessions. Data expressed in mean + SE; numbers above bars are sample sizes. (Reproduced from Reynolds and Jones, 1999.) 2.3.2. Gonad Development and Gametogenesis Ample laboratory and field evidence shows that chronic exposure to hypoxia can reduce gonad size and retard gametogenesis and gonad development in fish. The Gonadal Somatic Index (GSI) of adult carp (Cyprinus carpio) reared under hypoxia (1 mg O2/L, 12% saturation) for 8 weeks was reduced by some 40% and 33% in males and females, respectively (Wu et al., 2003). The GSI of male and female Atlantic croaker (Micropogonias undulatus) was reduced by 50% and 75%, respectively, after rearing under hypoxia (1.7 mg O2/L, 24% saturation) for 10 weeks (Thomas et al., 2006, 2007). Gulf killifish (Fundulus grandis) kept under hypoxia (1.34 mg O2/L, 19% saturation) for 1 month had a reduced number of eggs and amount of vitellogenin, and the GSI in females was also significantly lower (Landry et al., 2007). Notably, fish with a higher GSI also produced larvae of larger size with a higher rate of survival (Evans and GeVen, 1998). EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT Nest entrance (cm2) 3. 30 25 20 15 10 5 0 7.96 7.96 High oxygen Nest entrance before 10.47 99 22.91 Low oxygen Nest entrance after Fig. 3.7. Nest entrance size (mean  SE) of male sand goby (Pomatoschistus minutus) under low (n = 12) and high (n = 15) oxygen regimes. (Reproduced from Lissåker et al., 2003.) In male carp kept under hypoxia for 8 weeks, a significant reduction in the diameter of testes lobules was observed. Despite the fact that all stages of spermatogenesis can be observed in the testes, the number of spermatocytes (SPC) and spermatids (SPD) was significantly reduced, while a significantly higher number of spermatogonia (SPG) was found, indicating that testicular growth and sperm production was inhibited by hypoxia (Figure 3.9). Similar retardation of gonad development was also found in female carp. The size of gonads was reduced and less yolk deposition was found in each egg. Stage III oocytes were found in 83.3% of all hypoxic females; only 16.7% of hypoxic females carried stage IV oocytes and all hypoxic females failed to produce stage V oocytes. In contrast, eggs were visually observed in all normoxic females, and oocytes in 57.1% of normoxic females reached stage V (Wu et al., 2003). Retardation of gonad development and gametogenesis were found in zebrafish exposed to hypoxia (0.8 mg/L, 10% saturation) for 3 months. At 120 dpf, percentages of SPC and SPD were significantly reduced by hypoxia ( 46.6% and 36.6%, respectively), while SPG increased by three times in hypoxic males (Figure 3.9). Furthermore, mitosis was commonly observed in normoxic males, but was less common in hypoxic males. In females, oocytes were predominantly in vitellogenic and preovulatory stages in the normoxic fish but were mostly in previtellogenic and vitellogenic stages in hypoxic fish (Figure 3.10). It must be noted that since feeding was also reduced in fish under hypoxia (Zhou et al., 2001), the gonad retardation observed may, in part, be due to reduced feeding. A marked decrease in mature oocytes and the number of viable eggs was clearly evident when female Atlantic croakers (Micropogonias undulatus) were kept under hypoxia (2.7 ppm and 1.7 ppm, 38% and 24% saturation, respectively) in the laboratory for 10 weeks. Suppression of ovarian and 100 RUDOLF S. S. WU Total following time (s) A 300 * 200 100 0 Number of mating attempts B 3 2 1 0 C 4 Number of mating attempts Hypoxia 3 * Normoxia 2 1 0 Hypoxic Normoxic Test environment Fig. 3.8. EVect of oxygen on the mating behavior of male mosquito fish, Gambusia holbrooki, in a noncompetitive environment. (A) Total time males spent following females, (B) total number of attempted copulations, and (C) total number of successful copulations in 10 min. Data are mean  SE (n = 15). * p < 0.05. (Reproduced from Carter and Wilson, 2006.) 80 Normoxia Hypoxia *** 7.5 60 *** *** 5.0 2.5 20 *** *** 0.0 40 ** SPG SPC SPD Common carp 0 SPG SPC SPD Zebrafish B 300 Diameter (µm)/lobule 10.0 No. of cysts/lobule A 101 EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT Percentage at of each stage in testis 3. 200 *** 100 0 7.0 mg O2/L 1.0 mg O2/L Fig. 3.9. (A) Number of spermatogonia (SPG), spermatocytes (SPC), and spermatids (SPD) in the testis of common carp and zebrafish exposed to hypoxia. Values significantly diVerent from the control are indicated by asterisks (**, p < 0.01; ***, p < 0.001). (Wu et al., 2003; Shang et al., 2006) (B) Lobule diameter of testes of C. carpio upon exposure to 7.0 and 1.0 mg of O2/L (81% and 12% saturation, respectively) for 8 weeks (Zhou, 2001). Values significantly diVerent from the control are indicated by asterisks (n = 7–11, mean  SD) (***, p < 0.001). B Percentage at each stage in oocyte 80 *** 60 40 Normoxia Hypoxia *** 20 *** 0 Oo PreV Vit Ovarian cell stage *** PreO PNS Percentage of oocytes A CA PYS SYS TYS 100 80 60 40 20 0 Control 2.7 mg/L 1.7 mg/L Fig. 3.10. (A) Percentage of oogonia (Oo) and previtellogenic (PreV), vitellogenic (Vit), and preovulatory oocytes (PreO) in female zebrafish after 120 days of development upon exposure to normoxia (5.8 mg of O2/L, 75% saturation) and hypoxia (0.8 mg of O2/L, 10% saturation), (n = 12–15, mean  SD). Values significantly diVerent from the normoxic control are indicated by asterisks (t-test, ***, p < 0.001). (Reproduced from Shang et al., 2006.) (B) EVects of laboratory hypoxia exposure on ovarian development and endocrine function in female croakers. PNS, peri-nucleolus; CA, cortical alveoli; PYS, primary yolk; SYS, secondary yolk; TYS, tertiary yolk. (Reproduced from Thomas et al., 2007.) testicular growth was also found in Atlantic croakers collected from hypoxic areas (Thomas et al., 2006, 2007). The study of Landry et al. (2007) showed that daily egg production in F. grandis was significantly reduced after exposure to hypoxia (1.34 mg O2/L, 19% saturation) for 30 days (Figure 3.11). 102 RUDOLF S. S. WU 2.3.3. Quality of Sperm and Eggs Sperm motility is a reliable predictor for sperm quality and fertilization success (Au et al., 2002). After exposure to hypoxia for 12 weeks, sperm motility (measured by their curvilinear velocity VCL, straight-line velocity VSL, and angular path velocity VAP) was significantly decreased in male carp (Table 3.2), indicating that sperm quality was impaired. All of the normoxic and hypoxic male carp could be induced to spawn using carp pituitary extract; however, the percentage of spawning success in the hypoxic male carp was drastically reduced from 71.4% to 8.3%, clearly demonstrating that the sperm quality produced by males was impaired by hypoxia (Wu et al., 2003). Daily no. eggs/female 35 6.68 mg/L 30 1.34 mg/L 25 20 15 10 5 0 7 14 Lunar day 21 Fig. 3.11. Daily egg production per female Fundulus grandis (mean  SE) exposed to normoxia (6.68 mg O2/L, 93% saturation, n = 7) and hypoxia (1.34 mg O2/L, 19% saturation, n = 6) for a 30 days (Reproduced from Landry et al., 2007.) Table 3.2 Sperm motility of carp after exposure to normoxia (7.0 mg O2/L, 81% saturation) and hypoxia (1.0 mg O2/L, 12% saturation) for 12 weeks VCL VSL VAP 7.0 mg O2/L 1.0 mg O2/L 77.42  29.13 38.83  21.01 47.69  5.38 46.25  10.83* 10.65  3.89* 21.12  11.41* Mean  SD; n = 6. The velocity is expressed as micrometers per second. VCL, mean curvilinear velocity; VSL, mean straight-line velocity; VAP, angular path velocity (Wu et al., 2003). * Values significantly diVerent from the control (t-test: *, p < 0.05). 3. 103 EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 7.0 mgO2/L 1.0 mgO2/L 100 Percentage 80 60 *** ** 40 *** 20 *** 0 Fertilization Hatching Larval survivalOverall survival Fig. 3.12. Percentage of fertilization, hatching rate, larval survivorship, and overall survivorship (fertilized egg to 24 h post-hatching) after 12 weeks of normoxic and hypoxic exposure of the parent adult carp (mean  SD, n = 6) (t-test: **, p < 0.01; ***, p < 0.001). (Reproduced from Wu et al., 2003.) 2.3.4. Spawning, Fertilization Success, and Survival of Larvae The study of Wu et al. (2003) clearly related the decrease in GSI and impaired gametogenesis to sperm quality and subsequently reduced fertilization success and larval survivorship in hypoxic carps. Fertilization success was significantly reduced from 99.4% to 55.5% in hypoxic carps. 98.8% of the fertilized eggs produced by the normoxic carps hatched to larvae, while only 17.2% of fertilized eggs produced by the hypoxic group hatched to larvae. 93.7% of hatched larvae survived in the normoxic group, while larval survival decreased to 46.4% in the hypoxic group 24 h post-hatching. Overall, the survival of fertilized eggs through 24-h-old larvae decreased from 92.3% in the normoxic group to only 4.4% in the hypoxic group (Figure 3.12). JenkinsKeeran et al. (2001) further provided peripheral evidence that oxygen is an important factor in determining sperm motility in fish. Semen from striped bass stored for 48 h under oxygen had a significantly greater percentage of motile sperm (13%) than their counterpart stored under ambient air (9%) or nitrogen (4%). Hypoxia also significantly delayed the onset of spawning. Female Gulf killifish (Fundulus grandis) exposed to hypoxia (1.34 mg/L, 19% saturation) for 1 month showed a significant delay in their spawning (Landry et al., 2007). Wang et al. (2008) showed that although oocytes continued to develop when carp was exposed to long-term hypoxia (1 mg O2/L, 11% saturation for more than 2 months), the final oocyte maturation in hypoxic 104 RUDOLF S. S. WU females was significantly retarded, and both ovulation and spawning were inhibited in hypoxic female fish. This was correlated with a significant reduction in serum LH level, indicating that hypoxia may inhibit fish spawning through LH-dependent final oocyte maturation. 3. HYPOXIA AND FISH DEVELOPMENT It is generally accepted that embryonic and larval development (particularly the gastrula and blastula stages) are the most sensitive stage to stresses in the life cycle of fish (von Westernhagen, 1988; Johnson and Landahl, 1994; Cameron and von Westernhagen, 1997). Normal histogenesis and organogenesis during development rely on a series of intricate, programmed processes in which apoptosis and cell proliferation play a key role (Sanders and Wride, 1995; Jacobson et al., 1997; Vaux and Korsmeyer, 1999). In vitro and in vivo studies based on mammalian systems provide evidence that hypoxia can induce apoptosis and inhibit cell proliferation (Jung et al., 2001; Saed and Diamond, 2002; Liao et al., 2007; Poon et al., 2007; Lee et al., 2008). Conceivably, hypoxia may also alter cell proliferation and apoptosis in fish, thereby impairing development. In fish, stages of sex diVerentiation and sex determination have been shown to be particularly sensitive to endocrine disrupting chemicals (Strüssman and Nakamura, 2002). The fact that hypoxia is an endocrine disruptor suggests that hypoxia may also aVect sex diVerentiation and sex determination in fish. Surprisingly, the eVects of hypoxia on embryonic and larval development of fish remain largely unknown. 3.1. Regulation of Sex DiVerentiation, Sex Development, and Sex Determination Histogenesis and organogenesis during development primarily rely on cell proliferation and apoptosis (reviewed by Vaux and Korsmeyer, 1999; Su, 2000; Lossi et al., 2002). It is widely accepted that intracellular proteins of the Bcl-2 family are involved in the apoptotic signaling pathway; Bcl-2 and Bcl-xL are anti-apoptotic while Bax and Bad are pro-apoptotic (Reed et al., 1996). As such, the ratio of the anti-apoptotic Bcl-2 and the proapoptotic Bax is indicative of apoptotic potential (Martin et al., 1995; Misao et al., 1996; Kirshenbaum and de Moissac, 1997; Gross et al., 1998; Saikumar et al., 1998; Cook et al., 1999). Growth hormone appears to be an important factor in regulating development of teleost fishes. The secretion of growth hormones in fish, in turn, has been shown to be stimulated by neuropeptides, gonadotropin-releasing 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 105 hormone, growth hormone-releasing hormone, thyrotropin-releasing hormone, neuropeptide Y, serotonin, and pituitary adenylate cyclase-activating polypeptide (Holloway and Leatherland, 1998). Whether hypoxia may aVect fish development through directly aVecting the secretion of growth hormones or indirectly through aVecting their modulating hormones remains completely unknown and warrants further study. 3.2. Hypoxia Impairs Fish Development 3.2.1. Death and Sensitive Window Early gonad development and reproduction are the two sensitive windows, during which time the HPG axis is particularly susceptible to endocrine disruption caused by chemicals (Ankley and Johnson, 2004). In terms of mortality rate, however, there is no clear evidence to support that a certain life stage would be more susceptible to hypoxia. Survival times of larval bonefish (Albula sp.) in hypoxic sea water (0.68 mg O2/L, 10% saturation) decreased from 15 to 5 min over the period of metamorphosis, and this increased sensitivity to hypoxia has been attributed to an increased oxygen demand as metamorphosis advances (Pfeiler, 2001). Susceptibility of small-mouth bass (Micropterus dolomieui) to hypoxia also changed with the developmental stage. From the second day to the 10th day after hatching, larvae could not survive for 3 h when exposed to 1 mg O2/L (11% saturation), while the majority of the larvae survived under the same conditions after the 11th day (Spoor, 1984). Landman et al. (2005), however, found no significant diVerences in mortality rate between larval and juvenile rainbow trout (Oncorhynchus mykiss) and common bully (Gobiomorphus cotidianus) when exposed to acute hypoxia (48 h; LC50 values were 1.59– 1.62 mg O2/L, 16% saturation for rainbow trout parr and fry, and 0.77–0.91 mg O2/L, 8% saturation, for bully juvenile and fry). Dissolved oxygen above 4 mg O2/L (38% saturation) did not aVect survival of Chinook salmon (Oncorhynchus tshawytscha) embryos (Geist et al., 2006). Scyliorhinus canicula eggs 13–15 weeks old survived at 50% air saturation and normoxia for 10 weeks. Eggs exposed to 20% air saturation died after 3 weeks, while those exposed to anoxia for 2 h per day died after 10 weeks (Diez and Davenport, 1990). Increased larval mortality rate and reduced hatching success were found for nese (Chondrostoma nasus) embryos when exposed to 10% air saturation (Keckeis et al., 1996). Roussel (2007) noted that the survival from fertilization to the end of embryonic development in brown trout (Salmo trutta) decreased from 85% to 70% under hypoxia (3.0 mg O2/L, 26% saturation). Low oxygen levels (2.0–4.5 mg O2/L, 25% to 56% saturation) reduced the survival of embryos of Pacu, Piaractus brachypomus (17.3 % in hypoxia as compared with 68.5 % in normoxia) (Dabrowski et al., 2003). 106 RUDOLF S. S. WU Table 3.3 Viability of diVerent developmental stages of zebrafish in anoxia Period Cleavage Blastula Gastrula Segmentation Straightening Early Middle Hatching Hours after Fertilization* Percent alive after 24 h of anoxia (N) 2 4 6 13 83.1 (89) 83.2 (85) 97.7 (90) 98.8 (85) 25 30 50 64.0 (100) 4.4 (91) 0 (130) N is total number of embryos. * hpf when placed in the anoxic environment. Reproduced from Padilla and Roth (2001). The study of Padilla and Roth (2001) demonstrated that the susceptibility of zebrafish embryos to anoxia varies considerably with their developmental stage. Most zebrafish embryos before 25 hpf can survive 24 h of anoxia. Tolerance was reduced as embryos developed to the period of straightening (30 hpf), and fish after hatching (beyond 50 hpf) became very sensitive to anoxia (Table 3.3). A summary of the mortality rate of embryos and larvae of various fish species in response to hypoxia is given in Table 3.4. Clearly, hypoxic tolerance is species specific, which may be related to the ecology and natural habitat of the species. Despite the fact that certain life stages would be more sensitive to hypoxia for a given species, no generalization could be made on life stage specificity across fish species. Hypoxic tolerance also varies considerably according to oxygen levels and duration of exposure. Furthermore, it appears that the hypoxic window for death is very narrow. Above certain oxygen levels, the fish are able to make physiological and biochemical adjustments to survive, but death sets in very rapidly when oxygen levels fall below the threshold beyond which they are incapable of making these adjustments. 3.2.2. Development and Hatching Earlier studies have shown that hypoxia can retard embryonic development in the Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynus mykiss), leading to an increase in mortality rate and prematurely hatched embryos through stimulating chorionase secretion (Hamor and Garside, 1976). Subsequent studies have shown that hypoxia has a profound eVect on the rate of embryonic development in many fish species (Rombough, 1988). Table 3.4 Summary of mortality of embryo and larvae of diVerent fish species at diVerent developmental stages under hypoxia Fish species Dogfish (Scyliorhinus canicula L.) Nase (Chondrostoma nasus) Pacu (Piaractus brachypomus) Chinook salmon (Oncorhynchus tshawytscha) Brown trout (Salmo trutta) DO level 50% air saturation 20% air saturation 0% for 2h/day 10% air saturation 2.2  0.5 mg O2/L (27% saturation) 4 mg O2/L (38% saturation) 3 mg O2/L (26% saturation) DO, dissolved oxygen. Duration of exposure (days) Stage Mortality (%) Reference 13–15 weeks post-fertilization Diez and Davenport (1990) Fertilized egg to hatching Embryo 0 100 100 7.6 (at hatching)/37.7 (15 dph) 8.9 (at hatching)/98.5 (15 dph) 100 (at hatching) 6.2 (at hatching)/100 (15 dph) 100 (at hatching) ca. 80 40 Embryo 0 Dabrowski and Richard (2003) Geist et al. (2006) 110 From fertilization to end of embryonic development 30 Roussel (2007) 70 21 70 1.5 Fertilized egg to gastrula 3.5 Gastrula to eyed stage 5 8 Fertilized egg to eyed stage Eyed stage to hatching 13 N.A. Keckeis et al. (1996) 108 RUDOLF S. S. WU Padilla and Roth (2001) showed that although embryos of zebrafish, Danio rerio, can survive anoxia for 24 h, embryos entered into developmental arrest under anoxia, with all movement, cell division, developmental progression, and heart beats ceasing, presumably as adaptive features for energy conservation. No cells were arrested in mitosis, and flow cytometry analysis further revealed that blastomeres were arrested during the S and G2 phases of the cell cycle. Development of zebrafish embryos, however, resumed upon return to normoxia. Shang and Wu (2004) showed that development of zebrafish embryos was clearly delayed when kept under 0.5 mg O2/L (6.4% saturation), and took twice as long to develop when compared to the normoxic embryos. Similarly, development of freshwater medaka, Oryzias latipes, embryos was retarded upon exposure to hypoxia (0.8 mg O2/L, 10% saturation) (Cheung and Wu, 2006). Berntsen et al. (1990) showed that natural hatching in mature salmon eggs was induced by hypoxia, and Jobling (1995) further postulated that hypoxia caused by insuYcient diVusion of ambient oxygen across the chorion (to meet oxygen requirements of the developing embryo) may also trigger hatching. When embryos of the nase, Chondrostoma nasus, were exposed to hypoxia (10% of air saturation), the hatching period was prolonged from 2.7 days (in the normoxic control) to 4.2–5.3 days, and hatching success was also reduced (Keckeis et al., 1996). Roussel (2007) also reported that hatching in the brown trout, Salmo trutta, was delayed from 2–4 days to 5–10 days when the oxygen level was lowered to 3.0 mg O2/L (26% saturation). Hatching in the Chinook salmon, Oncorhynchus tshawytscha, was related to oxygen concentration; fish developed under 4 mg O2/L (38% saturation) required 6–10 days longer to hatch, and up to 24 days longer to emerge, when compared with the normoxic control (Geist et al., 2006). Delays in hatching were often accompanied by impaired development and a lower quality of the oVspring. Shang and Wu (2004) showed that the body length of zebrafish hatched under hypoxic conditions was significantly shorter than fish in the normoxic control, and further postulated that a smaller body size may possibly reduce the fitness of adult fish in their natural environment. Eggs of Syliorhinus canicula kept under anoxia and hypoxia (0% and 5% saturation) showed retarded growth and reduced proteolytic activities as compared to their normoxic counterpart (Diez and Davenport,1990). Massa et al. (1999) reported that not only was hatching delayed, but also a smaller body size of alevins, lower content of water, and lower yolk-sac conversion rate were found when brown trout (Salmo trutta) eggs were allowed to develop under low oxygen levels (3 mg O2/L) for 3 weeks after fertilization. The embryos of S. trutta grew more slowly and progressed through delayed hatching under hypoxia; however, both normoxic and hypoxic fish reached similar body sizes when yolk-sac absorption was completed. However, the 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 109 swimming activity of fish hatched from hypoxic embryos was reduced by 20% and suVered from a 14% higher predation rate compared with normoxic groups (Roussel, 2007) (Figures 3.13 to 3.15). 35 Number of emerging fish 30 25 20 15 10 5 0 60 65 70 75 80 85 90 95 100 105 110 115 120 Days after fertilization Fig. 3.13. Emergence time (in days after fertilization) of Salmo trutta alevins in each channel, for alevins incubated as normoxic embryos ( and solid lines) or hypoxic embryos ( and broken lines). Bold lines represent average profiles of emergence for each treatment. Vertical broken line indicates the date at which embryos were transferred into channels. (Reproduced from Roussel, 2007.) Length of embryos (mm) 40 35 30 25 20 15 10 5 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 Days after fertilization Fig. 3.14. Embryonic growth in brown trout (Salmo trutta) from hatching to complete yolk absorption when incubated under normoxia () or hypoxia (). Results are given as mean  SD, with von BertalanVy growth models plotted as solid curves. Broken line indicates the beginning of hatching. (Reproduced from Roussel, 2007). 110 RUDOLF S. S. WU A B 1.0 Proportion of fish eaten Proportion of fish swimming 1.0 0.8 0.6 0.4 0.2 0.0 0.8 0.6 0.4 0.2 0.0 Swimming activity Predation by Cottus gobio Fig. 3.15. (A) Proportion of alevins of Salmo trutta that swim in the water column, and (B) proportion of alevins eaten by the sculpin, Cottus gobio, in experimental channels, after being exposed to hypoxia (solid bars) or normoxia (open bars) as embryos (mean  SD). (Reproduced from Roussel, 2007.) The above results demonstrated that exposure to hypoxia during development can have carry-over eVects on later parts of the life cycle, and may reduce the fitness of adults in the natural habitat, although supporting field evidence is still not available. In many animals, both spawning and hatching is synchronized with environmental factors (e.g., temperature, food availability, density of predators) prevailing in the natural habitats so as to maximize the chance of survival for the juveniles (Smyder and Martin, 2002; Speer-Blank and Martin, 2004; Warkentin, 2007). The ecological consequence of delayed hatching caused by hypoxia is not known. 3.2.3. Malformation It is well known that hypoxia can cause deformities in fish. 100% of the hatched larvae of Chondrostoma nasus were deformed when the fertilized eggs were developed under 10% of air saturation from gastrula to eyed stage and from eyed stage to hatching (Keckeis et al., 1996). A high proportion (20–53%) of female eelpouts (Zoarces viviparous) showed developmental defects, including spinal and craniofacial defects, eye lesions or loss of eyes, in broods in Danish fjords receiving domestic and industrial eZuents resulting in serious oxygen depletion (Strand et al., 2004). It is also interesting to note that a higher occurrence of malformed fish larvae has been generally reported in polluted areas (Au, 2004), although this increase may not necessarily be attributable to hypoxia because polluted areas are also often contaminated with a variety of chemicals including teratogens and endocrine-disrupting chemicals. 3. 111 EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT Zebrafish embryos developing under hypoxia lost their normal synchronization, with their tails developing much faster than their heads. External abnormalities such as spinal deformity (predominantly manifesting as altered axial curvature) were also clearly evident (Figure 3.16). Many embryos also failed to develop their vascular systems after several days and died. After 96 h, the percentage of fish with malformations in the hypoxic treatment group was significantly higher than that of the normoxic control (Shang and Wu, 2004) (Figure 3.17). Most teratogens exert a marked eVect during certain stage(s) of embryonic development. Likewise, hypoxia may have diVerent eVects when Fig. 3.16. Typical examples of malformation in zebrafish caused by hypoxia (0.8 mg O2/L, 10% saturation) at 48 hpf, 72 hpf, and 96 hpf. (Reproduced from Shang, 2005.) 150 Malformation (%) 120 5.8 mg O2/L 0.8 mg O2/L ** * ** 90 60 30 0 48 72 96 Time (hpf) 120 168 Fig. 3.17. Percentage malformation in zebrafish embryos during diVerent developmental stages (48, 72, 96, 120, and 168 hpf) upon exposure to 5.8 and 0.8 mg O2/L (75% and 10% saturation, respectively). Values significantly diVerent from the normoxic control are indicated by asterisks (n = 100, mean  SD) (*, p < 0.05; **, p < 0.01). (Reproduced from Shang and Wu, 2004.) 112 RUDOLF S. S. WU administered at diVerent oxygen levels and developmental stages, and diVerent organs may have diVerent ‘‘critical windows’’ during which development is most sensitive and susceptible to hypoxic assault (Burggren, 1999). Conceivably, hypoxia occurring at early developmental stages (organogenesis) may aVect fish development more seriously than that occurring during histogenesis at later stages of development. Further study is required to determine the critical window of hypoxia that aVects the diVerent stages of gonad development, in order to provide a better understanding of the molecular basis of how hypoxia might aVect sex diVerentiation and determination in fish. During normal embryonic development, excess cells are commonly removed by apoptosis, and apoptosis is an essential mechanism for normal remodeling and morphogenesis. Hypoxia has been shown to induce in vitro apoptosis in a variety of cell types (Schroedl et al., 2002; Wang et al., 2004; Gozal et al., 2005; Lu et al., 2005; Lee et al., 2005; Zhao et al., 2007) and in vivo systems (Shin et al., 2004; David and Vert, 2004; Nagai et al., 2007). As such, disruption in apoptosis and change in apoptotic pattern may lead to subsequent malformation in fish. Shang and Wu (2004) demonstrated for the first time that patterns of apoptosis during fish development can be altered by hypoxia. Compared with the normoxic control, apoptotic cells in the tail of hypoxic embryos were significantly reduced ( 63.7%). In contrast, a significantly higher percentage (+116%) of apoptotic cells was found in the head region of hypoxic embryos as compared with control embryos (Figure 3.18). 150 5.8 mg O2/L * Apoptotic cells 120 0.8 mg O2/L 90 60 ** 30 0 Head Tail Fig. 3.18. Number of apoptotic cells at 24 hpf in zebrafish embryos upon exposure to 5.8 and 0.8 mg O2/L (75% and 10% saturation, respectively). Values significantly diVerent from the control are indicated by asterisks (n = 10, mean  SD) (t-test: *, p < 0.05; **, p < 0.01). (Reproduced from Shang and Wu, 2004.) 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 113 Concomitantly, a significantly higher ratio of Bax/Bcl-2 was found in the head and a lower ratio of Bax/Bcl-2 in the tail, thus oVering further molecular basis to support the observed malformation of hypoxic zebrafish found in the same study (Shang, 2005). The results clearly demonstrated that the apoptotic pattern in zebrafish embryos was altered by hypoxia, thus oVering a molecular basis to support the observed malformation in zebrafish caused by hypoxia. The mechanisms by which hypoxia induces apoptosis are not well understood. Malhotra and Brosius (1999) indicated that hypoxia can trigger apoptosis in diVerent cell types in a way similar to other stresses, and diVerent types of apoptosis (viz., phylogenetic apoptosis, morphogenetic apoptosis, and histogenetic apoptosis) and pathways (e.g., the mitochondrial pathways and the death receptor (Fas-Fasl) pathways) may be involved in histogenesis and organogenesis (Sun et al., 2002; Ribeiro et al., 2003; AdachiYamada and O’Connor, 2004; Laurikkala et al., 2006). Normal embryonic development (including brain development and spinal formation, which have been shown to be aVected by hypoxia) is regulated by an intricate process of cell proliferation and apoptosis. The fact that apoptosis was aVected by hypoxia implies that this intricate process might be disrupted, and provides an explanation of the observed deformities in hypoxic fish. However, in what way hypoxia may aVect apoptosis and also the exact relationship between alteration of apoptosis and delayed brain development and spinal deformities remains unclear (Shang and Wu, 2004). In sturgeons (Acipenser shrenckii) that exposed to hypoxia (15% saturation) for 30 min and recovered for 6 h and 30 h, the number of apoptotic cells in the retina, optic tectum, pituitary, and spinal cord showed a significant increase. However, the olfactory lobe, cerebellum, and pons/medulla had relatively few apoptotic cells, showing a diVerential pattern of apoptosis in response to hypoxia in the central nervous system of fish (Lu et al., 2005). Poon et al. (2007), however, found no change in apoptotic rate in liver after carps were exposed to hypoxia (0.5 mg O2/L, 6% saturation) for 42 days but extensive DNA damage was found in liver cells. Whether DNA damage resulting from hypoxic exposure may subsequently lead to malformation remains unknown. Hypoxia (10.3–16.6% saturation) occurring during somitogenesis can cause major vertebral deformity (centrum defect) in the red sea bream (Pagrus major), but the 2-cell stage to the blastula stage and gastrula stages were not sensitive to hypoxia (Hattori et al., 2004), thus lending support to the hypothesis that there is a critical window of hypoxic eVects on embryonic development. Centrum defects can also occur when eggs are exposed to extremely low oxygen concentrations, even for a brief period of time. For example, somitic disturbances were found in newly hatched larvae of Pagrus major upon exposure to anoxia and 10% saturation for only 10 and 120 min, respectively (Sawada et al., 2006) (Figures 3.19 and 3.20). 114 100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 360 240 120 60 osu re ti 30 10 me (min .) Incidence of somitic disturbances (%) Incidence of somitic disturbances (%) RUDOLF S. S. WU 0 ) (% o ti n Exp 5 0 10 25 entra c 50 con 75 xygen o 100 lved o s s Di Fig. 3.19. Incidence rate of somitic disturbances in newly hatched larvae of Pagrus major induced by exposure to hypoxic conditions. (Reproduced from Sawada et al., 2006.) Only limited studies have been carried out to decipher the underlying cellular and molecular mechanisms of hypoxia in embryonic development. Kajimura et al. (2005) showed that hypoxia strongly induced the expression of insulin-like growth factor binding protein (IGFBP)-1 in zebrafish, but not the expression of insulin-like growth factors (IGFs), IGF receptors, or other IGFBPs, showing that the target of hypoxic eVect is specific rather than general. Overexpression of IGFBP-1 resulted in retardation of growth and development under normoxia, while knockdown of IGFBP-1 significantly alleviated the hypoxia-induced growth retardation and developmental delay; the eVects were restored by reintroduction of IGFBP-1 to the IGFBP-1 knocked-down embryos. In vitro studies using cultured zebrafish embryonic cells showed that IGFBP-1 itself is not mitogenic but can inhibit IGF-1- and IGF-2-stimulated cell proliferation. This inhibitory eVect was removed when IGF-1 or IGF-2 was added, suggesting that IGFBP-1 inhibits embryonic development by inhibiting the activities of IGFs. In zebrafish larvae, cardiac activity was reduced and the formation of blood vessels in various tissues enhanced during early development upon 3. 115 EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 100 Somitic disturbances n = 300 80 60 Incidence (%) 40 20 0 20 Centrum defects n = 300 40 60 80 100 1 3 5 7 9 11 13 15 17 Somite and centrum number 19 21 23 Fig. 3.20. Incidence of somitic disturbances in newly hatched larvae of Pagrus major and centrum defects in juveniles exposed to hypoxia (10% saturation) for 240 min during somitogenesis. (Reproduced from Sawada et al., 2006.) chronic exposure to hypoxia (0.83 mg O2/L, 10% saturation) for 7 days (Pelster, 2002), probably due to the up-regulation of vascular endothelial growth factor (VEGF). The reduction in circulation may render the supply of oxygen and nutrient insuYcient for the metabolic demand of development, leading to developmental arrest. Shang and Wu (2004) found that the heart rate of zebrafish embryos reared under hypoxia (0.8 mg O2/L, 10% saturation) showed an initial increase at 96 hpf and became significantly lower than that of the control embryos at 288 hpf. However, Bagatto (2005) reported that development of zebrafish under severe hypoxia (0.8 mg O2/L, 10% saturation) showed a delayed onset of cardiovascular regulation. 3.2.4. Disruption of Hormones Using zebrafish as a study model, Shang et al. (2006) demonstrated that levels of T and E2, as well as the T/E2 ratio, which are critical in modulating developmental processes, can be aVected by hypoxia as early as 48 hpf, long before the occurrence of sex diVerentiation. At 60 dpf, T and E2 were reduced by 73.7% and 69.9%, respectively, in hypoxic males while no 116 RUDOLF S. S. WU significant diVerence in either hormone was observed between hypoxic and normoxic females. After 120 days, T concentrations increased by 57.4% in hypoxic females, while no change was observable between hypoxic and normoxic males. No significant diVerence in E2 could be found in either males or females from the normoxic control and the hypoxic treatment after 120 days. At 60 and 120 dpf, significant increases in the T/E2 ratio were clearly evident in hypoxic females (+90.9% and +175.4%, respectively). No change in T/E2 ratio, however, was found in male fish, showing that the hormonal disruption is sex specific (Table 3.1 and Figure 3.21). Studies on rainbow trout (Tanaka et al., 1992), medaka (Fukada et al., 1996), and tilapia (Chang et al., 1997) have shown that mRNA levels of CYP19 correlated well with aromatase activity, and changes in CYP19 gene expression in the gonad of zebrafish has been shown to associate with alterations of gonadal diVerentiation (Fenske and Senger, 2004). Sex differentiation begins Gonads differentiate into ovaries 3β-HSD (−) CYP11A (−) CYP19A (ND) CYP19B (−) 3β-HSD (−) CYP11A (−) CYP19A (−) CYP19B (−) Aromatase (−) (A) 10 dpf (B) 40 dpf 10–12 dpf 23–25 dpf Larval 3 dpf Sex differentiation/ reversal is completed Final maturation of the gonads Spawning Male 3β-HSD (−) CYP11A (−) CYP19A (−) CYP19B (NC) T/E2 (NC) Male 3β-HSD (−) CYP11A (−) CYP19A (+) CYP19B (+) T/E2 (NC) Female 3β-HSD (+) CYP11A (−) CYP19A (+) CYP19B (+) T/E2 (+) Aromatase (−) Female 3β-HSD (−) CYP11A (−) CYP19A (+) CYP19B (NC) T/E2 (+) Aromatase (−) (C) 60 dpf (D) 120 dpf 42 dpf 60 dpf 120 dpf Juvenile 30 dpf Adult 90 dpf Fig. 3.21. Summary diagram showing changes in expression of various sex hormone control genes, ratio of testosterone/estradiol (T/E2), and CYParom activity with respect to key stages of gonad development in zebrafish exposed to hypoxia (0.8 mg O2/L, 10% saturation) and normoxia (5.8 mg O2/L, 75% saturation) at 10, 40, 60, and 120 dpf. (+), significant increase in hypoxic treatment with respect to normoxic control; ( ), significant decrease in hypoxic treatment with respect to normoxic control; (ND), not detectable; (NC), no significant change between normoxic control and hypoxic treatment. (Modified from Shang et al., 2006.) 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 117 Shang et al. (2006) set out to test the hypothesis that hypoxia can disrupt genes controlling steroidogenic enzymes and sex hormones, thereby aVecting sex diVerentiation and sex determination in zebrafish. In their study, downregulation of CYP19B was found in hypoxic fish at 10 dpf, when gonads started to develop into ovaries, and down-regulations of both CYP19A and CYP19B as well as marked reduction of E2 production were found at 40 dpf during sex diVerentiation/reversal. Both 3b-HSD and CYP11A were significantly down-regulated by hypoxia at 10 and 40 dpf, suggesting a reduction in steroidogenesis during sexual diVerentiation and before sex determination is completed in the hypoxic group. Decreases in expression of steroidogenic enzymes and production of T and E2 provide a plausible mechanism for the retardation of gametogenesis, which was subsequently observed in both hypoxic males and females. The changes in sex hormone levels, expression levels of various genes controlling steroidogenesis, and aromatase activities with respect to each key stage of gonad development under hypoxia are summarized in Figure 3.21. Vitellogenin (VTG) production was markedly reduced in both male and female zebrafish developed under hypoxia for 60 and 120 days. In hypoxic females, VTG was markedly reduced by 84.6% at 60 dpf and by 97.6% at 120 dpf. Similarly, VTG was reduced by 78.9% at 60 dpf and 80.6% at 120 dpf in hypoxic males (Figure 3.22). The reduction of VTG correlated well with the retardation of oocyte development and egg production observed upon exposure to hypoxia in the same study (Shang, 2005). 3.2.5. Sex Differentiation, Sex Determination, and Sex Ratio It is generally believed that sex diVerentiation in fish is similar to mammalian systems whereby the presence or absence of a testis-determining factor directs male or female diVerentiation (Jobling, 1995). The balance of sex steroid hormones is important in determining sex diVerentiation (Kime, 1998), and phenotypic sex of fish may be influenced by various external factors and chemicals regardless of their genotypic sex (Jalabert et al., 2000), especially before gonadal diVerentiation. A specific ratio of T/E2 is required for sexual diVerentiation, and alteration of this ratio can impair gonadal development (Hileman, 1994). Shang et al. (2006) designed an experiment to test the hypothesis that hypoxia can alter the balance of sex hormones in fish, which subsequently aVects sex diVerentiation, sex determination, and sex ratio. In their experiment, zebrafish eggs were kept under normoxia (5.8 mg O2/L, 75% saturation) and hypoxia (0.8 mg O2/L, 10% saturation) for 4 months until they hatched and developed into sexually mature adults. The results showed that chronic exposure to hypoxia can aVect sex diVerentiation during development. 118 RUDOLF S. S. WU A VTG (ng/mL) 200 000 Normoxia Hypoxia 150 000 100 000 50 000 ** *** 0 60 120 Time (dpf) VTG (ng/mL) B 2000 Normoxia Hypoxia 1500 1000 *** 500 0 ** 60 120 Time (dpf) Fig. 3.22. VTG level in (A) female and (B) male zebrafish at 60 dpf and 120 dpf upon exposure to normoxia (5.8 mg O2/L, 75% saturation) and hypoxia (0.8 mg O2/L, 10% saturation), (n = 5, mean  SE). Values significantly diVerent from the normoxic control are indicated by asterisks (t-test: **, p < 0.01; ***, p < 0.001). (Reproduced from Shang, 2005.) Impairment of ovarian development and yolk deposition was found in hypoxic females, which was clearly associated with a decrease in E2 and VTG (Shang et al., 2006). Sex determination was altered, resulting in a malebiased population in the F1 generation (74.4% males in the hypoxic groups versus 61.9% males in the normoxic groups). The fact that no deaths occurred in the hypoxic treatment group after 7 days, long before sex diVerentiation occurred (10–12 dpf), indicated that the biased sex ratio under hypoxia was not due to diVerential mortality rates between diVerent sexes. Experimental evidence was further provided to show that the hypoxic eVect on sex change was mediated through down-regulations of various genes controlling the synthesis of sex hormones (i.e., 3b-HSD, CYP11A, CYP19A, and CYP19B), leading to changes in levels of T and E2 in female fish at key developmental stages (Figure 3.21). From 60 days onward, the 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 119 T/E2 ratio showed a significant increase in hypoxic females but not in hypoxic males, showing that females were more susceptible and that P450arom was inhibited by hypoxia. Taken together with (1) lower E2 and higher T levels found in hypoxic females as compared with normoxic females and (2) reductions in both E2 and T, while the T/E2 ratio remained unchanged in hypoxic males, it appeared that the disruption of the balance between E2 and T could be a major factor contributing to the observed malebiased population in the hypoxic treatment group. Another possible mechanism leading to a male-biased sex ratio may involve oocyte apoptosis. Both in vitro and in vivo studies on mammalian systems provide evidence that hypoxia can induce apoptosis (Jung et al., 2001; Saed and Diamond, 2002; Shin et al., 2004). Uchida et al. (2002) showed that the disappearance of large numbers of oocytes in male zebrafish during their normal transition from ovary-like tissue to testicular tissue was mediated through apoptosis. Likewise, large numbers of apoptotic early diplotene oocytes and ovarian follicles have been reported in developing male rainbow trout and Astyanax bimaculatus lacustris (Janz and Van der Kraak, 1997). The fact that hypoxia could alter the apoptotic pattern of zebrafish as early as 24 hpf (see Section 3.2.5; Figure 3.18) suggests that hypoxia may also alter the scheduled oocyte apoptosis in designated females during sex diVerentiation and favor the formation of testicular tissues, leading to a male-biased sex ratio in the F1 generation. In male medaka (Oryzias latipes) in which a sex-determining gene, DMY, has been found on the Y chromosome (Matsuda et al., 2002), 77% of genotypic XX females reared under hypoxia developed into phenotypic males, while sex change was not found in genotypic females in the normoxic control group (Cheung and Wu, 2006). This is consistent with the findings of Shang et al. (2006), who found that hypoxia caused sex change in zebrafish and that hypoxia can also alter sex diVerentiation and sex determination in species with a sex-determining gene. The studies of Matsuda (2003) and Hattori et al. (2007) showed that sexual development and determination in fish are, in part, determined by germ cell diVerentiation occurring at early embryonic stages. During sex diVerentiation, primordial germ cells may diVerentiate into oogonia or spermatogonia, while the supporting cells may diVerentiate into granulosa or Sertoli cells in the ovary and testis, respectively (Devlin and Nagahama, 2002). As such, the diVerentiation of germ cells and their supporting cells during developmental stages may play an important role in gonad diVerentiation and hence sex determination. Gimeno et al. (1996, 1997) showed that male common carp exposed to 4-tert-pentylphenol during the critical period of sex determination (24–51 dph) had a reduced number of primordial germ cells (PGC), which aVected gonadal structure, including the induction of 120 RUDOLF S. S. WU oviduct formation. Hypoxia may aVect the production and migration of PGC in a similar way, although this has not been demonstrated. Despite laboratory results showing that hypoxia can lead to a biased F1 generation in two diVerent species, field evidence showing that hypoxia may aVect sex determination and sex ratio of fish under naturally occurring hypoxia is not available. Furthermore, so far there has been no attempt to verify the eVects of hypoxia on fish development observed in the laboratory in the field. 4. SUPPORTING EVIDENCE FOR THE EFFECTS OF HYPOXIA ON REPRODUCTION AND DEVELOPMENT IN OTHER VERTEBRATES The scientific evidence provided in the above sections supports the notion that hypoxia is an endocrine disruptor and also a teratogen in fish. Similar to fish, reproductive processes in higher vertebrates are also modulated by sex hormones. The HPG axis and the genes and enzymes controlling steriodogenesis as well as the sex steroid hormones are highly conservative across diVerent vertebrate groups (Ankley and Johnson, 2004). For example, the amino acid sequences of StARs in fish, amphibians, avian species, and mammals are remarkably similar, and non-mammalian StARs share 63– 69% sequence identity with human StAR protein (Bauer et al., 2000). The structural organization of the fish receptors (TSHR, FSHR, and LHR) as deduced from the encoding cDNAs is highly homologous to the higher vertebrate receptors (Kumar and Trant, 2001). Thus, the endocrine-disrupting eVects of hypoxia found in fish, which subsequently lead to impairment of reproduction and development, may also occur in other vertebrates. Conversely, the eVects of hypoxia on reproduction and development revealed in other vertebrate groups may shed light on fish studies, although it must be noted that fish are generally more able to tolerate and adjust to hypoxic conditions than mammals (Ramirez et al., 2007). 4.1. In Vitro Evidence A number of in vitro studies provide supporting evidence that expression levels of genes controlling key steriodogenic enzymes and activities of steroidogenic enzymes are reduced in hypoxia. For example, physiologically realistic levels of hypoxia (66–123 torr, 12%–21% saturation) can specifically inhibit aldosteronogenesis in bovine adrenocortical cells in a dosedependent manner (RaV and Kohandarvish, 1990). Inhibition of the conversion of corticosterone to aldosterone (the step catalyzed by 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 121 P450c11AS) was found in rat adrenal cells exposed to hypoxia (10% O2, 48% air saturation) for 3 days. Importantly, change in other cytochrome P450 enzyme activities was not observed (RaV et al., 1996), showing that hypoxic inhibition is specific rather than a general down-regulation of steroidogenesis. A similar conclusion was arrived at in fish studies. Induction of CYP19 expression was found when trophoblast cells isolated from human placenta were maintained under hypoxic conditions (2% O2, 10% air saturation), and induction of CYParom mRNA associated with an increase in aromatase activity was clearly evident when hypoxic-treated trophoblasts were returned to normoxia (Jiang et al., 2000). Kurebayashi et al. (2001) found that the expression level of estrogen receptor (ERa) was significantly reduced by hypoxia (1% O2, 5% air saturation) in two human breast cancer cell lines (ML-20 and KPL-1). In addition, hypoxia markedly suppressed the induction of progesterone receptor (PgR) mRNA and protein by E2 in both cell lines. In vitro studies on steroidogenesis using human adrenal glands with aldosterone-secreting adenomas (RaV and Bruder, 2006) showed that hypoxia (40 mmHg, 5% air saturation) within the physiological range significantly inhibited cAMP- and ACTH-stimulated cortisol and dehydroepiandrosterone (DHEA) production, showing that steroidogenesis can be aVected by hypoxia. VEGF, which is inducible by hypoxia inducible factor-1 (HIF-1), can stimulate proliferation of the mouse TM3 Leydig cells and release of testosterone, while administration of anti-VEGF antibody inhibited the proliferation and release. The results suggest that hypoxia may stimulate cell proliferation and testosterone release in Leydig cells via an increase of VEGF production (Hwang et al., 2007). 4.2. In Vivo Evidence Similar to fish, chronic exposure to hypoxia (3.8 kPa, 18% saturation) delayed development and hatching in salamanders (Ambystoma sp.), and less developed and deformed embryos were produced upon hatching. Development and growth of Australian frog (Crinia georgiana) embryos were severely delayed at 2 kPa (10% saturation) and malformation was observed (Seymonr et al., 2000). In contrast, hypoxia did not aVect the developmental rate in the frog (Rana sp.), and hypoxic embryos hatched earlier than normoxic embryos, but a higher percentage of less developed embryos was found (Mills and Barnhart, 1999). The numbers of spermatogenic epithelial cells, Sertoli cells, and Leydig cells in the testicular tissue of male albino rats were significantly reduced after exposure to acute hypobaric hypoxia (Shevantaeva and Kosyuga, 2006). Seven-day-old rats exposed to fetal hypoxia (12% O2, 57% air saturation) 122 RUDOLF S. S. WU showed a decrease in plasma aldosterone but no eVects on steroidogenic enzyme expression (RaV et al., 2000). In male rats, chronic hypobaric hypoxia has been shown to reduce sperm output and aVect spermatogenesis. An increase in FSH and a decrease in LH followed by a decrease in testosterone were found (Farias et al., 2008). The above results support the findings in fish that hypoxia may aVect spermatogenesis through hormonal changes along the HPG axis. Similar to fish, mammalian studies showed that apoptosis, an important process in development, is also aVected by hypoxia. Apoptosis in human testicular germ cells was significantly suppressed below 10% oxygen (48% air saturation) (Erkkila et al., 1999). Using flow cytometry and TUNEL, Liao et al. (2007) demonstrated a significant increase in apoptotic germ cells in seminiferous tubules of the hypoxic Wistar rats, especially in spermatogonia and spermatocytes. Both expression level of Bax and the ratio of Bax to Bcl-2 was significantly higher in the hypoxic group after 30 days’ exposure, suggesting that chronic hypoxia promotes apoptosis of testicular germ cells in male rats by increasing Bax expression in the rat testis. The results support the findings in fish (Shang and Wu, 2004) that hypoxia can aVect apoptosis. 5. THE ROLE OF HYPOXIA-INDUCIBLE FACTORS Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that is highly conserved and has been found in many species from fish to mammal (Wang and Semenza, 1993; Bunn and Poyton, 1996; Guillemin and Krasnow, 1997; Nikinmaa and Rees, 2005). HIF-1 receives signals from the molecular oxygen sensor through redox reactions and/or phosphorylation (Bunn et al., 1998) and, in turn, regulates the transcription of a number of hypoxia-inducible genes responsible for necessary biochemical and physiological adjustments (Wu, 2002). Since the discovery of HIF-1 (Wang et al., 1995), cumulative evidence has shown that HIFs are the ‘‘master regulators’’ of many molecular responses to hypoxia and, to date, HIF-1 is known to either directly or indirectly regulate the transcriptions of more than 100 genes of diverse functions, including angiogenesis, erythropoiesis, glucose metabolism, vasodilation, cell growth, cell proliferation, transcriptional regulation, diVerentiation, migration, apoptosis, signaling, and cell fate decisions (Semenza et al., 1994; Arany et al., 1996; Okino et al., 1998; Lisy and Peet, 2008). For a detail review of HIFs, please see Chapter 10. Some of the above biological processes are fundamental to or indirectly related to reproductive processes as well as embryonic development. Conceivably, HIFs may also regulate certain genes controlling these processes, 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 123 through which fish reproduction and development are aVected, although this has not been clearly demonstrated. Indeed, HIF-1 is implicated in apoptosis (Piret et al., 2002) and reproduction (Park et al., 2007), and has been reported to down-regulate the activity of estrogen receptor (ERa) via the ubiquitinproteasome degradation pathway in human breast cancer cells (Cho et al., 2005). Deactivation of HIF-1a or HIF-1b in knock-out mice leads to embryonic lethality due to abnormal vascular development (Maltepe et al., 1997; Iyer et al., 1998). These peripheral evidences appear to indicate that some of the observed eVects of hypoxia on reproduction and development may be mediated through HIF-1a. It is interesting to note that mRNA expression of both HIF-1a and HIF-2a mRNA in ovaries of Atlantic croaker (Micropogonias undulatus) showed a significant increase after exposure to hypoxia (1.7–3.7 mg O2/L, 26%–56% saturation for 3 days to 3 weeks), while such up-regulations were not observable in muscle (Rahman and Thomas, 2007). This tissue-specific diVerential expression appears to suggest that HIFs may be involved in regulating reproductive function in fish, although this has yet to be tested. HIF-1 consists of two subunits, HIF-1a and HIF-1b, and the latter is the same as the aryl hydrocarbon receptor nuclear translocator (ARNT). The aryl hydrocarbon receptor (AhR) can be ligand-activated to heterodimerize with ARNT, leading to induction of the cytochrome P450 enzymes. Since both the AhR and HIF-1 a compete for ARNT, hypoxia could be expected to decrease the expression of cytochrome P450, which is also involved in steroidogenesis and therefore aVects sex hormone production, although there is no clear supporting evidence. In fish, HIF-1 has been shown to control VEGF and hence angiogenesis (Nikinmaa and Rees, 2005). Vascularization, an important process in fish embryonic development, is controlled by VEGF and many fish embryos reared under hypoxia failed to develop their vascular system and die (Shang and Wu, 2004), suggesting that HIF may also play a role in vascular development via VEGF in the early embryonic stages. It is not known whether cell proliferation, apoptosis, and development are regulated by HIF. Previous work has established that hypoxia can aVect key reproductive and developmental processes in fish through aVecting genes controlling steriodogenesis and production of hormones (Dabrowski and Richard, 2003; Shang et al., 2006; Thomas et al., 2006; Landry et al., 2007). Whether these disruptions are mediated through HIF or are independent events remains unknown. In vitro transfection studies using the H295R (a human adrenocortical carcinoma) cell line showed down-regulation of 3b-HSD1 and StAR in HIF-1a-overexpressed H295R cells but no change in genes controlling other steroidogenic enzymes, showing that hypoxia can specifically aVect certain target genes involved in steroidogenesis 124 RUDOLF S. S. WU Fig. 3.23. In situ localization of omTERT mRNA in testes of marine medaka. Scale bars = 50 mm; SG: spermatogonia; SC: spermatocyte; ST: spermatids; SP: spermatozoa. (A) Testis under normoxia (6.4 mg O2/L, 96% saturation). Expression of omTERT mRNA (blue) is strong in cysts containing spermatogonia and spermatocytes, moderate in diVerentiating spermatids, and absent in mature spermatozoa (red). (B) Testis exposed to hypoxia (1.8 mg O2/L, 27% saturation) for 96 h. Induction of omTERT mRNA (blue) is conspicuous in spermatogonia but less prominent in other testicular cells. (C) Adjacent testis section hybridized with an omTERT sense riboprobe, serving as a negative control. (Reproduced from Yu et al., 2006.) (Chu et al., 2006). Whether cell proliferation and apoptosis are regulated by HIF remains unclear. Using in situ hybridization, Yu et al. (2006) showed that hypoxia can induce telomerase reverse transcriptase (TERT) mRNA in the testis of marine medaka (Oryzias melastigma), and results of transfection assays further showed that overexpression of HIF-1a can induce the promoter activity of TERT (Figure 3.23). The results of this study support the notion that hypoxia can up-regulate TERT expression via HIF-1 in fish testis in vivo. Clearly, a systematic and comprehensive study is required to elucidate the possible role of HIF and its targets of regulation in fish with respect to hypoxic eVects on sex hormone production, reproductive impairment, and development. The functional role of HIF-2 is much less clear, but HIF-2a has been shown to regulate the DNA-damage-inducible alpha protein that induces G2 arrest and apoptosis (Hu et al., 2003). In embryonic stem cells, HIF2a protects cells against apoptosis during hypoxia (Carmeliet et al., 1998; Brusselmans et al., 2001). As such, HIF-2a may play a role in regulating hypoxic responses in specific cell types (Nangaku and Eckardt, 2007). The possibility may therefore exist that HIF-2a is involved in mediating the eVects of hypoxia on cell proliferation and apoptosis during fish development, although this hypothesis has never been tested. Studies have shown that HIF-3a can attenuate HIF-1a-mediated and hypoxia-mediated induction of HRE-driven reporter genes (Hara et al., 2001; Mazure et al., 2002), and may act as an internal repressor of HIF-1a (Makino et al., 2001). Again, whether HIF-3a is involved in mediating the eVects of hypoxia on cell proliferation and apoptosis during fish development remains unknown. 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 125 6. BIOLOGICAL AND ECOLOGICAL IMPLICATIONS Given the fact that (1) hormones and genes along the HPG axis, especially those involved in sex steroid synthesis, are highly conservative and (2) there is considerable plasticity in sex determination of fish, the impairment of reproduction and development observed in the few species studied may widely occur in other fish species. Reproductive impairment resulting from hypoxia, as manifested by reduction in reproductive output, quality of sperm and eggs, fertilization success, and larval survival, may have a significant eVect on natural fish populations. While reproductive impairment of individuals by hypoxia has been demonstrated in the natural environment, no field data are available to link the observed reproductive impairment to population decline thus far. Understandably, such data would be diYcult to collect since population size is often confounded by other factors such as pollution, over fishing, and natural variability prevailing in the same environment, which are diYcult to decipher. Laboratory experiments demonstrated that hypoxia can aVect fish development, leading to an increase in embryo mortality rates, delay in hatching, and malformation. It is likely that many of the malformed larvae/juveniles would not be able to survive and contribute to reproduction in the next generation. It is interesting to note that a higher occurrence of malformed fish larvae has been generally reported in polluted areas (Au, 2004), although the observed increase in malformed larvae may not necessarily be attributable to hypoxia because polluted areas are often also contaminated with a variety of chemicals including teratogens and endocrine-disrupting chemicals. Synchronization of the time of hatching with food availability in the natural habitat is important for many species (Eikenaar et al., 2003;Milione and Zeng, 2007), to ensure that natural prey items are available to the newly hatched larvae. Delayed hatching of fish larvae has been reported both in the laboratory and under field conditions (Ingendahl, 2001; Geist et al., 2006; Roussel, 2007); however, the ecological consequence of this occurrence has yet to be elucidated. Maintaining certain sex ratio is clearly important for ensuring reproductive encounters and hence reproductive success and sustainability of natural populations (Kokko and Brooks, 2003; Le Galliard et al., 2005; Rankin and Kokko, 2006). The laboratory findings that sex diVerentiation and sex ratio of zebrafish and medaka are aVected by hypoxia, resulting in a male-dominated F1 generation (Shang et al., 2006; Cheung and Wu, 2006) is of great environmental concern because this might potentially threaten species survival. A classic, parallel example is imposex in marine whelks (a phenomenon of which females snails exhibiting sexual characteristics of males) caused by 126 RUDOLF S. S. WU tributyltin contamination, which has resulted in a male-biased sex ratio, reproductive failure and extinction of natural populations over large areas worldwide (Bryan et al., 1986). It is important to note that the number of females is the primary limiting factor in determining the reproductive output of a population, and reduction in the number of females may increase competition between mating males and also reduce mating success (Kvarnemo and Ahnesjö, 1996; Jirotkul, 1999). Hypoxia may further reduce both the quantity and quality of gametes and hence reproductive success. The fact that hypoxia aVects large areas of aquatic systems worldwide (Diaz and Rosenberg, 1995; Wu, 2002) implies that the ecological consequences caused by hypoxia on natural fish populations could be potentially very serious. 7. CONCLUSIONS Hypoxia can impair reproduction, alter reproductive behaviors, aVect quality of sperm and egg, reduce fertilization success, delay development, reduce hatching success, and increase the incidence of malformation in fish. Results further showed that hypoxia may alter sex diVerentiation and sex determination. The severity of hypoxic eVects, however, depends on the developmental stage and level of oxygen, as well as duration and level of hypoxic exposure. There is good evidence to suggest that hypoxia impairs fish reproduction by aVecting multiple target sites along the HPG axis and enzymes controlling steroidogenesis. Importantly, hypoxia does not cause a general down-regulation of metabolism and reproductive functions, but targets specific hormones, neurotransmitters, and receptors along the HPG axis, as well as certain enzymes controlling steroidogenesis. There is emerging evidence to show that the molecular, hormonal, and behavioral responses of male and female fish to hypoxia may be diVerent. Hypoxia can arrest or delay fish development by aVecting the S and G2 phases of the cell cycle. However, other studies have shown that hypoxia may trigger hatching and development. Although it is well known that hypoxia can cause malformation, the underlying mechanism leading to malformation remains largely unclear. There is good evidence to suggest that this may be mediated through aVecting cell proliferation and apoptosis during the various developmental stages. Laboratory and field studies have shown that hypoxia can alter both the level and balance of androgens and estrogens in several fish species, thereby suppressing ovarian and testicular growth. In particular, hypoxia has been shown to down-regulate CYP19 and alter the ratio of testosterone to estradiol during early development in zebrafish, thereby favoring male 3. EFFECTS ON FISH REPRODUCTION AND DEVELOPMENT 127 development and leading to a male-biased F1 generation. In medaka, a large percentage of genotypic females (with XX chromosomes) showed testicular development and exhibited male phenotypic characteristics when embryos were allowed to develop under hypoxic conditions prior to sex determination. Scattered evidence appears to indicate that HIF-1 also regulates vascular development as well as some hypoxic responsive genes related to hormonal receptors, cell proliferation, and apoptosis. Whether HIF-1 also plays a role in mediating the observed reproductive and development impairments remain unclear. Transfection assays and knock-down experiments are required to verify the involvement of HIF-1 in mediating the eVects of hypoxia on fish reproduction and development. Since the hormones and regulation of the HPG axis, as well as the enzymes regulating steroidogenesis, are highly conservative in vertebrates, the eVects of hypoxia on fish reproduction and development may also occur in other (higher) vertebrates. Indeed, scattered results of in vitro and in vivo studies in higher vertebrates also lend support to this postulation. More comparative studies between hypoxic responses in diVerent vertebrate groups should be carried out to test this hypothesis and elucidate some common principles and mechanisms. Reproductive impairment and the adverse eVects on development caused by hypoxia revealed in this study suggests that hypoxia poses a significant threat to the sustainability of natural fish populations, especially when considering that hypoxia commonly occurs over very large areas worldwide, and the problem is likely to be exacerbated in the future. For many fish, reproduction is a seasonal event and may involve migration to specific environments, and hypoxia may determine when and where reproduction could occur in these species. Despite this, supporting field evidence is scarce, and the long-term eVects of hypoxia on natural fish populations remains virtually unknown. Finally, it must be cautioned that the vast majority of existing evidence on how hypoxia may aVect genes relating to reproduction and development has been based on mRNA transcripts, while the biochemical and physiological responses and adjustments of fish would depend upon the post-translational proteins. Notably, unlike many structural proteins and enzymes, a small change in the amount of sex hormones and neurotransmitters would be suYcient to cause major eVects in reproduction and development. Such a small diVerence may not be detectable by changes in the corresponding mRNA transcripts, especially noting that a two-fold change has been generally employed as a criterion in determining changes in gene profile in microarray studies. Using high-resolution 2-D gel electrophoresis, Bosworth et al. (2005) demonstrated that hypoxia did not change the general pattern of 128 RUDOLF S. S. WU protein expression, but only the amounts of six low-abundance proteins in the skeletal muscle of zebrafish. This result contradicts the widespread changes in mRNA levels in hypoxic fish reported in many studies, and the huge diVerence between the protein and mRNA expression patterns identified calls for a better understanding of proteomic changes in fish during hypoxic exposure. ACKNOWLEDGEMENTS I would like to thank Prof. David Randall and Sunny Lu for their comments on a draft of this review. I thank Helen Mok for her technical assistance in data collection and preparation of tables and figures. This work is supported by the Area of Excellence Scheme under the University Grants Committee of the Hong Kong Special Administration Region, China (Project No. AoE/ P-04/2004). REFERENCES Aarnio, K., BonsdorV, E., and Norkko, A. (1998). Role of Halicryptus spinulosus (Priapulida) in structuring meiofauna and settling macrofauna. Mar. Ecol. Prog. Ser. 163, 145–153. 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High correlation between microvillous olfactory receptor cell abundance and sensitivity to pheromones in olfactory nerve-sectioned goldfish. J. Comp. Physiol. A. 180, 39–52. 4 OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE HANS O. PÖRTNER GISELA LANNIG 1. Thermally Induced Hypoxemia in Fishes 1.1. Temperature-Dependent Oxygen Supply 1.2. Width of Thermal Window and Energy Budget 2. Temperature Adaptation: Role of Hypoxemia 2.1. Systemic Signaling Responses 2.2. Acid-Base and Ion Regulation 2.3. Hypoxemia-Related Cellular Stress and Signaling 3. Cellular Mechanisms of Thermal Adaptation 3.1. Capacity and EYciency of Mitochondria 3.2. Membrane Structure: Functional Implications and Costs 3.3. Calcium Homeostasis and Functioning 3.4. Energy Budget, Turnover, and Allocation 4. Perspectives: Hypoxia-sensitive Thermal Windows in Climate Sensitivity Temperature and hypoxia would traditionally be considered as diVerent environmental factors, with specific implications for whole organism functioning. Development of the concept of oxygen and capacity limited thermal tolerance in marine water breathers has revealed how these factors are intertwined. Thermal stress causes systemic hypoxemia and the interaction of temperature and thermally induced hypoxemia will thereby shape acclimation responses at various molecular to whole organism levels. The chapter discusses aspects such as temperature-dependent oxygen supply, width of thermal window and associated energy budget, hypoxemia related stress, and signaling, as well as the cellular mechanisms of thermal adaptation and associated costs including handling and role of calcium. The integration of these responses supports adjustment of metabolic and functional performance at cellular, 143 Hypoxia : Volume 27 FISH PHYSIOLOGY Copyright # 2009 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(08)00004-6 144 HANS O. PÖRTNER AND GISELA LANNIG tissue, and whole organism levels to within thermal limits. Thereby, processes involved in thermal acclimatization and adaptation counteract thermally induced hypoxemia in fish. Conversely, hypoxia and other stressors will aVect thermal tolerance limits and the processes involved in thermal acclimatization and adaptation. As a perspective, the specialization of whole organism functioning on limited temperature ranges emerges as a key element explaining current observations of climate change eVects on ecosystems. 1. THERMALLY INDUCED HYPOXEMIA IN FISHES Studies of temperature-dependent oxygen supply, mode of metabolism, and associated mechanisms of thermal adaptation in marine invertebrates and fishes across latitudes suggested a role of oxygen supply in thermal limitation. Initial evidence came from studies in marine invertebrates (annelids, sipunculids), which showed transition to anaerobic mitochondrial metabolism at both the low and the high end (called critical temperatures) of the thermal tolerance window (Zielinski and Pörtner, 1996; Sommer et al., 1997). These findings stimulated work in bivalves and fishes that demonstrated the onset of anaerobic succinate formation at high temperatures (van Dijk et al., 1999; Pörtner et al., 1999a; Peck et al., 2004). A more recent example confirmed the onset of anaerobic metabolism at low and high temperature extremes in cephalopod mantle tissue (Melzner et al., 2006). The transition to mitochondrial anaerobiosis was shown to result from the development of progressive hypoxemia in arterial haemolymph of a crustacean toward both sides of the thermal window, with an optimum range of maximum body fluid PO2 in between (Frederich and Pörtner, 2000). Once the critical PO2 of oxygen diVusion into cells and mitochondria was reached mitochondria started to respire anaerobically. These findings formed the basis of the concept of oxygen and capacity limited thermal tolerance as depicted in Figure 4.1. With it came the conclusion that in a systemic to molecular hierarchy of thermal tolerance the whole organism would experience functional limitations first before biochemical stress events would set in at tissue, cellular, or molecular levels (Pörtner, 2001, 2002; Figure 4.1). The concept implies that optimized oxygen supply to tissues between lower and upper pejus temperatures combined with the kinetic stimulation of performance rates by warming supports an optimum of performance close to upper pejus temperature. The excess oxygen available above oxygen demand for maintenance fuels the performance capacity of the animal and is reflected in its aerobic scope. Toward both edges of the thermal envelope oxygen supply capacity becomes limiting as oxygen demand of maintenance metabolism progressively exploits all of aerobic scope. This transition reflects 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 145 Stress hormones, adenosine, D redox Loss of performance Tp % Oxygen limited aerobic scope Acclimation in functional capacity Tp DEnergy consumers / DMitochondrial functions Acclimation in protection: Anaerobiosis Tc Tc +HIF-1 Hypoxemia (steady state) Denaturation Td 0 Stress hormones released Oxidative stress increased Anaerobic capacity O2 supply pathways Metabolic depression Td Acclimation in repair: + HSP, + antioxidants Optimum Rate of aerobic performance Hypoxia, CO2 0 Temperature Fig. 4.1. Oxygen and capacity limitation concept of thermal windows indicating the hierarchies (top) of functional limitation (beyond pejus temperatures, Tp), oxygen defiency, anaerobic metabolism and protection through metabolic depression (below and beyond critical temperatures, Tc), and denaturation, as well as repair (beyond denaturation temperatures, Td). These patterns of thermal limitation lead to a loss in functional capacity and the characteristic righttilted aerobic performance curve (bottom). Optimized oxygen supply to tissues between low and high pejus temperatures (top) combined with the kinetic stimulation of performance rates by warming supports a performance optimum (i.e., an optimum of aerobic scope) close to upper pejus temperature (bottom). Systemic (e.g., stress hormones, adenosine) and cellular signals (e.g., hypoxia inducible factor HIF-1a, and redox status) associated with temperature-induced hypoxemia contribute to the acclimation response, which leads to a shift in thermal tolerance windows. Ambient hypoxia frequently goes hand in hand with elevated CO2 levels; both cause a narrowing of thermal windows (Pörtner et al., 2005, Metzger et al., 2007). The graph has been modified and updated from Pörtner and Knust (2007). Note that the figure does not depict details of the signaling pathways involved. onset of thermal stress and causes an early loss of whole organism functional performance before biochemical stress events take place. A study in the eelpout, Zoarces viviparus, in fact demonstrated that the warming-induced decrement in aerobic scope matches the onset of a decrease in growth performance (Pörtner and Knust, 2007). The same thermal threshold is associated with a decrease in abundance of the species in the field, long before anaerobic metabolism sets in due to severe oxygen deficiency and before biochemical stress events take place. These findings clearly indicate that, at the limits of acclimatization capacity, the early onset of a performance decrement is suitable to cause a loss in fitness with the resulting consequences at ecosystem 146 HANS O. PÖRTNER AND GISELA LANNIG level. Physiological mechanisms setting performance at the whole organism level thus represent the long sought mechanistic link between climate and ecosystem change. Figure 4.1 distinguishes between the temperature range associated with a loss of performance (active range), the subsequent endurance of temperature extremes supported by metabolic depression (passive range), and the range of damage and repair, where protective mechanisms are being used and damaged molecules are accumulating for later removal or repair upon the return of temperatures to control conditions. To date, evidence of temperature-induced hypoxemia in fish builds on relatively few examples, with the study by Lannig et al. (2004) reporting temperature-dependent venous oxygen tensions, the study by van Dijk et al. (1999) addressing the transition to anaerobic metabolism. Pörtner and Knust (2007) and Pörtner et al. (2001, 2008) integrated these findings with those of temperature-dependent growth. The study by Mark et al. (2002) as well as Lannig et al. (2004) indicated a limited capacity of cardio-circulation to respond to warming beyond a certain limit, the pejus limit, while temperaturedependent oxygen demand increased upon warming. Excess ambient oxygen improves resistance to warming by shifting pejus limits (Weatherley, 1970; Mark et al., 2002; Figure 4.2). According to these findings hypoxemia results from a mismatch between oxygen supply capacity and oxygen demand, both processes being temperature dependent. Finally, the studies by Pörtner and Knust (2007) as well as Farrell et al. (2008) demonstrated the ecological relevance of the oxygen and capacity limitation concept. The study by Pörtner and Knust (2007) showed the link of thermally limited cardio-circulatory performance and aerobic scope to the onset of reduced growth performance and abundance in the natural environment, the German Wadden Sea. Oxygen supply limitations also play a key role in the thermal limitation of muscular exercise of migrating salmon and their inability to reach their spawning grounds in warming rivers (Farrell et al., 2008). 1.1. Temperature-Dependent Oxygen Supply The temperature-dependent functional capacity of oxygen supply systems (ventilation and cardio-circulation) thus appears crucial in setting whole organism thermal limits. Within the thermal window the capacities of these processes cover the oxygen demand of maintenance and aerobic scope. The level of temperature-dependent oxygen demand in relation to oxygen supply capacity will determine and is thus mirrored in the degree of oxygen saturation of body fluids. Together with blood flow velocity, body fluid oxygenation reflects the scope of oxygen supply to tissues. In the light of the limited number of studies addressing the oxygen limitation concept in fishes, the link between oxygen availability (through cardiac and ventilatory 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 147 Fig. 4.2. Hyperoxia eVects on oxygen consumption (A) and blood flow (B) in the aorta dorsalis of the Antarctic eelpout, Pachycara brachycephalum, under normoxia and hyperoxia with rising temperature. Under normoxia, MO2 showed a large exponential increment, which was eliminated under hyperoxia. At the same time, under normoxia, blood flow increased during warming to 7  C, and it remained constant and significantly elevated at higher temperatures. In contrast, blood flow remained fairly constant under hyperoxia. (Data by Mark et al., 2002.) performance) and whole organism aerobic performance still needs to be established on a quantitative basis. The temperature of optimum oxygen supply and the temperature of maximum energetic eYciency for aerobic performance is supposedly found close to upper pejus temperature (Figure 4.1). However, the maximum of venous PO2 in Atlantic cod (Lannig et al., 2004) apparently falls below the thermal optimum of growth performance in juvenile cod (Figure 4.3). This apparent discrepancy is alleviated when considering the clear allometry of thermal sensitivity, which 148 HANS O. PÖRTNER AND GISELA LANNIG TO TPI TPII Venous PO2(%) 100 TC 75 50 25 0 2 4 6 8 10 12 14 16 18 20 Temperature (°C) Body size Daily growth rate (%/day) 1.0 0.8 0.6 0.4 dW(T) = (A1*e−Ae1/T) − (A2*e-Ae2/T) 0.2 0.0 2 4 6 8 10 12 14 16 18 20 Acclimation temperature (°C) Fig. 4.3. Relative levels (% air saturation) in venous PO2 in Atlantic cod, Gadus morhua, acclimated to 10 C during acutely changing temperature at 1  C h-1 (A, data by Lannig et al., 2004). The acute thermal window of venous blood PO2 in cod displays a lower ‘‘optimum’’ than expected in relation to acclimation temperature and growth optimum of acclimated juvenile fish (B, data by Fischer, 2003). This apparent discrepancy may be due to various reasons: the figure compares diVerent body sizes such that diVerent optima result. An upward shift of the growth optimum likely occurs upon thermal acclimation. The role of hemoglobin (Hb) oxygen binding in blood oxygen transport in the warmth remains unclear (see text). The progressive increase in the use of Hb for oxygen transport may explain the tailing of venous PO2 toward warmer temperatures. causes a shift of growth (and likely, venous PO2) optima to lower temperatures in cod (Pörtner et al., 2008). This trend would be exacerbated by the 10% and 20% increase in male and female body mass, respectively, during the maturation process and prior to spawning. These trends may in fact explain the 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 149 sensitivity of the spawning population in the North Sea to warmer winter temperatures above 6 C or 7 C (Perry et al., 2005). Blood flow regulation and temperature-dependent functioning of hemoglobin also needs to be keyed into the picture of thermally limited oxygen supply (Lannig et al., 2004; Gollock et al., 2006, see Chapter 6). Blood volume is much lower in fish than in invertebrates with an open circulatory system, e.g., crustaceans of similar lifestyles. To compensate and support a higher metabolic rate, the amount of hemoglobin-bound oxygen per unit volume of blood is (more than) one order of magnitude larger in fish than the amount of pigment-bound oxygen in invertebrates. Oxygen transport via the pigment is thus more prominent in fish than in invertebrates. With constantly high arterial PO2 seen over the whole range of temperatures within the thermal window of cod (Sartoris et al., 2003) the maximum amount of oxygen released from hemoglobin at various temperatures depends on venous PO2 in relation to temperature-dependent oxygen binding. Cardio-circulation, which appears to comprise a large fraction of the energy budget (see below), will operate at lowest costs in relation to oxygen demand once oxygen release from hemoglobin is maximal. This is likely the case at lower rather than higher maximum venous PO2 values. Therefore, and in contrast to the respective patterns in invertebrates, the course of venous PO2 in fishes may not in itself provide the full picture with respect to the optimum of aerobic scope. This conjecture is corroborated by the observation that PvO2 did not change with acute warming above the thermal optimum for both resting and swimming sockeye salmon (Steinhausen et al., 2008). All of these considerations remain speculative as long as hemoglobin oxygen binding has not been investigated under in vivo conditions, with respect to its role and contribution to the window of thermal tolerance in fishes. The quantitative integration of temperature-dependent oxygen binding of hemoglobin (usually studied in vitro and in stripped hemoglobin) with the patterns and cost of circulation within and toward the edges of the thermal tolerance window are thus relevant issues, which, unfortunately, have not yet been explored under the framework of oxygen and capacity limited thermal tolerance. 1.2. Width of Thermal Window and Energy Budget Aerobic scope available for various ‘‘tasks’’ on various time scales (e.g., long-term: foraging, growth, reproduction, development versus short(er)-term: hunting, migration, escape) emerges as a key parameter shaping fitness. Aerobic scope is not only a matter of oxygen supply capacity but also a matter of energy eYciency, i.e., the reduction of baseline costs to maximize scope and the costeYcient collection, uptake, and use of available food and substrates. Growth rate, for example, is negatively influenced by the cost of foraging in fish. 150 HANS O. PÖRTNER AND GISELA LANNIG With respect to temperature-dependent baseline costs, whole organism oxygen demand is, on the one hand, set by the thermal responses of cellular energy consumers and the resulting level of cellular energy turnover (see Section 3.4). This includes cellular work in ventilatory or circulatory organs, which increases upon rising oxygen demand, even more so when oxygen is in short supply. Evidence for this conclusion comes from the observation that ventilation and circulation represent a significant cost in the energy budget of a fish. The cost to cover oxygen demand rises with the warming-induced increase in baseline oxygen requirements. Vice versa, this cost in itself contributes to whole organism baseline oxygen demand and standard metabolic rate. The contribution of ventilatory and circulatory costs to thermal limitation has been demonstrated through the alleviation of this thermal burden under the eVect of hyperoxia (Mark et al., 2002). On the other hand, functional capacities of cells and tissues co-define the warming-induced increment in the cost of ventilation and circulation and, thus, whole organism oxygen demand. Elevated functional capacity of an organ and of the circulatory system goes hand in hand with elevated baseline energy turnover, partly due to higher densities and maintenance costs of idling mitochondria and transmembrane ion exchange mechanisms, but also due to better capillarization of tissues and volume capacity of blood vessels. At elevated capacity, ventilation and cardio-circulation will find it ‘‘easier’’ to cover the temperature-induced increase in oxygen demand. In consequence, the increment in metabolic cost upon warming will be less and, thus, the onset of thermal stress is alleviated and shifted to higher temperatures. Conversely, baseline costs will be lower at reduced capacity but the cost increment upon warming will be higher at a lower scope, thereby leading to an early limitation. As a corollary, demand, capacity, and supply are intertwined in a way that functional capacity co-defines the width of the thermal window and, thus, the degree of thermal specialization of a species. This is one basic reason for a wider thermal tolerance window in more active species and a narrower window in sessile, hypometabolic species (Pörtner, 2004, 2006). These relationships are adequately illustrated by the contrasting characteristics of temperate and Antarctic marine fauna. In Antarctic waters, the evolutionary pathways of temperature adaptation can be understood from two points of view: Firstly, animals are exposed to an excess of ambient oxygen at cold temperature, due to high physical solubility of oxygen in ambient water and body fluids. This leads to a larger oxygen reserve than available in warmer waters or body fluids. The expression of intracellular lipid diVusion pathways for oxygen through high mitochondrial densities and networks strengthens this trend even further (Sidell, 1998; Sidell and O’Brien, 2006). This indicates a ‘‘relaxed’’ situation with respect to the eVort and energy demand of oxygen transport to tissues, which in turn can be set to lower 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 151 capacity and cost. This ‘‘relaxed’’ situation is also mirrored in the loss of functional protein in the oxygen transport system and in mitochondria (for review see Sidell and O’Brien, 2006; Pörtner, 2006). The energetic relaxation associated with reduced oxygen supply requirements supports a reduction in whole organism energy turnover and vice versa. Also, the minimized level of cold compensation of molecular, cell, tissue, and whole organism functioning allows minimizing energy turnover even further, such that overall oxygen demand is low, as low as expected from a normal Q10 eVect on the metabolic rate of a temperate zone fish with similar lifestyles (Clarke and Johnston, 1999). Energy-saving lifestyles in fact typify polar fishes. However, while lower oxygen supply capacity means a lower maintenance cost at cold habitat temperature as a benefit, the trade-oV inherent to low capacity is a drastic increment in cost upon mild warming and, as a consequence, an earlier limitation of scope in oxygen supply upon warming. Specialization of polar fishes on high oxygen levels at low ventilatory and circulatory capacities, as well as reduced overall energy turnover, will constrain their capacity to compensate for temperature-induced increments in energy turnover and will thus cause an early hypoxemia and a narrowing of thermal windows. These considerations give access to understanding the decrease in growth as a consequence of falling aerobic scope. While growth does not fully exploit the scope of aerobic energy turnover it still relies on both excess substrate and energy availability at low baseline costs. Figure 4.4 illustrates this relationship for the Antarctic eelpout by showing that cellular costs are likely minimal at about 4–5 C where growth is maximal. This minimum leaves an excess amount of aerobic energy for maximum growth, at a temperature when not only cellular but also systemic costs are low. However, the cellular minimum is not visible in whole organism oxygen consumption. This may indicate excess energy use for growth. Above this minimum baseline cellular cost rises; at the same time, ventilation and circulation costs rise as well. All of this likely removes excess energy and substrate in competition with longterm aerobic functioning like growth. These considerations indicate that a ‘‘relaxed’’ situation with respect to substrate supply and energy demand is required for the fuelling of long-term aerobic processes and their support on top of standard metabolism. Low cost in oxygen supply, avoidance of hypoxemia, and optimum long-term aerobic performance thus go hand in hand. It remains to be established whether the resulting thermal optima are similar to those seen when the animals actively exploit their aerobic scope for exercise at maximum cost of oxygen supply and at the edge of muscular hypoxemia due to high oxygen demand. Temperature-induced limitations in aerobic scope for exercise may also bear ecosystem level consequences as is illustrated by available data on Pacific salmon (Oncorhynchus nerka) entering the Fraser river, BC, during 152 HANS O. PÖRTNER AND GISELA LANNIG Fig. 4.4. Growth within the thermal window of the Antarctic eelpout, Pachycara brachycephalum. As growth determinations occur in thermally acclimated animals, acclimation shifted maximal growth rate to 4 C, a temperature above ambient. Maximum growth occurs at the low end of an acute exponential rise in whole animal oxygen consumption and at lowest cellular costs associated with optimal oxygen supply. (Based on data from Mark et al., 2002, 2005; Lannig et al., 2005; Brodte et al., 2006.) their spawning migrations (Farrell et al., 2008). Water temperatures in the stream in relation to the temperature window of aerobic scope define whether the salmon will successfully migrate upstream and spawn. This is a special example where a crucial singular event in the life cycle of a species depends upon that maximum capacity for exercise and local climate conditions match. In general, reproduction and early development are processes that rely on supportive climate conditions for any species. Supportive climate conditions are those that reduce the threat of thermally induced hypoxemia and allow keeping baseline costs at a minimum as well as cost-eYcient exploitation of aerobic scope for growth or reproduction. 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 153 Longer lasting processes like larval development, growth, or foraging activity are equally crucial in maintaining fitness under prevailing climate conditions. In this case, ambient temperature needs to remain below pejus limit for a significant fraction of time to allow these long-term processes to proceed. Such quantitative relationships between fitness levels, widths of the thermal tolerance windows, and ambient temperature variability and sensitivity to thermally induced hypoxemia remain to be established. 2. TEMPERATURE ADAPTATION: ROLE OF HYPOXEMIA The narrow windows of thermal tolerance that characterize many Antarctic fishes appear as a consequence of their long-term history of permanent cold adaptation over millions of years at minimal risk of exposure to hypoxia or thermally induced hypoxemia. For some zoarcid and notothenioid species, however, a limited but still exploitable capacity to acclimate to warmer conditions (around 5 C) has recently become apparent (Seebacher et al., 2005; Lannig et al., 2005). This matches the finding of inducible heat tolerance in some notothenioid fishes (Podrabsky and Somero, 2006). Warm acclimation capacity is still present in some fishes, in contrast to the situation in many Antarctic invertebrates. The growth optimum of such fauna may lie, in fact, above ambient temperature, as seen in the Antarctic eelpout, Pachycara brachycephalum, with an optimum of 4–5 C (Brodte et al., 2006). It needs to be emphasized here that, due to the length of time involved, growth analyses at a specific temperature always comprise eVects of prior acclimation if it occurs. From the point of view of acclimation capacity to changing temperatures, some fishes in the Antarctic may currently live in a slow lane, considering their potential to accelerate life functions and live long-term in warmer areas. Antarctic marine invertebrates may not have conserved this apparent capacity (e.g., Pörtner et al., 1999a, b; Peck et al., 2004). In contrast to Antarctic fauna, temperate fauna and many Arctic or sub-Arctic species or Arctic populations of widely distributed Northern hemisphere species are exposed to more unstable climate and temperature conditions, due to the open nature of Arctic oceans. These species shift thermal windows between seasons and do so for the sake of energy eYciency and savings (Pörtner, 2006). The fact that thermal windows do not match all seasons suggests that the shift of thermal windows may occur in response to or at the verge of thermal stress and associated hypoxemia. Thermal acclimation capacity and thus the capacity to respond to or avoid hypoxemia may vary among species and species populations. According to available data, the degree of cold acclimation capacity is larger in (sub)-Arctic populations of fish than in their Southern-more con-specifics (Lannig et al., 2003; Lucassen 154 HANS O. PÖRTNER AND GISELA LANNIG et al., 2006; Lurman, 2008). During cold acclimation, an increase in mitochondrial density and capacity contributes to eliminate the capacity limitations of ventilation and circulation; however, the associated rise in metabolic costs enhances sensitivity to warm temperatures as a trade-oV, due to the more rapid loss in aerobic scope upon warming. This conjecture could recently be confirmed for Atlantic cod (Z. Zittier, pers. comm.). As a result, the thermal window, i.e., both upper and lower limits of thermal tolerance, are shifted to colder temperatures (Pörtner et al., 2008). 2.1. Systemic Signaling Responses Seasonal temperature change and latitudinal diVerences in the temperature regime are well known to be associated with compensation processes at the cellular level including adjustments in the density and functional properties of mitochondria (see Section 3). These changes are associated with the respective shifts in thermal tolerance windows (Pörtner, 2002, 2006). Studies of mitochondrial densities and functional properties in response to temperature change therefore have a long history in the study of thermal adaptation (for review, see Pörtner et al., 2000a; Guderley, 2004). Studying the regulation of these responses provides access to the regulation of shifts in thermal tolerance and temperature-dependent performance optima. Apart from temperature, exhaustive exercise or hypoxia also induce adjustments of mitochondrial densities and functions (Leary and Moyes, 2000; Hood, 2001). While exercise causes mitochondrial proliferation, hypoxia elicits a decrease of mitochondrial capacities in fish (Johnston and Bernard, 1982; van der Meer et al., 2005). The full range of eVector(s) triggering such adjustments still needs to be identified. The key role of whole organism physiology in setting thermal tolerance as well as the concept of oxygen and capacity limited thermal tolerance is suggestive with respect to mechanisms eVective in thermal adaptation. At the systemic level central signals would be crucial in coordinating acclimatory responses of individual tissues and cells to temperature. Eckerle et al. (2008) studied the response of hepatocytes isolated from cold or warm acclimated eelpout to subsequent warming or cooling. Warm exposure of hepatocytes from cold acclimated fish led to reduced activities of cytochrome c oxidase (COX) and citrate synthase (CS), whereas cold incubation of hepatocytes from warm acclimated fish did not yield any changes in enzyme activities. The observed lack of metabolic cold adaptation of aerobic enzyme capacities at the cellular level in vitro might be due to the lack of systemic signaling and oxygen limitation in isolated cells. These observations corroborate that insuYcient oxygen supply and associated systemic events as observed in marine ectotherms during acute temperature change might be a 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 155 key trigger for compensatory adjustments (see Pörtner 2001, 2002). These systemic signals could involve hypoxemia, but also include the endocrine system. According to recent data (Eckerle et al., 2008) eelpout exposed to cooling accumulate adenosine in plasma and tissues. Cold-induced adenosine accumulation in Z. viviparus persisted for at least 3 days in plasma and even longer in liver. This period was similar to the time course of cold compensation in liver with respect to changes in the levels of RNA message and activities of mitochondrial enzymes (Lucassen et al., 2003). Early temperature change is well known to elicit undershoot or overshoot responses in the rate of oxygen consumption as an early ‘‘shock’’ response to cooling or warming, respectively (Cossins and Bowler, 1987). This may occur as a consequence of delays in the functional adjustment of molecules, membranes, cells, and tissues, including oxygen supply systems to temperature, with the potential consequence of early and transiently more severe hypoxemia. In fact, adenosine accumulates in animal tissues in response to hypoxia or anoxia (Lutz and Kabler, 1997; Reipschläger et al., 1997; Renshaw et al., 2002), as a result of a mismatch between ATP production and use. It is released into the extracellular space and can act as a central signal causing metabolic depression (Buck, 2004). At the cellular level adenosine causes various eVects, including reduced protein synthesis (Tinton et al., 1995), stimulation of anaerobic glycolysis (Lutz and Nilsson, 1997), and a decrease in oxygen consumption as seen in trout hepatocytes (Krumschnabel et al., 2000). Similar energetic disequilibria would also be involved in situations causing a change in mitochondrial densities or capacities, like during exercise or hypoxia. Associated metabolic signals involved and discussed to elicit or modulate mitochondrial proliferation are Nitric Oxide (NO) (Nisoli et al., 2004) and, most recently, adenosine (Eckerle et al., 2008). Hypoxemia also elicits and exacerbates oxidative stress. In fact hypoxemia developing in the animal toward both ends of the thermal window likely contributes to the pattern of oxidative stress as seen in temperate and Antarctic eelpout (Heise et al., 2007). The level of oxidative stress in response to hypoxemia likely acts as a systemic signal suitable to elicit an adaptive cellular response at the level of most if not all tissues (Section 2.3). Temperature changes also elicit the release of the classic stress hormones, catecholamines and corticosteroids, which may then influence the process of thermal adaptation (Wendelaar Bonga, 1997). The onset of systemic hypoxemia (cf. Pörtner 2001, 2002) may well be involved in the temperatureinduced release of stress hormones. The short-term stress response comprises the rapid release of the catecholamines epinephrine and norepinephrine from their storage site in the chromaYn cells of the head kidney (Reid et al., 1998; Fabbri et al., 1998). They are also rapidly removed from the plasma thereafter. In addition, longer-term accumulation of cortisol occurs from 156 HANS O. PÖRTNER AND GISELA LANNIG the inter-renal cells of the head kidney (Mommsen et al., 1999). Acute cold exposure caused an accumulation of catecholamines and cortisol in tilapia (Chen et al., 2002), whereas cold acclimation reversed these changes (Perry and Reid, 1994; van Ham et al., 2003; Davis, 2004). The release of cortisol was slightly delayed compared to that of the catecholamines, but the rise in plasma cortisol levels was more prolonged (Chen et al., 2002). Daily infusions of cortisol for 1 week caused an increase in CS activities in the liver, brain, and muscle of catfish (Tripathi and Verma, 2003). Cortisol treatment of isolated eelpout hepatocytes increased the mRNA expression of CS and of the nuclear encoded, but not of the mitochondrial encoded COX subunit. Enzyme activities remained unaVected (Eckerle, 2008). This resembles the early phase of cold acclimation in Z. viviparus where enzyme activities also remained unchanged when the message was increased (Lucassen et al., 2003). Cortisol may thus be involved during induction of the cold acclimation process and may be released in response to thermally induced hypoxemia. Cold acclimation from 20 C to 5 C enhanced the sensitivity of heart and liver in rainbow trout in response to accumulated epinephrine (Keen et al., 1993; McKinley and Hazel, 1993, 2000; Aho and Vornanen, 2001, Shiels et al., 2003). The increase in sensitivity to epinephrine is supported by a higher number of b-adrenoreceptors, as seen in hepatocytes from cold acclimated trout (McKinley and Hazel, 2000). In temperate zone eelpout winter acclimatization of the animals prior to hepatocyte preparation also appeared to enhance sensitivity to epinephrine, arguing again for a seasonal pattern. Epinephrine treatment of hepatocytes isolated from fish in winter caused an increase in the activities of both CS and COX (Eckerle, 2008). Furthermore, thyroid hormones were shown to increase the activities of CS in several tissues of catfish (Tripathi and Verma, 2003) and of COX in mullet (LeRay et al., 1970). It is presently unclear how and where thyroid hormones might fit into the general picture of the regulation of temperature adaptation in poikilotherms. The overall impression is, however, that hypoxemia may play a key role in initiating temperature acclimation mechanisms in fishes. 2.2. Acid-Base and Ion Regulation While metabolic capacities are adjusted to the prevailing temperature conditions, similar processes would have to adjust cellular and epithelial mechanisms of ion and acid-base regulation (Pörtner et al., 1998). Acidbase regulation is an energy-dependent process since some of the acid-base equivalents are transported by Hþ-ATPases or by secondary active processes, for example via the Naþ/Hþ exchanger, which depends upon the Na-gradient established by Naþ/Kþ-ATPase. It has recently been suggested that certain species are capable of modulating the cost of acid-base 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 157 regulation as a means of adjusting the rate of energy turnover to environmental requirements such as temperature change and hypoxia. This can occur acutely, e.g., in response to shifting setpoints of extracellular pH (Pörtner et al., 2000b) or long-term, by modulating the densities and capacities of responsible ion exchange mechanisms (Pörtner et al., 1998). This requires consideration that the setpoints of pH are temperature dependent. As protein functional capacity is influenced by pH, adequate pH regulation at fluctuating temperature may be required to reduce the risk of performance decrements including temperature-dependent hypoxemia. As a comprehensive concept, Reeves (1972) introduced the imidazole alphastat hypothesis stating that pH regulation in poikilotherms maintains the degree of protonation (a) of imidazole groups in proteins despite changes in body temperature (cf. Burton, 2002). A pH change of around 0.018 pH units  C 1 matches a
pK  C 1 of 0.018 and is expected to support the alphastat pattern and to support pH-dependent protein function at fluctuating temperature. Cameron (1989) proposed a ‘‘Z-stat’’ model where protein net charge Z is maintained rather than a in a protein with diverse histidine groups. This concept takes the variability between
pK  C 1 into account, which depends upon local charge configurations in the environment of the imidazole group as well as on ionic strength and, therefore, ranges between 0.016 and 0.024 C 1 for histidine and free imidazole compounds and between 0.0010 and 0.051 C 1 for histidine residues in proteins (Heisler, 1986). Available data, which are more comprehensive for intracellular than extracellular acid-base status, indicate a trend in line with the concept of alphastat regulation (Ultsch and Jackson, 1996). However, the variability in slopes of
pH  C 1 is larger than expected from the variability in
pK  C 1. The alphastat pattern of intracellular pH regulation could be confirmed for marine ectotherms (invertebrates and fish) exposed to various temperatures both depending on the season or in a latitudinal cline suggesting that deviation from the alphastat pattern is involved in or results from metabolic depression (review by Pörtner et al., 1998; Sartoris et al., 2003). Metabolic depression is a typical response to hypoxemia, e.g., at the edges of the thermal window. Consideration of the window of oxygen and capacity limited thermal tolerance in fact revealed that the slope of the pH/temperature line is linear only between critical temperatures (Sommer et al., 1997). Acidotic deviations at temperatures beyond that are caused by the transition to proton-producing anaerobic metabolism (cf. Pörtner, 1987). Concomitantly, a shift in pH regulation may occur during hypoxemia when an acidosis is induced and contributes to metabolic depression. This would be the reason for long-term deviations from the alphastat pattern, as any short-term disturbance would otherwise be compensated for by ion exchange mechanisms. The respective data are scarce in ectotherms if they exist. In this 158 HANS O. PÖRTNER AND GISELA LANNIG context, the use of pH-stat rather than alphastat conditions is a matter of debate in hypothermic surgery on humans. Compared to the elevation of pH according to alphastat the application of pH-stat perfusate to cooling tissue is equivalent to acidotic exposure and metabolic depression (Ohkura et al., 2004; Li et al., 2004). During surgery under severe hypothermia, further benefits of pH-stat include a relative rightward shift of the hemoglobin oxygen binding curve thereby supporting oxygen delivery. pH-stat causes increased cerebral blood flow and volume, associated with enhanced oxygen availability during circulatory arrest and a greater suppression of cerebral metabolic rate. In water breathers, the observed changes in pH with temperature are mostly elicited by non-respiratory mechanisms. A passive component is due to proton binding or release from intra- and extracellular buVers owing to the change in dissociation equilibria (pK-values) of the buVer components. In contrast to air breathers, active control of pH by means of ventilatory PCO2 adjustments is minimal; active ion exchange mechanisms predominate. In some species the passive contribution to pH regulation was found to be considerably below the alphastat value seen in vivo (van Dijk et al., 1997; Sartoris and Pörtner, 1997). The passive contribution accounted for only 35% of the temperature-induced pH shift in white muscle of the temperate eelpout, whereas it was close to 100% in the Antarctic eelpout. Lower passive pH shifts would lead to more acidic pH values in the cold and leave a larger contribution to ion exchange mechanisms to accomplish alphastat pH regulation. In general, the active component was larger in eurythermal than in stenothermal species (Pörtner and Sartoris, 1999). This indicates that energy savings in Antarctic stenotherms comprise a reduction in the level of active acid-base regulation. At the same time, living at high temperature variability includes the option to use the non-alphastat pH slope. This relative acidification would support metabolic depression as elicited by extreme temperature-induced hypoxemia (see above). In this case, large passive slopes would require active pH regulation to compensate for their eVect when more acidic pH values are to be maintained. In contrast, a low passive slope allows flexible adjustments of pH according to metabolic requirements. In the warmth this may be involved in metabolic depression as seen in freshwater burbot, Lota lota, during summer (Hardewig et al., 2004; Figure 4.1). On the other side of the temperature spectrum, animals living in large seasonal temperature variations frequently exhibit low pH values at low temperatures in the winter (Thebault and RaYn, 1991; Spicer et al, 1994). The shrimp Palaemon tends to be inactive at temperatures below 10 C, metabolic depression being reflected by a drop in intracellular pH below the alphastat pattern (Thebault & RaYn, 1991). Acidic pHi values were also reported by Whiteley et al. (1995) for winter crayfish, Austrapotamobius pallipes. One 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 159 might speculate that a capacity for metabolic depression in eurythermal animals is correlated with a reduced contribution of passive mechanisms to pH adjustment during temperature change. As a corollary, a larger active than passive component of alphastat regulation may not only be a prerequisite to colonize shallow coastal waters but may also allow for a variable adjustment of metabolic activity on a seasonal time scale and support flexible response to more frequent exposure of thermally induced hypoxemia under variable temperature conditions. 2.3. Hypoxemia-Related Cellular Stress and Signaling Temperature-induced metabolic adjustments at the cellular level are key to the maintenance of functioning and cellular energetics and thus survival of the organism. They are most crucial in shifts of the position and width of the thermal tolerance window on the temperature scale. Long-term compensatory adjustments in aerobic metabolism contribute to balance the temperature impact on metabolic processes and to support a new steady state in energy metabolism (see Section 3). For detailed information on the consequences of hypoxia/anoxia see Chapters 9 and 10. Regulation of thermal adaptation at the cellular level is likely strongly influenced and may even depend upon the response to systemic hypoxemia. The patterns of systemic signaling and oxidative stress in response to temperature-induced hypoxemia indicate a common response following temperature change (Figure 4.1). Both heat and cold exposure, in particular during the recovery phase at control temperatures, led to elevated oxidative stress parameters in hepatic tissue of Z. viviparus (Heise et al., 2006a,b), confirming that hypoxemia is suitable to elicit or at least exacerbate onset and eVects of oxidative stress. Excess ROS production in marine ectotherms may cause cellular damage (Abele et al., 1998, 2001) and impair cellular functioning (Chabi et al., 2008). Antioxidative defence may thus play an important role in setting passive tolerance to temperature extremes (Pörtner, 2002). Polar ectotherms are thought to be more vulnerable to cellular ROS production during warming than temperate ectotherms as their membranes rich in polyunsaturated fatty acids are easy targets for lipid radical formation (Brand et al., 1991; for review see Abele and Puntarulo, 2004). Strong antioxidative defence, for example, through vitamin E and specific derivatives is thus expressed in Antarctic fish (Dunlap et al., 2002; Heise et al., 2007). Oxidative stress on both sides of the thermal window may also play a role in shaping the pattern of thermal acclimation. In this context the activation of the hypoxia inducible transcription factor (HIF) as observed during temperature change in fish (Heise et al., 2006a,b, 2007) supports cellular and systemic stress resistance during temperature-induced oxygen shortage 160 HANS O. PÖRTNER AND GISELA LANNIG (Figure 4.5). At high temperatures hypoxic signaling and subsequent metabolic reorganization to counterbalance thermal oxygen limitation seems to be eVective only in the pejus temperature range, while it appears impaired at critical and even higher temperatures (cf. Figure 4.1). A strong reduction of the cellular redox potential (or a reduced glutathione redox ratio, 2GSSG/GSH) as A 16 Cold 14 12 Warm HIF-1 DNA binding activity (EMSA signal intensity, %) 10 8 6 4 2 0 Ctrl 18°C Recov. (12°C) 5 °C Recovery B 18 16 14 12 10 8 6 p < 0.05 4 2 0 0 −230 −240 −250 −260 −270 −280 Redox potential ΔE (mV) −290 Fig. 4.5. (A) Increased HIF-1 DNA binding in liver at extreme temperatures und during recovery from both cold and heat exposure. (B) Linear regression demonstrating increased HIF-1 DNA binding at a more reduced redox potential (i.e., a more reduced glutathione redox ratio in liver samples from Zoarces viviparus. (Data from Heise et al., 2006a,b.) 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 161 during severe hypoxia supports the binding activity of HIF (Figure 4.5). At present the diVerentiation between low and high temperatures with respect to the hypoxemia induced involvement of HIF is not clear. However, HIF-1 DNA binding activity was significantly higher at cold temperature, both in the polar and the temperate eelpout species, and when comparing winter and summer in temperate eelpout. In both the Antarctic cold and seasonal cold, the glutathione redox ratio was more oxidized when compared to the warmer condition. This indicates that HIF-1 might stimulate cold compensation mechanisms, but might operate diVerently in the warmth (Heise et al., 2007). HIF-1 could thus be involved in regulating the adjustment of tissue oxygenation at the border of the thermal envelope of a fish but no longer at more extreme temperatures. The heat shock response has long been studied in fish and in relation to temperature extremes (for review see Iwama et al., 1998, 2006). Comprehensive evidence on how the heat shock response keys into the general picture of oxygen limited thermal tolerance is not available for fish but comes from a comparative study on two Mediterranean bivalve species, the bearded mussel, Modiolus barbatus, and the Mediterranean mussel, Mytilus galloprovincialis (Anestis et al., 2007, 2008). Modiolus barbatus lives at depths between 8 and 15 m, experiences temperatures up to 21 C and M. galloprovincialis lives at depths between 0.5 and 5 m and at temperatures up to 26–28 C. At first sight it is puzzling that mortality sets in at about the same temperature of 26 C but is 20-fold higher in M. galloprovincialis at 26 C and more than 80-fold higher at 30 C than in M. barbatus. These apparently contradictory findings are resolved when considering that the heat shock response in M. barbatus sets in beyond 22 C and in M. galloprovincialis only beyond 26 C. Interestingly, M. barbaratus also displays a larger capacity to undergo metabolic depression than M. galloprovincialis. These findings would suggest an earlier limitation of aerobic scope in M. barbatus than in M. galloprovincialis. Extreme hypoxemia and anaerobiosis may also set in early and contribute to elicit the earlier onset of the heat shock response (Pörtner, 2002). However, the distribution of the two species clearly shows that the capacities to undergo metabolic depression and use the heat shock response do not a priori and exclusively define temperature-dependent distribution and heat-induced mortality. Mediterranean mussels (M. galloprovincialis), which regularly encounter water temperatures higher than 25 C, live near their incipient lethal temperature. Their extended aerobic range combined with their delayed and limited (compared to M. barbaratus) depression of metabolic rate likely support active survival and fitness at warmer temperatures. The earlier onset of the heat shock response in M. barbatus than in M. galloprovincialis likely mirrors an earlier loss in aerobic scope and onset of hypoxemia in the bearded mussel. This may precondition M. barbatus to 162 HANS O. PÖRTNER AND GISELA LANNIG passively tolerate more extreme temperatures than M. galloprovincialis, with the result of better passive survival, i.e., lower mortality of M. barbatus at extreme temperatures. At temperatures beyond 23 C the bearded mussel experiences constrained aerobic scope and metabolic depression, which will impair relevant physiological processes such as growth and reproduction and prevent long-term successful settling of shallower, warmer waters by this species. These data are in line with the systemic to molecular hierarchy of thermal tolerance postulated earlier where the heat shock response is interpreted to shape passive tolerance to thermal extremes, a feature highly relevant in intertidal organisms (Pörtner, 2002). The conclusions drawn for the mussels fully match those drawn for marine fish populations. In fact, abundance of eelpout in the German Wadden Sea begins to decline as soon as aerobic scope for growth is reduced (Pörtner and Knust, 2007; Wang and Overgaard, 2007). This occurs at the upper thermal limits of acclimation capacity. Maintenance of aerobic scope is thus most crucial for long-term survival of extreme temperature conditions in the field (cf. Pörtner and Knust, 2007). This also emphasizes that interpretation of laboratory data on tolerance benefits from consideration of a background of field data. 3. CELLULAR MECHANISMS OF THERMAL ADAPTATION In the following, we will focus in more detail on how temperatureinduced hypoxemia may influence temperature-related compensatory aspects in cell metabolism such as changes in mitochondrial capacities, in membrane structure, and in energy turnover and on the consequences for thermal tolerance. We will discuss temperature-dependent impact on cardiac calcium homeostasis as calcium is of great importance for cardiac performance, and may thus play an important role in setting windows of thermal tolerance in fish. All of these mechanisms will be interpreted in the light of how they might support the organism to overcome the threat of temperature-induced hypoxemia. 3.1. Capacity and EYciency of Mitochondria Temperature was shown to greatly aVect mitochondrial and enzymatic capacities and thermal plasticity of these parameters/factors was suggested to indicate thermal adaptation (DahlhoV and Somero, 1993; Weinstein and Somero, 1998; Pörtner et al., 1999a). Temperature adaptation of aerobic scope includes the adjustment of the scope of mitochondrial energy production and of the associated substrate oxidation capacity of mitochondria. 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 163 A large number of studies found mitochondrial capacities to fall with decreasing temperature, with the lowest capacities found at high mitochondrial densities in Antarctic fishes. Up-regulation of capacity seems to be restricted to cold exposed eurythermal species (for review see, e.g., Pörtner et al., 2000a, 2005b; Guderley and St-Pierre, 2002; Guderley, 2004; and references therein). ‘‘Increasing the volume and surface density of mitochondrial clusters is the primary mechanism’’ of supporting the aerobic capacity of muscle in cold-adapted Antarctic species (Johnston et al., 1994, 1998). In liver, cold compensation is mainly accomplished by a rise in tissue mass leaving mitochondrial protein per gram liver largely unchanged (Kent et al., 1988; Seddon and Prosser, 1997; Lannig et al., 2003, 2005). Enzyme activities were found increased in cold-acclimated and cold-adapted tissues, depending on enzyme and tissue (e.g., Guderley and Blier, 1988; Crockett and Sidell, 1990; Lannig et al., 2003, 2005; Kawall et al., 2002; Lucassen et al., 2003). Changes in the activities of membrane-bound enzymes are partly induced by changes of membrane structure (see Guderley and St-Pierre, 2002; Pörtner et al., 2005b; and below). The temperature dependence of mitochondrial functions most likely depends more on the integrity of mitochondrial membrane and membrane protein interactions than on protein stability per se (White and Somero, 1982; Guderley et al., 2008; see Section 3.2). The stable environments of the marine Antarctic supported extreme thermal specialization of its marine inhabitants (for review see Pörtner, 2006 and references therein). Accordingly, Antarctic fishes were regarded as being restricted in their ability to respond to temperature variation. However, Antarctic fish species maintained—at least to some degree—thermal plasticity of metabolic processes and whole animal performance as demonstrated for a zoarcid (Lannig et al., 2005, Brodte et al., 2006) and for a notothenioid (Seebacher et al., 2005). It is presently unclear how their mitochondrial functions are modified to support life in the ‘‘warmth.’’ In temperate fish warm acclimation goes hand in hand with reduced mitochondrial capacities (for review see Pörtner et al., 2005b). Warm-acclimated Antarctic eelpout, P. brachycephalum (Lannig et al., 2005) displayed unchanged capacities per milligram mitochondrial protein in liver and showed a clear reduction in mitochondrial capacities only at the whole organ level due to decreased liver size after long-term warm acclimation (5 C versus 0 C). Thus, in contrast to the finding of increased standard metabolic rate during acute warming (van Dijk et al., 1999; Mark et al., 2002) warm acclimated P. brachycephalum displayed a metabolic rate similar to coldacclimated specimens when measured at the respective acclimation temperature (0–6 C) (Brodte et al., 2006; Lannig G., unpublished data). Taken together, these findings indicate complete metabolic warm compensation due to a reduction in maintenance costs in the Antarctic eelpout. 164 HANS O. PÖRTNER AND GISELA LANNIG The more active Antarctic fish, Pagothenia borchgrevinki ‘‘displayed astonishing plasticity in cardiovascular response and metabolic control’’ to maintain locomotory performance at elevated temperatures (Seebacher et al., 2005). After 4–5 weeks of warm acclimation (4 C versus 1 C) activity of muscle lactate dehydrogenase and cytochrome c oxidase was significantly elevated indicating an up-regulation of enzymes involved in both anaerobic and aerobic energy production. Interestingly, the activities of other glycolytic and TCA cycle enzymes, like phosphofructokinase and citrate synthase, did not diVer in muscle tissue of cold- and warm-acclimated animals. The authors discussed the observed up-regulation of enzyme activities as a compensatory response to meet elevated maintenance costs at increased temperatures (Seebacher et al., 2005). They did not determine standard metabolic rate; however, in contrast to the respective observations in the eelpout, the enzymatic results indicate increased and thus uncompensated standard metabolism following warm acclimation of P. borchgrevinki. This assumption appears reasonable as, for comparison, reduced CS and unchanged COX activities in white muscle of North Sea cod, Gadus morhua, following warm acclimation (Lannig et al., 2003) were associated with similar standard metabolic rates in warm- and coldacclimated specimens, measured at the respective acclimation temperatures (Fischer, 2003). As a corollary, the polar zoarcid, P. brachycephalum, and the notothenioid, P. borchgrevinki, diVered in their compensatory response: the former reduced while the latter elevated metabolic rate upon warm acclimation. Both strategies involve successful avoidance of temperature-induced hypoxemia and likely shifted or expanded the thermal window toward higher temperatures. This conjecture, however, warrants further investigation. Mitochondrial proliferation in the cold compensates for the suppressing eVect of low temperature on metabolic and diVusion pathways (Tyler and Sidell, 1984; Egginton and Sidell, 1989; Egginton et al., 2002; O’Brien et al., 2003). In temperate zone species, mitochondrial proliferation causes a rise in cellular energy costs due to increased proton leak rates. The term proton leak describes an inherent proton permeability of the inner mitochondrial membrane. The futile cycle of proton pump and leak occurs without ATP production and can cover 25–50% of standard metabolism in endo- and ectothermal organisms (Brand et al., 1994; Rolfe and Brand, 1996; Brookes et al., 1998). Guderley and St-Pierre (2002) summarized the diVerent mitochondrial strategies of water breathers to cope with cold temperatures and emphasized the key role of proton leak in the regulation of respiratory capacity. In Antarctic species the finding of a high Arrhenius activation energy (Ea) for proton leak rates was thought to minimize dissipative proton flux despite high mitochondrial densities (Hardewig et al., 1999; Pörtner et al., 2000a). A high thermal response of proton leak rates results in Antarctic organisms. In consequence, a mismatch between aerobic ATP production 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 165 and demand and thus the need for complementary anaerobic ATP production will develop over a narrower temperature range than in eurytherms. The advantage of reduced energy costs for maintenance thereby contributes to an early loss in aerobic scope and thus reduced heat tolerance (Pörtner, 2006). These cellular phenomena complement the whole organism trade-oVs in energy budget described above (see Section 1.2). In this context the roles of uncoupling proteins (UCPs) and their adjustment with cold or warm acclimation and adaptation require consideration. UCPs are located in the inner mitochondrial membrane and uncouple ADP phosphorylation from substrate oxidation by increasing transmembrane proton conductance resulting in the short-circuiting of the redox reaction (Klingenberg et al., 2001). In endothermic mammals UCP1 is used in heat production (Klingenberg and Huang, 1999; Stuart et al., 1999a). UCP homologs (UCP2-5) found in ectothermic organisms suggest further functions unrelated to thermogenesis (Brand et al., 1999; Stuart et al., 1999b; Sokolova and Sokolov, 2005; Mark et al., 2006). To date three mechanisms are discussed: (1) involvement in fatty acid oxidation (Fleury et al., 1997; Samec et al., 1998; Ricquier and Bouillaud, 2000); (2) suppression of oxidative stress (Echtay et al., 2002; Liang et al., 2003; Mark et al., 2006); and (3) facilitation of metabolic flux by futile cycling (Mark et al., 2006). The production of reactive oxygen species (ROS) is significantly increased by mitochondrial electron transport when the proton electrochemical gradient (PEG) across the inner mitochondrial membrane is high. This correlation is explained by a putative feedback loop formed by a PEG-dependent inhibition of further electron flow down the electron transport chain, associated with situations of insuYcient ADP availability or reduced activity of ATP synthase. Mark et al. (2006) observed increased UCP2 expression in two zoarcid species, Z. viviparus (North Sea) and P. brachycephalum (Antarctic) upon cooling or warming, respectively. Nonetheless, mitochondrial proton leak rates remained unchanged (Lannig et al., 2005). Mark et al. (2006) suggested that UCP might balance both ATP turnover and ROS formation by controlling the mitochondrial membrane potential. Furthermore, UCP expression paralleled the increased HIF activity upon warming of the Antarctic eelpout and cooling of the temperate eelpout (Heise et al., 2007). Both may thus play a role in controlling tissue oxygenation, metabolic capacity, and oxidative stress. Further work is necessary to evaluate these hypotheses. 3.2. Membrane Structure: Functional Implications and Costs Membranes play a central role in temperature adaptation (White and Somero, 1982) as the fluidity of the membrane lipid bilayer responds immediately to temperature change and can seriously impair physiological 166 HANS O. PÖRTNER AND GISELA LANNIG function (Hazel, 1988; Hazel and Williams, 1990). Membrane-mediated processes such as ion and acid-base regulation depend on the maintenance of membrane structure and fluidity. When measured at a common temperature, membrane fluidity was found to decrease with increasing body temperature (see Hazel and Williams, 1990; and references therein). Membrane fluidity was highest in Antarctic fish and decreased in the following order: Antarctic fish ( 1 C) > Arctic fish (0 C) > goldfish (5 C) > goldfish (25 C) > pupfish (34 C) > rat (37 C), and is thereby related to thermal windows. When measured at the respective body temperature membrane fluidity was similar among animals, with slightly elevated values in the ‘‘warm animals’’ (homeoviscous adaptation, see Sinensky, 1974; Cossins et al., 1981; Hazel and Williams, 1990). In both cold-acclimated and cold-adapted animals cold exposure initiates a rise in the content of unsaturated fatty acids and in the number of double bonds to oVset the negative eVect of low temperatures on membrane fluidity (Guderley et al., 1997; Bock et al., 2001; for review see, e.g., Wodtke, 1981; Hazel and Williams, 1990; Cossins, 1994). In addition to homeoviscous adaptation McElhaney (1984a, b) introduced the term homeophasic adaptation, which refers to alterations in the lipid phase state after thermal compensation of membrane functioning has been observed in the absence of homeoviscous adaptation and vice versa (for more detail see Hazel and Williams 1990; Hazel, 1995). The level of membrane fluidity and enzyme activity of membrane-associated proteins, such as Naþ/Kþ or Ca2þ-ATPases are strongly correlated indicating that the surrounding lipid milieu is of great importance for enzymatic performance (Hazel, 1972; Cossins et al., 1981; Hazel and Williams, 1990; for review see Hoch, 1992). Mitochondrial membranes seem to exhibit stronger temperature-induced modification than membranes of other subcellular compartments (Cossins and Prosser, 1982; Hazel and Williams, 1990). As shown by Guderley et al. (2008) temperature-dependent mitochondrial respiration rates are strongly aVected by membrane fatty acid composition. Thermal impact on mitochondrial capacities was shown to diVer among fish fed with diets of diVerent fatty acid composition. Alterations in membrane composition, particularly in the degree of unsaturation of membrane lipids, might be associated with shifts in temperature-dependent breaks in Arrhenius plots of biochemical processes (known as Arrhenius Break Temperature, ABT). Following cold acclimation lower ABTs were determined e.g., for succinate oxidation by liver mitochondria of carp, Cyprinus carpio (Wodtke, 1976) or for Naþ/Kþ-ATPase activity in gill of eel, Anguilla anguilla (Thomson et al., 1977). Furthermore, ABTs of mitochondrial respiration rates, mainly observed for uncoupled respiration, correlated with the natural habitat temperature of the organism. Lower ABTs, albeit far above habitat temperature, were found in polar species (Weinstein and Somero, 1998). 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 167 Accordingly, the temperature-induced impairment and thus thermal sensitivity of cellular functioning is aVected by membrane composition. Hypoxemia may play a role or exacerbate these relationships by enhancing the level of oxidative stress and its impact on membrane structure. As changes in membrane properties occur without central input (Pearson et al., 1999) and in isolated cells (Koban, 1986; Tsugawa and Lagerspetz, 1990), membrane alterations seem, at least in part, to be regulated by cellular temperature alone. Response times diVer between warm- and cold-induced metabolic adjustments at least for membrane modification (see below). Short-term adjustments occur between 8 and 24 h and refer to rapid and mostly nonmaintained changes such as alterations in headgroup composition of phospholipids. Long-term adjustments occur more slowly (over days) and are considered responsible for the observed diVerences in membranes from ‘‘cold and warm’’ animals, e.g., diVerent levels of polyunsaturated fatty acids. The direction of thermal acclimation aVects the duration of longterm adjustments from 1 week during warm acclimation to several weeks during cold acclimation (for review see Cossins and Raynard, 1987; Hazel and Williams, 1990). The eYciency of homeoviscous adaptation varies with cell/organelle type and metabolic performance in a way that the degree of fluidity compensation was shown to be highest in mitochondria and decreased in the following order: mitochondria > synaptosomes > myelin (Cossins and Prosser, 1982). The observed heterogeneous response dependent on membrane type as well as the observed slightly lower eYciency of homeoviscous adaptation in cold-acclimated versus cold-adapted organisms led to the suggestion that ‘‘either the costs of perfect compensation are too high or the benefits too low to warrant such a pattern of adaptation‘‘ (see Hazel and Williams, 1990). Increased ratios of polyunsaturated over monounsaturated fatty acids result in increased molecular activity of membrane proteins. Hulbert and Else (1999) proposed that membrane acyl chain composition can therefore act as a pacemaker for standard metabolism since most of the processes relevant for metabolism such as ion pumps, proton leak, protein synthesis, or oxidative phosphorylation are carried out by membrane-bound systems. In fact, Pernet et al. (2008) showed that growth rates are highest with a reduction in standard metabolism and membrane unsaturation index. In the light of minimized proton leak rates in Antarctic animals we propose that this mechanism contributed to enable cold-adapted Antarctic stenotherms to benefit from a highly eYcient homeoviscous adaptation without concomitant increments in energy costs. This contributes to lower standard metabolic rates than observed in cold-adapted eurytherms. Higher growth rates seen in Antarctic than in Arctic zoarcids (Brodte et al., 2006) would be in line with these considerations. 168 HANS O. PÖRTNER AND GISELA LANNIG 3.3. Calcium Homeostasis and Functioning Calcium (Ca2þ) participates in numerous biochemical and physiological processes and adopts a central role in biological systems. It plays a key role in cardiac contraction and relaxation (Bers, 2002), which are critical in thermal tolerance due to their importance in oxygen delivery to tissues. In contrast to mammals where calcium is released from sarcoplasmic reticulum (SR Ca2þ release), cardiac function in fish strongly depends on extracellular calcium influx via sarcolemmal L-type calcium channels (SL Ca2þ influx) and Naþ/ Ca2þ exchange (Vornanen, 1997; 1999; Hove-Madsen and Tort, 1998; for review see Tibbits et al., 1992; Farrell, 1996; Lillywhite et al., 1999). However, the partitioning between intracellular versus extracellular Ca2þ handling for cardiac contraction seems to depend on the fish’s lifestyle. In more active fishes such as tuna or trout SR Ca2þ stores were shown to be more important (Shiels et al., 1999; Brill and Bushnell, 2001; Landeira-Fernandez et al., 2004). High rates of cardiac SR Ca2þ-ATPase activity and of SR Ca2þ uptake in tunas indicate ‘‘an important evolutionary step for the maintenance of higher heart rates . . .in bluefin tuna’’ (Castilho et al., 2007). Furthermore, sources of calcium appear to be diVerent between ventricular and atrial myocytes. In ventricular stripes of rainbow trout, O. mykiss, Keen et al. (1994) observed no contribution of SR Ca2þ-release channel in beat-to-beat regulation of cardiac contractility at routine heart rate (>0.6 Hz). In contrast, Aho and Vornanen (1999) observed significant ryanodine sensitivity (ryanodine = a specific and potent inhibitor of SR Ca2þ release, Rousseau et al., 1987) in trout atrium at physiological pacing rate. Inadequate calcium regulation during temperature change would lead to impaired cardiac performance followed by limited oxygen availability at the tissue level finally resulting in hypoxemia (see also Chapter 7). Thus several studies proposed a temperature-dependent alteration in the interplay between SL Ca2þ flux and SR Ca2þ flux to maintain adequate calcium levels to maintain cardiac performance during temperature change. At high temperatures significant ryanodine-induced impairment in cardiac performance was observed in rainbow trout, O. mykiss, at 15, 20, and 22 C (ventricular; HoveMadsen, 1992; Shiels and Farrell, 1997) and in skipjack tuna, Katsuwonus pelamis, at 25 C (atrial; Keen et al., 1992). In contrast, no ryanodine sensitivity was observed at low temperatures in ventricular myocytes of rainbow trout suggesting that cardiac contraction does not depend on SR Ca2þ release in the cold (Keen et al., 1994). In general, ryanodine insensitivity in trout ventricular cells was observed at temperatures below 15 C and might be linked to the thermal response of the SR Ca2þ-release channels, which tend to remain open at low temperatures (Sitsapesan et al., 1991; Hove-Madsen, 1992; Keen et al., 1992; Gesser, 1996; Shiels and Farrell, 1997). Interestingly, cold acclimation 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 169 did not change this situation. No eVect of ryanodine was observed at low compared to high test temperatures indicating that SR Ca2þ-release channels were expressed but played no significant role in ventricular contractility of trout at low temperatures (Keen et al., 1994; Vornanen, 1996). However, a significant contribution of SR Ca2þ flux to contractility following cold acclimation to 4 C was found at low temperatures in trout atrial myocytes (Aho and Vornanen, 1999; see also Gesser, 1996; Shiels et al., 1999). Furthermore, cold-induced compensation of Ca2þ handling capacity of the sarcoplasmic reticulum (SR) was also suggested for the ventricle of rainbow trout, O. mykiss (Keen et al., 1994; Aho and Vornanen, 1998). When measured at the same temperature Aho and Vornanen (1998) observed higher rates of SR Ca2þ uptake in ventricular homogenates of cold- compared to warm-acclimated fish indicating that SR compensated its capacity for calcium load in the cold, while ‘‘the Ca2þ release channels are not leaky in the cold.’’ In contrast to the suggested complete thermal compensation in ventricular SR Ca2þ uptake rate in trout, crucian carp showed reduced SR Ca2þ uptake rates at unchanged ryanodine sensitivity following cold acclimation (Vornanen, 1996; Aho and Vornanen, 1998). The authors linked the observed diVerence in SR Ca2þ sequestration to the life styles of the species. Fishes that remain rather active and need adequate cardiac functioning in the cold have higher Ca2þ-handling capacity in cardiac (ventricular) SR than less active and cold-dormant species like carp. Cold temperate species such as the burbot, Lota lota, showed significant cold-induced ryanodine sensitivity through a ryanodine-induced reduction of maximum cardiac force by 32  8% in atrial and by 16  3% in ventricular preparations when measured at 1 C (Tiitu and Vornanen, 2002a). This indicates that SR Ca2þ release is significantly involved in delivering calcium for cardiac contraction, thereby supporting active performance and associated cold tolerance in this cold-adapted species. SR Ca2þ release channels are modified in a way to oVset the observed increase in the open probability of the channels with acute cooling (Bers, 1987; Sitsapesan et al., 1991). Furthermore, Ca2þ-dependent activation of the ventricular SR Ca2þ release channels was found to similar degrees in burbot and rat suggesting that Ca2þ-induced Ca2þ release (CICR) is involved during excitation-contraction coupling in this cold-adapted fish (Vornanen, 2006). This process, however, is controversial because Shiels and coauthors (Shiels et al., 2006) suggested for the same species that Ca2þ is mainly provided via Na2þ/Ca2þ exchange (NCX) rather than via CICR. Furthermore, sarcolemmal Ca2þ flux also displayed cold compensation through elevated surface to volume ratios of smaller cardiac myocytes of burbot compared to larger cells of rainbow trout, resulting in reduced diVusion distances between SL and myofilaments (Tiitu and Vornanen, 2002b). 170 HANS O. PÖRTNER AND GISELA LANNIG Shiels et al. (2000) found similar thermal responses of SL Ca2þ influx and a Q10 of around 2 in mammals and fishes, suggesting that SR Ca2þ release or another mechanism may support SL Ca2þ flux to ensure cardiac functioning during temperature change and compensate for cold exposure. When the authors excluded SR Ca2þ flux by adding ryanodine they could show that in physiological situations such as during appropriate action potential waveforms and at the respective test temperatures, SL Ca2þ influx did not change with temperature in atrial myocytes of rainbow trout, O. mykiss. Temperature-induced modifications in the shape of APs that were proposed to be linked to the temperature-dependent expression of sarcolemmal Kþ channels (Vornanen et al., 2002), may contribute to maintain calcium homeostasis during an acute temperature change and may oVset the otherwise Q10-dependent decrease in SL Ca2þ influx (Shiels et al., 2000). In a consecutive study, the authors showed that the temperature dependence of SR Ca2þ cycling was also mediated by relevant stimulation via shape and frequency of action potentials (Shiels et al., 2002). Shape and frequency of action potentials thus appear to coordinate the role of SL and SR Ca2þ fluxes in cardiac contraction. Furthermore, hormones such as adrenaline, in particular, mediate SL and SR Ca2þ flux during temperature change. Several studies revealed the importance of adrenaline for calcium-dependent cardiac performance (Gesser, 1996; Shiels and Farrell, 1997; Rocha et al., 2007). Shiels and coauthors (2003) observed significant temperature-dependent sensitivity to adrenergic stimulation of sarcolemmal Ca2þ flux through the L-type Ca2þ channel. The response to adrenaline increased at decreasing test temperature. As an acute temperature decrease suppresses SL Ca2þ flux (see above) limited cardiac functioning will lead to hypoxemia in the cold in vivo if no rapid compensatory response sets in. This may contribute to the cold-induced undershoot phenomenon. Rapid compensation can be achieved through hormonal stimulation and reveals the importance of hormonal signaling to support cardiac performance during acute temperature stress in vivo and ameliorate the temperature-dependent alteration in Ca2þ flux (see Farrell, 1996). Long-term adjustments in calcium regulation through alterations in gene expression will oVset the temperature impact on calcium fluxes and support adequate cardiac performance and thus oxygen supply to tissues in a shifted thermal window. This may, however, involve a shift in the fractional energy demand of calcium signaling and homeostasis. All of these relationships remain largely unexplored. 3.4. Energy Budget, Turnover, and Allocation In general, the cellular as well as the organismal energy budget (as the sum of all cellular budgets) provides excess energy to growth and other functions only once the energy demand of maintenance and baseline 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 171 functioning of the organism is met (Wieser, 1994). Reductions in functional scope will occur once the scope for aerobic ATP supply via oxidative phosphorylation is reduced during temperature-induced hypoxemia. Vital cell functions were proposed to display a lower sensitivity to reduced ATP supply than accessory ones (Atkinson, 1977). Shifts in energy allocation to cellular processes might result, which are usually studied using respiration rates of isolated cells under the eVect of specific inhibitors. However, this methodological approach bears its risks and may only support qualitative conclusions (Mark et al., 2005). The various O2-consuming processes influence each other upon inhibition. Respiration was also shown to strongly depend on the concentrations of inhibitors as well as on the previous feeding regime of the experimental animals (see Krumschnabel and Wieser, 1994, Krumschnabel et al., 1997; Wieser and Krumschnabel, 2001). Interpretation of the respective findings and comparisons between studies should be carried out with adequate precaution. We confine this paragraph mainly to the two most prominent ATPconsumers of cells: Naþ/Kþ-ATPase and protein synthesis. Protein turnover and ion-motive ATPases represent key targets of hypoxia causing energy reallocation at the cellular level (Boutilier, 2001). This has implications for temperatures beyond the optimum range when thermally induced hypoxemia sets in. Furthermore, passive ion flux and active Kþ uptake via Naþ/KþATPase display largely diVerent kinetic responses to temperature. Active ion exchange displays a Q10 of 2–4 while passive ion flux is rather insensitive to temperature (Ellory and Hall, 1987; Gibbs, 1995). To overcome thermal disturbance of the coupling between active Kþ uptake and passive Kþ eZux two strategies have evolved at the cellular level: compensatory adjustment either of the passive Kþ leaks or of the active Naþ/Kþ-ATPase capacities and associated secondary active processes such as Naþ/Kþ/Cl exchanger that depend on ion gradients, respectively. The underlying mechanisms for the observed changes are still under debate. Temperatureinduced changes in Kþ eZux are thought to include: (1) alterations in membrane properties (homeoviscous adaptation, see Section 3.2); (2) down-regulation of ion channels as observed during environmental hypoxia (Péréz-Pinzón et al., 1992); and/or (3) changes in Kþ channel opening through changing concentrations of metabolite ligands (Dunne and Petersen, 1991; Hall and Willis, 1984). In Antarctic fishes with their increased serum osmolality, cost reductions by both the down-regulation of ion channels and reduced ion exchange capacities were suggested to contribute to the observed low rate of standard metabolism (Gonzalez-Cabrera et al., 1995; Pörtner et al., 1998; Guynn et al., 2002). Benefits are increased freezing resistance and decreased energy requirements to maintain the ionic gradient (Somero and DeVries, 1967; Prosser et al., 1970; O’Grady and DeVries, 1982). 172 HANS O. PÖRTNER AND GISELA LANNIG This fits the recent comparisons of active and passive pH regulation between stenothermal and eurythermal fish. They revealed that eurythermal fishes mainly use more costly active processes such as carriers dependent on Naþ/Kþ-ATPase, whereas cold-stenotherms rather depend on passive processes like nonbicarbonate buVering (Bock et al., 2001; Sartoris et al., 2003, see Section 2.2). Mark et al. (2005) observed a somewhat lower ouabain-sensitive respiration in hepatocytes of high-Antarctic compared to sub-Antarctic nototheniids indicating lower capacities for active ion regulation in the former. Following cold acclimation hepatocytes of rainbow trout, O. mykiss, showed no compensatory increase in Naþ/Kþ-ATPase but achieved a balanced ion regulation at a lower rate through a down-regulation of Kþ eZux (Krumschnabel et al., 1997). In contrast, hepatocytes of roach, Rutilus rutilus, showed near complete cold compensation of ion homeostasis following acclimation, through increased Naþ/Kþ-ATPase activity and increased Naþ/Kþ/Cl cotransport activity (Krumschnabel et al., 1997). The latter is insensitive to ouabain and represents a secondary active transport at the expense of Naþ and Cl gradient thereby saving ATP. The observed speciesspecific diVerences in ion regulation strategies with temperature acclimation were in line with previous findings by Schwarzbaum et al. (1992) and it was concluded that the diVerent strategies might depend on the level of eurythermy (more stenothermal salmonids versus more eurythermal cyprinids) (Krumschnabel et al., 1997). The diVerent ion regulation strategies correlated with cellular energy expenditure as seen in the study on rainbow trout (Krumschnabel et al., 1997). Similar and thus uncompensated total respiration rates were found between hepatocytes from cold- and warm-acclimated trout when measured at the same temperature. Nonetheless, diVerences were found in energy allocation. At low test temperatures oxygen consumption accounting for protein synthesis was increased by 10% likely through the benefit of reduced costs for ion regulation in cold- versus warm-acclimated fish (Krumschnabel et al., 1997). Available data on Antarctic fishes (Mark et al., 2005) revealed that a large fraction of about 28% of total cellular respiration (measured at 0 C and 0.1 mM cycloheximide) accounted for protein synthesis. An energy allocation to protein synthesis of between 20% and 25% was observed in hepatocytes of two other Antarctic fish species, Lepidonotothen kempi and P. brachycephalum (measured at 2 C and 0.03 mM cycloheximide; Langenbuch and Pörtner, 2003). Compensation of protein synthesis capacity for temperature is complete in polar ectotherms (Storch et al., 2003, 2005) and likely supports higher growth eYciency (Heilmayer et al., 2004). However, this point is still somewhat controversial. Conflicting results exist for protein synthesis costs at low temperatures and in polar versus boreal 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 173 animals, respectively (Pannevis and Houlihan, 1992; Whiteley et al., 1996; Marsh et al., 2001; Storch et al., 2003, 2005; Pace et al., 2004; for review see Fraser and Rogers, 2007). Furthermore, it seems that the costs for protein synthesis are negatively correlated with rates for protein synthesis such that at low synthesis rates the cost is elevated due to a suggested fixed cost component (Pannevis and Houlihan, 1992; Smith and Houlihan, 1995). To evaluate temperature eVects on energy allocation Mark et al. (2005) exposed isolated hepatocytes of various Antarctic fish species to an acute temperature rise (up to 15 C). Evidence for a temperature-dependent shift in ATP-consuming processes was minor. There was no eVect on energy partitioning between ion regulation (Naþ/Kþ-ATPase) or oxidative phosphorylation (Mark et al., 2005). The authors concluded that shifts in energy allocation might become eVective during systemic hypoxemia. Pannevis and Houlihan (1992) compared temperature-dependent respiration rates and absolute protein synthesis rates of cells obtained from 10 C acclimated rainbow trout, O. mykiss. The authors observed a thermal optimum of protein synthesis rates at intermediate temperatures: 40–50 ng mg protein-1 min-1 (14–18 C) compared to 15–30 ng mg protein-1 min-1 (5–10 C and 20 C, respectively). Interestingly, the authors noted no clear evidence for temperature-dependent diVerences in % cycloheximide inhibition of cellular respiration. The data may also suggest reduced protein synthesis at 20 C, which, however, was not visible when only cycloheximide-sensitive respiration rates were measured. Furthermore, respiration rates and the fraction of cellular protein synthesis were highest in cells from animals fed ad libitum (Krumschnabel et al., 1997) indicating that the condition of the fish influences the results making comparative approaches more complicated. Overall, the picture of temperature-dependent changes in cellular energy allocation is very unclear and presently does not support the elaboration of unifying principles. Clear control of the acclimation and feeding regime of the fishes as well as monitoring of cellular acclimation processes after isolation may provide a clearer picture. Further investigations are also needed depending on the lifestyle and physiology of the species studied (Wieser and Krumschnabel, 2001). For an appropriate evaluation of temperature-induced changes in cellular energy allocation, measurements should also be performed under simulated in vivo conditions, for example, at realistic levels of temperature-dependent tissue or venous PO2 values rather than at 100% air saturation, which is currently used in most investigations of cellular respiration. Figure 4.6 lists the various mechanistic aspects covered by the present chapter, with a focus on processes at the cellular level. For only a few of them the interaction between those processes and the interaction of temperature and hypoxemia eVects have been adequately explored. To further the study 174 HANS O. PÖRTNER AND GISELA LANNIG Fig. 4.6. Diagram listing key cellular parameters (I–IV) aVected by temperature change and associated responses to overcome the resulting impairment in cellular metabolism. Factors such as hormones, the shape of action potentials (APs), or blood and tissue oxygen levels (PO2) contribute to modulate the thermal impact on cellular processes. (SL = sacrolemmal and SR = sacroplasmic reticulum). of such interactions toward an integrative picture of temperature adaptation, inclusion of the role of hypoxemia still needs to be comprehensively developed, as a challenge for the years to come. For example, inadequate calcium regulation during temperature change would lead to impaired cardiac performance followed by impaired oxygen supply finally resulting in hypoxemia. Cardiac failure at critical temperatures may mainly depend on insuYcient oxygen supply to myocytes. As many fish species lack a coronary system the heart’s oxygen supply relies on venous PO2 (Farrell, 1993) and specific oxygen thresholds may exist where cardiac arrhythmia sets in (see Farrell and Clutterham, 2003 and references therein). Release of stress hormones, partly as a consequence of hypoxemia, ameliorates calcium regulation during acute temperature stress, thereby improving cardiac functioning and, in turn, alleviating hypoxemia eVects. One defence mechanism in response to hypoxic conditions is to reallocate cellular energy between energy consumers as aerobic ATP supply becomes limited, following a putative priority from less essential to more essential ATP-consuming processes. Furthermore, metabolic depression sets in, which involves a reduction in protein synthesis and in the cost of ion and acid-base regulation. Such cellular energy reallocation is likely more pronounced under temperature stress combined with oxygen limitation than under temperature 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 175 stress alone. Temperature and hypoxemia also come together in shaping the molecular signaling responses in the cell as exemplified in Figure 4.1. To overcome the threat of temperature-induced hypoxemia fishes either undergo metabolic depression (Hardewig et al., 2004) or maintain activity levels through metabolic acclimation to the new thermal regime (Seebacher et al., 2005; Lannig et al., 2005). Systemic, cellular, and molecular responses to hypoxemia at both sides of the thermal window need to be elaborated for a comprehensive understanding of thermal adaptation. Available data suggest a role for stress hormones, adenosine and hypoxia inducible factor HIF-1, and redox state in shaping temperature-dependent acclimation. Mechanisms involved in thermal adaptation include the homeoviscous adaptation of membranes and changes in mitochondrial capacities, as well as altered Arrhenius activation energies to compensate for the temperatureinduced alteration in aerobic energy metabolism. These mechanisms also support cardiac performance, in combination with alterations in calcium homeostasis. They include a cold-induced increase in the contribution of intracellular calcium cycling such as SR Ca2þ release to calcium homeostasis, which is otherwise mainly determined by sarcolemmal Ca2þ flux. Owing to the reliance of thermal tolerance on adequate oxygen supply for aerobic scope, those using mostly sarcolemmal Ca2þ flux are less tolerant to acute temperature changes. Further study is needed to qualify and quantify these interdependencies. 4. PERSPECTIVES: HYPOXIA-SENSITIVE THERMAL WINDOWS IN CLIMATE SENSITIVITY Temperature and hypoxia would traditionally be considered as diVerent environmental factors, each of which has its specific implications for whole organism functioning. Development of the concept of oxygen and capacity limited thermal tolerance has revealed how these factors are intertwined, since thermal stress causes systemic hypoxemia and the interaction of temperature and thermally induced hypoxemia shapes adaptive responses at various molecular to whole organism levels. The integration of these responses supports adjustment and maintenance of metabolic and functional performance at cellular, tissue, and whole organism levels. These principles play an important role in the context of climate change eVects on ecosystems. These are largely due to the current trends of warming in the world’s oceans caused by anthropogenic CO2 accumulation. On larger scales, eVects include shifts in geographical distribution such as the observed poleward shifts of phytoplankton, makroalgae, and marine-ectothermal 176 HANS O. PÖRTNER AND GISELA LANNIG animals along latitudinal clines (Lüning, 1990; Southward et al., 1995; Harrington et al., 1999; Walther et al., 2002; Parmesan and Yohe, 2003; Root et al., 2003; Perry et al., 2005). On smaller scales they also include local decreases in abundances of previously common species with the risk of local extinction of species or even ecosystems like coral reefs (Parmesan and Yohe, 2003; Thomas et al., 2004; Perry et al., 2005; Hoegh-Guldberg et al., 2007; Pörtner and Knust, 2007). In the German Wadden Sea, for example, the falling frequency of colder winters and increased occurrence of warmer summers are key in shaping population structure and community composition (Kröncke et al., 1998; Günther und Niesel, 1999; Pörtner and Knust, 2007). In general, observed ecosystem changes are related more to temperature anomalies (changes in thermal maxima or minima) than to changing temperature means (Stachowicz et al., 2002; Stenseth and Mysterud, 2002), with larger changes and eVects at high latitudes (Root et al., 2003). For a long time, the background and relevance of such observations has been obscured by the absence of an understanding of mechanistic cause and eVect (Jensen, 2003). The observations by Perry et al. (2005) include the finding that the Northward geographical shifts of various fish species (snake blenny, anglerfish, and cod) occur to various degrees. This may reflect diVerent thermal sensitivities of species coexisting in an ecosystem as a background for changes in community composition. Regime shifts may result like the one between colder years dominated by sardines and warmer years dominated by anchovies on the Pacific coast of Japan (Takasuka et al., 2007). In addition to direct eVects of temperature on individual species indirect eVects have to be identified at ecosystem level. Here, temperature-dependent changes in species interactions as in the food web may occur and exert their eVect at higher levels of the food cascade. As a prominent example, the shift in copepod faunal composition from larger Calanus finmarchicus to smaller Calanus helgolandicus in the Southern North Sea was seen as a major reason for the decrease in Atlantic cod (Gadus morhua) population, due to the reduced size of food particles available for juvenile cod (Beaugrand et al., 2003). More recently, Helaouët and Beaugrand (2007) showed that among the various parameters of the physical environment tested, temperature dominated the distribution of the copepod species, such that the warming trend caused the shift to the smaller species. These results indicate that direct physiological eVects of temperature on potential prey can then cause indirect eVects of temperature on the predator (cod). The principal understanding of direct temperature eVects on individual species is thus key to an understanding of climate-induced changes in species interactions at ecosystem level. This line of thought is in line with findings in terrestrial organisms (higher plants, insects, birds) where climate eVects on biogeography and biodiversity are independent from the position of the respective species in the food chain (Huntley et al., 4. OXYGEN AND CAPACITY LIMITED THERMAL TOLERANCE 177 2004). Thermal windows are thus decisive in shaping biogeography, a concept that warrants further analyses in aquatic ecosystems. Sensitivity to climate results from the specialization of species on climate regimes that include a limited range of temperatures which match a speciesspecific thermal window. In principle, this thermal window can be expected to match the temperature-dependent range of geographical distribution of a species and thereby defines the response to changing temperatures on both the cold and warm sides of the thermal window. The regime shifts from sardines during colder years to anchovies during warmer years (Takasuka et al., 2007) illustrate such eVects. In the case of Atlantic cod the warminginduced reduction of recruitment in the North Sea (Pörtner et al., 2001; Colosimo et al., 2003) and the Northward shift of the species (Perry et al., 2005) are mirrored in the warming-induced increase in recruitment on the cold side of its thermal window, in the Arctic Barents Sea. The direct eVects of cold versus warm temperatures on recruitment in various marine provinces strongly suggest that temperature directly influences individual species. Limited thermal windows of a species relate to limited windows of temperature-dependent growth. These limits are in close association with the temperatures causing climate-induced shifts at ecosystem level (Pörtner and Knust, 2007; Takasuka et al., 2007). In this context it needs to be considered that fluctuating food availability, as an indirect temperature eVect mediated through the food chain, may modulate the optimal temperature of growth. The seasonal timing of events is also being influenced by climate change and contributes to modulate ecosystem dynamics, as exemplified in the North Sea by later development of diatom blooms due to later grazing in previous years (Wiltshire and Manly, 2004), by earlier development of zooplankton (Greve et al., 1996), or by earlier migratory movements of, for example, squid (Loligo forbesi) into the North Sea (Sims et al., 2001). During a warming scenario such shifts in timing may be understood as an earlier entry of ambient temperature into species-specific thermal windows of performance during spring (in the case of zooplankton development or squid migration) or their later exit out of this window in the fall (in the case of later diatom grazing by zooplankton). As a corollary, the physical environment and especially temperature associated with temperature-induced hypoxemia exert large eVects on individual member species of a marine ecosystem. Changes in biogeographical distribution result, mirrored in shifts in abundance, species composition, and in species interactions, e.g., via changes in food web composition, at the edges of the thermal window of a species. A mechanistic analysis relies on an understanding of how these environmental conditions exert their direct 178 HANS O. PÖRTNER AND GISELA LANNIG limiting or supporting eVects on individual species and why species specialize on limited environmental windows. Such an understanding of some of the unifying principles of adaptation and limitation has emerged over recent years for eVects of temperature (for review see Pörtner et al., 2005a; Pörtner, 2001, 2002) and supported an understanding of the temperaturedependent evolution of animal species and phyla (Pörtner, 2004, 2006). The present chapter was intended to show that the principles of thermal adaptation and of temperature-dependent oxygen supply through circulation or ventilation are intertwined to an extent that oxygen supply capacity sets the earliest limits of thermal tolerance through the development of hypoxemia. Any factor depressing oxygen supply capacity will thus aVect thermal tolerance by narrowing thermal tolerance windows (Pörtner et al., 2005c). Ambient hypoxia reduces oxygen availability and thereby capacity and thermal tolerance. Elevated ambient CO2 levels frequently parallel aquatic hypoxia and independently but also synergistically cause enhanced sensitivity to thermal extremes (Metzger et al., 2007). However, these relationships remain unexplored in fishes. Altogether, such findings clearly show that cause and eVect analyses of past and future climate impacts on ecosystems need to take the synergistic physiological eVects of various environmental factors like temperature, carbon dioxide, and hypoxia into account. ACKNOWLEDGMENTS Supported by the MarCoPolI program of the Alfred-Wegener-Institute. REFERENCES Abele, D., and Puntarulo, S. (2004). Formation of reactive species and induction of antioxidant defence systems in polar and temperate marine invertebrates and fish. Comp. Biochem. Physiol. A 138, 405–415. Abele, D., Burlango, B., Viarengo, A., and Pörtner, H. O. (1998). 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Zielinski, S., and Pörtner, H. O. (1996). Energy metabolism and ATP free-energy change of the intertidal worm Sipunculus nudus below a critical temperature. J. Comp. Physiol. B 166, 492–500. 5 OXYGEN SENSING AND THE HYPOXIC VENTILATORY RESPONSE S. F. PERRY M. G. JONZ K. M. GILMOUR 1. Introduction 2. The Hypoxic Ventilatory Response 2.1. The Physiological Significance of the Hypoxic Ventilatory Response 3. O2 Sensing and O2 Sensors 3.1. Internally versus Externally Oriented O2 Chemoreceptors 3.2. Branchial versus Extra-Branchial O2 Chemoreceptors 3.3. Chemoreceptor Plasticity 4. Cellular Mechanisms of O2 Sensing 4.1. Cellular Models of O2 Sensing and Hypoxic Chemotransduction 4.2. O2 Chemotransduction in Fish Gill NECs 4.3. Neurotransmitters 5. Conclusions and Perspectives The hypoxic ventilatory response is arguably the single most important physiological response accompanying the exposure of fish to lowered ambient PO2. Increases in ventilation volume, driven by changes in breathing frequency and/or amplitude, serve to raise arterial PO2 and hence may delay the onset of transition from aerobic to anaerobic metabolism. In air-breathing fish, the hypoxic ventilatory response may consist of increases in aquatic and/or aerial respiration. Ventilatory responses in fish are initiated by O2 chemoreceptors able to detect changes in water and/or blood PO2. In zebrafish, the O2-sensing cells have been identified as neuroepithelial cells of the gill filament. In response to hypoxia, the neuroepithelial cells undergo membrane depolarization owing to an inhibition of outwardly directed 193 Hypoxia: Volume 27 FISH PHYSIOLOGY Copyright # 2009 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(08)00005-8 194 S. F. PERRY ET AL. potassium currents that ultimately elcits calcium entry and the release of neurotransmitter(s). 1. INTRODUCTION The two key factors that determine the rate of gas transfer in fish, and hence metabolic rate, are ventilation and perfusion. Thus, fish must be able to modify these convective processes to respond appropriately to changes in their environment or activity levels. The capacity of fish to mount appropriate cardiorespiratory responses, in turn, requires finely tuned sensory systems able to detect changes in the external and internal environments. During hypoxia, activation of the sensory or aVerent pathways initiates a suite of integrated reflexes aimed at promoting homeostasis. In this chapter, we focus on the hypoxic ventilatory response, arguably the single most important physiological reflex occurring during exposure to hypoxia; Chapter 7 in this volume reviews the cardiovascular responses to hypoxia. Obviously, these two chapters are complimentary and together provide a comprehensive overview of hypoxic cardiorespiratory reflexes in fish. In keeping with the tradition of previous volumes of Fish Physiology, we provide an extensive historical summary of the literature while also focusing on recent developments including the cellular mechanisms of oxygen sensing. 2. THE HYPOXIC VENTILATORY RESPONSE As Tables 5.1 and 5.2 demonstrate, fish exposed to hypoxia respond by hyperventilating (see also reviews by Shelton et al., 1986; Perry and Wood, 1989; Glass, 1992; Fritsche and Nilsson, 1993; Graham, 1997; Gilmour, 2001; Perry and Gilmour, 2002; Gilmour and Perry, 2007). This simple statement does not, however, do justice to the diversity of hypoxic ventilatory responses. Among species that are solely or predominately water-breathing, the vast majority respond to aquatic hypoxia by increasing the volume of water ventilated (V_ w) (Table 5.1), while bimodal (water and air) breathers typically increase reliance upon air-breathing (Table 5.2). However, the magnitude of the response, the water O2 tension (PwO2) threshold for hyperventilatory responses, and the mechanism through which ventilation is enhanced all vary greatly among species and, often, among diVerent studies on a single species. Ideally it would be possible to link interspecific diVerences in the hypoxic ventilatory response to factors such as hypoxia tolerance or ventilatory mechanics, but such comparisons are diYcult because experimental protocols have varied widely in the severity of hypoxia imposed, the rate of Table 5.1 Summary of (gill) ventilatory responses to hypoxia PO2 (Torr), time Species Agnathans Eptatretus stoutii Entosphenus tridentatus Lampetra fluviatilis Elasmobranchs Scyliorhinus stellaris Pacific hagfish Lamprey Lamprey Larger spotted dogfish S. canicula Sphyrna tiburo Dogfish Bonnethead shark Squalus acanthias Torpedo marmorata Spiny dogfish Ray (N = 1) Chondrosteians Acipenser baeri Siberian sturgeon 20, 20 min
V_ w (%) +149 40 30, 20 min 45, 30 min
Vf (%)
Vamp (%)
SV (%) +274 References +122 +155 +143 S. F. Perry, B. Vulesevic, M. Braun and K. M. Gilmour, unpublished Johansen et al., 1973 Claridge and Potter, 1975 Nikinmaa and Weber, 1984 Piiper et al., 1970 95, 1 h +52 ns 54, 1 h 80, 1 h 90, <60 min +19 +55 +264 ns ns 70, <60 min 35, 30 min 33, 20 min +765 +57 Short et al., 1979 Carlson and Parsons, 2003 +16 +67 +93 +111 Perry and Gilmour, 1996 Hughes, 1978 60, 1 h +121 +90 Nonnotte et al., 1993 40, 1 h +127 +110 (continued) Table 5.1 (continued ) PO2 (Torr), time Species A. naccarii A. transmontanus Adriatic sturgeon White sturgeon
Vf (%)
Vamp (%) 20, 1 h 30, 10 min 10, 30 min 19, 20 min +103 +124 38 +39 +165 +260 ns +130 81, 30 min 50, 30 min 35, 20 min 105, 1 h ns ns ns 31 +28 +26 +18 ns 60, 1 h 30, 1 h 69 71 10 23 Neopterygiians Amia calva Bowfin 35, 1 h 47, 15 min Teleosts Anguilla anguilla European eel 101 69 39 24 40 20, 2 h 40, <2 h A. japonica Japanese eel
V_ w (%) +67 +100 +58 +114 +150 +65 +146 18
SV (%) References Maxime et al., 1995 McKenzie et al., 1995 McKenzie et al., 1997 34 Burggren and Randall, 1978 60 68 +66 +80 Hedrick et al., 1991 McKenzie et al., 1991 Le Moigne et al., 1986 ns +5 20 +135 +43 11 Peyraud-Waitzenegger and Soulier, 1989 McKenzie et al., 2000 Chan, 1986 Apteronotus leptorhynchus Callionymus lyra Colossoma macropomum Cyprinus carpio Danio rerio Brown ghost knifefish Dragonet (N = 2) Tambaqui Carp Zebrafish Gadus morhua Atlantic cod Hoplerythrinus unitaeniatus Jeju (no access to air) 50, 30 min 50, 6 min 10, 10 min Mild 40 110, 1 h 75, 1 h 100, 30 min 90, 30 min 110, 20 min 70, 20 min 50, 20 min 20, 20 min 90, 20 min 50, 20 min 20, 20 min 80, 5 min 55, 5 min 35, 5 min 59, 10 min 46, 25 min 70, 1 h +10 +77 +287 +475 +83 +161 +59 +134 +39 +57 +250 +30 30 +63 +218 +41 +205 +179 +150 +30 +55 +73 +63 +49 +79 +54 +28 +39 +48 +35 ns +25 +120 +88 ns ns ns 59 ns M. Moorhead, M. Nguyen, J. Lewis, S. F. Perry, and K. M. Gilmour, unpublished data Hughes and Umezawa, 1968 Sundin et al., 2000 Itazawa and Takeda, 1978 Lomholt and Johansen, 1979 Glass et al., 1990 Soncini and Glass, 2000 Vulesevic et al., 2006 ns ns ns ns ns ns ns +11 Vulesevic and Perry, 2006 K. Borg, S. Sharam, and W. K. Milsom, unpublished data +50 +61 ns +37 +143 Kinkead et al., 1991 Oliveira et al., 2004 (continued) Table 5.1 (continued ) PO2 (Torr), time Species (access to air) Hoplias lacerdae H. malabaricus Hypostomus regani Ictalurus punctatus Katsuwonus pelamis Trairão Traira Ihering Channel catfish Skipjack tuna
V_ w (%) 50, 1 h 30, 1 h 20, 1 h 50, 15 min 40, 15 min 20, 15 min 20 100, 10 min 50, 10 min 25, 10 min 15, 10 min 20 11, 10 min 20, 30 min 20, 15 min 25, 5 h 27, < 2 h +575 +107 +102 105, 1 h 65, 1 h 46, 10 min 73, 5 min 50, 5 min 30, 5 min 100, 4 min 75, 4 min +106 +100 +460 +35 +118 +183 +209 +470 +800 +778 +287 +25 +45
Vf (%) +35 +37 +35 ns +24 ns +160 +8 +25 +38 +37 +40 +33 +51 +27 +20 +44 ns ns +33 +14 +16 +33
Vamp (%)
SV (%) References +371 +700 +643 +55 ns ns Perry et al., 2004 +160 +24 +74 +105 +126 +375 +88 +550 +170 +230 +91 +91 +93 +66 +154 +100 Rantin et al., 1992 Rantin and Johansen, 1984 Rantin et al., 1992 Sundin et al., 1999 Sakuragui et al., 2003 Perry et al., 2004 Mattias et al., 1998 Gerald and Cech, 1970 Burggren and Cameron, 1980 Burleson and Smatresk, 1990a Burleson et al., 2002 Sundin et al., 2003 Bushnell and Brill, 1992 Mugil cephalus Myoxocephalus scorpius Oncorhynchus mykiss Mullet Shorthorn sculpin Rainbow trout Ophiodon elongatus Opsanus beta Lingcod Gulf toadfish Oreochromis niloticus Tilapia 68 63, 30 min 31, 30 min 16, 30 min 38, 10 min +371 40 +119 80, 30 min 60, 30 min 40, 30 min 60, 5 min 75, 30 min 93, 20 min 47, 30 min 72, 30 min 90, 30 min 70, 25 min 50, 20 min 40, 20 min 60, 30 min 40, 20 min 45, 30 min 35, 10 min 40, 15 min +200 +500 +170 +582 +104 +113 +142 +192 +126 80 60 +50 +100 +58 ns ns ns +39 +149 +111 +211 +637 +57 +50 Turesson and Sundin, 2003 +760 +67 +144 +478 +16 ns ns ns ns +25 +35 ns +18 +25 ns +107 ns ns ns Cech and Wohlschlag, 1973 Shingles et al., 2005 Holeton and Randall, 1967a,b Hughes and Saunders, 1970 +489 +114 +119 +179 +145 +190 +44 +21 +85 +240 +390 +56 +50 +125 Davis and Cameron, 1971 Randall and Jones, 1973 Smith and Jones, 1982 Aota et al., 1990 Kinkead and Perry, 1990 Kinkead and Perry, 1991 Perry and Thomas, 1991 Bindon et al., 1994 Gilmour and Perry, 1994 Greco et al., 1995 Perry and Gilmour, 1996 Farrell and Daxboeck, 1981 S. F. Perry, M. D. McDonald, P. J. Walsh, and K. M. Gilmour, unpublished data Fernandes and Rantin, 1989 (continued) Table 5.1 (continued ) PO2 (Torr), time Species Orthodon microlepidotus Sacramento blackfish Parophrys vetulus English sole Piaractus mesopotamicus Pacu Platichthys flesus Flounder P. stellatus Starry flounder Plaice Pleuronectes platessa Prochilodus scrofa Rhinelepis strigosa Salminus maxillosus Curimbatá Cascudo preto Dourado
V_ w (%)
Vf (%)
Vamp (%)
SV (%) 20 22, 75 min 9, 75 min 90 +550 +300 +400 +50 +18 +33 +13 ns +400 +333 +500 +38 65 40 120, 2 h 88, 2 h 50, 2 h 20, 50 min 20, 15 min 30, 1 h 94, 1 h 62, 1 h 39, 1 h 50 +95 +195 +69 +203 +421 +90 +169 +72 +63 +106 +91 +200 ns +11 +8 +16 +33 +158 +103 ns ns +11 6 94, 1 h 62, 1 h 39, 1 h 10 26 +11 +33 +65 +130 +900 ns ns ns +209 +23 +14 +50 +92 +443 +557 70, 30 min 40, 30 min 20, 30 min +75 +190 +270 +17 +28 +21 ns +186 +264 References Kalinin et al., 1999 Campagna and Cech, 1981 Boese, 1988 +300 ns +65 +58 +83 +100 Leite et al., 2007 Perry et al., 2004 Kerstens et al., 1979 SteVensen et al., 1982 Watters and Smith, 1973 SteVensen et al., 1982 Fernandes et al., 1995 Takasusuki et al., 1998 De Salvo-Souza et al., 2001 Scophthalmus maximus Silurus glanis Turbot Sheatfish Thunnus albacares Yellowfin tuna T. obesus Bigeye tuna Tinca tinca Tench 60, 1 h 81, > 90 min 39, > 90 min 23, >90 min 100, 4 min 75, 4 min 100, 30 min 100, 4 min 75, 4 min 44, 30 min 25, 4 h +26 +70 +100 +279 +643 +100 Bushnell and Brill, 1992 +75 +37 +30 +75 Bushnell and Brill, 1991 Bushnell and Brill, 1992 +44 +500 Maxime et al., 2000 Forgue et al., 1989 +250 +68 Randall and Shelton, 1963 Eddy, 1974 The change in ventilation volume (
V_ w), ventilation frequency (
Vf), ventilation amplitude (
Vamp), or ventilatory stroke volume (
SV) under hypoxic conditions are expressed as a percentage of the normoxic value; thus, a negative value indicates a decrease while a positive value indicates an increase from the normoxic value. Percent changes were calculated from mean data reported in the original studies; changes that were not significant are noted as ns. The level of hypoxia to which the fish was exposed (PO2 in Torr) and the period of hypoxic exposure (time) are listed; in some studies, the period of hypoxic exposure was not noted. Table 5.2 Summary of gill ventilation and air-breathing responses to hypoxia in air-breathing fish PO2 (Torr), time Species Obligate air-breathers Channa argus Snakehead African Protopterus aethiopicus or lungfish P. dolloi P. annectens Lepidosiren paradoxa South American lungfish (N = 2) Facultative air-breathers A. calva Bowfin Amphipnous cuchia Cuchia eel Ancistrus chagresi Armoured catfish Clarias lazera H. unitaeniatus Jeju Pcrit (Torr) Air-breathing threshold (Torr) fAB (h 1) Peak gill ventilation (Torr) N H References 35, 3 h 61, 1 h ‐ ‐ NA NA ‐ 61 6.8 17 9.4 40 Glass et al., 1986 Jesse et al., 1967 23, 1 h 30 < 80 100 ‐ NA NA 70 ‐ 13.3 12.6 54 29 26.1 Babiker, 1979 Johansen and Lenfant, 1967 30 14 35, 1 h 47, 15 min 55 49, 8 h 15 ‐ ‐ ‐ ‐ ‐ ‐ ‐ NA NA ‐ NA NA NA NA 80 ‐ ‐ NA ‐ NA NA 9 5.5 0 0 1.4 1.2 9 30 12 3.2 5.1 3.5 7.2 16.1 0, 4 h 40* 33 – 0 14.4 30 70, 1 h 50, 1 h 100 40* NA 64 70 30* 19 0 63 0 5.5 Johansen et al., 1970 Randall et al., 1981 Hedrick et al., 1991 McKenzie et al., 1991 Hedrick and Jones, 1993 Hedrick and Jones, 1999 Lomholt and Johansen, 1974 Graham and Baird, 1982; Graham, 1983 Babiker, 1979 Oliveira et al., 2004 Hoplosternum littorale 30, 1 h 20, 1 h 60, 15 min 40, 15 min 30, 15 min 20, 15 min 10, 15 min 7.5, 3 h 105, 30 min 70, 1 h 40, 1 h 20, 1 h 10, 1 h 0, 4 h ‐ 40 40–50* 0 52.5 – 52.5 NA NA – 2 2 50* NA 20 0.5 – 60 – 10, 5.5 h 12, 1 h 34* – 50–65 NA Longnose gar Tarpon 20, 90 min 1, 1–6 h – – Pacific tarpon 45, 30 min 15, 20 min 45, 1 h Armoured catfish Hypostomus plecostomus H. regani Lepisosteus oculatus L. osseus Megalops atlanticus M. cyprinoides Armoured catfish Ihering Spotted gar Misgurnus anguillicaudatus Oriental weatherloach Neoceratodus forsteri Australian lungfish Odontamblyopus lacepedii Eel goby 14 18.5 0 2.5 12 36 36 19.1 28 Perry et al., 2004 McKenzie et al., 2007 Brauner et al., 1995 AVonso and Rantin, 2005 0 1 2.6 3.5 4.5 9.2 25* – 0 1 5.8 8.3 NA NA 75 37 3.5 1.4 11.7 7.7 Mattias et al., 1998 Smatresk and Cameron, 1982 Smatresk, 1986 Geiger et al., 2000 – – – 60 60 NA 60 – NA 0 1.8 10 43.8 31.8 20 0, 2 h 22, 60 min – – – 75 – 43 0 <1 45.6 5 Seymour et al., 2007 Clark et al., 2007 McMahon and Burggren, 1987 McNeil and Closs, 2007 Fritsche et al., 1993 40, 15 h 23, 2 h – – 58 78 70 – 0.1 0 0.3 7.9 Kind et al., 2002 Gonzales et al., 2006 7.5, 2 h Graham and Baird, 1982 10.8 (continued) Table 5.2 (continued ) PO2 (Torr), time Species Piabucina festae Rhinelepis strigosa Synbranchus marmoratus Cascudo preto South American swamp eel Pcrit (Torr) Air-breathing threshold (Torr) Peak gill ventilation (Torr) fAB (h 1) N H References 30 5 < 30 70* 20.5* – NA 22 30 – 25* 30 25 0 0 40.4 7 4.1 Graham et al., 1977 Takasusuki et al., 1998 Bicudo and Johansen, 1979 Trichogaster trichopterus Gourami 0, 4 h 75, 1 h 150* ‐ 33 NA NA – 0 12 4 20 Graham and Baird, 1984 Burggren, 1979 Umbra limi Mudminnow 37, 1 h 20, 90 min – 45 45 0 27 7 Gee, 1980 Pcrit, the partial pressure of O2 at which the transition from oxygen regulator to oxygen conformer occurs (* indicates that Pcrit was measured without access to air); Air-breathing threshold, the partial pressure of O2 at which air-breathing first occurs (species that breathe air under normoxic conditions are indicated with NA); Peak gill ventilation, the partial pressure of O2 at which ventilation of the gills was maximal (using V_ w where available, or Vf and/or Vamp as appropriate; species in which ventilation continued to increase to the most severe level of hypoxia examined are indicated with NA as it is not clear what impact even more severe hypoxic exposure would have had on ventilation); fAB, air-breathing frequency under normoxic (N) and hypoxic (H) conditions, where the level of hypoxia to which the fish was exposed and the period of hypoxic exposure are listed. Where variables were not measured in a particular study, a dash (–) is used. Additional data on thresholds for air breathing can be found in Table 1 of Takasusuki et al. (1998), while Graham (1997) provides comprehensive data both on air-breathing frequencies in normoxia vs. hypoxia (Table 5.2) and air-breathing thresholds (Table 6.1). 5. OXYGEN SENSING 205 change of PwO2, and the length of hypoxic exposure, as well as methods of assessing ventilation. Moreover, it is becoming increasingly clear that ventilatory responses to hypoxia within a species may be aVected by a multiplicity of conditions, including temperature (e.g., Spitzer et al., 1969; Watters and Smith, 1973; Campagna and Cech, 1981; Berschick et al., 1987; Gehrke and Fielder, 1988; Fernandes and Rantin, 1989; Glass et al., 1990; Fernandes et al., 1995; Geiger et al., 2000; Stecyk and Farrell, 2002, 2008; Cerezo and Garcia, 2004; Valverde et al., 2006), diet (e.g. McKenzie et al., 1997, 2000), perceived predation risk (e.g., Shingles et al., 2005; Randle and Chapman, 2005), developmental plasticity (Vulesevic and Perry, 2006), and prior history of hypoxic exposure (e.g., Kerstens et al., 1979; Lomholt and Johansen, 1979; Kramer and Mehegan, 1981; Graham and Baird, 1982, 1984; Graham, 1983; Nikinmaa and Weber, 1984; Taylor and Miller, 2001; Burleson et al., 2002; Routley et al., 2002; Timmerman and Chapman, 2004a, b; Vulesevic et al., 2006), yet in many cases our understanding of how and why these conditions aVect the hypoxic ventilatory response is less than complete. Under these conditions, it becomes diYcult to make generalizations about the hypoxic ventilatory response (beyond, of course, the obvious – that fish hyperventilate!) and this point should be kept in mind while considering the generalizations that follow. Table 5.1 summarizes data on the ventilatory responses to hypoxia of 46 species of fish from several taxonomic groups, focusing on species that are solely or predominantly water-breathing and studies that have measured V_ w and/or ventilation frequency (Vf) and stroke volume (or its commonly used proxy, ventilation amplitude; Vamp). With the exception of lamprey, for which only frequency data appear to have been collected, studies presenting data on Vf alone were not included. Table 5.1 includes four obligate ram ventilators (the bonnethead shark, Sphyrna tiburo, and three tuna species), which elevate V_ w by increasing gape (Figure 5.1) (Bushnell et al., 1990; Carlson and Parsons, 2003), as well as seven species that either lowered ventilation during hypoxia (Japanese eel, Anguilla japonica, and white sturgeon, Acipenser transmontanus) or for which data sets are incomplete (Pacific hagfish, Eptatretus stoutii, lamprey, Entosphenus tridentatus and Lampetra fluviatilis, starry flounder, Platichthys stellatus, and sheatfish, Silurus glanis). Of the remaining 35 species, 21 (60%) respond to hypoxia primarily or solely by increasing Vamp, 13 species (37%) employ increases in either or both of Vf and Vamp, and only one species (carp, Cyprinus carpio; 3%) increases Vf in the absence of amplitude or stroke volume adjustments. Included among species that increase Vf during hypoxia are those in which hypoxia promotes a transition from a pattern of episodic to continuous breathing (Figure 5.1) (e.g., Lomholt and Johansen, 1979; Smith et al., 1983; Gehrke and Fielder, 1988; Glass et al., 1990; Nonnotte et al., 1993; Maxime et al., 1995; 206 S. F. PERRY ET AL. A Inflow seawater PO2 (mmHg) 160 120 80 Gape (cm) 1.1 1.8 B 6s Fig. 5.1. Ventilatory responses to hypoxia. (A) An original recording of the increase in mouth gape that occurs in an obligate ram ventilator, yellowfin tuna (Thunnus albacares) as water O2 tension is lowered. Gape was measured as changes in impedance from electrodes attached to the upper and lower jaws. [Reproduced with permission from Bushnell et al. (1990).] (B) Ventilation in the sturgeon Acipenser baeri is episodic under normoxic conditions (left-hand panel) but becomes continuous upon exposure to hypoxic water (right-hand panel; PO2 = 60 Torr). Note the increase in ventilation amplitude that occurs in addition to the increases in frequency. The figure depicts original recordings of pressure changes associated with breathing in the branchial cavity, measured using a water-filled catheter connected to a pressure transducer. [Reproduced with permission from Nonnotte et al. (1993).] Fernandes et al., 1995; Reid et al., 2003; Vulesevic et al., 2006; Leite et al., 2007). This breakdown of responses provides support for the generalization that fish employ the energetically favourable strategy, given the density and viscosity of water as the ventilated medium, of increasing V_ w through large changes in ventilatory stroke volume coupled to more modest increases of Vf (Shelton et al., 1986; Perry and Wood, 1989; Gilmour, 2001). At the same time, it is clear that strategies for achieving hyperventilation among fish are diverse, at times even within a single species. For example, Vulesevic et al. (2006) reported that zebrafish (Danio rerio) responded to either hypoxia or hypercapnia (high water CO2 tension) by hyperventilating, but whereas hypoxia caused Vf to increase in the absence of changes in Vamp, the opposite was true of hypercapnia, where breathing amplitude increased but frequency was unaVected. Even more perplexing, the hypoxic hyperventilation in a diVerent group of zebrafish relied largely upon changes in Vamp (W. K. Milsom, personal communication; see Table 5.1). The hyperventilatory strategy may also depend upon the level of hypoxia. For example, 5. 207 OXYGEN SENSING moderate increases in V_ w in cod (Gadus morhua) were achieved by raising breathing frequency, whereas increases in stroke volume accounted for the greater hyperventilatory response to more severe levels of hypoxia (Kinkead et al., 1991). Typically, ventilatory responses to hypoxia are initiated very rapidly, i.e., as hypoxic water contacts the gill (Figure 5.2) (Bamford, 1974; Kinkead et al., 1991), and may be sustained for hours to days. Results from several studies that have monitored ventilation for 90 min to 24 h of hypoxia suggest that hyperventilatory responses to hypoxia are independent of exposure period during this time frame (Thomas and Hughes, 1982a,b; Forgue et al., 1989; Glass et al., 1990; Borch et al., 1993; Florindo et al., 2006), except possibly during exposure to near-anoxic conditions (Stecyk and Farrell, 2002). As exposure to hypoxia is prolonged, however, a suite of responses is initiated to enhance O2 uptake and delivery beyond that achieved by hyperventilation alone. These responses yield increases in hemoglobin– oxygen binding aYnity (e.g., Wood and Johansen, 1972; Wood et al., 1975; Tetens and Lykkeboe, 1981; Rutjes et al., 2007) and blood O2 carrying capacity (e.g. Wood and Johansen, 1973; Lai et al., 2006; Rutjes et al., 2007) that, together with other factors, enhance O2 transfer (see reviews by Perry and Wood, 1989; Nikinmaa, 2001) and might therefore lower the ventilatory convection requirement. Moreover, prolonged acclimation to hypoxic conditions also may influence the density, size, and morphology of gill neuroepithelial cells (Jonz et al., 2004; Vulesevic et al., 2006), the putative O2 chemosensors of the fish gill (see Sections 3.2.2 and 3.3), providing a mechanism through which acclimation-associated changes in ventilatory responses could be mediated. Acclimation to hypoxic conditions does appear to influence ventilation parameters. Under normoxic conditions, acclimation cmH2O Buccal 1 pressure 0 PO2 mmHg 150 75 0 0 5 10 s Fig. 5.2. An original recording of ventilation (measured as pressure changes in the buccal cavity associated with breathing using a water-filled catheter connected to a pressure transducer) and water O2 tension demonstrating that ventilatory responses to hypoxia are initiated within seconds of the hypoxic water contacting the gill. [Reproduced with permission from Bamford (1974).] 208 S. F. PERRY ET AL. to hypoxia eliminates episodic breathing in species that normally exhibit this breathing pattern (Lomholt and Johansen, 1979; Vulesevic et al., 2006), but is otherwise generally without eVect (Lomholt and Johansen, 1979; Burleson et al., 2002; Vulesevic et al., 2006). The impact of hypoxic acclimation on the hypoxic ventilatory response is more variable, with increased ventilatory sensitivity to hypoxia (
ventilation/
PO2) reported for some species (Kerstens et al., 1979; Burleson et al., 2002), but no change or reduced ventilation under hypoxic conditions reported for other species (Lomholt and Johansen, 1979; Nikinmaa and Weber, 1984; Vulesevic et al., 2006). There is clearly a need to investigate the time domains of the hypoxic ventilatory response (Powell et al., 1998) in fish in a more systematic fashion, to probe the underlying mechanisms, and to attempt to associate these time domains with the occurrence of other mechanisms that aVect branchial O2 transfer and blood O2 transport. Ventilatory responses to hypoxia typically reflect the severity of the hypoxic stimulus, with V_ w increasing as water PO2 falls (Table 5.1; Figure 5.3). Estimates of the maximum increase in V_ w depend not only on the species examined and the severity of hypoxia, but also upon the method used to determine V_ w and upon acclimation temperature. Notably, use of the Fick method leads to overestimation of V_ w, particularly at high levels of ventilation (Davis and Watters, 1970; Kalinin et al., 1999). Considering only the 26 studies listed in Table 5.1 in which V_ w was measured directly, maximum increases in V_ w during hypoxia range from 52% to 581% of the normoxic value (or V_ w maximally increases 1.5- to 6.9-fold during hypoxia). The maximum increase in V_ w also depends upon acclimation temperature, with higher ventilation volumes being achieved at higher acclimation temperatures. Although normoxic V_ w also increases with increasing acclimation temperature, presumably owing to the eVect of temperature on metabolic rate, enhanced ventilatory sensitivity to hypoxia (
ventilation/
PO2) in fish acclimated to higher temperatures typically results in greater percentage increases in V_ w during hypoxia (Table 5.3) (Spitzer et al., 1969; Campagna and Cech, 1981; Fernandes and Rantin, 1989; Glass et al., 1990; Cerezo and Garcia, 2004; Valverde et al., 2006). As with the eVects of hypoxic acclimation, the mechanisms underlying the impact of acclimation temperature on the hypoxic ventilatory response remain largely unexplored. The peak ventilatory eVort occurs in many studies at the lowest water O2 tension examined, but it is not uncommon to find that ventilation rises to a maximum and then falls as the level of hypoxia becomes more severe (Figure 5.3A). This fall in ventilation often seems to occur around the critical PO2 (Pcrit), i.e., the PO2 at which the transition from oxyregulator to oxyconformer occurs. Table 5.4 summarizes data for 10 species exhibiting this pattern, where the PO2 of peak ventilatory eVort was significantly correlated with Pcrit (correlation 5. 209 OXYGEN SENSING A 1600 50 1000 · Vw · MO 800 40 2 600 30 400 20 200 0 20 40 80 100 120 140 160 12 70 10 2500 60 · · Vw 2000 50 MO2 fASR 1500 40 1000 30 500 20 20 40 60 80 100 120 140 · 10 160 8 6 4 fASR (30 min–1) 80 3000 MO2 (mL O2 h–1 kg–1) 3500 0 2 0 PwO2 (Torr) 20 120 5000 110 4000 100 · · MO 3000 Vw 90 2 2000 80 fAB 70 1000 60 0 20 40 60 80 100 120 140 160 50 · 15 10 fAB (h–1) C Vw (mL min–1 kg–1) 10 PwO2 (Torr) B Vw (mL min–1 kg–1) 60 · MO2 (mL O2 h–1 kg–1) Vw (mL min–1 kg–1) 60 1200 MO2 (mL O2 h–1 kg–1) 70 1400 5 0 PwO2 (Torr) Fig. 5.3. The relationship between O2 uptake (MO2) and ventilation during exposure to hypoxia is depicted for three species of fish that make use of diVerent ventilatory strategies. (A) Gill ventilation volume (V_ w) and MO2 are plotted as a function of water O2 tension (PwO2) for the unimodal water-breather, Hoplias lacerdae. V_ w increases during hypoxia, peaking at a PwO2 of about 20 Torr, somewhat below the critical O2 tension (Pcrit), which was estimated to be 35 Torr. [Data replotted from Rantin et al. (1992).] (B) Data for pacu, Piaractus mesopotamicus, a species that utilizes aquatic surface respiration (ASR). In addition to V_ w and MO2, the frequency of ascents to the surface to perform ASR (fASR) is plotted as a function of PwO2. ASR was initiated at a PwO2 of 30 Torr, very close to the Pcrit of 34 Torr (measured without access to the water–air interface), while gill ventilation peaked at a somewhat lower PwO2 of 10 Torr. [Modified from Rantin et al. (1998).] (C) Data are presented for jeju, Hoplerythrinus unitaeniatus, a facultative air-breather. V_ w, MO2, and the frequency of air-breaths (fAB) are plotted as a function of PwO2. Air-breathing was absent until PwO2 dropped to 64 Torr, and then increased in frequency as PwO2 continued to fall. Pcrit in the absence of access to air was determined to be 40 Torr, while gill ventilation peaked at 30 Torr, again illustrating the appearance of an alternative ventilatory strategy at a PwO2 when metabolic rate would otherwise fall. [Modified from Oliveira et al. (2004).] Table 5.3 EVects of acclimation temperature on the hypoxic ventilatory response of water-breathing fish Species C. carpio Carp O. niloticus Tilapia O. microlepidotus Sacramento blackfish Temp ( C) Normoxic V_ w Max
V_ w (%)
V_ w/
PO2 10 20 20 25 50.2 241 100 200 161 222 1000 1.1 7.1 8.3 30 35 12 250 300 60 1300 1300 1133 400 21.7 27 34 2.7 20 28 200 360 1950 3167 4.3 12.7 References Glass et al., 1990 Fernandes and Rantin, 1989 Campagna and Cech, 1981 Normoxic V_ w, ventilation volume under normoxic conditions at the temperature indicated; max
V_ w, the maximum change in ventilation volume expressed as a percentage of the normoxic value;
V_ w/
PO2, the ventilatory sensitivity to hypoxia, i.e., the change in ventilation volume for a given change in water O2 tension. Only studies in which
V_ w was measured were included in the data set. 5. 211 OXYGEN SENSING Table 5.4 A comparison of critical PO2 (Pcrit) values with the PO2 of peak ventilation for water-breathing fish Pcrit (Torr) Species Peak gill ventilation (Torr) A. anguilla A. japonica D. rerio European eel Japanese eel Zebrafish 70 100–115 20a 40 80 43 H. lacerdae H. malabaricus Traira 35 20b 20 20–30 27.2 16 22c 30 Leiopotherapon unicolor O. mykiss Spangled perch Rainbow trout 49 40 P. mesopotamicus Pacu 34 P. flesus S. maximus Flounder Turbot 60–80 20–30 10 30 60 40 References Le Moigne et al., 1986 Chan, 1986 Vulesevic et al., 2006; Vulesevic and Perry, 2006 a Barrionuevo and Burggren, 1999 Rantin et al., 1992 Sundin et al., 1999; bRantin et al., 1992 Gehrke and Fielder, 1988 Perry and Gilmour, 1996; cOtt et al., 1980 Greco et al., 1995 Holeton and Randall, 1967b Rantin et al., 1998 Leite et al., 2007 SteVensen et al., 1982 Maxime et al., 2000 Pcrit, the partial pressure of O2 at which the transition from oxygen regulator to oxygen conformer occurs; Peak gill ventilation, the partial pressure of O2 at which ventilation of the gills was maximal (using V_ w where available, or Vf and/or Vamp as appropriate). coeYcient = 0.76; P = 0.01). A tendency for ventilation to fall as water O2 tension is lowered below the Pcrit probably explains the two entries in Table 5.1 for species in which ventilation fell with exposure to hypoxia, white sturgeon and Japanese eel. Ventilation parameters for Japanese eel were summarized in Table 5.1 for a PwO2 of 40 Torr, but Pcrit was estimated to be 100–115 Torr (Chan, 1986). White sturgeon appears to be a true oxygen conformer with an unusually high Pcrit, in that oxygen consumption declined with even very small reductions of water O2 tension (Burggren and Randall, 1978). Lowering breathing in this species during hypoxia may reduce the cost of ventilation per unit of oxygen uptake, particularly since oxygen extraction was maintained (Burggren and Randall, 1978). 212 S. F. PERRY ET AL. In some species, reductions in gill ventilation at severe levels of hypoxia are associated with the appearance of alternative ventilatory strategies, such as aquatic surface respiration (Table 5.5) or air-breathing (Table 5.2). Aquatic surface respiration (ASR) consists of using the thin zone (<0.5 mm; Burggren, 1982) of relatively oxygen-rich water at the air–water interface to ventilate the gills (e.g., Gee et al., 1978; Kramer and McClure, 1982; Table 5.5 PO2 thresholds for aquatic surface respiration (ASR) PO2 (Torr), time Species ASR Peak gill Pcrit threshold ventilation (Torr) (Torr) (Torr) Astatotilapia aeneocolor 3, 5 h 15* 15 3.4 A. ‘wrought-iron’ 3, 5 h 13.5* 15 15 Oscar 20, 2 h 50* 50 – Goldfish 0, 2 h 34*a 13.7 13.7 C. carpio Carp 0, 2 h 20*b 13.7 17.1 M. cephalus Mullet 16, 30 min 44*c 32 19.4 10, 30 min 34* 30 10 Astronotus ocellatus Carassius auratus P. mesopotamicus Pacu References Melnychuk and Chapman, 2002 Melnychuk and Chapman, 2002 Sloman et al., 2006 McNeil and Closs, 2007; a Fry and Hart, 1948 McNeil and Closs, 2007; b Ott et al., 1980 Shingles et al., 2005; c Nordlie and Lefler, 1975 Rantin et al., 1998 Pcrit, the partial pressure of O2 at which the transition from oxygen regulator to oxygen conformer occurs (* indicates that Pcrit was measured without access to the air–water interface); ASR threshold, the partial pressure of O2 at which ASR first occurs; Peak gill ventilation, the partial pressure of O2 at which ventilation of the gills was maximal (using V_ w where available, or Vf and/or Vamp as appropriate); the level of hypoxia to which the fish was exposed and the period of hypoxic exposure are also listed. Where variables were not measured in a particular study, a dash (–) is used. See in addition Table 2 in Gee and Gee (1991) for thresholds of ASR in 5 species of Eleotridae and 15 species of Gobiidae, Fig. 3 in Melnychuk and Chapman (2002) for thresholds of ASR and Pcrit values in 19 species of East African cichlids, Figs. 1 and 2 in McNeil and Closs (2007) for thresholds of ASR and PO2 of peak gill ventilation in 9 species commonly found in Australian billabongs, Fig. 5 in Soares et al. (2006) for thresholds of ASR and PO2 of peak gill ventilation in 8 Amazonian species, and Table 1 in Gee et al. (1978) for thresholds of ASR and PO2 of peak gill ventilation in 26 species from western Canada. 5. OXYGEN SENSING 213 Kramer, 1983; Gee and Gee, 1991; Melnychuk and Chapman, 2002; Soares et al., 2006; McNeil and Closs, 2007). Fish rise to the surface to skim the surface film of water, adopting a position in which the top of the head lies just at or below the surface of the water. Morphological adaptations such as flattened heads, upturned mouths (Lewis, 1970) and/or the development of lip protuberances during hypoxia (Winemiller, 1989; Sundin et al., 2000; Florindo et al., 2006) may facilitate or increase the eVectiveness of ASR. Although the initiation of ASR occurs at much lower O2 tensions than does hyperventilation, typically appearing at or below the PO2 of maximum gill ventilation (Table 5.5; Figure 5.3B), in most other respects trends in ASR are similar to those for gill ventilation. For example, ASR eVort (measured as time spent in ASR) increases as PO2 falls (Kramer and Mehegan, 1981; Kramer and McClure, 1982; Rantin et al., 1998), hypoxic acclimation reduces the use of ASR (Kramer and Mehegan, 1981; Timmerman and Chapman, 2004a), and acclimation to higher temperatures raises the threshold at which ASR appears (Gee et al., 1978). Unlike hyperventilation, the use of ASR is associated with an obvious predation risk because of the need to approach the surface of the water. More severe levels of hypoxia are required to elicit ASR in mullet (Mugil cephalus) in response to the threat of predation (Shingles et al., 2005). Similarly, small oscar (Astronotus ocellatus) surface for ASR at lower O2 tensions than larger oscar, which are less vulnerable to predation (Sloman et al., 2006). The eVect of perceived predation risk on the use of ASR emphasizes a need for investigation of the factors that regulate or modulate O2 chemosensory reflexes. Moreover, further elucidation of the stimuli that provoke ASR and the neural circuitry underlying this response is needed to confirm that ASR can be classified as an O2-chemoreflex (or hypoxic reflex) together with alteration of gill ventilation. ASR appears to be mediated by O2-sensitive chemoreceptors that are located in the orobranchial cavity and innervated by cranial nerve V, since it can be evoked by injection of sodium cyanide (see Section 3.1) into the bloodstream or ventilated water to stimulate internally oriented or externally oriented O2 chemoreceptors, respectively (Shingles et al., 2005) and it is eliminated by sectioning of the mandibular branches of cranial nerve V (Florindo et al., 2006). However, our knowledge of ASR as a chemosensory reflex is far less detailed than is the case for gill ventilation. Similar comments apply to air-breathing as an alternative ventilatory strategy during hypoxia, i.e., facultative air-breathing. Whether or to what extent obligate air-breathers respond to aquatic hypoxia is unclear, because several studies have reported eVects of aquatic hypoxia on air-breathing frequency (Table 5.2), yet others, often on the same species, have not (Johansen and Lenfant, 1968; Sanchez et al., 2001; Perry et al., 2005). Among facultative air-breathers that do not exhibit air-breathing under 214 S. F. PERRY ET AL. normoxic conditions (e.g., armoured catfish, Ancistrus chagresi, jeju, Hoplerythrinus unitaeniatus, ihering, Hypostomus regani, Pacific tarpon, Megalops atlanticus, cascudo preto, Rhinelepis strigosa; see Table 5.2), the onset of airbreathing coincides (approximately) with the Pcrit (measured without access to air) and/or the PO2 of maximum gill ventilation (Table 5.2; Figure 5.3C). Air-breathing frequency in facultative air-breathers in general increases as water PO2 falls (e.g., Johansen et al., 1970; Graham et al., 1977; Graham and Baird, 1982; Smatresk, 1986; McMahon and Burggren, 1987; Mattias et al., 1998; Takasusuki et al., 1998; Perry et al., 2004; Oliveira et al., 2004; AVonso and Rantin, 2005; Randle and Chapman, 2005), but the eVect of aquatic hypoxia on breath (tidal) volume is less clear, with both no eVect (Graham, 1983; McMahon and Burggren, 1987) and an increase in tidal volume (Lomholt and Johansen, 1974) having been reported. Few studies have examined the eVect of hypoxic acclimation on air-breathing patterns. However, acclimation to hypoxic conditions reduced air-breathing frequency at a given level of hypoxia in two species of armoured catfish (Graham and Baird, 1982), while at the same time increasing the duration and size of each air-breath so as to augment O2 extraction (Graham, 1983). Hypoxic acclimation was also found to raise the threshold for air-breathing in one study (Bicudo and Johansen, 1979), but not in others (Gee, 1980; Graham and Baird, 1982, 1984). Air-breathing frequency increases with increasing temperature (Johansen et al., 1970; Gee, 1980; Graham and Baird, 1982; McMahon and Burggren, 1987; Geiger et al., 2000) and, like ASR, is aVected by perceived predation threat. For example, air-breathing frequency during hypoxia in both gar (Lepisosteus platyrhincus; Smith and Kramer, 1986) and gourami (Colisa lalia; Wolf and Kramer, 1987) was reduced following exposure to, respectively, an avian or piscine predation threat. Similarly, hypoxic mudminnows (Umbra limi) subjected to a disturbance to simulate a predation threat tended to air-breathe in a synchronous fashion (Gee, 1980), while a higher air-breathing frequency under hypoxic conditions was reported for the African anabantoid fish, Ctenopoma muriei, when fish were held in groups permitting synchrony of air-breathing behavior (Randle and Chapman, 2005); synchronous air-breathing is thought to reduce vulnerability to aerial predation (Kramer and Graham, 1976). The existence of PO2 thresholds for air-breathing and the dependence of air-breathing frequency on PO2 suggest that air-breathing, like ASR and branchial ventilation, can be considered a hypoxic reflex. Some evidence links air-breathing to the activation of branchial O2-sensitive chemoreceptors. For example, air-breathing is stimulated by injection of cyanide into the bloodstream and/ or ventilatory water flow of gar (L. osseus) and bowfin (Amia calva) (Smatresk et al., 1986; Smatresk, 1986; McKenzie et al., 1991), as well as the bloodstream of the obligate air-breathing African lungfish (Protopterus aethiopicus) 5. OXYGEN SENSING 215 (Lahiri et al., 1970). In bowfin, O2 chemosensors linked to air-breathing reflexes may be located in the pseudobranch, since the combination of branchial denervation together with pseudobranch ablation was necessary to eliminate air-breathing responses to hypoxia (McKenzie et al., 1991; Hedrick and Jones, 1999). Branchial denervation attenuates air-breathing responses to cyanide in lungfish (Lahiri et al., 1970), and preliminary data suggest that the hypoxia-induced air-breathing reflex in gar is also eliminated by branchial denervation (Smatresk, 1994). In general, however, little is known of the O2 chemoreceptors and aVerent pathways that mediate airbreathing reflexes in fish. For a more detailed discussion of air-breathing in fish, Graham’s (1997) book on this topic should be consulted. 2.1. The Physiological Significance of the Hypoxic Ventilatory Response The ventilatory responses to hypoxia, namely gill hyperventilation, ASR and/or air-breathing, represent attempts to maintain O2 uptake in the face of declining (aquatic) O2 availability. The rate of O2 transfer across the gill is governed by diVusive conductance, convection (ventilation and perfusion), and the blood-to-water PO2 gradient (see reviews by Randall and Daxboeck, 1984; Malte and Weber, 1985; Perry and Wood, 1989; Randall, 1990; Piiper, 1998; Perry and Gilmour, 2002; Evans et al., 2005b). During hyperventilation, increased water flow across the gill decreases the inspired-expired PO2 diVerence, raising the mean blood-to-water PO2 gradient and resulting in an elevation of arterial PO2. Hyperventilation during hypoxia therefore serves to minimize the extent of the reduction in arterial PO2 (and hence arterial O2 content) that is the inevitable consequence of lowering water PO2 (e.g., Holeton and Randall, 1967b; Wood and Johansen, 1973; Eddy, 1974; Itazawa and Takeda, 1978; Burggren and Cameron, 1980; Forgue et al., 1989; Peyraud-Waitzenegger and Soulier, 1989; Glass et al., 1990; Nonnotte et al., 1993; Bindon et al., 1994; Greco et al., 1995; Maxime et al., 2000; Soncini and Glass, 2000). This benefit of hyperventilation becomes particularly important as arterial PO2 approaches the P50 value of hemoglobin (the PO2 at which hemoglobin is 50% saturated with O2), where the steep slope of the O2 equilibrium curve means that even small diVerences in arterial PO2 can have a relatively large impact on arterial O2 content. In addition to defending arterial PO2, hyperventilation during hypoxia produces a respiratory alkalosis as arterial PCO2 is lowered by equilibration of the arterial blood with ventilatory water of lower mean PCO2 [see Table 1 in Gilmour (2001) for examples]. Elevation of red blood cell pH following from the respiratory alkalosis can, in turn, increase the aYnity of hemoglobin for O2 via the Bohr eVect, thereby aiding O2 uptake (Jensen, 1991; Jensen et al., 1998). The benefit of the hypoxic hyperventilatory response, then, is increased branchial O2 transfer, which 216 S. F. PERRY ET AL. contributes to the maintenance of metabolic rate under hypoxic conditions in oxyregulators. The drawback of the hypoxic hyperventilatory response, on the other hand, is increased energy expenditure on ventilation, the cost of which is high even at rest in water-breathing fish (Cameron and Cech, 1970; Hughes and Saunders, 1970; Edwards, 1971; Jones and Schwarzfeld, 1974; SteVensen, 1985). The maintenance of metabolic rate under these circumstances becomes a battle of diminishing returns, in which the cost of increasing ventilation to maintain O2 uptake from an environment of reduced O2 availability eventually exceeds the benefits of the O2 so obtained. The switch to oxygen conforming, often with a concomitant reduction in V_ w (e.g., Table 5.4), will lower the energetic expenditure on ventilation. ASR or air-breathing provides an alternative strategy to augment O2 uptake under hypoxic conditions. The eVectiveness of these alternative hypoxic ventilatory responses as well as their physiological costs and benefits has, however, received little attention to date. Several studies have documented lower mortality rates under hypoxic conditions for fish allowed to perform ASR, indicating that this strategy has survival value (Lewis, 1970; Kramer and Mehegan, 1981; Kramer and McClure, 1982). The impact of ASR on O2 transfer was assessed by Burggren (1982), who found that goldfish (Carassius auratus) permitted to perform ASR under severely hypoxic conditions (water PO2 = 18 Torr) were able to maintain a significantly higher arterial PO2 than those denied access to the surface, or those given access to a water–N2 interface. Although the increase in arterial PO2 was small (about 1.2 Torr), it was eVective in doubling arterial blood O2 content because it occurred near the P50 value of goldfish hemoglobin (Burggren, 1982). Similarly, facultative air-breathers provided with access to air during aquatic hypoxia also exhibit improved blood O2 status (Perry et al., 2004). In jeju, the ability to defend arterial PO2 through air-breathing during hypoxia was suYcient to avoid catecholamine mobilization (Perry et al., 2004). Access to air during aquatic hypoxia reduces mortality (Huang et al., 2008) and enables facultative air-breathers both to attenuate the fall in O2 uptake at a given water PO2 and to sustain O2 uptake to more severe levels of hypoxia (see Figure 5.6 in Graham, 1997; as well as Graham et al., 1977). Enhanced pulmonary blood flow (Smatresk and Cameron, 1982; Fritsche et al., 1993), as well as increased pulmonary contribution to total O2 uptake (Johansen et al., 1967, 1970; Burggren, 1979; Smatresk and Cameron, 1982; Graham and Baird, 1984; see also Table 5.5 and Figure 5.7 in Graham, 1997), under hypoxic conditions also attest to the value of air-breathing as an alternative hypoxic ventilatory response. The above data, while somewhat sparse, lend support to the widely accepted view that ASR or air-breathing is an eVective means of augmenting O2 uptake during severe aquatic hypoxia. However, the costs associated with ASR or air-breathing largely remain to be 5. OXYGEN SENSING 217 determined. In both cases, to the cost of ventilation itself (which has yet to be assessed experimentally for air-breathing in fish) must be added the energetic expenditure to access and/or swim at the surface (i.e., costs of locomotion and buoyancy regulation) as well as time lost to other activities (e.g., feeding) and increased predation risk (Kramer, 1983). Theoretical considerations suggest that the travel costs of ASR and air-breathing are significant and will play an important role in determining PO2 thresholds for use of these alternative strategies (Kramer, 1983). 3. O2 SENSING AND O2 SENSORS The wealth of data demonstrating hypoxic hyperventilation in waterbreathing fish (see Tables 5.1 and 5.2), clearly attests to the presence of reliable O2-sensing mechanisms. However, unlike birds and mammals, which possess a predominant single site of O2 sensing (the O2 chemoreceptors of the carotid body), fish (and other lower vertebrates) may exhibit multiple sites of O2 chemoreception (Milsom and Burleson, 2007). Surprisingly few studies have been conducted to identify the sites of O2 chemoreceptors in fish, which has made it diYcult to formulate general principles, especially considering the marked intraspecific variability that exists in those few species that have been examined. Research has focused on two crucial issues: (1) whether the O2 chemoreceptors controlling breathing are oriented to sense the external or internal environments (or both); and (2) whether the chemoreceptors are branchial and/or extrabranchial. Elements of these issues have been dealt with in previous reviews (Hughes and Shelton, 1962; Randall, 1982; Shelton et al., 1986; Perry and Wood, 1989; Smatresk, 1990; Burleson et al., 1992; Fritsche and Nilsson, 1993; Burleson, 1995; Milsom et al., 1999; Perry and Gilmour, 1999, 2002; Gilmour, 2001; Gilmour and Perry, 2007; Milsom and Burleson, 2007). 3.1. Internally versus Externally Oriented O2 Chemoreceptors Ambient hypoxia causes a lowering of blood PO2 and, depending on the severity of the hypoxia and the P50 of hemoglobin, there may be associated reductions in arterial O2 content (CaO2) of variable severity. Thus, the hyperventilation accompanying hypoxia could reflect stimulation of receptors oriented to sense the external environment (so-called external receptors) and/or receptors localized to sense the PO2 or O2 concentration of the internal environment (so-called internal receptors). A variety of techniques has been used to assess the relative involvement of external and internal receptors. The most commonly used method is the selective application of 218 S. F. PERRY ET AL. respiratory stimuli (hypoxic media or cyanide) to the external and internal compartments by injections into the buccal cavity (to preferentially stimulate external receptors) or pre- or post-branchial blood (to preferentially stimulate internal receptors). Although conflicting results were obtained from trout [notably Eclancher and Dejours (1975) reported an absence of any eVect of external cyanide on ventilation in brown trout, Salmo trutta], all teleosts that have been studied exhibit hyperventilatory responses to both external and internal cyanide (rainbow trout, Oncorhynchus mykiss, Burleson and Milsom, 1995b; Reid and Perry, 2003; channel catfish, Ictalurus punctatus, Burleson and Smatresk, 1990b; traira, Hoplias malabaricus, Sundin et al., 1999; tambaqui, Colossoma macropomum, Sundin et al., 2000). Representative ventilation recordings from gulf toadfish (Opsanus beta) before and after administration of external or internal cyanide are depicted in Figure 5.4. Of the non-teleost species that have been examined, the sturgeon (A. naccarii, McKenzie et al., 1995) and bowfin (McKenzie et al., 1991) appear to posses both external and internal receptors whereas the gar (L. osseus) appears to lack external O2 receptors linked to hyperventilation (Smatresk et al., 1986). The injection of deoxygenated blood into the ventral aorta of rainbow trout (Bamford, 1974) or dorsal aorta of sea raven (Hemitripterus americanus) (Saunders and Sutterlin, 1971) yielded hyperventilatory responses further supporting the presence of internally oriented O2 chemoreceptors. Less frequently used techniques to assess the orientation of O2 chemoreceptors include the use of perfused preparations to selectively manipulate the external and internal compartments (Milsom and Brill, 1986; Burleson and Milsom, 1993) or impairment of blood O2 transport [induction of anemia (Smith and Jones, 1982) or exposure to carbon monoxide (Holeton, 1971a, 1977)]. Perfused gill preparations (Perry et al., 1984; Perry and Farrell, 1989), while inappropriate for studying most physiological functions (Perry et al., 1985a,b), have been useful in distinguishing external and internal branchial O2 chemoreceptors. Milsom and Brill (1986) and later Burleson and Milson (1993) recorded single nerve fiber activity originating from O2 chemoreceptors within the first gill arch of yellowfin tuna (Thunnus albacares) and rainbow trout, respectively. These challenging experiments (e.g., only 5% of the 800 fibers tested in trout were actually O2-sensitive) revealed the presence of three distinct populations of receptors, those exclusively responsive to external or internal hypoxic stimuli and those responsive to both. Interestingly, Burleson and Milsom (1993) reported that receptor discharge frequency declined when PO2 was lowered below approximately 40 Torr thereby suggesting a depressant eVect of severe hypoxia on O2 chemoreceptor activity. This abrupt switch from chemoreceptor stimulation to inhibition may, in part, underlie the attenuation of the hyperventilatory 5. 219 OXYGEN SENSING Opercular pressure (mmHg) A 10 8 6 4 2 0 −2 −4 Opercular pressure (mmHg) B 0 1 2 3 4 5 0 1 2 3 Time (min) 4 5 10 8 6 4 2 0 −2 −4 Fig. 5.4. Representative traces of opercular pressure (an index of ventilation amplitude) in Gulf toadfish (Opsanus beta) administered bolus injections of the O2 chemoreceptor stimulant sodium cyanide into (A) the buccal cavity (to preferentially stimulate external receptors) or (B) caudal vein (to preferentially stimulate internal receptors). The large vertical deflections were caused by brief periods of agitation. [S. F. Perry, M. D. McDonald, P. J. Walsh, and K. M. Gilmour, unpublished data.] response that is sometimes observed once a critical level of hypoxia is achieved (Figures 5.3 and 5.5; see also Table 5.4). Because rainbow trout hyperventilate in response to acute anemia (Smith and Jones, 1982) or externally applied carbon monoxide (Holeton, 1971b, 1977), it has been suggested that internal O2 chemoreceptors (or a subset of internal receptors) in fish may respond to changes in blood O2 content 220 S. F. PERRY ET AL. 400 * 350 * * Vf (min−1) 300 * 250 200 150 100 0 40 80 PwO2 (torr) 120 160 Fig. 5.5. The eVects of acute graded hypoxia on ventilation frequency (Vf) in adult zebrafish (Danio rerio) demonstrating a linear rise in Vf with increasing hypoxia until a threshold water PO2 (PWO2) is reached at which time Vf decreases. Significant diVerences from prehypoxia values are indicated by asterisks. [Data from Vulesevic and Perry (2006).] (see Randall, 1982). While it is diYcult to conceive of a cellular mechanism for sensing O2 content, it is possible that these receptors are responding to the rate of O2 delivery, which in turn is being influenced by blood O2 content (Randall, 1982). The increase in O2 receptor discharge frequency associated with cessation of perfusion in perfused tuna (Milsom and Brill, 1986) and to a lesser extent trout (Burleson and Milsom, 1993) gills is consistent with the notion of an O2 receptor responding to changes in the rate of O2 delivery. Alternatively, the receptor cells may simply be responding to a localized decrease in PO2 owing to continuing O2 consumption during the period of ischemia. Indeed, given the low O2 capacitance of the saline used in the perfused gill preparations, it seems unlikely that the stimulation of internal chemoreceptors with a lowering of saline PO2 could reflect a reduction in O2 content. An important caveat to consider when interpreting the results of experiments meant to distinguish between internal and external receptors is that stimuli such as cyanide, if added to the external or internal compartments, may still aVect cells within the multilayered epithelium separating these 5. OXYGEN SENSING 221 compartments. Thus, as discussed by Gilmour and Perry (2007), the O2 receptors apparently responding specifically to water- or blood-borne stimuli may indeed be the same receptors (see also Section 3.2.2). 3.2. Branchial versus Extra-Branchial O2 Chemoreceptors The gills are predominantly innervated by branches of the glossopharyngeal and vagus nerves (cranial nerves IX and X, respectively), both of which carry sensory nerve fibres (Nilsson, 1983). In teleosts, the first gill arch is innervated by IX and X whereas arches 2–4 are supplied only by branches of X. Thus, bilateral sectioning of these nerves is typically used to reveal the presence of branchial O2 chemoreceptors and their role in the regulation of breathing. The pseudobranch also is innervated by branches of the glossopharyngeal nerve and has been considered as a possible site of O2 chemoreception in those species known to possess a pseudobranch (see Section 3.2.1). The invasiveness of the surgery itself may compromise normal breathing and blood gases (e.g., Saunders and Sutterlin, 1971) and it may be for this reason that so few species have yielded useful data on the eVects of bilateral gill denervation on the ventilatory responses to hypoxia. Indeed, data from a mere six species have been reported (tench Tinca tinca, Hughes and Shelton, 1962; sea raven, Saunders and Sutterlin, 1971; channel catfish, Burleson and Smatresk, 1990a; traira, Sundin et al., 1999; bowfin, McKenzie et al., 1991 and tambaqui, Sundin et al., 2000; Milsom et al., 2002). With the exception of channel catfish, each species retained some capacity to respond normally to hypoxia after bilateral gill denervation, supporting the presence of extrabranchial receptors. In some instances, there was an obvious involvement of both branchial and extra-branchial receptors in promoting the overall response. For example, in the tambaqui, branchial denervation prevented the increase in breathing frequency associated with hypoxia, indicating an exclusive branchial location for these receptors (Sundin et al., 2000). On the other hand, extra-branchial receptors appeared to be responsible for the increase in breathing amplitude during hypoxia. 3.2.1. Extra-branchial Receptors The pseudobranch (Bridges et al., 1998) has been implicated in O2 sensing (Laurent and Rouzeau, 1972; Jones and Milsom, 1982) on the basis of its morphology and the responsiveness of in vitro perfused preparations to hypoxic perfusate (Laurent and Rouzeau, 1972). Specifically, Laurent and Rouzeau (1972) demonstrated two patterns of neural discharge from aVerent vagal branches of the trout pseudobranch; medium amplitude (50–200 mV) impulses of approximately 2 msec duration (termed Type A impulses) and lower amplitude (< 50 mV) impulses of relatively long (> 4 msec) duration 222 S. F. PERRY ET AL. (termed Type B impulses). While both discharge activities were sporadic and/ or irregular under normoxic conditions, there was a significant increase in Type B activity with increasing levels of hypoxia. Although suggestive of a role for the pseudobranch in O2 sensing, the response characteristics and sensitivity of the nerve activity to hypoxia diVered markedly from nerve activities measured from mammalian carotid body or tuna first gill arch (see Milsom and Brill, 1986). Moreover, neither bilateral sectioning of pseudobranch aVerent nerve fibers (Randall and Jones, 1973) nor pseudobranch removal (Bamford, 1974) altered the ventilatory response of trout to external hypoxia. Additionally, many species known to exhibit robust ventilatory responses to hypoxia lack a pseudobranch (e.g., channel catfish). Thus, at present, there is no conclusive evidence to support a role for the pseudobranch as an extra-branchial site of O2 sensing. The results of several studies have indirectly implicated the central nervous system (brain) as a site of O2 chemoreception. Most notably, infusion of hypoxic blood into the dorsal aorta of otherwise normoxic sea raven elicited hyperventilation (Saunders and Sutterlin, 1971) and injections of deoxygenated blood into the ventral aorta caused hyperventilation but only after a significant latency period (Bamford, 1974; Eclancher and Dejours, 1975). The latency period was thought to reflect the time required for the hypoxic blood to travel to central receptors. Subsequent studies have employed a more direct approach to assess the possible role of the brain in O2 sensing in which the brain is superfused in situ with hypoxic saline. The results of these studies performed on tambaqui (Milsom et al., 2002) and bowfin (Hedrick et al., 1991) failed to provide any evidence to support the existence of central O2 chemoreceptors in fish. In the absence of any direct data to support a role for the pseudobranch or central nervous system in O2 sensing, the persistence of ventilatory responses to hypoxia in fish experiencing gill denervation may reflect the presence of extra-branchial receptors within the orobranchial cavity (Milsom et al., 2002). An alternate and not mutually exclusive hypothesis (Randall and Taylor, 1991) is that ventilatory responses attributed to extra-branchial O2 receptors may in fact arise from the release of catecholamines (adrenaline and noradrenaline) into the circulation. The principle evidence that led to the theory of a supporting role for circulating catecholamines in stimulating breathing is that these hormones are released into the bloodstream during acute hypoxia (Butler et al., 1978; Boutilier et al., 1988; Ristori and Laurent, 1989) coupled with reports that intravascular injections of catecholamines can evoke hyperventilatory responses in European eel, A. anguilla (PeyreaudWaitzenegger, 1979; Peyreaud-Waitzenegger et al., 1980). However, an examination of all available data (Table 5.6), clearly demonstrates that a myriad of responses can be elicited by injections of exogenous Table 5.6 The eVects of exogenous catecholamines on ventilation volume (V_ w), ventilation frequency (Vf), and ventilation amplitude (Vamp) or stroke volume (SV) in a variety of teleost and non-teleost species Species O. mykiss O. mykiss O. mykiss O. mykiss Injected dose or circulating levelsa 1 V_ w Vamp or SVb Comments # NC # Injected during moderate hypoxia (90 Torr) Injected during moderate hypercapnia (4.5 Torr) Injected during hyperoxia (640 Torr) 30 min infusion of A # # # 30 min infusion of NA # NC Single injection of A Single injection of NA Single injection of A # NC Single injection of A NC NC Single injection of A NC " Single injection of NA # " Single injection of NA NC NC Single injection of NA 15 nmol l NA; 75 nmol l 1 A # 12 nmol l 1 NA; 140 nmol l 1 A # 5 nmol l 1 NA; 75 nmol l 1 A 1.4 nmol l 1 NA; 164 nmol l 1 A 200 nmol l 1 NA; 5.8 nmol l 1 A 3.2 nmol kg 1 A 3.2 nmol kg 1 NA 2.1 nmol l 1 NA; 22 nmol l 1 A 4.9 nmol l 1 NA; 57 nmol l 1 A 9.4 nmol l 1 NA; 278 nmol l 1 A 15 nmol l 1 NA; 2.5 nmol l 1 A 71 nmol l 1 NA; 4.4 nmol l 1 A 207 nmol l 1 NA; 21.8 nmol l 1 A Vf NC # "!# References Kinkead and Perry, 1991 Kinkead and Perry, 1990 Playle et al., 1990 Aota and Randall, 1993 (continued) Table 5.6 (continued ) Species O. mykiss O. mykiss A. anguilla Injected dose or circulating levelsa 1 5 nmol kg 1 5 nmol kg 5.6 nmol kg kg 1 1 100 nmol kg Comments Single injection of A " NC " NC NC NC NC NC A " " Single injection of A Single injection of NA Single injection of NA Single injection of catecholamine (A/NA) cocktail during moderate hypoxia Single injection of A during summer NA " " A # # NA # # # # A # # Single injection of NA during winter Single injection of catecholamine (A/NA) cocktail Single injection of A A # # Single injection of A A; 9.4 nmol NA 10 nmol kg Vamp or SVb NC 1 1 Vf NC 1 5 nmol kg 5 nmol kg C. macropomum A 100 nmol kg 1 A 5 nmol kg 1 NA 100 nmol kg 1 NA 60 nmol kg 1 A; 15 nmol kg 1 NA 5 nmol kg G. morhua V_ w 1 1 # Single injection of NA during summer Single injection of A during winter References Burleson and Milsom, 1995b Perry and Gilmour, 1996 PeyreaudWaitzenegger, 1979; PeyreaudWaitzenegger et al., 1980 PeyreaudWaitzenegger et al., 1980 Perry et al., 1992 Milsom et al., 2002 A. calva A. nacarii S. acanthias 5 nmol kg 1 A 5 nmol kg 1 NA 30 nmol kg 1 NAd 38 nmol kg 38 nmol kg 1 1 A; NA NCc NCc Single infusion of A NC " " " Single infusion of A Single injection of NA # NC Single injection of catecholamine (A/NA) cocktail during moderate hypoxia McKenzie et al., 1991 McKenzie et al., 1995 Perry and Gilmour, 1996 a Wherever possible, we report actual measured levels of circulating catecholamines because of the inherent problems associated with comparing injected doses to levels actually achieved during acute hypoxia. b Vamp was estimated in a variety of ways including measurement of opercular or buccal pressures or linear opercular deflections determined from impedance measurements. SV was determined only in those experiments measuring true ventilation volumes and respiratory frequencies. c Data were not statistically significant when analyzed by ANOVA but paired t-tests revealed significant increases at 2.5 min postinfusion. d This dose is our own estimate based on the mean weight of the fish used in the entire study. NC, no change 226 S. F. PERRY ET AL. catecholamines. In those studies in which ventilation volumes were directly measured, the predominant response to catecholamine injection is hypoventilation (Kinkead and Perry, 1990, 1991; Playle et al., 1990; Perry et al., 1992). Of particular interest are the results of those experiments in which catecholamines were administered under pre-existing conditions of hyperventilation associated with moderate hypoxia (not severe enough, however, to elicit endogenous catecholamine release). In such cases, sudden elevation of circulating catecholamines caused abrupt hypoventilation (rainbow trout; Kinkead and Perry, 1991), a lowering of breathing frequency (spiny dogfish, Squalus acanthias; Perry and Gilmour, 1996), or no change (rainbow trout; Perry and Gilmour, 1996). With the exception of two species that show obvious hyperventilatory responses to exogenous catecholamines (European eel in summer months only, Peyreaud-Waitzenegger, 1979; Adriatic sturgeon, A. nacarii, McKenzie et al., 1995), most fish that have been examined either exhibit hypoventilation (decreased frequency and/ or amplitude) or are unresponsive to injected catecholamines (Table 5.6). Thus, there are no strong data to support a general stimulatory role for circulating catecholamines in the control of breathing during hypoxia, although in certain species it is conceivable that they play a supplementary role at very severe levels of hypoxia when O2 chemoreceptors may be inhibited (Burleson and Milsom, 1993). Extra-branchial receptors involved in the mediation of hypoxic ventilatory responses may also be found in the air-breathing organ (ABO) of air-breathing fish. Obligate air-breathers, while probably unresponsive to aquatic hypoxia (see Section 2), typically hyperventilate when exposed to aerial hypoxia (e.g., Johansen and Lenfant, 1968; Burggren, 1979; Glass et al., 1986; Sanchez et al., 2001; Perry et al., 2005, 2008; Alton et al., 2007). This eVect may be mediated by internally oriented branchial O2 chemoreceptors (see Section 3.1), a possibility for which there is some, albeit sparse, experimental evidence (Lahiri et al., 1970; see Section 2). An alternative possibility is that O2 chemoreceptors are present in the ABO. Physiological evidence supporting the existence of fish ABO chemoreceptors is mixed, with Graham et al. (1995) citing the rapidity of a gas-voiding reflex in Monopterus albus as evidence for the presence of an ABO chemosensor, but Alton et al. (2007) finding little evidence of ABO O2 chemoreceptors in Trichogaster leeri. However, histochemical evidence suggests that pulmonary neuroepithelial cells (NECs) may be present in air-breathing fish that utilize lungs (Zaccone et al., 1989, 1997; Adriaensen and Scheuermann, 1993; Kemp et al., 2003). Additional work is needed to resolve the physiological function of such pulmonary NECs in fish. 5. OXYGEN SENSING 227 3.2.2. Branchial Receptors The NECs (Figure 5.6) of the gill are considered by many to be suitable candidates for the branchial O2 chemoreceptors that mediate ventilatory reflexes in fish exposed to hypoxia (Dunel-Erb et al., 1982; Bailly et al., 1992; Sundin et al., 1998; Jonz and Nurse, 2003; Vulesevic et al., 2006; Burleson et al., 2006; Coolidge et al., 2008). The observation that these cells degranulate in response to severe hypoxia (< 10 Torr) likely was the first experimental evidence linking NECs and O2 sensing in fish (Dunel-Erb et al., 1982). Morphologically, NECs are reminiscent of O2-chemoreceptive carotid body type I cells and neuroepithelial bodies (NEBs) in mammals (González et al., 1994; Cutz and Jackson, 1999), and are considered to be phylogenetic precursors of these cells. In addition, NECs are highly conserved in fish, as they have been identified in the gills of every species in which these cells have been investigated (see Table 5.7). NECs contain neurotransmitters (see Section 4.3), particularly serotonin (5-hydroxytryptamine, 5-HT), and cytoplasmic synaptic vesicles in which these chemicals are stored (Dunel-Erb et al., 1982; Jonz and Nurse, 2003; Saltys et al., 2006). Furthermore, these vesicles are concentrated at the plasma membrane near adjacent nerve fibres. Nervous innervation of gill NECs has been documented at the ultrastructural level (Dunel-Erb et al., 1982; Bailly et al., 1992), and the association of entire populations of NECs with nerve fibres was observed in whole-mount gills of zebrafish using confocal microscopy, allowing visualization of complex innervation patterns in this tissue (Jonz and Nurse, 2003; Saltys et al., 2006). It is apparent from ultrastructural and histological studies that NECs of the filaments receive multiple sources of innervation (Dunel-Erb et al., 1982; Bailly et al., 1992; Sundin et al., 1998; Jonz and Nurse, 2003; Zaccone et al., 2006) and a component of this innervation includes sensory nerve fibres. In addition, during zebrafish development, innervation of NECs in filament primordia coincided with a significant increase in the hyperventilatory response to environmental hypoxia in lightly anaesthetized larvae, suggesting that these nerve fibres were sensory (Jonz and Nurse, 2005). NECs, some of which contain 5-HT, have also been identified in the secondary lamellae of the gill, but their innervation appears to be species specific (Jonz and Nurse, 2003; Zaccone et al., 2006; Saltys et al., 2006; Coolidge et al., 2008). The role of innervated lamellar NECs in sensing hypoxia is questionable, at least in zebrafish, because lamellae are not even present during early larval stages, when O2-sensory pathways of the gill filaments are established and the larvae can respond to hypoxia (Jonz and Nurse, 2005). Any potential role in the hypoxic response played by noninnervated NECs of the lamellae may be that of paracrine release of stored chemicals and subsequent eVects on surrounding tissue (Coolidge et al., 2008). 228 S. F. PERRY ET AL. Table 5.7 Neurochemicals (or other associated markers) identified in gill neuroepithelial cells of fish by immunohistochemistry Species Neurotransmitter Serotonin Neuropeptide Endothelin References A. calva Bowfin A. anguilla Blennius sanguinolentus C. auratus Eel Blenny Goldfish D. rerio Zebrafish Dicentrarchus labrax G. morhua Heteropneustes fossilis H. lacerdae H. malabaricus I. melas I. nebulosus I. punctatus L. osseus Sea perch Atlantic cod Indian catfish Trairão Traira Black bullhead Bullhead Channel catfish Gar Micropterus dolomieui O. mykiss Black bass Rainbow trout O. massambica Perca fluviatilis P. annectens Salmo trutta S. stellaris Tilapia Perch African lungfish Brown trout Dogfish A. calva Bowfin Conger conger H. fossilis S. canicula Sea eel Indian catfish Dogfish GoniakowskaWitalinska et al., 1995 Zaccone et al., 1992 Zaccone et al., 1992 Saltys et al., 2006; Coolidge et al., 2008 Jonz and Nurse, 2003; Jonz and Nurse, 2005; Saltys et al., 2006 Bailly et al., 1992 Sundin et al., 1998 Zaccone et al., 1992 Coolidge et al., 2008 Coolidge et al., 2008 Bailly et al., 1992 Zaccone et al., 1992 Burleson et al., 2006 As cited by Zaccone et al., 1997 Bailly et al., 1992 Bailly et al., 1992; Saltys et al., 2006; Coolidge et al., 2008 Bailly et al., 1992 Bailly et al., 1992 Zaccone et al., 1992 Zaccone et al., 1992 As cited by Zaccone et al., 1997 GoniakowskaWitalinska et al., 1995; Zaccone et al., 1996 Zaccone et al., 1996 Zaccone et al., 1996 Zaccone et al., 1996 (continued) 5. 229 OXYGEN SENSING Table 5.7 (continued) Species References S. trutta Brown trout T. marmorata A. calva Electric ray Bowfin A. anguilla B. sanguinolentus H. fossilis I. nebulosus Lampetra japonica Eel Blenny Indian catfish Bullhead Lamprey L. osseus Gar Neuropeptide Y P. annectens S. trutta O. mossambica African lungfish Brown trout Tilapia PACAP 27 and 38a H. fossilis Indian catfish Pangasus hypothalamus H. fossilis Vietnamese catfish Indian catfish S. pavo Blenny As cited by Zaccone et al., 2006 H. fossilis Indian catfish Zaccone et al., 2006 L. osseus Gar Neuronal nitric oxide synthasec H. fossilis Indian catfish Tyrosine hydroxylasec I. punctatus Channel catfish As cited by Zaccone et al., 1997 Mauceri et al., 1999; Zaccone et al., 2003 Burleson et al., 2006 Enkephalins Vasoactive intestinal polypeptide Biosynthetic enzyme Endothelial nitric oxide synthaseb a Zaccone et al., 1996; Mauceri et al., 1999 Zaccone et al., 1996 GoniakowskaWitalinska et al., 1995 Zaccone et al., 1992 Zaccone et al., 1992 Zaccone et al., 1992 Zaccone et al., 1992 As cited by Zaccone et al., 1992 As cited by Zaccone et al., 1997 Zaccone et al., 1992 Zaccone et al., 1992 As cited by Zaccone et al., 1994 As cited by Zaccone et al., 2006 As cited by Zaccone et al., 2006 Zaccone et al., 2003 Pituitary adenylate cyclase-activating polypeptide. Biosynthetic enzyme involved in production of nitric oxide. c Biosynthetic enzyme involved in production of catecholamines. Note that in some studies NECs were not serotonergic. Parts of this table were compiled from Zaccone et al., 1994 and 1997. b 230 S. F. PERRY ET AL. Fig. 5.6. Confocal image of neuroepithelial cells (NECs; green) of the gill filaments (arrow) and lamellae (arrowheads) from zebrafish labelled with antibodies against the neurotransmitter, serotonin (5-HT). Nerve fibers (red) are labeled with antibodies against a neuron-specific antigen. Scale bar 50 mm. [Modified from Jonz and Nurse (2003).] Notably, 5-HT has been described to have a paracrine role in the rat carotid body (Nurse, 2005). The O2-sensitivity of innervated or noninnervated lamellar NECs, however, has not been tested. 3.3. Chemoreceptor Plasticity If gill NECs are indeed the O2 chemosensors responsible for triggering cardiorespiratory responses to hypoxia, then it should be possible to link changes in chemoreceptor number, i.e., chemoreceptor plasticity, to diVerences in the hypoxic ventilatory response (plasticity of respiratory control), and vice versa. This possibility has been explored both during early development and in adult fish using zebrafish. During zebrafish development, a hyperventilatory response to hypoxia can be detected as early as 2 days post‐fertilization (dpf ), but increases significantly at the point (7 dpf ) 5. OXYGEN SENSING 231 where innervation of NECs of the filament primordia occurs (Jonz and Nurse, 2005), a coincidence of events that supports a critical role for filament NECs in initiating O2 ventilatory reflexes. Furthermore, chemoreceptor plasticity in adult zebrafish appears to be associated with alteration of ventilatory responses. For example, adult zebrafish acclimated to hyperoxic water for 28 days exhibited a significant reduction in the density of gill filament NECs together with a diminished ventilation frequency response to either hypoxia or cyanide (Vulesevic et al., 2006). Acclimation of zebrafish to hypoxic conditions for 28–60 days did not aVect the density of 5-HTcontaining NECs (Jonz et al., 2004; Vulesevic and Perry, 2006), nor were ventilatory responses to cyanide or acute hypoxia altered (Vulesevic and Perry, 2006). Interestingly, chronic hypoxia elicited proliferation of non-5HT-containing NECs and induced morphological changes of 5-HT-containing NECs, including increases in cell size and the growth of neuron-like processes (Jonz et al., 2004), but the functional significance of these changes remains to be determined. Plasticity of respiratory control has also been investigated in zebrafish, by exposing fish during the first week of development to hypoxia or hyperoxia, then assessing the hypoxic ventilatory response of these fish as adults (Vulesevic and Perry, 2006). Although early rearing under hypoxic conditions did not aVect adult ventilatory responses, zebrafish exposed to hyperoxia for the first week of development exhibited blunted ventilatory responses to both hypoxia and cyanide (Vulesevic and Perry, 2006). The study of Vulesevic and Perry (2006) did not examine NECs, but clearly future studies should attempt to link such developmental plasticity of ventilatory responses to changes in chemoreceptor number, morphology, and/or function. 4. CELLULAR MECHANISMS OF O2 SENSING 4.1. Cellular Models of O2 Sensing and Hypoxic Chemotransduction It is well established that both prokaryotic and eurkaryotic cells are sensitive to changes in oxygen (Bunn and Poyton, 1996). However, O2 chemoreceptors in vertebrates are specialized cells of the periphery that respond to acute changes in O2 tension and initiate appropriate cardiorespiratory responses. Much of what is known about the cellular mechanisms of O2 sensing has come from decades of research on O2-sensitive cells from mammalian systems. These include type I (or glomus) cells of the carotid body, neuroepithelial bodies (NEBs) of the lung epithelium, adrenal medullary chromaYn cells (AMCs) of neonates, and vascular smooth muscle cells. Prior to discussing a model for O2 chemoreception in the fish gill 232 S. F. PERRY ET AL. (see Section 4.2), the current working hypotheses of cellular mechanisms of O2 sensing in vertebrates will be briefly summarized, i.e., how O2 is actually ‘‘sensed’’ within the cell and how the hypoxic stimulus results in a cellular response, such as membrane depolarization and neurotransmitter release. For a more detailed account of O2 sensors and mechanisms of chemotransduction, beyond the scope of this chapter, the reader is referred to other recent review articles (López-Barneo et al., 2001, 2004; Weir et al., 2005; Lahiri et al., 2006; Kemp, 2006; Prabhakar, 2006; Buckler, 2007; Dinger et al., 2007; López-López and Pérez-Garcı́a, 2007; Peers and Wyatt, 2007; Wyatt and Evans, 2007). At present, a ‘‘universal’’ O2 sensor common to all O2-chemosensory cells has not been identified. Moreover, the molecular identity of a sensor in the carotid body, the primary model for O2 sensing, still remains a controversial issue. Two hypotheses have been proposed to explain how O2 is sensed by chemoreceptors: the ‘‘membrane hypothesis,’’ originally proposed for the carotid body (López-Barneo et al., 1988), predicts that O2 is sensed at the plasma membrane (i.e., membrane-delimited); whereas in the ‘‘mitochondrial hypothesis,’’ detection of hypoxia is linked to changes in oxidative phosphorylation and/or levels of reactive oxygen species (ROS). There is a general consensus in the mammalian literature that chemotransduction of the hypoxic stimulus is primarily mediated by inhibition of plasma membrane K+ channels and consequent depolarization (see above referenced reviews). This depolarization is believed to result in an increase in cytosolic Ca2+ levels, due to activation of voltage-gated Ca2+ channels, and the subsequent release of neurotransmitters (Weir et al., 2005). The type of K+ channel involved in the hypoxic response, however, is specific to species and cell type, and there may even be more than one type of O2-sensitive K+ channel within a single chemoreceptor. For example, chemotransduction channels include background (or leak) K+ channels (KB) that are O2-sensitive in type I cells of the rat carotid body (Buckler, 1997), Ca2+-dependent K+ channels (KCa) that are believed to play a role in hypoxic chemotransduction in rat type I cells (Peers, 1990), rat AMCs (Thompson and Nurse, 1998) and NEBs of rabbit (Fu et al., 1999), and O2-sensitive voltage-dependent Kv channel subtypes that are present in the carotid bodies of other mammals (López-López and Pérez-Garcı́a, 2007). KB channels, as found in carotid body chemoreceptors, are particularly interesting because they function independently of changes in membrane potential and conduct K+ ions at resting levels, when other K+ channels may be closed. These channels make a significant contribution to setting the membrane potential and excitability (i.e., input resistance) of the cell (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Lesage, 2003). O2 sensitivity of KB channels may then be advantageous in chemoreceptors because KB inhibition by hypoxia, which presumably occurs when the cell is 5. OXYGEN SENSING 233 at resting membrane potential, is not dependent on the channel first being activated at a particular voltage. Evidence supporting the membrane hypothesis of O2 sensing in carotid body type I cells was recently presented. Hemoxygenase-2 (HO-2) was identified within the O2-sensitive KCa channel complex in rat type I cells and was reported to act as an O2 sensor that mediated KCa channel activity through production of carbon monoxide (CO) (Williams et al., 2004), which itself activates these channels (Wang and Wu, 1997; Riesco-Fagundo et al., 2001). However, in HO-2 knockout mice, O2 sensitivity in carotid body type I cells and AMCs was unaVected (Ortega-Sáenz et al., 2006), suggesting species specificity of this putative sensing mechanism. An alternative model argues that sensing of hypoxia may occur through production of ROS, such as hydrogen peroxide (H2O2), by a membrane-bound NADPH oxidase or the mitochondrion. In NEBs of the rabbit lung, O2-sensitive K+ currents are potentiated by H2O2 and NADPH oxidase activation (Wang et al., 1996; Fu et al., 2000), and O2 sensitivity of similar channels is abolished in transgenic mice deficient in the NADPH oxidase subunit, gp91phox (Fu et al., 2000). However, in carotid body type I cells NADPH appears instead to mediate repolarization (He et al., 2002; Dinger et al., 2007), and O2 sensing in oxidase-deficient mice remains unaltered in pulmonary arterial myocytes (Archer et al., 1999) and neonatal AMCs (Thompson et al., 2002). In the latter case, evidence suggests that O2 sensing in neonatal AMCs occurs at a rotenone-sensitive site (possibly complex I of the mitochondrial electron transport chain) that couples to decreased ROS production during hypoxia and inhibition of O2-sensitive K+ channels at the plasma membrane (Thompson et al., 2007; Buttigieg et al., 2008). However, controversy surrounds ROS as a mediator of chemotransduction, and whether acute hypoxia increases or decreases ROS (Weir et al., 2005). Two models predict how mitochondrial O2 sensing, via a decrease in oxidative phosphorylation during hypoxia, may be coupled to regulation of plasma membrane K+ channels in carotid body type I cells. The first proposes that since ATP enhances the activity of a specific type of KB (‘‘TASK-like’’) channel (Williams and Buckler, 2004), during hypoxia a decrease in ATP would lead to its inhibition (Wyatt and Buckler 2004; Varas et al., 2007). The second model proposes that a fall in ATP production during hypoxia leads to an increase in the cytosolic AMP/ATP ratio, followed by subsequent activation of AMP-activated protein kinase and inhibition of O2-sensitive K+ channels, such as KB and KCa, by phosphorylation (Evans et al., 2005a; Wyatt et al., 2007; Wyatt and Evans, 2007). A recent study proposed that hydrogen sulphide (H2S) may act as a sensor or transducer of O2 and mediate responses to hypoxia in vascular smooth muscle of vertebrates, including fish and mammals (Olson et al., 2006). In this 234 S. F. PERRY ET AL. model, the concentration of intracellular H2S, an endogenous signaling molecule (Wang, 2002), is controlled by H2S production and its oxidation by available O2. The model proposes that decreased availability of O2 during hypoxia leads to reduced oxidation of H2S and its subsequent accumulation. Since H2S has been shown to have eVects on membrane potential similar to those of hypoxia, this may lead to appropriate vasoactive responses, which may be vasoconstriction or vasodilation depending on the tissue and species (Olson et al., 2006). While a definitive link between H2S and changes in membrane potential under conditions of hypoxia has not been established, H2S has been shown to activate K+ channels in vascular smooth muscle cells and induce hyperpolarization (Zhao et al., 2001; Cheng et al., 2004). To summarize, while there is little doubt that ion channels play a major role in O2 chemotransduction at the plasma membrane, a diversity of explanations as to how hypoxia is actually sensed by O2 chemoreceptors currently exists. Rather than viewing these many possibilities as contradictory, the ‘‘chemosome hypothesis’’ (Prabhakar, 2006) proposes that multiple O2 sensors are involved in the cellular response to hypoxia, thus allowing for responses across a broad range of PO2 levels with temporal flexibility. 4.2. O2 Chemotransduction in Fish Gill NECs Little is known of the process of hypoxic chemotransduction in NECs of the fish gill. However, recent studies of the response of membrane ion channels to hypoxia in NECs (Jonz et al., 2004) appear to match data from mammalian studies (Buckler, 1997). In NECs isolated from zebrafish gills, a plasma membrane K+ current was recorded that was reversibly inhibited (mean of 16% at 25 Torr) by a decrease in extracellular PO2 (Figure 5.7). Pharmacological characterization of this current demonstrated that it was resistant to traditional blockers of voltage-dependent K+ channels (tetraethylammonium, TEA; and 4-aminopyridine, 4-AP) but inhibited by quinidine, which blocks several membrane conductances, including those of voltage-independent KB channels. Sensitivity of an ion conductance to quinidine but not TEA and 4AP has previously been used to identify KB channels in other O2-sensitive cells (Buckler, 1997; O’Kelly et al., 1999; Campanucci et al., 2003). Modeling of the current-voltage relationship of the O2-sensitive current itself produced results in agreement with pharmacological data: that the O2-sensitive K+ current was carried by KB channels (Jonz et al., 2004). Importantly, this inhibition of KB by hypoxia in zebrafish NECs led to a membrane depolarization of about 6 mV. An O2-sensitive (presumably K+) current was also reported in isolated NECs of channel catfish (Burleson et al., 2006). However, in this study hypoxia produced either inhibition or potentiation of an O2-sensitive current, 5. 235 OXYGEN SENSING Nox/Rec 200 150 I (pA) Hox 100 50 0 −50 −100 −80 −60 −40 −20 0 20 40 V (mV) Fig. 5.7. Whole-cell voltage-clamp recording of an isolated neuroepithelial cell (NEC) from a zebrafish (Danio rerio) gill filament showing reversible inhibition of an outward K+ current during acute reduction of PO2 from normoxia (Nox, 150 Torr) to hypoxia (Hox, 25 Torr). During the recording, the membrane potential was progressively changed from 100 mV to 50 mV to induce the current. Rec, recovery. [Modified from Jonz et al. (2004).] and this may have been due to recording from separate cell populations (Burleson et al., 2006). Generally, a chemotransduction mechanism similar to that of mammalian O2 chemoreceptors (López-Barneo et al., 2001), such as carotid body type I cells, can be postulated for gill NECs: a decrease in tissue PO2, from either environmental or arterial hypoxia, causes membrane depolarization followed by Ca2+-dependent neurotransmitter release and activation of sensory nerve fibres. This proposed model is summarized in Figure 5.8. A number of steps of this putative pathway remain to be identified in gill NECs. Specifically, the presence of plasma membrane Ca2+ channels necessary for Ca2+dependent neurosecretion has not been investigated, and there is no convincing evidence (but see Section 3.2.2 for anecdotal reports) that depolarization following acute hypoxia does, in fact, cause release of 5-HT or any other neurotransmitter. Nevertheless, it would appear that regulation of KB channels by hypoxia is a fundamental mechanism that has been relatively conserved and may have appeared early in vertebrate evolution. 236 S. F. PERRY ET AL. Water PO2 1 Low O2 5 K+ 2 ↓ Vm 3 Ca2+ ↑ Ca2+ 6 ··· ↑ Firing rate 4 Blood PO2 Fig. 5.8. Proposed model for oxygen sensing by gill neuroepithelial cells in fish. A schematic diagram illustrates that gill neuroepithelial cells (NECs) may respond to a decrease in internal (blood) or external (water) PO2. A decrease in PO2 is sensed by the NEC and leads to (1) inhibition of membrane-bound K+ channels. This causes (2) a reduction in the membrane potential (Vm) and (3) subsequent activation of voltage-gated Ca2+ channels. An influx of Ca2+ across the membrane will (4) increase intracellular Ca2+ levels, which will (5) induce secretion of neurotransmitter(s) from cytoplasmic synaptic vesicles into the extracellular (synaptic) space. Neurosecretion from the presynaptic NEC will (6) cause activation of receptors on a postsynaptic (sensory) neuron leading to an increase in firing rate. See text for further details and Table 5.7 for putative neurotransmitters. A putative O2 sensor for NECs of the gill filaments has not yet emerged. Hypoxic sensitivity of the O2-sensitive KB current over a range of membrane potentials in NECs (Jonz et al., 2004) appears to resemble that of rat carotid body type I cells (Buckler, 1997). It is, therefore, tempting to speculate that KB channels in NECs may also be susceptible to regulation by cytoplasmic components, or linked to the mitochondrion, as proposed for mammalian chemoreceptors. However, in whole-cell patch-clamp recordings in zebrafish, NECs were dialyzed with an intracellular recording solution, suggesting that inhibition of KB channels occurred in the absence of native cytoplasmic modulators (Jonz et al., 2004), thus favoring a membrane-delimited mechanism (membrane hypothesis). Thus, as is the case for mammalian models of O2 sensing, determination of whether O2 sensing in NECs of the fish gill occurs via a membrane-delimited or mitochondrial mechanism (or both) awaits further experimentation. Such studies may point to an O2 sensor that is present in both anamniotes and amniotes, that has been conserved throughout vertebrate phylogenesis, and that may be universal among all O2 chemoreceptors. 5. OXYGEN SENSING 237 4.3. Neurotransmitters Most studies characterizing the morphology and distribution of gill NECs have conveniently exploited the expression of 5-HT in these cells for their identification (see Table 5.7). However, not all gill NECs contain 5-HT. A relatively small proportion of NECs have been described in the gill filaments and respiratory lamellae of zebrafish, goldfish, and trout that are not serotonergic but contain neurosecretory synaptic vesicles (Jonz and Nurse, 2003; Saltys et al., 2006). In addition, 5-HT-negative NECs have been reported in the gills of other fish species (Zaccone et al., 1994). It seems prudent to suggest that in these studies, 5-HT-negative NECs store an unidentified chemical substance, perhaps a neurotransmitter, and that such chemicals may play a role in neurotransmission between O2 chemoreceptor and sensory nerve fibre, or in paracrine pathways, within the gill. The role of nonserotonergic NECs as O2 chemoreceptors has not been confirmed, however, since only NECs containing monoamines (e.g., 5-HT) could be identified for patch-clamp recording and tested for O2 sensitivity (Jonz et al., 2004). Many histochemical studies have identified the presence of neurochemicals other than or in addition to 5-HT in gill NECs that may potentially contribute to chemical neurotransmission from O2 chemoreceptors, or paracrine eVects on surrounding tissue, during the hypoxic response. These are summarized in Table 5.7 and include neuropeptides (Zaccone et al., 1992, 1994, 1996, 1997, 2006; Goniakowska-Witalinksa et al., 1995; Mauceri et al., 1999) and biosynthetic enzymes (Zaccone et al., 1997, 2003, 2006; Mauceri et al., 1999; Burleson et al., 2006) involved in the synthesis of neurotransmitters, such as nitric oxide and catecholamines. It is evident that there is a wide variety of neurochemicals found in gill NECs, even within a single species. NECs of the Indian catfish (Heteropneustes fossilis), for example, contain 5-HT, endothelin, enkephalins, and both endothelial and neuronal nitric oxide synthase (Zaccone et al., 1992, 1996; Mauceri et al., 1999). However, information regarding the colocalization of specific neurochemicals to NECs, and whether these cells play a role in aVerent signaling (via innervation) or paracrine regulation during hypoxia, is incomplete. There is, unfortunately, very little available evidence in support of neurochemical transmission between NECs and sensory nerve fibres in the fish gill, and so it is diYcult to propose what events occur after NECs are depolarized by hypoxic stimulation and what signals are received by sensory nerves. Evidence from the mammalian carotid body suggests that a variety of neurochemicals, such as acetylcholine (ACh), ATP, catecholamines (dopamine, norepinephrine), 5-HT, GABA, and neuropeptides, play excitatory, inhibitory, and modulatory roles in O2 sensing (González et al., 1994; 238 S. F. PERRY ET AL. Nurse, 2005; Prabhakar, 2006; Lahiri et al., 2006). In addition, secretion of 5-HT from pulmonary NEBs stimulated by hypoxia has been demonstrated (Fu et al., 2002). It is then reasonable to predict that similar chemical signals may be utilized in the fish gill. Tyrosine hydroxylase (TH), which is involved in the biosynthetic pathway of catecholamines, has been localized to NECs of the channel catfish (Burleson et al., 2006). In addition, preliminary evidence supports the presence of cells in the gills of trout and goldfish that contain TH and VAChT, the vesicular transporter that mediates loading of ACh into synaptic vesicles (C. S. Ciuhandu and W. K. Milsom, personal communication). Interestingly, exogenous application of ACh, 5-HT, and dopamine to isolated perfused gill arch preparations in rainbow trout produced an increase in discharge frequency of aVerent glossopharyngeal fibres, suggesting activation of hypoxia-sensing pathways (Burleson and Milsom, 1995a). Although not yet supported by data at the cellular level, histochemical and physiological evidence suggests that the neurochemical basis of O2 chemoreception in the gill may involve multiple populations of NECs (i.e., 5-HT-positive and -negative NECs of the filament and lamellae), multiple neurotransmitters or neuropeptides, and perhaps a diversity of excitatory, inhibitory and modulatory mechanisms. 5. CONCLUSIONS AND PERSPECTIVES The hypoxic ventilatory response of fish has been studied for more than half a century. Yet despite the wealth of data that has been accumulated on how fish respond to hypoxia (see, e.g., Tables 5.1 and 5.2), a surprising number of questions remains. It is clear that hyperventilation is the dominant response to aquatic hypoxia, at least in unimodal water-breathers and facultative air-breathers. However, the devil is in the details, and understanding the diversity of hypoxic ventilatory responses both within andamong species will require a more systematic approach to assessing the responses and a concerted eVort to attribute diVerences in thresholds, magnitude, sensitivity, and timing of ventilatory responses to underlying diVerences in branchial gas transfer and blood gas transport. The hypoxic ventilatory reflex is clearly linked to O2-sensitive chemoreceptors with the gill being the predominant site of O2 chemosensing. Again, however, greater insight will come from pinning down details of the specific location(s) and orientation of chemosensory cells, and by making explicit links between phenomena such as chemoreceptor plasticity and modulation of hypoxic ventilatory reflexes. Moreover, little is known of the central pathways through which information from peripheral O2 sensors is integrated to elicit ventilatory chemoreflexes, nor have the neurotransmitters and receptors involved in the aVerent, central, or 5. 239 OXYGEN SENSING eVerent pathways been described in any detail (see reviews by Gilmour and Perry, 2007; Sundin et al., 2007). The chemonsensory pathways underlying ASR and air-breathing are in particular need of elucidation. Finally, while recent years have witnessed significant advances in our understanding of the cellular basis of O2 chemosensing in fish, much work remains to fully characterize the stimulus transduction mechanisms present in fish O2-sensitive chemoreceptor cells. Arguably, elucidation of these mechanisms could shed light on the current uncertainty and often conflicting views concerning the mechanisms of chemotransduction present in mammalian cells. 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N-methyl-D-aspartate receptors mediate chemoreflexes in the shorthorn sculpin Myoxocephalus scorpius. J. Exp. Biol. 206, 1251–1259. Valverde, J. C., Lopez, F.-J. M., and Garcia, B. G. (2006). Oxygen consumption and ventilatory frequency responses to gradual hypoxia in common dentex (Dentex dentex): Basis for suitable oxygen level estimations. Aquaculture 256, 542–551. Varas, R., Wyatt, C. N., and Buckler, K. J. (2007). Modulation of TASK-like background potassium channels in rat arterial chemoreceptor cells by intracellular ATP and other nucleotides. J. Physiol. 583, 521–536. Vulesevic, B., and Perry, S. F. (2006). Developmental plasticity of ventilatory control in zebrafish, Danio rerio. Respir. Physiol. NeuroBiol. 154, 396–405. Vulesevic, B., McNeill, B., and Perry, S. F. (2006). Chemoreceptor plasticity and respiratory acclimation in the zebrafish, Danio rerio. J. Exp. Biol. 209, 1261–1273. Wang, R. (2002). Two’s company, three’s a crowd: Can H2S be the third endogenous gaseous transmitter? EMBO J. 16, 1792–1798. Wang, R., and Wu, L. (1997). The chemical modification of KCa channels by carbon monoxide in vascular smooth muscle cells. J. Biol. Chem. 272, 8222–8226. Wang, R., Youngson, C., Wong, V., Yeger, H., Dinauer, M. C., Vega-Saenz de Miera, E., Rudy, B., and Cutz, E. (1996). NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines. Proc. Natl. Acad. Sci. USA 93, 13182–13187. 5. OXYGEN SENSING 253 Watters, K. W., and Smith, L. S. (1973). Respiratory dynamics of the starry flounder Platichthys stellatus in response to low oxygen and high temperature. Mar. Biol. 19, 133–148. Weir, E. K., López-Barneo, J., Buckler, K. J., and Archer, S. L. (2005). Acute oxygen-sensing mechanisms. N. Eng. J. Med. 353, 2042–2055. Williams, B. A., and Buckler, K. J. (2004). Biophysical properties and metabolic regulation of a TASK-like potassium channel in rat carotid body type I cells. Am. J. Physiol. 286, L221–L230. Williams, S. E., Wootton, P., Mason, H. S., Bould, J., Iles, D. E., Riccardi, D., Peers, C., and Kemp, P. J. (2004). Hemoxygenase-2 is an oxygen sensor for a calcium-sensitive potassium channel. Science 306, 2093–2097. Winemiller, K. O. (1989). Development of dermal lip protuberances for aquatic surface respiration in South American Characid fishes. Copeia 382–3901989. Wolf, N. G., and Kramer, D. L. (1987). Use of cover and the need to breathe: The eVects of hypoxia on vulnerability of dwarf gouramis to predatory snakeheads. Oecologia 73, 127–132. Wood, S. C., and Johansen, K. (1972). Adaptation to hypoxia by increased HbO2 aYnity and decreased red cell ATP concentration. Nature 237, 278–279. Wood, S. C., and Johansen, K. (1973). Blood oxygen transport and acid-base balance in eels during hypoxia. Am. J. Physiol. 225, 849–851. Wood, S. C., Johansen, K., and Weber, R. E. (1975). EVects of ambient PO2 on hemoglobinoxygen aYnity and red cell ATP concentrations in a benthic fish, Pleuronectes platessa. Respir. Physiol. 25, 259–267. Wyatt, C. N., and Buckler, K. J. (2004). The eVect of mitochondrial inhibitors on membrane currents in isolated neonatal rat carotid body type I cells. J. Physiol. 556, 175–191. Wyatt, C. N., and Evans, A. M. (2007). AMP-activated protein kinase and chemotransduction in the carotid body. Respir. Physiol. NeuroBiol. 157, 22–29. Wyatt, C. N., Mustard, K. J., Pearson, S. A., Dallas, M. L., Atkinson, L., Kumar, P., Peers, C., Hardie, D. G., and Evans, A. M. (2007). AMP-activated protein kinase mediates carotid body excitation by hypoxia. J. Biol. Chem. 282, 8092–8098. Zaccone, G., Tagliafierro, G., Goniakowska-Witalinska, L., Fasulo, S., Ainis, L., and Mauceri, A. (1989). Serotonin-like immunoreactive cells in the pulmonary epithelium of ancient fish species. Histochemistry 92, 61–63. Zaccone, G., Lauweryns, S., Fasulo, S., Tagliafierro, G., Ainis, L., and Licata, A. (1992). Immunocytochemical localization of serotonin and neuropeptides in the neuroendocrine paraneurons of teleost and lungfish gills. Acta Zool. 73, 177–183. Zaccone, G., Fasulo, S., and Ainis, L. (1994). Distribution patterns of the paraneuronal endocrine cells in the skin, gills and the airways of fishes as determined by immunohistochemical and histological methods. Histochem. J. 26, 609–629. Zaccone, G., Mauceri, A., Fasulo, S., Ainis, L., Lo Cascio, P., and Ricca, M. B. (1996). Localization of immunoreactive endothelin in the neuroendocrine cells of fish gill. Neuropeptides 30, 53–57. Zaccone, G., Fasulo, S., Ainis, L., and Licata, A. (1997). Paraneurons in the gills and airways of fishes. Microsc. Res. Tech. 37, 4–12. Zaccone, G., Ainis, L., Mauceri, A., Lo Cascio, P., Lo Guidice, F., and Fasulo, S. (2003). NANC nerves in the respiratory air sac and branchial vasculature of the Indian catfish, Heteropneustes fossilis. Acta Histochem 105, 151–163. Zaccone, G., Mauceri, A., and Fasulo, S. (2006). Neuropeptides and nitric oxide synthase in the gill and the air-breathing organs of fishes. J. Exp. Zool. 305A, 428–439. Zhao, W., Zhang, J., Lu, Y., and Wang, R. (2001). The vasorelaxant eVect of H2S as a novel endogenous gaseous KATP channel opener. EMBO J 20, 6008–6016. 6 BLOOD-GAS TRANSPORT AND HEMOGLOBIN FUNCTION: ADAPTATIONS FOR FUNCTIONAL AND ENVIRONMENTAL HYPOXIA RUFUS M. G. WELLS 1. Introduction 2. The Hb System 2.1. Concepts of Oxygen Transport 2.2. Oxygen-Carrying Capacity Responses 3. Proton Load May Improve Oxygen Delivery: Bohr and Root Effects 3.1. Responses to Respiratory and Metabolic Acidosis 3.2. Evidence for Visual Impairment in Functional Hypoxia 4. Environmental Temperature: Oxygen Supply and Demand 5. Endothermic Fishes: Stabilizing Internal Oxygen Tensions 6. Expression and Significance of Multiple Hb Components 7. Role of Other Globins in Hypoxia 7.1. Myoglobin: Intracellular Oxygen Transfer 7.2. Neuroglobin: Protection of Neural Tissues 8. Erythrocyte Responses to Hypoxia 8.1. Phosphate Regulation of Hb-Oxygen AYnity 8.2. Allosteric EVects of Chloride and Water 8.3. Integrative Functions of the Erythrocyte 9. Role of b-Adrenergic Receptors in Erythrocyte Oxygen Transfer 10. Novel Molecular Mechanisms for Hypoxia Protection 10.1. Putative Role of Hb-Nitric Oxide Binding 10.2. Post-Translational Modification of Hb Function 11. Hypoxia Inducible Factor HIF-1a: Evidence for Role in Hypoxic Resistance 12. Conclusions and Commentary The physiological diversity of blood oxygen transport traits in fishes appears designed to maintain tissue oxygenation under challenges from both metabolic demand and environmental oxygen supply. Brief episodes of functional hypoxia may occur during strenuous exercise when aerobic 255 Hypoxia: Volume 27 FISH PHYSIOLOGY Copyright # 2009 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(08)00006-X 256 RUFUS M. G. WELLS metabolism cannot be maintained and, when ambient oxygen tensions are low, more persistent environmental hypoxia may result. Hypoxic responses may be acclimatory (phenotypic plasticity) or adaptational (evolutionary plasticity). Fish adapted for an athletic lifestyle do not generally thrive under environmental hypoxia. Highly active species tend to have high O2carrying capacities, relatively low blood O2-aYnities, sigmoidal binding curves, marked Bohr and Root eVects, and O2-aYnity is modulated by adenosine triphosphate (ATP). Fish living in habitats that are periodically low in oxygen may also have high oxygen-carrying capacity, but generally have high blood O2-aYnities, low Hill coeYcients, and hemoglobin (Hb) function is modulated by both guanosine triphosphate (GTP) and ATP. Hb function is further regulated by erythrocyte surface adrenoceptors when present. Multiple Hb components are functionally diVerentiated in some species, but not in others, and are not generally altered by acclimation. Low heterogeneity in Antarctic fish does not appear to be an adaptation for environmental stability. The O2-binding properties of purified Hbs are diYcult to interpret ecologically and consideration of the erythrocyte environment is critical to sensible interpretation of physiological traits. The Bohr eVect depends on an ateriovenous pH gradient sustained by respiratory acidosis (CO2), whereas the Root eVect is activated by fixed acid (lactate) and, unless localized in specific retial tissues, may seriously compromise eVective oxygen transport in hypoxic situations. Reduced temperature sensitivity of Hb–O2 binding occurs in endothermic fishes that encounter thermal shifts at the gill exchange surface. Recent progress has been made in understanding the environmental thresholds for expression of factors compensating for hypoxia. These include globin synthesis, a role for Hb in regulation of the paracrine vasodilator NO, and changes in gene expression of HIF targets. Responses to hypoxia may be species specific, and comparisons become more diYcult to interpret with increasing phylogenetic distance. The challenge for the future is to place research findings in the context of physiological ecology and behavior. 1. INTRODUCTION The most successful group of vertebrates, both in terms of species diversity and habitat distribution, are the bony fishes. The diversity of modern fishes represents a long evolutionary history and complex phylogeny and any attempt to interpret adaptive features of the oxygen transport system requires not only an understanding of the inter-relationship of extant fishes, but consideration of historical selection pressures. On the basis of spiracular anatomy from Devonian fossil fishes, Clack (2007) proposed that lungs were 6. BLOOD-GAS TRANSPORT 257 present in most of the early bony fishes, whether of freshwater, estuarine, or marginal marine origin. While air-gulping supports facultative air breathing and presented evolutionary opportunities that led to the tetrapods (Graham, 1997), the hypoxic condition of aquatic habitats in the mid–late Devonian indicates widespread environmental hypoxia (Berner, 2006). The subsequent evolution of the swim bladder in teleost fishes was a major innovation permitting vertical exploitation of the water column, but presented new challenges for obligate aquatic breathers in coping with hypoxia. The evolutionary success of teleost fishes is largely due to an oxygen secretion mechanism involving special hemoglobins (Hbs) that are unique to this group. These Hbs, called Root eVect Hbs, are present in most teleosts and enable smart vision and buoyancy control (Pelster and Randall, 1998; Berenbrink et al., 2005). Oxygen secretion into the avascular eye is important for high-performance visual discrimination (Herbert et al., 2002), and for adjusting gas volumes in the swim bladder without having to visit the air– water interface to gulp air (Graham, 1997). Along with the critical importance of gas secretion, an eYcient oxygen transport system is required for aerobic performance. It is worth noting that the most athletic fish easily outperform any mammal both in terms of maximum sustained and sprint speeds, and distances travelled during migrations. This is all the more remarkable considering the density of the aquatic medium, and the much lower oxygen content of water. Fish therefore have the potential to suVer oxygen deprivation both as a result of strenuous exercise (functional hypoxia), and when living in water of low and variable oxygen content (environmental hypoxia). How then, do fish cope with hypoxia? Several reviews emphasize diVerent aspects of this question and are recommended for further reading. Molecular approaches to structure– function relationships in fish Hbs have been reviewed by Jensen et al. (1998), Weber and Fago (2004), and de Souza and Bonilla-Rodriguez (2007). The unique properties of fish Hbs manifested through the Root eVect with an emphasis on molecular interpretation have been reviewed by Brittain (2005, 2008). Nikinmaa (2006) has reviewed the role of erythrocyte membrane exchangers and pH regulation of blood-gas transport, and the eVect of temperature on oxygen transport has been reviewed by Jensen et al., (1993). Brauner and Val (2006) reviewed oxygen transport in tropical fishes and included adaptations of the gas exchange organs. Adaptive mechanisms contributing to the final step in the oxygen cascade from erythrocyte to mitochondrion have been reviewed by Wilhelm Filho (2007). The present chapter attempts to deal with the question of how fish cope with hypoxia in the context of the blood oxygen transport system, both in terms of adaptations and phenotypic adjustments (acclimation). The 258 RUFUS M. G. WELLS evolutionary plasticity of the Hb molecule and its functional interactions within the complex environment of the erythrocyte allows for adaptation to both environmental and functional hypoxia. In addition, so-called phenotypic plasticity permits individual scope for habitat exploitation under variable oxygen conditions. In earlier reviews, Wells (1990, 1999) commented on the diYculty of comparing physiological adaptations in divergent species, and on the absence of information on environmental thresholds for expression of factors compensating for hypoxia. Considerable progress has been made in the last decade. In addition to the central role of Hb, further consideration is given to other members of the globin family, and to recently discovered special roles of Hb in hypoxia protection. 2. THE Hb SYSTEM 2.1. Concepts of Oxygen Transport The design of the blood oxygen transport system in fishes is expected to have suYcient resilience to maintain an adequate oxygen supply to tissues both in the face of short-term functional hypoxia, where internal oxygen pressures fall as a result of strenuous exercise, and under transient or permanent levels of environmental hypoxia. The protein Hb is critical in meeting these expectations, because it enables oxygen to diVuse across the gas exchange membrane against its concentration gradient. Approximately 1.35 mL oxygen can be bound by 1 g Hb, and so the higher the Hb concentration, the higher the oxygen-carrying capacity of blood becomes, although optimum capacity is limited by the viscosity of blood at high hematocrit (Wells and Baldwin, 1990). Measurements of blood Hb concentration, however, do not tell the full story. The concentration of Hb within the erythrocyte appears to vary considerably among fish species and is quantified by mean cell Hb concentration (MCHC) calculated by dividing [Hb] by the hematocrit (the fraction of blood volume occupied by erythrocytes). Although there does not appear to be a systematic review of MCHC in fish, the parameter seems to correlate with both activity levels and environmental temperature (see Wells, 2005). The adjustments of the Hb system required to compensate for hypoxia are more subtle than simply increasing the erythrocyte mass. It is the oxygenbinding properties of Hb that impart the functional diversity of this remarkable protein to match oxygen supply and demand. These properties are conventionally described by the oxygen equilibrium curve (OEC), which describes the relationship between the partial pressure of oxygen (PO2) and the fraction of Hb-bound oxygen. OECs range in shape from essentially 6. BLOOD-GAS TRANSPORT 259 hyperbolic through to sigmoidal and the shape is quantified by Hill’s coeYcient, n, which will have values from 1.0 where the OEC is hyperbolic, to approximately 3 for a strongly sigmoidal OEC (Figure 6.1A). The diagnostic parameter is the P50 or PO2 at which half of the Hb content is oxygenated. The P50, however, is increased by both temperature and protons (where reduced pH expresses the Bohr eVect), and by phosphate compounds in the erythrocytes (Figure 6.1B). The principal erythrocyte organic phosphate compounds in fish are adenosine and guanosine triphosphates (ATP, GTP), which bind to Hb and decrease its aYnity for oxygen (Weber and Wells, 1989; Val, 2000). Fish blood diVers from that of other vertebrates in that it may show an extreme reaction to low pH such that full saturation in air is impossible. This phenomenon is known as the Root eVect (Figure 6.1C). These relationships are potentially confusing because the OEC is an in vitro determination and not a characteristic of a living fish. Nonetheless, there are strong reasons for believing that features of the OEC have physiological significance when considering how fish adapt to hypoxia. An example of how the OEC appears adaptive to either environmental hypoxia or to functional hypoxia is shown in Figure 6.2. Here, the catfish, which is adapted to a low oxygen environment, has a relatively high bloodoxygen aYnity, low sigmoidal coeYcient (Hill’s n-value), and blood that is relatively insensitive to pH; these features depend on the ability of tissues to function at low internal PO2, and favor the loading of oxygen in the gills. The trout in contrast, has a relatively low blood-oxygen aYnity, strongly sigmoidal OEC, and sensitivity of the P50 to pH; these features favor tissue unloading and the maximum oxygen loading and unloading occurs over the steep part of the OEC thus delaying the onset of functional hypoxia during exercise. 2.2. Oxygen-Carrying Capacity Responses The correlation between Hb concentration and the potential for functional hypoxia in fast-swimming teleost fishes is well established. For example, when pelagic and benthic tropical reef fishes are compared, the most active fishes had approximately twice the Hb content of inactive species (Wells and Baldwin, 1990). Hb concentration in elasmobranchs does not, however, correlate with their propensity for functional hypoxia (Baldwin and Wells, 1990). Fish living at sub-zero temperatures in the Antarctic seas have extremely low metabolic rates and have either very low Hb contents or none at all (Wells et al., 1980). Athletic fish, however, do not maintain a permanently high oxygen-carrying capacity, but during exercise release additional erythrocytes from the adrenergicallystimulated spleen into the circulation (Wells and Weber, 1990). Big gamefish such as tuna and marlin have 260 RUFUS M. G. WELLS Fig. 6.1. Variations in the shape and position of theoretical blood oxygen equilibrium curves (OEC). (A) Comparison of two OECs with similar half-saturation values (P50), but contrasting 6. BLOOD-GAS TRANSPORT 261 extensive Hb reserves that may result in transient hematocrit values that exceed 70% during extreme activity (Wells et al., 1986). Exercise training in rainbow trout, Oncorhynchus mykiss, however, did not lead to an increase in either oxygen-carrying capacity or P50, and optimization of O2 transport occurred through improved microcirculation (Davie et al., 1986). The question of whether exposure to environmental hypoxia leads to similar acclimatory responses is more complex. Initial exposure to environmental hypoxia in rainbow trout, O. mykiss, resulted in an increase in Hb concentration through release of erythrocytes from the spleen, but under persistent hypoxia, an erythropoietin-mediated synthesis of new erythrocytes increased the oxygen-carrying capacity of the blood (Lai et al., 2006). Oxygen-carrying capacity may also be increased following brief exposure to environmental hypoxia (about 30% saturation) via hemoconcentration and in this case a reduction in plasma volume occurred (Tervonen et al., 2006). Chronic exposure to extreme hypoxia in the sailfin molly, Poecilia latipinna, initially resulted in aquatic surface breathing behavior that diminished with time, though increased Hb concentration was maintained throughout the 6-week period of hypoxic exposure (Timmerman and Chapman, 2004). It is not clear whether the capacity responses of trout are typical of teleosts. Acclimation for 40 days under three levels of chronic hypoxia did not result in physiologically significant changes to O2-carrying capacity in turbot, Scophthalamus maximus, or sea bass, Dicentrachus labrax, although capacity in the more active sea bass under normoxic conditions was already twice that of the inactive benthic species (Pichavant et al., 2003). The tambaqui, Colossoma macropomum, is an inhabitant of the Amazonian floodplain and subjected seasonally not only to hypoxia, but also to hypercapnia and elevated levels of sulfide to which it seems tolerant. The initial response to hypoxia was a temporary increase in Hb concentration, and levels were readjusted toward normal carrying capacity within a few days as respiratory and metabolic processes compensated to maintain oxygen delivery (AVonso et al., 2002). Carter and Wilson (2006) demonstrated cooperativity coeYcients (Hill’s n-value) showing high cooperativity (n ¼ 2.4) where oxygen may be loaded or unloaded over a narrow range of PO2 on the steep part of the curve, and low cooperativity (n ¼ 1.2) where oxygen may be eVectively loaded or unloaded over a broader range of PO2. (B) The rightward shift of the OEC (increased P50) resulting from either a fall in pH (Bohr factor  =
logP50/
pH), an increase in temperature, or a high erythrocyte ATP content. These three factors improve oxygen delivery to tissues, although saturation in the gills may be compromised if environmental PO2 falls much below 80 mmHg. (C) The exaggerated rightward shift of the OEC in response to proton load (low pH) in some fish may result in failure to saturate the blood even at high PO2, and indicates a Root eVect. 262 RUFUS M. G. WELLS Fig. 6.2. A comparison of the main operational features of whole blood OEC in a fish adapted to environmental hypoxia (e.g., catfish; Grigg 1969), and an athletic fish adapted for functional hypoxia (e.g., trout; Eddy et al., 1977). The former is characterized by a relatively high O2 aYnity, low cooperativity, and small Bohr factor thus favoring O2 uptake in the gills. The latter is characterized by lower O2 aYnity, strongly cooperative O2 binding, and a larger Bohr factor favoring O2 unloading to tissues. improved sexual fitness of hypoxia-acclimated male mosquito fish, Gambusia holbrooki, largely as a result of elevated oxygen-carrying capacity. It therefore seems that increasing Hb content is a useful short-term acclimatory strategy to cope with transient environmental hypoxia, but that persistent exposure requires responses that do not compromise the substantial increase in viscosity of blood expected from an excess of erythrocytes in the circulation. These responses involve regulation of Hb-oxygen aYnity and are discussed in Section 8.1. Fish living permanently in low-oxygen environments appear to have comparatively high Hb contents. Chapman et al. (2002) introduced the concept of physiological refugia in populations of aquatic-breathing indigenous cichlid fishes in African lakes. These species had high Hb contents (>100 g l-1) and their populations were maintained in deep, hypoxic swamp refugia despite impacts from invasive species not so well adapted to environmental hypoxia. 6. BLOOD-GAS TRANSPORT 263 3. PROTON LOAD MAY IMPROVE OXYGEN DELIVERY: BOHR AND ROOT EFFECTS 3.1. Responses to Respiratory and Metabolic Acidosis When the demand for oxygen exceeds supply, the pH of the internal environment falls as a result of both accumulated dissolved carbon dioxide from respiration, and the activation of anaerobic energy production leading to lactic acid. Under these conditions of environmental or functional hypoxia, the excess of protons arising from the hydration reaction of CO2 and dissociation of lactic acid is largely buVered by the histidine groups of muscle proteins (Abe et al., 1985) and fish that are more athletic tend to have higher buVering capacities than less active species (Dickson and Somero, 1987; Wells et al., 1988). The buVering power of surface histidine components of fish Hbs, however, is rather poor with the consequence that pH eVects on Hb function may be quite dramatic (Jensen, 2004). Protons that bind to Hb signal a conformational change in the protein that results in a reduced oxygen aYnity, such that more oxygen is released in response to higher proton load. This response is called the Bohr eVect and may be quantified by  ¼
log P50/
pH (see Figure 6.1B). Values are typically negative in the physiological pH range because the blood-oxygen aYnity parameter, P50, increases with decreasing pH. Values of  are themselves pH-dependent and at very low pH, oxygen aYnity starts to increase, signaling the positive acidBohr eVect (Pelster and Weber, 1990; Weber, 2000). Negative and positive Bohr eVects are sometimes referred to as alkaline and acid Bohr eVects, respectively. The positive (acid) Bohr eVect may be of interest to protein chemists, but is probably without physiological significance. Nevertheless, teleost fish have some of the biggest negative Bohr eVects in the animal kingdom (Jensen, 1989) and the molecular structures responsible for this strong pH response appear diVerent from those in mammals or in primitive fish groups such as the agnathans (Qiu et al., 2000; Nikinmaa, 2004). Many fish show a decrease in Hb-oxygen binding capacity at low pH, even when blood PO2 is high. This phenomenon, the Root eVect, is exclusive to fishes and saturation may not even be possible under an atmosphere of pure oxygen. Counter-current multipliers in the choroid rete of the eye, and in the gas gland of the swim bladder, secrete Hb-bound oxygen and assist vision and buoyancy (Pelster and Randall, 1998). There is a characteristic loss of Hb-cooperativity at low pH and n-values may on occasion be <1.0. This feature, together with an absence of an acid-Bohr eVect, may be used to confirm the presence of a Root eVect Hb (Brittain, 2008). The eVect is in part due to an extreme conformational shift in the Hb molecule to the low aYnity state, and in part due to modification of the quaternary architecture of Hb by 264 RUFUS M. G. WELLS allosteric eVectors in the presence of protons (Bonaventura et al., 2004; Brittain, 2005). Further complexities arise from the interactions of anion exchangers and carbonic anhydrase in modulating the Bohr eVect in the complex intra-erythrocytic environment whereby carbonic anhydrase catalyzes the fast hydration of CO2 in the capillaries, thereby activating the Bohr eVect (Jensen, 2004). We should therefore view the Bohr eVect as operational under an arterial–venous pH diVerence generated by respiratory acidosis, and the Root eVect as operational under a localized circulatory metabolic acidosis. This distinction is obviously important when comparing environmental and functional hypoxias. Physiological interpretation of the adaptive significance of Bohr and Root eVects at the level of protein function, however, are fraught with diYculty because the Bohr eVect can only be measured by reference to full saturation (P100) and is quantified from the pH-induced change in P50. In practice, P100 is generally assumed when blood or Hb is in equilibrium with air at a specified pH (and possibly PCO2) and temperature. By contrast, the Root eVect can only be evaluated at a particular pH by reference to P100, or oxygen-carrying capacity determined under diVerent experimental conditions, and there is no agreed parameter for its quantification. For example, Berenbrink et al. (2005) quantified the Root eVect as the reduction in saturation of an air-equilibrated hemolysate at pH > 8.0 when the pH was dropped to pH ¼ 5.5. Accordingly, some physiologists consider the Root eVect to be a distinct characteristic of fish blood in which oxygen bound to Hb can be released by protons at constant high PO2, whereas others see it as an exaggerated Bohr eVect (Wells, 1999; Weber and Fago, 2004; Brittain, 2005). Although from a physiological viewpoint, it is diYcult to know how large a Bohr eVect needs to be before it becomes a Root eVect, the magnitude of both eVects across species is highly correlated, and it has been suggested that the Root eVect originally evolved as an extension of the Bohr eVect by varying the number of surface histidine substitutions (Berenbrink et al., 2005). There appears no simple molecular explanation at the protein structural level to explain the Root eVect. The failure to transpose a Root eVect through suspected key residues into recombinant mutant human HbA (Nagai et al., 1985), and the production of chimaeric Hbs by site-directed mutagenesis (Unzai et al., 2009) have thus far failed to demonstrate the expected structure–function correlation. The present view is that the Root eVect arises from several evolutionary pathways, each producing species-specific synergistic clusters of many residues that contribute quantitatively to saturation inhibition (Bonaventura et al., 2004; Brittain, 2008). A further diYculty in the physiological interpretation of Bohr and Root eVects arises from a consideration of the origin of the proton burden when a fish becomes hypoxic. When a respiratory acidosis generates protons from 6. BLOOD-GAS TRANSPORT 265 the hydration of CO2 and subsequent dissociation of carbonic acid (as determined from the Henderson-Hasselbalch relationship), the reaction is reversed at the gas exchange surface when the lower external PCO2 enables release of the gas to the surrounding medium, and the postbranchial eVerent blood pH rises. This is important because an operational CO2-Bohr eVect depends on the arterial–venous pH diVerence from respiratory acidosis. However, in the case of a metabolic acidosis, the acid is ‘‘fixed’’ in the sense that the quantity of protons from dissociated lactic acid cycles between venous and arterial networks without the necessary pH change required to elicit a functional Bohr eVect – in other words, blood-oxygen aYnity theoretically remains approximately the same at both the uptake and delivery sites, and continues until lactate is recycled through the gluconeogenic pathway. The proton burden from functional hypoxia is likely to be vastly in excess of the proton load from aerobic respiration. Hb function may, however, be largely insulated from this excess through the action of the erythrocyte surface Naþ/Hþ exchangers that occur in most teleost fishes (see Section 9). As a result of these conceptual diYculties, and because some investigators include the Root eVect component within measurements of the Bohr eVect, a comparison of the pH sensitivity of blood oxygen transport systems among species with diVerent athletic traits and with fish from oxygen-labile habitats does not yield a consistent picture (see Jensen et. al., 1998; Pelster and Randall, 1998; Jensen, 2004; Pelster and Weber, 2004). Acclimation of the cyprinid fish, Tinca tinca, to environmental hypoxia resulted in a marked increase in blood-oxygen aYnity brought about by a sharp decrease in erythrocyte GTP (Jensen and Weber, 1982). That there was no change in the Bohr factor remains puzzling because erythrocyte phosphates should enhance the Bohr factor. We can expect, however, that aerobically active fishes should have both marked Bohr and Root eVects, and fishes that generate significant metabolic acidosis are unlikely to thrive in hypoxic environments unless they have recourse to air-breathing. 3.2. Evidence for Visual Impairment in Functional Hypoxia The Root eVect correlates with the presence of a dense capillary network in the fish eye known as the choroid rete. This structure supports retinal oxygen flux through secretion of lactic acid in an essentially closed system, thus liberating oxygen via the Root eVect to the highly aerobic retinal cells. Berenbrink et al. (2005) surveyed the Root eVect in diverse groups of fishes and found that a fall in pH from 8.5 to 5.5 reduced Hb saturation by around 10% in sharks and lungfish, but most teleost species showed a depression of around 40% with values ranging from 2% in the catfish Silurus sp. that are 266 RUFUS M. G. WELLS benthic dwelling and perhaps have limited need for visual acuity to 80% in the cod, Gadus morhua, which are mesopelagic species. Anecdotal evidence suggests that captured and highly agitated fish suVer visual dysfunction and often appear unable to navigate solid objects. A likely explanation is that the high fixed acid load in the circulation generated by anaerobic burst activity switches the Root Hbs into the extreme low-aYnity state, thus impeding oxygen supply to the eye. There is some experimental evidence that visual discrimination is altered in functionally hypoxic fish possessing Root eVect Hbs (Herbert and Wells, 2002; Herbert et al., 2002). Vision is likely to be one of the first systems aVected during exposure to hypoxia. 4. ENVIRONMENTAL TEMPERATURE: OXYGEN SUPPLY AND DEMAND There is a close relationship between aquatic hypoxia and temperature, for as temperature increases, the obligatory decrease in oxygen solubility results in less dissolved oxygen available for respiration. Compounding the reduced availability of oxygen, metabolism—and hence biological oxygen demand—increases at warmer temperatures creating the potential for substantial hypoxic eVects. Fish living in shallow, freshwater, and estuarine regions are likely to be most aVected. The oxygen transport systems of fishes appear to be geared for hypoxic acclimation resulting from elevated temperature due to the exothermic nature of Hb-O2 binding, where the blood-oxygen aYnity decreases as temperature rises. This has the consequence of releasing more oxygen to tissues as metabolic demand increases in tandem with temperatures. The eVect may be quantified by the van’t HoV relationship:

DHa ðT1 T2 Þ=ðT2 T1 Þ
log P150 =P250 where
H is the heat of oxygenation and T1 and T2 are the lower and higher 1 2 and P50 are the Hb-oxygen aYnity coeYcients at temperatures in K, and P50 T1 and T2, respectively. Values close to zero indicate relative temperatureindependence of oxygen binding (Weber and Wells, 1989). Accordingly, fish living in thermally stable or seasonally changing habitats tend to show normal temperature sensitivity with negative values for
H (Weber and Wells, 1989) although
H may itself be temperature-dependent (Fago et al., 1997). The cyprinid fishes, Carassius carassius and C. auratus, showed a decrease in blood-oxygen aYnity following exposure to higher temperatures, and a concomitant increase in gill surface area, thus ensuring that oxygen turnover to tissues was maintained (Sollid et al., 2005). Thermal acclimation to warmer temperature partly reverses the change in oxygen aYnity so that oxygen 6. BLOOD-GAS TRANSPORT 267 uptake in the gills can be improved (Albers et al., 1983). In an extreme stenothermal example, whole blood oxygen equilibrium studies with Antarctic fish acclimated to 1.5 C and 4.5 C revealed
H values comparable to those expected from seasonal adjustments in temperate fishes, though with low P50 values (Tetens et al., 1984). A seasonal decrease in blood-oxygen aYnity is not due solely to the entropy of Hb–O2 binding. The allosteric modulator GTP also increased in summer-acclimated eels, Anguilla anguilla, further assisting oxygen turnover to actively metabolizing tissues (Andersen et al., 1985). Powers et al. (1979) compared the temperature eVects on oxygen binding of Hbs and whole blood from a range of tropical and temperate teleosts, and, interestingly, diVerences in the thermal sensitivity of O2 binding could not be shown for purified Hb, but only in the presence of phosphate cofactors. This study emphasizes the need for care in ecological interpretation of data from purified Hb. A high value for
H in the cool temperate teleost, Odax pullus was hypothesized to compromise the warmer limits of its geographic range (Brix et al., 1998a). By contrast, intertidal triplefin fishes subjected to rapid fluctuations in temperature showed low values of
H at low pH, suggesting that oxygen transport is maintained despite rapid thermal shifts (Brix et al., 1999). 5. ENDOTHERMIC FISHES: STABILIZING INTERNAL OXYGEN TENSIONS A diVerent thermal problem arises for the several groups of fishes that have developed partial endothermy. Tunas, swordfishes, and lamnid sharks generate heat in the red, mitochondrial-rich swimming musculature that is exchanged via counter-current multiplication in order to generate more thermodynamically eYcient processes in swimming, and sensory information processing (Block and Carey, 1985; Altringham and Block, 1997; Block et al., 2001; Fritsches et al., 2005). When cool blood with a negative
H (as is usually the case) meets warm blood in the gills, there is a potential for loss of oxygen from the arterial to venous circuit. The Hbs of fish with internal temperature gradients of 10–20 C appear to show temperature-insensitive oxygen binding as indicated by values of
H that are close to zero. For example, the albacore tuna, Thunnus alalunga, had a reversed temperature eVect (
H = +1.72) from 10 C to 30 C, closely matching the maximal thermal gradient from ambient water to core body temperature (Cech et al., 1984). Interestingly, a strong Bohr factor ( = 1.17) was found, yet negligible Root and cooperativity eVects (Hill’s n = 1.1) were also found. These results were contested by Jones et al. (1986) who reported marked 268 RUFUS M. G. WELLS sigmoidal whole blood binding curves (Hill’s n ¼ 1.72) and a smaller Bohr eVect ( ¼ 0.59) for the kawakawa, Euthynnus aYnis. The discrepancy could be explained either if full saturation was not experimentally achieved in T. alalunga or if the much smaller species E. aYnis is not significantly endothermic. The reverse temperature eVect in Hb component I isolated from bluefin tuna, Thunnus thynnus, is explained by a large Bohr factor in which the protons bind endothermically resulting in a positive value for
H (Ikeda-Saito et al., 1983). The temperature eVects on whole blood from T. thynnus were similar to those seen in Hb solutions, suggesting that erythrocyte ATP or other cofactors were not responsible for mediating the observed temperature insensitivity (Brill and Bushnell, 2006). Recent work has furthermore demonstrated that
H itself is temperaturedependent in tunas. Clark et al. (2008) working with southern bluefin tuna, Thunnus maccoyii, showed that the temperature eVect was reversed at low temperatures, but whole blood oxygen binding was essentially independent of temperature at warmer temperatures. The investigators proposed that this was to avoid premature O2 oV-loading around the heat exchanger. The bigeye tuna, Thunnus obesus, faces a unique hypoxic conflict. In addition to extreme depth excursions exposing the gills to a temperature shift from as high as 28 C down to 7 C, the fish spends considerable periods in the hypoxic oxygen minimum zone and thus faces both functional and environmental hypoxia (Lowe et al., 2000). The authors found that unlike other tunas, T. obesus blood has in addition to temperature insensitivity an exceptionally high aYnity for oxygen, coupled with a large Bohr factor, thus appearing to optimize both uptake in the gills and release to warmer tissues. Close examination of the functional properties of Hb in the porbeagle shark, Lamna nasus, has revealed that the temperature eVect is saturation dependent, with a normal temperature eVect at low saturation (deoxygenated, venous blood), and a reverse temperature eVect at high saturation (reflecting arterial blood), thus oxygen transport is protected despite the thermal gradient (Larsen et al., 2003). This special feature is not, however, solely mediated via the intrinsic properties of Hb, but induced by ATP–Hb binding. A comparable mechanism seems to operate in other endothermic fishes such as the striped marlin, Tetrapterus audax (Weber and Jensen, 1988). 6. EXPRESSION AND SIGNIFICANCE OF MULTIPLE Hb COMPONENTS Nearly all animals have more than one kind of Hb present in their erythrocytes. Multiple forms of Hb are particularly common in ectothermic animals, especially in fish that are expected to cope with fluctuating conditions 6. BLOOD-GAS TRANSPORT 269 of environmental oxygen and temperature (Weber, 1990; Wells, 1999). Our knowledge of the functional significance of multiple Hbs is largely due to the continuing investigations of R. E. Weber and colleagues, and the search for examples of Hb heterogeneity that might suggest adaptation to hypoxia in fish has attracted considerable interest (for reviews see Weber and Jensen, 1988; Weber and Wells, 1989; Weber, 1990, 1996, 2000; Weber and Fago, 2004). Two groups of freshwater fishes have received special attention. The anguilliform eels are likely to be found in water low in dissolved oxygen and subjected to environmental hypoxia. Hb components isolated from the European eel, Anguilla anguilla, can be broadly resolved by electrophoresis into anodic Hbs that have low oxygen aYnities accompanied by marked Bohr and Root eVects, and cathodic Hbs that lack significant pH eVects on either oxygen aYnity or cooperativity. The cathodic Hbs are assumed to confer protection against hypoxia and acidosis (Fago et al., 1995; Tamburrini et al., 2001). Hemoglobins isolated from other eels, the common moray, Muraena helena (Pellegrini et al., 1995), brown moray, Gymnothorax unicolor (Tamburrini et al., 2001), and the conger eel, Conger conger (Pellegrini et al., 2003), show a similar pattern of functional heterogeneity. The deep-sea eel, Symenchelis parasitica, while showing similar heterogeneity, possesses very little of the cathodic component and this feature is consistent with a more stable environment (Weber et al., 2003). Salmonid fishes, and in particular rainbow trout, Oncorhynchus mykiss (formerly Salmo gairdneri), have also been well researched. In contrast to eels, salmonids tend to inhabit well-aerated habitats and are athletic fish. Accordingly, trout are expected to develop some degree of functional hypoxia during strenuous exercise (McKenzie et al., 2004). As in eels, trout possess multiple Hbs that resolve into anodal and cathodal components whereby the latter are largely insensitive to pH and allosteric eVectors (Weber et al., 1976a). Though the Hb system of rainbow trout, O. mykiss, has been studied for decades, only recently have further functionally heterogeneous components been detected, bringing the current total from four to nine (Fago et al., 2002). A comparison of the Hb system in eels and trout does not suggest that Hb multiplicity is a particular characteristic of fish living in oxygen-deficient habitats. Plaice, Platessa platessa, and flounder, Platchthys flesus, contain 8 Hbs apiece, though there is little functional diVerentiation and their predominantly anodic components appear adapted for hypoxia through low sensitivity to phosphates and pH (Weber and de Wilde, 1976). Cathodic Hbs are not therefore a prerequisite for hypoxia adaptation. Hb multiplicity also occurs in cartilaginous fishes (Dafre and Reischl, 1997; Galderisi et al., 1996) and in primitive teleosts such as the sturgeons (Luk’yanenko, 1978). Hb multiplicity was compared in three phylogenetically distant teleosts inhabiting the same oxygen-deficient tropical billabongs: the Hb system in 270 RUFUS M. G. WELLS the osteoglossiform saratoga, Scleropages jardinii, was characterized by a single Hb component, the elopiform tarpon, Megalops cyprinoides (a facultative air breather) by one major and one minor component, and the perciform barramundi, Lates calcarifer, by seven components (Wells et al., 1997). The latter species is a more advanced teleost. GTP was the principal modulator of Hb function in the water breathers, and ATP in the active, airbreathing tarpon. Stronger evidence for the adaptive value of functionally heterogeneous Hbs is to be found among closely related species occupying diVerent ecological niches. A functional analysis of Hb components isolated by isoelectric focusing from congeneric species of triplefin fishes (Family Tripterygiidae) found that Hbs less sensitive to pH and temperature occurred in species found in the oxy-labile and thermally unstable habitats of rock pools (Brix et al., 1999). Further, the functional attributes of Hb components from marine kyphosid fishes appeared to support the distinction in temperature sensitivities of oxygen binding in relation to the thermal habitats of the diVerent species investigated (Brix et al., 1998). In another example, the African cichlid fishes of Lake Victoria have rapidly evolved into specialized niches, and both Hb heterogeneity and the Hb–O2 binding characteristics of hemolysates appeared to correlate with hypoxia (Verheyen et al., 1986), although cichlids generally lacked the pH-insensitive cathodic components (Weber, 1990). The eel pouts (Family Zoarcidae) are inactive, benthic, and generally deep-sea species occurring at all latitudes. In an extreme example, Thermarces cerberus is associated with deep sea hydrothermal vents where temperatures are high and oxygen content is very low; its Hbs are not functionally diVerentiated and showed pH and phosphate sensitivity, but much higher aYnities than other zoarcids (Weber et al., 2003). By contrast, nototheniid fishes from the cold, thermally stable Antarctic seas where oxygen content is generally high showed far less Hb heterogeneity than temperate and tropical fishes, and their Hbs showed marked phosphate and pH-sensitive oxygen binding (reviewed by di Prisco et al., 1998; Wells, 2005). Five components have been isolated from the cryopelagic species, Pagothenia borchgrevinki (Cocca et al., 2000), and three from Pleuragramma antarcticum (Tamburrini et al., 1997). The Hbs from P. borchgrevinki are functionally distinct and are presumed to support the more active behavior of this species (Riccio et al., 2000). Other benthic notothenioids have either a single Hb component (Kunzmann, 1991), or an additional minor component comprising less that 5% total Hb (di Prisco et al., 1991). Interestingly, the nototheniid, Notothenia angustata, occurs at much lower latitudes in southern New Zealand and also shares this pattern of low Hb diversity (Fago et al., 1992).The minor fraction is expressed in greater amounts in Gobionotothen gibberifrons and has been correlated with hypoxic adaptation (Marinakis 6. BLOOD-GAS TRANSPORT 271 et al., 2003). The Antarctic species Anotopterus pharao and Macrourus holotrachys are neither endemic nor members of the Notothenioidei, and have four to five Hb components (Kunzmann, 1991). The number of Hb components present in Arctic fishes is generally higher than that of Antarctic fishes (di Prisco and Tamburrini, 1992; D’Avino and di Prisco, 1997). Two hypotheses have been advanced to explain these observations. Arctic fishes are frequently distributed in a latitudinal cline covering significant thermal variation whereas Antarctic fishes are thermally isolated by the circumpolar seas and thus face less environmental perturbation in oxygen supply and demand. Alternatively, the Arctic fishes are pleisiomorphic, and contrast with the monophyletic origins of the dominant nototheniods of Antarctica leading to lower diversity of Hb components (see reviews by Wells, 2005; Verde et al., 2006). A comparison of Hb multiplicity in several groups of fish is shown in Figure 6.3 and does not suggest any correlation between athletic fish groups likely to experience functional hypoxia, those likely to experience environmental hypoxia, or phylogenetic relationships. At the population level, there is scant evidence for Hb polymorphisms that may be linked to environmental oxygen tensions, though polymorphisms appear common in salmonids and sturgeons (Giles, 1991; Soldatov, 2002). Studies with Arctic and cold temperate fishes have revealed Hb polymorphisms in Atlantic cod, Gadus morhua (Fyhn et al., 1994; Brix et al., 1998b), and turbot, Scophthalamus maximus (Imsland et al., 2000), but links Selachiformes (7) Anguilliformes (9) Pleuronectiformes (4) Perciformes (20) Scombroids (7) Salmonids (4) Notothenioids (14) 0 1 2 3 4 5 6 7 8 9 10 11 12 Hb components Fig. 6.3. Number of hemoglobin (Hb) components in representative species from distinct phylogenies (mean  S.D.). The taxonomic hierarchies are not equivalent and the number of species represented within each group is in parentheses. 272 RUFUS M. G. WELLS to environmental oxygen are circumstantial. A predominance of low pHsensitive Hb components may be linked with more variable environmental temperatures in the turbot (Samuelsen et al., 1999). Hb multiplicity in the Atlantic croaker, Micropogon undulates, revealed a complex polymorphism with functional phosphate-mediated diVerences in O2 binding, but no clear ecological interpretation emerged (Shelly and Mangum, 1997). On the basis of a ‘‘division of labor’’ into pH- and phosphate-insensitive high-aYnity cathodic components, and generally lower aYnity anodic components with marked phosphate and Bohr eVects, a loose categorization of fish into Type I species having only anodic Hb and Type II with both anodic and cathodic Hb (incorporating an oxygen reserve for hypoxic episodes via cathodic Hb) has been proposed (Weber, 1990, 2000; Weber et al., 2003). The mudfish, Labeo capensis, is an exception, in which both anodal and cathodal components are phosphate-sensitive (Frey et al., 1998). Despite the large number of studies documenting the presence of multiple Hbs in fish, however, caution should be exercised before accepting that any isolated component reflects the in vivo condition, for Hbs are fragile molecules easily modified through the sometimes harsh purification treatments. Characterization of Hb components is most often dependent on electrophoretic mobility corresponding with diVerences in molecular surface charges; neither neutral substitutions nor internal structural diVerences may be revealed. In addition, the formation of hybrid globin complexes might not represent the Hb species in the intact erythrocyte (see Soldatov, 2002). Physiological interpretation based on the functional properties of isolated, purified Hb components should be made with caution. Giardina et al. (2004) pointed out that in vivo functionality may be markedly diVerent where the pH range under which Bohr and Root eVects operate are modulated by red cell organic phosphates, and the operational range is shifted to a higher pH range. Unusual whole blood eVects may also arise from the interactions of diVerent Hb isoforms with functionally distinct binding characteristics to produce cooperativity coeYcients with values less than unity (Deker and Nadja, 2007). At present, there is insuYcient evidence for adaptive diVerences in fishes from diVerent oxygen environments with respect to interspecies variance in ratios of cathodal to anodal components. There remain, however, convincing ecological associations with the functional attributes of the dominant Hb component. A key question is, of course, whether the pattern of Hb components alters when a fish is exposed to chronic hypoxia. Surprisingly, there is very little evidence for acclimatory adjustments in Hb isoforms that might compensate hypoxic exposure. Marinsky et al. (1990) chemically rendered rainbow trout, O. mykiss, anaemic and then observed diVerences in the Hb pattern of fish recovering in controlled normoxic and hypoxic environments. Acclimation 6. BLOOD-GAS TRANSPORT 273 of goldfish, Carassius auratus, to temperature cycling also resulted in a shift in the Hb isomorph profile (Houston and Gingras-Bedard, 1994). The authors considered a rearrangement of the globins rather than de novo synthesis of new Hbs, which might be adaptive to the altered oxygen demands. Exposure to hypoxia or increased temperature of the mudfish, Labeo capensis, resulted in raised Hb concentration, but aVected neither the pattern of Hb multiplicity nor the intrinsic Hb oxygen-binding properties, indicating that the principal acclimatory mechanism of the Hb system to hypoxia is elicited through phosphate–Hb interactions (Frey et al., 1998). More recently, several species of African cichlid fishes were raised from an early stage in development under hypoxic conditions, and diVerences in the Hb systems contrasted with normoxic controls (Rutjes et al., 2007). The authors demonstrated the expected increases in oxygen-carrying capacity, and phosphate shifts increasing Hb-O2 aYnity, but have critically shown that the synthesis of high-aYnity Hb isomorphs occurs and a clear case of adaptive response seems justified. The most recent study of changes in the Hb system following hypoxic exposure has been conducted by Campo et al. (2008). The gilthead sea bream, Sparus auratus, has two Hb components thatshare a common b-globin gene. Under normoxic conditions (7 mg L 1 oxygen), the components are present in approximately equal proportions, but these change progressively as fish are rendered hypoxic (2.5 mg L 1 oxygen). Campo et al. (2008) were not, however, able to demonstrate functional diVerences between the two components, either in intrinsic oxygen aYnity, ATP modulation, Bohr and Root eVects, or cooperativity, and hence the acclimatory value of the component ratio is unknown. 7. ROLE OF OTHER GLOBINS IN HYPOXIA 7.1. Myoglobin: Intracellular Oxygen Transfer It is evident from the appearance of fish fillets in the supermarket that more athletic fishes have both more red muscle and intense colour, indicating higher myoglobin (Mb) content. The concentration of monomeric heme protein, Mb, correlates with mitochondrial oxygen demand in a variety of muscle tissues and in the oxy-form diVuses at about 1/20th the rate of diatomic oxygen, thereby contributing usefully to the oxygen cascade (Wittenberg, 2007). The presence of Mb in both locomotory and ventricular muscles suggests a role in maintaining aerobic metabolism when activity increases. The swimming muscles in fish are diVerentiated into fast-contracting fibers deployed during anaerobic burst activity, and slow-contracting fibers 274 RUFUS M. G. WELLS rich in mitochondria and Mb that are used predominantly during sustained aerobic swimming. Direct experimental evidence obtained using optical fiber sensors points to a role for Mb in providing improved intramuscular oxygen tensions during strenuous swimming in free-swimming rainbow trout, Oncorhynchus mykiss (McKenzie et al., 2004). Moreover, the authors showed that the PO2 in the diVerentiated fish muscle was significantly higher than that typically found in other vertebrates. The presence of myoglobin in the fish ventricle appears to be an insurance against myocardial hypoxia. Functional studies on perfused hearts of the myoglobin-rich sea raven, Hemitripterus americanus, and the myoglobinpoor ocean pout, Macrozoarces americanus, showed that the former species was better able to maintain oxygen consumption under hypoxia, but failed to do so following chemical blocking of Mb (Bailey and Driedzic, 1986). However, electrically paced ventricular strips from the sculpin, Myoxocephalus octodecimspinosus, suggested that Mb did not play a critical role in maintaining performance under normoxia (Canty and Driedzic, 1987). Assumptions about tissue hypoxia based on dissolved oxygen levels in a perfusate are also problematic in in vitro studies of this kind. Further, Mb is not expressed in a range of unrelated species of sedentary fish, suggesting that Mb plays a role in protection against functional rather than environmental hypoxia (Grove and Sidell, 2002). Further research is needed in order to see whether these observations can be extrapolated to include elite swimmers such as carangids or gamefish. However, Mb concentration increased with cardiac growth in tuna (Thunnus thynnus), and showed a breakpoint increase at the phase of development coinciding with high-performance swimming (Poupa et al., 1981). Current research using molecular tools has focused on questions about the adaptive significance of globin gene expression. Fraser et al. (2006) demonstrated that hypoxia-tolerant common carp, Cyprinus carpio, increased expression of Mb under chronic hypoxia. Roesner et al. (2008) have extended these observations using real-time PCR and report that only Mb, but not a- or b-globin, neuroglobin or cytoglobin, expression is altered under hypoxic challenge in the carp. Enhanced expression of Mb was also observed in zebrafish, Danio reria, acclimated to severe hypoxic conditions for 3 weeks (van der Meer et al., 2005). The plasticity of Mb expression in Atlantic cod, Gadus morhua, was also evident during temperature acclimation when both demand for oxygen and its solubility are altered, and in addition to modulating oxygen diVusivity, the Mb appeared also to play a role in scavenging nitric oxide (Lurman et al., 2007; see Section 10.1). In the chronically cold environment of the Antarctic seas, the loss of both Mb and Hb expression in icefishes (Family Channichthyidae) has occurred, but there are associated costs of reduced cardiac performance and a requirement to re-engineer the circulatory system (Sidell and O’Brien, 2006). 6. BLOOD-GAS TRANSPORT 275 Gracey et al. (2001) measured the critical oxygen tension for the hypoxiatolerant and burrow-dwelling goby, Gillichthys mirabilis, then determined the transcriptional response to hypoxia using cDNA microarray technology. The authors noted diVerential patterns of gene expression in various tissues directed toward closing down a number of major energy-requiring pathways and changes to heme metabolism. Further studies of gene expression under appropriate physiological conditions are required for other species of fish that regularly experience either functional or environmental hypoxia. Functional studies on the oxygen-binding characteristics of fish myoglobins are sparse. Nichols and Weber (1989) correlated Mb-oxygen binding aYnities with demand for oxygen in fish from diVerent habitats. Mb extracted from the red swimming muscles of various fish species point to adaptive diVerences according to the activity of the species, such that inactive species living at low environmental temperatures (e.g., buValo sculpin, Enophrys bison) had comparatively lower aYnities when compared to more active species (e.g., yellowfin tuna, Thunnus albacares) living at warmer temperatures. This diversity of function is supported by Marcinek et al. (2001) who compared highly athletic endothermic and ectothermic fishes and related functional diVerences to the requirement to transfer oxygen at normal muscle operating temperatures. Optimization of Mb-O2 aYnity to tissue temperatures suggests that most animals are capable of adaptive adjustments to oxygen availability (Wittenberg, 2007). The implication of these studies on Mb is that hypoxia has a major eVect on the PO2 of the mitochondrial environment and that Mb may serve to buVer against such changes. 7.2. Neuroglobin: Protection of Neural Tissues The presence of novel heme compounds in the neural tissues of vertebrates has recently been described (Burmester et al., 2000) and has led to questions about their role in protecting neurons against either too much, or too little oxygen. These neuroglobins (Ngb), together with Hb and Mb, appear to be expressed in zebrafish (Danio rerio) in response to activation of hypoxia-inducible genes (Roesner et al., 2006). Using real-time PCR, the authors noted marked increases in Ngb mRNA in brain but not retinal tissues, and Mb mRNA in cardiac tissue when fish were exposed to hypoxic insult. These observations are consistent with the diVerential eVects of hypoxia whereby the anoxic crucian carp brain (Carassius carassius) maintained functionality (Nilsson, 2001), but the fish suVered severe visual impairment (Johansson et al., 1997). Whether Ngb function parallels the role of Mb in myotome or is involved in detoxification of reactive oxygen species and prevention of apoptosis is not 276 RUFUS M. G. WELLS known. However, Hundahl et al., (2006) described the oxygenational characteristics of Ngb and reported non-cooperative binding and the absence of a Bohr eVect. These characteristics allow for a role in either oxygen storage or enhanced diVusivity of oxygen. On this basis the authors proposed that neural excitability was extended under hypoxia. Ngb from zebrafish has an exceptionally high aYnity for oxygen (P50 ¼ 1 mmHg) and is co-located with mitochondria (Fuchs et al., 2004). Up-regulation of Ngb after extended hypoxia in mice suggests a universal mechanism of neural protection from hypoxic injury (Hundahl et al., 2005). Recently discovered Globin-X, occurring only in fishes and amphibians, shares a common origin with Ngb and appears to be an ancient globin arising at the divergence of bony fishes and tetrapods (Roesner et al., 2005). The functions of Globin-X, together with ubiquitous cytoglobins (Burmester et al., 2002), remain unclear in relation to their expression under hypoxic challenge. 8. ERYTHROCYTE RESPONSES TO HYPOXIA 8.1. Phosphate Regulation of Hb-Oxygen AYnity The erythrocyte phosphates are a main line of both acclimatory and adaptational defence in hypoxia protection. ATP is present in all fish erythrocytes, but fish regularly exposed to aquatic hypoxia also tend to have high proportions of GTP; an alternative strategy is present in air-breathing fish that have 2,3-diphosphoglyceric acid (DPG) (e.g., armoured catfish, Pteroglopichthys spp.) or lungfish, Protopterus spp. that have inositol phosphates (IP) (Val, 2000). These phosphates bind to specific sites in the central cavity of the Hb tetramer and stabilize the structure in the low-aYnity, deoxy conformation. Known as allosteric regulation, this feature of Hb provides for adjustments to oxygen aYnity via changes in the molar ratio of phosphate cofactor: Hb. A decrease in phosphates results in increased Hb oxygen aYnity, thus favoring full saturation in the gills. The mechanism was first described for the eel (Wood and Johansen, 1972) with the cofactor GTP exerting the main influence (Weber et al., 1976b). Subsequently, many fish living in hypoxic waters have been shown to use a similar survival strategy (Weber, 1996; Val, 2000). The distribution of phosphate cofactors across species suggests a correlation with environmental hypoxia. GTP appears to predominate in species such as eels that are regularly subjected to environmental hypoxia, whereas ATP predominates in fish such as trout that are more likely to experience functional hypoxia. The distinction between ATP and GTP on the one hand 6. BLOOD-GAS TRANSPORT 277 and DPG and IP on the other is important because their synthesis proceeds according to aerobic and anaerobic pathways, respectively. Trinucleotide production is linked to oxidative phosphorylation, whereas DPG and IP production is linked to the glycolytic pathway. Thus, under hypoxic challenge, shifting from aerobic to anaerobic metabolism with accompanying acidosis favors the sharp reduction of ATP and GTP, or, if present, an increase in DPG. This in turn results either in an increase in blood-oxygen aYnity (reduced ATP, GTP), favoring oxygen uptake in the gills, or a decrease in blood-oxygen aYnity (increased DPG, IP), promoting eYcient oxygen unloading to tissues. One further role for NTP regulation deserves mention. Associated with the mass-specific reduction in oxygen requirements with increasing size in fish is a concomitant increase in oxygen aYnity. In the piranha, Serrasalmus rhombius, the growth-correlated reduction in P50 is due entirely to reduced erythrocyte GTP rather than changes to the Hb isoforms (Wood et al., 1979). It is not known whether larger individuals have less capacity for hypoxia acclimation than do smaller individuals. Weber (1996) has reviewed the distribution of phosphate compounds in the erythrocytes of some Amazonian fish. However, the control mechanisms signaling regulation of erythrocyte organic phosphates remain unclear (Nikinmaa, 2002). In addition to the direct allosteric control of Hb-O2 aYnity by phosphates, both ATP and GTP modulate the Bohr eVect in, for example, the anodic Hb of the eel, Gymnothorax unicolour (Tamburrini et al., 2001). The enhanced binding of protons to the anodic Hb component of the eel, Anguilla anguilla, in the presence of GTP reveals the obverse of the Bohr eVect—i.e., the Haldane eVect (Brauner and Weber, 1998). The binding of ATP or GTP to the anodic components of several species of fish is essential for expression of the Root eVect; reduction in the NTP:Hb ratio during hypoxic acclimation, however, is not suYcient to diminish the strength of the Root eVect (Pelster and Weber, 1990). The presence of high concentrations of the cofactor DPG in erythrocytes is typically associated with mammals, but also occurs in the air-breathing Amazonia catfish, Hoplosternum littorale (Weber et al., 2000). In addition, lungfish erythrocytes contain the potent modulator inositol polyphosphate (IP; Val, 2000). Both DPG and the typical fish cofactors ATP and GTP are present in the erythrocytes of the catfish. Weber et al. (2000) reported pronounced but diVerentiated phosphate eVects on the cathodal Hb, DPG<ATP<GTP, and concluded that the catfish Hb system appeared to impart no selective advantage for DPG binding given the lower sensitivity to DPG. An alternative interpretation considers the strategies for regulation of red cell organic phosphates under hypoxic pressure, whereby the modified pathway of red cell glycolysis leads to an accumulation of DPG, and via suppression of oxidative phosphorylation, a marked reduction of ATP and 278 RUFUS M. G. WELLS other trinucleotides (see Val, 2000). Accordingly, it may be predicted that the typical air-breathing strategy of elevated DPG or IP modulation under hypoxia thus decreases Hb-oxygen aYnity thereby securing adequate tissue oxygen delivery, and contrasts with the hypoxic reduction of ATP/GTP content alone that would increase Hb-oxygen aYnity, thus ensuring maximal oxygen uptake at the exchange surface. Clearly, the catfish is a worthy candidate for experiments on acclimation to hypoxia under appropriate physiological conditions. In the primitive erythrocytes of jawless agnathan fishes (lampreys and hagfish) the response to hypoxia is cell swelling. This results in an increased Hb-O2 aYnity through favoring the dissociation of the monomer–oligomer Hb complex (Nikinmaa, 2001). Cell swelling also occurs in teleosts under hypoxia, but the mechanism is entirely diVerent. Here, the regulatory role of the organic phosphate–Hb complex is involved and the equilibrium favors the free unbound state resulting in increased oxygen aYnity with erythrocyte swelling (see Nikinmaa, 2001 for review). 8.2. Allosteric EVects of Chloride and Water Whereas red cell organic phosphates have been clearly shown to play a part in hypoxia regulation of Hb oxygen transport, the reaction of Hb with numerous other cytosolic and protoplasmic factors complicates an adaptive interpretation of allosteric regulation at the subcellular level (Weber and Voelter, 2004). Chloride ions have been shown to play an allosteric role in the eel, Gymnothorax unicolour; Cl increases cooperativity thereby maximizing oxygen uptake and delivery over a precisely delineated change in PO2 (Tamburrini et al., 2001). The activity of water molecules in oxygen-linked allosteric regulation of Hb function is also widespread, and a characteristic of the anodic Hb components of the eel, Anguilla anguilla, and rainbow trout, O. mykiss (Hundahl et al., 2003). Water decreases oxygen aYnity by a diVerent mechanism in the agnathan hagfish, Myxine glutinosa, by stabilizing the oligomeric state of Hb in the erythrocytes (Müller et al., 2003). Both water and chloride ions are ubiquitous and hence unlikely to play an adaptive role in modulating oxygen aYnity in response to hypoxic challenge. 8.3. Integrative Functions of the Erythrocyte The fish erythrocyte is a functional unit of oxygen transport. Studies on whole blood or intact erythrocytes in relation to hypoxia are likely to be of higher ecophysiological relevance than those of the reduced elements of the oxygen transport system because the Hb is operating under realistic in vivo conditions. This is not to say, however, that the extensive literature on Hb 6. BLOOD-GAS TRANSPORT 279 structure-function is not critical to our interpretation of how the integrated system works. Nonetheless, it has proved far easier to collect and freeze blood samples from fish with interesting phylogenies living in interesting habitats, and to study the purified Hbs at a time convenient for the investigator. Recent whole blood studies are few and far between because the intact erythrocytes cannot be stored for more than a few hours without substantial degradation of erythrocyte metabolites and Hb integrity. Caldwell et al. (2006) found that erythrocytes from O. mykiss can be stored for up to 96 h with almost no eVect on the magnitude of the erythrocyte b-adrenergic response. Comparing the oxygen transport functions of purified lysates with whole blood from three phylogenetically distant fish living in the hypoxic billabongs of northern Australia revealed discrepancies that led to the conclusion that an adaptive interpretation of isolated Hbs was unwarranted (Wells et al., 1997). The athletic, facultative air-breathing tarpon, Megalops cyprinoides, had the lowest whole blood-oxygen aYnity and largest Bohr factor, whereas the water-breathing saratoga, Scleropages jardinii, had the highest aYnity and smallest Bohr factor. However, in purified lysate, barramundi, Lates calcarifer, showed the highest aYnity and saratoga the strongest Bohr eVect. The respective erythrocyte environments therefore modify the eVective properties of Hb that adapt these fish for hypoxic habitats. In an integrated study, Yang et al. (1992) compared the suite of metabolic and respiratory adaptations in two scorpaenid fishes that diVered in their depth distribution. Scorpaena guttata lives in relatively shallow water on the upper continental shelf, whereas Sebastolobus alascanus occurs at depths in excess of 1000 m in the oxygen minimum zone. The authors found that the species adapted to the hypoxic zone had a higher whole blood-oxygen aYnity compared to its shallow water counterpart. Both species showed cooperative oxygen binding with similar Hill coeYcients, and similar hematocrit values suggested that oxygen-carrying capacities were similar. These features suggest that the principal adaptive feature of the oxygen transport system is the P50, and that this serves in S. alascanus to maintain full saturation in the gills when the fish is exposed to low environmental oxygen. Whether these functional diVerences are due to the intrinsic properties of the Hb or the erythrocyte ATP/GTP modulators has not yet been determined. While regulation of blood-oxygen aYnity in the face of hypoxia seems a sensible adaptation for fish living in habitats that become periodically depleted in oxygen, one would not expect to find similar hypoxic responses in inactive species living in a well-oxygenated environment. Curiously, the Antarctic fish, Pagothenia borchgrevinki, showed a robust acclimatory response to hypoxia that included increased oxygen-carrying capacity and increased whole blood-oxygen aYnity arising from down-regulation of 280 RUFUS M. G. WELLS erythrocyte ATP production (Wells et al., 1989). The authors suggested that the hypoxic response represents a generalized, phenotypic plasticity that is likely to be present in most fish species, rather than a specific adaptation to environmental hypoxia. The facultative air-breathing tarpon, Megalops cyprinoides, and the salmon catfish, Arius leptaspis, both inhabit oxygen-poor billabongs. Comparison of whole blood oxygen binding in the two species showed that the former had a low aYnity blood with marked Bohr eVect and cooperativity, and allosteric modulation by ATP; by contrast, the catfish had high aYnity blood, smaller Bohr eVect and cooperativity, with regulation by GTP (Wells et al., 2005). Thus, high aYnity, reduced Bohr and Hill coeYcients, and GTP modulation of Hb function seem adaptive for water-breathers living under chronic hypoxia. Acclimation studies in the obligate air-breathing lungfish, Protopterus amphibius, showed a similar response. The oxygen aYnity of whole blood increased sharply during aestivation in response to low oxygen in the near environment of the mud cocoon and regulation of aYnity was eVected by reduced erythrocyte GTP (Johansen et al., 1976). Initial expectations that air-breathing fishes would have higher blood oxygen aYnities than water-breathers and in view of the frequently hypercapnic conditions of many oxygen-deficient habitats, reduced Bohr and Root eVects have not been confirmed in a survey of the blood oxygen transport properties of many air- and water-breathing species (reviewed by Graham, 1997). There was some evidence to support the hypothesis when comparing closely related osteoglossid genera, but the correlation disappeared with phylogenetic distance. Nonetheless, a high oxygen-carrying capacity appears to be a feature of most air-breathing fishes (Graham, 1997). Acclimation of the sedentary benthic turbot, Scophthalamus maximus, and the more active sea bass, Dicentrarchus labrax, to several levels of environmental hypoxia did not result in changes to blood-O2 aYnity; the species diVerences in O2-binding properties under normoxia revealed a smaller Bohr factor and lower Hb concentration in the turbot, and these characteristics appear suYcient to deal with reduced O2 loading in the gills while maintaining eYcient transfer to tissues (Pichavant et al., 2003). Studying the mechanisms of hypoxia tolerance in rainbow trout, O. mykiss, Boutilier et al. (1988) showed that whereas the ATP:Hb ratio decreased upon hypoxic exposure, so too did the pH gradient across the erythrocyte membrane, so that the integrated eVect was no change in bloodoxygen aYnity. This study calls into question the assumed adaptive significance of allosteric compensation for hypoxia, since the presumed Bohr eVect oVsets the ATP eVect. It also calls into question the relevance of interpreting physiological measurements on the basis of plasma pH when the erythrocytic 6. BLOOD-GAS TRANSPORT 281 proton–Hb relationship is unknown. Subsequent investigation into the transfer of oxygen from blood to myoglobin-rich red swimming muscle in O. mykiss supports the hypothesis that red muscle in teleost fishes leads to higher intramuscular PO2 as a result of the sigmoidal oxygen equilibrium curve (McKenzie et al., 2004). The authors came to this conclusion by exposing trout to mild environmental hypoxia (50% water saturation) and monitoring red muscle PO2 using O2-sensitive optical fiber sensors while exercising the fish. They further discounted a role for the Root eVect Hb in oxygenating the muscle. A recent study found unexpected diVerences in blood-oxygen aYnity between four unrelated cold-temperate marine teleost fishes that appeared unrelated to the likelihood of functional hypoxia or to the predicted responses of each species to environmental hypoxia (Herbert et al., 2006). The authors advised against overestimating the adaptive functional properties of Hb when comparing unrelated species. Sturgeons are primitive, chondrostean fishes (Family Acipenseridae) that diVer from teleost fishes in their hypoxic responses. Under the acute hypoxic challenge of either moderate (50% saturation) or severe (20%) environmental hypoxia, the typical teleostean response of increased oxygen-carrying capacity did not occur; hematocrits in this group under normoxic conditions were not notably high and compensation was respiratory and metabolic rather than hematological (Baker et al., 2005). Sturgeons, however, despite their benthic cruising behavior, have much lower whole blood oxygen aYnities than are typical of teleosts with robust hypoxic tolerances (Crocker and Cech, 1998), although the oxygen equilibrium properties of the anadromous green sturgeon, Acipenser medirostris, seem typical of fish adapted to environmental hypoxia: modest Bohr eVect, low Hill coeYcient (1.4–1.5), and GTP modulation (KauVman et al., 2007). Furthermore, oxygen binding is temperature sensitive with values of
H tending to become more positive with increasing temperature (
H = 34.2 kJ mol 1 at 11 C; 6.7 at 24 C). 9. ROLE OF b-ADRENERGIC RECEPTORS IN ERYTHROCYTE OXYGEN TRANSFER Exposure to low environmental oxygen or exercise challenge triggers the release into the circulation of a hormonal flush of catecholamines and corticosteroids (Randall and Perry, 1992; Gamperl et al., 1994; Lowe and Wells, 1996). Although this is a typical vertebrate response to stress, teleost fishes are unusual in having erythrocyte surface receptors that bind adrenaline and noradrenaline (Nikinmaa and Heustis, 1984). These receptors are of ancient evolutionary origin and are thought to predate the fish–tetrapod 282 RUFUS M. G. WELLS division (Nikinmaa, 2003). In teleost fishes, b3-adrenergic receptors control the function of a sodium-proton pump in the erythrocyte membrane so that intracellular protons are exchanged for extracellular sodium ions (Nickerson et al., 2003). The eVect of the Na+/H+ exchanger is twofold: first, the reduction of proton activity inside the erythrocyte raises the pH of the erythrocyte and thereby increases Hb-O2 aYnity via the Bohr eVect; and second, the inward flow of sodium is balanced by a parallel water flux with the result that the erythrocyte swells (Nikinmaa, 2002). The low buVering capacity of lamprey and teleost fish Hbs, coupled with large Bohr and Haldane eVects, means that the Na+/H+ exchanger plays a critical role in regulating blood-oxygen aYnity in response to acute hypoxia (Nikinmaa, 1997, 2001). These reactions were first noted by Nikinmaa (1982, 1983) in erythrocytes from rainbow trout (Salmo gairdneri, now known as Oncorhynchus mykiss). Evidence for an adrenergic eVect during functional or environmental hypoxia is most obviously seen from a sharp rise in hematocrit that cannot be explained by an increase in the numbers of erythrocytes released under concomitant adrenergic stimulation of the spleen (Wells and Weber, 1990). This has the additional eVect of an increase in Hb-O2 aYnity due to dilution of the cell contents and dissociation of the phosphate–Hb complex. These processes can result in a rapid increase in blood-oxygen aYnity, thus securing adequate oxygen-binding in the gills when the fish is subjected to hypoxia. Perry and Reid (1992) suggested that a decrease in arterial PO2 below the P50 was required to trigger the catecholamine response. Specific examples are the response to environmental hypoxia by rainbow trout, O. mykiss (Tetens and Christensen, 1987), and exercise-induced functional hypoxia in O. mykiss (Primmett et al., 1986) and striped bass, Morone saxatilis (Nikinmaa et al., 1984). This hypoxic response is also influenced by temperature because of the direct eVect on metabolism, and the reduced availability of oxygen at warmer environmental temperatures. The seasonal response in Arctic charr, Salvelinus alpinus, is an up-regulation of the erythrocyte adrenergic system at low seasonal temperatures (Lecklin and Nikinmaa, 1999). Jensen (2001) hypothesized that the low buVer values for Hb in teleosts might be a necessary prerequisite for the regulation of erythrocyte pH via the Na+/H+ exchanger. The observation that three species of tuna all showed low Hb-specific buVer values, despite the remarkable metabolic acidosis that develops during burst swimming, supported the hypothesis (Jensen, 2001). With plasma pH reduced by as much as 0.4 units, it might be supposed that the erythrocyte response to catecholamines would be greater than that in other less active species. Lowe et al. (1998) found, however, that the responses of two tuna species were similar to those in less active teleosts, 6. BLOOD-GAS TRANSPORT 283 and that adaptations to extreme functional hypoxia do not occur at the level of erythrocyte function. Until recently, there was little evidence for activation of a Na+/H+ exchanger in response to functional hypoxia in sharks or other cartilaginous fishes. Brill et al. (2008), however, have shown that anaerobic exercise in the sandbar shark, Carcharhinus plumbeus, is not accompanied by a strongly decreased blood-O2 aYnity because the metabolic acidosis is compensated by alkalinization of the erythrocytes following activation of the Na+/H+ exchanger. Moreover, the significant increase in hematocrit reflected not only erythrocyte swelling, but a real increase in oxygen-carrying capacity. The Na+/H+ exchanger is absent from the Osteoglossomorpha, representing primitive teleost fishes. b-Adrenergic stimulation of erythrocytes from the primitive agnathan fish, Lampetra fluviatilis, resulted in volume change via chloride channels rather than the Na+/H+ exchanger, which appears to be absent in this group (Nikinmaa et al., 2001). The swelling of erythrocytes under hypoxia might be expected to impede the flow of blood through the capillary circulation. This does not appear to be the case, however, and b-adrenergic stimulation of trout (O. mykiss) erythrocytes actually decreased the shear-dependence of blood viscosity (Wells et al., 1991). That the adrenergic mechanism really does improve oxygen transport under functional hypoxia was demonstrated experimentally by testing visual function in the b-blocked trout, O. mykiss (Herbert and Wells, 2002). Retinal function is highly oxygen dependent, and in the absence of the adrenergic mechanism, severe visual impairment is likely when fish become hypoxic. 10. NOVEL MOLECULAR MECHANISMS FOR HYPOXIA PROTECTION 10.1. Putative Role of Hb-Nitric Oxide Binding Much interest has followed the discovery that nitric oxide (NO) released from vascular endothelium may act as a paracrine vasodilator to relax vascular smooth muscle, allowing for improved blood flow (JaVrey and Snyder, 1995). How widespread across the vertebrate groups this eVect is has not yet been determined. Since NO is both short-lived and neurotoxic in excess, its production and distribution must be closely regulated. While NOsynthase plays an important role in its production, the observation that NO binds to the thiol groups of proteins (JaVrey and Snyder, 1995) has invited speculation concerning a new role for Hb (Reischl et al., 2007). Accordingly, Weber and Fago (2004) considered whether oxygenation-linked NO binding 284 RUFUS M. G. WELLS might form the basis of NO transport and release to induce more widespread vasodilation under conditions of tissue hypoxia. This speculation assumes further significance for fish living in hypoxic freshwater habitats where nitrite levels are often high. Jensen (2003) suggested that Hb may play a role in nitrate reduction and production of NO thereby improving circulation under conditions of low environmental oxygen. Under hypoxia, the higher proportion of deoxyhaemoglobin promotes greater nitrite reductase activity, and hence NO production (Jensen, 2008). In fishes, the NO-mediated regulation of vascular dilation appears well developed in the branchial vasculatures of the eel, Anguilla anguilla (Pellegrino et al., 2002), and the Atlantic salmon, Salmo salar (Ebbesson et al., 2005). A role for Hb in NO regulation has also been suggested for the endothelial lining of the swimbladder, and in lungs of air-breathing fishes (Zaccone et al., 2006). Surprisingly, the Antarctic icefish, Chionodraco hamatus, has a welldeveloped NO-synthase mechanism, but lacks Hb altogether (Pellegrino et al., 2004; Amelio et al., 2006). Sidell and O’Brien (2006) suggested that icefish have larger blood vessels as a result of NO not being scavenged by Hb. Thus, NO has both acute acclimatory and adaptational possibilities for regulation of oxygen transport. Few physiological experiments have been undertaken to resolve the role of NO-Hb in hypoxic fishes. Swenson et al. (2005) noted a rapid up-regulation of NO production in hypoxic dogfish, Squalus acanthias, that appeared to promote vasodilation. The authors favored the view that Hb plays a role as a NO scavenger since the physiological eVects of NO were most marked in Hb-free preparations. These observations in sharks are supported by those from a teleost fish; channel catfish, Ictalurus puncatus, when subjected to severe hypoxia showed an increase in nitrergic nerve fibres in the branchial region (Zaccone et al., 2006). It seems reasonable to conclude that hypoxia will up-regulate NOsynthase expression in fishes, allowing for NO-mediated vasodilation. In the meantime, Hb–NO interactions remain poorly understood (Fago et al., 2003) and although current opinion supports the role of Hbs from fish and other animals in both releasing NO upon deoxygenation and in thiolated removal of NO, the results of new research in this field are eagerly awaited. 10.2. Post-Translational Modification of Hb Function The possibility of nongenetic modifications to Hb function in fishes seems not to have been considered. Yet, there is growing evidence that this is a common phenomenon in avian and mammalian Hbs where glycosylation, glutathionylation, and deamidation of b-globins may result in altered functionality (Di Simplico et al., 1996; Dafre and Reischl, 1998; Niwa et al., 2000; 6. BLOOD-GAS TRANSPORT 285 Henty et al., 2007). Glutathionylation appears to occur in the scalloped hammerhead shark, Sphyrna lewini (Dafre and Reischl, 1997) but there is no evidence yet that these mechanisms could provide phenotypic compensation for hypoxia. 11. HYPOXIA INDUCIBLE FACTOR HIF-1a: EVIDENCE FOR ROLE IN HYPOXIC RESISTANCE The search for adaptive responses to hypoxia through changes in gene expression has gathered considerable momentum in recent years. Oxygen sensing via hypoxia inducible factors (HIFs) and molecular responses to hypoxic challenge has been demonstrated throughout the animal kingdom (Hoogewijs et al., 2007). Fish are at the forefront of this research because of comparative species diVerences in responses to hypoxia from fish inhabiting diverse aquatic habitats. The HIF-1a protein in fish is an important transcription factor that mediates a range of responses to hypoxia through the expression of genes controlling the oxygen transport system, and possible HIF targets are erythropoietin, globin synthesis, angiogenesis, and gill surface area (reviewed by Nikinmaa and Rees, 2005). Suppression of apoptosis and metabolic arrest in the hypoxia-resistant crucian carp, Carassius carassius, resulted in a significant increase in gill surface area when the fish was chronically exposed to hypoxia (Sollid et al., 2006). Both seasonal and latitudinal diVerences in water temperature have a significant eVect on oxygen solubility with oxygen content being much reduced in warmer waters. The organismal response to increased temperature is generally a higher metabolic demand for oxygen. These two opposing factors interact to exacerbate internal hypoxia. Rissanen et al. (2006) have shown that temperature has a marked eVect on HIF-1a expression in C. carassius suggesting possible adaptation to temperature-induced hypoxia. Recent studies with the polar zoarcids Zoarces viviparous and Pachycara brachycephalum provide further evidence for a transcriptional control mechanism for oxygen transport in temperature acclimation of extreme poikilotherms (Heise et al., 2006, 2007). Oxidative defence mechanisms in both cold-adapted and acclimated fishes therefore appear to be mediated by HIF-1a expression. A critical question remains as to whether sequence variation in the HIF-1a gene can be linked to intraspecific diVerences in hypoxia defence. Current research by Scandinavian investigators is attempting to address this question by comparing both phylogenetically similar species with diVerent hypoxic tolerances, and unrelated species with similar oxygen requirements (Rytkönen et al., 2007). The investigators are thus potentially able to 286 RUFUS M. G. WELLS distinguish adaptive from evolutionarily neutral changes. Adaptive responses of HIF-1a expression allow fish to delay the onset of metabolically ineYcient anaerobiosis. It is not yet clear, however, at which point in the HIF-1a pathway species-specific diVerences occur. 12. CONCLUSIONS AND COMMENTARY Environmental hypoxia is common in aquatic habitats at all latitudes, and under natural conditions is associated with increased carbon dioxide, ammonia, hydrogen sulfide, and reduced pH. Apart from the oxygen-minimum zone and areas around deep sea thermal vents, the marine environment is not generally associated with hypoxic habitats, yet even coral reef fish show significant mechanisms of hypoxia tolerance (Nilsson and Renshaw, 2004; Nilsson and Östlund-Nilsson, 2006). It is not often enough emphasized that the first responses of fish to environmental hypoxia are generally behavioral and include reduced locomotion, feeding, and reproductive activities, and, where possible, an attempt to seek out cooler water (Randall et al., 2006). Species adapted to persistent environmental hypoxia often seem to have high Hb concentrations, as do populations compensating for reduced oxygen availability. To a large extent, this increased oxygen-carrying capacity compensates for the reduced turnover of oxygen to tissues caused by apparently adaptive increases in blood-oxygen aYnity under hypoxia. Brauner and Wang (1997) calculated that increased oxygen-carrying capacity was significantly more beneficial to tissue oxygen delivery during environmental hypoxia than were changes in blood-oxygen aYnity. There is, therefore, some uncertainty concerning the adaptive value of acclimatory shifts in Hb-O2 aYnity, particularly in the absence of more complete information on compensatory adjustments in the cardiovasular system and metabolic processes. In the meantime, it may be useful to evaluate ‘‘benefit’’ in the sense that an increase in Hb concentration in response to environmental hypoxia and strenuous exercise benefits perfusion limited situations, whereas allosteric eVects modulating Hb function, and perhaps myoglobin, benefit diVusion limited gas exchange. Adaptations to functional hypoxia are more obviously manifested through oxygen transport characteristics. The role of the spleen as a reservoir of erythrocytes in raising oxygen-carrying capacity during exercise is well understood, as are the advantages of low blood-O2 aYnity favoring unloading to tissues, a robust Bohr factor permitting oxygen turnover in response to demand, and highly cooperative Hb allowing for rapid loading and unloading of oxygen and carbon dioxide over comparatively narrow pressure gradients. 6. BLOOD-GAS TRANSPORT 287 Oxygen transport systems appear geared toward maintaining the oxygen cascade from the gas exchangers facing the environment, to the cytochrome oxidase enzymes in the mitochondria of working tissues where oxygen pressures may be <1 mmHg. Accordingly, much of our adaptive interpretation of blood oxygen transport has been toward optimizing this gradient. It seems counterintuitive then that the evolution of an oxygen transport system may not solely be directed toward maximizing oxygen supply. There is growing evidence that a major constraint is the requirement to protect internal tissues against reactive oxygen species, especially in high performance fish where mitochondrial densities in the red swimming muscles are among the highest in vertebrates, and antioxidant protection is essential to prevent oxidative damage (Wilhelm Filho, 2007). Furthermore, the cascade should be interpreted against a historical background of extensive fluctuations in atmospheric oxygen levels over the Phanaerozoic era, when bimodal breathing became common (Berner et al., 2007; Flück et al., 2007). Fish have a long evolutionary history in coping with fluctuations in oxygen availability, and have emerged as the most functionally diverse vertebrate group. In present day habitats, hypoxia is very common and an important determinant of species distribution. Recent research has reinterpreted the role of Hb as a cellular oxygen sensor inducing a cascade of changes in response to hypoxia (Wu, 2002). These include transcription of factors such as HIF-1a, glycolytic and phosphorylation pathways, and globin synthesis. The role of Hb as an oxygensensing mechanism linking K+ flux in erythrocytes of trout, O. mykiss, has also been proposed (Berenbrink et al., 2000). The interaction of deoxygenated Hb with the cytoplasmic domains of Band 3 erythrocyte membrane proteins suggests further modulation of several metabolic functions, including glycolysis, the pentose phosphate pathway, and ion exchanges (Weber et al., 2004). The importance of the Band 3 protein in fish is not well characterized, given the lesser role of glycolysis in nucleated fish erythrocytes, but could play a part in hypoxia acclimation in air-breathing fish such as armoured catfish, Pteroglopichthys spp., and lungfish, Protopterus spp., which possess the glycolytic O2 aYnity modulators DPG and IP, respectively (Val, 2000). Much of what we know about functional adaptations of the oxygen transport system to hypoxia in fishes has not been the result of acclimation or field experiments, but has been inferred from laboratory-based studies on isolated Hbs and erythrocytes. Despite our detailed understanding of the molecular basis of functional adaptations in fish Hbs (see Weber and Fago, 2004), the physiological significance of Hb function at the whole organism level remains less well understood. Considerable plasticity has been shown in the respiratory system in response to changes in oxygen supply (environmental hypoxia) and demand (functional hypoxia). As pointed out by Bavis 288 RUFUS M. G. WELLS et al. (2007) it is diYcult to predict the responses of an animal based on the plasticity of a single system. This lesson is also important in interpreting the oxygen transport system since that in turn is comprised of several diVerent elements. Nonetheless, it seems that contributing to respiratory plasticity are: adjustments to oxygen-carrying capacity, Hb isoforms, modulation of Hb-oxygen aYnity, regulation of the internal erythrocyte environment, and expression of hypoxia inducible factors. Results from expression profiling in the long-jaw mudsucker, Gillichthys mirabilis, are likely to be typical of most fish that are routinely exposed to hypoxia (see Nikinmaa and Rees, 2005), and showed that a large number of genes are both induced and suppressed during hypoxic exposure, revealing the diVerent roles of specific tissues during hypoxia (Gracey et al., 2001). Changes in hypoxic response during development are less well understood. Exposure of zebrafish larvae (Danio rerio) to a low oxygen environment during development resulted in the stimulation of convective oxygen transport (Jacob et al., 2002), but the capacity for the Hb system to adapt is unknown. Although continued whole-organism research into how fish cope with demand for oxygen under restricted supply is likely to remain a productive area, diVerences in habitat and activity level need to be related to hypoxiarelated gene expression in order to more fully understand the molecular basis for adaptation to oxygen fluctuations. Emerging research on hypoxia inducible factors, the role of NO, and erythrocyte surface receptors emphasizes the integrative approach to understanding hypoxic responses at the organismal level. ACKNOWLEDGMENTS The author wishes to thank Thomas Brittain for useful discussion. REFERENCES Abe, H., Dobson, G. P., Hoeger, U., and Parkhouse, W. S. (1985). Role of histidine-related compounds to intracellular buVering in fish skeletal muscle. Am. J. Physiol. 249, R449–R454. AVonso, E. G., Polez, V. L. P., Corrêa, C. F., Mazon, A. F., Araujo, M. R. R., Moraes, G., and Rantin, F. T. (2002). 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(1979). Respiratory properties of blood and hemoglobin solutions from the piranha. Comp. Biochem. Physiol. 62A, 163–167. Wu, R. S. S. (2002). Hypoxia: From molecular responses to ecosystem responses. Mar. Poll. Bull. 45, 35–45. Yang, T.-H., Lai, N. C., and Somero, G. N. (1992). Respiratory, blood, and heart enzymatic adaptations of Sebastolobus alascanus (Scorpaenidae; Teleostei) to the oxygen minimum zone: A comparative study. Biol. Bull. 183, 490–499. Zaccone, G., Mauceri, A., and Fasulo, S. (2006). Neuropeptides and nitric oxide synthase in the gill and air-breathing organs of fishes. J. Exp. Zool. 305A, 428–439. 7 CARDIOVASCULAR FUNCTION AND CARDIAC METABOLISM A. KURT GAMPERL W. R. DRIEDZIC 1. Introduction 2. Hypoxic Effects on In Vivo Cardiovascular Function 2.1. Acute Hypoxia 2.2. Chronic Hypoxia 3. Cardiac Energy Metabolism 3.1. Creatine Phosphate and ATP Levels 3.2. Decreasing ATP Demand 3.3. The Potential of Enhanced Oxygen Utilization Under Hypoxia 3.4. Anaerobic Energy Metabolism 4. Additional Insights 4.1. Interactive EVects of Temperature and Hypoxia 4.2. Preconditioning 5. Concluding Remarks Next to extremes in temperature, hypoxia is arguably the most significant environmental challenge faced by fishes. This is because of the disruptions/ consequences it has for the fish’s physiology, reproduction, and survival, and the fact that hypoxia is an increasing problem in aquatic systems worldwide. The cardiovascular system is critical for the eVective uptake of oxygen from the environment and the distribution/transport of oxygen and nutrients to the tissues, and its proper functioning is paramount to activities such as locomotion and digestion, and to the capacity to deal with environmental pertubations. In this chapter, an overview of cardiovascular responses to hypoxia in fishes, of some of the mechanisms that influence/mediate the eVects of hypoxia on the fish’s cardiovascular system, and of how myocardial energy metabolism is regulated under hypoxia (this aspect is critical to the continued functioning of the heart during periods of oxygen shortage) is 301 Hypoxia: Volume 27 FISH PHYSIOLOGY Copyright # 2009 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(08)00007-1 302 A. KURT GAMPERL AND W. R. DRIEDZIC provided. Information is reviewed on the eVects of both acute and chronic hypoxia and on interspecific variation in the magnitude and timing of responses, and covers life stages from embryo to adult and levels of biological organization from gene expression to the whole animal. Further, where possible, recent advances in our understanding of the influence of hypoxia on fish cardiovascular function are highlighted, and unresolved issues are identified. It is expected that this chapter will become a valued resource for those interested in the interplay between hypoxia and cardiovascular function, and will stimulate research in this interesting area of fish physiology. 1. INTRODUCTION Aquatic habitats are subject to many environmental variations and one of the most important parameters aVecting non air-breathing vertebrates is dissolved oxygen. Hypoxia, or oxygen depletion, is a phenomenon that occurs in a wide variety of aquatic environments, from the Amazon drainage basin, to iced-over shallow water bodies in winter, and ever increasingly, to coastal marine areas around the world (including the Black and Baltic Seas, the Gulf of Mexico, and the Gulf of St. Lawrence). The response of fish to hypoxic environments includes complex behavioral changes such as decreased locomotion and predator avoidance capacity (Dalla Via et al., 1998; Lefrançois and Domenici, 2006; Behrens and SteVensen, 2007) or movement away from/avoidance of areas of low dissolved oxygen (Pihl et al., 1991; Claireaux et al., 1995) (see Chapter 2). When escape from the hypoxic stress is not possible, a variety of physiological adjustments may be invoked to compensate for low oxygen availability, thus allowing fish to withstand short-term (acute) hypoxic exposure (Jensen et al., 1993; Val et al., 1998) or to eventually allow for the restoration of essential activities such as feeding, reproduction, and escape from predators (Jensen et al., 1993) (see Chapter 10). Given the critical role that cardiovascular function plays in blood oxygen transport and substrate delivery, and that fish heart function is solely or partially (for those with coronary arteries, or with lungs or accessory breathing organs) dependent on whatever oxygen is left in the venous blood after it has traversed the fish’s other tissues, it is not surprising that fish cardiovascular function during hypoxia has been an active area of research for over five decades. In this chapter we provide an overview of cardiovascular responses to hypoxia in fishes (with the caveat that only minimal reference is made to the crucian carp, which is the focus of Chapter 9), some of the mechanisms that influence/mediate the eVects of hypoxia on the cardiovascular system, and how myocardial energy metabolism is regulated under hypoxic conditions. 7. CARDIOVASCULAR FUNCTION 303 Further, where possible, we emphasize specific areas where recent advances in our understanding of the influence of hypoxia on fish cardiovascular function have been realized, and where unresolved issues remain. This chapter was challenging to put together given the wide range of hypoxia tolerance exhibited amongst fish species, and the large variations in the severity and duration of hypoxia, and temperature, used in experimental protocols. For example, the common carp (Cyprinus carpio) can withstand rapidly induced anoxia for 2–24 h (depending on temperature; Stecyk and Farrell, 2007), while the Atlantic cod (Gadus morhua) only tolerates exposure to a water PO2 of 10 mmHg (at 10 C) for a matter of minutes (Petersen and Gamperl, unpublished data). Water O2 content varies inversely with temperature, while metabolic demand generally goes up by a factor of 2–3 with each 10 C increase in temperature, and thus hypoxia-tolerance for a given species is temperature dependent. Finally, many authors have examined the eVects of hypoxia on fish cardiovascular function by rapidly exposing fish to 8–12 min of anoxia/severe hypoxia, whereas others have used protocols where water oxygen levels were gradually reduced over several hours, fish were rapidly exposed to severe hypoxia and held at this level of oxygenation for extended periods (h), or fish were maintained at moderate levels of hypoxia (i.e., 35–60 mmHg) for several weeks. Given the methodological complexity of the available literature, we divided the information contained in this chapter into two broad categories for ease of presentation (although not all research falls easily within either category; e.g., see data from Stecyk and Farrell, 2007 in section 4.1): acute hypoxia referring to research that exposed fish to minutes to hours of lowered water O2; and chronic hypoxia indicating reductions in water O2 levels that lasted days or weeks, i.e., long enough to have changes in gene and protein expression. Further, we used oxygen partial pressure (mmHg) as our unit of water O2 measurement throughout the chapter. This was largely done to facilitate multispecies comparisons (i.e., the reader can easily judge the severity of hypoxia for a species at a given temperature based on oxygen availability; fully saturated water having a PO2 of approximately 155 mmHg at sea level), and because arterial blood PO2, which generally reflects water PO2, has a number of potential implications for hypoxia tolerance. For example, hemoglobin– oxygen binding aYnity is expressed in mmHg as the P50 (PO2 at which hemoglobin is 50% saturated with oxygen), and there is evidence that the release of catecholamines from the chromaYn tissue, which stimulates a number of physiological alterations that would improve hypoxia tolerance [e.g., oxygen uptake, blood oxygen transport, and cardiac function; e.g., see Farrell and Jones (1992) and Randall and Perry (1992)], appears to occur near the fish’s P50 value (Reid and Perry, 1994). 304 A. KURT GAMPERL AND W. R. DRIEDZIC 2. HYPOXIC EFFECTS ON IN VIVO CARDIOVASCULAR FUNCTION 2.1. Acute Hypoxia 2.1.1. Heart Rate The vast majority of research on the eVects of hypoxia on fish cardiovascular function has investigated the eVects of a few minutes or hours of exposure to reduced oxygen levels. In fishes, the most common cardiac response to hypoxia is reflex bradycardia (a decrease in heart rate, fH), and a recent review by Farrell (2007) provides a comprehensive overview of the data in this area and proposes several direct benefits of hypoxic bradycardia to the fish heart. These benefits include: (1) improved cardiac contractility through the negative force-frequency eVect; (2) enhanced oxygenation of the myocardium due to an increase in the diastolic residence time of blood in the lumen of the heart (i.e., increased time for oxygen diVusion), and stretching of the myocardium (i.e., decreased diVusion distance), in species that respond to hypoxia with a concomitant increase in stroke volume (SV, see below); (3) a reduction in myocardial oxygen demand due to a decrease in the rate of ventricular pressure development (dP/dt); and (4) an increase in coronary blood flow, and thus a diminished reliance on the oxygen content and partial pressure of venous blood, due to an extended diastolic period (diastole the portion of the cardiac cycle where the majority of coronary blood flow occurs; 75–85%; Davie and Franklin, 1993; Gamperl et al., 1995). Thus, in this chapter, we only provide a brief summary of the eVect of acute hypoxia on heart rate in fishes, of how temperature and hypoxia tolerance influence the onset of bradycardia, and what control mechanisms may account for species and other diVerences. First, there are some taxonomic groups that do not exhibit changes in heart rate when exposed to hypoxia or where no clear response pattern to hypoxia has been established. For example, since reflex bradycardia is primarily mediated by vagal cardioinhibitory tone, and hagfishes lack autonomic cardiac innervation (Nilsson, 1983), it is not surprising that heart rate ( fH) in this taxa remains unchanged in response to severe hypoxia (Axelsson et al., 1990). Bradycardia is absent in all three genera of lungfish when exposed to aquatic hypoxia (Neoceratodus, Fritsche et al., 1993; Leptidosiren, Sanchez et al., 2001; Protopterus, Perry et al., 2005). This finding is likely related to the absence of external gill O2 chemoreceptors or external O2 chemoreceptors that are unresponsive/very insensitive to changes in water O2 levels in this taxa (Perry et al., 2005), and suggests that the loss of hypoxic bradycardia may have coincided with the evolution of air-breathing in fishes. However, the picture is not so clear when the fH response of other air-breathing fishes to 7. CARDIOVASCULAR FUNCTION 305 aquatic hypoxia is examined. For example, the jeju (Hoploerythrinus unitaeniatus) developed hypoxic bradycardia when hypoxia began to compromise oxygen consumption (approx. 40 mmHg; Oliveira et al., 2004), fH in two species of facultative air-breathing Amazonian armoured catfish (Liposarcus pardalis and Glyptoperichthyes gibbceps) (MacCormack et al., 2003a) did not change significantly (although average fH decreased by 15 and 40 beats min-1, respectively) at dissolved oxygen levels down to 1 mg l-1, and the garfish (Lepisosteus oculatus; Smatresk and Cameron, 1982) and Synbranchus marmoratus (Skals et al., 2006) showed modest tachycardia during exposure to aquatic hypoxia (approx. 12 mmHg and 50 mmHg, respectively). Further, the interpretation of these latter data is complicated because: (1) the garfish and S. marmoratus were allowed access to air, and the increase in fH with aquatic hypoxia (even during periods of aquatic ventilation) may have resulted because inflation of their accessory breathing organs overrode the drive for hypoxic bradycardia initiated by O2 receptors in the gills (e.g., see Graham, 1997; Skals et al., 2006); and (2) experiments with two water-breathing Amazonian fish species have shown that internally oriented gill O2 chemoreceptors (i.e., those sensing changes in blood oxygen) can exclusively (in Hoplias malabaricus; Sundin et al., 1999b) or in combination with externally oriented O2 receptors (in Colossoma macropodum; Sundin et al., 2000) elicit bradycardia. Clearly, in the absence of other measurements of changes in fH with exposure to aquatic hypoxia, it is not possible to determine to what extent air-breathing fishes have lost/retained the capacity for hypoxia-induced bradycardia, or whether the loss of capacity for bradycardia in at least some air-breathing fishes is related to the concurrent absence of external O2 receptors. The final group in which there is no clear picture with regards to the presence/absence of hypoxic bradycardia is Antarctic fishes. Despite the fact that these species live in a cold stenothermal environment with very stable water oxygen levels, the response to acute hypoxia varies among species, studies, and individuals ranging from no eVect, to slight tachycardia, to a clear hypoxic bradycardia. The variable results for red-blooded Antarctic species (i.e., Trematomus bernachii and P. borchgrvinki) may be due to a high and variable cholinergic tone on the heart (where only individuals with low cholinergic tone show substantial decreases in heart rate) and those for the icefish (Chaenocephalus aceratus) may be due to diVerences in experimental protocols or hypoxic thresholds (Axelsson, 2005). However, as with the airbreathing fishes, clarification of the fH response of Antarctic taxa to environmental hypoxia requires careful study, where the rate of hypoxic initiation is consistent (i.e., gradual or abrupt) and where oxygen levels in the water are lowered to such an extent as to preclude diVerential fH responses due to varied responsiveness of the O2 chemoreceptors that trigger hypoxic bradycardia. 306 A. KURT GAMPERL AND W. R. DRIEDZIC With regards to the majority of water-breathing fishes, it is clear that diminished water oxygen levels lead to hypoxic bradycardia, and that hypoxia tolerance and temperature influence the water oxygen level (PO2) at which the reduction in heart rate is initiated. To illustrate how PO2 influences the onset of bradycardia we have plotted the fH–water PO2 relationships for 10 species of water-breathing fishes that were acclimated to either 22–25 C or 8–12 C and exposed to an experimental protocol that involved the gradual reduction of water oxygen levels; this latter criteria was used because rapid versus gradual decreases in water O2 levels can aVect the fH response to hypoxia (e.g., see Butler and Taylor, 1971; Figure 7.1). What can be seen at both water temperatures is that there is a large range of PO2 values at which fH becomes noticeably reduced; fH reductions occurring at PO2 levels as high as 110mm Hg in the dourado (Salminus maxillosus) and 70 mmHg in the rainbow trout (Oncorhynchus mykiss) and Japanese eel (Anguilla japonica), to as low as 25 mmHg for Hoplias lacerdae, 35 mmHg for the cod (Gadus morhua), and <40 mmHg for the tench (Tinca tinca L.). What factors determine the PO2 at which bradycardia is initiated has not been extensively studied but data from a number of investigations suggest that it is related to a fish’s lifestyle and hypoxia tolerance. For example, Rantin et al. (1993) performed a direct comparison of H. lacerdae (an Amazonian species that inhabits well-oxygenated rivers) and H. malabaricus (considered to be well adapted to hypoxic conditions), and the critical PO2 (the PO2 at which routine oxygen consumption can no longer be maintained) and PO2 at which bradycardia was initiated were approximately 35 and 20 mmHg in the two species, respectively. Furimsky et al. (2003) showed that largemouth bass (Micropterus salmoides), which prefer shallow/weedy areas (i.e., a habitat prone to large fluctuations in dissolved oxygen), initiate bradycardia at least 45 mmHg later than the smallmouth bass (M. dolomieu), which inhabits deeper and colder waters. They also showed that this delayed onset of bradycardia was associated with several physiological variables (e.g., a lower P50 value for hemoglobin–oxygen binding) that would have allowed for enhanced hypoxia tolerance in the former species (although our analysis of the available data failed to reveal a significant relationship between the PO2 at which bradycardia was initiated during graded hypoxic exposure and literature values for a species’ P50 value for hemoglobin– oxygen binding). Finally, the dourado and rainbow trout, which are very active species that normally inhabit well-oxygenated waters, have thresholds for the induction of bradycardia of >70 mmHg while those for the hypoxiatolerant carp (C. carpio) and tench are <40 mmHg. With respect to temperature, although it appears from Figure 7.1 that this parameter does not constrain the range of PO2 values over which hypoxiatolerant and hypoxia-sensitive species initiate bradycardia, there are several studies which show that the hypoxic threshold for bradycardia increases with 7. 307 CARDIOVASCULAR FUNCTION A 120 Hoplias malabaricus Hoplias lacerdae Common carp Dourado Japanese eel 100 80 60 40 Heart rate (bpm) 20 0 70 60 50 B Wolffish Tench Lingcod Trout Atlantic cod 40 30 20 10 0 0 50 100 150 PO2 (mmHg) Fig. 7.1. Relationship between water oxygen level (PO2) and heart rate (fH) for various teleost species acclimated to temperatures of 22–25oC (A) and 8–12oC (B). Data for Hoplias malabaricus, Hoplias lacerdae, and common carp (Cyprinus carpio) from Rantin et al. (1993); Dourado (Salminus maxillosus) from De Salvo Souza et al. (2001); Japanese eel (Anguilla japonica) from Chan (1986); Tench (Tinca tinca) and trout (Oncorhynchus mykiss) from Marvin and Heath (1968); Lingcod (Ophiodon elongates) from Farrell (1982); Atlantic cod (Gadus morhua) from Petersen and Gamperl (unpublished data); wolYsh (Anarhichas lupus) from Joaquim and Gamperl (unpublished data). 308 A. KURT GAMPERL AND W. R. DRIEDZIC temperature; the PO2 at which bradycardia was initiated increased from 20 to 60 mmHg in spangled perch (Leiopotherapon unicolor) acclimated to 10 and 30 C (Gehrke and Fielder, 1988) and from <40 to >120 mmHg in dogfish (Syliorhinus canicula) at seasonal temperatures of 7 and 17 C (Figure 7.2A) (Butler and Taylor, 1975). While this appears to be the ‘‘typical’’ relationship between temperature and the water PO2 at which bradycardia occurs, and makes sense given the similar relationship between water temperature and in vivo hemoglobin–oxygen aYnity (e.g., see Perry and Reid, 1994), recent experiments by Mendonca and Gamperl (unpublished data) indicate that this relationship does not apply to all teleost species. These authors acclimated winter flounder (Pleuronectes americanus) to 8 and 15 C and then exposed them to a gradual hypoxic challenge by decreasing water O2 levels by 10% air saturation (approximately 15 mmHg) per hour. Surprisingly, while the onset of bradycardia was 90 mmHg in flounder acclimated to 8 C, fH remained constant in fish acclimated to 15 C down to a PO2 of at least 30 mmHg (i.e., the response was opposite to that observed in the spangled perch and dogfish)(Figure 7.2B). Further, this result does not appear to be peculiar to the particular experimental conditions utilized by Mendonca et al. (unpublished data) as Cech et al. (1977) showed that exposure of this species to water of 45% air saturation (i.e., a PO2 of approx. 65 mmHg) had no eVect on fH at 10 C. Flatfish lack adrenergic cardiac innervation (Santer, 1972; Donald and Campbell, 1982) and cholinergic tone on the heart increases (see Sureau et al., 1989), not decreases, with temperature as has been shown for other teleosts such as the rainbow trout (e.g., see Wood et al., 1979). Thus, one could speculate that these diVerences in cardiac nervous control are responsible for the temperature-dependent diVerences in the response of the flounder versus the dogfish and spangled perch to graded hypoxia; the hypothesis being that a higher cholinergic tone in flounder at 15 C precluded increases in vagal tone from mediating a decrease in fH in response to lowered water oxygen levels. This explanation is unlikely, however, as elasmobranchs also lack cardiac adrenergic innervation, and Taylor et al. (1977) showed that cholinergic tone on the heart increases in the dogfish with temperature. At present we have no physiological mechanism to explain why there is no bradycardia in 15 C-acclimated winter flounder down to water oxygen levels of 30 mmHg. However, we cannot preclude the possibility that fH did not decrease in flounder at this higher temperature due to some, as yet unexplained, ability to avoid myocardial dysfunction. For example, MacCormack and Driedzic (2002) demonstrated that ventricle strips of the yellowtail flounder (Limanda ferruginea) show a transient increase in force development when subjected to anoxia (i.e., N2 gassing). Sundin et al. (2000) showed that hypoxia still induced bradycardia in tambaqui (C. macropomum) after 7. 309 CARDIOVASCULAR FUNCTION A 45 40 7 ⬚C 12 ⬚C 17 ⬚C 35 30 25 Heart rate (bpm) 20 15 B 8 ⬚C 15 ⬚C 70 60 50 40 30 20 10 20 40 60 80 100 PO2 (mm Hg) 120 140 160 Fig. 7.2. The eVect of acclimation temperature on the relationship between water PO2 and heart rate for (A) the dogfish (Scyliorhinus canicula; Butler and Taylor, 1975) and (B) the winter flounder (Pleuronectes americanus; Mendonca and Gamperl, unpublished data) Values for the flounder are means  S.E. sectioning of cranial nerves IX and X to the gill arches and pretreatment with atropine, although the bradycardia was only approximately 30% of that seen in ‘‘intact’’ animals; i.e., there is a non-neural component to hypoxia-induced bradycardia in some fish species. Finally, Rantin et al. (1995) showed that C. carpio has an unusual pattern of fH changes in response to hypoxia 310 A. KURT GAMPERL AND W. R. DRIEDZIC (i.e., fH increasing by approx. 25 beats min-1 prior to the onset of bradycardia at 35 mmHg; see Figure 7.1A) and that changes in the electrocardiogram (ECG) of this species were decidedly diVerent as compared to three other tropical fish species examined. In C. carpio, the direction of the ECG reversed from + to – with the onset of severe hypoxia, as compared to no change (in Piaractus mesopotamicus) or a – to + transition in H. malabaricus and H. lacerdae, and there was only minimal change in the amplitude of the T-wave of C. carpio with graded hypoxia as opposed to a 1.8- to 4-fold increase in this parameter in the other three species. To this point, we have confined our discussion of the eVects of acute hypoxia on fH to adult fishes. However, there are several papers that have now examined the ontogeny of fH control in fishes under hypoxic conditions. From these studies it is clear that hypoxia-induced reductions in fH and cardiac activity during early development are a direct eVect of severe oxygen shortage on the cardiac myocytes, and that the exact nature of fH responses to acute hypoxia depends on developmental stage and the degree of hypoxia. While the lack of a hypoxia-induced bradycardia in early larval stages has been noted for the rainbow trout (Holeton, 1971) and Arctic charr (Salvelinus alpinus)(McDonald and McMahon, 1977), it is recent work on the zebrafish (Zebra danio) that is the primary basis for these conclusions. For example, although Padilla and Roth (2001) showed that 4 h of anoxic exposure reduced the fH of zebrafish by 40% at 29 hours post-fertilization, Jonz and Nurse (2005) reported that gill neuroepithelial cells (NEC, considered to be the gill’s O2 chemoreceptors) are not expressed until 5 days post-fertilization (dpf) and not innervated until 7 dpf, and Schwerte et al. (2006) indicated that nervous cholinergic tone on the heart is not established until approximately 12 dpf. Further, bradycardia is absent before 20 dpf at rearing temperatures between 25 and 31 C when PO2 does not fall below 10 mmHg during a graded hypoxic challenge (see Figure 7.3; Barrionuevo and Burggren, 1999). With regards to temperature and developmental eVects on the degree and onset of hypoxiainduced bradycardia in zebrafish, the results of Barrionuevo and Burggren (1999) are diYcult to interpret as temperature also aVects developmental rate. However, it appears that fH is more susceptible to hypoxia-induced reductions at warmer rearing temperatures at any given developmental stage (the examined range 0–100 dpf) (e.g., see Figure 7.3). While these studies have greatly advanced our understanding of heart rate development in teleosts, they also raise one intriguing question. If both NEC and the heart are innervated by 12 dpf in zebrafish, and cholinergic receptors are functional in the zebrafish heart by 5 dpf (Schwerte et al., 2006), why does bradycardia not develop until >20 dpf ? The answer to this question will require further study, but the most logical interpretation is that sensitivity of the NEC to reductions in water oxygenation, or of myocardial 7. 311 CARDIOVASCULAR FUNCTION 200 31 ⬚C 28 ⬚C 150 25 ⬚C 100 Day 20 50 0 20 40 60 80 100 120 140 31 ⬚C 28 ⬚C 25 ⬚C 200 Heart rate (beats·min−1) 150 100 Day 30 50 0 20 40 60 80 100 120 140 200 31 ⬚C 28 ⬚C 150 25 ⬚C 100 Day 40 50 0 20 40 60 80 100 120 140 200 31 ⬚C 28 ⬚C 150 25 ⬚C 100 Day 50 50 0 20 40 60 80 100 PO2 (mm Hg) 120 140 Fig. 7.3. Influence of acute hypoxic exposure on heart rate in zebrafish (D. rerio) larvae (day 20 post‐fertilization) and juveniles (days 30, 40, and 50 post‐fertilization) reared at various temperatures. Mean values  SE are plotted; n ¼ 10 for each plotted developmental stage at all three temperatures. [Modified from Barrionuevo and Burggren (1999) with the permission of the American Journal of Physiology.] 312 A. KURT GAMPERL AND W. R. DRIEDZIC cholinergic receptors to nervous stimulation, is low during early life-history stages and increases with development. 2.1.2. Cardiac Output, Stroke Volume, andVenous Tone With the exception of hagfish (Axelsson et al., 1990), which have an extremely low cardiac output (Q) and power output (PO) at rest (Axelsson et al., 1990; Forster et al., 1991), and do not exhibit hypoxia-induced bradycardia, all fish examined to date show an increase in stroke volume (SV) when hypoxia-induced bradycardia develops. The diVerence among species, however, lies in the level of hypoxia at which they initiate increases in SV and the extent that increases in SV compensate for the eVect of hypoxic-induced bradycardia on cardiac output (Q). In general, there are three patterns that are exhibited by fishes, and these are illustrated in Figure 7.4 using the Atlantic cod, Atlantic wolYsh (Anarhichas lupus), and winter flounder as examples. In the first response pattern, as seen in the Atlantic cod and rainbow trout (also see Wood and Shelton, 1980; Sandblom and Axelsson, 2005), SV starts to increase prior to hypoxic bradycardia leading to an initial increase in Q, and SV is initially able to compensate for hypoxia-induced decreases in fH before Q eventually falls. In the second pattern, increases in SV are either concomitant with the onset of bradycardia (e.g., dourado—de Salvo Souza et al., 2001; sea bass, Dicentrarchus labrax—Axelsson et al., 2002; lingcod—Farrell., 1982; Japanese eel—Chan, 1986) or begin after the bradycardia is initiated (often near the limit of hypoxia tolerance, e.g., in wolfish; smallmouth bass; Furimsky et al., 2003). However, these increases in SV are inadequate to compensate for decreases in fH and Q falls almost continuously (albeit slower than fH) with the severity of hypoxia. Finally, in some fishes, for example the winter flounder (Figure 7.4), dogfish shark (Scyliorhinus canicula; Butler and Taylor, 1971), and sturgeon (Acipenser naccarii; Agnisola et al., 1999) (at least down to a PO2 of 35 mmHg), increases in SV initiated during hypoxia are able to fully compensate for the drop in fH with hypoxia, such that Q is maintained. These latter two patterns, if one utilizes the same terminology as applied to the oxygen consumption–water PO2 relationship, are characterized as representing conformers and regulators, respectively, with regards to their Q responses. Given the limited number of species on which direct measurements of Q and SV have been performed under well-controlled experimental conditions, it is not possible to determine to what extent interspecific diVerences in the responses of these parameters to hypoxia are related to diVerences in activity, lifestyle, and hypoxia tolerance. For example, although data on the flounder and sturgeon suggest that hypoxia-tolerant species can maintain Q through increases in SV until low oxygen levels, data on the eel (Anguilla anguilla) indicate that this hypoxia-tolerant species (critical water O2 tension 25 mmHg 7. 313 CARDIOVASCULAR FUNCTION A 45 40 Heart rate (bpm) 35 30 25 20 15 Winter flounder 10 Wolffish Atlantic cod 5 B 1.1 Stroke volume (ml kg−1) 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 Cardiac output (ml min−1 kg−1) C 50 40 30 20 10 0 0 20 40 60 80 100 PO2 (mmHg) 120 140 160 Fig. 7.4. Relationship between water PO2 and various cardiac parameters for the winter flounder (Pleuronectes americanus; Mendonca and Gamperl, unpublished data), wolYsh (Anarhichas lupus; Joaquim and Gamperl, unpublished data), and Atlantic cod (G. morhua; Petersen and Gamperl, unpublished data) at temperatures of 8–10oC. Values are means  S.E. 314 A. KURT GAMPERL AND W. R. DRIEDZIC at 25 C; Cruz-Neto and SteVensen, 1997) does not elevate SV in response to bradycardia at 40 mmHg O2 (Peyraud-Waitzenegger and Soulier, 1989). Further, we have a less than complete picture of what mechanisms, other than the increase in filling time and filling pressure (resulting from the pooling of central venous blood) that are concomitant with bradycardia (Farrell, 1991; Altimiras and Axelsson, 2004), enable fish to elevate SV in response to aquatic hypoxia. However, evidence has accumulated over the past decade that the active regulation of venous tone and cardiac filling are equally important for regulating SV in fishes, including during hypoxia. For example, Sandblom and Axelsson (2005) showed that venous pressure (Pven) and SV increase in rainbow trout at water oxygen levels that do not elicit bradycardia (Figure 7.5). Sandblom and Axelsson (2006) showed, using venous capacitance curves, that some of the venous blood volume is actively shifted into the stressed vascular compartment by an increase in venous smooth muscle tonus during hypoxia, and that this results in an elevated mean circulatory filling pressure (Figure 7.6). Sandblom and Axelsson (2006) showed that hypoxiainduced changes in trout venous capacitance are primarily under a-adrenergic control, and that this regulation has both neural and hormonal components [based on the diVerential eVects of the a-adrenergic agonist prazosin and the neuronal blocking agent bretylium on the mean circulatory filling pressure (MCFP); see Figure 7.6]. Finally, Skals et al. (2006) showed the venous system plays an important regulatory role with regards to cardiac filling and SV in the air-breathing swamp eel (Synbranhus marmoratus) during hypoxia, and that this control is dependent upon both a- and b-adrenergic mechanisms. This latter study is important because it shows that venous tone may be controlled by similar mechanisms across a range of teleost species. Although venous tone appears to be a major factor controlling cardiac filling and SV/Q during hypoxia, several other mechanisms may be involved. These include: (1) diminished myocardial force development; (2) hypoxiamediated changes in gill vascular resistance, potentially leading to alterations in cardiac afterload and end-systolic volume; and (3) local (regional) alterations in vascular tone resulting in reduced systemic vascular resistance (Rsys) and a decreased arteriovenous pressure gradient (Sandblom and Axelsson, 2005). Although we will examine the eVect of anoxia/severe hypoxia on myocardial function/contractility later in the chapter, the next two sections discuss the eVect of hypoxia on branchial and systemic resistance, and evaluate their capacity to contribute to changes in arterial blood pressure. 2.1.3. BranchialVascular Responses to Hypoxia Most teleosts and elasmobranchs respond to severe hypoxia with an increase in branchial vascular resistance (Rgill)(Butler and Taylor, 1975; Farrell, 1982; Pettersson and Johansen, 1982; Sundin and Nilsson, 1997; 7. 315 CARDIOVASCULAR FUNCTION A 0.4 Pven (kPa) 0.3 0.2 * 0.1 0.0 B 200 180 SV (%) 160 140 120 * 100 80 60 ƒH (beats min−1) C 80 60 40 Mild hypoxia 5 min 20 Fig. 7.5. EVect of 8 min of mild hypoxia (PO2 85 mmHg; section located between dotted lines) on central venous pressure (Pven), stroke volume (SV), and heart rate (fH) in rainbow trout (Oncorhynchus mykiss). Statistically significant diVerence between the average value for the normoxic period and the average value of the last 2 min of the hypoxic period. [Modified from Sandblom and Axelsson (2005).] Stensløkken et al., 2004), and the mechanisms mediating changes in pressure and flow within the gill have been studied using a number of techniques, including epi-illumination microscopy. Once blood reaches the teleost gill from the heart it flows through the aVerent filament arteries (AFA), is oxygenated in the secondary lamellae (SL), and then reaches the eVerent 316 A. KURT GAMPERL AND W. R. DRIEDZIC Blood volume (%) A 140 Untreated 120 100 80 * * * * * 60 Blood volume (%) B 140 Prazosin 120 ¶ 100 ¶ *¶ 80 60 Blood volume (%) C 140 Bretylium 120 * * 100 * 80 60 −0.2 * * 0 0.2 0.4 0.6 MCFP (kPa) 0.8 1 1.2 Fig. 7.6. Mean values ( S.D.) of mean circulatory filling pressure (MCFP) at 80–120% of total blood volume in untreated (n ¼ 11) (A), prazosin-treated (1 mg/kg; n ¼ 9) (B), and bretyliumtreated (10 mg/kg; n ¼ 9) (C) rainbow trout (O. mykiss). Solid lines represent normoxia and broken lines represent hypoxia (70 mmHg). *Statistical diVerence between normoxia and hypoxia; and } statistical difference between prazosin‐ and bretylium-treated normoxic values compared with corresponding untreated normoxic values (P < 0.05). [Reproduced from Sandblom and Axelsson (2006) with permission from the American Journal of Physiology.] filamentous artery (EFA). At this point, however, it has two pathways that it can follow, and control of gill blood flow during hypoxia dramatically alters the distribution between these two pathways (Figure 7.7). Under resting normoxic conditions, the majority of blood leaving the SL enters the EFA, with only 5–30% of the blood flow entering the arteriovenous anastomoses (AVA) and nutritive vasculature (NV) where it is returned to the venous system through the branchial vein (BV) (Hughes et al., 1982; Ishimatsu et al., 1988; Sundin and Nilsson, 1992). In contrast, during severe hypoxia, 7. 317 CARDIOVASCULAR FUNCTION CNS X SC BN ACh 5-HT Adr Adr EFA Adr Sph NV CVS AFA 5-HT ? Adr ELa AVAs Ado SL Ala Fig. 7.7. Working model for autonomic control of teleost gill vasculature, demonstrating potential sites for control of vascular resistance and thereby blood flow distribution between the arterioarterial and arteriovenous pathways. Sphincter (represented by dark oblong shapes) at base of the eVerent filamental arteries (EFA) is a key site aVecting blood pressure and flow in the gill vasculature. Cholinergic and serotonergic innervation of the sphincter produce constriction, whereas adrenergic innervation may cause dilation, acting through b-adrenoceptors. Other potential sites for blood distribution control are the nutritive vasculature (NV) and arteriovenous anastomoses (AVAs). Adrenergic innervation of NV produces vasoconstriction via a-adrenoceptors, and this may also apply to AVAs, whereas serotinergic innervation and locally released adenosine (Ado) have been shown to cause dilation. AFA, aVerent filamental artery; ALa, aVerent lamellar arteriole; SL, (secondary) lamella; AVAs, anastomoses between EFA, eVerent lamellar arteriole, and central venous system (CVS); ELa, eVerent lamellar arteriole; BN, branchial nerve; ACh, Adr, and 5-HT putative nerve types (cholinergic, adrenergic, and serotonergic, respectively); CNS, central nervous system; SC, sympathetic chain; Sph, sphincter at base of EFA; X, vagus nerve. [Modified from Sundin and Nilsson (1997), with permission of the American Journal of Physiology.] 318 A. KURT GAMPERL AND W. R. DRIEDZIC blood flow in the BV increases considerably (by about 1.5-fold in cod; Sundin, 1995), and a greater proportion of oxygenated blood is returned to the venous circulation. This redistribution of blood flow potentially plays a critical role in hypoxia tolerance by facilitating the energy-demanding work of ion-transporting cells that are located in the filamental epithelium, and of the heart by raising the oxygen content and partial pressure of the venous blood that supplies the myocardium (at least the spongy myocardium). Control of flow and resistance in the teleost and elasmobranch gill vasculature has been studied using a number of pharmacological agonists and antagonists (both in normoxia and hypoxia)( Pettersson and Nilsson, 1979; Nilsson, 1984; Sundin, 1995; Sundin and Nilsson, 1996; Sundin and Nilsson, 1997; Smith et al., 2001; Stensløkken et al., 2004), and although abrupt (rapid) hypoxic exposures have been used exclusively in these investigations (and thus extension of this knowledge to the eVects of moderate or gradual hypoxic exposures is unclear), we have a fairly comprehensive understanding of the control of gill vascular tone and blood flow under these conditions. First, although vasoactive compounds such as acetylcholine (Sundin and Nilsson, 1997) and adenosine (Sundin and Nilsson, 1996) can constrict the distal parts of the EFA and/or AFA, no constriction of these vessels has been observed during hypoxia using in vivo epi-illumination microscopy (Sundin and Nilsson, 1997; Stensløkken et al., 2004). This suggests that the net eVect of all mechanisms that mediate the hypoxic response does not involve vasoconstriction of the distal vasculature on either the aVerent or eVerent side. Second, alterations in neurohormonal control of AVA/NV and EFA resistance work in concert to re-direct flow into the teleost gill’s venous circulation during hypoxia (Sundin, 1995; Sundin and Nilsson, 1997). For example, it appears that during normoxia (and possibly moderate hypoxia) b-adrenergic mediated dilation of the sphincters located at the base of the EFA (and distal to the AVA opening), and a-adrenergic mediated constriction of the AVA/ NV predominate, and thus the majority of blood flow enters the arterioarterial system. Whereas, during severe hypoxia, cholinergic and serotinergic nerves cause constriction of the sphincter, while adenosine (mediated though A1 receptors) and increases in seritonergic nerve activity dilate the AVA and/ or NV: the result of these neurohormonal changes is an increase in Rgill and enhanced flow through the AVA and NV (i.e., arteriovenous system). Third, although hypoxia can directly constrict the arterioarterial pathway (i.e., the EFA in teleosts; Sundin et al., 1995; Smith et al., 2001), it appears that hypoxia-induced vasoconstriction of the EFA is normally balanced by the dilatory eVects of norepinephrine released from adrenergic nerves (Sundin et al., 1995). Finally, although the branchial vascular anatomy of elasmobranchs diVers significantly from that of teleosts, it appears from the study of Stensløkken et al. (2004) that cholinergic-mediated constriction of the EFA 7. CARDIOVASCULAR FUNCTION 319 sphincter and adenosine-induced dilation of gill longitudinal vessels (the functional equivalent of the AVA in teleosts) also play a role in gill vasomotor responses to hypoxia in this taxa. 2.1.4. Systemic Vascular Resistance and Changes in Arterial Pressures When examining the literature relating to these parameters, it is diYcult to make even the most basic generalizations about how they are aVected by hypoxia. There are several reasons for this. First, the majority of experiments have involved the rapid exposure of fish to hypoxia of limited duration (i.e., 8–12 min), and many of the cardiovascular responses to this type of experimental protocol are transitory or inconsistent. For example, Axelsson and Fritsche (1991), Sundin (1995), and Fritsche and Nilsson (1989) all exposed Atlantic cod to a water PO2 of 40–50 mmHg for 8–10 min and report that while Rsys increased initially by 20–50%, Rsys returned to prehypoxic levels within the first 5–6 min of hypoxia (i.e., the response of Rsys was likely an acute stress response due to the protocol and not associated with hypoxia itself; e.g., see Ristori and Laurent, 1989). There does not appear to be a clear relationship between the severity of hypoxia and Rsys, as the Rsys of rainbow trout increases by approximately 10% at a PO2 of 85 mmHg, decreases by approximately 30% at a PO2 of 50 mmHg, but is essentially unchanged by exposure to severe hypoxia (PO2 <10 mmHg) (Sundin and Nilsson, 1997; Sandblom and Axelsson, 2005). Finally, it is diYcult to predict how dorsal aortic pressure (PDA) and ventral aortic pressure (PVA) will change when exposed to rapid hypoxia, because of the three major responses of Q. Also, Q can vary considerably even at the same level of hypoxia within a species [e.g., the Q of Atlantic cod did not change in Fritsche and Nilsson (1989) but increased by 50% in Axelsson and Fritsche (1991 and Sundin (1995)]. Second, although isolated vessels of the rainbow trout respond to hypoxia, whether the vessels are refractory, constrict, or dilate depends on the type of vessel and the nature of pre-existing stimulation, and other factors (e.g., season or other environmental factors) appear to play a modulatory role in conditioning the response of the vessels (Smith et al., 2001). Third, the response of PDA and PVA to graded hypoxia is species-dependent among teleosts; the rainbow trout showing significant increases in both parameters starting at approximately 70–80 mmHg (Holeton and Randall, 1967), tuna showing no change in either parameter down to 50 mmHg (Bushnell and Brill, 1992), whereas both the Japanese eel (Anguilla japonica; Chan, 1986) and lingcod (Farrell, 1982) become hypotensive somewhere between 75 and 35 mmHg. It is obvious from the preceding discussion that our knowledge of the control of systemic vascular resistance and blood pressure in fishes is extremely limited, and that carefully designed experiments using appropriate 320 A. KURT GAMPERL AND W. R. DRIEDZIC acclimation times/conditions and varied species will be required before our understanding is significantly enhanced. However, there are a few points that are worth making at this time. Based on the limited species examined to date, it appears that elasmobranchs and sturgeons regulate systemic vascular resistance in a diVerent way from teleosts. Systemic vascular resistance decreases in the dogfish (Butler and Taylor, 1971) and sturgeon (Agnisola et al., 1999) at PO2s less than 60 mmHg, while it increases in those teleosts examined to date, including the rainbow trout (Holeton and Randall, 1967), lingcod (Farrell, 1982), and Japanese eel (Chan, 1986). Two studies have investigated the eVects of hypoxia on gastrointestinal (GI) blood flow (Axelsson and Fritsche, 1991; Axelsson et al., 2002), and provide important insights into the regulation of Rsys and blood flow distribution during hypoxia (e.g., see Figure 7.8). (1) Resistance in vessels supplying the GI 20 6 P VA 4 2 * * P DA qCoA/qMeA (% change) Pressure (kPa) 8 −60 80 40 RMeA/RCoA (% change) * Q̇ 40 20 0 −20 * −40 20 40 VS Rs (% change) ƒH (beats min−1) −20 600 480 360 240 120 0 −60 60 60 qCoA qMeA * −80 0 Q/VS (% change) 0 * RMeA * RCoA 0 −40 −80 Fig. 7.8. The response of Atlantic cod (Gadus morhua) cardiovascular parameters to 8 min of hypoxic exposure (PO2 30–40 mmHg) (N = 8–12). Data are means  S.E. Asterisks indicate statistically significant (P <0.05) diVerences compared with normoxic values. PVA, ventral aortic pressure; PDA, dorsal aortic pressure; fH, heart rate; Q, cardiac output; VS, stroke volume; qCoa, flow in coeliac artery; qMeA, flow in mesenteric artery; RCoA, resistance in coeliac artery; RMeA, resistance in mesenteric artery; Rsys, systemic vascular resistance. [Modified from Axelsson and Fritsche (1991).] 7. CARDIOVASCULAR FUNCTION 321 vessels of cod increases by 150–320% when exposed to severe hypoxia (30–40 mmHg), and this vasoconstriction is dependent on both nervous and humoral adrenergic mechanisms; these two mechanisms playing varied, but important, roles in controlling overall systemic vascular resistance in cod and rainbow trout at rest and during exercise and hypoxic exposure (Randall and Daxboeck, 1982; Smith et al., 1985; Axelsson and Nilsson, 1986; Fritsche and Nilsson, 1990). (2) The relative proportion of Q reaching the gut was reduced by approximately 50% and 20% in unfed versus fed seabass, respectively, when exposed to hypoxia (Axelsson et al., 2002). This study shows that locally released vasoactive substances can oVset the eVects of hypoxia on GI vascular resistance and blood flow. (3) The observation that these large increases in resistance of the GI circulation did not lead to increases in Rsys, and that GI blood flow as a proportion of cardiac output fell by 50% during hypoxia indicates that the somatic vasculature is significantly dilated at these water oxygen levels and that there is a redistribution of blood flow from the GI circulation to the somatic circulation. These direct measurements of blood flow contradict earlier measurements using microspheres, which suggested that the proportion of Q directed to the muscles and visceral organs does not change during hypoxia (Cameron, 1975). Finally, it has been recently reported that concentrations of H2S (hydrogen sulphide) in rainbow trout plasma reach levels that produce vasoactive eVects in isolated vessels (Dombkowski et al., 2004), that H2S is constitutively synthesized by vascular smooth muscle and cellular concentrations are determined by a simple balance between H2S production and the amount of O2 available for H2S (hydrogen sulphide) oxidation (Olson, 2008), and that hypoxia and H2S evoke the same response in vertebrate isolated blood vessels, irrespective of whether the response is a contraction, relaxation, or is multiphasic (see Fig. 1 in Olson, 2008). Given the extremely varied response of isolated trout vessels to hypoxia (Smith et al., 2001) and the data on H2S to date, it appears that H2S acts as an ‘‘oxygen sensing molecule’’ in smooth muscle cells (e.g., see Olson et al., 2006). Further research eVorts in this area will significantly enhance our understanding of the regulation of blood flow and vascular resistance in fishes during both hypoxia and hyperoxia. 2.2. Chronic Hypoxia In contrast to the large body of literature that exists on the eVects of acute hypoxia on fish cardiovascular function, the eVects of chronic (days to weeks) hypoxia has been largely overlooked; with the exception of the crucian carp (see Chapter 9). However, there are now a few studies that have looked at the eVects of chronic hypoxia on cardiovascular function in developing zebrafish and adult fish. 322 A. KURT GAMPERL AND W. R. DRIEDZIC 2.2.1. Hypoxic Effects on Zebrafish Cardiovascular Development/Function To date, four studies have investigated the eVect of chronic hypoxia on cardiovascular development, and although the use of diVerent temperatures, levels of hypoxia, and strains complicates some interpretations, these studies reveal novel insights into how chronic hypoxia modifies cardiovascular function and development in the early life history stages of this species. In the first study on the eVects of chronic hypoxia on zebrafish cardiac function, Jacob et al. (2002) placed embryos into moderately hypoxic (approx. 75 mmHg O2) water and measured heart function using videomicroscopy from 1 to 12 dpf at temperatures ranging from 25 to 31oC. This study revealed some temperature-dependent diVerences in the response of cardiac function to hypoxia (i.e, when dpf changes appeared), but clearly showed that chronic hypoxia increases Q as early as 3–4 dpf (at 25 and 28oC) and that this elevated Q was due to both increases in fH and end-diastolic volume (Figure 7.9). This is a very interesting finding, as adrenergic receptors do not appear in the normoxic zebrafish heart until 5–6 dpf (Bagatto, 2005; Schwerte et al., 2006), exposure to severe hypoxia (15 mmHg) apparently delays the appearance of adrenergic receptors in the heart by 2 days at 25oC (Bagatto, 2005), and vagal tone on the heart is not established until 12 dpf at 28oC (Schwerte et al., 2006). Further, the direct eVect of hypoxia on vertebrate cardiomyocytes is normally a decrease in activity, and chronic exposure of zebrafish embryos/larvae to more severe hypoxia results in a sustained decrease, not increase, in fH (Figure 7.10, Bagatto, 2005; Moore et al., 2006). Collectively, these results suggest that cardiac function was increased in the study by Jacob et al. (2002) because convective oxygen transport becomes important in fish larvae when the gradient for bulk oxygen diVusion (i.e., from the environment to the tissues) is reduced, that the aVerent nervous system is capable of sensing hypoxic conditions very early in life, and that central control units are active and can indirectly stimulate the heart via some presently unidentified hormone. While the increase in fH was most likely due to stimulation of the cardiac pacemaker or the cardiomyoctyes, Jacob et al. (2002) do not provide any explanation for the increase in end-diastolic volume with hypoxia. However, this could be due to an increase in ventricle size (a parameter not measured in any of the studies), ventricular remodeling (but see Marques et al., 2008), or an increase in venous tone/pressure and thus cardiac filling (this homeostatic mechanism clearly established for adult fish; see Sandblom and Axelsson, 2005, 2006). With regards to the eVects of chronic hypoxia on the zebrafish’s vasculature, a number of eVects were also noted. These included increases and decreases in blood flow distribution to the muscle (by approx. 350%) and GI tract (by approx. 45%), respectively, 7. 323 CARDIOVASCULAR FUNCTION A (T = 25⬚C) Heart rate (beats min−1) 300 * * 200 100 0 2 3 4 5 6 7 B Cardiac output (µl min−1) 0.10 * 0.08 * * 4 5 0.06 0.04 0.02 0 2 3 6 7 C Cardiac volume (nl) 0.8 * * Diastole 0.6 0.4 Systole 0.2 0 2 3 4 5 6 Time of development (dpf) 7 Fig. 7.9. Cardiac activity [heart rate (A); cardiac output (B); systolic and diastolic ventricular volumes (C)] in zebrafish (D. rerio) larvae raised under normoxia (PO2 ¼ 150 mmHg) and under chronic hypoxia (PO2 ¼ 75 mmHg) at a temperature of 25 C (n ¼ 10). dpf ¼ days post‐ fertilization.*Significantly diVerent from controls (P <0.05). [Modified from Jacob et al. (2002), with permission of the American Journal of Physiology.] 324 A. KURT GAMPERL AND W. R. DRIEDZIC 200 A 180 Heart rate (beats mini−1) C 160 140 C 120 A 100 Control (normoxia) Development in hypoxia 80 60 0 2 10 4 6 8 Age (days post-fertilization) 12 14 Fig. 7.10. Mean resting heart rate (beats min 1) during development in the zebrafish (D. rerio) at 25  C when reared under normoxia and hypoxia (PO2 <10 mmHg). The letter C indicates the first significant negative chronotropic response to cholinergic agonists during development, and the letter A indicates the first significant positive chronotropic response to adrenergic agonists. All data points are significantly diVerent from the corresponding controls. Data shown are means  S.E. [Modified from Bagatto (2005).] of 12–15 dpf zebrafish (Schwerte et al., 2003), and changes in the size of major arteries and veins (although these latter results were study and age dependent: Bagatto, 2005; Moore et al., 2006). An increase in blood flow to the muscle (especially superficial red muscle) would distribute blood from the core to the surface of the larvae, and thus potentially enhance cutaneous oxygen uptake and internal oxygen convection during hypoxic conditions. While the mechanism(s) facilitating the redistribution of blood flow in hypoxic zebrafish larvae are unknown, a-adrenergically controlled precapillary sphincters are present in the intersegmental muscle tissue of zebrafish by 8 dpf (Schwerte and Pelster, 2000) and Fritsche et al. (2000) showed that nitric oxide can cause vasodilation in the zebrafish as early as 5 dpf. However, the redistribution of blood flow is clearly not due to changes in angiogenesis (increased/decreased vascularization) as Schwerte et al. (2003) failed to show any eVect of chronic hypoxia (PO2 65 mmHg) on vascularization of the tail muscle or the gut. While the above results are novel, and informative, the work of Moore et al. (2006) may be the most important as it sets the stage for potentially 7. 325 CARDIOVASCULAR FUNCTION transformative discoveries relative to how genetics and environmental challenges during the embryonic/larval period influence adult cardiovascular function and morphology. This study tested for family-specific diVerences in the response of an integrated set of cardiovascular traits to severe hypoxia (approx. 15 mmHg) and reported that considerable variation in the degree of familial response to hypoxia exists in cardiovascular traits that relate to Q (Figure 7.11). While these authors only measured these traits at 4 dpf, and thus it is not known whether diVerences in traits at this stage of development translate into diVerences in the juvenile or adult, there is indirect evidence A B 110 1 100 0.75 80 Volume (nl) Heart rate (beats/min) 90 70 60 Family I Family J Family K Family L Family M 50 40 30 C Hypoxia 0.25 0 Normoxia Hypoxia D 0.8 0.7 Normoxia 60 Cardiac output (nl/min) 50 0.6 Stroke volume (nl) 0.5 0.5 0.4 0.3 40 30 20 0.2 10 0.1 0 0 Hypoxia Normoxia Environment Hypoxia Normoxia Environment Fig. 7.11. Cardiac performance in zebrafish (D. rerio) as aVected by family and environment (hypoxia, <10 mmHg; normoxia). In B, solid lines and symbols represent means for end diastolic volume (EDV) while intermittent lines and open symbols represent end systolic volume (ESV). Values are means  1 S.E. Family and family  environment interactions were significant sources of variation in all five traits, while a direct eVect of environment was only significant in heart rate and ESV. [Reproduced from Moore et al. (2006).] 326 A. KURT GAMPERL AND W. R. DRIEDZIC that genetic variation in response plasticity may provide the basic ingredient for adaptation to variable environments. For example the work of Gamperl et al. (Gamperl et al., 2004; Faust et al., 2004; Overgaard et al., 2004b) shows that there is considerable variation in the inherent myocardial hypoxia tolerance of rainbow trout from diVerent hatcheries, and that this influences their capacity to be preconditioned (see below). 2.2.2. Effects on Adult Cardiovascular Function Surprisingly, to date, there is only one publication on the eVects of chronic hypoxia on in vivo cardiovascular function. Burleson et al. (2002) acclimated channel catfish to normoxia and hypoxia (PO2 75 mmHg) for 1 week, and reported that fH in hypoxia-acclimated fish was 15–20% higher as compared with normoxia-acclimated individuals when tested under both normoxia and hypoxia. However, Petersen and Gamperl (unpublished data) recently made the first measurements of fish cardiorespiratory function during exercise (from rest to critical swimming speed) under hypoxia (PO2 approx. 60 mmHg), and of how acclimation of cod to this same level of hypoxia for 6–12 weeks influenced resting and exercise-induced cardiac function under both hypoxic and normoxic conditions. This study confirmed the findings of Burleson et al. (2002) with regards to chronic hypoxia increasing resting fH (see below; also Figure 7.12), and suggests that this is a regulated response. This is because Petersen and Gamperl (unpublished data) report that hypoxia-acclimated cod had significantly lower values for resting and maximum SV and Q in both swim tests, and a significantly lower scope for SV when swum under hypoxic conditions, as compared with the normoxia-acclimated group (Figure 7.12). There are at least three potential explanations for the poor pumping capacity of hearts from hypoxia-acclimated fish. First, it is possible that the cod myocardium was damaged by constant exposure to low oxygen conditions. Such a conclusion would be consistent with the findings of Lennard and Huddart (1992) who reported that cardiomyocytes in flounder (Platichthys flesus) subjected to 3 weeks of hypoxia (water PO2 35 mmHg) showed striking changes in mitochondrial morphology (decreased size, budding, and necrosis) and evidence of myofibril degeneration. However, the level of hypoxia utilized in the Petersen and Gamperl (unpublished data) (approx. 60 mmHg) was not nearly as severe as that used by Lennard and Huddart (1992), and several studies have shown that, at least in the trout heart, acute (<30 min) exposure to severe anoxia (perfusate PO2 <1 kPa) does not result in myocardial necrosis or a disruption in myocardial energetic and enzymatic status (Faust et al., 2004; Overgaard et al., 2004a,b). These data, thus, question whether myocardial damage/necrosis was experienced by the hypoxia-acclimated cod. Second, it is possible that the hearts of hypoxia-acclimated cod were merely ‘‘stunned’’ (i.e., experiencing mechanical dysfunction related to a decrease in myocardial 7. 327 CARDIOVASCULAR FUNCTION Normoxic swim Hypoxic swim 55 55 Ucrit test Heart rate (beats min−1) 50 * 45 * ** 50 ** Recovery * 45 * * * 40 40 35 35 30 30 25 25 1.4 1.4 * 1.2 Stroke volume (ml kg−1) Ucrit test * * Recovery 1.0 0.8 *** * ** 1.2 * * ** ** 1.0 * * 0.8 * 0.6 * * * *** * 0.6 * 0.4 0.4 0.2 0.2 60 60 Cardiac output (ml min−1 kg−1) * * 50 * 40 50 ** 40 30 ** * 20 * * * 20 10 10 0 Oxygen consumption (mg O2 h−1 kg−1) * 30 0 250 250 * 200 200 150 150 * 100 50 0 0.0 * *** * * 100 * 50 Normoxic acclimated Hypoxic acclimated 0 0.5 1.0 1.5 Swimming speed (body length sec−1) 25 125 Time (min) 0.0 0.5 1.0 1.5 Swimming speed (body length sec−1) 25 125 Time (min) Fig. 7.12. Cardiac parameters and oxygen consumption in normoxia- (N ¼ 10) and hypoxiaacclimated (PO2 60 mmHg, N ¼ 12) Atlantic cod (Gadus morhua) during critical swimming speed (Ucrit) tests, and during postexercise recovery. All fish were swum in normoxic water on day 1 and hypoxic water on day 2, but recovery was performed in normoxic water for both swims. *Indicates a significant diVerence (P <0.05) between the normoxia- and hypoxia-acclimated groups at a particular swimming speed (Petersen and Gamperl, unpublished data). 328 A. KURT GAMPERL AND W. R. DRIEDZIC calcium sensitivity; see Bolli and Marban, 1999). However, this seems unlikely as Driedzic et al. (1985) showed that 4–6 weeks of hypoxic acclimation (at 45 mmHg) enhanced the contractility of normoxic myocardial strips from Zoarces vivparous under conditions of elevated calcium. Further, Gamperl and Petersen (unpublished data) have shown that while the maximum in situ cardiac performance of hearts from hypoxia-acclimated cod is reduced by a similar amount to that measured in vivo under normoxic conditions (by approx. 25%; see Figure 7.12), they can maintain maximum cardiac function under severe hypoxia (PO2 5–10 mmHg) longer than hearts from normoxia-acclimated cod, and show enhanced post-hypoxia recovery of maximum cardiac function as compared to their normoxia-acclimated counterparts. Finally, it is possible there was no myocardial damage or significant dysfunction in the hearts of hypoxiaacclimated cod, and that hypoxia-induced myocardial remodeling reduced the maximum SV of the heart. Although, the similar relative ventricular mass (RVM) in hypoxia- and normoxia-acclimated cod (in contrast to the increase in RVM after trout are made hypoxemic by repeated injections of phenylhydrazine; Simonot and Farrell, 2007) provides some evidence against extensive cardiac remodeling in chronically hypoxic cod, Marques et al. (2008) showed that acclimation of zebrafish and the cichlid (Haplochromis piceatus) to 75 mmHg O2 increased cardiac myocyte density (presumably though hyperplasia) and that this resulted in a smaller ventricular outflow tract and reductions in the size of the central ventricular cavity and lacunae (Figure 7.13). Such a decrease in the capacity of the ventricle to fill with blood would certainly explain why maximum in vivo SV was reduced by 28% in the hypoxia-acclimated cod, and raises the possibility that cardiac remodeling caused by hypoxic acclimation aims to reduce the wall tension required to eject blood from the ventricle (i.e., see Law of LaPlace) and the workload of individual cardiomyocytes. Clearly, more research needs to be conducted before the mechanism(s) mediating the diminished pumping capacity of hearts from hypoxia-acclimated fishes can be understood. Interestingly, despite the diminished SV and elevated resting fH, hypoxiaacclimated cod were able to increase fH by a similar amount in the normoxic swim (i.e., the scope for fH was not altered by hypoxia acclimation), and they were able to elevate fH during the hypoxic swim to levels measured during normoxia (Figure 7.12). This latter result allowed them to have a significantly greater scope for fH (12.6 vs. 5.8 beats min 1 in normoxia-acclimated fish) when tested under hypoxic conditions, and to achieve the same maximum Q as compared to normoxia-acclimated fish when swum at 60 mmHg O2. The mechanism(s) resulting in the diVerential regulation of fH in the two groups when swum under hypoxic conditions cannot be ascertained from the work of Petersen and Gamperl (unpublished data) or the literature. However, as the fH of in situ hearts from hypoxia-acclimated cod was similar to that of 7. 329 CARDIOVASCULAR FUNCTION A B Normoxia 0.2 mm Hypoxia 0.2 mm Fig. 7.13. Histological changes in cichlid (Haplochromis piceatus) hearts after exposure to chronic constant hypoxia (PO2 ¼ 15 mmHg for 3 weeks). (A) Heart of normoxia-acclimated individual. (B) Heart of hypoxia-acclimated individual. Hearts were sectioned and stained with hematoxylin-eosin. [Modified from Marques et al. (2008).] normoxia-acclimated hearts at resting or maximal Q (Petersen and Gamperl, unpublished data), it is clear that the enhanced capacity of hearts from hypoxia-acclimated fish to elevate fH is under neural and/or hormonal control. These results, in combination with recent data showing that rainbow trout at 24 C can maintain Q even when fH is cut in half using the pharmacological agent zetabradine (Gamperl et al., 2008), highlight the tremendous plasticity in how fish cardiorespiratory physiology responds to environmental challenges and that our understanding of control mechanisms that mediate myocardial function and adaptation in fishes is far from complete. 3. CARDIAC ENERGY METABOLISM Earlier reviews summarize the control of energy metabolism in fish hearts (Driedzic, 1992; Driedzic and Gesser, 1994). In addition, Farrell and Stecyk (2007) provide a more recent discussion of rates of ATP turnover under normoxia and hypoxia and how they relate to the heart’s power output requirements, especially in hagfish, carps, and the rainbow trout. They point out that there are two general strategies in the cardiac response to hypoxia. The first is to meet routine power output (ATP requirement) with a much enhanced anaerobic ATP production (i.e., glycolysis); the second is to reduce 330 A. KURT GAMPERL AND W. R. DRIEDZIC power output (ATP demand). In either situation, however, energy generation could be supported by an enhanced oxygen extraction. This section builds upon these contributions and addresses more recent issues of how metabolism under hypoxic conditions is regulated and serves to extend survival time. Earlier papers are referred to only in the development of key points. 3.1. Creatine Phosphate and ATP Levels The primary function of energy metabolism in the heart is to maintain ATP levels to support the ATPases of the contractile apparatus and of ion pumping. Studies with whole animals and with in vitro preparations reveal that total heart ATP levels are generally well defended under hypoxic conditions and that cardiac failure ensues before any substantive drop in total tissue ATP (see Driedzic and Gesser, 1994). This is especially true if ATP demand is low (Arthur et al., 1992; Driedzic and Gesser, 1994; Overgaard and Gesser, 2004). In contrast, Creatine Phosphate (CP) levels often fall to extremely low levels under hypoxic conditions, and although rarely measured, this would result in concomitant increases in the free phosphate pool (an increase in phosphate reportedly one of the mechanisms responsible for contractile failure; Allen et al., 1985; Godt and Nosek, 1989). For instance, in the perfused rainbow trout heart performing basal levels of work, CP decreased by 80% when exposed to anoxia while ATP levels remained constant (Arthur et al., 1992). Interestingly, microarray experiments reveal 3- to 4-fold decreases in heart creatine kinase mRNA levels as a result of hypoxic acclimation. This was shown for Gillichthys mirabilis held for 6 days (Gracey et al., 2001) and for zebrafish held for 21 days, both at 20 mmHg oxygen (Marques et al., 2008). The functional significance of this remains to be ascertained, but it could result in slower rates of CP discharge (and thus phosphate accumulation) in hypoxiaadapted fish when faced with an acute hypoxic challenge or slower rates of CP replenishment following hypoxic episodes. Ventricle preparations functioning at reduced levels of work under hypoxia can often increase force development in response to Ca2+ application (Driedzic and Gesser, 1994; Bailey et al., 2000). This is an important finding since it suggests that ATP production mechanisms need not be operating at the maximum rates under some hypoxic conditions, and therefore that the rate of ATP production is not necessarily the limiting factor to performance. 3.2. Decreasing ATP Demand A hallmark of anoxic tolerance is the ability to decrease ATP demand. As discussed earlier in this review, the hypoxia-induced bradycardia shown by some species will decrease demands on myofibrillar ATPase. Further, 7. CARDIOVASCULAR FUNCTION 331 Vornanen et al. (see Chapter 9) discuss myosin isoforms that change from summer to winter in crucian carp, and energy conservation under hypoxia occurs through other processes as presented below. Hypoxia is associated with decreased demands of ion pumping mechanisms. The reader is again referred to Chapter 9 for a discussion of the relationship between electrical activity and Ca2+ management. Crucian carp (C. carassius) acclimated to <6 mmHg oxygen showed a 30% decrease in maximal in vitro Na+ K+ ATPase activity (Paajanen and Vornanen, 2003). The authors propose that this is related to the decreased number of action potentials associated with slower heart rates, and that this alone could contribute to reduced ATP usage. Surprisingly, there was no change in properties of the inwardly rectifying K+ current, a major leak and repolarizing pathway. However, it was stated (although not documented) that in a few recordings sarcolemmal KATP (sarcKATP) channel activity was observed and this occurred more frequently in hypoxic than normoxic myocytes. SarcKATP channels have been identified in crucian carp and rainbow trout myocytes, and these channels increase their open probability in vitro in response to lack of ATP or complete metabolic inhibition (oxygen stripping and glycoytic poisoning) (Paajanen and Vornanen, 2002). Total ATP levels generally remain relatively stable under hypoxic conditions; however, it is likely that there are subcellular microenvironments in which the ATP/ADP ratio decreases. This could in turn open sarcKATP channels resulting in K+ eZux that serves to shorten the duration of the action potential, limiting Ca2+ influx, and consequently reducing contractility. There is compelling evidence that the opening of these channels is important in some species. Application of anoxia (NaCN plus N2) to rainbow trout ventricle strips resulted in a rapid loss of force associated with a transient decrease in action potential duration (Gesser and Høglund, 1988). In the goldfish heart, hypoxia results in a decrease in action potential duration in association with the opening of sarcKATP channels and the opening of these channels improves hypoxic cell viability. Furthermore, the opening of sarcKATP channels appears to be mediated by nitric oxide activation of guanylyl cyclase (Cameron et al., 2003; Chen et al., 2005). This is presumably followed by phosphorylation of the channel protein via protein kinase G (Han et al., 2001). Regardless of the mechanism, the opening of sarcKATP channels under hypoxia could serve to decrease demands on Na+ K+ ATPase, Ca2+ ATPases and indirectly on myofibrillar ATPases. In eVect, there is an elegant feedback mechanism at the metabolic level whereby a small decrease in ATP could by itself reduce further ATP demand. Mitochondrial KATP (mitKATP) channels may also be involved in the hypoxia defense mechanism, both through decreasing contractility and maintaining mitochondrial integrity. Treatment of hypoxic ventricle strips 332 A. KURT GAMPERL AND W. R. DRIEDZIC from yellowtail flounder (Limanda ferruginea) with the drug diazoxide, a mitKATP channel opener, exacerbated the loss of twitch force presumably conserving ATP (MacCormack and Driedzic, 2002) and extending the hypoxic viability of isolated heart cells from goldfish (Cameron et al., 2003). In contrast, force development was increased under hypoxia in ventricle strips from the Amazonian armoured catfish, Liposarcus pardalis, when treated with 5-hydroxydecanoic acid (5HD), a mitKATP channel blocker (MacCormack et al., 2003b), and consistent with this finding 5HD decreased the protective eVect of channel opening in hypoxic goldfish hearts (Chen et al., 2005). How the opening of mitKATP channels during hypoxia can alter force development in not known, but activation of mitKATP channels results in depolarization of the mitochondrial membrane and altered mitochondrial Ca2+ uptake and release (Holmuhamedov et al., 1998). Since force development is intimately associated with calcium levels, any alteration in calcium cycling could aVect twitch force development. Regardless, the physiological implications of mitKATP regulation appear to be important although the mechanisms of action remain to be resolved. Aside from contractile aspects, dos Santos et al. (2002) argue that in the ischemic rat heart, open mitKATP channels maintain mitochondrial volume and the tight structure of the intermembrane space, and that this prevents ATP hydrolysis by mitochondria under oxygen limitation. If this occurs in fish hearts, it would be a further mechanism for extending energy reserves. Adenosine is well recognized as an agent that protects the mammalian heart under oxygen limitation through a variety of actions including reduced cardiac performance (for extensive reference to the mammalian literature see MacCormack and Driedzic, 2007; Stecyk et al., 2007). Adenosine is formed from the breakdown of the adenylate pool, and serves as a signaling molecule that links ATP supply with ATP demand. For example, the injection of adenosine resulted in a decrease in heart rate in the normoxic Antarctic notothenioid Pagotehenia borchgrevinki (Sundin et al., 1999a) and the epualette shark (Hemiscyllium ocellatum) (Stensløkken et al., 2004). Also, under normoxic conditions adenosine caused a decrease in heart rate in isolated, whole heart preparations and a decrease in force developed by electrically paced ventricle strips from rainbow trout (Aho and Vornanen, 2002). However, adenosine control does not appear to be generally important for the hypoxic myocardium. Short-horned sculpin subjected to hypoxia for up to 6 h showed marked bradycardia but no change in heart adenosine levels, despite the finding that adenosine levels increased following 30 min of reoxygenation (MacCormack and Driedzic, 2004). Atropine treatment resulted in a release of bradycardia and an increase in Q under hypoxia, but there was no further eVect of an adenosine blocker suggesting that the heart of the hypoxic short-horned sculpin is not under adenosine control 7. CARDIOVASCULAR FUNCTION 333 (MacCormack and Driedzic, 2007). In L. pardalis, a species that does not exhibit bradycardia (MacCormack et al., 2003a), there was no change in heart adenosine content as a function of hypoxia; however, adenosine levels were significantly higher in fish maintained in laboratory aquaria than in fish sampled directly from a pond (MacCormack et al., 2006). The studies with short-horned sculpin and L. pardalis, two species that show quite diVerent cardiac responses to hypoxia, are important in revealing that heart adenosine concentrations can vary under diVerent conditions, but at least in these fish, adenosine does not play a role in protecting the heart under hypoxia. Similar conclusions were reached in experiments with crucian carp where injection of the adenosine receptor antagonist aminophylline did not release hypoxiainduced bradycardia or change Q. Furthermore, the application of aminophylline had no impact on contractile failure of ventricle strips caused by NaCN (Vornanen and Toumennoro, 1999; Stecyk et al., 2007). Finally, injection of aminophylline did not alter hypoxia-induced bradycardia in the epualette shark (Stensløkken et al., 2004). The only study that suggests adenosine control plays a role under hypoxia is with common carp (Cyprius carpio). Heart rate in normoxic water at 5 C was about 8 bpm and Q about 4 mL min 1 kg-1, and these values decreased to about 4 bpm and 2.3 mL min 1 kg-1 under hypoxia. Adenosine receptor blockade with aminophylline increased these values to only about 5 bpm and 3.3 mL min 1 kg-1 (Stecyk et al., 2007). Collectively, this body of work eliminates the appealing conjecture that adenosine control plays an important role in reducing ATP demand by the hypoxic fish heart, although the possibility of a modest contribution in the case of the common carp cannot be ruled out. Next to contraction and ion pumping, protein synthesis accounts for the greatest ATP demand in the fish heart. Under hypoxia, protein synthesis is decreased by about 50% in both crucian carp (C. carassius) and oscar (Astronotus ocellatus) (Smith et al., 1996; Lewis et al., 2007). These studies followed the incorporation of radiolabeled phenylalanine into the protein pool and thus reflect decreases in rates of translation. How this decrease in protein synthesis is achieved is unknown but a decrease in pH is a likely possibility. 3.3. The Potential of Enhanced Oxygen Utilization Under Hypoxia One potential metabolic strategy to cope with hypoxia would be to extend the lower limit at which oxygen extraction from the extracellular space is possible. It is well established across diVerent species that the presence of myoglobin (Mb), at least in acutely challenged isolated hearts, results in a better maintenance of ATP levels, oxygen consumption, and performance under hypoxia (Driedzic and Gesser, 1994; Acierno et al., 1997). As such, an increase in heart Mb content is a potential adaptive response to hypoxia. 334 A. KURT GAMPERL AND W. R. DRIEDZIC Indeed, Mb protein levels increased by 20% in hearts of zebrafish maintained for 48 h at 22% oxygen (Roesner et al., 2006). However, an increase in Mb content does not appear to be a common response. There was no change in Mb content in hearts of Zoarces viviparous held under hypoxia for 4–6 weeks (Driedzic et al., 1985). Changes in Mb expression were also not observed in microarray studies on G. mirabilis and zebrafish (Gracey et al., 2001; Marques et al., 2008), yet given the conservative nature of the protein it should have appeared in these experiments. More recently, Hall et al. (unpublished data) showed no change in Mb mRNA levels in hearts of Atlantic cod held at 40% oxygen for 3 or 6 days (Figure 7.14). In contrast, a low temperature challenge does result in an increase in both protein and Mb transcript level in hearts of Atlantic cod (Lurman et al., 2007), showing that the modulation of the content of this protein can occur. If Mb is so powerful in allowing enhanced oxygen extraction, we question why 1.0 Mb 0.8 0.6 0.4 0.2 0.0 2.0 GLUT1 1.6 1.2 0.8 * * 0.4 0.0 2.0 GLUT4 1.6 1.2 0.8 0.4 0.0 2.5 HK 2.0 * 1.5 1.0 0.5 0.0 3 6 Time (days) Fig. 7.14. Relative gene expression levels, determined by qPCR, in the heart of Atlantic cod (G. morhua) held under normoxic (black bars) or hypoxic (grey bars) conditions (60 mmHg oxygen) (J. R. Hall, unpublished data). 7. CARDIOVASCULAR FUNCTION 335 substantially increased levels of this protein are not observed in the hypoxic heart. The answer may reside in the penetrating commentary of Sidell and O’Brien (2006). These authors point out that Mb is a nitric oxide-oxygenase, and as such, any increase in Mb content could result in a decrease in nitric oxide (NO). In some fish hearts, NO has a negative inotropic eVect (Tota et al., 2005). In the context of the hypoxic fish heart, an elevation of Mb could decrease NO levels and in turn result in increased energy demand. Thus, there may be a complex compromise between the benefits of increased oxygen extraction through high Mb levels and the trade oV of increased ATP demand due to loss of the inhibitory actions of NO. This conjecture remains to be tested. It is theoretically possible that increased mitochondrial surface area could allow for more eVective uptake of oxygen at low concentration gradients. For example, flounder (Platitchthys flesus) subjected to hypoxic conditions for 3 weeks showed microscopic evidence of mitochondrial necrosis, but in addition, many mitochondria were greater in length and the number of cristae in each mitochondrion appeared to have increased (Lennard and Huddart, 1992). However, three separate studies involving hypoxic acclimation have failed to show any increase in marker mitochondrial enzymes including citrate synthase, malate dehydrogenase, and cytochrome C oxidase (Z. viviparous, Driedzic et al., 1985; Hoplias microlepsis, Dickson and Graham, 1986; Astronotus crassipinnis, Chippari-Gomes et al., 2005). It is therefore unlikely that modification of mitochondrial properties occurs with respect to eVective utilization of available oxygen and metabolic fuels under hypoxia. However, as discussed under mitKATP channels and hexokinase (HK), there may be other changes to mitochondrial function under hypoxia that are critical. 3.4. Anaerobic Energy Metabolism Under hypoxic conditions heart glycogen stores and blood-borne glucose are called upon as anaerobic energy sources with lactate accumulating as the end product (e.g., Bailey et al., 2000; Vornanen and Paajanen, 2004; Overgaard and Gesser, 2004; MacCormack et al., 2006). 3.4.1. Acute Response 3.4.1.1. Glycogen Breakdown. Heart glycogen is mobilized under oxygen limiting conditions, and although the availability of extracellular glucose curtails glycogen utilization in American eel (Anguilla rostrata) and Atlantic cod heart preparations, it does not prevent glycogen breakdown (Bailey et al., 2000; Clow et al., 2004). Glycogen metabolism is generally a well understood phenomenon related to activation of the glycogen phosphorylase cascade. Contrary to what would be anticipated, however, goldfish subjected to 24 h of anoxia showed a decrease in % phosphorylase-a (Storey, 1987). 336 A. KURT GAMPERL AND W. R. DRIEDZIC As presented in Driedzic and Gesser (1994), this may be related to exhaustion of heart glycogen at that time. But, this issue remains to be resolved. 3.4.1.2. Glucose is Essential for Heart Performance Under Hypoxia. The necessity for glucose to support heart performance is well established. For example, isolated, perfused heart preparations from American eel and Atlantic cod could sustain 50% of normoxic power development for 2 h under hypoxia if glucose was available in the medium, whereas hearts from both species failed within 40 min without glucose (Bailey et al., 2000; Clow et al., 2004). Ventricle strips from rainbow trout subjected to anoxia for 30 min showed better force development and maintenance of resting tension when glucose was available in the medium than without glucose when glycogen was partially depleted by prior challenge (Gesser, 2002). Finally, isolated, perfused rainbow trout hearts can sustain sub-basal levels of performance under hypoxia with glucose in the medium, but with reduced levels of ATP turnover based on oxygen consumption and lactate production measurements (Arthur et al., 1992; Farrell and Stecyk, 2007). Interestingly, the use of glucose under hypoxia may involve features beyond the provision of fuel for total ATP production, including a direct interplay between glucose metabolism and ion balance. This issue is addressed in the section on hexokinase (HK) below. Regardless, it is clear that glucose utilization is required for ATP production to support the contractile apparatus under oxygen limitation, especially if glycogen stores are compromised. How this is achieved is addressed in the following sections. 3.4.1.3. Glucose Concentration Gradient Increases Under hypoxia. Glucose enters cells by facilitated diVusion, and uptake is determined by the glucose concentration gradient and the abundance of GLUTs (glucose transporter proteins). Hypoxia places increased demands on the glucose transport system as anaerobic metabolism is highly activated (unless the tissue is entering a severe hypometabolic state), and often results in an increase in blood glucose (Table 7.1) that may help support the concentration gradient from the extra- to the intracellular space [e.g. flounder (Jørgensen and Mustafa, 1980); goldfish (Shoubridge and Hochachka, 1983); Atlantic cod (Claireaux and Dutil, 1992); rainbow trout (Haman et al., 1997); A. crassipinnis (Chippari-Gomes et al., 2005); Amazonian armoured catfish (L. pardalis) (MacCormack et al., 2006), and many other species of Amazonian fishes (see Table 10.2 in Val et al., 2006)]. In rainbow trout an increase in plasma glucose under the initial stages of hypoxia is associated with a transient increase in the rate of glucose appearance, presumably from liver glycogen, without a change in the rate of whole animal glucose disappearance (Haman et al., 1997). For glucose uptake though, it is the glucose gradient that is critical and not blood glucose levels per se. Table 7.1 presents values for blood/plasma Table 7.1 Blood and heart glucose levels and heart lactate levels under normoxic and hypoxic conditions Glucose normoxia Species a Flounder Goldfishb Atlantic codc African lungfishd Rainbow troute Armoured catfishf Short-horned sculping a Glucose hypoxia Lactate Blood/plasma Heart Gradient Blood/plasma Heart Gradient Normoxic Hypoxic Conditions 1.33 1.39 8.40 0.23 12.50 2.20 0.23 2.14 1.94 4.48 0.96 8.80 2.50 0.35 –0.81 –0.55 3.92 –0.73 3.70 –0.30 –0.12 2.91 7.21 11 2 7.3 5 0.6 1.58 4.43 3.84 1 5.8 2.9 0.7 1.33 2.78 7.16 1 1.5 2.1 –0.1 6.2 0.42 1.84 1.9 0.71 0.05 8 10.2 11.35 17.55 5.2 6.2 20.2 14 29 h; 15 mmHg; 10 C 60 h; anoxia; 4 C 6 h; 30 mmHg; 5 C 12 h; 22 C 3 h; 20 mmHg; 4 C 3 h; <45 mmHg; 26 C 5 h; <33 mmHg; 8 C Platichthys flesus; Jorgensen and Mustafa (1980). C. carrassius; Shourbridge and Hochachka (1983). c Gadus morhua; Claireaux and Dutil (1992). d Protopterus aethiopicus; Dunn et al. (1983). e O. mykiss; Dunn and Hochachka (1986). f Liposarcus pardalis; MacCormack et al. (2006). g Myxocephalus scorpius; MacCormack et al. (2006). Air breathing lungfish and armoured catfish were denied access to air. Glucose values expressed as mmol glucose/mL blood or plasma. Glucose and lactate in heart is expressed as mmol/g wet weight. Glucose gradient was calculated as the blood/plasma value minus the heart value. No correction was made for extracellular space. All values are taken directly from published papers. b 338 A. KURT GAMPERL AND W. R. DRIEDZIC glucose and heart glucose. No attempt has been made to calculate intra- and extracellular glucose so this analysis must be viewed as a first approximation only. In each of the seven cases, fish were subjected to a hypoxic challenge suYcient to at least result in elevated average levels of heart lactate. Under normoxic conditions five species showed higher levels of heart glucose than blood glucose, which may be an artifact of the lack of precise values for intraand extracellular glucose. However, an alternate explanation is that heart glucose values are high due to mobilization of glycogen during the sampling period and/or gluconeogenesis, both of which would require an active glucose 6-phosphatase. These contentions remain to be tested and are beyond the scope of this review. More importantly, in the context of this analysis, is that under hypoxia all of the species show a positive gradient for glucose diVusion from the extra- to the intracellular space with the exception of shorthorn sculpin, where there is essentially no diVerence between plasma and cellular levels. Further, in most cases, the hypoxic challenge was associated with a more favorable inward glucose gradient. An increase in the inward diVusion gradient in association with hypoxia suggests that the removal of glucose (i.e., by phosphorylation) is elevated to a greater extent than glucose entry into the cell. This may be a cue to one of the features of maintaining anaerobic metabolism. 3.4.1.4. Glucose Uptake is Increased Under Hypoxia via Enhancement of Facilitated DiVusion. Injection of anoxic goldfish with C14-labeled glucose resulted in C14 glucose-specific activity in the heart that was equivalent to that measured in the blood after 3 h. Lactate was also labeled, and although the data are limited, it appears that the specific activity in heart was higher than in blood (Shoubridge and Hochachka, 1983). This is an important study in that it provides evidence for glucose uptake, equilibration of glucose between the blood and intracellular space, and the production of lactate. Thereafter, it was shown in isolated, perfused rainbow trout hearts that glucose uptake, as assessed by C14 2-deoxyglucose, was stimulated 10-fold in NaCN-treated preparations relative to normoxic hearts performing low levels of work (West et al., 1993). The stimulation of glucose uptake under hypoxia was subsequently confirmed in American eel ventricle strips (45% increase) (Rodnick et al., 1997) and in isolated, perfused Atlantic cod hearts (approx. 3-fold increase) (Clow et al., 2004). In American eel ventricle strips, cytochalasin B, a general inhibitor of GLUTs, prevented both anoxia-stimulated and contraction-stimulated increases in glucose uptake (Rodnick et al., 1997). In isolated, perfused hearts of Atlantic cod, the inclusion of cytochalasin B in the medium, during hypoxia, resulted in a significant decrease in glucose uptake. This was associated with a consistent trend of lower levels of performance, lower levels of tissue glucose, and increased glycogen breakdown (Figure. 7.15) 339 CARDIOVASCULAR FUNCTION 2-DG uptake (µmol g−1 15 min−1) 7. 7 b 6 c 5 4 3 2 a 1 0 120 a % Power 100 ab 80 b 60 40 20 0 Glycogen (µmol glucose g−1) 1.0 a 0.8 0.6 b 0.4 b 0.2 0.0 Glycogen (µmol glucose g−1) 14 a 12 10 8 b 6 4 b 2 0 Normoxic Hypoxic Hypoxic + cytochalasin B Fig. 7.15. Hearts isolated from Atlantic cod (G. morhua) that were perfused under normoxic or hypoxic conditions with media containing cytochalasin B to inhibit glucose transporter proteins. In all cases the media contained 5 mM glucose. 2-Deoxyglucose (2-DG) uptake was determined following 15 min of perfusion. % Power output shows values following 30 min of perfusion when at least 7 of 8 preparations were viable. Glucose and glycogen levels were assessed following 120 min of perfusion or immediately after heart failure. Values that do not share a common letter are significantly different. [Data are taken from Clow et al. (2004).] 340 A. KURT GAMPERL AND W. R. DRIEDZIC (Clow et al., 2004). All of these features are consistent with an inhibition of hypoxia-induced enhancement of facilitated glucose diVusion. There is only one study dealing with the impact of hypoxia on heart glucose uptake at the whole animal level (MacCormack et al., 2007). Shorthorn sculpin were subjected to approximately 30 mmHg oxygen for up to 4 h, and these authors report a 30% reduction in heart rate (with no change in stroke volume), and that the hypoxic challenge did not result in an increase in glucose uptake despite an increase in plasma glucose (Note, however, that levels of glucose in this species are extraordinarily low at <0.6 mM.) The simplest explanation for this finding is that aerobic metabolism, even at reduced oxygen availability, was able to support ATP demand. Consistent with this interpretation is the high Mb level in hearts of shorthorn sculpin (Driedzic and Stewart, 1982). In addition, treatment with atropine under hypoxia resulted in increases in fH, Q, and glucose uptake (MacCormack et al., 2007), again implying that the maximal rate of glycolysis associated with ATP production is not necessarily the limiting factor in performance of the hypoxic myocardium. 3.4.1.5. Glucose Transporters. In mammals, facilitated glucose transport is achieved primarily via four Na-independent proteins (Wood and Trayhurn, 2003). Homologs of mammalian GLUTs 1, 2, 3, and 4 have been characterized in Atlantic cod (Hall et al., 2004, 2005, 2006). Similar to well-studied mammalian systems GLUT1 is found in most tissues, GLUT2 is the predominant liver isoform, GLUT3 is in kidney/spleen, and GLUT4 is in heart and muscle. Rainbow trout hearts shows high expression levels of GLUT1 (Teerijoki et al., 2000), while the tilapia (O. nilotica) heart shows high amounts of both transcript and protein (Wright et al., 1998). GLUT4 is abundant in red and white skeletal muscle of brown trout (Salmo truta) but only at very low levels in heart (Planas et al., 2000). In the rat heart, ischemia results in the movement of GLUT4 protein from intracellular vesicles to the T-tubular membrane and sacrolemma, and provides a beneficial eVect at high glucose levels (Ramasamy et al., 2001; Davey et al., 2007). We are not aware of any investigations of this nature in the fish heart but this certainly should be assessed as a potential mechanism for defense against acute hypoxia. The low level of GLUT4 mRNA in the heart of brown trout is particularly provocative as it might be associated with the relative hypoxia sensitivity of salmonid hearts. 3.4.1.6. Hexokinase Activity. Hexokinase (HK) is a regulated enzyme that catalyzes the first step in the use of glucose according to the following reaction: glucose + ATP ! G6P + ADP. HK may be critical for the maximum utilization of glucose through its direct catalytic activity and its role in maintaining low intracellular glucose, and thus maximizing the glucose diVusion gradient. In addition, HK binds to mitochondria where it may have functions in addition to simple catalysis. 7. CARDIOVASCULAR FUNCTION 341 A number of studies suggest that HK catalytic activity per se in heart may be important under hypoxia. The calculated anaerobic ATP yield based on in vitro HK activity matches total ATPase activity for many ectothermic and endothermic vertebrates (Driedzic and Gesser, 1994). There is a linear relationship between total HK and LDH in hearts of ectothermic vertebrates (Driedzic and Gesser, 1994). In three species of Amazonian fish (but not north temperate species), force development of ventricle strips under NaCN treatment rank orders with maximal HK activity (Bailey et al., 1999; West et al., 1999). The maximal activity of HK in the fish heart is high by mammalian and avian standards (Driedzic and Gesser, 1994), and although there may be a correlation between HK activity and rates of ATP production, it should be appreciated that the maximal rate of glucose utilization in heart preparations, as estimated from glucose uptake, is only a small fraction of the total maximal HK activity assessed with in vitro assays. For example, following correction for assay temperatures, heart glucose uptake amounts to only 1.3, 2.8, and 8.2% of maximal HK activities for American eel, rainbow trout, and Atlantic cod, respectively (West et al., 1993; Driedzic and Gesser, 1994; Rodnick et al., 1997; Clow et al., 2004). Similarly, maximal in vitro rates of HK activity are much higher than rates of lactate production in hypoxic heart preparations from rainbow trout or L. pardalis (Overgaard and Gesser, 2004; Overgaard et al., 2004; Treberg et al., 2007). We continue to query why there are such high levels of HK in fish hearts. The binding of HK to the outer mitochondrial membrane may be a specific control feature of glucose utilization. In the rat heart there are two major isoforms of HK, HKI and HKII. A proportion of both isoforms is bound to mitochondria under normoxic conditions and the level of binding increases under ischemia (Zuurbier et al., 2005; Southworth et al., 2007). The binding of HK to the particulate fraction was assessed in hypoxia-resistant ventricle strips of the fish L. pardalis. Heart preparations were subjected to 2 h of anoxia, which was suYcient to result in an increase in lactate from 2.5 to 12.7 mmol g 1. Simultaneous measurements of HK and citrate synthase activities in cell fractions revealed that a much higher proportion (>4 times) of HK is associated with the mitochondrial pellet in L. pardalis than in rat hearts. Following hypoxia HK binding to mitochondria tended to increase (P ¼ 0.08) on the basis of HK/CS ratio, and using an alternative approach of assessing generalized binding to a particulate fraction via sucrose dilution there was a substantial and significant increase in enzyme binding (Treberg et al., 2007). Similar to L. pardalis, the proportion of HK activity in the particulate fraction of goldfish heart, and in the mitochondrial enriched pellet of eel heart, is high by mammalian standards (Duncan and Storey, 1991; Rodnick et al., 1997). This might explain why hypoxia did not increase HK binding in either of these two species. On balance it appears that in fish hearts, even under normoxic 342 A. KURT GAMPERL AND W. R. DRIEDZIC conditions, there is a high proportion of HK bound to the mitochondrial membrane and in some species this may increase with a hypoxic challenge. As data are available for only three species of hypoxia-tolerant fishes, it would be interesting to determine the level of enzyme binding in hearts from hypoxiasensitive species to assess the generality of this feature. Enzyme binding may be a response to defend against ischemic/hypoxic insults on kinetic grounds. HK is generally inhibited by glucose-6-phosphate (G6P) as has been shown for American eel heart (Rodnick et al., 1997), and in rat brain mitochondrial inhibition of HK by G6P is decreased when the enzyme is bound (Wilson, 2003). In addition, HK binds to voltage-dependent anion channels (VDACs), and mice lacking VDACs have a reduced capacity to metabolize glucose (Anflous-Pharayra et al., 2007). The function of enzyme binding though is probably much more complex than a relationship to simple enzyme activity per se. For instance, VDACs serve as conduits for metabolite movement, including adenylates, across the outer mitochondrial membrane. In rat liver mitochondria the binding of HK closes the transition pore, reducing Ca2+ release and possibly the release of cytochrome c that leads to apoptotic cell death (Azoulay-Zohar et al., 2004). We suggest that HK binding and subsequent activity in association with VDACs could translate into a localized decrease in the ATP/ADP ratio within the intermembrane space, and in turn, open mitKATP channels. If this is the case, it would link two of the key elements in the hypoxia defense process. 3.4.1.7. Phosphofructokinase. Phosphofructokinase (PFK) is activated during the early stages of oxygen limitation as evidenced by cross-over analysis of metabolite levels. Fish heart PFK, similar to other systems, is inhibited by low pH, ATP, and citrate while activators include AMP, Pi, and fructose-2,6,-diphosphate (F-2,6-P2). F-2,6-P2 levels increase in the heart of anoxic goldfish and may function as a potent activator (Storey, 1987; Driedzic and Gesser, 1994). Subcellular binding of PFK may also be important in glycolytic control. Following 21 h of anoxia the percentage of PFK bound to the particulate fraction of goldfish heart increased from 35% to 48% (Duncan and Storey, 1991). Similar increases from 20% to 45% bound enzyme were noted for isolated ventricle sheet preparations of L. pardalis subjected to 2 h of severe hypoxia (Treberg et al., 2007). Although not shown directly in these studies, it is most likely that PFK is binding to myofibrils. In the bound configuration inhibitors are less eVective and activators are more eVective, thus catalytic activity is enhanced (Brooks and Storey, 1995). The binding of PFK to the particulate fraction under normoxia is higher in goldfish and L. pardalis than rat and mice; moreover, under hypoxia the percentage of bound PFK approaches 50% in the fish species but only 25% in rat heart (see Treberg et al., 2007 for details). As such, this may also represent a key feature in the hypoxia defense mechanism of the fish heart. 7. CARDIOVASCULAR FUNCTION 343 3.4.1.8. Pyruvate Kinase and Lactate Dehydrogenase. There is no increase in binding of pyruvate kinase (PyK) or lactate dehydrogenase (LDH) to the particulate fraction as there is for HK and PFK in hypoxic ventricle sheets (Treberg et al., 2007). PyK is likely activated by increases in the activator F-1,6-P2 under oxygen limitation (see Driedzic and Gesser, 1994). However, there are no obvious correlations between the tolerance of isolated preparations to anoxia and either maximal in vitro activity of PyK or LDH, or in LDH kinetics (e.g., isozyme assembledge, Km for pyruvate, activity ratios) (see Driedzic and Gesser, 1994; Bailey et al., 1999; West et al., 1999). 3.4.1.9. General Model for Activation of Glycolysis Under Acute Hypoxia. A substantial body of literature exists on the impact of hypoxia on heart metabolism, as detailed above, and there is now suYcient information to propose a generalized model that results in anaerobic energy generation in response to acute hypoxia in the fish heart. Foremost, CP is utilized to maintain cellular ATP levels; a metabolic corollary is that Pi levels increase. The impact of the rise in Pi on contractility and its possible role in controlled down-regulation of heart performance, however, warrants further investigation. Although total ATP levels remain relatively constant we cannot rule out the possibility of localized decreases in ATP/ADP ratio that would open sarcKATP channels leading to reduced ATP demand. Hypoxia leads to glycogen mobilization in both liver and heart. Glucose released from the liver results in an increase in blood glucose that creates a more favorable gradient for glucose diVusion into the myocytes. G6P produced in the heart from glycogen can enter glycolysis directly. Glucose transport is activated, and this might be due to movement of the GLUT4 isoform to the sarcolemmal membrane (untested hypothesis). PFK binds to contractile fibrils and is activated through increases in Pi and F-2,6-P2. Activation of PFK would serve to decrease G6P levels. HK binds to the mitochondrial membrane and becomes less susceptible to inhibition by G6P. Binding of HK may also play a role in mitKATP channel regulation. An activated HK would decrease intracellular glucose, and thus, improve glucose entry. The detailed aspects of the sequelae of events is yet to be proven, especially the causes of enzyme binding as our picture is drawn from many parts of a puzzle. The challenge is to intellectually connect altered gene expression and subsequent alterations in protein levels to changes in Ca2+ levels that trigger contraction and ATP levels that provide energy for contraction, and to do it in a living animal as opposed to reductionist preparations. 3.4.2. Chronic Response This section deals with changes in processes of anaerobic metabolism that may occur after exposure to hypoxia for days to weeks, that is, a time frame long enough to result in changes in gene expression and protein levels. Our 344 A. KURT GAMPERL AND W. R. DRIEDZIC understanding of the events at this level, however, is poor and rests primarily on a few papers dealing with heart performance, enzyme activity levels, and gene expression. Z. viviparous were acclimated to hypoxia (35 mmHg ) for 4–6 weeks. Thereafter, ventricle strips were challenged under anoxia with and without glucose in the medium. All preparations failed to a similar extent under anoxia, and after 30 min developed about 65% of their initial force. Then, an increase in extracellular Ca2+ (from 1 to 5 mM) was used to assess maximal force development, and this resulted in a substantial and sustainable increase in force development only with glucose in the medium, and only in hearts from hypoxia-adapted animals (Driedzic et al., 1985; see Driedzic and Bailey, 1999 for further discussion). Interestingly, however, there was no change in the maximal activity levels of PFK or PyK following hypoxia acclimation. This experiment suggests that there is an adaptable feature(s) in the hypoxic response that may be related to glucose utilization perhaps at the level of glucose entry or HK. In this context, although no change was noted in GLUT1 expression in hearts of Atlantic cod held at 40–45% O2 (approx. 60 mmHg) after either 6 h (Hall et al., 2004) or 24 h (Hall et al., 2005), more recent studies show an increase following either 3 or 6 days (Figure 7.14; Hall et al., unpublished data). Further, the mean values for GLUT4 mRNA and HK mRNA increased, with the increase in HK expression after 6 days of hypoxia reaching significance. It would be of interest to determine if these changes result in increased rates of glucose uptake. In killifish (Fundulus grandis) following 4 weeks at 30 mmHg oxygen, the maximal activity of total homogenate HK, triose phosphate isomerase, and PK increased by 27, 18, and 30%, respectively (Martı́nez et al., 2006). In contrast, there was no change in the activity of eight other glycolytic enzymes including PFK and LDH, and Dickson and Graham (1986) showed no significant change in PK or LDH in Hoplias microlepis held under hypoxic conditions for 16–25 days. The extremely hypoxia-tolerantAmazonian cichlid (A. crassipinnis) presents an interesting case study. At 6% oxygen (approx. 10 mmHg) these fish can maintain an MO2 of about 60% of that measured during normoxia, and a stepwise decrease to 1% oxygen (1.5 mmHg) over 2 days results in a decrease in PyK and an increase in LDH maximal in vitro activity (Chippari-Gomes et al., 2005). Microarray studies also provide us with contrasting results. For example, Gracey et al. (2001) exposed G. mirabilis to 15 mmHg O2 for 6 days and showed a down-regulation in heart levels of transcripts for two glycolytic enzymes, enolase and glyceraldehyde-3-phosphate dehydrogenase, with no change in LDH-A mRNA. In contrast, in zebrafish held under hypoxia for 21 days there was an up-regulation of heart PK and aldolase (Marques et al., 2008). The results from the enzyme activity and gene microarray studies do not present a consistent or coherent response to chronic hypoxia. What is missing 7. CARDIOVASCULAR FUNCTION 345 in the field at present are comprehensive and integrative studies that relate the elements of gene expression, enzyme activity, glucose transport, lactate production, myocardial performance, etc. Hopefully these will be performed soon, and will add significantly to our understanding of the biochemical and metabolic responses of the fish heart to chronic hypoxia. 4. ADDITIONAL INSIGHTS Thus far, we have attempted to synthesize the available literature on the eVects of chronic and acute hypoxia on in vivo cardiovascular function, and on aspects of biochemistry and metabolism related to cardiac hypoxia tolerance and performance under hypoxia. Although we cannot cover all topics related to hypoxia and cardiovascular physiology/biochemistry in this chapter, there are several important/interesting aspects that still need to be addressed. 4.1. Interactive EVects of Temperature and Hypoxia While it is clear that hypoxia suppresses cardiac function in fishes, the degree to which cardiac function is aVected is related to both the severity of hypoxia and water temperature. With regards to the interactive eVects of temperature and severe hypoxia (anoxia) on aspects of cardiac function, there are several studies that provide particularly relevant information in addition to that presented in Section 2.1. First, Overgaard et al. (2004a) acclimated rainbow trout to 10 C and evaluated the capacity of their hearts to maintain basal in situ cardiac function during severe hypoxia/anoxia (PO2 5 mmHg), and to recover maximal cardiac function when returned to normoxia, when tested at 5, 10, 15, or 18 C. This study showed that, although hearts at 5 C could maintain cardiac performance throughout 20 min of severe hypoxia and maximal cardiac performance recovered fully after the severe hypoxic period, there was a significant increase in functional impairment during anoxia and recovery from anoxia as temperature increased (i.e., in situ heart performance during severe hypoxia and upon recovery was inversely related to temperature) (Figure 7.16). Further, they showed that this functional impairment (of both SV and fH) at elevated temperatures occurred even though cardiac glycolytic enzyme activity and the rate of lactate production were increased proportionally with temperature, and there was no evidence of myocardial necrosis or diVerences in biochemical and energetic parameters between groups. These results lead to two important conclusions: (1) That while a decrease in cardiac performance with severe hypoxia at any particular temperature results from insuYcient anaerobic energy production to meet demand (e.g., see Arthur et al., 1992), the inverse 346 A. KURT GAMPERL AND W. R. DRIEDZIC A 5⬚C 10⬚C 15⬚C 18⬚C Cardiac power output (mW g−1) 2.0 1.5 1.0 * 0.5 0.0 * 5 10 Time (min) 15 20 12 10 Cardiac power output (mW g−1) * * 0 B * * * Pre-anoxia maximal Post-anoxia maximal 8 6 * 4 * * 2 0 5⬚C 10⬚C 15⬚C 18⬚C Fig. 7.16. EVect of test temperature on changes in trout (O. mykiss) basal cardiac power output during 20 min of anoxic perfusion (A), and on maximum power output after recovery from 20 min of anoxic perfusion as compared with preanoxic (normoxic) values (B). In both A and B an asterisk (*) indicates a value diVerent from preanoxic values at a given temperature. Values are means  S.E.M; N = 6–7. Trout were all acclimated at 10oC. [Modified from Farrell and Stecyk (2007); original data from Overgaard et al. (2004a).] relationship between cardiac performance during severe hypoxia and temperature was due to a faster accumulation of waste products, in particular, intracellular phosphate and protons. Indeed, cardiac failure during hypoxia/anoxia appears to be caused by increased levels of intracellular inorganic phosphates and reduced intracellular pH 7. CARDIOVASCULAR FUNCTION 347 (Turner and Driedzic, 1980; Allen et al., 1985; Godt and Nosek, 1989; Arthur et al., 1992). (2) That decreased postanoxic performance of the trout heart is due to myocardial stunning (mechanical dysfunction that persists after reoxygenation/reperfusion due to a reduction in Ca2+ responsiveness caused by damage of the contractile apparatus by oxygen radicals and/or Ca2+ overload; see Bolli and Marban, 1999), and that this loss of Ca2+ responsiveness is related to temperature and the duration of flow deprivation. Second, while the above in situ study on rainbow trout suggests that reductions in cardiac function with anoxic exposure are gradual, with the rate of decrease depending on temperature, Stecyk et al. (2007) show that the in vivo cardiovascular response of the common carp (C. carpio) to anoxia is triphasic, and that not all changes are directly related to temperature. For example, these authors showed that the rate of loss of cardiac function (mainly as a result of changes in fH, as opposed to both fH and SV) during phase 1 (acute phase) was slower at 10 C than at 5 C (with 15 C being intermediate), that in the middle (prolonged) phase the heart achieved minimal levels of cardiac activity that were temperature independent (Q10 ¼ 1.2), and that in phase 3 (expiratory phase) cardiac activity temporarily increased at all temperatures before the carp approached death. These data suggest that complex cardiorespiratory control mechanisms are utilized by the common carp to survive anoxia (these were revealed in a later study; Stecyk and Farrell, 2006), and that elevations in cardiac function to meet the needs of the whole animal may ultimately lead to cardiac damage or failure. For example, Stecyk et al. (2007) suggest that the increase in cardiac activity during phase 3 of anoxia was related to the heart’s role in transporting nutrients to, and wastes from, the tissues. Third, it is well established that aquatic hypoxia is not the only situation where the heart’s oxygen supply may be limited (e.g., severe anemia, exhaustive exercise), and that adrenergic nervous tone and circulating catecholamines stimulate positive inotropic and chronotropic responses that allow the fish heart to maintain or elevate its performance under conditions that would normally compromise myocardial function (Gesser et al., 1982; Farrell, 1984; Stecyk and Farrell, 2006; Hanson et al., 2006). Given the importance of circulating catecholamines and adrenergic nervous tone (e.g., see Stecyk and Farrell, 2006) to cardiac function under conditions that result in hypoxia or hypoxemia, and that adrenergic sensitivity of the trout myocardium is decreased at elevated temperatures due in large part to a decrease in cell-surface b-adrenoreceptor density (Keen et al., 1993), Hanson and Farrell (2007) assessed the hypoxic threshold for maximum in situ cardiac performance at 18 C under conditions that simulated venous blood composition during 348 A. KURT GAMPERL AND W. R. DRIEDZIC exhaustive exercise [e.g., acidosis, pH 7.5; hyperkalemia, K+, 5 mM; and maximal adrenergic stimulation, 500 nM adrenaline) and compared it to previous data collected at 10 C (Hanson et al., 2007). Hanson and Farrell (2007) found that complete cardiac failure occurred at a perfusate PO2 of 38 mmHg at 18 C, an oxygen tension that far exceeded the hypoxic threshold at 10 C (15 mmHg for diminished cardiac function and 7.5 mmHg for heart failure; Hanson and Farrell, 2006) and venous oxygen partial pressures measured in salmonids swimming maximally at temperatures between 6 and 16 C (PO2 7–28 mmHg; SteVensen and Farrell, 1998; Farrell and Clutterham, 2003). Collectively, this research suggests that the capacity of adrenergic mechanisms to support cardiac function is diminished at high temperatures, and that this may limit myocardial hypoxia tolerance in vivo under conditions requiring elevated cardiac performance. 4.2. Preconditioning Thus far we have discussed the influence of environmental (hypoxic)– genetic interactions during development/rearing on cardiac function (see Section 2.2.1), and shown that reductions in SV caused by chronic hypoxia are compensated/partially compensated for by an enhanced capacity to elevate fH (see Section 2.2.2). While these studies highlight the capacity of the myocardium to respond/adapt to prolonged low oxygen conditions, it is clear from studies of preconditioning that even short-term exposure to hypoxia (i.e., minutes) can have profound implications for the fish heart’s capacity to deal with oxygen deprivation. Preconditioning has been studied extensively in the mammalian heart, and is a phenomenon whereby prior exposure of the mammalian heart to a physiological insult (ischemia, hypoxia, acidosis, stretch, rapid pacing, etc.) or biologically active molecules (adenosine, adrenaline, bradykinin, etc.) protects the myocardium from damage or loss of function resulting from a subsequent hypoxic/ischemic episode (e.g., see reviews by Downey et al., 2007; Gross and Gross, 2007). Gamperl et al. (2001) provided the first evidence (using hypoxia-sensitive trout) that preconditioning exists in fishes, and thus that preconditioning is a mechanism of cardioprotection that appeared early in the evolution of vertebrates (see Figure 7.17A). Further, research in this area to date has shown that: (1) increased anaerobic glycolysis, fueled by exogenous glucose, is associated with preconditioning (Gamperl et al., 2001); (2) trout hearts with inherent myocardial hypoxia tolerance cannot be preconditioned (Figure 7.17B, Gamperl et al., 2004; Overgaard et al., 2004b); (3) unlike mammalian cardiac cells, fish myocardial cells are not irreversibly damaged (i.e., do not die) following exposure to periods of oxygen deprivation  30 min. (Gamperl et al., 2001; Overgaard et al., 2004b); and (4) preconditioning is not limited to myocardium that normally receives 349 CARDIOVASCULAR FUNCTION A B a a,b 0.8 0.7 0.9 0.6 0.8 0.9 b b 1.1 a,b a 1.0 b a,b 0.9 0.7 b c 0.8 a a a a a 1.1 0.9 1.0 0.8 0.9 0.7 0.8 H yp H ox yp ia C ox − l on ia ow tro −h w l ig or h k Pr wo ec rk on d. Before/after a a a 0.8 1.0 a 1.0 b 1.0 Before/after 1.1 a,b a a a m Co n i 30 n h tro m ypo l in x hy ia po Pr xi ec a on d. Before/after 0.9 5 7. Fig. 7.17. Comparison of the ability of preconditioning (5 min of hypoxic pre-exposure) to protect (A) hypoxia-sensitive (Gamperl et al., 2001) and (B) hypoxia-tolerant (Gamperl et al., 2004) trout (O. mykiss) hearts from the myocardial dysfunction that follows more prolonged exposure to hypoxia. In A, 5 min of hypoxic pre-exposure completely eliminated the loss of myocardial function that normally followed the ‘‘Hypoxia-high workload’’ protocol. In B, preconditioning with 5 min of hypoxia either did not aVect, or increased, the amount of myocardial dysfunction following exposure to ‘‘30 min of hypoxia.’’ Top panels, maximum cardiac output; middle panels, maximum stroke volume; bottom panels, heart rate. Note that the hypoxia-tolerant trout hearts in B required twice the duration of hypoxia (15 vs. 30 min), and 6 times the workload, as compared with hypoxia-sensitive hearts (A) to achieve a comparable (15–20%) decrease in posthypoxic myocardial function. Values were obtained by comparing maximum in situ cardiac function before and after the treatment protocols. All values are means  S.E.M; N = 7–9. Dissimilar letters indicate a significant diVerence at P<0.05, as determined by one-way ANOVA. Hypoxia in these experiments was defined as perfusate PO2 = 5–10 mmHg. Control hearts were only exposed to oxygenated saline. [Reproduced from Gamperl and Farrell (2004), with permission from the Journal of Experimental Biology.] 350 A. KURT GAMPERL AND W. R. DRIEDZIC highly oxygenated blood from the coronary circulation (Gamperl and Genge, unpublished data; see Fig. 6 in Gamperl and Farrell, 2004). These studies provide important insights into fish myocardial hypoxia tolerance, and provide indirect evidence that the cellular mechanisms/signaling pathways involved in providing protection to the myocardium following short-term (acute) and long-term (chronic) oxygen deprivation are similar. This hypothesis is consistent with the mammalian literature (e.g., see Kolář and Ostadal, 2004), though it is not known which of the multitude of pathways/mechanisms that have been identified in the mammalian heart to have cardioprotective eVects are involved in fishes. 5. CONCLUDING REMARKS In this chapter we have shown that our understanding of some aspects of fish cardiovascular responses to hypoxia (e.g., heart rate responses to acute hypoxia, control of branchial resistance/blood flow) is fairly advanced. However, it is also obvious from the information provided that our knowledge of many, even basic, aspects of cardiovascular function/control under hypoxic conditions is extremely limited. For example, information on the control of systemic vascular resistance during hypoxia and on the eVects of chronic hypoxia is less than extensive. Unlike the eVects of temperature (e.g., see Shiels et al., 2002), we have little idea of whether excitation-contraction coupling and calcium dynamics are aVected by myocardial hypoxia in fishes (with the exception of the crucian carp; Vornanen and Paajanen, 2004). Finally, we have only begun to understand how complex changes in gene expression, protein levels/function/subcellular localization, signaling cascades, and the control of oxidative and anaerobic metabolism result in intra- and interspecific diVerences in myocardial hypoxia tolerance or mediate the phenomenon of preconditioning. However, this is an extremely active field of scientific investigation which, along with continued advances in in vivo physiological measurement techniques, cellular imaging, molecular cloning, and functional genomics, will reveal many novel insights into myocardial plasticity and adaptation in fishes (vertebrates), and the molecular and biochemical pathways that protect the heart from environmental insults that might normally lead to cardiac dysfunction, myocardial damage, and eventually death. ACKNOWLEDGMENTS We would like to thank Marc Bolli, Juan Perez-Casanova, and Connie Short for their assistance in putting this body of work together. This contribution and the research programs of the authors are supported by grants from the Natural Sciences and Engineering Research 7. CARDIOVASCULAR FUNCTION 351 Council of Canada (NSERC), the Canadian Institutes of Health Research (CIHR), and funds made available through the Canada Foundation for Innovation. 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Effects of Hypoxia and Digestive State on Oxygen Transport 6. Effects of Hypoxia on Appetite 7. Assimilation Efficiency 8. Effects of Hypoxia on Growth in Air-Breathing Fishes 9. Conclusions and Perspectives Here we review how hypoxia affects growth and digestion in fish. Thus, the growth eVects of hypoxia are explained in terms of reductions of energy intake (appetite) and assimilation eYciency as well as in terms of the costs of digestion or specific dynamic action. It is clear that the most commonly documented cause of hypoxia-related growth retardation is through loss of appetite and the regulatory physiology of this eVect is discussed. Finally, the 361 Hypoxia: Volume 27 FISH PHYSIOLOGY Copyright # 2009 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(08)00008-3 362 TOBIAS WANG ET AL. eVects of hypoxia on the growth of air-breathing fish are reviewed and the most promising areas for future research on oxygen’s role as a limiting factor for fish growth are highlighted. 1. INTRODUCTION The ultimate goal of an animal, in Darwinian terms, is to propagate its genes by maximising lifetime reproductive output. Growth and reproduction are tightly linked in fish as fecundity increases with body mass (Wootton, 1998). Thus, for an animal to reproduce maximally, it must maximize its ‘‘energy surplus,’’ which is the excess of energy after having covered household costs such as heart function, ion regulation, and ongoing synthesis of proteins, etc., which can then be converted into tissue growth and reproduction. Several factors obviously influence the amount of energy available for growth in an animal. In the following discussion of the eVects of hypoxia on growth, these factors are included using the framework for abiotic influences classified by Fry (1971), and for biotic factors as proposed by Brett (1979). Biotic factors such as interactions with conspecifics (i.e., competition for food) or other species (i.e., prey–predator interactions) can aVect the amount of food that the animal has access to. In fish, especially under laboratory or aquaculture conditions, food availability is of course mostly determined by researchers (or managers), and huge eVorts have been put into studying the eVect of food quality and stocking density, since these parameters clearly often aVect growth. However, correlations between growth and quality/density can be blurred. For instance, schooling fish may well be aVected in a diVerent way by density than fish that are normally solitary. Also, food ‘‘quality’’ is clearly species dependent and composition must be tailored to the needs of the individual species. Food availability is another limiting biotic factor, simply because food equates to energy. Abiotic factors such as temperature determine the amount of energy spent on maintenance, as most biological processes (for instance protein synthesis and degradation) are temperature dependent. Temperature has accordingly been classified as a controlling abiotic factor and can have both positive and negative eVects on growth. Other abiotic factors such as salinity are classified as ‘‘masking’’ because they change the costs of specific aspects of metabolism. Oxygen availability, which is the focus of this section, is classified by Fry (1971) as a limiting factor. Oxygen is the key electron accepter in aerobic respiration and thus directly limits the amount of energy that can be metabolized by an animal. Hypoxia occurs naturally and on a regular basis in many habitats. As an example, hypoxia can occur regularly and predictably as a result of the lack of photosynthesis at night, but occurs more unpredictably as a result of 8. THE EFFECTS OF HYPOXIA ON GROWTH AND DIGESTION 363 eutrophication, stagnant water, or ice cover (Nilsson and Östlund-Nilsson, 2008). Though a naturally occurring phenomenon, the frequency, abundance, and severity of hypoxic events have increased due to anthropogenic organic and inorganic nutrient loading, and also the much discussed global warming (Diaz, 2001; see Chapter 1). Hypoxia is also a widespread problem in aquaculture, where stocking density is high, requiring the expenditure of large amounts of energy in aeration. It is therefore not surprising that most studies relating hypoxia to growth performance and digestion have been conducted on commercially important species such as Atlantic cod, trout, and catfish. 2. ENERGETIC CONSIDERATIONS FOR GROWTH 2.1. EVects of Hypoxia on Metabolism Hypoxia exerts its general influence on growth by disturbing metabolic pathways and the reallocation of energy resources. An organism’s metabolism is normally divided into basal or standard metabolic rate (SMR) and routine metabolism (RMR). SMR represents the energy expenditure to maintain basic life functions, including the maintenance of ion gradients, osmoregulation, and constitutive rates of protein synthesis, and thus represent the minimum cost of living. This notion of a SMR is obviously somewhat artificial because it changes with the condition of the animal and tends, for example, to decrease progressively during food deprivation (e.g., Van Dijk et al., 2002; Wang et al., 2006). Furthermore, it is diYcult to measure experimentally (see SteVensen, 1989). Nevertheless it serves as a useful conceptual tool to quantify environmental or physiological changes, such as those imposed by hypoxia or the animal’s feeding state. Under natural conditions, as well as in aquaculture, the metabolism of an animal is considerably higher than its SMR because of the energy expended on physical activity, food digestion, or reproduction. This metabolic rate is normally referred to as the routine metabolic rate (RMR), while the maximal oxygen uptake of an animal, typically measured during strenuous exercise, is denoted VO2max. As explained in more detail below, RMR for a given individual will be higher the more food is being digested because the metabolic cost of digestion increases proportionally with ration. Hypoxia leads to reductions in all three levels of metabolism, but the thresholds will normally diVer, so that VO2max is the most sensitive followed by RMR, while SMR is the least sensitive. These eVects of hypoxia are illustrated in Figure 8.1, which shows how VO2max can be expected to decline (e.g., Claireaux and Lagardère, 1999) as oxygen availability is reduced. The exact manner by which VO2max decreases is dictated by a complex interplay 364 TOBIAS WANG ET AL. Aerobic scope (postprandial) nc on tin uu m Digestion of “large” meal xy ge Aerobic scope (fasting) itin go Digestion of “small” meal Lim Rate of oxygen consumption VO2max (normoxia) Standard metabolic rate (fasting) Pcrit fasting Pcrit during digestion of “large” meal Oxygen availability Fig. 8.1. A schematic representation of the limitation imposed by hypoxia on maximal oxygen consumption (VO2max). Fasting fish at standard metabolic rate generally are able to sustain oxygen consumption in hypoxia until a critical level (Pcrit), which corresponds to the interception with the line predicting VO2max at a given oxygen availability. Digestion, by virtue of elevating the rate of oxygen consumption, increases Pcrit and reduces the aerobic scope, which is defined as the diVerence between oxygen consumption at rest and exercise. [Modified from Claireaux and Lagardère (1999).] of blood oxygen-binding characteristics and the abilities of the gills and the cardiovascular systems to transport oxygen to the metabolizing tissue (e.g., Jones et al., 1970; Webb, 1994). In resting fish at SMR, the eVects of hypoxia are much less pronounced, but at some level of hypoxia, oxygen delivery no longer satisfies metabolic needs and aerobic metabolism will decline (critical oxygen tension, Pcrit). As predicted in the model represented in Figure 8.1, an elevation of metabolism is associated with an increase in Pcrit. Thus, as metabolism rises during digestion (discussed in more detail below), the organism becomes more sensitive to hypoxia. 2.2. Basic Energy Balance, Metabolism, and Allocation to Growth In energetic terms, the amount of energy available for growth in a nonreproducing fish is given as the diVerence between the energy ingested through food minus the sum of energy spent on metabolism and amount of energy that is excreted in urine and faeces: Egrowth ¼ Efood Emetabolism ðEfeces þ Eurine Þ ð1Þ 8. THE EFFECTS OF HYPOXIA ON GROWTH AND DIGESTION 365 The amount of energy that can be allocated to growth (Egrowth) represents the diVerence between the energy in the food that is consumed (Efood) and the total amount of energy that is used for standard metabolic rate, physical activity, and digestion (Emetabolism) and the energy lost in feces and urine (Efeces and Eurine, respectively). The amount of energy lost in the feces represents food items that were not assimilated over the gut, while the energy excreted as waste products in urine (or over the gills) stems from breakdown of absorbed nutrients. Growth is typically measured as the specific growth rate (SGR), which can be calculated as follows: SGR ¼ lnWt lnW0 t ⋅100% ð2Þ Where t is time, normally in days, and Wt is the body mass after t days. W0 is the initial body mass. Growth is thus expressed as the percentage of the initial body mass gained by the fish per day, and usually lies in the range of 0% day-1 to 4% day-1 (see Table 8.1), but it can also be negative in situations where food intake is insuYcient to balance energy expenditure, which can occur during fasting and starvation as well as anorexia imposed by hypoxia or other challenging situations (Brett and Groves, 1979). Another useful term when considering allocation of energy to growth is the food conversion eYciency (also called the gross conversion eYciency, K1), which is the amount of the energy consumed that is allocated into growth: K1 ¼ Egrowth ⋅100% Efood ð3Þ The net conversion eYciency (K2) is also defined: K2 ¼ Egrowth ⋅100% Efood ERMR ð4Þ The accuracy of this determination depends on the accuracy with which the routine metabolic rate can be measured, and as such adds little knowledge that cannot be gained from measuring the gross conversion eYciency (Brett and Groves, 1979). A third useful term is the assimilation eYciency (AsE), which is the amount of energy consumed that is actually absorbed by the animal, i.e., it is the energy content of the feces subtracted from the energy consumed: AsE ¼ Efood ðEfaeces þ Eurine Þ ⋅100% Efood ð5Þ The assimilation eYciency influences growth in that an animal with low assimilation eYciencies will have to eat more of a particular food to absorb Table 8.1 Specific Growth Rates (SGR) at DiVerent Levels of Dissolved Oxygen Common name Latin name Initial Salinity (‰) Mb(g) Feeding level Temp Light cycle ( C) O2 (%) SGR FI (g day–1 –1 b fish–1) (% day ) a Winter flounder Pseudo-pleuronectes americanus 1.54 25.5 Ad lib. ‘‘Natural’’ 20.7 87.0 2.5 – Silver catfish Rhamdia quelen 4.99 – 5 % 1 d–1 – 22.4 28.6 77.5 1.2 1.5c – – 24.3 65.6 52.2 39.1 24.7 100 1.7 1.2 0.9 0.9 1.4d – – – – – 10.0 70 30 93 1.1 0.8 0.8e – – 33f 84 75 65 56 45 122.9 81.4 50.8 33.9 92.3 0.9 0.7 0.8 0.6 0.5 0.8g 0.9 0.7 0.4 1.0h 32 25 27 22 15 128 141 92 67 – 64.1 44.9 0.5 0.5 – – Channel catfish Atlantic cod Ictalurus punctatus Gadus morhua 15.0 728.2 – 28 1 d–1 3 wk–1 ‘‘Natural’’ ‘‘Natural’’ Spotted wolYsh Anarhichas minor 68.5 – 1 d–1 6D:18L 8.0 Turbot Scophthalmus maximus 120 34 2 d–1 8D:16L 17.0 Reference Bejda et al. (1992) Braun et al. (2006) Buentello et al. (2000) Chabot and Dutil (1999) Foss et al. (2002) Pichavant et al. (2000) Turbot European sea bass Nile tilapia Scophthalmus maximus Dicentrarchus labrax 66.3 60.8 34 34 2 d–1 Restricted 2 d–1 8D:16L 8D:16L 8D:16L 17.0 94.9 2.0h – 17.0 17.0 57.7 41.0 93.3 94.9 1.1 0.7 0.7 0.8h – – – – 57.7 41.0 93.3 >68.5 0.5 0.3 0.3 4.47 – – – 2.15 <47.9 >68.5 <47.9 84.7 23.9 118.8 99.0 84.2 69.3 59.4 49.5 39.6 118.8 99.0 84.2 69.3 59.4 49.5 39.6 3.55 2.19 1.63 0.61i 0.15 4.05 j 4.00 3.90 4.00 2.90 2.00 –0.05 1.65 1.45 1.30 1.55 0.95 0.15 – 1.54 6.08 4.43 – – – – – – – – – – – – – – – – – Restricted 2 d–1 8D:16L 12D:12L 17.0 32.3 140.9 – 2 d–1 12D:12L 32.3 9.4 16 3 d–1 Not stated 26.6 – 1 h–1 (light) 12D:12L 15 1 d–1 15 Oreochromis niloticus 20.0 Silver bream Sparus sarba Rainbow trout Oncorhynchus mykiss 100 12D:12L Pichavant et al. (2001) Pichavant et al. (2001) Tran–Duy et al. (2008) Chiba (1983) Pedersen (1987) (continued) Table 8.1 (continued ) Common name Turbot Latin name Scophthalmus maximus Initial Salinity (‰) Mb(g) 54.5 34 Feeding level 2 d–1 Light cycle Temp ( C) SGR FI (g day–1 O2 (%)a (% day–1)b fish–1) 6D:18L 17.0 100.1 1.75 0.91k 2.00 2.02 1.02 1.00 1.00 – European sea bass Dicentrarchus labrax 19 37 2% 1 d–1 12D:12L 22 147.4 223.6 86 White sturgeon Acipenser transmontanus <1 – 3 d–1 12D:12L 15 40 84 0.78 1.6 – – 14.8 58 84 58 84 58 84 58 84 58 84 58 94.7 0.6 2.6 2.0 2.9 2.3 1.6 1.4 2.2 2.0 3.2 3.0 2.0l – – – – – – – – – – – – 14.7 51.7 31.9 94.6 1.3 0.3 1.5 – – – 50.7 31.6 0.5 0.3 – – 20 25 Striped bass Morone saxatilis 3 d–1 <1 12D:12L 15 20 25 Plaice Dab Pleuronectes platessa Limanda limanda 22.8 23.5 32 32 3–8% 2 d–1 3–8% 2 d–1 10D:14L 10D:14L Reference Person–Le– Ruyet et al. (2002) Thetmeyer et al. (1999) Cech et al. (1984) Cech et al. (1984) Petersen and Pihl (1995) Petersen and Pihl (1995) Southern flounder Atlantic manhaden Paralichthys lethostigma Brevoortia tyrannus 1.8 7.7 15 15 2 d–1 1 d–1 10D:14L 10D:14L 25.0 85.9 1.9 – 25.0 62.6 36.9 78.9 3.0 3.5 2.5m – – – 52.6 26.3 19.7 87.0 58.0 29.0 21.7 78.9 1.7 1.5 0.9 2.6 2.7 2.5 1.1 2.2 – – – – – – – – 52.6 26.3 19.7 87.0 58.0 29.0 21.7 99.3 2.4 2.2 1.5 1.5 2.0 2.0 0.1 1.44 – – – – – – – – 69.5 29.8 20.9 80.1 5.7 0.87 0.68 –0.24 1.4 0.1 – – – – – 30.0 Spot Leiostomus xanthurus 7.1 15 1 d–1 10D:14L 25.0 30.0 Sockeye salmon Common carp Oncorhyncus nerka Cyprinus carpio 5.6 57.5 – – 4 d–1 2% d–1 ‘‘Natural’’ 12D:12L 15.0 22.0 Taylor and Miller (2001) McNatt and Rice (2004) McNatt and Rice (2004) Brett and Blackburn (1981) Zhou et al. (2001) (continued) Table 8.1 (continued ) Common name Channel catfish Latin name Ictalurus punctatus Initial Salinity (‰) Mb(g) 60.0 – Feeding level 3% d–1 Ad lib. Light cycle Temp ( C) SGR FI (g day–1 O2 (%)a (% day–1)b fish–1) – 26.6 100 5m 26.6 60 36 100 60 36 5 3 6 5 3 – Reference Andrews et al. (1973) Mb ¼ body mass mgO2 L 1 a ⋅100%assuming standard barometric pressure and accounting for If not stated in %, the saturation was calculated as mgO2 L 1 ð100%saturationÞ temperature and salinity. lnW1 lnW0 b ⋅100% SGR ¼ t c Readings from figures presented in the paper. d Final weight (W1) was calculated from the weight gain increment presented in the figure from the paper. The wait gain was an average for the total period (12 weeks). e Final weight was calculated from the presented change in body mass, which was averaged for the entire measurement period (84 days). f Readings from figure presented in the paper. g Readings from figures presented in the paper. h Calculated from mean final and mean initial weight read of the figure in the paper. i Averages of SGR calculated from final and initial weights, selected tanks in the data table, 6 high oxygen and 6 low. j Only the growth rate for the highest and intermediate feeding ratio is presented. k [Food intake (% BW day–1) * mean weight] /100 l Readings from figures presented in the paper. Only the average of the entire period is presented here. ðW1 W0 Þ=Wmean m ⋅100% Growth rate calculated as t 8. THE EFFECTS OF HYPOXIA ON GROWTH AND DIGESTION 371 a certain amount of energy (Jobling, 1993). Under natural conditions lower assimilation eYciency will therefore be a cost to the animal in terms of hours spent feeding. Hypoxia may aVect all components of the energy equation (1). Thus, as will be reviewed below, hypoxia inhibits appetite of fish causing Efood to decrease, leaving less energy available for growth. Hypoxia may also aVect assimilation eYciency and hence Efeces. These eVects are obviously interrelated and factors that influence the amount of food eaten are likely to aVect the assimilation eYciency and any factor that influences the rate of the digestive processes and assimilation is likely to have an influence upon the amount of food ingested. 2.3. The Relationship between Metabolism, Aerobic Scope, and Growth The eVects of hypoxia are often interpreted in the context of ‘‘Fry’s paradigm’’ (Fry, 1971; Kerr, 1976, 1990), where an animal’s ability to perform activity is dictated by the influence of environmental factors on metabolism. In short, controlling factors such as temperature determine the rate of biochemical processes comprising metabolism and thus dictate both maximum and standard metabolic rates. Limiting factors, such as hypoxia, reduce oxygen supply and constrain aerobic metabolism (see Figure 8.1), while masking factors, such as salinity, may aVect SMR by altering the energy expenditure associated with key metabolic processes. In addition to showing how oxygen availability limits standard and maximal oxygen uptake, Figure 8.1 also illustrates how hypoxia and digestion aVect the aerobic scope. Aerobic scope is defined as the proportional change in oxygen uptake between SMR and VO2max. In a digesting animal, however, the aerobic scope is reduced because RMR becomes greatly elevated above SMR thus limiting the extent to which aerobic metabolism can be increased during physical activity. The environmental conditions that maximize the aerobic scope are often interpreted as being optimal for growth (e.g., Brett, 1979), but have only been evaluated in terms of temperature (Lefebvre et al., 2001; Mallekh and Lagardère, 2002; Claireaux and Lefrançois, 2007). An example of the correlation between aerobic scope and maximal feeding rate, which presumably translates into maximal growth, is presented in Figure 8.2 for turbot (Scophthalmus maximus). Increased temperature leads to an elevation of both SMR and VO2max, but because VO2max stabilizes at the higher temperature interval, feeding rate and presumably growth is maximal at approximately 18 oC. Although temperature and hypoxia most likely aVect growth and feeding behavior through diVerent mechanisms, it has been argued that the reduced appetite and growth rates in hypoxia represent an adaptive behavioral response 372 B Metabolic scope (mgO2 kg−1 h−1) A Oxygen uptake (mgO2 kg−1 h−1) TOBIAS WANG ET AL. 250 SMR MMR 200 150 100 50 0 180 160 140 120 100 80 0.9 Feeding ratio (% day−1) C 0.8 0.7 0.6 0.5 0.4 5 10 15 Temperature (degrees C) 20 Fig. 8.2. Standard and maximal oxygen uptake (A), metabolic scope (B), and feeding ratio (C) in turbot (Scophthalmus maximus) at diVerent temperatures and in normoxic water. The metabolic scope is the diVerence between standard metabolic rate (SMR) and maximum metabolic rate (MMR) (VO2max), and attains its highest value at approximately 18  C. At temperatures above 18  C, SMR continues to increase, while VO2max levels oV, causing metabolic scope to decrease. Maximal feeding rate was observed at 18  C where metabolic scope is highest. [Data from Mallekh and Lagardère (2002).] 8. THE EFFECTS OF HYPOXIA ON GROWTH AND DIGESTION 373 to protect the aerobic scope for activity (Claireaux et al., 2000; Claireaux and Lefrançois, 2007). This suggestion is certainly intuitively appealing, and while the physiological mechanisms remain to be characterized, there is certainly a compelling relation between scope for activity and overall growth performance in all species studied so far (Claireaux et al., 2000; Mallekh and Lagardère, 2002; Claireaux and Lefrançois, 2007). 3. THE RISE IN METABOLISM DURING DIGESTION: SPECIFIC DYNAMIC ACTION (SDA) Digestion causes metabolism to increase in all animals including fish. The metabolic increment is almost exclusively aerobic and has been termed ‘‘heat increment of feeding’’ and ‘‘calorigenic eVect,’’ but ‘‘specific dynamic action of food’’ (normally abbreviated as SDA) is currently the most common term (Rubner, 1902; Kleiber, 1961; Jobling, 1981). In its strictest sense, SDA only includes the metabolic costs involved with digestion, absorption, and utilization of food, whereas the ‘‘apparent SDA’’, measured as the change in metabolic rate throughout the postprandial period, also includes other costs associated with feeding, such as prey handling, as well as structural or functional remodeling of the digestive organs. It can be diYcult to separate the individual components, and virtually all studies on energetic responses to feeding report apparent SDA responses. The SDA response is generally characterized by a rise in oxygen consumption within minutes or hours after ingestion, followed by a gradual decline to the resting level over many hours or days. The total amount of extra energy spent during digestion, i.e., the integral of the postprandial metabolism minus resting metabolic rate, is a measure of the energy expenditure associated with digestion. It can be useful to express the energy expenditure for digestion relative to the amount of ingested energy as the SDA coeYcient, allowing a quantitative evaluation of the cost of digestion in relation to input. The SDA coeYcient of fish normally ranges between 5% and 20% (e.g., Jobling, 1983; Eliason et al., 2007), and while the SDA response may be viewed as a substantial bioenergetic ‘‘cost of growth’’ (Jobling, 1981), this cost is a prerequisite for assimilation and should not be regarded as a simple metabolic loss (Mallekh and Lagardère, 2002). The contribution of the various digestive processes to the total SDA response, i.e., prey capture, muscular contraction of the stomach and gut motility, secretion of digestive juices and mucosal absorption of nutrients, and digestion, is likely to vary with food composition and meal size, and to vary among species (e.g., Jobling, 1981; Wang et al., 2006; McCue, 2006). The mechanical component of digestion seems to be rather small. Thus, meals consisting of inert kaolin, which stimulates gastrointestinal motility without being attended by 374 TOBIAS WANG ET AL. postabsorptive processes, produced minor changes in metabolism in plaice (Platessa platessa), whereas protein-rich food elicited marked and swift metabolic responses (Jobling and Davies, 1980; cf. Tandler and Beamish, 1979, 1980). Furthermore, infusion of amino acids directly into the blood stream, which induces metabolic responses similar to those elicited by feeding and inhibition of protein synthesis, completely abolished the SDA response in vivo. Thus, it seems that the biochemical transformation of food and de novo protein synthesis in the postabsorptive state are the major contributors to the SDA response (Brown and Cameron, 1991a,b; Bureau et al., 2002). Both the magnitude and the duration of the SDA response increases with meal size (e.g., Fu et al., 2005; Andrade et al., 2005). Large meals may elicit many-fold increases of the RMR, lasting for many days. Some fishes have a discontinuous feeding pattern, where they fast for long periods followed by the ingestion of large meals. An example is the Atlantic cod (Gadus morhua) where the peak VO2 during SDA may represent up to 90% of VO2max (Soofiani and Hawkins, 1982; Claireaux et al., 2000). The SDA response also varies with food composition (Jobling and Davies, 1980), body size (Hunt von Herbing and White, 2002), and environmental factors, but both its magnitude and duration correlates with the rate and amount of food passing through the gastrointestinal system. Thus, factors that prolong the digestive processes, such as lowered body temperature, prolong the duration of the SDA response associated with lower maximal values, while the SDA coeYcient generally remains unaVected (e.g., Jobling and Davies, 1980; Soofiani and Hawkins, 1982). Chabot and Claireaux (2008) note that in the common sole (Solea solea), which has a small stomach where maximum meal size is less than 3% of body mass, neither the peak value nor the duration of postprandial metabolism are aVected until hypoxia becomes very severe (<30% saturation). The eVects of hypoxia on the SDA response was recently characterized in Atlantic cod (Gadus morhua) exposed to 5% O2 or normoxia (Jordan and SteVensen, 2007; Figure 8.3). This level of hypoxia did not aVect RMR, but the SDA response to a meal of approximately 5% of body mass was significantly prolonged in 5% O2 compared to normoxia and was associated with a lower maximal rate of oxygen consumption. It was not verified whether the prolongation of the SDA response was associated with increased retention time and a slower rate of digestion, but it is likely that reduced oxygen delivery to the gastrointestinal organs and the liver delayed the digestion and assimilation. Clearly, it would be informative to perform similar studies on other species, preferably over a range of meal sizes and at diVerent temperatures, and correlate the changes in the SDA response with temporal changes in nutrient assimilation. Also, it would be of considerable interest to establish whether the levels of hypoxia that aVect the SDA response of a given species correlates with the oxygen levels that retard growth and reduce appetite. 8. 375 THE EFFECTS OF HYPOXIA ON GROWTH AND DIGESTION Oxygen uptake (mg O2 kg−1 h−1) 140 Normoxia (19.8 kPa ~ 95%) Hypoxia (6.3 kPa ~ 30%) 120 100 80 60 24 48 72 96 120 144 168 192 216 240 264 288 Time since feeding (hours) Time to max O2 uptake (h) 200 180 160 140 120 100 80 60 40 20 0 250 35 30 25 20 15 10 5 0 25 SDA coefficient (%) Duration of SDA response (h) Peak O2 uptake (mg O2 kg−1 h−1) 0 200 150 100 50 0 20 15 10 5 0 Normoxia Hypoxia Normoxia Hypoxia Fig. 8.3. The eVects of digestion of a meal corresponding to 5% of body mass on oxygen uptake in Atlantic cod (Gadus morhua) maintained in normoxic or hypoxic water. Duration of SDA response, time to peak VO2, and SDA coeYcient are increased under hypoxia, while the peak VO2 is reduced. [Data are means  S.E. from Jordan and SteVensen (2007).] 376 TOBIAS WANG ET AL. 4. GENERAL EFFECTS OF HYPOXIA ON GROWTH, APPETITE, AND ASSIMILATION The eVects of hypoxia on growth have been characterized in a number of studies in diVerent fish, and the findings from many of these studies are collated in Table 8.1. These diVerent growth experiments have been conducted under very diVerent abiotic and biotic regimes (e.g., diVerent temperatures, water composition, feeding rates, and levels of hypoxia), but it is evident that hypoxia inevitably stifles growth and that this hypoxia-related reduction in growth is primarily a result of reduced food intake (Davis, 1975; Brett, 1979). Two examples are shown in Figures 8.4 and 8.5 for silver bream (Sparus sarba) and the Atlantic cod (Gadus morhua). In both species growth is reduced under hypoxia, and while ingestion rate decreases in both species, growth is also reduced as a result of impaired food conversion eYciency in the silver bream (Chiba, 1983; Chabot and Dutil, 1999). In some species, severe hypoxia is even associated with weight loss as the reduced food intake results in a negative energy balance where basic metabolic needs are covered by internal stores. It is evident, however, that the specific level of hypoxia that retards growth varies among species and is likely to depend on the individual species’ ability to compensate physiologically for the reduction in available oxygen. Thus, species with high oxygen aYnities and robust cardiorespiratory responses to hypoxia are likely to be less aVected than the more hypoxia-sensitive species. As extraordinary exceptions to this rule, two species of cichlids (Astatoreochromis alluaudi and Haplochromis ishmaeli) from Lake Victoria have similar growth rates in normoxia and at a PO2 of approximately 2kPa (10%) for over a year (Rutjes et al., 2007). While it is possible that these cichlids were fed on a restricted ration and that eVects of hypoxia would be evident if the fish were fed to satiety, the study by Rutjes et al. (2007) shows that very hypoxia-tolerant fish can complete digestive processes and grow under extraordinary hypoxic conditions. Growth eVects of hypoxia also depend on the amount of available food, i.e., the food ration or feeding levels (equation 1). This is illustrated in Figure 8.6, where growth and food intake were measured in rainbow trout (Oncorhynchus mykiss) at 15  C at various degrees of hypoxia and diVerent feeding levels (Pedersen, 1987). Specific growth rate decreased with decreasing feeding levels (Figure 8.6A), and lowered food consumption seemed to explain most of the growth reduction (Figure 8.6B). Thus, the reduction in appetite was evident at all feeding levels, but growth retardation in hypoxia was most pronounced at the lower feeding levels. All of the growth studies presented have been performed in captivity under more or less controlled conditions and given the obvious diYculties of performing growth studies in the wild, very little data is available from 8. THE EFFECTS OF HYPOXIA ON GROWTH AND DIGESTION A 377 Growth rate (%) 40 b 30 c 20 10 a 0 8 Feeding rate (%) B b 6 4 b a 2 C Food conversion efficiency (%) 0 80 b b 50 Oxygen saturation (%) 85 60 40 a 20 0 24 Figure 8.4. Growth rates, feeding rates, and conversion eYciencies at 27  C in silver bream (Sparus sarba) at diVerent dissolved oxygen levels. DiVerent letters denote significantly diVerent means. It can be seen that growth, feeding rate, and conversion eYciency are impaired at the lowest oxygen level. [Data are means  S.E. from Chiba (1983).] natural habitats. Hypoxic episodes in south-east Kattegat have been correlated with the abundance of smaller plaice (Pleuronectes platessa) and dab (Limanda limanda), indicating that hypoxia also limits growth under natural conditions (Petersen and Pihl, 1995). Also, a recorded decrease in growth in flathead flounder (Hippoglossoides dubius) was found to be correlated with an occasional decrease in dissolved oxygen levels in Funka Bay, Japan (Kimura et al., 2004). Under natural conditions, hypoxia is likely to occur in combination with hypercapnia and will often be associated with elevated 378 TOBIAS WANG ET AL. c cb -1 Specific growth rate (% day ) 1.0 0.8 cb cb ab 0.6 a 0.4 0.2 -1 30 -1 Ingestion rate (g day fish ) 0.0 35 25 20 15 10 5 0 35 Efficiency (%) 30 25 20 15 10 5 0 40 50 60 70 80 90 100 Oxygen saturation (%) Fig. 8.5. Growth rates, ingestion rates, and conversion efficiency in Atlantic cod (Gadus morhua) reared at 10  C under different oxygen levels. Letters indicates significantly different values, thus growth was less at the lowest oxygen level. There was a significant correlation between ingestion rate and oxygen level (R2 = 0.93). There was a significant linear correlation between conversion efficiency and oxygen (R2 = 0.92), if the efficiency at 93% saturation was excluded. [Data are means estimated from Chabot and Dutil (1999), and S.E. can therefore not be provided.] temperatures. These additional stressors are likely to exacerbate the adverse eVects of hypoxia and it would be interesting to see if future studies could assess the roles of disturbed acid–base balance and/or temperature challenge on growth and digestive performance in hypoxia. 8. 379 THE EFFECTS OF HYPOXIA ON GROWTH AND DIGESTION 6 Specific growth rate (% day−1) 5 FL 1.0 4 3 FL 0.6 2 1 FL 0.2 0 −1 Food consumption (cal (kcal fish)−1 day−1) 120 FL 1.0 100 80 FL 0.6 60 40 FL 0.2 20 0 20 40 60 80 100 Oxygen saturation (%) 120 140 Fig. 8.6. Specific growth rates (A) and food intake (B) in rainbow trout (Oncorhyncus mykiss) at 15  C under diVerent amounts of dissolved oxygen and at diVerent relative feeding levels (FL). [Data are means estimated from Pedersen (1987), and S.E. can therefore not be provided.] 4.1. Adaptation of Growth during Long-Term Hypoxia While hypoxia consistently lowers growth rate, long-term adaptations to prolonged hypoxia may alleviate the negative eVects of insuYcient oxygen. The temporal changes in growth performance during long-term hypoxia have been studied in a few species (see Table 8.2 and Figure 8.7). In general, Table 8.2 Specific Growth Rates at DiVerent Durations of Hypoxia Common name Latin name Initial Salinity (‰) Mb(g) Feeding level O2 Time SGR lLght cycle Temp ( C) (%)a (day) (% day–1)b Spotted wolYsh Anarhichas minor 68.5 – 1 day–1 6D:18L 8.0 Dab Limanda limanda 23.5 32 3–8% 2 day–1 10D:14L 14.7 Plaice Pleuronectes platessa Paralichthys lethostigma 22.8 32 3–8% 2 day–1 10D:14L 14.8 1.8 15 Ad lib. 2 day–1 10D:14L 25.0 Southern flounder Turbot Scophthalmus maximus 120 34 2 day–1 8D:16L 17.0 Turbot Scophthalmus maximus 120 34 2 day–1 8D:16L 17.0 34 0–32 33–55 56–76 30 0–10 11–20 94.7 0–10 11–20 36.9 0–14 15–21 62.6 0–14 15–21 45 0–15 16–30 31–45 64 0–15 16–30 31–45 0.35 0.35 0.63 –0.1 0.6 –0.4 0.8 1.6 2.2 2.5 3.5 0.3 0.5 0.7 0.3 0.5 0.7 Reference Foss et al. (2002) Petersen and Pihl (1995) Petersen and Pihl (1995) Taylor and Miller (2001) Pichavant et al. (2000) Pichavant et al. (2000) mgO2 L 1 ⋅100%, assuming standard barometric pressure and accounting for If not stated in %, the saturation was calculated as 1 mgO2 L ð100%saturationÞ temperature and salinity. lnW1 lnW0 b ⋅100% SGR ¼ t a A B Specific growth rates (% day−1) THE EFFECTS OF HYPOXIA ON GROWTH AND DIGESTION Specific growth rates (% day−1) 8. 381 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0 0.8 0.6 0.4 0.2 0 Day 0−32 Day 33−55 Day 56−76 Fig. 8.7. Specific growth rates in juvenile turbot (Scophthalmus maximus) at (A) 95% saturation and (B) 45–65% saturation, both at 17 C. In normoxia the SGR decreases with time, while it increases in hypoxia. [Data are means estimated from Pichavant et al. (2000), and S.E. can therefore not be provided.] fish adapt to the hypoxic conditions and increase growth rate progressively over time, and some species even reverse an initial weight loss to a weight gain (e.g., Petersen and Pihl, 1995). The mechanisms that underlie this adaptation to long-term hypoxia are likely to involve the common physiological responses to hypoxia, which include increased blood O2 aYnity, blood volume, and hemoglobin concentrations, as well as increased capillary density, etc. These physiological adaptations would increase oxygen transfer from the hypoxic water to the metabolizing tissue. Some studies have measured the hematocrit of animals grown in diVerent oxygen concentrations, but with unclear results. In some experiments the hematocrit is slightly elevated in animals grown at lower oxygen levels (Taylor and Miller, 2001) while in others there is no significant diVerence (Andrews et al., 1973). A study by Chabot and Dutil (1999) revealed no diVerence among groups reared at diVerent oxygen saturations, but the hematocrit was larger in the 382 TOBIAS WANG ET AL. control group at the beginning of the experiment. However, a causal relationship between the classic physiological responses and the recovery of appetite and growth remains to be established. 4.2. EVects of Dynamic Changes in Oxygen Levels on Growth While the eVects of chronic hypoxia have been characterized in some detail, the consequences of fluctuating O2 levels for growth and digestion have been investigated more rarely. In brook trout (Salvelinus fontinalis), coho salmon (Oncorhynchus kisutch), and largemouth bass (Micropterus salmoides), growth seems to be more aVected by fluctuations in O2 concentrations than by exposure to constant intermediate O2 concentrations (reviewed by Brett, 1979). Adverse eVects on the growth to O2 oscillations compared to constant concentrations [2.8–6.2 mg L-1 (~38–84%) and 4.74 mg L–1 (~65%), respectively] have recently been demonstrated in juvenile southern flounder (Paralichthys lethostigma) and related respiratory adjustments were described (Taylor and Miller, 2001). In comparison, growth performances of European sea bass juveniles (Dicentrarchus labrax) are not significantly aVected by repetitive O2 oscillations of between 6 (~85%) and 3 mg L–1 O2 concentrations (~42%) compared to constant O2 concentrations of 6 or 3 mg L–1, respectively (Thetmeyer et al., 1999). The eVects of dynamic changes in oxygen levels are likely to diVer drastically depending on the duration and severity of the hypoxic insults. Thus, as expanded on below, acute exposure to severe hypoxia can induce vomiting, and the reduction in appetite is likely to persist for many hours upon return to normoxia. Furthermore, given that some of the physiological responses to hypoxia, such as synthesis of additional red cells and angiogenesis, are accompanied by energetic costs, it is likely that dynamic changes in oxygen levels would increase RMR and reduce the amount of energy available for growth (see equation 1). 4.3. The EVect of Interactions between Temperature, Salinity, and Hypoxia on Growth Rate The solubility of oxygen decreases as water temperature increases, while the stimulation by temperature of metabolic processes increases the need for oxygen delivery. Recently, it has been emphasized that the capacity for the cardiorespiratory system is an important determinant of temperature tolerance (e.g., Portner and Knust, 2007; see Chapter 4). Growth generally increases with temperature, as illustrated for striped bass (Morone saxatilis) and white sturgeon (Acipenser transmontanus) in Figure 8.8, which also illustrates the typical Q10 of 2 (Cech et al.,1984). Besides illustrating the profound eVect of temperature on growth, this example also illustrates the 8. THE EFFECTS OF HYPOXIA ON GROWTH AND DIGESTION A 3.5 3.0 SGR (% day−1) 383 84 % 58% M. saxatilis 84 % 58% A. transmontanus 2.5 2.0 1.5 1.0 0.5 0 B 3.5 SGR (% day−1) 3.0 2.5 2.0 1.5 1.0 0.5 0 15.0 25.0 20.0 Temperature (degrees C) Fig. 8.8. Specific growth rates at diVerent temperatures and dissolved oxygen levels in (A) striped bass (Morone saxatilis) and (B) white sturgeon (Acipenser transmontanus). Measurements were conducted over 30–34 days. It can be seen that SGR increases with temperature for both species at both normoxia and hypoxia, and that the SGR is lower in normoxia compared to hypoxia. [Data are means estimated from Cech et al. (1984) and S.E. can therefore not be provided.] species-specific variation in sensitivity to, for instance, hypoxia. However, growth typically decreases when the optimal body temperature is exceeded. This negative eVect might be directly caused by the eVects of temperature on proteins, but the eVects at more moderate temperatures are likely to include an inability to maintain suYcient oxygen delivery to the gastrointestinal organs during digestion. In natural systems, the incidence of hypoxia does increase with temperature as evident from the variety of air-breathing fish in the tropics. It would be interesting to characterize the eVects of hypoxia at diVerent temperatures in species from temperate and tropical regions. In some areas, e.g., fjords and estuaries, the eVect of hypoxia will, besides interactions with temperature, also be aVected by varying salinity. Owing to the cost of osmoregulation in higher salinities, growth may be influenced more by hypoxia in higher salinities than at lower salinities. 384 TOBIAS WANG ET AL. 5. EFFECTS OF HYPOXIA AND DIGESTIVE STATE ON OXYGEN TRANSPORT Apart from the rise in metabolism, digestion is associated with a number of physiological changes, such as changes in acid–base status caused by gastric acid secretion, and elevated nitrogen excretion in connection with the increased protein metabolism and changes in water balance as the food items are degraded to osmotically active nutrients (e.g., Taylor et al., 2007; Wood et al., 2007). Along with the metabolic changes, the physiological challenge of digestion alters the manner in which fish can respond to hypoxia. Digestive state also aVects the responses to other situations with elevated metabolism increases and the energetic burden imposed by digestion, for example, aVects swimming ability (Blaikie and Kerr, 1996; Alsop and Wood, 1997). Most markedly, the rise in metabolism associated with digestion and the need to increase perfusion of the gastrointestinal tract to facilitate absorption of nutrients requires cardiovascular responses that include an increase in cardiac output, through increments of heart rate and stroke volume, as well as a redistribution of blood flows to the digestive organs (Wang et al., 2005). In fasting fish, blood flow to the gastrointestinal tract accounts for 10–30% of total cardiac output at rest, but this proportion increases drastically within hours after feeding, and may constitute 60–70% of cardiac output in the postprandial period (Axelsson et al., 1989, 2000; Axelsson and Fritsche, 1991; Thorarensen et al., 1994; Farrell et al., 2001; Eliason et al., 2008; Altimiras et al., 2008). The rise in gastrointestinal blood flow seems to depend on meal size, rather than species diVerences. As in other vertebrates, blood flow to the gastrointestinal organs is normally reduced during hypoxia to prioritize oxygen-sensitive organs such as the heart and brain (e.g., Axelsson and Fritsche, 1991; Axelsson et al., 2002). A reduction in blood flow to the gastrointestinal organs may compromise digestive functions and is likely to lower absorption eYciency and prolong the digestive process. Axelsson et al. (2002) measured gastrointestinal blood flow during hypoxia in fasting and digesting European sea bass (Dicentrarchus labrax). The fasting sea bass exhibited the typical piscine cardiovascular response to hypoxia, consisting of a reduction in heart rate and a reduction in gastrointestinal blood flow (Figure 8.9). Feeding causes gastrointestinal blood flow to increase, primarily through an increased heart rate, and the proportion of cardiac output allocated to gut increases from 24% to 35%. When the sea bass was challenged by hypoxia in the postprandial period, the proportion of blood flow directed to the gastrointestinal system remained elevated although cardiac output decreased as in the fasting sea bass. As discussed by Axelsson et al. (2002), the maintenance of the relative gut perfusion is 8. 385 THE EFFECTS OF HYPOXIA ON GROWTH AND DIGESTION A 70 Postprandial sea bass Heart rate (min−1) 60 50 40 Fasting sea bass 30 20 10 0 B Cardiac output (ml min−1 kg−1) 60 50 40 30 20 10 0 25 Total gut blood flow (ml min−1 kg−1) C 20 15 10 5 Relative gut blood flow (%) 0 D 50 40 30 20 10 0 0 5 10 PO2 (kPa) 15 20 Fig. 8.9. The eVects of progressive hypoxia on heart rate (A), cardiac output (B), total gut blood flow (C), and relative gut blood flow (C) in fasting and postprandial European sea bass (Dicentrarchus labrax). The relative gut blood flow is the total gut blood flow relative to the cardiac output. Both fasting and digesting fish respond to hypoxia with a bradycardia, but digesting fish maintain a higher gut blood flow at all hypoxic conditions. [Data from Axelsson et al. (2002).] 386 TOBIAS WANG ET AL. likely to stem from local release of signal transmitter substances causing relaxation of the vascular beds in the stomach and intestine associated with digestion, rather than a lack of control over gastrointestinal blood flow. Mechanical stretch of the stomach may be one of the signals causing such release of vasoactive substances, but chemical stimuli are also likely to play an important role (Seth et al., 2008). Studying the same species, Altimiras et al. (2008) recently showed that gastrointestinal blood flow is decreased during swimming in both fasting and postprandial animals, suggesting that the regulation of the blood distribution diVers between muscular exercise and hypoxia. 6. EFFECTS OF HYPOXIA ON APPETITE Hypoxia causes significant reductions in appetite and the resulting reduction in ingested food constitutes the major part of the hypoxia-induced growth reduction (equation 1), which is illustrated by the feed intake (FI) values in Table 8.1. Acute exposure to hypoxia immediately aVects the digestive processes and digesting Atlantic cod (Gadus morhua), for example, immediately void their stomach when exposed to hypoxia (Claireaux et al., 2000). As such, the vomiting response may be viewed as part of the normal stress response to an immediate hypoxic challenge, and is likely to reflect the fact that organ systems other than those involved in digestion are prioritized during hypoxia. Using a similar teleology, it seems advantageous to reduce food intake to lessen the metabolic burden associated with digestion during long-term hypoxia, leaving more of the aerobic scope for physical activity (Figure 8.1). The mechanisms by which mild hypoxia reduces appetite over longer time scales have not been studied in fish or even in mammals, where considerable attention has been paid to understanding the mechanisms underlying anorexia and the associated weight loss at altitude (e.g., Vats et al., 2007). As in other vertebrates, long-term regulation of food intake in fish is controlled by a complex interplay of stretch and chemoreceptors within the stomach and intestine as well as hormones released either centrally or from the gastrointestinal organs (VolkoV et al., 2005; Gorissen et al., 2006). Most of the satiety-inducing inputs are transmitted to the nucleus lateralis tuberis within the hypothalamus, which through the nucleus preopticus is involved in regulation of appetite and the release of growth hormone. In general, appetite is stimulated by the orexigenic peptide hormone ghrelin, which is released from the fasting stomach and acts directly on the pituitary causing release of growth hormone. Satiety, on the other hand, seems more complex and involves many diVerent hormones and signal molecules, such as cholecystokinin (CCK), as well as gastric and intestinal satiety signals induced 8. THE EFFECTS OF HYPOXIA ON GROWTH AND DIGESTION 387 primarily by stretch of the stomach as well as the presence of nutrients in the intestine (VolkoV et al., 2005; Gorissen et al., 2006; Maljaars et al., 2007). More long-term eVects include hormones such as leptin, which is released from adipose tissue and provides information on energy state of the organism. While many of these hormones have been characterized in fish (e.g., VolkoV et al., 2005; Gorissen et al., 2006; Canosa et al., 2007), circulating levels of these hormones have not been measured during hypoxia and their role remains to be studied. The eVects of hypoxia on the central regulation of appetite have been studied in rainbow trout (O. mykiss) through measurements of forebrain corticotropin-releasing factor and urotensin (Bernier and Craig, 2005). The forebrain concentration of both peptides increased in hypoxia and after treatment with an antagonist to inhibit the receptors for corticotropinreleasing factor (Bernier and Craig, 2005). It was concluded that the hypophysiotropic factors stimulate the hypothalamic–pituitary–adrenal axis in fish and that this part of the stress response plays a role in reducing food intake during hypoxia. The piscine stomach is innervated by stretch receptors that relay information on the presence and amount of food in the stomach, as well as chemoreceptors in the intestine providing feed-back on the presence of food in the gut (e.g., Grove et al., 1978, 1985; Grove and Holmgren, 1992a,b). A significant part of the appetite reduction occurring during hypoxia may arise from prolonged stimulation of the gastric and intestinal stretch and chemoreceptors. Thus, as hypoxia prolongs the digestive processes, as indicated from the extended SDA response (Jordan and SteVensen, 2007; Figure 8.3), the stimulation of stretch receptors and chemoreceptors persists for a longer period of time extending the sensation of satiety. Certainly gut emptying time is prolonged and gut motility is inhibited by hypoxia in mammals (Yamaji et al., 1996; Yoshimoto et al., 2004), and similar eVects are likely to be present in fish. 7. ASSIMILATION EFFICIENCY Assimilation eYciency refers to the amount of ingested food that is assimilated over the gut (see equation 5). Assimilation eYciency is also referred to as absorption eYciency (Jobling, 1993) or digestion eYciency. In humans, assimilation seems to be reduced in severe hypoxia, but contributes only slightly to the weight loss at altitude (Westerterp et al., 1994; Westerterp et al., 2000). This is also the case in invertebrates where slight (3%) reductions in assimilation eYciency are only seen during very severe hypoxia (McGaw, 2008). In this study, the gut clearance time of the 388 TOBIAS WANG ET AL. Dungeness crab, Cancer magister, increased as PO2 fell below 10.5 kPa, whereas assimilation eYciency was only slightly aVected at the lowest PO2 of 1.6 kPa, indicating that digestion was prolonged rather than directly impaired by hypoxia. As in these other animal groups, there are few studies that have addressed the eVects of hypoxia on assimilation eYciency in fish and in this group contradicting results have been seen. Thus, in the Nile tilapia (Oreochromis niloticus), assimilation eYciency was reduced from ~80% in normoxia to ~54% under severe hypoxia (Tsadik and Kutty, 1987). In contrast, Pedersen (1987) found that assimilation in rainbow trout (O. mykiss) was unaVected in hypoxia. It is clear that this is an area for further research on a greater variety of fish species and at diVerent oxygen levels. In particular, it would be interesting to investigate whether increased passage time is a general compensation for a less eVective absorption during hypoxia. 8. EFFECTS OF HYPOXIA ON GROWTH IN AIR-BREATHING FISHES Diurnal hypoxic events are particularly common in small and stagnant water bodies in the tropics where air-breathing fish often dominate the piscine fauna. The eVects of hypoxia on growth rate, feed intake, and digestion have, however, only been studied in a few species of air-breathing fishes. This may be because obligate air-breathers, which, by virtue of their ability to obtain the vast majority of their oxygen from the air, are unlikely to be strongly aVected by aquatic hypoxia (e.g., Sanchez et al., 2001). Facultative air-breathers, on the other hand, are aVected by aquatic hypoxia and the few existing studies point to clear eVects on energetics and growth. The obligate air-breathing fish Ophiocephalus striatus (now Channa striatus) maintains routine metabolic rate in hypoxic water by increasing surfacing and air-breathing frequency to extract oxygen from the air (Vivekanandan, 1977). Many air-breathing fish species increase the frequency of surfacing during digestion (Vivekanandan, 1977; Ponniah and Pandian, 1977; Figure 8.10A). Furthermore, Pandian and Vivekanandan (1976) observed that fed fish in unaerated water had a lower food consumption than fish in aerated water, which might indicate that the cost of elevating the air-breathing frequency at some point exceeds the gain of keeping a certain metabolic rate, leaving scope for activity and digestion. This is also apparent from starved individuals of C. striatus having a higher hanging frequency and duration in deeper aquariums, than more shallow aquariums Vivekanandan (1977). Vivekanandan argues that the fed fish had higher food consumption in deeper aquariums as a result of the increased surfacing costs necessary to 8. 389 THE EFFECTS OF HYPOXIA ON GROWTH AND DIGESTION A Surfacing frequency (day−1) 2500 2000 Starved Fed 1500 1000 500 0 B Food intake (g cal g−1 day−1) 300 250 200 150 100 50 C Food conversion efficiency (%) 0 30 25 20 feeding rate 15 10 5 0 2.5 5.0 15.5 Depth (cm) 31.0 40.0 Fig. 8.10. Surfacing frequency (A), food intake (B), and food conversion eYciency (C) in Snakehead fish (Channa striatus) at 27 C, at diVerent depths. The oxygen level was not controlled very accurately, resulting in higher O2 levels in the starved group; this might explain the higher surfacing frequency in fed fish. Food intake increases with depth in fed fish. Surfacing frequency was independent of depth in starved fish. The food conversion eYciency is highest at small depths, but independent of depth below 15 cm. The fish had a body mass of 750  70 mg and were measured over a period of 21 days. [Data are means  S.D. from Vivekanandan (1977).] 390 TOBIAS WANG ET AL. obtain the necessary oxygen for their RMR (see Figure 8.10B). In the facultative air-breather Heteropneustes fossilis, the surfacing frequency also increased with increasing distance to the surface (Pandian and Vivekanandan, 1976). This compensation apparently only holds up to a limit where the costs of surfacing exceed the gain, and it is more profitable for the fish to rely solely on aquatic respiration, which is not possible in C. striatus (see also Kramer, 1987). A possible consequence of the elevated surfacing frequency during digestion and feeding is that the SDA (specific dynamic action) will be higher in air-breathing species than in non-airbreathers (Krishnan and Reddy, 1989), but further studies on this topic are needed to evaluate this hypothesis. Juvenile air-breathing fish tend to depend on aquatic respiration until their air-breathing organ has been suYciently developed. Therefore, growth in juvenile air-breathing fish is probably more dependent on dissolved oxygen levels than in adults. Even the giant South American obligate air-breather Arapaima gigas, which as an adult drowns within 10 min if prevented from access to air, is entirely water-breathing from hatching until about 9 days old after which a transition begins where the gill lamellae disappear and the airbreathing organ develops (Brauner et al., 2004). It is likely that hypoxia slows down this transition as well as aVecting growth itself. In support of this theory, Ebeling and Alpert (1966) found in paradise fish (Macropodus opercularis) that the air-breathing organ developed more slowly under hypoxic conditions than under normoxia. Finally, this ontogeny of air-breathing is probably aVected by other factors such as the need to minimize predation risks in juveniles, which may be why a number of air-breathers perform synchronous surfacing behaviors (Kramer and Graham, 1976). The growth eVects of hypoxia have been largely overlooked in air-breathing fish and given their fascinating position in the evolution from water to land and their increasing commercial importance, this aspect is worthy of further study. 9. CONCLUSIONS AND PERSPECTIVES Hypoxia is common in natural areas and fish have evolved a number of physiological responses to tolerate large variations in oxygen availability. A substantial number of studies show that hypoxia limits growth primarily through a reduction in appetite, and it seems that the anorexic response and growth retardation occur at relatively mild levels of hypoxia. The overall eVects of hypoxia on growth were already well established when the eVects of hypoxia were reviewed by Brett for Fish Physiology in 1979 (see also Davis, 1975). Thus, as concluded 30 years ago, the impaired growth during hypoxia is primarily caused by a depression of food intake. More recent studies 8. THE EFFECTS OF HYPOXIA ON GROWTH AND DIGESTION 391 including cardiorespiratory and metabolic measurements have reinforced the link between aerobic scope and maximal growth. The causal link between scope of metabolism and appetite, however, still needs to be established. Thus future studies that provide in depth analysis of the hormonal profile along with blood flow and metabolic measurements would be very useful. 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EVects of feeding and starvation on growth and swimming activity in an obligatory air-breathing fish. Hydrobiologia 49, 33–39. Pedersen, C. L. (1987). Energy budgets for juvenile rainbow trout at various oxygen concentrations. Aquaculture 62, 289–298. Petersen, J. K., and Pihl, L. (1995). Responses to hypoxia of plaice, Pleuronectes platessa, and dab, Limanda Limanda, in the south-east Kattegat: Distribution and growth. Environ. Biol. Fish. 43, 311–321. Person-Le Ruyet, J., Pichavant, K., Vacher, C., Le Bayon, N., Sévère, A., and Boeuf, G. (2002). EVects of oxygen supersaturation on growth and metabolism in juvenile turbot (Scophthalmus maximus). Aquaculture 205, 373–383. Pichavant, K., Person-Le Ruyet, J., Le Bayon, N., Sévère, A., Le Roux, A., Quéméner, L., Maxime, V., Nonnotte, G., and Boeuf, G. (2000). EVects of hypoxia on growth and metabolism of juvenile turbot. Aquaculture 188, 103–114. Pichavant, K., Person-Le Ruyet, J., Le Bayon, N., Sévère, A., Le Roux, A., and Boeuf, G. (2001). Comparative eVects of long-term hypoxia on growth, feeding and oxygen consumption in juvenile turbot and European seabass. J. Fish Biol. 59, 875–883. Ponniah, A. G., and Pandian, T. J. (1977). Surfacing activity and food utilization in the airbreathing fish Polyacanthus cupanus exposed to constant PO2. Hydrobiologia 53, 221–227. Portner, H.-O., and Knust, R. (2007). Climate change aVects marine fishes through the oxygen limitation of thermal tolerance. Science 315, 95–97. Rubner, M. (1902). In ‘‘Die Gesetze des Energie verbrauchs bei der Ernährung’’ Frank Dauticke, Leipzig & Vienna. Rutjes, H. A., Nieven, M. C., Weber, R. E., Witte, F., and Van den Thillart, G. E. E. J. M. (2007). Multiple strategies of Lake Victoria cichlids to cope with lifelong hypoxia include hemoglobin switching. Am. J. Physiol. 293, R1376–R1383. 8. THE EFFECTS OF HYPOXIA ON GROWTH AND DIGESTION 395 Sanchez, A., Soncini, R., Wang, T., Koldkjær, P., Taylor, E. W., and Glass, M. L. (2001). The diVerential cardio-respiratory responses to ambient hypoxia and systemic hypoxaemia in the South American lungfish, Lepidosiren paradoxa. Comp. Biochem. Physiol. 130, 677–687. Seth, H., Sandblom, E., Holmgren, S., and Axelsson, M. (2008). EVects of gastric extension on the cardiovascular system in rainbow trout (Oncorhynchus mykiss). Am. J. Physiol. 294, R1648–R1656. Soofiani, N. M., and Hawkins, A. D. (1982). Energetic costs at diVerent levels of feeding in the juvenile cod, Gadus morhua. J. Fish Biol. 21, 577–592. SteVensen, J. F. (1989). Some errors in respirometry of aquatic breathers: How to avoid and correct for them. Fish Physiol. Biochem. 6, 49–59. Tandler, A., and Beamish, F. W. H. (1979). Mechanical and biochemical components of apparent specific dynamic action in largemouth bass, Micropterus salmoides Lacepede. J. Fish Biol. 14, 343–350. Tandler, A., and Beamish, F. W. H. (1980). Specific dynamic action and diet in largemouth bass, Micropterus salmoides (Lacepede). J. Nutr. 110, 750–764. Taylor, J. C., and Miller, J. M. (2001). Physiological performance of juvenile southern flounder, Paralichthys lethostigma (Jordan and Gilbert, 1984), in chronic and episodic hypoxia. J. Exp. Mar. Biol. Ecol. 258, 195–214. Taylor, J. R., Whittamore, J. M., Wilson, R. W., and Grosell, M. (2007). Postprandial acid-base balance and ion regulation in freshwater and seawater-acclimated European flounder, Platichthys flesus. J. Comp. Physiol. B 177, 597–608. Thetmeyer, H., Waller, U., Black, K. D., Inselmann, S., and Rosenthal, H. (1999). Growth of European sea bass (Dicentrarchus labrax L.) under hypoxic and oscillating oxygen conditions. Aquaculture 174, 355–367. Thorarensen, H., Gallaugher, P. E., Kiessling, A. K., and Farrell, A. P. (1994). Intestinal blood flow in swimming chinook salmon Oncorhynchus tshawytscha and the eVects of hematocrit on blood flow distribution. J. Exp. Biol. 179, 115–129. Tran-Duy, A., Schrama, J. W., van Dam, A. A., and Verreth, J. A. J. 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Wood, C. M., Kajimura, M., Bucking, C., and Walsh, P. J. (2007). Osmoregulation, ionoregulation and acid-base regulation by the gastrointestinal tract after feeding in the elasmobranch (Squalus acanthias). J. Exp. Biol. 210, 1335–1349. Wootton, J. R. (1998). In ‘‘Ecology of Teleost Fishes’’ 2nd ed, Kluwer, Dordrecht. Yamaji, R., Sakamoto, M., Miyake, K., and Nakano, Y. (1996). Hypoxia inhibits gastric emptying and gastric acid secretion in conscious rats. J. Nutr 126, 673–680. Yoshimoto, M., Sasaki, M., Naraki, N., Morhi, M., and Miki, K. (2004). Regulation of gastric motility at simulated high altitude in conscious rats. J. Appl. Physiol. 97, 599–604. Zhou, B. S., Wu, R. S. S., Randall, D. J., and Lam, P. K. S. (2001). Bioenergetics and RNA/DNA ratios in the common carp (Cyprinus carpio) under hypoxia. J. Comp. Physiol. B 171, 49–57. 9 THE ANOXIA-TOLERANT CRUCIAN CARP (CARASSIUS CARASSIUS L.) MATTI VORNANEN JONATHAN A.W. STECYK GÖRAN E. NILSSON 1. Introduction 1.1. Distribution and Habitat of Crucian Carp 1.2. The Need for Oxygen in the Vertebrate Brain and Heart 2. Mechanisms of Hypoxic and Anoxic Survival 2.1. Ethanol Production 2.2. Gill Remodeling 2.3. Cardiorespiratory Adjustments 2.4. The Heart in Anoxia 2.5. The Brain in Anoxia 3. Seasonality of Crucian Carp Physiology: Preparing for Winter Anoxia 3.1. Seasonal Changes of Glycogen Stores 3.2. Seasonality of the Heart 3.3. Seasonality of Brain 4. Summary The crucian carp is probably the most anoxia-tolerant fish there is, surviving without oxygen for days to months depending on temperature. The anoxia tolerance has evolved in response to over-wintering in ponds and small lakes that can become anoxic for months during the winter. The exceptional anoxia tolerance of the crucian carp is based on special physiological traits that are either constitutively expressed or seasonally primed. A key to its anoxia tolerance is its constitutive ability to produce ethanol as the major anaerobic end product. The ethanol production is supported by massive stores of glycogen in various tissues, and these stores are largest in the autumn before the onset of wintertime anoxia. Metabolic depression is less pronounced than in anoxia-tolerant turtles and there is no major 397 Hypoxia: Volume 27 FISH PHYSIOLOGY Copyright # 2009 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(08)00009-5 398 MATTI VORNANEN ET AL. down-regulation of membrane permeability in brain ("channel arrest"), possibly with the exception of reduced NMDA receptor function. Increased levels of the inhibitory neurotransmitter GABA and low levels of the excitatory transmitter glutamate together with a modest activation of glycolysis probably ensure energy balance to the anoxic brain and aid to maintain normal ion gradients across neuronal membranes. The heart has been found to sustain cardiac output in anoxia, possibly to allow for substrate transport and a sufficient rate of ethanol release to the water. Like the brain, the heart also shows few signs of reduced ion permeability in anoxia. However, a lack of compensatory temperature acclimatization suggests that it is utilizing the low winter temperature to suppress its energy needs during anoxia. 1. INTRODUCTION While the vast majority of vertebrates need a constant supply of oxygen to survive, there are a few examples of vertebrates that can survive long periods of anoxia – the complete absence of oxygen. The best studied examples of such animals are some species of North American freshwater turtles and two cyprinid fishes: the crucian carp (Carassius carassius L.) and the closely related goldfish (Carassius auratus L.). Most studies on goldfish have been made on fish obtained from the aquarium trade, where goldfish have been cultured for more than a millennium, and where the main selection pressures have been for readily breeding in captivity, and for various colors and shapes, rather than for anoxia tolerance. Thus, many of the original traits promoting anoxia tolerance may have been lost. Indeed, goldfish do not display the same extreme anoxia tolerance as the crucian carp, which survives days of anoxia at room temperature and several months of anoxia at temperatures close to 0 C (Blazka, 1958; Blazka, 1960; Piironen and Holopainen, 1986) (Figure 9.1). In this chapter we will focus on the crucian carp, although some references to studies on goldfish will be given. Additionally, when relevant, we will contrast and draw parallels between the mechanisms of anoxia tolerance that the unrelated crucian carp and freshwater turtle display. 1.1. Distribution and Habitat of Crucian Carp The crucian carp has a wide distribution in central Asia and Europe, ranging from the Arctic Circle in Scandinavia to central France and the Black Sea in the south, and from England to the Lena River in Russia (Holopainen et al., 1997b). Crucian carp exist in two distinct forms; both evolved to reduce or avoid predation (Nilsson, 1855). When crucian carp coexist with piscivorous fish in lakes, the presence of predators induces a 9. 399 ANOXIA-TOLERANT CRUCIAN CARP Temperature (⬚C) 20 15 10 5 0 0 50 100 150 Anoxia tolerance (days) 200 250 Fig. 9.1. Anoxia tolerance of crucian carp at diVerent temperatures measured as lethal time to 50% mortality (LT50). Tolerance to anoxia decreases exponentially as a function of temperature. [Data from (Piironen and Holopainen, 1986).] change in body morphology from a shallow and long-bodied fish to a much deeper bodied form, which decreases predation eYciency of gape-limited piscivores such as pike (Brönmark and Milner, 1992; Holopainen et al., 1997a), and enhances the escape locomotor performance of crucian carp (Domenici et al., 2008). An alternative survival strategy of crucian carp is based on an equally prominent trait that allows it to completely avoid predatory fish: they are able to inhabit small ponds where anoxic periods exclude the presence of other fish species. In the fall and winter seasons, these ponds progressively become hypoxic because oxygen consumption by the inhabitants exceeds the photosynthetic oxygen production by plants, and ice and snow cover prevents diVusion of oxygen from the atmosphere (Nagell and Brittain, 1977). The hypoxic period lasts until the spring and frequently includes totally anoxic periods (Figure 9.2). It is the latter survival strategy that has resulted in the evolution of physiological traits that promote anoxia tolerance (Blazka, 1958; Nilsson, 2001). The ponds in which crucian carp reside also experience large and regular seasonal changes in temperature (Figure 9.2). Consequently, crucian carp are also known for their excellent eurythermicity with thermal tolerance ranging from 0 to 38 C, with a high thermal optimum of 27 C (Horoszewicz, 1973). 1.2. The Need for Oxygen in the Vertebrate Brain and Heart An animal that survives prolonged anoxia, like the crucian carp, has to overcome a major problem: to protect its tissues from energy deficiency. The stop in oxidative phosphorylation during anoxia leaves the animal with glycolysis as the only major ATP-producing process. Glycolysis has an ATP yield that is only about 6–10% of that of the oxidation of glucose during normoxic conditions (depending on how well the mitochondria are coupled during 400 MATTI VORNANEN ET AL. 10 25 8 Temperature (⬚C) 20 Temperature Oxygen 15 6 10 4 5 2 0 Oxygen content (mg/L) Ice cover 0 Jun Aug Oct Dec Feb Apr Jun Fig. 9.2. Seasonal changes in temperature and oxygen content in a typical habitat of crucian carp in Eastern Finland at the latitude of 62 41/N. [Data from (Vornanen and Paajanen, 2004).] normoxia; Hochachka and Somero, 2002). Consequently, it is widely accepted that the high intrinsic rates of ATP use make the vertebrate brain and heart particularly sensitive to low oxygen levels, as they will rapidly run out of ATP when the sole source of ATP is anaerobic glycolysis. In mammals, most of the ATP turnover in brain is devoted to neural signaling, particularly to the ionpumping needed to maintain the membrane potentials of electrically active neurons (Erecinska and Silver, 1994). While fish bodies only consumes about one tenth of the energy used by mammalian bodies, fish brains do not diVer much from mammalian brains when it comes to ATP use (Nilsson, 1996), and it is likely that electric activity is also the main energy consumer in the fish brain. In the majority of vertebrates, the brain will rapidly fail to function in anoxia. Key events of the anoxic brain failure in mammals include a loss of ion homeostasis, which can be detected as a rapid rise in extracellular K+ levels, and a subsequent outflow of glutamate – the major excitatory neurotransmitter in brain. Both these events are also seen in anoxic rainbow trout (Oncorhynchus mykiss) brain within about half an hour in anoxia at 10 C (Nilsson et al., 1993a; Hylland et al., 1995). The depolarization and the glutamate out-flow triggers the opening of Ca2+ channels and a massive rise in intracellular Ca2+, which in turn activates several degenerative pathways (Arundine and Tymianski, 2003). Like the brain, the heart has an ATP requirement greatly exceeding that of most other tissues. For the heart to function as a muscular pump, a continual ATP supply is needed for powering the myofilament sliding by the myosin ATPase. Also, ATP is needed to power the various ATP-dependent ion-motive pumps (i.e., Na+/K+-ATPase and Ca2+-ATPases) essential for repeated action potential generation, intracellular Ca2+ homeostasis, and membrane ion transport (Aho and Vornanen, 1997; Rolfe and Brown, 1997; Huss and Kelly, 2005; 9. ANOXIA-TOLERANT CRUCIAN CARP 401 Taha and Lopaschuk, 2007). For mammalian hearts, mechanical activity has been estimated to account for 75–85% of the cardiac ATP demand and the Na+/ K+-ATPase and Ca2+-ATPases for 15–25% (Schramm et al., 1994; Rolfe and Brown, 1997), and it is assumed that 2% of the cellular ATP pool is consumed in each heart beat (Balaban, 2002). Thus, like the brain, if ATP supply is not matched to ATP demand, the heart’s ATP pool will quickly be depleted and a catastrophic sequence of events will occur, including failure of the ATPdependent ion-motive pumps, disruption of cellular membrane potentials, and a loss of ionic integrity of cellular membranes. This will lead to cardiomyocyte death by necrosis, cardiac failure, and, ultimately, organismal death (Hochachka, 1986; Boutilier, 2001). The cardiac ATP budget in the carp may diVer from mammals due to diVerences in cellular structure and Ca2+ management (Santer, 1985; Aho and Vornanen, 1997). Even so, the energetic cost of mechanical work likely remains the greater fraction of total cardiac ATP expenditure because myofibrillar volume density of cardiomyocytes is similar for both fish and mammals (Santer, 1985; Aho and Vornanen, 1997). An additional contributing factor to anoxic cardiac failure is the accumulation of protons from anaerobic metabolism (acidosis). Acidosis dramatically decreases the ability of the heart to pump blood by reducing contractile force and promoting fatal ventricular arrhythmias (Williamson et al., 1976; Gesser and Jørgensen, 1982; Orchard and Kentish, 1990). Obviously, the crucian carp brain and heart constitute striking exceptions to those of most vertebrates, as these organs continue to function for days to months in anoxia. Such a feat is possible because the hypoxia- and anoxiatolerance of crucian carp depend on both (1) innate or constitutively expressed traits, e.g., ethanol production and gill remodeling, that can be recruited any time when oxygen shortage sets in (these mechanisms are described in section 2), and (2) induced traits, e.g., glycogen content of various tissues and remodeling of cardiac function, which are seasonally primed by environmental cues and set the ultimate limits for anoxia tolerance (the latter are described in Section 3). 2. MECHANISMS OF HYPOXIC AND ANOXIC SURVIVAL 2.1. Ethanol Production The crucian carp has the exotic ability to produce ethanol as the major end-product of anaerobic metabolism. Among vertebrates, this trait has only been found in two close relatives: the goldfish (Carassius auratus) and the bitterling (Rhodeus amarus) (Shoubridge and Hochachka, 1980; Mourik et al., 1982; Johnston and Bernard, 1983; Holopainen et al., 1986; Nilsson, 1988; 402 MATTI VORNANEN ET AL. Brain and other organs Glucose Glucose Lactate Blood Lactate Muscle LDH Pyruvate PDH Acetaldehyde ADH Ethanol Mitochondria Excretion through the gills Fig. 9.3. The ethanol-producing pathway in anoxic crucian carp and goldfish. Ethanol is only produced in muscle tissue, while all other organs produce lactate during anoxia. In muscle, lactate is turned into pyruvate by lactate dehydrogenase (LDH). Pyruvate is further converted to acetaldehyde by pyruvate dehydrogenase (PDH), which in contrast to other vertebrates leaks out acetaldehyde during anoxia. The acetaldehyde is turned into ethanol by alcohol dehydrogenase (ADH), an enzyme that only occurs in muscle of crucian carp and goldfish. Ethanol readily passes biological membranes, and after it leaks out into the blood it will leave the fish over the gills. In this way, a buildup of the anaerobic end-product is avoided. Wissing and Zebe, 1988). The enzyme system used to produce ethanol is confined to skeletal muscle (red and white) and is a three-step process. Lactate is turned into pyruvate by lactate dehydrogenase (LDH), pyruvate is turned into acetaldehyde by Enzyme 1 (also called pyruvate decarboxylase) of the pyruvate dehydrogenase (PDH) complex, and acetaldehyde is turned into ethanol by alcohol dehydrogenase (ADH) (Figure 9.3). Only the skeletal muscle of crucian carp and goldfish contains ADH, so all other tissues, including the brain and heart, will produce lactate in anoxia, which has to be transported in the blood to the muscle, where it is transformed to ethanol. The ethanol, which readily passes through cellular membranes, quickly diVuses out into the blood for transport to the gills through which it easily diVuses into the 9. ANOXIA-TOLERANT CRUCIAN CARP 403 ambient water. Consequently, provided internal convection continues, the level of ethanol in blood does not rise high enough to significantly suppress nervous activity (the steady state level is below 10 mM; Van Waarde et al., 1993). The unusual distribution of ADH in crucian carp (all in muscle and none in liver) is retained also during the summer, when the crucian carp is unlikely to encounter anoxia (Nilsson, 1990a). While the ethanol production allows the crucian carp to endure long-term anoxia without suVering lactic acidosis, it has a clear energetic drawback: ethanol, a very energy-rich hydrocarbon is released to the water and forever lost. Therefore, to allow for long-term survival in anoxia, the crucian carp accumulates enormous glycogen stores prior to the winter months (see Section 3.1) and the only factor that appears to limit its anoxia endurance is the total depletion of the main glycogen store in the liver (Nilsson, 1990b). In contrast, in animals where lactate is accumulated, it can later either be used as a fuel or used to synthesize glycogen when O2 becomes available (if the animal survives the anoxic episode). It is likely that the ability of the crucian carp to sustain a considerable neural, cardiac, and physical activity during anoxia is closely linked to its ability to produce ethanol as the major end-product of anaerobic metabolism, allowing it to avoid self-poisoning from production of lactate and the associated H+. Freshwater turtles, the other well-studied example of anoxia-tolerant vertebrates, cannot produce any other anaerobic endproduct than lactate, which forces them to resort to a drastic depression of energy metabolism in anoxia to reduce lactate production as much as possible. Still, lactate levels as high as 200 mM may be reached in turtles, which they need to buVer with calcium carbonate from their bones and shell (Jackson, 2002). It is highly unlikely that any fish would be able to tolerate such high lactate levels as they, as water breathers, have relatively low CO2 levels in their tissues, and therefore a low pH buVering capacity. Thus, ethanol production is probably a key prerequisite for long-term anoxic survival in crucian carp. 2.2. Gill Remodeling Even if the crucian carp can survive without any oxygen, the steady depletion of glycogen stores and loss of the energy-rich ethanol is costly. Thus, getting access to oxygen is highly desirable from an energetic point of view. The crucian carp can reversibly adjust the morphology of its gills to match its oxygen needs (Figure 9.4) (Sollid and Nilsson, 2006; Nilsson, 2007). Sollid et al. (2003) showed that normoxic crucian carp kept in relatively cold water (20 C or less) have gills that lack protruding lamellae. But when the crucian carp is exposed to hypoxia, it starts remodeling the gills, resulting in a 7-fold increase in the respiratory surface area and reducing diVusion 404 MATTI VORNANEN ET AL. Fig. 9.4. Scanning electron micrographs of gill filaments from crucian carp kept in normoxic water at 8 C (A–B), in hypoxic water at 8 C (C), or in normoxic water at 25 C (D). Scale bars are 150 mm in A and 50 mm in B–D. [Data from (Sollid et al., 2003, 2005a).] distances, thereby boosting the ability to take up oxygen. The lamellae are actually present in normoxic crucian carp, but they are covered by a nonvascularized cell mass situated outside the gill epithelium that completely occupies the space between the gill lamellae. The mitotic and apoptotic activity in this interlamellar cell mass (ILCM) varies with ambient oxygen levels (Sollid et al., 2003). Thus, mitosis dominates in normoxic water, causing ILCM to fill up the interlamellar space, while in hypoxic water, apoptosis prevails and the ILCM nearly disappears, exposing the respiratory epithelium to the water. Goldfish (Carassius auratus) also have the same capacity for remodeling the gills when kept at low temperature (7 C) (Sollid et al., 2005a). In addition to hypoxia, increasing the water temperature to 25 C causes gill remodeling (within hours) in crucian carp (Figure 9.4D). This indicates that an elevated oxygen demand is a key trigger for the remodeling process (Sollid et al., 2005a). In contrast, a total absence of oxygen does not induce the remodeling, which makes functional sense since there is no oxygen to take up (Sollid et al., 2005b). The mechanistic reason for this may be that the apoptosis needed to remove the ILCM could be oxygen dependent. 9. ANOXIA-TOLERANT CRUCIAN CARP 405 With regard to the molecular signals involved in the gill remodeling, little is known. Although hypoxia-inducible factor-1a (HIF-1a) increases in hypoxic crucian carp gills (coinciding with a reduction in the ILCM), the level of this transcription factor is also increased by a fall in temperature (which coincides with increased ILCM) (Rissanen et al., 2006; Sollid et al., 2006). This makes an involvement of HIF-1a less likely. 2.3. Cardiorespiratory Adjustments In addition to remodeling the gills, crucian carp utilize other strategies to maintain oxygen transfer at the gills and tissues in the face of declining oxygen availability. Foremost, crucian carp hemoglobin has an extremely high oxygen aYnity at high temperature (P50 = 1.8 mmHg at pH 7.7 and 20 C) that increases markedly with decreasing temperature (P50 = 0.8 mmHg at 10 C; Sollid et al., 2005a). Further, like other fish exposed to hypoxia, carp immediately hyperventilate to maximize oxygen uptake. With the onset of anoxia, ventilation frequency (fR) nearly doubles (Stecyk et al., 2004b) (Figure 9.5). Concurrently, ventilation amplitude also increases (J. A. W. Stecyk, K- O. Stensløkken, G. E. Nilsson, unpublished observation). With prolonged anoxia exposure, fR returns to the control normoxic rate. The immediate cardiovascular response of most fish to oxygen deprivation is bradycardia (slowing of heart rate; fH) (Farrell, 2007), and the crucian carp is no exception in this regard (Vornanen 1994a; Vornanen and Toumennoro 1999; Stecyk et al., 2004b)(Figure 9.5). Hypoxic bradycardia may benefit oxygen uptake at the gills, although supporting evidence is equivocal in other fish species, and is believed to aVord a number of direct benefits to the heart, such as increasing the residence time of blood in the heart lumen, therefore allowing a greater time for oxygen diVusion, and improving cardiac contractility through the negative force-frequency eVect (see Farrell, 2007 for review). However, the bradycardia in crucian carp is transitory. By 48 h of anoxia at 8 C, cardiac output (Q), fH, cardiac power output (PO) and cardiac stroke volume all return to control normoxic levels where they remain stable for at least 5 days (Stecyk et al., 2004b). Concurrently, ventral aortic blood pressure and peripheral resistance decrease by 30% and 40%, respectively, signifying vasodilation in peripheral tissues. The ability of crucian carp to maintain cardiovascular status at normoxic levels during prolonged anoxia is unique among the vertebrates, and the normal Q during anoxia has been proposed (Stecyk et al., 2004b; Farrell and Stecyk, 2007) to be perhaps essential for shuttling ethanol to the gills for excretion and rapidly distributing glucose from the crucian carp’s large liver glycogen stores (Holopainen and Hyvärinen, 1985; Hyvärinen et al., 1985) to metabolically active tissues. Likewise, the peripheral vasodilation during anoxia 406 MATTI VORNANEN ET AL. A Power output (% Control) 150 Cardiac output (% Control) 175 150 125 100 75 50 B 125 100 75 50 Heart rate (min−1) C 20 15 10 5 Stroke volume (% Control) D 175 150 125 100 75 Ventral aortic blood pressure (kPa) 2.5 Respiration rate Peripheral resistance (min−1) (% Control) E 0.0 F G 2.0 1.5 1.0 * * * * 0.5 * * * * 125 100 75 50 30 ** * 15 0 0 5 10 15 20 40 60 80 100 120 Anoxia exposure time (h) Fig. 9.5. Chronological changes of cardiorespiratory status in 8 C-acclimated crucian carp during 5 days of anoxia exposure. (A) Cardiac power output, (B) cardiac output, (C) heart rate, (D) stroke volume, (E) ventral aortic blood pressure, (F) peripheral resistance, and (G) respiration rate. Dashed lines indicate the control normoxic level for each measured parameter. 9. ANOXIA-TOLERANT CRUCIAN CARP 407 may reflect an increased perfusion of skeletal muscle where the conversion of lactate to ethanol takes place. The cardiovascular responses of the crucian carp to anoxia are regulated by the autonomic nervous system. Cardiac inhibitory cholinergic and excitatory b-adrenergic tones, as well as a tonic a-adrenergic vasoconstriction have been revealed in anoxic crucian carp by injections of pharmacological blockers (Vornanen and Toumennoro, 1999; Stecyk et al., 2004b). However, it remains to be determined if autonomic cardiovascular control persists beyond 5 days of anoxia and at colder acclimation temperatures. Thus, autonomic cardiovascular controls remain intact in the crucian carp during anoxia exposure at both warm- and cold-acclimation temperatures, consistent with its brain remaining functional (Lutz and Nilsson, 1997; Nilsson, 2001). In comparison, autonomic cardiovascular control is blunted in 5 Cacclimated anoxic turtles, which suppress brain activity in anoxia (Hicks and Farrell, 2000; Stecyk et al., 2004a) (see Section 2.5). 2.4. The Heart in Anoxia 2.4.1. Balancing ATP Supply and Demand The crucian carp’s ability to maintain cardiac activity during anoxia may be possible because the cardiac ATP demand of their routine PO has been suggested to be below the maximum glycolytic potential (i.e., ATP supply) of ectothermic vertebrates (Farrell and Stecyk, 2007). In other words, during anoxia, anaerobic ATP generation is suYcient to power the heart to pump as in normoxia. This cardiac ATP conservation strategy comes about through a low arterial blood pressure compared to other teleosts, including another cyprinid fish, the common carp (Cyprinus carpio) (Farrell and Jones, 1992; Stecyk and Farrell, 2006). Also important for limiting cardiac activity and thus ATP demand is the low temperature during cold wintertime anoxia and inverse thermal acclimatization that prepares the heart for winter anoxia (see Section 3.2). If it is that the crucian carp does not necessarily need to reduce cardiac ATP demands during anoxia to match energy use to supply, it is not surprising that anoxic channel arrest is not a strategy utilized by anoxic crucian carp. Channel arrest is a hypothesized energy-conserving strategy, first proposed by Lutz et al. (1985) and Hochachka (1986), in which the number and/or open probability of functional ion channels is reduced with either oxygen limitation or low temperature to diminish the metabolic cost of For cardiac variables, significant diVerences (asterisks) are only indicated between normoxic control (time zero) and hours 48, 72, 96, and 120. For respiration (ventilation) rate, all significant diVerences from the normoxic control are indicated. Values are means  S.E.M. from 6 to 18 fish. [Adapted from Stecyk et al. (2004b).] 408 MATTI VORNANEN ET AL. ion pumping to maintain ion gradients. For the heart, channel arrest could primarily be expected to involve decreases in Na+ current and K+ currents, which would then reduce demands by the Na+/K+-ATPase, and a decrease in Ca2+ current, which would reduce demands by Ca2+-ATPases and Na+/K+ATPase. Specifically, reducing Na+ inflow during an action potential upstroke means that less Na+ has to be extruded afterwards by Na+/K+ATPase. Similarly, reduced Ca2+ influx means that less Na+ is needed to extrude Ca2+ via the Na+/Ca2+-exchanger. K+ channels represent a K+ leakage pathway across the sarcolemma, allowing for continuous K+ eZux during diastole and/or systole and placing continuous demands on the Na+/ K+-ATPase (Roden et al., 2002). Therefore, a down-regulation of K+ channels would limit K+ leakage and also lower ATP demand. However, it has been discovered that crucian carp cardiac electrophysiology is largely unaVected by severe hypoxia and anoxia (reviewed by Stecyk et al., 2008). Thus, the channel arrest hypothesis does not appear to be valid for the crucian carp heart. Specifically, the amplitude and kinetics (whole-cell conductance, single-channel conductance, and open probability) of ventricular inward rectifier K+ current are unaVected after 4 weeks of severe hypoxia exposure (<0.4 mg O2 L-1) at 4 C (Paajanen and Vornanen, 2003). Even so, sarcolemmal Na+/K+-ATPase activity is reduced by one third within 4 days of anoxia exposure at 4 C as well as with the onset of hypoxic conditions in the natural environment (Aho and Vornanen, 1997). This change likely conserves ATP, but is at odds with the channel arrest hypothesis because it is not accompanied by a concomitant reduction of K+ current. Further, the number of ventricular L-type Ca2+ channels and the density of Ca2+ current do not change with the seasonal decrease in water oxygen content (Vornanen and Paajanen, 2004). Therefore, cardiac down-regulation of L-type Ca2+ channels is not triggered by seasonal anoxia in the natural environment. 2.4.2. Cardiac Protection Against Anoxia In the vertebrate heart, several molecular mechanisms have evolved to protect the heart against hypoxic or ischemic insults. These mechanisms include opening of the ATP-sensitive K+ channels in sarcolemma and mitochondria of cardiac myocytes and release of adenosine, a general negative feedback regulator of cardiac function (Mubagwa and Flameng, 2001; Zingman et al., 2007). 2.4.2.1. ATP-sensitive K+ channels. ATP-sensitive K+ channels are formed as heteromultimers of inwardly rectifying K+ channel Kir6.2 and sulfonurea receptors SUR2A and provide cardiac protection during metabolic stress by virtue of the direct coupling between channel opening and myocyte energy balance. When cellular phosphorylation potential is high, ATP-sensitive K+ channels are kept closed by ATP binding to the Kir6.2 proteins, whereas under metabolic stress the intrinsic ATPase of the SUR2A 9. ANOXIA-TOLERANT CRUCIAN CARP 409 hydrolyses ATP and subsequent Mg-ADP binding to SUR2A overrides the inhibition of ATP on the Kir6.2 (Nichols, 2006). Binding of ATP to the Kir6.2 proteins occurs at two orders of magnitude lower ATP concentrations (Kd = 10–30 mM) than is the bulk ATP concentration (6–10 mM) of wellenergized cells. Therefore, a creatine kinase and adenylate kinase systems are needed to couple energy state of the myocyte to the opening of ATP-sensitive K+ channels in the diVusion-restricted subsarcolemmal space (Zingman et al., 2007). ATP-sensitive K+ current shortens the duration of cardiac AP, and thereby limits sarcolemmal Ca2+ influx and reduces cardiac contractility, which results in energy savings and possible rescue of cardiac myocytes from the anoxic cell death. ATP-sensitive K+ channels also play a role in cardiac preconditioning, a process where short periods of ischemia provide cardiac protection against subsequent ischemic insults or reperfusion injury. It might be expected that in anoxia-tolerant vertebrates, cardiac protection by ATP-sensitive K+ channels would be particularly well developed in order to allow survival under severe hypoxic and anoxic stress. However, comparison of the cardiac ATP-sensitive K+ current in several vertebrate species indicates almost the opposite. Density of the ATP-sensitive K+ current is much larger in a mammal and a land-dwelling lizard than in an aquatic frog and two fish species (Paajanen and Vornanen, 2002). Among six vertebrate species spanning a wide range of hypoxia tolerance, crucian carp had the second smallest ATP-sensitive K+ current, with only rainbow trout having a smaller current (Figure 9.6). The relatively small current is not, however, a limiting factor for cardiac protection, since it still clearly exceeds the densities of other outward K+ currents and is suYcient to shrink the AP duration to almost nil (Paajanen and Vornanen, 2004). More importantly, relatively extreme measures are needed to open ATP-sensitive K+ channels in isolated cardiac myocytes from crucian carp, especially in myocytes from cold-acclimated (4 C) fish. In warm-acclimated fish (18 C), inhibition of the aerobic metabolism with an oxygen scavenger, 0.1 mM Na2S2O3, is suYcient to activate the ATP-sensitive K+ current, whereas in ventricular myocytes of cold-acclimated fish, inhibition of both aerobic and anaerobic (5 mM iodoacetate) metabolism is necessary to induce the current. In excised crucian carp hearts, blocking of the aerobic metabolism with cyanide increases the duration of contraction and prolongs ventricular AP, indicating that ATP-sensitive K+ channels are not opened in this multicellular preparation either (Vornanen and Tuomennoro, 1999). However, a small (15.1%) shortening of AP has been recorded in hypoxic ventricular myocytes of the warmacclimated (21 C) goldfish, a close relative to crucian carp, and the opening was prevented by an inhibitor of nitric oxide synthesis, L-NAME (Cameron et al., 2003; Chen et al., 2005). Whether this represents a real diVerence in regulation of ATP-sensitive K+ channels between closely related species or is caused by diVerences in experimental conditions, is not clear. 410 MATTI VORNANEN ET AL. IK,ATP (nS/pF) 1.6 1.2 0.8 R. norv. R. temp. IK,ATP (pA/pF) L. vivip. R. norvegicus 0.4 0.0 X. laevis wa C.carass. ca O.mykiss 150 R. temporaria wa O.mykiss ca C. carass. L. vivipara 50 X. laevis ca C. carassius wa C. carassius ca O. mykiss mV −90 −60 30 −30 wa O. mykiss −50 Fig. 9.6. The size of ATP-sensitive K+ current (IK,ATP) in ventricular myocytes of six vertebrate species. Right side of the figure shows linear current–voltage relations when metabolism of the cells was compromised with 0.1 mM Na2S2O3 and/or 5 mM iodoacetate. Mean ( S.E.M.; N ¼ 4–27) conductance of the ATP-sensitive K+ current for the six species is shown on the left. The species are: rat (Rattus norvegicus), frog (Rana temporaria), clawed frog (Xenopus laevis), European common lizard (Lacerta vivipara), crucian carp (Carassius carassius) and rainbow trout (Oncorhynchus mykiss). The abbreviations wa and ca denote warm-acclimated and coldacclimated, respectively. [Data from (Paajanen and Vornanen 2002).] The high resistance of crucian carp ATP-sensitive K+ channels to opening is even more surprising considering their regulatory properties. ATP-sensitive K+ channels open when MgADP binding to the SUR2A overrides ATPdependent inhibition of the Kir6.2 channels. In crucian carp and goldfish hearts, ATP-sensitive K+ channels have a very low aYnity to ATP (Kd = 1.35–1.85 mM in crucian carp), and therefore relatively small changes in cellular ATP concentration would be expected to remove ATP-dependent inhibition without SUR2A ATPase activity (Ganim et al., 1998; Paajanen and Vornanen, 2004). The general ability of anoxia-tolerant organisms to maintain high [ATP]i in anoxia does not explain the resistance to opening, since intracellular perfusion of myocytes with a ATP-free solution cannot open the ATP-sensitive K+ channels in crucian carp ventricular myocytes. These findings suggest that bulk ATP concentration alone is insuYcient to signal the opening of ATP-sensitive K+ channels even though ATP aYnity of the channel is low. Current evidence suggests that the ATP-sensitive K+ channels of the crucian carp heart are not primarily involved in anoxia protection, but rather associated with cardioprotection against severe heat stress (Ganim et al., 1998; Paajanen and Vornanen, 2004). 2.4.2.2. Adenosine. In many animals, various states of energy deficiency, including hypoxia, quickly results in increased levels of adenosine as a result of a net breakdown of phosphorylated adenosines (ATP, ADP, and AMP), and it is 9. ANOXIA-TOLERANT CRUCIAN CARP 411 well established that both the rise in adenosine and the fall in ATP can initiate an array of mechanisms aimed at restoring energy levels (Lipton, 1999). In mammals, adenosine is able to balance cardiac function under energy-limited conditions by reducing work load of the heart and improving glycolytic energy supply through stimulation of cellular glucose uptake (Mubagwa et al., 1996). Adenosine is released in the circulation when oxygen demand of the cardiac muscle exceeds circulatory supply of oxygen, e.g., in hypoxia and anoxia. Adenosine improves blood flow in the coronary vessels by vasodilatation, reduces the velocity of impulse conduction over the heart, and weakens contractile force of atrial and ventricular myocardia (Mubagwa et al., 1996). By these actions, adenosine is thought to rebalance energy demand and supply, thereby providing protection for the heart during hypoxia. The significance of adenosinergic control of the heart under oxygen deficient conditions is still poorly documented in fish and especially the mechanisms of action are largely unexplored (Rotmensch et al., 1981; Meghji and Burnstock, 1984; Lennard and Huddart, 1989; Sundin et al., 1999; MacCormack and Driedzic, 2004). In the crucian carp heart, adenosine is a weak modulator of cardiac function and unlikely to have any major role in anoxia protection of the heart (Vornanen and Tuomennoro, 1999; Stecyk et al., 2007). Adenosine at the concentration of 0.1 mM or lower has no eVect on contractile function of isolated atrial and ventricular muscle of the crucian carp heart, and 1 mM adenosine causes a small increase of force in ventricular muscle and a small decrease of force in atrial muscle (Vornanen and Tuomennoro, 1999). Electrophysiological eVects of adenosine are often mediated by activation of the ligand-gated inward rectifier K+ channels, which generate an outward K+ current (IKAdo) that shortens the duration of cardiac AP, especially in atrial myocytes (Belardinelli et al., 1995). Adenosine neither aVects cardiac AP nor activates IKAdo in crucian carp atrial and ventricular myocytes at a concentration of 0.1 mM, which is an eVective dose in the hypoxia-sensitive trout heart (Vornanen and Tuomennoro, 1999; Aho and Vornanen, 2002). Consistent with the findings from multicellular cardiac preparations and cardiac myocytes, intra-arterial injection of aminophylline, an adenosine receptor blocker, to 5-day anoxic crucian carp did not change their cardiovascular status (Stecyk et al., 2007). 2.5. The Brain in Anoxia 2.5.1. Suppression of Brain Activity A fundamental factor in the anoxic survival strategy of both the crucian carp and the freshwater turtles is their remarkable ability to maintain brain ATP levels when exposed to anoxia (Figure 9.7) (Lutz et al., 1984; Johansson 412 MATTI VORNANEN ET AL. 3 [AMP] Total adenylate phosphates [ADP] Energy charge [ATP] 2 2 1 1 0 Energy charge Concentration (µmol . g−1) 3 0 Normoxia in situ Brain slice 20 h in O2 Brain slice 20 h in N2 Fig. 9.7. Energy charge is maintained in the anoxic crucian carp brain. The graph shows levels of ATP, ADP, AMP, total adenylate phosphates (ATP+ADP+AMP) and energy charge (EC) in crucian carp brain and in brain slices (telencephalon) kept for 20 h in normoxic or anoxic Ringer at 12 C. EC = ([ATP] + ½ [ADP])/([ATP] + [ADP] + [AMP]). Values are means  S.E.M. from 6 fish. [Reproduced from (Johansson et al., 1995).] and Nilsson, 1995; Johansson et al., 1995), whereby many of the detrimental processes initiated by anoxia in other vertebrate brains are avoided. However, the crucian carp and the turtles seem to diVer in the way they achieve this. For one, the turtles become virtually comatose in anoxia, with a brain that is nearly completely electrically silent (Fernandes et al., 1997). The accompanying deep metabolic depression is thought to be the major factor enabling the turtle brain to maintain its energy charge (Nilsson and Lutz, 2004; Bickler and Buck, 2007). By contrast, the crucian carp must uphold much of its brain functions since it remains physically active in anoxia (Nilsson et al., 1993b; Nilsson, 2001), although the activity level is reduced and senses like hearing and vision have been found to be suppressed in goldfish and crucian carp (Suzue et al., 1987; Johansson et al., 1997). Also, whole body metabolism, measured as heat production, is more suppressed in the turtle than in the crucian carp. Wholebody metabolic rate of warm-acclimated turtles (20 C–24 C) is depressed to 15–18% of the normoxic metabolic rate during prolonged anoxia exposure (Jackson, 1968; Herbert and Jackson, 1985). For cold-acclimated turtles 9. ANOXIA-TOLERANT CRUCIAN CARP 413 (3 C), the decreases in metabolic rate is even greater, reaching values less than 10% of the normoxic metabolic rate at 12 weeks of anoxia exposure (Herbert and Jackson, 1985). No such measurements have been done in crucian carp, but in the closely related goldfish there is only a 70% reduction in heat production during 3 h of anoxia at 20 C (Van Waversveld et al., 1989). With regard to the crucian carp brain, microcalorimetric measurements on brain slices have indicated a modest 30% reduction in ATP turnover during anoxia, and that the anoxic crucian carp brain has to increase its glycolytic rate 3-fold to defend its ATP levels (Johansson et al., 1995). Measurements of the Na+/K+-ATPase activity in the brain of turtles and crucian carp also point toward a more severe energetic depression in turtles. Na+/K+-ATPase is the major ATP consumer in the brain and is responsible for establishing the electrochemical gradients of Na+ and K+ across the plasma membrane, which are necessary for negative resting membrane potential, electrical excitability, neurotransmitter uptake, and osmotic balance of neuronal cells. For example, in the mammalian brain, 50–80% of the total energy budget is devoted to the Na+/K+-ATPase (Erecinska et al., 2004). Therefore, a depression of Na+/K+-ATPase activity would lead to ATP conservation. After 24 h of anoxia at 20 C, a 30% fall in the activity of Na+/K+-ATPase is seen in major parts of turtle brain (Figure 9.8) (Hylland et al., 1997). Conversely, anoxia exposure does not decrease the number of Na+/K+-ATPase alpha subunits or the activity of this enzyme in the crucian carp brain (Figure 9.8) (Hylland et al., 1997; Vornanen and Paajanen, 2006). In support of these findings, determinations of [3H]ouabain binding and Na+/K+-ATPase activity in fish caught directly from the wild around the year have not indicated a depression of the Na+/K+-ATPase activity during the anoxic season (Vornanen and Paajanen, 2006). Another indication of maintained brain activity in anoxia is the sustained doubling in brain blood flow, starting within the first minutes of anoxia and lasting at least over a 6 h period of anoxia at 10 C (Nilsson et al., 1994). The increased brain blood flow is probably needed to shuttle glucose to, and lactate from, the brain to support the increased glycolytic activity. In turtle brain, blood flow also doubles initially in anoxia but falls back to preanoxic levels within the first hours of anoxia (Hylland et al., 1994; Stecyk et al., 2004a), which probably corresponds to the entrance into deep neural suppression and a reduced need for glucose supply. 2.5.2. Mechanisms of Neural Depression in Anoxia 2.5.2.1. Ion channels and protein synthesis. The diVerences displayed by crucian carp and turtles in physical and metabolic activity are also reflected in the mechanisms employed to suppress brain energy demands in anoxia. A major diVerence appears to be the utilization of channel arrest. This 414 MATTI VORNANEN ET AL. 7 Normoxia 6 Crucian carp Anoxia 5 Recovery Na/K-ATPase activity (µmol min−1 g−1) 4 3 2 1 0 4 Normoxia Trachemys turtle Anoxia 3 Recovery 2 * * 1 0 Telencephalon + Cerebellum Brain stem + Fig. 9.8. Brain Na /K -ATPase activity is maintained in anoxic crucian carp but not turtle. Both species were exposed to 24 h of anoxia at 20 C followed by 24 h of reoxygenation. Values are means  S.E.M. from 6 to 7 animals. [Reproduced from (Hylland et al., 1997).] mechanism appears to play a major role in suppressing neural excitability and ATP use in turtles (Bickler and Buck, 2007), but, like for the crucian carp heart (see Section 2.4), there is so far very little evidence that brain channel arrest plays any major role in the anoxia tolerance of crucian carp. In anesthetized crucian carp, brain K+ permeability appears to be unaVected by anoxia, as measured by the eZux of K+ to the extracellular space when Na+/K+ATPase is blocked with ouabain (Johansson and Nilsson, 1995). Crucian carp brain slices also display a similar lack of change in Ca2+ permeability, at least during the initial hours of anoxia (Nilsson, 2001). However, there are some indications of a limited "channel arrest" in crucian carp brain: measurements of the expression of excitatory neurotransmitter receptors show that most receptors are relatively unaVected by anoxia, but that mRNA levels of certain NMDA receptor subunits, including the ubiquitous NR1 subunit, are 9. ANOXIA-TOLERANT CRUCIAN CARP 415 depressed by about 50% after a week of anoxia at 10 C (Ellefsen et al., 2008). Moreover, a recent study utilizing the whole-cell patch clamp technique on telencephalic brain slices from goldfish show that acute (40 min) anoxia exposure causes a 40–50 % fall in the NMDA receptor activity (Wilkie et al., 2008). The NMDA receptor is a major glutamatergic receptor with a large conductivity for Ca2+, and such changes could function to reduce neural excitability. Indeed, there is good evidence for a reduced NMDA receptor function in the turtle brain (Bickler and Buck, 2007). However, studies of ion channel gene expression in crucian carp have also revealed that mRNA levels of various subunits of voltage-gated Na+ and Ca2+ channels are maintained, or even increased, in the brain of crucian carp kept in anoxia for a week (Ellefsen et al., 2008). These voltage-gated channels are responsible for the generation of action potentials and neurotransmitter release and could therefore be important targets for a channel arrest strategy involving reduced gene expression, but apparently such a strategy is not utilized by the crucian carp. Like for the heart, a possible explanation for the absence of any major channel arrest in crucian carp brain is oVered by the low metabolic rate induced by the low temperature of the anoxic season (see Section 3.3), combined with the fact that the ethanol-producing pathway relieves the crucian carp of the problem of having to minimize lactate accumulation. With regard to protein synthesis in crucian carp, measurements utilizing [3H] phenylalanine incorporation have revealed maintained rates in brain, as opposed to liver (>95% suppression), muscle (50% suppression), and heart (50% suppression) (Figure 9.9) (Smith et al., 1996). This unequal suppression of protein synthesis makes sense since only about 3% of the energy used by the crucian carp brain goes to protein synthesis, so not much would be saved from shutting it down. By contrast, the extreme suppression of protein synthesis in the crucian carp liver could make a significant contribution to energy savings on the whole body level, as protein synthesis can make up more than 50% of the energy use of the fish liver (Smith et al., 1996). However, apparently the freshwater turtle has again turned to a more radical strategy as its rate of protein synthesis in all tissues, including brain, is virtually at a halt in anoxia (Fraser et al., 2001). 2.5.2.2. Neurotransmitters and neuromodulators. Presently, most evidence points toward neurotransmitters and neuromodulators as responsible for suppressing the electric activity of the anoxic crucian carp brain. The major inhibitory neurotransmitter in the vertebrate brain is gamma-aminobutyric acid (GABA), and microdialysis studies on anesthetized crucian carp show that extracellular [GABA] rises in the brain (telencephalon) during anoxia (Hylland and Nilsson, 1999). At the same time, extracellular glutamate levels remain low (Hylland and Nilsson, 1999), which of course makes the 416 MATTI VORNANEN ET AL. 1.5 15 Normoxia 1 10 * 0.5 5 * * Protein synthesis rate (%/day) (liver) Protein synthesis rate (%/day) (all tissues except liver) Anoxia 48 h * 0 Liver White muscle Red muscle Heart Brain 0 Fig. 9.9. Rates of protein synthesis in vivo in various tissues of crucian carp exposed to anoxia for 48 h at 9 C. Values are means  S.E.M. from 12 to 16 fish. [Data from (Smith et al., 1996).] anoxic crucian carp brain strikingly diVerent from anoxic mammalian brains, and also diVerent from the anoxic rainbow trout brain (Hylland et al., 1995), which all show substantial rises in extracellular glutamate. However, compared to the massive (80-fold) increase in extracellular [GABA] seen in the anoxic turtle brain (Nilsson and Lutz, 1991), the rise in extracellular [GABA] in the crucian carp brain (telencephalon) is relatively modest: on average it is doubled after 6 h of anoxia (Figure 9.10) (Hylland and Nilsson, 1999). There is also a considerable individual variation in the extracellular [GABA] rise during anoxia, varying from no change to a 6-fold increase, which indicates that the release of GABA is fine-tuned to the need for neural suppression. The most direct evidence for a role of GABA in metabolic depression comes from studies using inhibitors of the GABA synthesizing enzyme glutamate decarboxylase (isoniazid or 3-mercaptopropionic acid), or a 9. 417 ANOXIA-TOLERANT CRUCIAN CARP 250 Start of anoxia Extracellular concentration (% of normoxic level) 300 200 GABA 150 100 Glutamate 50 –100 0 100 200 Time (min) 300 400 Fig. 9.10. Changes in the extracellular brain levels of GABA and glutamate in crucian carp brain, measured with a microdialysis probe placed in the telencephalon of anesthetized fish kept at 10 C. Values are means  S.E.M. from 6 to 8 fish. [Data from (Hylland and Nilsson, 1999).] blocker of GABAA receptors (securinine). Such manipulations make crucian carp release up to three times more ethanol to the water during anoxia (while normoxic oxygen consumption is unaVected), suggesting a profound inhibition of metabolic depression (Figure 9.11) (Nilsson, 1992). The crucian carp may not only utilize GABA as a metabolic depressant under normal anoxic conditions, but may also employ a more massive GABA release as a last line of defense during severe neural energy deficiency. In the crucian carp brain (telencephalon), the potential for releasing GABA appears to be higher than for releasing glutamate. By running a high-[K+] Ringer through the microdialysis probe, to depolarize the surrounding tissue, Hylland and Nilsson (1999) found a 14-fold increase in extracellular [GABA], while extracellular [glutamate] was barely doubled. Similarly, when neural ATP levels are forced to fall by superfusing the crucian carp telencephalon with the glycolytic inhibitor iodoacetate, the resultant increase in extracellular [GABA] was found to be both faster and more massive (a 10fold increase after 30 min) than that of extracellular [glutamate] (a 3-fold increase after 2 h) (Hylland and Nilsson, 1999). There is a close metabolic interrelation between GABA and glutamate, which is interesting from an anoxia-defense perspective. GABA is 418 MATTI VORNANEN ET AL. A Isoniazid 500 mg Kg–1 Isoniazid 250 mg Kg–1 Control 3 2 1 0 Rate of ethanol production (mmol kg–1 h–1) B 50 100 150 200 250 300 3.0 2.5 2.0 1.5 1.0 3-mercaptopropionic acid 0.5 Control 0.0 C 0 0 100 200 300 3 2 1 Securinine Control 0 0 50 200 100 150 Time in anoxia (min) 250 300 Fig. 9.11. Evidence for a role of GABA in controlling anoxic metabolic rate. The graphs show the eVects of anti-GABAergic agents on the rate of ethanol release to the water by anoxic crucian carp kept at 18 C. Isoniazid (250–500 mg kg-1 i.p.) and 3-mercaptopropionic acid (200 mg kg-1 i.p.) both block the GABA-synthesizing enzyme glutamate decarboxylase. 3-mercaptopropionic acid also inhibits neuronal GABA release. Securinine (20 mg kg-1 i.p.) blocks GABAA receptors. Values are means  S.E.M. from 4 to 6 fish. [Reproduced from (Nilsson, 1992).] 9. ANOXIA-TOLERANT CRUCIAN CARP 419 synthesized from glutamate in a single oxygen-independent step, catalyzed by glutamate decarboxylase. In contrast, both the synthesis of glutamate and the breakdown of GABA depend on oxygen-dependent processes that will stop in anoxia. As a result, brain tissue shows a steady increase in [GABA] and a corresponding fall in [glutamate] during anoxia (Siesjö, 1978; Nilsson and Lutz, 1993). Interestingly, GABA is the major inhibitory neurotransmitter and glutamate the major excitatory neurotransmitter in all vertebrates as well as many invertebrates (Gerschenfeld, 1973; Usherwood, 1978; Koopowitz and Keenan, 1982; McGeer and McGeer, 1989; Restifo and White, 1990). It has been hypothesized that hypoxia could be the underlying selection pressure that is responsible for maintaining GABA inhibitory and glutamate excitatory throughout animal evolution, because it provides a system where a fall in oxygen will automatically make the inhibitory neurotransmitter levels rise and the excitatory fall, thereby inducing and maintaining hypoxic metabolic depression (Nilsson and Lutz, 1993). In the goldfish, which, as mentioned, appears to be somewhat less well adapted to anoxia than the crucian carp (a possible side eVect of long domestication), elevated extracellular [glutamate] has been seen in brain during anoxia, probably as a consequence of a poorly maintained ATP level (Van Ginneken et al., 1996). In this species, a release of glutamate during energy deficiency may initiate protective mechanisms mediated by one class of glutamate receptors, the group II metabotropic glutamate receptors (Poli et al., 2003). 2.5.2.3. Adenosine. The role of adenosine in protecting the anoxic brain has also been investigated in anoxia-tolerant vertebrates. In the turtle (Trachemys) brain there is an almost immediate, substantial rise in extracellular adenosine during the initial phase of anoxia, linked to the simultaneous fall in ATP (Nilsson and Lutz, 1992). However, unlike the turtle, microdialysis experiments have so far failed to detect an increase in extracellular adenosine in the anoxic crucian carp brain (P. Hylland and G. E. Nilsson, unpublished data), but that may reflect limitations of the microdialysis method, because other evidence points at a role for adenosine in both stimulating brain blood flow and reducing metabolic rate in crucian carp during anoxia. First, the sustained doubling of cerebral blood flow in crucian carp is probably adenosine mediated since it can be blocked by superfusing the brain with the adenosine receptor blocker aminophylline (Nilsson et al., 1994) (Also, the temporary elevation in cerebral blood flow seen in turtles appears to be adenosine mediated; Hylland et al., 1994). Second, blocking adenosine receptors with aminophylline in anoxic crucian carp causes a 3-fold increase in the rate of ethanol release to the water (while it is without eVect on normoxic oxygen consumption), suggesting that adenosine causes a significant inhibition of metabolic rate in anoxia (Nilsson, 1991). It could also be mentioned that in goldfish hepatocytes, adenosine has a powerful depressant eVect on 420 MATTI VORNANEN ET AL. both protein synthesis and Na+/K+-ATPase (Krumschnabel et al., 2000), and that adenosine suppresses K+ stimulated Ca2+-dependent glutamate release in goldfish cerebellar slices (Rosati et al., 1995). 3. SEASONALITY OF CRUCIAN CARP PHYSIOLOGY: PREPARING FOR WINTER ANOXIA Although anoxia tolerance of the crucian carp greatly surpasses that of most other vertebrates, it is not a fully fixed trait, but includes an inducible component that varies according to the season (Piironen and Holopainen, 1986). Experiments on seasonally acclimatized fish indicate that anoxia tolerance of adult crucian carp follows an exponential dependence on temperature and extrapolation of the curve to zero temperature suggests a theoretical maximum of 235 days for anoxia tolerance (see Figure 9.1). There are two reasons for the seasonal diVerence in anoxic survival time. Firstly, since metabolic rate of organisms increases with increasing temperature, anoxic survival time of ectothermic animals correlates inversely with ambient temperature. At low temperature, energy stores and essential nutrients last longer and there is less production and accumulation of toxic end-products of metabolism. Secondly, crucian carp metabolism and organ function is altered seasonally to reflect the three main phases in the year of a crucian carp: growth and multiple breeding episodes in early summer, accumulation of energy reserves for winter in late summer, and anoxic overwintering (Holopainen et al., 1997). Thus, crucian carp physiology becomes primed to be beneficial for the survival of winter anoxia. In the habitat of crucian carp, anoxia is a regular and well predictable seasonal condition that is accompanied by several environmental cues, most notably temperature (Nagell and Brittain, 1977; Vornanen and Paajanen, 2004). In fact, changes in oxygen content and temperature occur almost in parallel (Vornanen and Paajanen, 2004; see Figure 9.2). Thus, ambient temperature can function as an environmental cue that entrains the fish for winter anoxia and reoxygenation in spring. 3.1. Seasonal Changes of Glycogen Stores In the absence of molecular oxygen, fats are unsuitable for energy production and the animal must rely on the catabolism of carbohydrate reserves in the body. Consistent with this, lipid content of the crucian carp is low (ca. 2% of wet weight) and the lipid metabolism of the tissues (e.g., liver) is active only for a short period in summer from May to September (Blazka, 1958; Piironen and Holopainen, 1986; Lind, 1992). Further, carp increase the size of glycogen stores in the body in preparation for winter anoxia. 9. 421 ANOXIA-TOLERANT CRUCIAN CARP 3.1.1. Liver and Muscle Glycogen In the vertebrate body, the liver and the skeletal muscle have the largest glycogen reserves. Glucose from liver glycogen can be released to the blood and exploited elsewhere in the body. In muscle tissue, however, glycogen is for local use since muscle lacks the glucose-6-phosphatase needed to release glucose into the circulation. The liver of crucian carp has an exceptional ability to store glycogen, which appears in enormous seasonal changes in the size of liver and glycogen concentration of the liver tissue (Figure 9.12). In winter, the liver constitutes 12–15% of the whole body mass and glycogen concentration of the liver can be 35% of the liver wet weight or 4.5% of the fish body mass (Hyvärinen et al., 1985; Holopainen and Hyvärinen, 1985). In July, when glycogen stores are smallest, the liver mass and glycogen concentration of the liver are 1.5% and 2%, respectively. Thus, there is about a 15-fold seasonal variation in liver glycogen content. White myotomal muscle has similar annual glycogen dynamics as the liver, but the stores are maximally about 4% of the muscle wet weight (Hyvärinen et al., 1985). In winter, liver and muscle glycogen together form about 6% of the fish body mass. Crucian carp begin to prepare for hypoxia/anoxia in late July by accumulating glycogen deposits in the liver. The increase in liver size continues as long as the fish forage. When water temperature drops, and when the hypoxic period sets in, crucian carp begin a fast, which can last almost half a year from November to May (Penttinen and Holopainen, 1992). It is not known whether depletion of glycogen reserves causes mortality in crucian carp 30 14 Glycogen (% of liver wet weight) Liver weight (% of body mass) 12 10 20 8 15 6 10 4 5 0 Liver size (%) Glycogen content (%) 25 2 Apr May Jun Jul Aug Sep Oct Nov 0 Fig. 9.12. Seasonal changes in liver size (% of body mass) and liver glycogen content (% of liver wet weight) of crucian carp. [Data taken from (Holopainen and Hyvärinen, 1985).] 422 MATTI VORNANEN ET AL. populations. It seems that the total glycogen stores of the crucian carp body exceed the metabolic demands of the anoxic fish, as significant amounts of liver glycogen (about 20%) are still present at the end of April when the hypoxic/anoxic period is over. The remaining glycogen stores may represent a surplus of the safety margin, which is subsequently used in preparation for breeding. Indeed, the activity of liver glycogen phosphorylase, a glycogen hydrolyzing enzyme, peaks in May during the maturation of gonads (Holopainen and Hyvärinen, 1985). Thus, the glycogen stores can also be important for successful breeding. Still, in an experiment where crucian carp were starved for 18 days (at 8 C, before the breeding season), they did not utilize the liver glycogen store, apparently saving it for anoxic periods (Nilsson, 1990b). 3.1.2. Brain Glycogen Glycogen stores of the vertebrate brain are usually small giving an impression that the brain tissue is unable to store significant amounts of carbohydrates. High concentrations of glycogen have been found in the lamprey (Petromyzon marinus L.) brain (Rovainen, 1970), indicating that brain glycogen might be an important energy source at least in some animal species. Considering the excellent anoxia tolerance of crucian carp, it is not surprising that their brains store more glycogen than mammalian brains (Schmidt and Wegener, 1988), but that the concentration of brain glycogen is comparable to the glycogen stores of the white skeletal muscle is quite impressive (Vornanen and Paajanen, 2006). The brain glycogen content of the winter-acclimatized crucian carp greatly surpasses brain glycogen stores reported for any other vertebrate species. In frogs and reptiles, including anoxia-tolerant turtles, the concentration of brain glycogen varies between 8 and 18 mmol/g, while in crucian carp the winter average is 204 mmol/g (3.3% of wet brain mass) (Table 9.1). The lamprey brain approaches the crucian carp brain with a value of 137 mmol/g (Rovainen, 1970). Like in its other tissues, the glycogen stores in the crucian carp brain are smallest in June to July and reach a maximum in December to February (Figure 9.13). The diVerence in brain glycogen content between summer and winter, like the liver, is about 15-fold (Vornanen and Paajanen, 2006). The large seasonal variation in the brain glycogen content suggests that glycogen is important for the anoxic survival of the brain. It is unclear, however, whether brain glycogen functions as an immediate energy source when anoxia sets in or whether it is an emergency reservoir that is recruited under prolonged anoxia if the circulation fails to provide suYcient glucose to meet the brain’s demands. In this respect, it is notable that under natural conditions brain glycogen is not used during moderately hypoxic periods but only under total oxygen shortage (Vornanen and Paajanen, 2006). As the Na+/K+-ATPase is Table 9.1 Glycogen concentration (glucosyl units, mmol/g wet weight) in brain, heart and liver of crucian carp in comparison with those of other vertebrates Species Crucian carp, Carassius carassius, in summer Crucian carp, Carassius carassius, in winter Goldfish, Carassius auratus Rainbow trout, Oncorhynchus mykiss Frog, Rana ridibunda Frog, Rana temporaria Turtle, Chrysemys picta belli Turtle, Trachemys (Pseudemys) scripta Rat, Rattus norvegicus Brain Heart Liver References 13 18-86 123 Vornanen & Paajanen, 2004 and 2006; Vornanen, 1994; Hyvärinen et al., 1985 204 493 2160 13-20 0.5 142 25-60 ca 800 110 Vornanen and Paajanen, 2004 and 2006; Vornanen, 1994; Hyvärinen et al., 1985 McDougal et al., 1968; Schmidt and Wegener, 1988; Merrick, 1954 Polakof et al., 2008; Bernier et al., 1996; Gesser, 2002; French et al., 1981 ca 8 18 315 284-413 80-180 156 285 ca 120 300-600 L’vova and Plotnikov, 1978 Donohoe and Boutilier, 1998 Packard and Packard, 2005; Daw et al., 1967; Beall and Privitera, 1973 McDougal et al., 1968; Warren et al., 2006; Warren and Jackson, 2008 2-12 15 68 L’vova and Plotnikov, 1978; Støttrup et al., 2006; Cruz and Dienel, 2002 16 References: Beall, R. J., and Privitera, C. A. (1973). EVects of cold exposure on cardiac metabolism of the turtle Pseudemys (Chrysemys) picta. Am. J. Physiol. 224, 435–441; Bernier, N. J., Fuentes, J., and Randall, D. J. (1996). Adenosine receptor blockade and hypoxia-tolerance in rainbow trout and pacific hagfish. II. EVects of plasma catecholamines and erythrocytes. J. Exp. Biol. 199, 497–507; Cruz, N. F., and Dienel, G. A. (2002). High glycogen levels in brains of rats with minimal environmental stimuli: Implications for metabolic contributions of working astrocytes. J. Cereb. Blood Flow Metab. 22, 1476–1489; Donohoe, P. H., and Boutilier, R. G. (1998). The protective eVects of metabolic rate depression in hypoxic cold submerged frogs. Resp. Physiol. 111, 325–336; French, C. J., Mommsen, T. P., and Hochachka, P. W. (1981). Amino acid utilisation in isolated hepatocytes from rainbow trout. Eur. J. Biochem. 113, 311–317; Gesser, H. (2002) Mechanical performance and glycolytic requirement in trout ventricular muscle. J. Exp. Biol. 293, 360–367; Hyvärinen, H., Holopainen, I. J., and Piironen, J. (1985). Anaerobic wintering of crucian carp (Carassius carassius L.) - I. Annual dynamics of glycogen reserves in nature. Comp. Biochem. Physiol. 82A, 797–803; L0 vova, S. P., and Plotnikov, V. P. (1978). Content of glycogen and glucose in tissues of the frog and some reptiles. Zh. Evol. Biokhim. Fiziol. 14, 400–402; Mandic, M., et al. (2008). Metabolic recovery in goldfish: A comparison of recovery from severe hypoxia exposure and exhaustive exercise. Comp.Biochem.Physiol.C (in press); McDougal, D. B., Jr., et al. (1968). The eVects of anoxia upon energy sources and selected metabolic intermediates in the brains of fish, frog and turtle. J. Neurochem. 15, 577–588; Merrick, A. W. (1954). Cardiac glycogen following fulminating anoxia. Am. J. Physiol. 176, 83–85; Packard, M. J., and Packard, G. C. (2005). Patterns of variation in glycogen, free glucose and lactate in organs of supercooled hatchling painted turtles (Chrysemys picta). J. Exp. Biol. 208, 3169–3176; Polakof, S., Mı́gues, J. M., and Soengas, J. L. (2008). Changes in food intake and glucosensing function of hypothalamus and hindbrain in rainbow trout subjected to hyperglycemic or hypoglycemic conditions. J. Comp. Physiol. A. 194, 829–839; Schmidt, H., and Wegener, G. (1988). Glycogen phosphorylase in fish brain (Carassius carassius) during hypoxia. Biochem. Soc. Trans. 16, 621–622; Støttrup, N. B., et al. (2006). L-glutamate and glutamate improve haemodynamic function and restore myocardial glycogen content during postischaemic reperfusion: A radioactive tracer study in the rat isolated heart. Clin. Exp. Pharm. Physiol. 33, 1099–1103; Vornanen, M. (1994). Seasonal adaptation of crucian carp (Carassius carassius L.) heart: Glycogen stores and lactate dehydrogenase activity. Can. J. Zool. 72, 433–442; Vornanen, M., and Paajanen, V. (2004). Seasonality of dihydropyridine receptor binding in the heart of an anoxia-tolerant vertebrate, the crucian carp (Carassius carassius L.). Am. J Physiol. 287, R1263–R1269; Vornanen, M., and Paajanen, V. (2006). Seasonal changes in glycogen content and Na+-K+-ATPase activity in the brain of crucian carp. Am. J. Physiol. 291, R1482–R1489; Warren, D., and Jackson, D. (2008). Lactate metabolism in anoxic turtles: An integrative review. J. Comp. Physiol. B 178, 133–148; Warren, D., Reese, S., and Jackson, D. (2006). Tissue glycogen and extracellular buVering limit the survival of red eared slider turtles during anoxic submergence at 3 C. Physiol. Biochem. Zool. 79, 736–744. Daw, J. C., Wenger, D. P., and Berne, R. M. (1967). Relationship between cardiac glycogen and tolerance to anoxia in the western painted turtle. Chrysemys picta bellii. Comp. Biochem. Physiol. 22, 69–73. 9. 425 ANOXIA-TOLERANT CRUCIAN CARP 25 8 20 6 2 4 1 0 Jun Aug Oct Dec Feb Apr Jun Oxygen (mg/L) Glycogen (% of wet weight) 3 10 15 10 2 5 0 0 Temperature (⬚C) Heart glycogen Brain glycogen Temperature Oxygen Fig. 9.13. Seasonal changes in glycogen concentration of crucian carp heart and brain. [Data from (Vornanen and Paajanen, 2004, 2006).] preferentially fueled by ATP produced from glycolysis (see DharChowdhury et al., 2007), glycogen might protect neurons against anoxic depolarization by securing ATP demand of this vital ion pump. 3.1.3. Heart Glycogen In winter, activity of the crucian carp heart is probably fairly low due to cold temperature and absence of positive thermal compensation in contractile activity of the heart (Matikainen and Vornanen, 1992; Tiitu and Vornanen, 2001). Even so, the heart functions continuously and has a steady need for energy also during the long hypoxic/anoxic period. Functionality and anoxia tolerance of the crucian carp heart must be dependent on anaerobic glycolysis and therefore on the glycolytic capacity of the tissue, i.e., the amount of cardiac glycogen stores, glycolytic enzymes, and sarcolemmal glucose uptake for an eYcient use of exogenous glucose. Unlike mammalian cardiac muscle, crucian carp heart does not store fats, but instead has massive glycogen stores (Figure 9.13). In mid-winter, glycogen content of the heart in small fish (about 10 g) can be as high as 8% of the wet heart weight, while a minimum of 0.3% occurs in May (Vornanen, 1994a; Vornanen and Paajanen, 2004). Thus, there can be a 26-fold seasonal diVerence in cardiac glycogen reserves. Glycogen phosphorylase activity of the heart is similar as in the red muscle but higher than in the white muscle (Hyvärinen et al., 1985). Lactate dehydrogenase (LDH) activity of the crucian carp heart is only half of the 426 MATTI VORNANEN ET AL. LDH activities measured in some other hypoxia-resistant fishes, e.g., South American lungfish (Lepidosiren paradoxa) and synbranchid eel (Synbranchus marmarotus) (Hochachka and Hulbert, 1978; Hochachka et al., 1978) and there is little seasonal change in cardiac LDH activity (Vornanen, 1994a). In fact, a small depression of LDH activity is apparent toward winter (Lind, 1992; Vornanen, 1994a). It is possible that the moderate activities of glycogenolytic and glycolytic enzymes and the absence of positive thermal compensation in enzyme activities cannot sustain a fast rate of glycolytic energy production in the cold, and that the activity of the crucian carp heart has to be modest in winter. Although the glycogen stores of the crucian carp heart are large in comparison to those of other vertebrates and the cardiac energy demand is presumably rather low in winter, the heart must be largely dependent on blood glucose for energy supply, due to the small size of the organ. Uptake of exogenous glucose by the crucian carp heart has not been determined. 3.2. Seasonality of the Heart Crucian carp stop feeding in winter (Penttinen and Holopainen, 1992) and are diYcult to catch in the anoxic season, which is indicative for a relatively inactive lifestyle during anoxic winter that should reduce the demands on circulation. Therefore, it is expected that seasonal temperature acclimatization in the form of lowering temperatures could also prepare the heart for winter anoxia (Tiitu and Vornanen, 2001). Several findings indicate that the heart of crucian carp, unlike those of many other fish species, does not compensate for the depressive eVects of low temperature in its function. Consequently, activity of the heart and circulation of blood will probably be depressed at the low temperatures of the anoxic winter season, although direct demonstration of this in the wild is still lacking. 3.2.1. Heart Size and Heart Rate The rate of circulation is determined by cardiac output, which is the product of heart rate and stroke volume. The relative size of the heart is an important determinant for heart function as it directly aVects the stroke volume (Graham and Farrell, 1989). The ventricle size of crucian carp heart is approximately 0.08% of the body mass, which is a quite typical value for a teleost fish, although less than in some more active fish species (Wilber et al., 1961; Farrell et al., 1992; Tiitu and Vornanen, 2002). Coldinduced enlargement of the heart, which is characteristic for many cold-active fish species, does not occur in seasonally acclimatized crucian carp. Instead there is marked decrease in cardiac water content in late autumn, probably due to accumulation of glycogen in the heart (Aho and Vornanen, 1997). 9. ANOXIA-TOLERANT CRUCIAN CARP 427 Considering that cold-induced hypertrophy of the heart should compensate for temperature-dependent depression of cardiac contractility and increased viscosity of the blood, the absence of this compensation in crucian carp is suggestive for temperature-dependent reduction of circulation. Heart rate is a significant determinant of cardiac output and it is strongly modified by both acute and chronic temperature changes in fish (Farrell and Jones, 1992). In summer, normoxic heart rate of crucian carp varies from 8 beats/min at 4 C to slightly over 100 beats/min at 30 C and acclimatization to winter strongly depresses heart rates at temperatures above 10 C, while the rate at 4 C is approximately the same in summer and winter (Matikainen and Vornanen, 1992). The heart rate of winter-acclimatized crucian carp (at 4 C) is less than one third of the rate of cold-acclimated rainbow trout (Oncorhynchus mykiss) and burbot (Lota lota) hearts (about 30 beats/min). Thus, the low heart rate and absence of positive thermal compensation in beating rate typical for many temperate fish species, probably keeps cardiac ATP demands low in preparation for anoxic conditions. 3.2.2. Cardiac Contractility Myocardial contractility describes the performance of cardiac muscle and is defined as the intrinsic ability of a cardiac muscle tissue to contract at a given sarcomere length. Adjustment of cardiac contractility to new conditions happens at the level of individual myocytes in the properties of myofilaments or in the management of intracellular Ca2+ concentration. The duration of ventricular twitch, especially the relaxation phase, is much longer in winter-acclimatized crucian carp than in summer-acclimatized fish (Vornanen, 1994b), suggesting seasonal diVerences in cardiac contractility. The rate of cardiac contraction is determined by attachment and detachment rate of cross bridges, i.e., by myosin ATPase activity and by the rate of activation induced by Ca2+ ions and their removal (Hoh et al., 1988), and evidence is accumulating that both Ca2+ management and cardiac myosins are modulated by seasonal acclimatization in crucian carp. These changes probably contribute to the anoxia tolerance of the heart. The force of cardiac contraction is directly related to the amount of free intracellular Ca2+. Electrical excitation of the sarcolemma grades the size of intracellular Ca2+ in a process of excitation-contraction (e-c) coupling to produce adequate amounts of force and power for pumping of blood (Bers, 2002). As a part of a physiologically integrated entity, contractility and e-c coupling of the fish heart must be fine-tuned to correspond to the overall physiological demands under varying environmental conditions, and accordingly we can expect modifications at the organ, cell, and molecular level in fish exposed to prolonged anoxia. 428 MATTI VORNANEN ET AL. DHPR (pmol/mg) DHPR Temperature Oxygen 10 25 8 20 6 1.0 4 0.5 Jun Aug Oct Dec Feb Apr Jun Oxygen (mg/L) 1.5 15 10 2 5 0 0 Temperature (⬚C) In most fish hearts, the major part of Ca2+ comes from the extracellular space through L-type Ca2+ channels and Na+–Ca2+ exchange (NCX), and may trigger a further release of Ca2+ from the sarcoplasmic reticulum (SR) via the SR Ca2+ release channels. Contraction ends when Ca2+ is returned from myofilaments back to the extracellular space and into the lumen of the SR by NCX and SR Ca2+-pump, respectively (Tibbits et al., 1992; Vornanen et al., 2002b; Hove-Madsen et al., 2003; Shiels and White, 2005). Sarcolemmal Ca2+ influx through both Ca2+ channels and NCX is critically dependent on the shape of AP, especially on plateau height and duration, and therefore any current that has influence on the shape of AP may aVect voltage-dependent Ca2+ transport across the SL and, accordingly, e-c coupling (Edman and Johannsson, 1976). Function of the sarcolemmal K+ currents is especially important, since they regulate the duration of cardiac AP (Vornanen et al., 2002b; Schotten et al., 2007). L-type Ca2+ current and NCX are the principal Ca2+ pathways in the crucian carp cardiac myocytes (Vornanen, 1997; Vornanen, 1999). Seasonal changes in the number of pore-forming alpha subunits of the L-type Ca2+ channels (DHPRs, dihydropyridine receptors) in crucian carp heart have been measured by [methyl-3H]PN200-110 binding (Vornanen and Paajanen, 2004) and show that the number of Ca2+ channels are approximately doubled for a relatively short period of time in mid-summer (May– July), i.e., for the major part of the year the density of Ca2+ channels is low (Figure 9.14). Furthermore, the change in the number of Ca2+ channels can Fig. 9.14. Seasonal changes in the number of dihydropyridine receptors (DHPR; alpha subunits of the L-type Ca2+ channels) in the crucian carp heart. [Data from (Vornanen and Paajanen, 2004).] 9. ANOXIA-TOLERANT CRUCIAN CARP 429 be triggered by temperature acclimation (Tiitu and Vornanen, 2003). Functionally, these changes appear as 74% larger Ca2+ current (at 11 C) in summer-acclimatized hearts in comparison to winter-acclimatized hearts, and when measured in seasonally relevant temperatures (4 C and 18 C) the current is 6.1 times larger in summer than in winter (Vornanen and Paajanen, 2004). Even if the lengthening of ventricular AP from about 1.3 s to 2.8 s in the cold (Paajanen and Vornanen, 2004) is taken into account, sarcolemmal Ca2+ influx through L-type Ca2+ channels is at least three times larger in summer than in winter. Although seasonal changes in e-c coupling proteins of the crucian carp heart, except myosin heavy chains and L-type Ca2+ channels, have not been examined yet, temperature acclimation under laboratory conditions indicate that several ion transport mechanisms are depressed by cold-acclimation. The density of Na+ current, which determines the rate of impulse propagation in the heart, is strongly depressed to one-fifth of that of warm-acclimated fish with cold-acclimation (Haverinen and Vornanen, 2004). Thapsigargin (a specific blocker of SR Ca2+-pump)-sensitive Ca2+ uptake of the cardiac SR is also decreased in cold-acclimated crucian carp (Aho and Vornanen, 1998). Assuming that cold-acclimation primes the heart of crucian carp for winter, those findings suggest that several steps of the cardiac e-c coupling are downregulated for winter and that the activity of the heart is depressed in the absence of positive thermal compensation in the cold winter waters. Indeed, tissue level experiments indicate that the kinetic properties of atrial and ventricular contraction are strongly depressed by cold-acclimation, which should appear as strongly reduced cardiac power output in the cold (Tiitu and Vornanen, 2001). Interesting exceptions to the inverse thermal compensation of the crucian carp cardiac function are sarcolemmal K+ currents (Haverinen and Vornanen, 2008). Two major K+ currents of the fish heart are the inward rectifier K+ current (IK1), which maintains the negative resting membrane potential and contributes to the final rate of AP repolarization, and the rapid component of the delayed rectifier K+ current (IKr), which is important in the regulation of plateau duration (Vornanen et al., 2002a). Densities of these K+ currents are increased by cold-acclimation in atrial and ventricular myocytes of the crucian carp heart so that the sizes of the currents are similar in cold-acclimated fish at 4 C and in warm-acclimated fish at 18 C (Haverinen and Vornanen, 2008). Still, the duration of AP is 2.6 and 2.8 times longer at 4 C than at 18 C for ventricular and atrial muscle, respectively. Obviously positive temperature compensation in the density of K+ currents is not suYcient to prevent the lengthening of cardiac AP in winter. Thus, our current knowledge of cardiac ion currents of the crucian carp indicates that the inward Na+ and Ca2+ currents are depressed and the 430 MATTI VORNANEN ET AL. outward K+ currents are enhanced in the cold-acclimatized winter fish. Inward currents are excitatory in that they promote contraction, while outward currents tend to stabilize membrane to the negative equilibrium potential of K+ ions. Therefore, opposite changes in inward and outward currents are likely to reduce excitability of the cardiac muscle. Cardiac contractility is also aVected by composition and function of myofibrillar proteins. Two myosin heavy chains have been demonstrated in crucian carp ventricle by SDS-PAGE (Vornanen, 1994b). Only one myosin heavy chain isoform is expressed in the hearts of winter-acclimatized fish and is therefore called ‘‘winter’’ myosin, whereas the hearts of summer-acclimatized fish express both winter and ‘‘summer’’ isoforms. In June and July both isoforms are almost equally represented in the ventricular muscle, but the amount of summer myosin decreases already in August and cannot be resolved any more in September. Small amounts of summer myosin appear again in May, when waters warm up (see Figure 9.2). The physiological significance of this seasonal pattern in myosin heavy chain composition probably lies in the diVerent myosin-ATPase activities of the two isoforms: the activity is much greater in summer than in winter (Vornanen, 1994b; Tiitu and Vornanen, 2001). It is well known that the contraction of slow myosins occurs with less energy expenditure than the contraction of fast myosins (Alpert and Mulieri, 1982). Therefore, the exclusive reliance on the slow myosin in winter would improve energetic economy of the heart under conditions where energy production is severely limited by oxygen shortage. In heart function, this should appear as a reduced cardiac power output, which might be well tolerated due to reduced circulatory demands. The slow myosin may also be useful in tuning the rate of myofilament sliding to the low heart rate and the long duration of cardiac action potential in the cold. Taken together, inverse thermal compensation seems to be typical for the crucian carp, with the exception of sarcolemmal K+ currents, which will result in temperature-dependent depression of cardiac function in cold and anoxic winter waters. 3.3. Seasonality of Brain 3.3.1. Brain Na+ /K + -ATPase As described above, anoxia exposure does not decrease the number of Na+/K+-ATPase alpha subunits or their molecular activity in the crucian carp brain (Hylland et al., 1997; Vornanen and Paajanen, 2006). In contrast, determinations of [3H]ouabain binding and Na+/K+-ATPase activity in crucian carp caught directly from the wild have revealed a strong temperature dependence of Na+/K+-ATPase (Vornanen and Paajanen, 2006). As a result, 9. ANOXIA-TOLERANT CRUCIAN CARP 431 the activity of the sodium pump in winter is only 10–15% of its activity in summer, suggesting a considerable down-regulation of brain activity with cold acclimation. In fact, a small positive compensation of the Na+/K+ATPase activity is seen in mid-winter, which might be needed to prevent the brain from depressing into a comatose state. The positive compensation in brain Na+/K+-ATPase activity is attained without increase in the number of pump units by a decrease in temperature dependence (Q10) of the enzyme catalysis (Vornanen and Paajanen, 2006). Seasonal changes in phosphatidylethanolamine and phosphatidylserine phospholipids of the neuronal membrane might explain the reduced temperature dependence of the sodium pump (see below) (Käkelä et al., 2008). 3.3.2. Brain Lipids The brain of crucian carp retains a significant level of functionality in complete anoxia (Nilsson, 2001) and is assumed to sustain considerable nerve function under cold and hypoxic/anoxic winter conditions (see Section 2.5). Many crucial neuronal activities occur in the plasma membrane where proper function of ion channels, ion pumps, and membrane receptors is essential for electrical excitability and neurotransmission. Those molecules are embedded in the phospholipid membrane, which should provide a favorable matrix to the integral membrane proteins under all environmental conditions. Temperature has a particularly strong eVect on the physical properties of biological membranes and in many ectotherms temperature acclimation strongly modifies membrane lipids to maintain the semifluid state of the plasma membrane (Sinensky, 1974). Hypoxia, ischemia, and reperfusion also aVect the biochemical composition of the lipid membrane and may damage the membrane (Cao et al., 2007). In the typical habitat of crucian carp, low temperature and hypoxia/anoxia occur simultaneously and therefore the brain membranes have to cope with both thermal and hypoxic stresses, which may aVect brain lipids diVerently and which may require diVerent adaptations. Considering the large seasonal changes in abiotic conditions of the crucian carp habitat, it is not surprising that profound seasonal changes in the composition of brain lipids occur (Käkelä et al., 2008). Comparison of membrane lipids from fish acclimated in laboratory to diVerent temperatures and fish collected from the wild in diVerent seasons indicates that fatty acid composition of the brain lipids display similar temperature responses in laboratory-acclimated and seasonally acclimatized crucian carp (Figure 9.15A). At low temperatures, the brains contain lower levels of saturated fatty acids, higher levels of polyunsaturated fatty acids, and the average length of the monounsaturated fatty acid chain is shorter (Käkelä et al., 2008). All these changes are compatible with the model of 432 MATTI VORNANEN ET AL. Average double bond number of a chain 1.8 r 2 = 0.89, P < 0.001 1.6 1.4 Seasonal acclimatization Laboratory acclimation 1.2 0 5 10 15 20 25 30 Temperature (⬚C) 25 PS di-PUFA PS di-22:6n-3 PE di-PUFA PE di-22:6n-3 ⬚C 25 20 15 15 10 10 5 mol% 20 5 May 02 Jul 02 Aug 02 Nov 02 Jan 03 Mar 03 Temperature (⬚C) 30 0 Fig. 9.15. Seasonal- and temperature-induced changes in crucian carp brain lipids. Temperature dependence of the average double bond number of the acyl and alkenyl chains in the total lipids of the crucian carp acclimated in the laboratory at three diVerent temperatures for 4 weeks (A). Seasonal changes in the total di-PUFA and di-22:6n-3 phosphatidylserine (PS) and phosphatidylethanolamine (PE) in the brain of crucian carp collected from the wild (B). Values are means  S.D. from 5 to 7 fish. [Data from Käkelä et al. (2008).] homeoviscous adaptation of membrane fluidity (Sinensky, 1974) and suggest compensation for the direct eVects of temperature to maintain the proper fluidity of the brain membranes. Acclimation/acclimatization causes little changes in the phospholipid class composition, i.e., the relative contents of phosphatidylethanolamines, phopshatidylcholines, phosphatidylserines, phosphatidylinositols, and plasmalogens, but induces marked changes in molecular species composition 9. ANOXIA-TOLERANT CRUCIAN CARP 433 (diVerent fatty acid combinations) of the brain phospholipids (Figure 9.15B). Most notably a large increase of the di-22:6n-3 phosphatidylserine and phosphatidylethanolamine species (DHA, docosahexaenoic acid estrified to carbon-1 and carbon-2 of the phospholipids) appears in the cold. Since the increase of DHA in the total fatty acyl pool of the brain is small, the formation of di-DHA aminophospholipid species appears to be a specific molecular rearrangement for winter. Plasma membranes of eukaryotic cells are highly asymmetric, with most phosphatidylethanolamines and all phosphatidylserines residing in the inner membrane leaflet (Virtanen et al., 1998), and therefore di-DHA changes should have a significant impact on this membrane compartment. Such highly unsaturated species are needed to maintain adequate membrane fluidity in the vicinity of ion transporters and other integral membrane proteins. More specifically, these changes may be behind the noticed decrease in temperature-dependence of Na+/K+-ATPase in winter, since DHA-containing phospholipids activate Na+/K+-ATPase in excitable membranes (Turner et al., 2003). On the other hand, DHA-containing lipids also protect against ischemia, oxygen deprivation, and reperfusion injuries in mammals (Strokin et al., 2006; Cao et al., 2007). In particular, DHA-containing lipids alleviate ischemia-associated decrease in Na+/K+-ATPase activity and thereby reduce brain infarct size. The brain lipidome of the crucian carp is strongly modified by seasonal acclimatization and the seasonal changes are in many respects similar, although not identical, to changes induced by temperature acclimation. This suggests that ambient temperature is the main environmental cue that primes brain lipids for winter stresses. The responses in seasonal acclimatization are sometimes smaller than in laboratory acclimation, which may represent combined eVects of low temperature and hypoxia on brain lipids. 4. SUMMARY The crucian carp employs a number of innate survival strategies to tolerate prolonged anoxia exposure. With the onset of oxygen deprivation, respiration rate is augmented and the gills remodeled. These responses, in conjunction with an extremely high hemoglobin oxygen aYnity, should extend the period that oxygen can be extracted from the water when the crucian carp is faced with a steady fall in ambient oxygen levels during the early winter. A concurrent severe bradycardia likely benefits myocardial oxygen supply. When oxygen is no longer available, crucian carp produce ethanol as the major anaerobic end-product to avoid self-poisoning by lactate and H+ ions, and increase brain blood flow to deliver fermentable fuel. Maintained cardiac activity during prolonged anoxia may be possible 434 MATTI VORNANEN ET AL. because the ATP demand of the heart lies below the maximum glycolytic potential for ATP production and is believed to be necessitated for transport of fermentable fuels and ethanol among tissues. Unlike the equally anoxiatolerant freshwater turtle, the anoxic crucain carp sustains neural activity. During anoxia, there is no depression of brain Na+/K+-ATPase activity and K+ and Ca2+ permeability remains unchanged, but some indication of limited ‘‘channel arrest’’ involving NMDA receptors exists. In addition to constitutively expressed characteristics, a seasonal induction of numerous physiological traits is imperative for preparing the carp for long-term anoxic survival in the winter. In the habitat of crucian carp, anoxia is a regular and predictable seasonal condition that is accompanied by several environmental cues, most notably temperature. 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Evidence of anoxia-induced channel arrest in the brain of the goldfish (Carassius auratus). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 148, 355–362. Williamson, J. R., Safer, B., Rich, T., SchaVer, S., and Kobayashi, K. (1976). EVects of acidosis on myocardial contractility and metabolism. ActaMedScandSuppl 587, 95–112. Wissing, J., and Zebe, E. (1988). The anaerobic metabolism of the bitterling Rhodeus amarus (Cyprinidae, Teleostei). Comp. Biochem. Physiol. B 89, 299–303. Zingman, L. V., Alekseev, A. E., Hodgson-Zingman, D. M., and Terzic, A. (2007). ATPsensitive potassium channels: Metabolic sensing and cardioprotection. J. Appl. Physiol. 103, 1888–1893. 10 METABOLIC AND MOLECULAR RESPONSES OF FISH TO HYPOXIA JEFFREY G. RICHARDS 1. 2. 3. 4. Introduction The Metabolic Challenge of Hypoxia Exposure The Concept of Time in the Metabolic Responses to Hypoxia Metabolic and Molecular Responses to Hypoxia 4.1. Increases in O2 Transport 4.2. O2-Independent ATP Production 4.3. Metabolic Rate Suppression 5. Coordinating the Metabolic and Molecular Responses to Hypoxia 5.1. AMP-Activated Protein Kinase as a Metabolic Coordinator 5.2. Hypoxia Inducible Factor 6. Conclusions and Perspectives Hypoxia survival requires a well-coordinated response to either secure more O2 from the depleted environment or to defend against the metabolic consequences of too little O2 at the mitochondria, which limits aerobic ATP production. Inhibition of aerobic ATP production during hypoxia exposure imposes a substrate-limited cap on the duration of survival because O2independent ATP production (anaerobic) is far less eYcient than aerobic ATP production. It has long been held that hypoxia-tolerant animals are able to extend the period of survival under severely hypoxic conditions through a depression of basal metabolic rate, which limits the extent of activation of O2-independent pathways of ATP production. This contention appears to be supported by the available literature; however, more studies measuring metabolic rate during hypoxia exposure are needed before a definitive outcome can be decided. Duration of hypoxia exposure is also an important component to consider when assessing the responses to hypoxia. Long-term hypoxia exposure (> a few hours in some cases) can result in large changes in 443 Hypoxia: Volume 27 FISH PHYSIOLOGY Copyright # 2009 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(08)00010-1 444 JEFFREY G. RICHARDS gene expression, which underlie acclimation/acclimatization and potentially enhance hypoxic survival. Hypoxia-mediated changes in gene expression are likely regulated by the transcription factor, hypoxia inducible factor (HIF), which is well characterized in mammalian systems, but has only recently been examined in fish. Hypoxia inducible factor appears to be regulated in a similar fashion in fish as in mammals, but to date, there does not appear to be a direct link between HIF function and hypoxia tolerance in fish. 1. INTRODUCTION Environmental hypoxia is a common, naturally occurring phenomenon in many aquatic ecosystems, the prevalence of which is increasing due to anthropogenic nutrient loading and eutrophication (reviewed in Chapter 1). In light of these O2 fluctuations in the aquatic environment, it is perhaps not surprising that among all vertebrates, fish boast the largest number of hypoxia-tolerant species; hypoxia has clearly played an important role shaping the evolution of many unique adaptive strategies for hypoxic survival. Previous chapters in this volume have outlined a myriad of physiological and biochemical strategies that facilitate O2 uptake under hypoxic conditions including changes in behavior, ventilation, hemoglobin-O2 binding characteristics, and cardiovascular function. These strategies work to sustain metabolic function by maximizing O2 extraction from the environment. Of importance to the present chapter, however, are the biochemical and molecular strategies that are responsible for defending against the metabolic consequences of O2 levels that fall below a threshold where metabolic function is aVected or cannot be maintained. Paramount to this defense strategy is a well coordinated response to maintain cellular ATP turnover, albeit at reduced levels, and the ability for hypoxic acclimation to ‘‘enhance’’ cellular and whole animal function under O2 limiting conditions. Metabolic and molecular responses to hypoxia are critical to enhance survival at O2 levels below a species critical oxygen tension (Pcrit). In the context of this chapter, Pcrit is defined as the environmental O2 tension at which an organism’s O2 consumption rate transitions from being independent of environmental O2 to being dependent on environmental O2 (see Figure 10.1A; Pörtner & Grieshaber 1993). As such, Pcrit represents a whole-animal measure of O2 extraction capacity from the environment and is considered by many researchers as an indicator of hypoxia tolerance (Chapman et al., 2002). Many physiological adjustments can aVect Pcrit, and the majority of these have been outlined in previous chapters in this volume. For example, increases in O2 extraction capacity through modifications to ventilation (see Chapter 5), O2 transport systems (see Chapter 6), 10. 445 METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA O2 consumption rate A Routine O2 consumption Pcrit Environmental O2 Reproduction Feeding/digestion Ventilation Swimming/movement O2 transport/delivery Reproduction Feeding/digestion O2 consumption rate or metablic rate B 1 Metabolic rate 2 O2 consumption 3 Pcrit Environmental O2 Fig. 10.1. Metabolic responses of fish to changes in environmental O2. (A) A species’ critical oxygen tension (Pcrit) is the point at which O2 consumption rate transitions from being independent of environmental O2 levels (often referred to as O2 regulation) to being dependent on environmental O2 (often referred to as O2 conforming). Pcrit can be increased, detrimentally aVecting hypoxia tolerance by increasing energetically expensive processes such as reproduction, growth, or digestion. Pcrit can also be decreased, enhancing hypoxia tolerance through changes in respiration (VE and gill perfusion), O2 transport/delivery (changes in hemoglobin–O2 binding aYnity and cardiovascular responses), or through reductions in energetically expensive processes such as reproduction, digestion, and swimming. (B) At O2 levels below Pcrit, survival is dependent upon the ability of an animal to suppress basal metabolic rate to limit the extent of the activation of O2-independent pathways of ATP production. See text for more detail. or O2 delivery systems (see Chapter 7) can theoretically result in a decrease in Pcrit, and thus an enhancement of hypoxia tolerance. In contrast, increases in whole animal metabolic demands associated with, for example, gonad development and reproduction (see Chapter 3) as well as during digestion 446 JEFFREY G. RICHARDS and allocation of energy to growth (see Chapter 8) can cause an increase in Pcrit, and a decrease in hypoxia tolerance. Thus, suppression of reproduction, digestion, and growth during hypoxia exposure reduces metabolic demands and enhances hypoxia tolerance and survival (Figure 10.1A). 2. THE METABOLIC CHALLENGE OF HYPOXIA EXPOSURE At O2 levels below Pcrit, the fundamental challenge is one of metabolic energy balance. Greater than 95% of the O2 consumed by a fish in normoxia is used as the terminal electron acceptor by the mitochondrial electron transport chain for ATP production (via oxidative phosphorylation). If environmental hypoxia leads to hypoxemia (i.e., physiological mechanisms to enhance O2 uptake are insuYcient to protect the animal from its environment and blood O2 content is reduced), then there is the potential for an O2 limitation at the mitochondrion, which imposes limitations on the capacity for ATP production. Under these conditions, ATP can only be generated by processes such as glycolysis yielding lactate production or through direct phosphate transfer from phosphorylated intermediates such as creatine phosphate (CrP). These processes of direct phosphate transfer from a substrate to ADP forming ATP are termed substrate-level phosphorylation. Although these processes of ATP generation can occur during periods of O2 lack, the amount of ATP produced per mole of substrate consumed is approximately 15- to 30-fold lower than if mitochondrial respiration occurs. For example, aerobic catabolism of 1 mole of glucose yields 30 moles of ATP, while the anaerobic catabolism of glucose, involving only glycolysis and lactate production, produces 2 moles of ATP. A reduction in the ability of an organism or cell to generate suYcient ATP to meet metabolic demands presents a problem for the maintenance of cellular energy balance. Hypoxiasensitive animals quickly succumb to hypoxia due to an inability to maintain cellular energy balance and a loss of cellular [ATP] (Boutilier, 2001). Thus, during hypoxia the inhibition of O2-based mitochondrial ATP production imposes a potential substrate-limited cap on the duration of survival. Under these O2 limiting conditions duration of survival is dictated by two, interrelated factors: (1) the ability to reduce metabolic demands through a controlled metabolic rate suppression; and (2) the availability of substrate for O2-independent ATP production. Illustrated in Figure 10.1B is a conceptual framework to understand the relationship between metabolic rate suppression and capacity for O2-independent ATP production. At environmental O2 tensions below Pcrit, hypoxia tolerance is likely to be dictated by the degree of metabolic rate suppression, which extends the length of time a fixed quantity of fermentable substrate can support cellular function. For example, scenario 1 would 10. METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA 447 represent a severely hypoxia-sensitive fish, where at O2 tensions below Pcrit, the animal attempts to maintain metabolic rate, which can only be accomplished by a large activation of O2-independent pathways of ATP production (largely glycolysis) thus utilizing fermentable fuels at a high rate (indicated by the large curved arrow). If the quantity of fermentable fuels is limited, then the animal will quickly succumb to hypoxia and die. On the other hand, scenarios 2 and 3 represent increasing levels of hypoxia tolerance, where decreases in metabolic rate limit the magnitude of the activation of O2-independent ATP production (shorter curved arrows) and extend the period of time that can be supported by substrate-level phosphorylation. Thus, it seems reasonable to hypothesize that there should be a relationship between hypoxia tolerance, the magnitude of the hypoxia-induced metabolic rate suppression, and the availability of fermentable fuels to support O2-independent ATP production. At the cellular level, the precise mechanism of hypoxia-induced death is not known; however, it is clear that hypoxic death in fish is associated with catastrophic loss of substrate, failure of essential ATP consuming processes, accumulation of toxic levels of waste products (protons and lactate), and cellular necrosis. Underlying all of these potential causes of hypoxia-induced death is an inability of the animal to maintain metabolic energy balance. Boutilier and St-Pierre (2000) analyzed the available literature and proposed an elegant hypoxia-induced (and hypothermia-induced) cascade of events that yield necrotic cell death. In hypoxia-sensitive animals, hypoxia exposure leads to an inability to generate suYcient ATP to meet the metabolic demands of cellular ion regulation, protein synthesis, and other metabolic processes – a mismatch between ATP supply and demand – therefore, cellular [ATP] falls to levels that are insuYcient to maintain the activity of these energy-consuming processes. Boutilier and St-Pierre (2000) pointed to cellular ion regulation to be the most critical aspect of cell survival and proposed that a loss of ATP limits the capacity of a cell to maintain transmembrane potential resulting from net Na+ influx and K+ eZux. This results in depolarization of plasma and organelle membranes, Ca2+ accumulation in the cytosol from organelles and extracellular fluid, the activation of phospholipases and Ca2+-dependent proteases, and the rupture of membranes, ultimately resulting in necrotic cell death. It has been proposed that hypoxiatolerant animals are able to stave oV these catastrophic events by initiating regulated metabolic rate suppression and stabilizing cellular [ATP]. Stable cellular [ATP] during hypoxia exposure is often accepted as the hallmark measure of a hypoxia-tolerant animal (Hochachka et al., 1996; Boutilier, 2001); however, this has been demonstrated to be an over simplification. Numerous studies have shown a substantial disruption of cellular energetics during hypoxia exposure even in hypoxia-tolerant organisms (van den Thillart et al., 1980,1989; Borger et al., 1998; Hallman et al., 2008; 448 JEFFREY G. RICHARDS Jibb and Richards, 2008; Richards et al., 2008;) and changes in cellular [ATP] appear to be tissue specific. For example, in muscle, numerous studies have demonstrated that cellular [ATP] is not aVected by hypoxia/anoxia exposure (van den Thillart et al., 1980; Richards et al., 2007, 2008); while in liver, [ATP] decreases initially upon hypoxia exposure and then stabilizes at a lower concentration (Figure 10.2A) (J. Dalla Via et al., 1994; Jibb and Richards, 2008; van den Thillart et al., 1980). These results are in general agreement with the results of Busk and Boutilier (2005) who showed in isolated eel hepatocytes that anoxia exposure caused an initial decrease in [ATP], followed by a stabilization at a new, lower level. In contrast, Krumschnabel et al. (1997) demonstrated that exposure of isolated goldfish hepatocytes to anoxia did not result in a decrease in [ATP], while the same preparation exposed to chemical anoxia, using NaCN, showed a decrease in [ATP]. This latter decrease in [ATP] was modest when compared with the large decreases of [ATP] observed in anoxia-exposed hepatocytes isolated from the hypoxia-intolerant rainbow trout (Krumschnabel et al., 1997). It has been postulated that the reason for the diVerences in response in [ATP] between muscle and liver is related to the tissue [CrP]. Muscle [CrP] are much higher than measured in liver (20 to 50 versus <5 mmol/g wet tissue, respectively); thus, in liver, there is a lack of capacity to buVer [ATP] during the onset of hypoxia. Whether tissue [ATP] is aVected by hypoxia or not, intracellular acidosis and CrP hydrolysis result in an accumulation of [ADPfree] and [AMPfree], causing increases in [ADPfree]/[ATP] and [AMPfree]/[ATP] and substantial losses of cellular phosphorylation potential (Figure 10.2; Hallman et al., 2008; Jibb and Richards, 2008; Richards et al., 2008; van den Thillart et al., 1989). This disruption of cellular energy status plays several important roles in the cell during hypoxia exposure. First, decreases in phosphorylation potential may aVect rates of cellular ATP production and substrate oxidation. For example, hypoxia exposure was associated with a significant drop in the free energy of ATP hydrolysis (
fG’; Figure 10.2C) (Hallman et al., 2008; Jibb and Richards, 2008; Richards et al., 2008). Estimates of the critical limit of
fG’ for the maintenance of cellular function suggest that below a threshold of 52 kJ/mol, cellular processes such as ion pumping can no longer derive suYcient energy from ATP hydrolysis to be maintained (Hardewig et al., 1998; Jansen et al., 2003). Second, changes in cellular [ADPfree]/[ATP] and [AMPfree]/[ATP] are important signals coordinating the metabolic responses to hypoxia. For example, increases in [ADPfree]/ [ATP] are known to allosterically activate glycolysis, increasing O2-independent ATP production and more recent evidence indicates that increases in [AMPfree]/[ATP] may be vital to overall coordination of metabolic rate suppression in certain tissues of hypoxia-tolerant fish (Jibb and Richards, 2008). 10. 449 METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA A ATP (µmol • g−1 wet tissue) 3 2 1 0 B [ADP free]/[ATP] 40 30 20 10 0 C −66 −64 ∆f G⬘ATP (kj/mol) −62 −60 −58 −56 −54 −52 −50 N 1 2 4 8 Time in hypoxia (h) 12 Fig. 10.2. Liver [ATP] (A), calculated [ADPfree]/[ATP] (B), Gibbs free energy of ATP hydrolysis (C) (
fG0 ATP) in goldfish exposed to normoxia and 12 h of hypoxia (<0.5% air saturation). Horizontal dashed lines through normoxia are shown as a reference. [Data from Jibb and Richards (2008) with permission.] 450 JEFFREY G. RICHARDS 3. THE CONCEPT OF TIME IN THE METABOLIC RESPONSES TO HYPOXIA When considering the physiological and biochemical responses to hypoxia, environmental O2 levels are not the only factor to consider: the length of time spent in hypoxia can have dramatic eVects on the responses to O2 lack. Upon exposure to hypoxia, immediate survival is dependent upon the ability of the fish to quickly modify existing physiological and biochemical systems in an attempt to maintain metabolic function. If these immediate responses are suYcient for survival of the onset of hypoxia, then an animal has the opportunity to acclimate or acclimatize, which, for the most part, is thought to be of benefit in enhancing the ability of an animal to survive hypoxic exposure. At the heart of any acclimation response are changes in gene expression, which can alter the capacity of an animal to endure hypoxia. Changes in gene expression, if they are translated into functional changes in protein amount or possibly protein turnover rates, can aVect hypoxia survival by either increasing or decreasing the amounts of specific proteins in a metabolic pathway. For example, large increases in the expression of the lactate dehydrogenase gene (ldh) and increases in LDH activity have been observed during both long- and short-term hypoxia exposure in fishes (e.g., Amazonia cichlid) (Almeida-Val et al., 1995, 2006). In addition, selective changes in gene expression can result in protein isoform switching, which in some cases has been shown to enhance survival to environmental perturbation (Schulte, 2004). Environmental hypoxia is well known to aVect gene expression patterns in fish with several microarray studies showing changes in the transcription of genes involved in O2 uptake, energy turnover, growth and development, immune responses, cell signaling, and stress (Figure 10.3 and Table 10.1; Gracey et al., 2001; Ton et al., 2003). Thus, almost every physiological and biochemical response discussed earlier in this volume is regulated, at least in part, by changes in gene expression. Regulation and coordination of changes in gene expression in response to hypoxia exposure are mediated largely by the transcription factor hypoxia inducible factor (HIF), which has been characterized in mammalian and fish systems. The remaining portion of this chapter is divided into two parts. The first section outlines the metabolic and molecular responses of fish to hypoxia exposure. Combining the metabolic and molecular gene expression changes is meant to emphasize that the changes observed in metabolic phenotype are also controlled to a degree by changes in gene expression, which underlie acclimation responses. The second portion of this chapter examines how these processes are coordinated at the biochemical and molecular level with emphasis on HIF as a regulator of hypoxia-induced changes in gene expression. 451 METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA A D Liver G.seta sketetal cardiac LDH-A Triosephosphate isomerase Triosephosphate isomerase PFK-2/FBP-2 Enolase GAPDH Glucose-6-phosphatase Glycogen myophosphorylase Creatine kinase Creatine kinase Cylochrome b Cylochrome b Cylochrome c oxidase I Cylochrome c oxidase I B E Transferrin Transferrin IGFBP-1 IGFBP-1 Transducer of Erb-B2 Transducer of Erb-B2 B-cell translocation gene 1 Antisense RBP2 Antisense RBP2 MAPK-phosphatase 1 Control 8h 24h 72h 144h 8h 24h 72h 144h cardiac α-tropomyosin α-tropomyosin Myosin heavy chain Myosin heavy chain Myosin regulatory light chain 2A Skeletal α-actin Skeletal α-actin β-actin β-actin C Elongatin factor 2 Ribosomal protein S19 Ribosomal protein L24 Ribosomal protein L11 Ribosomal protein S23 Ribosomal protein P1 Ribosomal protein L6 Ribosomal protein S13 Ribosomal protein S15 Ribosomal protein S8 Ribosomal protein S8 Ribosomal protein L23 Ribosomal protein L22 Ribosomal protein S2 Ribosomal protein S2 >8x 4x Fold repressed 1:1 4X Fold induced >8x G.seta Heme oxygenase Ferritin H3 Ferritin H3 Hemopexin Hemopexin Transferrin Transferrin Muscle sketetal Liver G.mirabilis Control 8h 24h 72h 144h 8h 24h 72h 144h 72h exp.2 144h exp.2 24h acute 8h 24h 72h 144h 8h 24h 72h 144h G.mirabilis Muscle Control 8h 24h 72h 144h 8h 24h 72h 144h 72h exp.2 144h exp.2 24h acute 10. F Tyrosine aminotransferase Tyrosine aminotransferase Tyrosine aminotransferase Alanine-glyoxylate aminotransferase Alanine-glyoxylate aminotransferase Cystathione-β-synthase Cystathione-β-synthase S-adenosylmethionine synthase S-adenosylmethionine synthase Formyltetrahydofolate dehydrogenase Cysteine dioxygenase Glutamine synthetase G IgE/mannose receptor, C type 2 apolipoprotein A4 precursor apolipoprotein A1 precursor Selenium-binding protein Insulin-induced protein Hypoxia-inducible gene Cathepsin L Serpin Hypothetical gene AF266210 Cytochrome P450-cc24 EST AW777 145 EST AW777 145 EST AW777 144 EST AW777 140 EST AW777 099 EST AW777 133 EST AW777 104 EST AW777 142 EST AW777 111 EST AW777 148 EST AW777 128 Fig. 10.3. Changes in gene expression assessed using cDNA microarray in two species of mudsucker during hypoxia exposure. Genes are categorized on the basis of their probable biological role: (A) ATP metabolism; (B) locomotion and contraction; (C) protein translation; (D) iron metabolism; (E) antigrowth and proliferation; (F) amino acid metabolism; and (G) cryptic role. [Reproduced from Gracey et al. (2001) with permission.] 4. METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA Many excellent reviews have summarized the metabolic and molecular responses of fish and other lower vertebrates to hypoxia exposure (AlmeidaVal et al., 2006; Bickler and Buck, 2007; Nikinmaa and Rees, 2005). Many of the chapters in this book outline numerous responses to hypoxia including responses that work to increase O2 uptake and the metabolic adjustments Table 10.1 Molecular responses to hypoxia Common name Longjaw mudsucker Zebrafish Scientific name Gillichthys mirabilis Danio rerio Temp. ( C) 15 28 Hypoxia a 10% air sat. 5% oxygen 23% air sat. Duration 8, 24, 72, and 144 h 24 h Whole animal or tissue Gene expression changes Reference Liver " glycolysis (7) " amino acid metabolism " iron and Hb metabolism (8) " anti-growth & cell proliferation (10) # aerobic metabolism (4) Muscle " glycolysis (4) # glycolysis & CK (5) # aerobic metabolism (4) # locomotion and contraction (9) # protein synthesis (15) 48 h postfertilization embryos " glycolysis (6) Ton et al. " cell signaling (5) (2003) # aerobic metabolism (10) # creatine kinase (2) # cell structure & mobility (20) # ion transporting ATPases (5) # protein synthesis (6) # iron & Hb metabolism (5) # cell division (5) # cell/organism defense (5) Gracey et al. (2001) Zebrafish Danio rerio 28 10% air sat. 21 daysb Gills " glycolysis (2) " disease defense (12) " phosphatases (6) " chapterones (8) # aerobic metabolism (31) # protein synthesis (54) " elongation factors (2) # stress response (6) # apoptosis (8) # locomotion and contraction # growth regulation (12) # innume response (9) # proteosome degradation (6) van der Meer et al. (2005) The numbers of genes indicated may have either increased or decreased in expression, but overall the authors concluded that the changes indicated would yield an overall change in biological outcome indicated by the arrow. a Pcrit of this species is 1.2 mg O2/L and hypoxia exposure was 0.8 mg O2/L b Decrease in oxygen occurred gradually over 4 days. 454 JEFFREY G. RICHARDS associated with hypoxia exposure in the heart (see Chapter 7) and the metabolic aspects yielding the extraordinarily anoxia-tolerant crucian carp (Carassius carassius; see Chapter 9). From all of the preceding chapters it has become evident that there are three principal aspects that function to maintain cellular energy balance and these include: (1) increased O2 uptake from the hypoxic environment to sustain a modicum of aerobic ATP production; (2) strong activation of an O2-independent means of ATP production; and (3) a reduction in metabolic demands through regulated metabolic rate suppression, which is described in more detail below. 4.1. Increases in O2 Transport As outlined previously in this volume, the physiological and biochemical responses that yield increases in O2 transport capacity are important adaptations to survive hypoxia. Indeed, recent work by Mandic et al. (2008) showed in a group of closely related intertidal fish species (sculpins from the family Cottidae) that approximately 75% of the variation in hypoxia tolerance (assessed as Pcrit) could be explained by variation in physiological attributes aVecting O2 uptake (Hb–O2 binding aYnity or gill surface area) or O2 use (routine metabolic rate). However, since other chapters have explicitly dealt with the physiological responses that increase O2 uptake from the environment and O2 delivery to the tissues, in this section I will solely focus on the O2dependent changes in gene expression that may form the foundation of possible acclimation responses. In fact, almost every microarray study performed to date has shown an eVect of hypoxia exposure on mRNA levels for proteins involved in Hb metabolism and oxygen transport (Figure 10.3 and Table 10.1). As pointed out in Chapter 6, modifications to Hb–O2 binding aYnity and blood Hb content are important responses to hypoxia. At the gene expression level, Gracey et al. (2001) showed dramatic changes in the expression of genes involved in heme metabolism in liver of the mudsucker in response to hypoxia exposure. Several genes involved in iron-heme catabolism and heme protein turnover were all induced by hypoxia exposure. These general changes in genes involved in iron and heme metabolism could be linked with hypoxia-induced erythropoietin (EPO) or erythropoiesis and increased demand for iron from hemoglobin synthesis. By contrast, zebrafish embryos exposed to hypoxia show a general decrease in the expression of genes involved in Hb metabolism. Specifically, Ton et al. (2003) showed large decreases in mRNA levels for globin, bA1, hemoglobin b chain, globin a-embryonic, globin 2 a-embryonic, and, oddly, erythropoietin. The probable explanation for these counterintuitive decreases in mRNA levels for proteins involved in blood O2 transport is that the very small zebrafish embryos do not require blood flow for survival and O2 uptake is mostly via diVusion. 10. METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA 455 In mammals, hypoxia is a powerful regulator of the production of erythropoietin (Semenza and Wang, 1992), which causes an increase in red blood cell production leading to increases in Ht and increases in blood O2-carrying capacity. There is no published data directly linking hypoxia exposure and changes in EPO gene expression in fish, although injection of human EPO into goldfish stimulates red blood cell production (Taglialatela and Della Corte, 1997) demonstrating that if EPO is synthesized it could enhance red blood cell production. It must be pointed out, however, that the available data on hypoxia-induced EPO regulation in fish is not clear in its conclusion. When Fugu EPO gene and promoter region constructs (6 kb) are transfected into human carcinoma cell lines transcription is not hypoxia responsive (Chou et al., 2004) and this is supported by a lack of an HIF-binding hypoxia response element (HRE) in the promoter region of Fugu. However, when these same promoter region constructs were transfected into fish cell lines, increased expression of an alternatively spliced EPO transcript was observed in cells subjected to hypoxia (Fraser et al., 2006), suggesting at least some degree of hypoxia regulation of EPO in fish. Hypoxia-induced changes in myoglobin (Mb) expression have recently received considerable attention in fish and have been discussed by Wells (see Chapter 6) and Gamperl and Driedzic (see Chapter 7). Typically, Mb is expressed at high levels in red-skeletal and cardiac muscle, but recent evidence has shown a hypoxia-induced expression of Mb in nonmuscle tissues of the hypoxia-tolerant common carp (Cyprinus carpio; Fraser et al., 2006) and in the gills of zebrafish (van der Meer et al., 2005). In the carp, increases in Mb mRNA were observed during 1–8 days of hypoxia exposure in the liver, gill, and brain. Increases in mRNA were reflected in increased protein expression determined using 2D gel electrophoresis, which suggests that the increase in Mb expression may enhance O2 diVusion into tissues during hypoxia exposure. Enhanced expression of Mb in the gills of zebrafish (van der Meer et al., 2005), suggests a potential generalized role for Mb in facilitating O2 transport in fish tissues; however, it is interesting to reiterate that the expression of Hb genes were not aVected in the same gills during hypoxia exposure. A brain-specific myoglobin was also identified in the common carp, distinct from neuroglobin, but it was not hypoxia responsive at the transcript level. Additional details on Mb and neuroglobin expression in fish can be found in Chapter 6 of this volume. 4.2. O2-Independent ATP Production Hypoxia exposure in fish elicits a strong activation of substrate-level phosphorylation via glycolysis and CrP hydrolysis and a decrease in aerobic metabolism. Endogenous glycogen typically serves as the carbohydrate store 456 JEFFREY G. RICHARDS for glycolysis, thus the levels of tissue glycogen are indicative of the capacity of a tissue to support ATP turnover via glycolysis. Furthermore, due to the suppression of appetite and digestive function during hypoxia (see Chapter 8) endogenous stores of fermentable fuels represent the only source of substrate to support ATP production. As illustrated in Table 10.2, hypoxia-tolerant animals such as carp, goldfish, killifish, and oscar typically have higher levels of tissue glycogen relative to animals considered to be hypoxia sensitive (e.g., rainbow trout). Thus, it seems reasonable to conclude that across broad taxonomic groups of fish, those animals with more glycogen will be able to produce more ATP for longer periods of time at lower O2 levels (see Figure 10.1B). Another striking feature illustrated by Table 10.2 is the very large glycogen stores that occur in liver compared with those observed in other tissues including the heart, brain, and skeletal muscle. Liver glycogen is thought to serve as a repository of glucose that can be used by other tissues to support glycolytic ATP production during hypoxia exposure; however, for this to occur the glucose liberated from liver glycogen must be transported between tissues. The details of glucose transport during hypoxia exposure are outlined in Chapter 7. At the molecular level, every cDNA microarray study performed on fish has shown a typical hypoxia-induced metabolic switch, that is, a reduction in mRNA levels for proteins involved in aerobic metabolism and an increase in the mRNA levels for proteins involved in anaerobic metabolism (see Table 10.1; glycolysis, creatine kinase, and aerobic metabolism). For example, in zebrafish embryos, Ton et al. (2003) showed a decrease in the expression of mRNA coding for genes involved in the TCA cycle, including succinate dehydrogenase, malate dehydrogenase, and citrate synthase, and an increase in expression of genes involved in glycolysis including phosphoglycerate mutase, phosphoglycerate kinase, enolase, aldolase, and lactate dehydrogenase (details of hypoxia exposure given in Table 10.1). Similarly, in gills of zebrafish exposed to hypoxia, the levels of mRNA for proteins involved in the TCA cycle and electron transfer chain were all decreased signifying an overall decline in mitochondrial ATP production (van der Meer et al., 2005). Simultaneously, increases in mRNA coding for proteins involved in glycolytic ATP production were noted in the gill during hypoxia exposure, including increases in glycogen phosphorylase and aldolase. Further, there was a general decrease in the expression of genes that code for proteins involved in fat metabolism, cellular uptake, and transport, including acyl-CoA dehydrogenase, intestinal fatty acid binding protein, and other metabolite binding proteins. Also associated with the metabolic switch from aerobic to anaerobic metabolism were highly sensitive changes in the expression of pyruvate dehydrogenase kinase, which was up-regulated in muscle of killifish (Fundulus heteroclitus) during hypoxia exposure, but Table 10.2 Glycogen content in brain, liver, and muscle of fish Common name Scientific name Brain Liver Skeletal muscle Crucian carp Carassius carassius 13 to 204 123 to 2160 Goldfish Carassius auratus 13 to 20 800 Rainbow trout Oncorhynchus mykiss 0.5 110 Killifish Fundulus heteroclitus N/A 299 to 550 10 to 40 Oscar Astronotus ocellatus 203 to 279 25 to 30 Blue discus Symphysodon aequifasciatus 100 15 Tilapia African Lungfish Pacu Silver catfish Oreochromis mossambicus Protropterus dolloi Piaractus mesopoamicus Rhamdia quelen 175 mg/g protein 98 to 180 ca. 500 Glycogen content is reported in mmol glucosyl units/g wet tissue unless otherwise stated. 30 Heart References 18 to 493 Voranen et al. (Chapter 9) Voranen et al. (Chapter 9); (Mandic et al. (In Press) Voranen et al. (Chapter 9) Fangue et al. (2008); Richards et al. (2008) Chippari-Gomes et al. (2005); Richards et al. (2007) Chippari-Gomes et al. (2005) Chang et al. (2007) Frick et al. (2008) Moraes et al. (2006) 142 25 to 60 8 to 10 15 21 N/A 50 to 60 458 JEFFREY G. RICHARDS these changes did not yield measurable changes in PDK protein content (Richards et al., 2008). Associated with the large increases in the expression of glycolytic enzymes, increases in mRNA levels for proteins involved in amino acid catabolism have been demonstrated. In the liver of longjaw mudsucker, Gracey et al. (2001) noted increases in S-adenoylmethionine synthase and cystathione synthease, which catalyze steps in methionine degradation as well as several aminotransferases. Consistent with the induction of aminotransferases was the coexpression of glutamine synthetase, which catalyzes the major liver ammonia detoxification reaction of the synthesis of glutamine from glutamate. Catabolism of gluconeogenic amino acids, such as tyrosine and serine, yields either pyruvate or TCA cycle intermediates, both of which can serve as carbon skeletons for gluconeogenesis. Further evidence linking amino acid catabolism with hypoxia-induced gluconeogenesis is that the expression of glucose-6 phosphatase was strongly induced in response to hypoxia. Glucose-6 phosphatase catalyzes the dephosphorylation of glucose-6 phosphate to glucose, which can be transported in the circulation to other tissues to fuel glycolysis. Thus, for the longjaw mudsucker, amino acid catabolism coupled with gluconeogenesis in the liver may represent a mechanism to maintain blood glucose levels during hypoxia and may contribute to maintaining whole animal energy balance. Changes in the mRNA levels for several metabolite transporters have also been noted in many studies. For example, mRNA for MCT4, a membrane-bound lactate/pyruvate transporter, increased in response to hypoxia exposure in zebrafish (Ton et al., 2003). Furthermore, there were dramatic increases in the expression of glucose transporters (GLUT) in eye, gill, and kidney of grass carp during exposure to hypoxia (up to 170 h at 0.6% air saturation; Zhang et al., 2003). Changes in both of these transporters in response to hypoxia exposure indicate an overall increase in the movement of substrates for glycolysis and waste products (lactate). Tissue-specific eVects of hypoxia exposure have been noted in several studies suggesting that not all tissues respond similarly to hypoxia. DiVerential gene expression responses have been noted in the liver and muscle of the longjaw mudsucker during hypoxia exposure (Gracey et al., 2001). In the liver of the mudsucker, there was an overall increase in mRNA levels for proteins involved in glycolysis, with large increases observed in mRNA for LDH-A, triosephosphate isomerase, PFK-2/FBP-2, enolase, and glucose 6-phosphatase. Smaller, yet significant increases in mRNA were also noted for cytochrome b and cytochrome c oxidase, which are proteins of the mitochondrial electron transport chain, and possibly point to an enhancement of overall capacity for the mitochondria in liver to sustain at least some level of ATP production. In muscle tissue, however only minor increases in 10. METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA 459 mRNA for the glycolytic enzymes (LDH-A and PFK-2) were observed and in direct contrast to the response observed in liver, substantial decreases in mRNA levels for the glycolytic enzymes enolase, GAPDH, and glucose 6-phospate dehydrogenase, as well as creatine kinase were observed. Furthermore, unlike the eVects of hypoxia on liver mRNA levels, there were substantial increases in the expression of cytochrome b and cytochrome c oxidase I in muscle. In general, the tissue-specific responses observed in the mudsucker at the mRNA level is consistent with the tissue-specific eVects of 4 weeks of hypoxia exposure (15% air saturation) in Fundulus grandis (Martinez et al., 2006). This study clearly demonstrated that enzyme activities of glycolysis and glycogen metabolism were strongly suppressed by hypoxia exposure in skeletal muscle, while in liver there was evidence for increases in several enzymes involved in glycolysis and carbohydrate oxidation (Figure 10.4). Fewer changes in glycolytic and glycogen enzymes were observed in the heart and brain compared with the liver and muscle and those that did change, did so with a smaller magnitude. Interestingly, among the tissues that showed general increases in enzymes within the glycolytic pathway, the enzymes that increased were not always the same enzymes. Martinez et al. (2006) speculated that tissue-specific diVerences in the responses to long-term hypoxia in Fundulus grandis reflect the balance of energetic demands, metabolic role, and oxygen supply to the tissues. More studies are needed to examine the tissue-specific eVects of hypoxia exposure on metabolic energy supply. 4.3. Metabolic Rate Suppression The ability to suppress cellular ATP demand to match the limited capacity for O2-independent ATP production has emerged as the unifying adaptive strategy ensuring hypoxia survival (Hochachka et al., 1996). Because ATP turnover rates cannot be measured directly in vivo, those interested in measuring metabolic rate must use indirect measures. The two typical indirect measures for metabolic rate is O2 consumption and heat loss. Measurements of O2 consumption only determine the contributions of aerobic metabolism to overall ATP turnover and therefore during periods of metabolic stress that lead to increases in substrate-level phosphorylation O2 consumption can underestimate total ATP turnover or metabolic rate. The best indirect measure of metabolic rate (often referred to as the ‘‘direct’’ measure of metabolic rate to demonstrate its superiority) is the measurement of heat loss. Metabolic heat production is proportional to ATP turnover, therefore a reduction in heat loss can be directly linked with a reduction in total ATP turnover and metabolic rate suppression. 460 JEFFREY G. RICHARDS 100 Skeletal muscle Liver Heart Brain 50 Percent change in hypoxia 0 n.d. −50 −100 100 50 0 HK PGI PFK ALD TPI GAPDH PGK PGM ENO PYK LDH −100 HK PGI PFK ALD TPI GAPDH PGK PGM ENO PYK LDH −50 Enzyme Fig. 10.4. EVects of long-term hypoxia exposure on glycolytic enzyme activities (i.u. mg-1 protein) in tissues of Fundulus grandis. The y axis represents the percentage change in the mean value for each enzyme measured from hypoxic fish relative to the normoxic value for (A) skeletal muscle, (B) liver, (C) heart, and (D) brain. HK, hexokinase; PGI, phosphoglucoisomerase; PFK, phosphofructokinase; ALD, aldolase; TPI, triose phosphate isomerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PGK, phosphoglycerokinase; PGM, phosphoglyceromutase; ENO, enolase; PYK, pyruvate kinase; LDH, lactate dehydrogenase. [Data from Martinez et al. (2006) with permission.] 4.3.1. Evidence of Metabolic Rate Suppression in Fish Heat production in fish during hypoxia/anoxia exposure has been assessed in several species including goldfish (Carassius auratus; Stangl & Wegener, 1996; van Waversveld et al., 1988a,b; van Ginneken et al., 2004), crucian carp (Carassius carassius; Johansson et al., 1995), tilapia 10. METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA 461 (Oreochromis mossambiscus; van Ginneken et al., 1997, 1999), European eel (Anquilla anquilla; van Ginneken et al., 2001), zebrafish (Brachydanio rerio; Stangl & Wegener, 1996), and in isolated hepatocytes from rainbow trout (Oncorhynchus mykiss; Rissanen et al., 2006) (see Table 10.3). Interestingly, across a very broad range of fish species including those considered to be hypoxia tolerant (crucian carp, goldfish, and tilapia) and tissues from those considered to be intolerant (rainbow trout), all species show the capacity to decrease metabolic rate in response to hypoxia exposure. The most impressive reductions in metabolic rate, however, still occurred in the goldfish, tilapia, and European eel with an 70% decrease in metabolic rate during hypoxia exposure (Table 10.3). Hepatocytes isolated from rainbow trout showed a decreased metabolic rate to a lesser degree than seen in more hypoxia-tolerant animals such as goldfish and tilapia (whole-animal measurements), but comparisons between isolated tissues and whole animals are diYcult to make because of tissue-specific responses to hypoxia. Oddly, zebrafish exposed to severe hypoxia (<6% air saturation) for only 50 min showed a progressive increase in heat production indicating an overall increase in metabolic rate during hypoxia exposure (Stangl and Wegener, 1996). This increase in metabolic rate may represent increased costs associated with hypoxia-induced movement and escape behavior. Although metabolic rate suppression is clearly a response of fish to hypoxia/anoxia exposure, due to the limited number of studies available it is not possible to comment with any certainty on the direct association between the degree of metabolic rate suppression and overall hypoxia tolerance. 4.3.2. Mechanisms of Metabolic Rate Suppression The question of how organisms are able to reduce metabolic rate below routine levels has received considerable attention over the past several decades. Original work in this field using hepatocytes isolated from the anoxia-tolerant turtle (Chrysemys picta), demonstrated that a 94% suppression in metabolic rate during anoxia exposure was achieved through the dramatic down-regulation of Na pumping, protein turnover, urea synthesis and gluconeogensis (Buck & Hochachka, 1993; Buck et al., 1993a,b; Land et al., 1993; Hochachka et al., 1996; Hochachka and Lutz, 2001). It is now clear that cellular mechanisms underlying metabolic rate suppression are similar across broad taxonomic groups with metabolic rate suppression involving the controlled arrest of processes involved in membrane ion movement (Buck and Hochachka, 1993; Richards et al., 2007), protein synthesis (Lewis et al., 2007; Wieser and Krumschnabel, 2001), RNA transcription, urea synthesis, gluconeogensis, and other anabolic pathways (Hochachka et al., 1996). Table 10.3 Maximum recorded decreases in metabolic rate in fish during hypoxia/anoxia exposure Common name Goldfish Goldfish Goldfish Goldfish Goldfish Crucian carp Tilapia Tilapia European eel Rainbow trout Zebrafish a Scientific name Carassius auratus C. auratus C. auratus C. auratus C. auratus Carassius carassius Oreochromis mossambicus Oreochromis. mossambicus Anquilla anquilla Oncorhynchus mykiss Brachydanio rerio Metabolic Rate Suppression (% decrease from normoxia) Whole animal or tissue Temperature ( C) Whole animal 20 Anoxia 2 to 3h 70 Whole animal Whole animal Whole animal Whole animal 20 20 20 20 3h 3h 3h 5h 59 53 70 to 85 55 Brain slices 12 10% air sat. 5% air sat. Anoxia 3% air sat. Progressive Anoxia van Waversveld et al. (1988, 1989a) van Waversveld et al. (1989a) van Waversveld et al. (1989a) Stangl and Wegener (1996) van Ginneken et al. (2004) 20 h 37 Johansson et al. (1995) Whole animal 20 5% 8h 55 van Ginneken et al. (1997) Whole animal 20 1h 64 van Ginneken et al. (1999) Whole animal 20 3% air sat. Progressive Anoxia 1h 70 van Ginneken et al. (2001) 4% air sat. Progressive 6% air sat. 6 to 12 min 46 Rissanen et al. (2006) 50 min Increase by 50b Stangl and Wegener (1996) Hepatocytes Whole animal 201a 25 Hypoxia Duration References Animals were acclimated to 12 C and metabolic rate was measured in isolated hepatocytes at 20 C. Metabolic rate increased upon exposure to hypoxia. In studies with more than one level of hypoxia shown, the degree of metabolic rate suppression for the most severe level of hypoxia is shown. b 10. METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA 463 The suppression of protein synthesis has been described in both isolated hepatocytes and fish in vivo in species ranging from the crucian carp (Smith et al., 1996; see Chapter 9), to goldfish (Jibb and Richards, 2008), to the Amazonian oscar (Astronotus ocellatus; Lewis et al., 2007). In the oscar, severe hypoxia exposure (10% air saturation) caused tissue specific decreases in protein synthesis rates that varied from 27% decreases in protein synthesis rate in brain to 60% decreases in heart. In the crucian carp, Smith et al. (1996) also demonstrated substantial decreases in protein synthesis rates in heart, liver, and muscle in response to anoxia exposure and these decreases were, in part, mediated by decreases in RNA transcription rates (Smith et al., 1999). In the goldfish, hypoxia exposure (<0.5% air saturation) caused a very rapid (within 0.5 h) 70% decline in liver protein translation rate (assessed in cellfree isolates). These decreases in protein synthesis rates in the hypoxic goldfish were mediated through specific phosphorylation of eukaryotic elongation factor-2 (Jibb and Richards, 2008), which halts protein elongation during translation (Figure 10.5). Few studies have examined how other ATP-consuming processes besides protein synthesis are modified during hypoxia exposure in fish, but some modifications to ion pumping have been noted. In particular, hypoxia-induced decreases in the activity of Na+/K+-ATPase as observed in some studies (Bogdanova et al., 2005) could represent a substantial ATP saving, but results are conflicting. The crucian carp does not decrease brain Na+/K+ATPase activity during anoxia exposure (reviewed in detail in Chapter 9; Hylland et al., 1997) despite increases in the inhibitory neuromodulators GABA (Nilsson, 1992) and adenosine (Nilsson, 1991). This lack of an eVect of anoxia/hypoxia exposure on Na+/K+-ATPase activity in the brain of crucian carp is unlike the response observed in turtles, which suppress the activity of Na+/K+-ATPase. The diVerential responses observed in the two champions of anoxia tolerance is probably associated with the fact that crucian carp remains active during anoxia exposure, unlike the comatose turtle (see Chapter 9). Recent work by Richards et al. (2007) demonstrated a substantial decrease in gill Na+/K+-ATPase activity in the oscar exposed to hypoxia (5% air saturation) and it was speculated that this decrease was achieved by a post-translational modification to the Na+/K+-ATPase protein. A similar eVect of hypoxia exposure was observed in isolated trout hepatocytes, where hypoxia caused a transient down-regulation of Na+/K+ATPase activity (Bogdanova et al., 2005). These authors speculated that decreases in Na+/K+-ATPase activity in response to hypoxia may be accomplished by local changes in reactive oxygen species, but no precise mechanism was given. 464 JEFFREY G. RICHARDS A Phoshpo-eEF2 at Thr56 (Normalized to total eEF2) 3 * * * * 2 1 0 N 1 2 4 8 Time in hypoxia (h) 0.5 1 2 4 Time in hypoxia (h) 12 B Phospho-eEF2 Total eEF2 N Protein synthesis rate (Pmol leucine . mg total protein−1 . h−1) C 8 12 5 4 3 2 * * 1 * * * 4 8 Time in hypoxia (h) 12 * 0 N 1 2 Fig. 10.5. Liver phospho-eEF2 (A), representative phosphoThr56-eEF2 and eEF2 Western blots (B), and protein synthesis rate (C) in goldfish exposed to normoxia and 12 h of hypoxia. [Data from Jibb and Richards (2008) with permission.] 10. METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA 465 4.3.3. Molecular Responses that Facilitate Metabolic Rate Suppression As outlined above, decreases in protein synthesis rates are an important response to hypoxia exposure reducing ATP demands and facilitating whole animal metabolic rate suppression. To this end, across a number of tissues, including muscle, liver, and gills, cDNA microarray studies have demonstrated dramatic decreases in mRNA coding for proteins involved in protein synthesis. In muscle, the levels of mRNA coding for elongation factor 2 and several ribosomal proteins have all been shown to be substantially reduced in response to hypoxia exposure (Table 10.1; Gracey et al., 2001). Similarly, in the gills of zebrafish exposed to hypoxia, decreases in mRNA coding for ribosomal proteins have been shown; however, the same study showed a curious accumulation of mRNA coding for elongation factors (van der Meer et al., 2005). Metabolic energy saving can also be realized through a reduction in movement (Chapter 2) and the maintenance of cellular machinery for movement. Genes involved in muscle contraction including a-tropomyosin, myosin heavy chain, myosin regulatory light chain 2A, skeletal muscle a-actin, and b-actin were, for the most part, all strongly suppressed in response to hypoxia exposure in the mudsucker (Gracey et al., 2001). A similar response was also noted in zebrafish embryos exposed to hypoxia with decreases in mRNA coding for proteins involved in contraction, extracellular matrix, and cytoskeletal proteins (Ton et al., 2003). Cell growth and proliferation is generally suppressed during hypoxia exposure as a mechanism for ATP conservation. Gracey et al. (2001) observed mRNA increases for a number of genes involved in the suppression of cell growth and proliferation. For example, elevated levels of mRNA for insulin-like growth factor binding protein 1 (IGFBP-1), which regulates the availability of insulin-like growth factors in circulation, were observed in liver. Increases in MAP-kinase phosphates were also observed, including MKP-2, which attenuates the activity of the ERK group of MAP kinases. These kinases are phosphorylated in response to the binding of growth factor to cell-surface receptors and activate a signaling cascade that stimulates cell growth. The importance of inhibition of cell growth as an adaptive response to hypoxia exposure is best illustrated by the elegant work of Sollid and Nilsson (Sollid and Nilsson, 2006; Sollid et al., 2006; see Chapter 9). Briefly, in the crucian carp, hypoxia exposure causes a dramatic increase in gill surface area, mediated primarily by a decrease in cell division and increase in apoptosis in the intralamellar space. However, hypoxia does not yield an increase in mRNA consistent with an increase in cellular apoptosis in zebrafish gills (van der Meer et al., 2005). 466 JEFFREY G. RICHARDS Ton et al. (2003) showed repression of several genes involved cell division such as cyclin G1 and proliferating cell nuclear antigen in zebrafish embryos, which is consistent with observations that hypoxia causes these embryos to undergo developmental arrest and enter a state of suspended animation (Padilla and Roth, 2001). 5. COORDINATING THE METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA Cell survival during hypoxia exposure requires a metabolic reorganization to decrease ATP demands to match the reduced capacity for ATP production and these metabolic responses must be coordinated temporally otherwise hypoxia exposure will lead to cell death. Several signal transduction cascades have been shown to be activated in response to hypoxia exposure in mammals and other vertebrates (Storey and Storey, 2004), but considerably less work has been done in fishes. In the remaining part of this chapter, I will outline recent advances in the role of one specific signal transduction cascade, the AMP-activated protein kinase, and its role in coordinating the metabolic responses to hypoxia followed by the role of HIF in coordinating the gene expression responses described in this chapter and others. 5.1. AMP-Activated Protein Kinase as a Metabolic Coordinator Recent evidence has suggested that AMP-activated protein kinase (AMPK) may play a critical role in coordinating the metabolic responses to hypoxia in the hypoxia-tolerant goldfish. AMPK is a heterotrimeric protein kinase comprised of a catalytic subunit (a) and two regulatory subunits, and phosphorylation of AMPK at Thr-172 on the a-subunit activates the protein (Carling, 2004). Activation of AMPK in mammals inhibits energetically expensive anabolic processes including protein synthesis (Horman et al., 2002), glycogen synthesis (Nielsen et al., 2002), and fatty acid synthesis (Hardie and Pan, 2002) rates. Furthermore, activation of AMPK increases skeletal muscle hexokinase activity, GLUT-4 glucose transporter expression (Holmes et al., 1999), and translocation to the membrane (Kurth-Kraczek et al., 1999), and increased phosphofructokinase-2 (PFK-2) activity in rat cardiomyocytes (Marsin et al., 2000), all of which could enhance O2-independent ATP production. Combined, these actions have led to AMPK being termed the cellular ‘‘energy gauge’’ because of its critical role in maintaining cellular energy balance. 10. METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA 467 Jibb and Richards (2008) demonstrated that AMPK was activated in the liver of goldfish exposed to severe hypoxia and that there was a close temporal change in [AMPfree]/[ATP] and AMPK activity. Increases in AMPK activity in the liver were associated with an increase in the percent phosphorylation of a well-characterized target of AMPK, eukaryotic elongation factor-2 (eEF2), and decreases in protein synthesis rates measured in liver cell-free extracts (Figure 10.5) suggesting that a disruption of cellular energy status is important for the activation of mechanisms involved in metabolic rate suppression. AMP-activated protein kinase, however, was not activated in muscle, brain, heart, or gill during 12 h of severe hypoxia exposure in goldfish suggesting a tissue-specific regulation of AMPK and metabolic responses to hypoxia (Jibb and Richards, 2008). 5.2. Hypoxia Inducible Factor Hypoxia-regulated gene expression was described some decades ago, but it wasn’t until 1992 that the O2-regulated transcription factor, HIF-1a, was identified as a key regulator of hypoxia-regulated gene expression (Semenza and Wang, 1992). Since its discovery, HIF has been viewed as the molecular master factor of the hypoxic response and a great deal of information is now available on the genes and gene families regulated by HIF (Semenza, 2007; Gardner & Corn, 2008). Many excellent reviews of hypoxia-regulated gene expression and HIF are present in the literature (e.g., Kenneth and Rocha, 2008) including several on fish (e.g., Nikinmaa and Rees, 2005). In the remaining part of this chapter, I will outline HIF regulation in mammals and then describe what is known of HIF function in fish using the work done in mammals as a point of reference. Hypoxia inducible factor is a heterodimeric transcription factor composed of two subunits; an O2-sensitive HIF a subunit and an O2 stable HIF b (also referred to as the aryl hydrocarbon receptor nuclear translocator; ARNT). Hypoxia inducible factor a and b subunits are both members of a very large family of transcription factors known as bHLH/PAS domainproteins, named because all members of this family contain a basic helixloop-helix (bHLH) domain as well as one or several PAS domains (domain named after its first members Per, ARNT, and Sim). The bHLH/PAS domain-containing transcription factors constitute a superfamily of transcription factors that are capable of forming homo- and heterodimers through the bHLH and PAS domain and have been implicated in regulating the transcription of genes involved in circadian rhythm, central nervous system development, and induction of hydrocarbon metabolizing enzymes, as well as the cellular responses to hypoxia. Hypoxia inducible factor a, 468 JEFFREY G. RICHARDS unlike HIF b, contains an O2-dependent degradation domain (ODD), rendering these proteins labile in the presence of O2. In mammalian systems, HIF is regulated through the post-translational modifications of HIF a, which aVects both protein stability and transactivation (Figure 10.6). The O2-dependent control of HIF a is provided by the actions of two proteins, prolyl hydroxylase (PHDs) and factor inhibiting HIF (FIH; Mahon et al., 2001), both of which are members of the 2oxoglutarate-dependent dioxygenase superfamily of hydroxylases. These proteins both require iron and 2-oxoglutarate as cofactors or substrates (Schofield et al., 1999), and possess many of the features of an O2-sensitive control mechanism (Land and Hochachka, 1995). Under conditions of normal cellular O2 tensions, HIF a and HIF b are continuously transcribed and translated; however, HIF a is rapidly hydroxylated at two conserved proline residues in the ODD by PHD. HIF a proteins containing hydroxylated O2 Hypoxia Nomoxia Anoxia O2 FIH PHD HIF 1 α HIF 1 β FIH PHPHD HIF 1 α ODD cTAD P300/CBP PAS-B PAS-A bHLH HIF 1 β OH PAC HIF 1 α HIF 1 β PAS OHOH P300/CBP bHLH HIF 1 α VHL mRNA transcription VHL OH OH OH HIF 1 α Degraded by proteosome tacgtc n bq n bq Fig. 10.6. Regulation of hypoxia inducible factor by O2. In normoxic cells, propyl hydroxylases (PHD) and factor inhibiting HIF (FIH) enzymes use O2 to hydroxylate key resides on the HIF a subunit in the oxygen-dependent domain (ODD). Hydroxylation of the ODD signals the von Hippel-Lindau (VHL) protein binding leading to ubiquitination and subsequent degradation by the proteosome. The stability of HIF b is not aVected by O2 levels. During periods of cellular hypoxia, PDH and FIH are inhibited resulting in the stabilization of HIF a and as HIF a accumulates it dimerizes with HIF b, recruits other co-activators (e.g. p300/CBP) and activates the transcription of genes containing hypoxia response elements in their promoter region. 10. METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA 469 proline residues are recognized by an E3 ubiquitin ligase, the von HippelLindau protein (VHL), which promotes Lys48-linked ubiquitination and targets HIF a for rapid degradation by the cellular proteasome. HIF a is also hydroxylated at a conserved asparagine residue in the C-terminaltransactivation domain (cTAD) by FIH, which prevents the recruitment of the p300/CBP transcriptional coactivators leading to a reduced ability of HIF a to transactivate and an overall suppression of HIF regulated gene transcription (Linke et al., 2004). Thus, under normoxic conditions, HIF a protein is continually made but prevented from accumulating or initiating transcription through the PHD-mediated ubiquitin-proteasome degradation and the FIH inhibition of transactivation. In a normoxic cell, HIF a has a half-life of approximately 5–10 min. The onset of cellular hypoxia leads to an inactivation of PHD and FIH and a lack of HIF a hydroxylation. The lack of HIF a proyl hydroxylation prevents pVHL from recognizing HIF a and initiating the ubiquitinregulated protein degradation. Thus, cellular hypoxia leads to an almost instantaneous stabilization and accumulation of HIF a, which migrates into the nucleus and dimerizes with HIF b. The HIF a/HIF b dimer then binds with the p300/CBP coactivator, and the complete complex binds to specific hypoxia-response elements (HRE) in the promoter regions of target genes. The absence of asparagine hydroxylation by FIH is permissive for the HIF dimer to interact with transcriptional coactivators and initiate transcription (Lando et al., 2002). Numerous genes have been reported to possess HRE and associated elements in their 50 promoter regions and their HIF regulation has been described; the known hypoxia-induced gene expression response in fish is described below. 5.2.1. HIF Isoforms 5.2.1.1. HIF a Isoforms. Three HIF a subunit isoforms have been identified in mammals (designated HIF 1a, HIF 2a, HIF 3a; Gu et al., 1998) and some diVerences between these isoforms have been described, although the precise function of these isoforms has not been fully elucidated. Hypoxia inducible factor 1a and 2a both contain transactivation domains (cTAD domains), while HIF 3a appears to lack the cTAD and, as such, it has been proposed that HIF 3a may act as an inhibitor of HIF 1a and HIF 2a (Bardos and Ashcroft, 2005). HIF 1a and 2a have been shown to have non-redundant functions in the cell, and although HIF 1a is the best-studied isoform, recent studies in mammals have illuminated important roles for HIF 2a in cancer tumor growth (Carroll and Ashcroft, 2006; Hu et al., 2006). HIF 2a has also been shown to be expressed at high levels in certain cell types such as vascular endothelial cells, kidney fibroblasts, hepatocytes, glial cells, interstitial cells of the pancreas, and epithelial cells of the intestinal lumen (Jain et al., 1998). 470 JEFFREY G. RICHARDS The first fish HIF a sequence was determined for the rainbow trout by Soitamo et al. (2001) and since that point, a total of 38 HIF a gene sequences have been identified in fish either through direct sequencing (Powell & Hahn, 2002; Law et al., 2006; Rahman and Thomas, 2007; Rojas et al., 2007; Rytkönen et al., 2007) or as part of genome sequencing projects (see Ensembl genome projects for Zebrafish, Fugu, Tetradon, Medaka, and Stickleback; Figure 10.7). For the most part, fish HIF a protein sequences are slightly shorter than their counterparts in tetrapods. For example, the length of HIF 1a in fish is between 699 and 778 amino acids while in tetrapods HIF 1a is between 800 and 836 amino acids long. Phylogenetic analysis of available tetrapod, bird, and fish HIF a sequences indicate that the three major classes of HIF a sequences seen in mammals are represented in fish (Figure 10.7). For the most part, within each isoform class, fish sequences group closely together and are distinct from their tetrapod and bird counterparts. The one exception is for HIF 1a from the Russian sturgeon (Acipenser gueldenstaedtii), which groups more closely with the birds (chicken, Gallus gallus) and tetrapods. This grouping of the more pleisiomorphic sturgeon HIF 1a with tetrapods and birds suggests that the more derived teleost fish species may have a faster changing HIF 1a sequence. Several HIF 4a isoforms have been identified in fish (Law et al., 2006; also see orange-spotted grouper; Epinephelus coioides), however their identification at the time was based upon a lack of similarity to the scant fish and tetrapod HIF sequences. Since that time, the proliferation of available HIF a sequences and the phylogenetic analysis performed in Figure 10.7 suggests that previously identified HIF 4a sequences are in fact HIF 3a sequences. Among the isoforms identified in fish, all the appropriate functional domains can be identified. For example, sequence analysis of deduced amino acid sequence of fish HIF 1a genes reveals the presence of four major functional domains including the bHLH domain, two PAS domains (PAS-A and PAS-B), ODD domain, and the DNA-binding domain termed cTAD. These four major functional domains are the same as those seen in tetrapod and bird HIF 1a sequences. Sliding window analysis of 11 fish HIF 1a gene sequences clearly demonstrates that the amount of amino acid sequence variability between fish species is lowest at the four major functional domains (Figure 10.8). These analyses suggest that the amino acid sequence of the important functional domains is well conserved across fish species. Sites showing a high degree of sequence variability occur in areas that have not been identified as important for HIF 1a function. Given the high degree of similarity between fish, tetrapod, and bird sequences it seems reasonable to generalize that HIF 1a functions in fish in much the same way as it does in tetrapods (Figure 10.6). 10. 471 METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA Stizostedion incioperca Gymnocephalus cernuus Perca fluvitilis Pachycara brachycephalum 42 99 Zoarces viviparus 39 Gacterosteus aculeatus 13 Epinephelus coioides 35 Oryzias melastigma Platichys flesus 17 53 Micropogonias undulatus Dicentrarchud labrax 27 Takifugu rubripes 93 76 Tetraododn nigroviridis Esox lucius Oncorhynchus mykiss 99 Thymallus thymallus 66 Ictalurus punctatus 48 Ctenopharyngodon idella 100 Aspius aspius Danio rerio 99 Carassius carassius 17 Cyprinus carpio 46 17 Gymnocypris przewalskii 98 99 54 42 95 HIF-1 α Mustelus canis Rana temporaria Xenopus laevis Acipenser gueldenstaedtii 94 30 Gallus gallus 40 94 Bos taurus Bos grunniens 61 Pantholops hodgsonii Homo sapiens 31 95 Macaca fascicularis Oryctolagus cuniculus 32 98 100 99 Spermophilus tridecemlineatus 91 Rattus norvegicus Mus musculus 73 Microtus oeconomus Spalax judaei 57 98 Eospslax baileyi Eospslax cansus 81 Rattus norvegicus 87 99 Mus musculus 100 Bos taurus 90 99 83 Coturnix cotumix Ctenopharyngodon idella Ictalurus punctatus Trematomus hansoni 99 HIF-2 α Chionodraco myersi 65 Oryzias latipes Fundulus heteroclitus Micropogonias undulatus Takifugu rubripes 85 Rattus norvegicus 100 100 Mus musculus Bos taurus 51 Homo sapiens Ctenopharyngodon idella 100 Danio rerio Ictalurus punctatus Epinephelus coioides 65 Oryzias latipes 53 38 99 56 94 99 HIF-3 α Takifugu rubripes Tetraodon nigroviridis 0.1 Fig. 10.7. Phylogenetic analysis of HIF a isoform amino acid sequences from fish, tetrapods, and birds. The phylogeny was created from deduced amino acid sequences from GenBank or Ensembl: Oncorhynchus mykiss HIF 1 (AF304864); Thymallus thymallus HIF 1(ABO26714); 472 JEFFREY G. RICHARDS 5.2.1.2. HIF b Isoforms. At least two HIF b isoforms have been identified in tetrapods; HIF 1b, which is ubiquitously expressed in most tissues, and HIF 2b, which is primarily restricted to nervous system and kidneys at specific developmental stages (Hirose et al., 1996). In fishes, a total of 14 HIF b isoforms have been identified and in general they group closely with other known HIF b isoforms identified in tetrapods and birds (Figure 10.9). At the sequence level, HIF b is similar to HIF a in that it is a member of the bHLH/ PAS group of transcription factors and both isoforms possess bHLH and PAS domains. HIF b also possesses a terminal activation domain (AD). Sequence analysis of all available fish HIF b sequences reveals a high degree of sequence conservation in these well-identified functional domains (Figure 10.10). Overall, this high degree of sequence similarity and the conservation of important regulatory and functional domains suggest that fish HIF b probably functions in a similar fashion to its tetrapod and bird orthologs. Esox lucius HIF 1(ABO26715); Micropogonias undulates HIF 1 (ABD32158); Perca fluviatilis HIF 1 (ABO26717); Stizostedion lucioperca HIF 1 (ABO26718); Gymnocephalus cernuus HIF 1 (ABO26716); Pachycara brachycephalum HIF 1 (AAZ52828); Zoarces viviparous HIF 1 (AAZ52832); Dicentrarchus labrax HIF 1 (AAZ95453); Epinephelus coioides HIF 1 (AAW29027); Gasterosteus aculeatus HIF 1 (ABO26719); Oryzias latipes HIF 1 (ENSORLT00000004404); Rattus norvegicus HIF 1 (NP_075578); Tetraodon nigroviridis HIF 1 (ENSTNIG00000017339); Takifugu rubripes HIF 1 (ENSTRUG00000012093); Oryzias melastigma HIF 1 (ABC47310); Ctenopharyngodon idella HIF 1 (AAR95697); Platichthys flesus HIF 1 (ABO26720); Ictalurus punctatus HIF 1 (AAZ75952); Danio rerio HIF 1 (AAQ91619); Cyprinus carpio HIF 1 (ABV59209); Carassius carassius HIF 1 (ABC24677); Gymnocypris przewalskii HIF 1 (AAW69834); Aspius aspius HIF 1 (ABO26713); Acipenser gueldenstaedtii HIF 1 (ABO26712); Rana temporaria HIF 1 (ABY86629); Mustelus canis HIF 1 (ABY86628); Gallus gallus HIF 1 (NP_989628); Xenopus laevis HIF 1 (ABF71072); Rattus norvegicus HIF 1 (NP_077335); Eospalax baileyi HIF 1 (ABB17537); Eospalax cansus HIF 1 (ABQ53550); Microtus oeconomus HIF 1 (AAY27087); Spalax judaei HIF 1 (CAG29396); Spermophilus tridecemlineatus HIF 1 (AAU14021); Mus musculus HIF 1 (BAA20130); Oryctolagus cuniculus HIF 1 (NP_001076251); Pantholops hodgsonii HIF 1 (AAX89137); Bos grunniens HIF 1 (ABH06559); Mus musculus HIF 1 (AAH26139); Bos taurus HIF 1 (NP_776764); Homo sapiens HIF 1 (AAF20149); Macaca fascicularis HIF 1 (BAE01417); Fundulus heteroclitus HIF 2 (AAL95711); Micropogonias undulates HIF 2 (ABD32159); Takifugu rubripes HIF 2 (ENSTRUT00000013648); Ctenopharyngodon idella HIF 2 (AAT76668); Bos taurus HIF 2 (BAA78676); Mus musculus HIF 2 (NP_034267); Trematomus hansoni HIF 2 (AAZ52830); Ictalurus punctatus HIF 2 (ABK27926); Chionodraco myers HIF 2 (AAZ52827); Coturnix coturnix HIF 2 (AAF21052); Ctenopharyngodon idella HIF 4 (AAR95698); Danio rerio HIF 3 (AAQ94179); Ictalurus punctatus HIF 3 (AAZ75953); Epinephelus coioides HIF 4 (AAW29028); Rattus norvegicus HIF 3 (NP_071973); Mus musculus HIF 3 (NP_058564); Bos taurus HIF 3 (NP_001098812); Homo sapiens HIF 3 (NP_690008); Takifugu rubripes HIF 3 (ENSTRUT00000021549); Tetraodon nigroviridis HIF 3 (ENSTNIT00000009762); Oryzias latepes HIF 3 (ENSORLT00000002500). Sequences were aligned using ClustalW and phylogenetic analysis was performed using the neighbor-joining methods with complete deletion of gaps using MEGA2 software (Kumar et al., 2001). The support for each node was assessed using 500 bootstrap replicates and are presented at each branch point. Bold-face type indicates fish sequences. 10. 473 METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA A cODD bHLH PAS-A PAS-B ODD cTAD HIF 1α Variability (%) B 100 80 60 40 20 0 0 100 200 300 400 500 Amino acid 600 700 800 Fig. 10.8. Structural analysis of HIF 1a amino acid sequence. (A) Relative position of the four major functional domains of HIF 1a including the basic helix-loop-helix (bHLH) domain, two PAS domains (Per/ARNT/Sim), the O2-dependent domain (ODD), and Cterminal transactivation domain (cTAD). (B) Percentage variability among 11 HIF 1a isoforms from fish as determined by sliding window analysis on predicted amino acid sequences. Sliding window analysis quantifies the variation between aligned sequences by counting the average number of diVerences between isoforms within overlapping windows. For the present analysis, an overlapping window of 20 amino acids was used. Sliding window analysis was performed using MEGA software (version 1.02). Arrows point to the relative sequence variability of the major functional domains. Sliding window analysis of HIF 1a was performed using sequences from the following species: Perca fluviatilis, Stizostedion lucioperca, Gymnocephalus cernuus, Pachycara brachycephalum, Zoarces viviparous, Dicentrarchus labrax, Gasterosteus aculeatus, Oryzias latipes, Tetraodon nigroviridis, Takifugu rubripes, and Tetraodon nigroviridis. In addition to its role in hypoxic signaling, HIF 1b, or rather ARNT, is known to play an important role in regulating gene expression changes in response to toxic aryl-hydrocarbon exposure (Hahn et al., 2006). A number of the gene expression responses to aryl-hydrocarbon exposure are similar to those observed in response to hypoxia exposure including increases in lactate dehydrogenase gene expression. HIF 1b regulates aryl hydrocarbonmediated changes in gene expression through the binding of the aryl hydrocarbon to a specific receptor, the aryl hydrocarbon receptor (AHR). The AHR then binds to its partner, HIF 1b, and the AHR/HIF 1b heterodimer moves to the nucleus where it binds to xenobiotic responsive elements (XREs). Binding of the AHR/HIF 1b heterodimer to XRE regions adjacent to aryl hydrocarbon-inducible genes increases their transcription. Because HIF 1b is known to be involved in the responses to hypoxia and arylhydrocarbon exposure, possible interactions between responses may exist. Kraemer and Schulte (2004) demonstrated an antagonistic interaction 474 JEFFREY G. RICHARDS 63 Bos taurus 74 Sus scrofa Mus musculus 78 49 96 Canis familiaris Pan troglodytes Homo sapiens Gallus gallus Phalacrocorax carbo 99 Monodelphis domestica Xenopus tropicalis 96 Xenopus laevis 100 65 Ctenopharyngodon idella Danio rerio 98 73 67 46 Fundulus heteroclitus Gasterosteus aculeatus Micropogonias undulatus Oryzias latipes Takifugu rubripes Gasterosteus aculeatus 80 Micropogonias undulatus 65 Oryzias latipes 81 Takifugu rubripes 100 Tetraodon nigroviridis 100 Oncorhynchus mykiss Danio rerio 42 100 Xenopus laevis Ornithorhynchus anatinus 88 100 66 46 Mus musculus Rattus norvegicus Peromyscus maniculatus Pongo abelii 92 Bos taurus 62 36 33 Phoca sibirica Cavia porcellus Oryctolagus cuniculus 0.02 Fig. 10.9. Phylogenetic analysis of HIF b isoform amino acid sequences from fish, tetrapods, and birds. The phylogeny was created from deduced amino acid sequences from GenBank or Ensembl: Pongo abelii HIF 1b (NP_001125275); Gasterosteus aculeatus HIF 1b 10. METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA 475 between exposure to PCBs (3,30 ,4,40 -tetrachlorobiphenyl) and hypoxia (15% air saturation) in killifish and suggested that prior PCB exposure could make these fish less tolerant of environmental hypoxia. 5.2.1.3. PHD Isoforms. Four PDH isoforms have been identified in mammals, numbered PDH 1 to 4, and so far only PDH 1, 2, and 3 have been shown to hydroxylate HIF. Biochemical analysis has shown PHD 2 to have a higher aYnity for HIF 1a, whereas PHD 1 and PDH 3 have higher aYnity for HIF 2a (AppelhoV et al., 2004). Prolyl hydroxylase sequences have been found in fish as a result of genome sequencing projects, but to date no study has explicitly characterized the sequence or function of PHD isoforms in fish. This will undoubtedly be an important and fruitful area of research in the next few years as PHDs are now considered the cellular O2 sensors responsible for initiating the HIF response. 5.2.2. Regulation of HIFActivity in Fish The O2-dependent regulation of HIF in fish has received remarkably little attention since the literature was reviewed by Nikinmaa and Rees (2005). However, given the available data on sequence similarity between tetrapod and fish HIF a and b sequences it seems reasonable to speculate that the same or similar mechanisms of O2-dependent regulation of HIF shown in Figure 10.6 are at play in fish. Specifically, as pointed out by Rahman and Thomas (2007) for Atlantic croaker (Micropogonias undulates) and shown in (ENSGACG00000011686); Oryzias latipes HIF 1b (ENSORLG00000010551); Takifugu rubripes HIF 1b (ENSTRUG00000014504); Ornithorhynchus anatinus HIF 1b (XP_001517995); Peromyscus maniculatus HIF 1b (AAN52084); Bos taurus HIF 1b (ABG67008); Mus musculus HIF 1b (AAH12870); Oryctolagus cuniculus HIF 1b (NP_001075675); Phoca sibirica HIF 1b (BAE16957); Cavia porcellus HIF 1b (BAF02596); Rattus norvegicus HIF 1b (AAO89090); Xenopus laevis HIF 1b (NP_001082130); Oncorhynchus mykiss HIF 1b (AAC60052); Micropogonias undulates HIF 1b (ABD32160); Fundulus heteroclitus HIF 2b (AAD09750); Micropogonias undulates HIF 2b (ABD32161); Ctenopharyngodon idella HIF 2b (AAT70730); Danio rerio HIF 2b (NP_571749); Mus musculus HIF 2b (BAA09799); Rattus norvegicus HIF 2b (AAB05247); Canis familiaris HIF 2b (XP_850172); Gallus gallus HIF 2b (XP_413854); Phalacrocorax carbo HIF 2b (BAF44221); Bos taurus HIF 2b (XP_612854); Sus scrofa HIF 2b (XP_001926107); Monodelphis domestica HIF 2b (XP_001367955); Pan troglodytes HIF 2b (XP_001156233); Homo sapiens HIF 2b (NP_055677); Xenopus tropicalis HIF 2b (NP_001093686); Xenopus laevis HIF 2b (AAQ91608); Danio rerio HIF 2b (AAG25919); Gasterosteus aculeatus HIF 2b (ENSGACG00000013947); Xenopus laevis HIF 2b (NP_001083622); Oryzias latipes HIF 2b (ENSORLG00000019479); Takifugu rubripes HIF 2b (ENSTRUG00000007832); Tetraodon nigroviridis HIF b (ENSTNIG00000008064). Sequences were aligned using ClustalW and phylogenetic analysis was performed using the neighbor-joining methods with complete deletion of gaps using MEGA2 software (Kumar et al., 2001). The support for each node was assessed using 500 bootstrap replicates and is presented at each branch point. Bold-face type indicates fish sequences. 476 JEFFREY G. RICHARDS A bHLH PAS AD HIF 1β Variability (%) B 100 80 60 40 20 0 0 100 200 300 400 500 Amino acid 600 700 800 Fig. 10.10. Structural analysis of combined HIF 1b and 2b amino acid sequence. (A) Relative position of the four major functional domains of HIF b including the basic helix-loop-helix (bHLH) domain, a PAS domain (Per/ARNT/Sim), and the activation domain (AD). Panel B shows the percentage variability among all available HIF 1b and HIF 2b isoforms available in fish as determined by sliding window analysis on predicted amino acid sequences. Figure 10.8, there is a high degree of sequence similarity in the core O2dependent degradation domain regions of fish HIF a sequences, suggesting a similar mechanism of HIF degradation to that in other vertebrate species. The first and only study to address the issue of O2-dependent regulation of HIF in fish was that of Soitamo et al. (2001), which demonstrated that although HIF 1a was present under normoxic conditions (air saturation) in rainbow trout and salmon cell lines, the levels of HIF 1a protein increased during hypoxia exposure. Oddly, however, the maximum levels of HIF 1a protein were noted in cells cultured at 5% O2, which as the authors pointed out is similar to typical venous PO2. These data suggest that in vivo, HIF 1a may accumulate under what should be considered as normoxic conditions in tissues. Additional research is needed to understand how HIF functions in fish cells and whether there are diVerences in O2 sensitivity in HIF 1aregulated gene expression among fish that vary in hypoxia tolerance. 5.2.3. Hypoxia-regulated HIF a mRNA Expression Unlike in mammals, where there appears to be little or no regulation of HIF at the mRNA level, hypoxia-induced changes in HIF a mRNA and protein expression have been noted in several fish species. Law et al. (2006) examined the mRNA and protein levels of two HIF a isoforms (1 and 3; note that these authors incorrectly named HIF 3a as HIF 4a) from the hypoxiatolerant grass carp (Ctenopharyngodon idella) and showed substantial increases in HIF 1a mRNA in gill and kidney after 4 h exposure to 7% air saturation compared with normoxia-exposed fish. In the same fish, no or 10. METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA 477 few changes in HIF 1a were noted in brain, eye, gill, heart, kidney, liver, and muscle. On the other hand, substantial increases in the HIF 3a isoform (identified as HIF 4a) were observed during hypoxia exposure in all tissues examined. Similarly, Rahman and Thomas (2007) demonstrated that both HIF 1a and HIF 2a from the hypoxia-tolerant Atlantic croaker were hypoxia responsive in ovaries during short-term (3–7 days at 20% air saturation) and longer-term hypoxia exposure (3 weeks at 20–40% air saturation). There does not, however, appear to be a good relationship between hypoxia tolerance and HIF a expression, although the available data are limited. Specifically, HIF 1a mRNA levels have also been shown to increase in the liver of the hypoxia-sensitive sea bass (Dicentrarchus labrax; Terova et al., 2008) during both acute hypoxia exposure (4 h at 20% air saturation) and 15 days of chronic hypoxia (50% air saturation). 5.2.4. Relationship Between HIF Function and Hypoxia Tolerance in Fish The fact that there is enormous variation in hypoxia tolerance among fish species raises the question of whether there is a relationship between HIF function and hypoxia tolerance. In fact, careful comparisons among fish species known to vary in hypoxia tolerance open the possibility of elucidating which aspects of HIF function are adaptive and thus potentially most important in dictating hypoxia tolerance. Surprisingly however, most of our current understanding of HIF regulation and function comes from mammalian models, which typically only experience hypoxia as a result of disease such as cancer (Gort et al., 2008). To begin to address the question of whether HIF structure or responsiveness to hypoxia diVer among hypoxia-sensitive and hypoxia-tolerant fish species, Rytkönen et al. (2007) sequenced HIF 1a from nine species of fish that varied in lifestyle related to O2 requirements (hypoxia tolerance was not quantified). Analysis of sequence variation among the available fish HIF 1a amino acid sequences showed that there was no clear protein signature associated with O2 requirements (Rytkönen et al., 2007). Further analysis of these sequences and others revealed that the overall evolutionary rate in teleost HIF 1a was approximately twice as fast as the predicted evolutionary rate in mammalian HIF 1a (Rytkönen et al., 2008). Despite the faster sequence divergence, however, crucial functional domains in HIF 1a (Figure 10.6) were found to be under stringent purifying selection in all vertebrates. As a result, the faster sequence divergence occurred in the less crucial areas of sequence. Some evidence for positive selection on HIF 1a amino acid sequence was observed, but was not associated with sequence variation in the O2 sensitive ODD, but was associated with the bHLH/PAS domains. 478 JEFFREY G. RICHARDS 5.2.4. Oxygen-dependent Gene Expression Hypoxia exposure in fish is well known to initiate a complex suite of gene and protein expression responses, many of which have been outlined above (see Table 10.1; Gracey et al., 2001; Ton et al., 2002, 2003; Bosworth et al., 2005; van der Meer et al., 2005). However, in many cases a direct link between changes in gene or protein expression and the transcriptional regulator HIF has not been directly assessed, therefore the reader is cautioned against assuming all responses described above are mediated by HIF. In reality, remarkably few studies, especially in fish, have focused on identifying functional HRE in the promoter regions of the hypoxia-responsive genes. The only definitive studies conducted in fish that have shown a direct relationship between HIF and hypoxia-regulated gene expression is for insulin-like growth factor binding protein in zebrafish (Kajimura et al., 2005, 2006). In mammalian and carcinoma cell lines, however, HIF has been directly implicated in regulating the expression of genes involved in a number of physiological and biochemical responses to hypoxia (outlined above). Patterns of gene expression in response to hypoxia exposure can vary between tissues and in some cases the diVerences can be dramatic. Ju et al. (2007) using an 8046 gene microarray showed substantial tissue-specific gene regulation and few consistent responses between tissues. In response to hypoxia exposure, 501 genes in the brain, 442 genes in the gills, and 715 genes in the liver were diVerentially expressed in hypoxia-exposed medaka (Oryzias latipes) and there were a number of pathways aVected (Table 10.1). Among the up-regulated genes there were remarkably few overlapping genes with 24, 21, and 20 genes showing the same expression patterns between brain and gill, brain and liver, and gill and liver, respectively (Figure 10.11). Of the genes that were shown to be down-regulated, 65, 24, and 26 genes were common between brain and gill, brain and liver and gill and liver, respectively (Ju et al., 2007). Only nine genes in total changed in a consistent fashion across all tissues examined. Of all the tissues examined, liver showed the greatest number of diVerentially expressed genes. 6. CONCLUSIONS AND PERSPECTIVES Hypoxia survival requires a rapid reorganization of physiological and biochemical systems to either maximize O2 uptake from the hypoxic environment to support the maintenance of a routine metabolic rate or cellular adjustments to function under O2-limiting conditions. Survival under O2limiting conditions requires a cellular metabolic reorganization to reduce ATP consumption through a regulated metabolic rate suppression to 10. 479 METABOLIC AND MOLECULAR RESPONSES TO HYPOXIA A Brain 209 B Gill 96 24 Brain 149 202 65 2 21 Gill 7 20 24 26 342 273 Liver Liver Fig. 10.11. Venn diagram showing diVerentially expressed genes in medake during hypoxia exposure. (A) Number of up-regulated genes in response to hypoxia exposure; (B) number of down-regulated genes in response to hypoxia exposure. [Data from Ju et al. (2007) with permission.] match the limited capacity for O2-independent ATP production. As outlined above, controlled metabolic rate suppression is essential to extend the length of time that can be supported by the limited levels of fermentable fuels. Thus, it appears reasonable to speculate that the degree of metabolic rate suppression and the quantity of stored fermentable fuel is likely strongly selected for in hypoxia-tolerant fishes. Indeed, this chapter has outlined and summarized the available information on the degree of metabolic rate suppression in a variety of fish species as well the quantity of tissue glycogen and, broadly speaking, there was a reasonable relationship between fish lifestyle (that being sluggish, hypoxia-tolerant carp species c.f. athletic, intolerant salmonid species, for example) and stored fermentable fuels, but the relationship between metabolic rate suppression and hypoxia tolerance is, however, oddly not clear. This is primarily because of the scant data available on the topic. Further still, the study of HIF in fish and hypoxia-regulated gene expression has been fruitful in demonstrating that HIF function in fish appears at least superficially similar to that observed in mammals, but the relationship between HIF function and hypoxia tolerance is still lacking. Despite the wealth of information available on the metabolic and molecular responses of a variety of fish species to hypoxia, we are still far from a unified concept of the important adaptations underlying hypoxia tolerance. However, fish provide an incredibly tractable system to understand the evolution of hypoxia tolerance because of the incredible diversity of fishes as well as their diverse O2 habitats. 480 JEFFREY G. RICHARDS REFERENCES Almeida-Val, V. M., Chippari-Gomes, A. R., and Lopes, N. P. (2006). Metabolic and physiological adjustments to low oxygen and high temperature in fishes of the amazon. In ‘‘The Physiology of Tropical Fishes Vol. 21’’ (Val, A. L., Almeida-Val, V. M., and Randal, D. J., Eds.), pp. 443–491. Elsevier, San Diego. Almeida-Val, V. M. F., Farias, I. P., Paula-Silva, M. N., and Duncan, W. P. (1995). Biochemical adjustments to hypoxia by Amazon cichlids. Braz. J. Med. Biol. Res. 28, 1257–1263. AppelhoV, R. J., Tian, Y. M., Raval, R. 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Simuultaneous direct and indirect calorimetry on normoxic and anoxic goldfish. J. Exp. Biol. 142, 325–335. Wieser, W., and Krumschnabel, G. (2001). Hierarchies of ATP-consuming processes: Direct compared with indirect measurements, and comparative aspects. Biochem. J. 355, 389–395. Zhang, Z. P., Wu, R. S. S., Mok, H. O. L., Wang, Y. L., Poon, W. W. L., Cheng, S. H., and Kong, R. Y. C. (2003). Isolation, characterization and expression analysis of a hypoxiaresponsive glucose transporter gene from the grass carp, Ctenopharyngodon idellus. Eur. J. Biochem. 270, 3010–3017. 11 DEFINING HYPOXIA: AN INTEGRATIVE SYNTHESIS OF THE RESPONSES OF FISH TO HYPOXIA ANTHONY P. FARRELL JEFFREY G. RICHARDS 1. Scope of the Chapter 2. Defining Hypoxia 2.1. Environmental Hypoxia 2.2. Functional Hypoxia 2.3. Exposure Time in Defining Hypoxia 3. Considerations for the Future This chapter attempts to synthesize the responses of fish to hypoxia presented in this Fish Physiology volume. The previous chapters are built on by diVerentiating between environmental hypoxia and functional hypoxia, and by outlining the possible compensatory mechanisms that fish use to counteract these forms of hypoxia. Environmental hypoxia is most simply defined as the water PO2 when physiological function is compromised, thus the definition of environmental hypoxia is dependent upon the physiological system under examination. Hypoxia-induced decrements in maximal oxygen consumption and thus reduced aerobic scope occur at higher water O2 levels than changes in routine oxygen consumption, which when compromised, is quantified as the critical oxygen tension (Pcrit). At water O2 levels below Pcrit, duration of survival is dependent upon the capacity to reduce metabolic demands to match the limited supply of fermentable fuels. Functional hypoxia, on the other hand, occurs during situations where tissue O2 demands exceed circulatory O2 supply, which can be evident during exercise, temperature extremes, anemia, acidosis, and changes in gill structure, but the physiological strategies used to survive environmental hypoxia are not necessarily utilized to endure functional hypoxia. 487 Hypoxia: Volume 27 FISH PHYSIOLOGY Copyright # 2009 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(08)00011-3 488 ANTHONY P. FARRELL AND JEFFREY G. RICHARDS 1. SCOPE OF THE CHAPTER While a complete picture of the consequences of hypoxia in fishes will require much work to finalize, several central messages have emerged, which are detailed in the preceding chapters of this volume. The aim here is to synthesize these messages, where possible, and point to where future research might be most valuable. 2. DEFINING HYPOXIA Simply put, hypoxia is a shortage of O2. Anoxia is a complete lack of O2. In its simplest context, regulators define aquatic hypoxia as dissolved O2 concentrations below 2–3 mg O2/L in marine and estuarine environments and below 5–6 mg O2/L in freshwater environments. With these thresholds, regulators are aiming to protect the environment of the most sensitive fish species and for North American and European freshwaters this is often a salmonid. However, as pointed out by Diaz and Breitburg (see Chapter 1), this is clearly an oversimplification. Indeed, in a recent meta-analysis of toxicological literature (lethal and sublethal indicators of hypoxia), fish were found to be generally the most sensitive of marine taxa (Vaquer-Sunyer and Duarte, 2008). Furthermore, the current literature range for defining hypoxia of 0.2 to 4.0 mg O2/L, with a mean of 2.1 mg O2/L, fails to adequately protect sensitive species. Instead, Vaquer-Sunyer and Duarte (2008) suggest that 4.6 mg O2/L may be more appropriate to protect the 90th percentile of the distribution of mean lethal O2 concentrations. Clearly, using an environmental concentration of O2 is a poor way to describe hypoxia. Foremost, what is functionally hypoxic for one fish is most certainly not functionally hypoxic for all fish. Indeed, Vaquer-Sunyer and Duarte (2008) show environmental thresholds for sublethal responses to hypoxia in fishes ranging from 2 to 10 mg O2/L. Furthermore, the O2 concentration in water tells us relatively little about what is happening in the fish itself. As pointed out in many of the preceding chapters, hypoxia develops for a variety of reasons. Consequently, hypoxia can be described as several forms. Wells (see Chapter 6) highlights and diVerentiates between environmental hypoxia and functional hypoxia, while Pörtner and Lannig (see Chapter 4) further expand on functional hypoxia by highlighting temperature-induced hypoxia. Temperature is a very important consideration when evaluating responses to hypoxia in fish because, not only does it have dramatic eVects on the fish’s O2 demands (Q10 values of up to 4 to 6 have been reported for O2 consumption), but it also aVects the amount of dissolved O2 available in the 11. DEFINING HYPOXIA 489 water (see Chapter 1). The temperature dependence of O2 solubility in water is clearly another reason for not relying on O2 concentration alone to define environmental hypoxia. Within physiological temperature ranges for most fish (0 to 40C), there is a 10 and 20% decrease in dissolved O2 for every 10C increase in temperature. Here we attempt to bring together all forms of hypoxia into an integrated framework, building upon the more detailed knowledge and citations in the individual chapters. In doing so, we rely on the partial pressure of O2 (PO2) in our definitions of hypoxia since this is what drives oxygen diVusion and, in part, determines O2 concentration. 2.1. Environmental Hypoxia In the broadest possible context, environmental hypoxia can be defined as the water PO2 when physiological function is first compromised, i.e., a sublethal eVect in toxicological terms. From a physiological perspective, environmental hypoxia can be defined as any water PO2 that decreases the arterial blood O2 concentration (CaO2), because such a decrease has the potential to decrease the arterial O2 transfer factor [TaO2 = the product of cardiac output (Q) and CaO2]. At these water PO2 levels, the fish is limited in its capacity to acquire O2 from the environment and its blood is hypoxemic. Even so, hypoxemia does not mean that the tissues are hypoxic; routine O2 needs can still be met through compensatory mechanisms. In using the above definition of environmental hypoxia, it becomes clear how a resting fish can initially maintain TaO2 by compensating for the decrease in water PO2 and the arterial hypoxemia. Compensations include increasing gill ventilation (to deliver more, but O2-depleted water to the gills if this is a short fall), increasing gill perfusion (if O2 transfer across the gill secondary lamellae is perfusion rather than diVusion limited, which is often the case), increasing Q (to deliver more blood to tissues), increasing the blood hemoglobin (Hb) concentration (to increase the O2-carrying capacity of blood usually through splenic red blood cell (rbc) release), or increasing tissue O2 extraction (to remove more O2 from the arterial blood and increase the arterial-venous O2 diVerence). However, a fish’s metabolic state must also be taken into consideration when assessing the relative importance of these compensatory responses. Full expression of these compensatory mechanisms may be possible only in resting fish. Indeed, if fish are exercising at maximum MO2 before the hypoxic challenge started, all these compensatory mechanisms may have been already fully utilized to support the elevated O2 requirement of the locomotory skeletal muscles. Thus, exercising fish are more sensitive than resting fish to decreasing water PO2 (Figure 11.1) and hypoxemia. Postprandial fish likely lie somewhere in between these two extremes. 490 ANTHONY P. FARRELL AND JEFFREY G. RICHARDS Hypoxemia Locomotion Maximal MO2 duction O2 consumption rate Repro Growth Routine MO2 Basal MO2 Hypoxic Pcrit Normoxic Water PO2 Fig. 11.1. Metabolic responses of fish to environmental hypoxia. Solid lines indicate O2 con_ 2 to support maximal, routine, and basal metabolic rates. Dashed lines show sumption rates MO the theoretical eVects of decreases in environmental O2 tension on physiological processes. The critical O2 tension (Pcrit) is defined here as the point at which routine O2 consumption transitions from being independent of environmental PO2 to being dependent upon environmental PO2. The _ 2 is the aerobic scope, which decreases with difference between routine and maximal MO increasing levels of hypoxia. See text for more details. Even when water PO2 values are well below 100% saturation, there is an additional safety factor before hypoxemia sets. It is possible for most fishes to achieve 85–95% O2 saturation of Hb at PO2 values <15 kPa (see Chapter 6). Thus, fish have a zone of insensitivity to environmental hypoxia that is determined by the O2-binding aYnity of Hb. However, once water PO2 falls below this zone of insensitivity, the zone of environmental hypoxia begins. Mechanistically, the zone of environmental hypoxia begins when arterial O2 saturation falls below that seen in normoxia, the four compensatory mechanisms mentioned above kick in, and functional activities start to be compromised (Figure 11.1). Clearly, the O2 binding aYnity of Hb is the principal factor setting the exact water PO2 when integrated function begins to be lost. Consequently, since the O2-binding aYnity of Hb varies enormously among fishes, it is fairly obvious why any definition of environmental hypoxia using water Po2 can be done only in a species-specific context. A simple index of fish’s resilience to a low water PO2 is its Hb P50 (the PO2 at which arterial blood is 50% saturated); a low P50 value reflects a high Hb–O2 binding aYnity. Fish with a low P50 will curtail activities at a lower water PO2 than fish with a higher P50. This raises the interesting point of why, in an environment where large changes in water O2 saturation are commonplace (see Chapter 1), any fish 11. DEFINING HYPOXIA 491 would have a low Hb–O2 binding aYnity if a high aYnity Hb can confer such an advantage? Wells (Chapter 6) makes the point that fishes adapted to environmental hypoxia have high-aYnity Hbs whereas fishes adapted for functional hypoxia (exercise in this case) have low aYnity Hbs. In other words, hypoxia-tolerant fishes are rarely athletic and vice versa. Low-aYnity Hbs also result in a higher arterial PO2 and this favors a faster rate of O2 diVusion during unloading at tissues. Thus, P50 should vary inversely with maximum MO2, an idea that could be easily tested among the great diversity of fish species. Unlike Hb aYnity, elevated Hb and even tissue myoglobin (Mb) concentrations are adaptations common to both environmental and functional hypoxia (see Chapter 6). In both situations an elevated Hb concentration conveys the advantages of increased O2-carrying capacity, TaO2, buVering capacity, and CO2 capacity. The zone of environmental hypoxia can also be defined as beginning at a water PO2 when aerobic scope must begin to decrease with declining water PO2 (Figure 11.1). As water PO2 decreases further, a point is ultimately reached when there is no aerobic scope and only routine metabolic activities can be maintained. Above this water PO2, a minimum routine O2 consumption is maintained and below this water PO2, routine O2 consumption must conform to water PO2 The water PO2 at which this transition occurs is defined in the context of this chapter as the Pcrit. Simply put, Pcrit is the water PO2 at which fish transitions its O2 consumption rate (MO2) from being independent of water PO2 to being dependent upon water PO2 and _ 2) is zero. _ 2-routine MO aerobic scope (maximal MO In reality, however, Pcrit can be more broadly used for as the water PO2 when any physiological function changes as a function of water PO2. For example, Pcrit is widely used to indicate the initiation of the hypoxic bradycardia response common in fishes (but not other vertebrates; Farrell, 2007a). The Pcrit for bradycardia, however, is unlikely to be analogous to the Pcrit determined for MO2. This is because bradycardia has variable eVects on Q and hence TaO2 that are species-specific (see Chapter 7). For species such as trout, cod, flounder, sharks, and sturgeon, bradycardia is associated with an increase in stroke volume to maintain or slightly increase routine Q, and MO2. Hence, the Pcrit for bradycardia in these species lies within the zone for hypoxia but above the Pcrit for MO2. However, the decrease in Q, despite associated increases in stroke volume with bradycardia in sea bass, lingcod, and eels, can only indicate either a state of collapse or oxygen conformity. Species diVerences in Pcrit must then reflect interspecific diVerences in Hb–O2 binding aYnity and P50 values. Indeed, recent work by Mandic et al. (2008) has shown a tight relationship between Pcrit and Hb P50 among several species of intertidal fishes from the family Cottidae. This then allows for a simpler definition of the zone of environmental hypoxia: a range of water 492 ANTHONY P. FARRELL AND JEFFREY G. RICHARDS PO2 values over which aerobic scope progressively falls to zero. Obviously, this is the zone where physiologists have focused much of their study because many fascinating behavioral, physiological, biochemical, and molecular changes occur as a fish tries to either compensate and maintain functional activities (oxyregulating), or curtail functions and reduce O2 demand (oxyconforming). These responses might be loosely termed stress responses, although the primary stress hormones, such as epinephrine and norepinephrine are not typically released until arterial PO2 levels fall below the blood P50 (Perry and Reid, 1992). For all fishes, the zone of environmental hypoxia is characterized by a progressive loss of physiological function as the hypoxic state deepens and aerobic scope declines. The progressive loss of physiological function is likely an orderly aVair. However, we know of no one who has fully characterized the exact order of these functional losses. Consequently, the order and degree of loss represented in Figure 11.1 is educated guesswork on our part. We do know that fish cannot swim as fast in hypoxia as in normoxia. But does this mean that avoidance behaviors are more likely to be powered anaerobically as Pcrit is approached? Or is a small component of aerobic swimming capacity retained until just before Pcrit for such purposes? Hypoxic fish also cease feeding. Mechanistically, this could be because blood flow is diverted away from the gut to more needy or O2-sensitive tissues, as we know that absolute gut blood flow certainly decreases in hypoxic fish, even if hypoxia occurs after they have been fed. However, the physiological mechanisms integrating such a response are unknown. Hypoxia can also act as an emetic, which would be a serious concern in open sea cage aquaculture operations if hypoxic conditions were prevalent. In particular, anoxic deepwater up-wellings, eutrophication, and even elevated water temperature could dramatically lower water PO2 and potentially impair digestion and growth well before lethal oxygen levels are reached. Reproductive development is also suspended at some point with environmental hypoxia. Reproduction may resume without serious fitness consequences for iteroparous fishes when either normoxia is restored or fish acclimate to the hypoxic conditions. However, for semelparous fishes such as salmon, which only have one opportunity to spawn, hypoxic suspension of reproductive development could have serious fitness consequences. Consideration of this potential concern may be all the more important with global warming, given that high temperature is known to halt the spawning migrations of adult salmon in the Columbia and Fraser Rivers, likely as a result of a state of functional hypoxia (more on this below). A state of physiological anoxia exists at a water PO2 when O2 no longer loads onto either Hb or tissue Mb. Experimentally, this state is diYcult to achieve and confirm, especially when the P50 values for hypoxia-tolerant fishes are <1 kPa. Therefore, to reflect this uncertainty, researchers often 11. DEFINING HYPOXIA 493 use the term severe hypoxia when it is clear that an animal has switched to a state of temporary anaerobic existence that is supported by glycolytic metabolism. At O2 levels below Pcrit, the basic challenge is one of balancing metabolic energy demand with supply. The principal problem is that an inhibition of O2dependent mitochondrial ATP production imposes a substrate-limited cap on the duration of survival and as Richards points out (see Chapter 10), the duration of survival under these O2-limiting conditions is likely dictated by two key factors: compensations, which is the ability to reduce basal metabolic demands through a controlled metabolic rate suppression; and provisions, which is the amount of substrate available for O2-independent ATP production. A third factor, which is apparently evident only in the cyprinid family (see Chapter 9), is one of eVective waste handling. In the long term, acidosis may become a more serious physiological challenge than anoxia if the fish has provisioned an extensive glycogen store and has down-regulated metabolism so that these energy stores can be metered out at a slower rate. As Vornanen and colleagues point out in Chapter 9, the conversion of lactate to ethanol and the associated consumption of protons limit the development of acidosis in the anoxia-tolerant crucian carp. An analogous situation exists in anoxic freshwater turtles that buVer protons and lactate using their shell. 2.2. Functional Hypoxia Functional hypoxia can come in various forms, but it always reflects a situation where tissue O2 demands exceed circulatory supply. Below we will discuss four physiological states that result in functional hypoxia: exercise, anemia, acidosis, and changes in gill structure, plus the confounding eVects of temperature on the development of functional hypoxia. 2.2.1. Exercise The most obvious example of functional hypoxia is exercise. There are numerous examples showing that as fish swim faster (increasing the O2 demand of locomotory muscle), there comes a point when anaerobically powered locomotion takes over and lactate starts appearing in the tissues and blood. The precise mechanisms underlying this aerobic/anaerobic transition are not entirely resolved (Farrell, 2007b). Some hold that it’s an O2 supply limitation whereby the heart has reached its maximum anatomical (stroke volume) and physiological (heart rate and power development) pumping capacity, meaning internal convection can no longer deliver O2 to the working muscle at the rate required to sustain aerobic metabolism. This internal convection limitation typically manifests itself as a plateau in Q during incremental swimming trials, as in salmonids (e.g. Steinhauser et al., 494 ANTHONY P. FARRELL AND JEFFREY G. RICHARDS 2008). O2 supply can also become limited if Hb is not fully oxygenated as it passes through the secondary lamella (as with environmental hypoxia), which could be a product of either an external convection limitation (gill ventilation), or a limitation in gill O2 diVusion (see below). Still others view muscle capillarity as being a limiting factor for O2 supply to working muscle (a muscle O2 diVusion limitation). Other research groups support the notion that the aerobic/anaerobic transition during exercise in fish is due to metabolic inertia and an energy demand that outpaces the capacity for aerobic metabolism (e.g., Richards et al., 2002). Specifically, the reason lactate accumulates during intense exercise is simply that the rate of ATP production from oxidative phosphorylation cannot keep pace with the high ATP demands of contracting white muscle. As a result, pathways of ATP production shift from the slow mitochondrial pathway to the much faster glycolytic pathways and even faster PCr hydrolysis pathway. These faster metabolic pathways likely become increasingly important when tailbeat or fin sculling frequencies increase with swimming speed. The maximum contraction frequency of fish skeletal muscle is set, in part, by their ATP production pathways, as well as the kinetic properties of their contractile proteins and supporting excitationcontraction processes. Given that aerobic metabolism isn’t really even an option due to the low mitochondrial content in white muscle, this means O2 delivery to white muscle may not be an important component of why white muscle makes lactate and why measurements of white muscle mitochondrial NADH/NAD+ indicate no O2 limitation (Richards et al., 2002). What is also clear in this debate is that as fish swim faster, they ultimately ‘‘shift muscular gears,’’ The well-capillarized, mitochondrial-rich red muscle is no longer able to power locomotion. Instead, the 20-times more abundant, and therefore mechanically more powerful, white muscle takes over. What is remarkable in this transition is that around 50% of a salmon’s body mass is switched into a fully functional state with apparently little initial impact on either Q or venous PO2 (PvO2). This observation is consistent with PvO2 reaching a lower plateau state as salmonids approach maximum prolonged swimming speed. The most parsimonious explanation for the PvO2 plateau is that O2 diVusion to white muscle becomes diVusion limited under extreme exercise states. Whether red muscle also becomes functionally hypoxic due to a perfusion limitation is unknown, however. Unravelling the ‘‘chicken and egg’’ conundrum of O2 supply during exercise will be on going for some time to come since species diVerences undoubtedly exist. Furthermore, the potential role that body mass may play in determining which step in the O2 cascade limits O2 delivery in fish will require consideration beyond the recent review of Nilsson and OstlundNilsson (2008). Although Nilsson and Ostlund-Nilsson (2008) generally 11. DEFINING HYPOXIA 495 concluded that body size had little impact on the dynamics of O2 uptake from hypoxic environments, other anecdotal evidence suggests body size may be an important consideration in O2 delivery. In this regard, a fascinating discovery made over 30 years ago was that Mb in the tuna ventricle increases abruptly once the fish reaches about 20 kg in body mass (Poupa et al., 1981), suggesting that body mass plays a role in O2 delivery in fishes. Myoglobin increases tissue O2 storage and high Mb concentrations are thought to be adaptations for both environmental and functional hypoxia. However, as pointed out by Gamperl and Driedzic (see Chapter 7), the potential role of Mb in facilitating O2 diVusion in fish hearts is not entirely resolved. 2.2.2. Anemia This may come about as a result of accidental and experimental blood loss, pathologies (e.g., hemorrhagic septicemia), and adaptations. A low Hb concentration reduces CaO2 and potentially TaO2. EVects on CaO2 can be compensated for by increases in Q, which over time appear to stimulate an increase in relative ventricular mass. These compensatory responses of Q and relative ventricular mass to low Hb concentrations have been seen with experimentally induced anemia (Simonot and Farrell, 2007), as well as with adaptations to anemia. For example, some flatfishes have naturally low Hb concentrations, as do polar fishes (apparently as an adaptation to coldinduced increases in blood viscosity), and both have high Q. Nevertheless, while hemoglobin-free Antarctic icefishes have a Q and ventricular mass that _ 2, heart rate, nor cardiac rivals that of tunas, neither their maximum MO power production come close to those of tunas (Axelsson, 2005). Consequently, the fish heart is a very plastic organ, one that responds over experimental and evolutionary time scales to ensure adequate oxygen supply. Nevertheless, there is a clear tradeoV in terms of functional ranges and aerobic scope is considerably lower in these anemic fish compared with tunas. 2.2.3. Acidosis Many fish Hbs have a high Bohr coeYcient and Root eVect with some of the largest values among vertebrates (see Chapter 6). These eVects potentially increase O2 unloading from Hb as a result of CO2 and H+ release from tissues during capillary blood transit. Consequently, a high Bohr coeYcient and Root eVect are seen as adaptive for athletic fish. Added to the Bohr and Root eVects is the eVect of catecholamine release, which is a primary stress response during hypoxia (probably when CaO2 falls below the Hb P50) and stimulates the rbc Na+/H+ exchanger, elevating rbc pH and securing O2 uptake at the gills during acidosis, but negatively aVecting O2 delivery to tissues. Thus, if fish can maintain PaO2 during exercise, it would seem prudent not to release catecholamines during exercise at least in terms of tissue O2 delivery. 496 ANTHONY P. FARRELL AND JEFFREY G. RICHARDS Collectively, a large Bohr and Root eVect may cause a decrease in Hb–O2 binding aYnity, visualized as a right-shift in the Hb–O2 dissociation curve, which increases the PO2 gradient for unloading O2 at tissues, enhancing the rate and amount of O2 diVusion. Despite such benefits, the right-shifted Hb– O2 dissociation curve reduces the PO2 gradient driving O2 diVusion across the gill lamellae, potentially impairing both the rate and the amount of O2 diVusion. Of course, such impairments would be manifest only if gill O2 diVusion was diVusion limited. Implicit to these adaptations that modulate Hb function is that a diVusion limitation of O2 transfer at the gills is more unlikely for athletic fishes than for hypoxia-tolerant fishes. Indeed, this appears to be the case as the epithelial barrier of the secondary lamellae of eels is considerably thicker than that of tuna, for example. Species comparisons of gill diVusion capacitance might then be revealing in terms of hypoxia tolerance. A cautionary note in this regard is that such morphological changes may reflect other challenges of hypoxic environments rather than a primary modulation of gill O2 diVusion. 2.2.4. Changes in the Gill Lamellae The lamellae of the gills are the primary O2 exchange sites for all waterbreathing fishes heavier than about 5 g. Here, the O2 gradient between water and blood, the lamellar surface area, and the thickness of the lamellar epithelial barrier are the primary determinants of the rate of O2 diVusion. Various lamellae adaptations among fish have been extensively quantified (see Volume 10 in the Fish Physiology series). In addition, it is also clear that the lamellar morphology is extremely plastic. Functional changes in gill morphology are well documented for hypoxia, low temperature, and a variety of toxicant exposures. Hypoxia can increase the lamellar surface area and reduce the blood to water diVusion barrier. In the scaleless carp these changes, which clearly benefit O2 diVusion at the gills, can occur rapidly in 12–24 h (Matey et al., 2008). On the other hand, low temperature exposure decreases lamellar surface area through mitotic expansion of the filament epithelial cell between adjacent lamella (see Chapter 9). These changes decrease the diVusive capacity of the gills for all molecules, presumably as a protective mechanism to limit diVusive ion movement, but at the same time decreases in lamellar surface area reduce aerobic scope. The so-called osmorespiratory compromise (Gonzalez and McDonald, 1992) highlights the potential tradeoVs of having a multifunctional gill (that being an organ involved in both ion regulation and O2 transfer) and that modifications to the gill epithelium that limit molecule movement are beneficial for ion regulation, but detrimental for gas exchange, in some cases inducing functional hypoxia. It seems probable that O2 conformity during hypoxia relaxes the need to optimize gill 11. DEFINING HYPOXIA 497 diVusing capacity perhaps decreasing ion loses (in freshwater) or gain (in seawater), decreasing toxicant uptake from water, and increasing the protective barrier for pathogen entry. 2.2.5. Temperature-induced Hypoxia The recent discovery that temperature extremes lead to hypoxic states is generating considerable interest in ecologists, physiologists, biochemists, and molecular biologists. Pörtner and Lanning (see Chapter 4) detail the evidence for, and consequences of, this form of hypoxia. While temperature-induced hypoxia obviously reflects an environmental change, it would be misleading to characterize this as a form of environmental hypoxia, as there is good evidence that some species maintain TaO2 at high temperature but are still functionally hypoxic when temperature exceeds its optimum (Steinhausen et al., 2008). In this case, it is the routine O2 demand that increases as a result of the Q10 eVect on routine metabolic rate. In fact, much like exercise, increasing temperature increases the tissue O2 demand until it eventually outstrips the ability of the heart to deliver O2. Once TaO2 is maximal, further increases in temperature may lead to cardiac collapse as revealed by a declining Q and cardiac arrhythmias (Clark et al., 2008). The mechanism(s) for cardiac collapse at high temperature is (are) still under scrutiny (see Chapter 7). One possible mechanism explaining cardiac collapse at high temperatures relates to the fact that at least 50%, if not all, of the ventricular muscle in fishes depends on the venous PO2 as the pressure driving O2 diVusion to cardiac mitochondria. Thus, a hypothetical temperature-induced hypoxic spiral to death might start with Q first reaching its anatomical (maximum stroke volume) and physiological (maximum heart rate and power) limits. Any increase in O2 extraction from arterial blood to meet the increased peripheral tissue O2 demands would ultimately lower venous PO2, which then limits cardiac O2 supply, restricting cardiac performance and TaO2. Attempts by fish to exercise at high temperature, and perhaps even the energetic requirements of avoidance behaviors, could easily make matters worse by increasing O2 extraction by the muscle and further reducing PvO2. Further work into the causes of cardiac collapse is urgently needed. Fish appear to have evolved a number of strategies to avert or delay the hypoxic cardiac spiral. Foremost, and as noted above, venous PO2 may be maintained above a threshold level in one of several ways. One is that unloading of O2 at peripheral tissues may eventually become diVusion limited once internal convection has reached its maximum capacity and the arterial to venous O2 diVerence has been fully exploited. This may be inevitable if the metabolic demand of white muscle, which has an inherent tissue O2 diVusion limitation, takes on an increasing portion of the overall metabolic rate as temperature increases. In addition, the temperature-induced 498 ANTHONY P. FARRELL AND JEFFREY G. RICHARDS decrease in Hb–O2 binding aYnity potentially elevates PvO2 with increasing temperature, enhancing tissue O2 delivery, including that to the heart. If, however, tissue O2 uptake becomes diVusion limited, as suggested, the temperature-induced decrease in Hb aYnity would increase tissue O2 delivery without decreasing PvO2. Indeed, acute warming can result in decreases or no change in PvO2 when TaO2 has been maximized (Steinhausen et al., 2008). Lastly, a coronary circulation represents a more secure O2 supply for the ventricle because it brings arterial blood directly from the gills to at least the outer part of the ventricle. The phylogenetic distribution of the coronary circulation in relation to temperature is unknown. Those teleosts that have a coronary circulation tend to be athletic or hypoxia tolerant, and so temperature tolerance may not be a primary factor determining the presence or absence of a coronary circulation in fish. Interestingly, all elasmobranchs have a coronary circulation, but most teleosts do not. While it may seem reasonable to conclude that temperature-induced hypoxia is best categorized as a state of functional hypoxia, there are intriguing functional parallels with environmental hypoxia that need to be further explored. In characterizing the responses to environmental hypoxia (Figure 11.1), we identified a zone of independence of O2 consumption from water PO2, where aerobic scope was preserved. Is this zone homologous or just functionally analogous with the zone for optimal temperature described by Pörtner and Lannig (see Chapter 4)? Similarly, is the zone of hypoxia functionally homologous to the pejus temperatures and the Pcrit functionally homologous to the critical temperatures? 2.3. Exposure Time in Defining Hypoxia An important component of defining hypoxia is exposure time. The rate of change in water PO2 during the development of hypoxia is crucial for both the type of physiological and biochemical responses initiated as well as animal survival. The faster the rate of change in water PO2, the more likely dire consequences will follow. This is primarily because the capacity for responding to hypoxia over short time periods is dictated by functioning physiological and biochemical systems that are in place at the time hypoxia is imposed. If, for example, during progressive hypoxia exposure the physiological mechanisms for maintaining O2 extraction from the environment do not maintain pace with the falling environmental levels, then the fish falls into a cascade toward eventual death. As mentioned previously in this chapter, this transition point is classically defined at Pcrit. At water PO2 levels below Pcrit, the time until death will be dictated primarily by two interlinked biochemical responses: (1) reductions in basal metabolic demands to match the limited capacity for ATP production; and (2) the availability of 11. DEFINING HYPOXIA 499 substrate for O2-independent ATP synthesis. There is another possible metabolic option that can be employed to extend survival at O2 levels far below Pcrit and that is the capacity to modify the mitochondria to function at reduced O2 tensions, but unfortunately little work has examined this option in fish. Acclimation or acclimatization to hypoxia exposure involves processes that are distinct from the acute response, but are likely initiated simultaneously with hypoxia exposure, and involve the restructuring of physiological and biochemical responses to enhance function and extend survival. As Richards points out in Chapter 10, all cDNA microarray studies performed to date with hypoxia-exposed fish show a suite of gene expression changes that are consistent with optimizing O2 uptake from the environment (e.g., gill apoptotic factors causing a thinning of the gill; see also Chapter 9), O2 distribution and circulation (e.g., changes in heme synthesis and iron metabolism; see also Chapters 5 and 6), and metabolic energy balance (e.g., up-regulation of glycolysis and suppression of energy consumption; see Chapters 7, 9, and 10). Hypoxia-induced changes in gene expression in fish are likely regulated in a similar fashion to mammals and by the hypoxia inducible factor (HIF; see Chapter 10), but remarkably, a direct linkage between HIF function and hypoxia tolerance among fish species has not been demonstrated at this time. Overall, acclimation occurs over hours, days and weeks, and involves changes predominately in gene expression, which facilitate the reorganization of the physiological process of O2 uptake and distribution and biochemical processes of cellular energy metabolism. While the gill secondary lamella and cardiac tissues are both extremely plastic in fishes, secondary lamellae can be altered after as little as 12 h of hypoxia, but it takes at least 2 weeks for the heart to increase in mass during anemia. Temperature may also play a role in how quickly these compensations take place, and temperature may even trigger for hypoxic acclimations. The interaction between cold acclimation and hypoxic acclimation is worth considering since cooling of the water of course precedes the progression to hypoxia in ice-covered lakes. Although few, if any studies, have examined the eVects of hypoxic acclimation on Pcrit, it is likely that acclimation would yield a decrease in Pcrit brought about by changes in gill structure, cardiac function, and Hb profiles. Acclimation may also shift O2 thresholds for the display of avoidance behaviors. At water PO2 levels below Pcrit, the advantage of acclimation responses in up-regulating tissue-specific capacities for O2-independent ATP production is reasonably clear, which can dramatically aVect survival time. Clearly, prolonged exposure to environments depleted in O2 can aVect the hypoxic response and must be considered when deciding whether a particular fish species is hypoxic or not. 500 ANTHONY P. FARRELL AND JEFFREY G. RICHARDS When considering the potential impacts of acclimation/acclimatization on hypoxia tolerance, not only should chronic depletions in water PO2 be considered, but also oscillations in water PO2. Many of the most hypoxiatolerant fish species inhabit environments that undergo diurnal fluctuations in O2 that are due primarily to plant respiration (see Chapter 1 for more detail). Some of the best examples are fish found in the Amazon (see Volume 21 on Tropical Fishes in the Fish Physiology series) and fishes found within intertidal environments. It is presently unknown if oscillations in water PO2 achieve the same degree of acclimation as chronic hypoxia, but it is likely that some degree of environmental entrainment occurs. The diVerentiating eVects of oscillations in water PO2 from chronic hypoxia exposure is the need to recover from the oscillating hypoxia. Hypoxic recovery has not been extensively studied, but in general is has been shown to occur at a faster rate than recovery from exercise (Hallman et al., 2008) and involves an enhancement of _ 2 to facilitate an aerobic recovery from the anaerobic period. Further MO work into hypoxia recovery is needed. It has long been assumed that the prevalence of environmental hypoxia in the aquatic environment has been a powerful evolutionary pressure resulting in the selection of the traits described throughout this volume. Indeed, fish species represent the ideal ‘‘model’’ system to understand the selection of traits underlying hypoxia tolerance because of the highly specious nature of fish and the extremely O2 diverse environments they inhabit. Certainly, the study of diverse fish groups has led to the general consensus of the selection-driven traits associated with hypoxia tolerance, but the capacity to address questions of adaptation is far more limited. With the advent of the phylogenetic comparative method, comparative physiologists can now attempt to identify the repeated evolution of a trait correlated with one or more putatively selective variables while factoring out the possible confounding eVects of shared ancestry among species (Felsenstein, 1985). The application of phylogenetically independent contrasts assists in identifying selection-driven traits but requires an understanding of the phylogenetic relationships among the species under study. A phylogenetic comparative approach was recently taken by Mandic et al. (2008) using a group of fish from the family Cottidae (sculpins), which are distributed along the marine intertidal zone and experience oscillating hypoxia to varying degrees. In these fish, there was a phylogenetically independent relationship between Pcrit and routine metabolic rate, total gill surface area, and rbc Hb–O2 binding aYnity, such that variation in these components accounted for 75% of the variation in Pcrit (Mandic et al., 2008). More studies that take into account phylogenetic relationships are needed to isolate adaptation from phylogenetic signal. Ultimately, beyond a critical PO2 only a passive, anaerobic existence is possible, one that is rarely exploited among fishes. Carps and hagfishes may 11. DEFINING HYPOXIA 501 be the important exceptions in this regard. However, sessile invertebrates inhabiting the extremely variable intertidal environment use metabolic depression, anaerobic energy production, and stress protection mechanisms to provide short- to medium-term tolerance of this extremely challenging environment. Mobile intertidal fishes, on the other hand, employ a complex suite of behavioral, physiological, and biochemical strategies for long-term hypoxic survival, which are influenced to a large degree by many ecological factors (see Chapter 2) such as perceived risk of predation and availability of cover. The use of simple whole-animal measures, such as Pcrit, will undoubtedly be of benefit to examine the relationships between environment, ecology, and hypoxia tolerance among the numerous species of small fishes that inhabit challenging environments. 3. CONSIDERATIONS FOR THE FUTURE We are entering a golden age for comparative physiology, propelled by the development of tools to dissect the responses of fish from gene through to the ecosystem and to place these responses into an evolutionary and ecological context. Although we have accumulated a wealth of information on how many fish species respond to hypoxia (both environmental and functional) and the potential adaptations underling hypoxia tolerance, there remain several areas of study that have yet to be adequately explored. Furthermore, the ability to compare among studies is paramount, but as pointed out above, hypoxia has no simple definition and therefore mechanisms for comparisons among studies must be developed. We oVer the following suggestions for consideration to anyone interested in hypoxic research. 1. Because ‘‘hypoxia’’ must be considered in light of the species under study, a water PO2 that is hypoxic for one fish species could conceivably have no eVects on another fish species. For example, what might be considered hypoxic for a rainbow trout may have almost no measurable eVect on an extremely hypoxia-tolerant fish such as the crucian carp. Thus, as pointed out earlier in this chapter, reporting the water PO2 at which a physiological response occurs is more useful if it is put into the context of the organism under study. Being able to standardize hypoxia exposures across species is essential to understanding processes of adaptation. Expressing hypoxia exposures relative to Pcrit, as defined by the transition from an oxyregulator to an oxyconformer, may provide an overall framework to allow researchers to standardize responses observed in diverse groups of fish. 2. There is a need for more studies that better replicate environmental conditions. Studies of chronic hypoxic exposure are in short supply, as 502 ANTHONY P. FARRELL AND JEFFREY G. RICHARDS are oscillations that might reflect diurnal rhythms. Synergistic responses between hypoxia and other environmental changes such as hypercarbia (elevated water CO2), acidosis, and temperature would be worthwhile as multiple loadings are predicted to decrease aerobic scope. Furthermore, studies that integrate across levels of biological organization and put behavioral, physiological, biochemical, and molecular responses into an ecological context are essential. 3. Additional curiosity driven research should explore whether the ultimate outcomes of environmental hypoxia exposure and functional hypoxia exposure in fish are similar, particularly at the biochemical and molecular level. Some research has demonstrated that responses to environmental hypoxia and functional hypoxia induced by exercise diVer, but the precise reasons for these diVerences are not known. Does, for example, exercise-induced hypoxia elicit the same gene expression responses in muscle as environmental hypoxia? ACKNOWLEDGMENTS The authors would like to thank the Natural Sciences and Engineering Council of Canada for financial support of their research. REFERENCES Axelsson, M. (2005). The circulatory system and its control. In ‘‘Fish Physiology, Polar Fishes’’ (Farrell, A. P., and SteVensen, J. F., Eds.), Vol. 22, pp. 239–280. Academic Press, San Diego. Clark, T. D., Sandblom, E., Cox, G. K., Hinch, S. G., and Farrell, A. P. (2008). Circulatory limits to oxygen supply during an acute temperature increase in the Chinook salmon (Oncorhynchus tshawytscha). Am. J. Physiol. 295, R1631–R1639. Farrell, A. P. (2007a). Tribute to P. L. Lutz: A message from the heart – why hypoxic bradycardia in fishes? J. Exp. Biol. 210, 1715–1725. Farrell, A. P. (2007b). Cardiorespiratory performance during prolonged swimming tests with salmonids: A perspective on temperature eVects and potential analytical pitfalls. Phil. Trans. R. Soc. B. 362, 2017–2030. Felsenstein, J. (1985). Phylogenies and the comparative method. Am. Nat. 125, 1–15. Gonzalez, R. J., and McDonald, D. G. (1992). The relationship between oxygen consumption and ion loss in a freshwater fish. J. Exp. Biol. 163, 317–332. Hallman, T. M., Rocha, A., Jones, D. R., and Richards, J. G. (2008). Metabolic recovery from exercise and hypoxia exposure measured using 31P- and 1H-NMR in the common carp, Cyprinus carpio. J. Exp. Biol. 211, 3237–3248. Mandic, M., Todgham, A. E., and Richards, J. G. (2008). Mechanisms and evolution of hypoxia tolerance in fish. Proc. Roy. Soc. B. DOI: 10.1098/rspb.2008.1235. Matey, V., Richards, J. G., Wang, Y. X., Wood, C. M., Rogers, J., Semple, J., Murray, B. W., Chen, X.-Q., Du, J., and Brauner, C. J. (2008). The eVect of hypoxia on gill morphology and 11. DEFINING HYPOXIA 503 ionoregulatory status in the endangered Lake Qinghai scaleless carp, Gymnocypris przewalskii. J. Exp. Biol. 211, 1063–1074. Nilsson, G. E., and Ostlund-Nilsson, S. (2008). Does size matter for hypoxia tolerance in fish? Biol. Rev. Camb. Philos. Soc. 83, 173–189. Poupa, O, Lindstrom, L., Maresca, A., and Tota, B. (1981). Cardiac growth, myoglobin, proteins and DNA in developing tuna (Thunnus thynnus thynnus L.). Comp. Biochem. Physiol. A 70, 217–222. Perry, S. F., and Reid, S. D. (1992). Relationship between blood O2-content and catecholamine levels during hypoxia in rainbow trout and American eel. Am. J. Physiol. 263, R240–R249. Richards, J. G., Heigenhauser, G. J. F., and Wood, C. M. (2002). Glycogen phosphorylase and pyruvate dehydrogenase transformation in white muscle of trout during high-intensity exercise. Am. J. Physiol. 282, R828–R836. Simonot, D. L., and Farrell, A. P. (2007). Cardiac remodelling in rainbow trout Oncorhynchus mykiss Walbaum in response to phenylhydrazine-induced anaemia. J. Exp. Biol. 210, 2574–2584. Steinhausen M. F., Sandblom E., Eliasson E., Verhille C., and Farrell A. P. (2008). The effect of acute temperature increases on the cardiorespiratory performance of resting and swimming sockeye salmon (Oncorhynchus nerka). J. Exp. Biol. 211, 3915–3926. Vaquer-Sunyer, R., and Duarte, C. M. (2008). Thresholds of hypoxia for marine diversity. PNAS 105, 15452–15457. INDEX A acid—base and ion regulation, 156–9 Acipenser baeri, 195, 206 Acipenser medirostris, 281 Acipenser naccarii, 47, 50, 196, 226 Acipenser shrenckii, 113 Acipenser transmontanus, 196, 211, 368, 382 Acipenseridae, 281 adenosine accumulation, eelpout, 155 Crucian carp, 410–11, 419–20 heart rate, control under hypoxia, 332–3 Adour river, south‐west France, 83 Adriatic sturgeon, (Acipenser naccarii), 47, 50, 196, 226 Aequidens coeruleopunctatus, 30 aerobic scope, 371–3 African lungfish see lungfish air‐breathers eVects of hypoxia on growth, 388–90 facultative and obligate, 388 see also aquatic surface respiration (ASR) air‐breathing organ (ABO), 39 Albula sp., 105 alcohol dehydrogenase, 402 alphastat pattern, 157 Amazonia catfish (Hoplosternum littorale), 203, 277 Ambloplites rupestris, 30 Amia calva, 42, 196, 202, 214, 222, 226, 229, 230 Ammodytes, 47 Ammodytidae, 47, 52–3 AMP‐activated protein kinase (AMPK), 466 Amphipnous cuchia, 202 Amphiprion melanopus, 55 Anabantoideae, 53, 214 (Ctenopoma damasi), floating foam nest, 55 Anabas testudineus, 53 anaerobiosis chronic hypoxic response, 344 energy sources, 335–6 ethanol production in carp, 401–4 glucose to support heart performance, 336 glycogen phosphorylase cascade, 335–6 mitochondrial, 144 succinate formation, 144 Anarhichas lupus, 307, 312, 313 Anarhichas minor, 366, 380 anchovy (Engraulis mordax), increase in speed under hypoxia, 62 Ancistrus chagresi, 42, 61, 202, 214, 276 androstenediol, 86(fig.) anemonefish (Amphiprion melanopus), 55 Anguilla anguilla, 196, 211, 224, 226, 230, 267, 269, 277, 460–2 Anguilla japonica, 196, 211, 306, 307, 319 Anguilla rostrata, 335–6, 338 Anotopterus pharao, 271 anoxic tolerance ability to decrease ATP demand, 330–2 cardiac activity (carp), 407–8 Antarctic eelpout (Pachycara brachycephalum), 147(fig.) growth optimum, 153 growth within thermal window, 152(fig.) hyperoxia eVects on oxygen consumption and blood flow, 147(fig.) oxygen transport in temperature acclimation, 285 protein synthesis, 172 505 506 Antarctic marine fauna energy‐saving lifestyles, 151 Hb components, 270–1 myoglobin expression, 274 presence/absence of hypoxic bradycardia, 305 vs temperate fauna, 150–3 see also icefish anthropogenic CO2 accumulation, 175 anthropogenic nutrient enrichment eutrophication, 1, 6–7 influence on oxygen budgets, 6 apoptosis, 104–5, 113 disruption in, 112 types of apoptosis, 113 apoptotic cell death, 342 appetite and assimilation, 376–81, 386–8 hypoxia eVects on central regulation, 387 Apteronotus leptorhynchus, 197 aquaculture, fish kills, 81 aquatic surface respiration (ASR), 28–38, 39–43, 212–14 air‐breathing responses to hypoxia in air‐breathing fish, 202–4(tab.) energetics, 60 facultative air‐breathers, 214 obligate air‐breathers, 224 PO2 thresholds, 212(tab.) response to extreme hypoxia, 64 risk of predation, increased prey vulnerability, 60 synchronous air breathing, 61 tendency to surface at lower DO thresholds, 61 threshold in brooding females, 56 travel costs of ASR and air‐breathing, 219 vertical distribution of prey, 60 Arapaima gigas, 390 Arctic charr (Salvelinus alpinus), 282, 310 Arctic marine fauna energy‐saving lifestyles, 151 Hb components, 271 widely distributed Northern hemisphere species, 153 Arius leptaspis, 280 Arrhenius activation energies, 175 arterial O2 transfer factor [TaO2], 489 INDEX aryl hydrocarbon receptor (AHR), 123, 473 aryl hydrocarbon receptor nuclear translocator (ARNT), 123, 467 Ascelichthys rhodorus, 53 assimilation, and appetite, 376–81, 386–8 assimilation eYciency (AsE), 365 Astatoreochromis alluaudi, 31 Astatotilapia aeneocolor, 31, 212 Astatotilapia velifer, 31 Astatotilapia ‘wrought‐iron’, 31, 64, 212 Astronotus crassipinnis, 335, 344 Astronotus ocellatus, 28, 31, 37, 59, 212, 213, 333, 457, 463 Astyanax bimaculatus lacustris, 119 Atchafalaya River, Louisiana, 83 Atlantic cod (Gadus morhua), 47, 48, 147–9, 154, 176–7, 197, 226, 229, 266 anaerobiosis, 335–6 blood and heart glucose levels and heart lactate levels, 337(tab.) cardiac parameters and oxygen consumption, 327(fig.) cardiovascular parameters to, 8 min of hypoxic exposure, 320(fig.) acute stress response to protocol, 319 cardiovascular responses to hypoxia, 303 cytochalasin B and glucose transporter proteins, 339(fig.) diVerent oxygen saturations and temperatures, 48 eVects of exogenous catecholamines on ventilation volume, frequency, and amplitude, 225–6(tab.) fH‐water PO2 relationships, 306, 307, 328 gene expression levels, 334(fig.) glucose uptake under hypoxia, 338 Hb polymorphisms, 271 hypoxia, appetite and assimilation, 376–81, 386–8 hypoxia‐related reduction in growth, 376, 378(fig.) myoglobin, 274 specific dynamic action (SDA), 374–5(fig.) specific growth rates (SGR), 366(tab.) thermal optimum of growth performance, 147–9, 154 ventilatory responses to hypoxia, 206 visual acuity, 266 507 INDEX warming‐induced reduction of recruitment in North Sea, 177 water PO2 and various cardiac parameters, 313(fig.) Atlantic croaker (Micropogonias undulatus), 90, 96, 99 Hb polymorphisms, 272 HIF‐1 and HIF‐2 123 injection with GnRHa, 91 sex hormones, 93–5(tab.) Atlantic herring (Clupea harengus), 47 schooling, 50, 62 Atlantic salmon (Salmo salar) embryonic development, 106–7 inability to reach spawning grounds in warming rivers, 146 NO‐mediated regulation of vascular dilation, 284 ATP normoxia vs, 12 h of hypoxia (goldfish), 449(fig.) stable cellular [ATP], hypoxia tolerance, 447–8, 449(fig.) ATP conservation, 465 ATP production (glycolysis), 329–31, 343–4, 399–400 decrease in demand, 330–2 general model for activation, 329–31 glucose to support heart performance, 336 mitochondrial electron transport chain, 446 O2 ‐independent, 455–7 SarcKATP channels, 331 ATP supply, via oxidative phosphorylation, reduction during temperature‐ induced hypoxemia, 171 ATP‐dependent ion‐motive pumps, 400 Aurelia aurita, 60 Austrapotamobius pallipes, 159 B Barbus spp., 32 barramundi (Lates calcarifer), 270, 278 bass, see also sea bass (Dicentrarchus labrax) bass, largemouth (Micropterus salmoides), fluctuating O2 levels for growth, 382 bass, small‐mouth (Micropterus dolomieui), 105, 229 f H—water PO2 relationships, 306, 307 larval survival times, 105 bass, striped (Morone saxatilis), 36(fig.), 282, 368, 382–3 Bcl‐2 and Bcl‐xL, anti‐apoptosis, 104 behavioral responses, 25–81 beta‐adrenergic receptors of erythrocytes, 281–3 Betta splendens, 54, 64 bimodal respiration, 40 biomass (kg wet wt. h‐1 trawling), and oxygen concentration, 82(fig.) bitterling (Rhodeus amarus), 401 black bass see bass, small‐mouth (Micropterus dolomieui) Black Sea, 6 blacknose (Carcharhinus acronotus), 47, 51 blennies, 41 (Blennius pholis), spontaneous emersion, 53 (Blennius sanguinolentus), 229, 230 (Helcogramma medium), 53 Blennioidiae, 41 blood, viscosity, 258 blood oxygen transport, 255–301 blue discus (Symphysodon aequifasciatus), 457 (tab.) body size, eVect on hypoxia tolerance, 59 bonefish (Albula sp.), larval survival times, 105 bonnethead (Sphyrna tiburo), 51 Botia sidthimunki, 32 bowfin (Amia calva), 42, 196, 202, 214, 222, 226, 229, 230 Brachydanio albolineatus, 32 brain lipids, Crucian carp, 431–3 branchial O2 chemoreceptors, 228–30 bream, silver (Sparus sarba), 367 brook stickleback (Culaea inconstans), 62, 63 brook trout (Salvelinus fontinalis), fluctuating O2 levels for growth, 382 brown trout (Salmo trutta), 87, 229 expression of CYP19 92 hatching, 108, 109(fig.) mortality of embryo and larvae, 107(tab.), 110(fig.) survival through embryonic development, 105 buValo sculpin (Enophrys bison), 275 buVer components, dissociation equilibria (pK‐values), 158 bullhead (Ictalurus nebulosus), 229, 230 508 INDEX bully (Gobiomorphus cotidianus), fry survival, 105 burbot (Lota lota) cold‐induced ryanodine sensitivity, 169 metabolic depression, 158 C Ca‐induced Ca2þ release (CICR), 169 Ca‐induced Ca2þ‐dependent neurosecretion, 235 Calanus finmarchicus, 176 Calanus helgolandicus, 176 calcium channels sarcolemmal L‐type (SL), 168–72 sarcolemmal reticular (SC), 168–72 calcium regulation, inadequate, during temperature change, 168 Callionymus lyra, 197 Cancer magister, 387 Carassius auratus see goldfish Carassius carassius see carp, Crucian carp (Carassius carassius) Carcharhinus acronotus, 47, 51 cardiac activity, during anoxia, 407–8 cardiac contractility, rainbow trout (Oncorhynchus mykiss), 168 cardiac coronary circulation, elasmobranchs and teleosts, 498 cardiac energy metabolism, 329–43 cardiovascular responses to hypoxia, 301–62 branchial vascular resistance, 314–19 cardiac energy metabolism, 329–43 cardiac output, stroke volume and venous tone, 312–14 chronic hypoxia in adult fish, 326 control of flow and resistance, 318–19 heart rate, 304–12 mean circulatory filling pressure (MCFP), 314, 316(fig.) model for autonomic control of teleost gill vasculature, 317(fig.) preconditioning, 348, 349(fig.) presence/absence of hypoxic bradycardia, 304–6 systemic vascular resistance and blood pressure, 319–21 water oxygen level (PO2) and heart rate ( fH), 307(fig.) carotid body chemoreceptors, 232–4 carotid body neurochemicals, 237–8 carp, see also goldfish (Carassius auratus) carp, common (Cyprinus carpio), 32, 93–4, 98, 197 adenosine, control under hypoxia, 333 cardiovascular responses to hypoxia, 303, 310, 347 eVects of acclimation temperature on hypoxic ventilatory response, 210(tab.) fertilization, hatching rate, larval survivorship, and overall survivorship, 103–5 Gonadal Somatic Index (GSI), 98 hypoxia‐induced expression of Mb in nonmuscle tissues, 455 myoglobin, 274 normoxia vs hypoxia, spermatogonia, 101(fig.) PO2 thresholds for aquatic surface respiration (ASR), 212 specific growth rates (SGR), 370(tab.) sperm motility, 102(tab.) predictor for sperm quality, 102–3 spermatogenesis, 99 testosterone, estradiol and triiodothyronine disruption, 93, 94 carp, Crucian carp (Carassius carassius), 44, 266, 275, 285, 397–444 anoxia‐tolerance, 397–444 at diVerent temperatures, 399(fig.) seasonality, 420–33 blood and heart glucose levels and heart lactate levels, 337(tab.) brain activity during anoxia, 411–13, 430–3 adenosine, 419–20 Naþ/Kþ ATP‐ase activity, 331, 430 cardiac activity, 405–7 cardiac activity during anoxia, 407–8, 425–30 adenosine, 410–11 ATP‐sensitive Kþ channels, 408–10 distribution and habitat, 398–400 ethanol as major end product of anaerobiosis, 401–2 gill remodeling, 403–4, 465–6 glycogen stores, 403, 420–6, 423(tab.) INDEX in brain, liver, and muscle, 457(tab.) winter/summer, 423(tab.) hemoglobin, 405 HIF‐1alpha expression, 285 hypoxia cardiovascular response, 405–7 protein synthesis, 333 reduced need for O2 399–400 maximum recorded decreases in metabolic rate, 462 metabolic rate suppression, 460–1 neuroglobin, 275 neurotransmitters and neuromodulators, 415–17 phosphatidylethanolamines, 431, 433 seasonality of anoxia‐tolerance, 420–33 brain, 430–3 brain glycogen, 422–5 glycogen stores, 420–6, 423(tab.) heart glycogen, 425–6 heart size, rate and stroke volume, 426–7 liver and skeletal muscle glycogen, 421–2 myocardial contractility, 427–30 suppression of neural excitability, 413–15 carp, grass (Ctenopharyngodon idella), 476 Ca2þ‐ATPase, 400 cascudo preto (Rhinelepis strigosa), 201, 204, 214 catecholamines, 155 circulating, stimulating breathing, 224 evoking hyperventilatory responses, 224 catfish, Amazonian (Hoplosternum littorale), 203, 277 catfish, armoured (Ancistrus chagresi), 42, 61, 202, 214, 276 catfish, armoured (Glyptoperichthys gibbceps), 305 catfish, armoured (Liposarcus pardalis), 305, 333, 337, 341 blood and heart glucose levels and heart lactate levels, 337(tab.) hexokinase activity, 340–1 PFK binding, 342 catfish, armoured (Pterogoplichthys spp.), 276, 287 catfish, channel (Ictalurus punctatus), 222, 230, 366 chronic hypoxia eVect on cardiovascular responses, 326 growth rates, 370 509 NO in exposure to hypoxia, 284 steroidogenic enzymes, 87 ventilation during exposure to hypoxia, 198 catfish (Hypostomus regani), 198, 203, 214 catfish, Indian (Heteropneustes fossilis), 229, 230, 389 NECs, 237 catfish, salmon (Arius leptaspis), 280 catfish (Silurus glanis), 201, 266 catfish, silver (Rhamdia quelen), 366(tab.) catfish, upside‐down (Synodontis nigriventris), 28 catfish, Vietnamese (Pangasius hypothalamus), 230 Catostomidae, 30 Catostomus commersoni, 30 cDNA microarray studies, 456 cell arrest, in mitosis, 108 cell proliferation and apoptosis, 104–5 cellular stress and signaling, 159–62 Centrarchidae, 30 Centropomidae, 30 cephalopod mantle tissue, transition to mitochondrial anaerobiosis, 144 Chaenocephalus aceratus, 305 Channa argus, 42, 202 Channa striatus, 388, 389 Channichthyidae, 274 Characidae, 30 characoid (Hepsetus odoe), floating foam nest, 55 chemoreceptors, see also oxygen‐sensing mechanisms chemosome hypothesis, 235 chinook salmon (Oncorhynchus tshawytscha), 87, 105 embryonic development, 105 mortality of embryo and larvae, 107 (tab.), 108 Chionodraco hamatus, 284 cholecystokinin (CCK), 386 cholesterol convertsion to pregnenolone, 85 inner mitochondrial membrane, 85 Chondrostoma nasus, 105, 107, 108, 110 chorionase, stimulation of secretion, 106 Chrosomus eos, 32 Chrysaora quinquecirrha, 60 Cichlasoma biocellatum, 31 510 cichlids, 30–1(tab.) Astatotilapia aeneocolor, 31, 212 Astatotilapia velifer, 31 Astatotilapia ‘wrought‐iron’ 31, 64, 212 Astronotus crassipinnis, 335, 344 dominance hierarchies, 64 Haplochromis piceatus, 55, 328 histological changes in heart in chronic hypoxia, 329 Hoplias microlepis, 335, 344 moving young to oxic habitat, 55 physiological refugia, 262 Pseudocrenilabrus multicolor victoriae, mouth brooding, 56 see also oscar Clarias, 42 Clarias lazera, 203 Clarias liocephalus, 61 climate change, eVects on ecosystems, 175–8 climbing perch (Anabas testudineus), 53 Clinocottus globiceps, 53 Clupea harengus, 47, 50, 62 cod (Gadus morhua) see Atlantic cod coho salmon (Oncorhynchus kisutsch), fluctuating O2 levels for growth, 382 cold acclimation, 156, 172–3 see also thermal tolerance Colisa lalia, 214 Colossoma macropomum, 28, 29, 197, 222, 223, 226, 261, 305 Conger conger, 229, 269 Copella, 36 copepod species, 176 coral reef crevices, 6 coral reef gobies, 58 Coregonus clupeaformis, 35 coronary circulation, elasmobranchs and teleosts, 498 corticosteroids, 155 corticotropin‐releasing factor, 387 Cottidae, 29, 38, 41, 53 Cottus gobio, 110 crab (Cancer magister), 387 crayfish (Austrapotamobius pallipes), 159 creatine kinase (CK) levels, 330 Crucian carp (Carassius carassius) see carp crustacean, progressive hypoxemia in arterial haemolymph, 144 Ctenopharyngodon idella, 476 Ctenopoma damasi, 55 INDEX Ctenopoma mureiri, 214 Culaea inconstans, 34, 62, 63 cyanide, hyperventilatory responses by all teleosts, 220–1 CYP19A and CYP19B, 117 Cyprichromis leptosoma, 31 Cyprinidae, 32–3 Cyprinodontidae, 33 cytochrome P450 enzymes, 85–7 expression, 123 D dab (Limanda limanda), 368 hypoxia‐related reduction in growth, 377 specific growth rates at diVerent durations of hypoxia, 380(tab.) damselfish (Pomacentrus amboinensis), cortisol levels, 96 Danio rerio see zebrafish dead zones, 81 death, mechanism of hypoxia‐induced death, 447 death receptor (Fas‐Fasl) pathways, 113 decomposition of organic matter, 2 demersal fish, and pelagic fish, shift in dominance, 82 density stratification of water column, 2 development, 104–20 cell proliferation and apoptosis, 104–5 delays in hatching, 107–9 disruption in apoptosis, 112 embryonic development, 106–7 gonads, 98–101 hormonal disruption, 115–17 implications, impairment and population decline, 125–6 insulin‐like growth factor binding protein (IGFBP)–1 114 malformations, 110–15 organogenesis, 112 neuroepithelial cells (NECs), early larval stages, 231 other vertebrates, 120–2 sperm motility, predictor for sperm quality, 102–3 teratogens, 110–15 511 INDEX developmental stages, viability in anoxia, 106(tab.) Devonian aquatic habitats, hypoxic condition, 257 Dicentrarchus labrax, 62, 229, 261, 280, 367–8, 382, 384, 385 digestion appetite and assimilation, 376–81, 386–8 assimilation eYciency, 387–8 hypoxia, 384–6 specific dynamic action of food (SDA), 373 vomiting response, 386 dissociation equilibria (pK‐values), buVer components, 158 dissolved oxygen (DO), and temperature, 266–7 dissolved oxygen (DO) (freshwater), 487 air vs water, 2 nomogram (10oC and, 30oC), 3 threshold value, 2 dissolved oxygen (DO) (marine and estuarine), 487 aquatic surface respiration (ASR), tendency to surface at lower DO thresholds, 61 nomogram (10oC and, 30oC), 3 OMZs, 4 threshold value, 2 DMY (Y‐specific DM domain) gene, 87 docosahexaenoic acid, 433 dogfish, larger spotted (Scyliorhinus stellaris), 195, 229 dogfish (Scyliorhinus canicula) acclimation temperature and heart rate, 309(fig.) eggs, 13–15 weeks old survival, 105 hatching, 108 mortality of embryo and larvae, 107(tab.) systemic vascular resistance, 320 water acclimation and bradycardia, 308 dogfish, spiny (Squalus acanthias), 195, 284 eVects of exogenous catecholamines on ventilation volume, frequency, and amplitude, 226(tab.) NO production, 284 dopamine (DA), inhibiting LH secretion, 85 dourado (Salminus maxillosus), 306, 307 Dover sole (Solea solea), 49 dragonet (Callionymus lyra), 197 E ecological interactions and hypoxia, 57–64 eel, American (Anguilla rostrata), 335–6 glucose uptake under hypoxia, 338 eel, common (Anguilla anguilla), 196, 211, 229, 230 anodic Hb component, 277 catecholamines evoke hyperventilatory responses, 224 eVects of exogenous catecholamines on ventilation volume, frequency, and amplitude, 225–6(tab.) Hb components, 269 maximum recorded decreases in metabolic rate, 462(tab.) metabolic rate suppression, 460–1 NO‐mediated regulation of vascular dilation, 284 oxygen turnover, 267 eel, conger (Conger conger), 229, 269 eel, deep‐sea (Symenchelis parasitica), Hb components, 269 eel (Gymnothorax unicolour), 277, 278 eel, Japanese (Anguilla japonica), 196, 211, 306, 307, 319 eel, swamp (Synbranchus marmoratus), 42, 204, 305, 314 eelpout, Antarctic see Antarctic eelpout eelpout (Zoarces viviparus), 110, 145, 155–6, 159–60, 285, 328, 334–5 acclimation to hypoxia, glucose utilization, 344–5 adenosine accumulation, 155 cold acclimation, 156 increased cardiac myocyte density, 328 developmental defects, 110 myoglobin, 334 oxidative stress parameters in hepatic tissue, 159 oxygen and capacity limitation concept, 145(fig.) recovery from both cold and heat exposure, 160(fig.) eels, Hb polymorphisms, 271(fig.) elasmobranchs, coronary circulation, 498 endothelin, identified in gill neuroepithelial cells, 229–30(tab.) endothermy, partial, 267–8 512 INDEX energy, available for growth, 364–71 energy budget, turnover, and allocation, 170–5 Engraulis mordax, 62 enkephalins, identified in gill neuroepithelial cells, 230(tab.) environmental hypoxia, 489–93 metabolic responses of fish, 490(fig.) epinephrine and norepinephrine, 155–6 Epiplatys dageti, 33 Eptatretus stoutii, 195 Erythrinus, 42 erythrocytes adaptational defence in hypoxia protection, 276–8 integrative functions, 278–9 organic phosphate compounds, 259 phosphates and Bohr factor, 265 role of beta‐adrenergic receptors, 281–3 escape response, fast‐starts, 61–2 Esox lucius, 33, 59 ethanol as major end product of anaerobiosis, 401 production in carp, 401–4 Etheostoma exile, 34 Etheostoma nigrum, 34 Euthynnus aYnis, 268 eutrophic lakes, summer oxygen depletion, 4 eutrophication, defined, 6 evolution of adaptive strategies, 4 eye choroid rete Counter‐current multipliers, 263 retinal oxygen flux through secretion of lactic acid, 265 visual acuity, 265–6 Florida flagfish (Jordanella floridae), 55 Florida smoothhound shark (Mustelus norrisi), 51 flounder, see also winter flounder (Pleuronectes americanus) flounder (Platichthys flesus), 200, 211, 269 blood and heart glucose levels and heart lactate levels, 337(tab.) mitochondrial degeneration in chronic hypoxia, 335 myofibril degeneration in chronic hypoxia, 326 flounder, Southern (Paralichthys lethostigma), 369 fluctuating O2 levels for growth, 382 specific growth rates at diVerent durations of hypoxia, 380(tab.) flounder, yellowtail (Limanda ferruginea), 308 food conversion eYciency (gross conversion eYciency, K1), 365 food/feeding food consumption, and increased surfacing costs, 388 specific dynamic action (SDA), 373–5, 387, 390 see also digestion France, SW, Adour river, 83 freshwater burbot (Lota lota), metabolic depression, 158, 169 freshwater medaka see medaka frog, glycogen stores, 423(tab.) functional hypoxia, 493–8 Fundulus grandis, 29, 60, 93, 98, 102–4, 459 Fundulus heteroclitus, 38, 456–7 G F factor inhibiting HIF (FIH), 468 Facultative air‐breathers, 53 fast‐starts, 61–2 fathead minnow (Pimephales promelas), 29, 58, 62 fertilizer use, and rise of dead zones, 7(fig.) flathead flounder (Hippoglossoides dubius), 377 floodplains, 6 G‐protein‐coupled membrane GtH receptors (GtH‐Rs), 84–5 GABA, inhibition of synthesis by glutamate decarboxylase, 416–19 Gadus morhua see Atlantic cod Galaxidae, 34 Gambusia holbrooki, 35, 59, 100, 262 gametogenesis, 85–7, 98–101 gar (Lepisosteus oculatus), 61, 203, 305 hypoxic bradycardia, 203, 305 synchronous air breathing, 61 INDEX gar (Lepisosteus osseus), 61, 203, 229, 230 synchronous air breathing, 61 gar (Lepisosteus platyrhincus), 43, 214, 229 lack of external O2 receptors linked to hyperventilation, 221 gar (Synbranchus marmoratus), 42, 204, 305, 314 gene expression profile, 67–9 genes muscle contraction, 465 O2‐dependent expression, 478 up‐ and down‐regulated genes in response to hypoxia exposure, Venn diagram, 479(fig.) see also hypoxia‐inducible factors Germany, Wadden Sea, 176 ghrelin, 386 gill remodeling, 499 Crucian carp, 403–4 gill ventilation and air‐breathing responses to hypoxia in air‐breathing fish, 202–4(tab.) branchial O2 chemoreceptors, 228–9 carotid body type I cells, 235 control of flow and resistance, 318–19 eVect of bilateral gill denervation on ventilatory responses to hypoxia, 222 model for autonomic control of teleost gill vasculature, 317(fig.) neuroepithelial cells (NECs), 207, 218(fig.), 228–30, 236(fig.) oxygen chemotransduction, 235–7 respiratory surface, scaling relationship, and metabolic rate, 59 responses to hypoxia, 195–205(tab.) Gillichthys mirabilis, 89, 288, 330, 451–2, 458 Gillichthys seta, 89 gilthead sea bream (Sparus auratus), Hb components, 273 glucose concentration gradient and abundance of GLUTs, 336 and heart performance, 335–6 hexokinase activity, 340–1 increased uptake in hypoxia via enhancement of facilitated diVusion, 338 glutathione redox ratio, 2GSSG/GSH, 160 513 GLUTs (glucose transporter proteins), 336, 338, 340, 466 glycogen phosphorylase cascade, 335–6 glycolysis see ATP production (glycolysis) Glyptoperichthys gibbceps, 305 Gnathonemus victoriae, 34 Gobionotothen gibberifrons, 270 Gobiosoma bosc, 60 goby, common percentage of time spent fanning, 98(fig.) (Pomatoschistus microps), parental care, 55 goby, coral reef gobies, 58 goby, land‐locked, (Rhinogobius sp.), 55 goby, mudsucker (Gillichthys mirabilis), 89, 288, 330, 451–2 myoglobin, 334 goby, naked, (Gobiosoma bosc), 60 goby, sand, (Pomatoschistus minutus), male parental care, 49, 55 golden grey mullet (Liza aurata), 38 fast‐starts, 61–2 goldfish (Carassius auratus), 32, 38, 212, 217, 229, 266 anoxia‐tolerance, 398 ATP, normoxia vs, 12 h of hypoxia, 449 (fig.) blood‐oxygen aYnity, 266 glycogen stores, 423(tab.) in brain, liver, and muscle, 457(tab.) liver phospho‐eEF2 464(fig.) metabolic rate suppression, 460–2 neuroepithelial cells (NECs), containing neurosecretory synaptic vesicles, 237 phosphofructokinase (PFK), 342 temperature cycling, 273 gonad development, 98–101 sperm motility, predictor for sperm quality, 102–3 Gonadal Somatic Index (GSI), adult carp, 98 gonadotropin‐releasing hormone (GnRH), 84–5, 90–1 gourami (Colisa lalia), 214 gourami (Macropodus opercularis), 54 Great Barrier Reef, 58 growth appetite and assimilation, 376–81, 386–8 assimilation eYciency (AsE), 365 514 INDEX growth (continued ) eVects of dynamic changes in oxygen levels, 382–3 energy available for growth, 364–71 food conversion eYciency (gross conversion eYciency, K1), 365 net conversion eYciency K2, 365 prolonged hypoxia, 379–82 specific growth rate (SGR), 365 temperature, salinity and hypoxia, 382–3 growth hormones, secretion, 104 Gulf killifish (Fundulus grandis), 29, 459 acclimation to hypoxia glucose utilization, 344–5 glycolytic enzyme activities, 460(fig.) daily egg production per female, 102(fig.) hormonal disruption, 93, 98 onset of spawning, 103–4 Gulf toadfish (Opsanus beta), opercular pressure (index of ventilation amplitude), 216(fig.) Gymnocorymbus, 30 Gymnothorax unicolour, 277 Gymnotus, 42 H hagfish (Myxine glutinosa), 278 cardiac output (Q) and power output (PO), 312 haplochromines, moving young to oxic habitat, 55 Haplochromis piceatus, 55, 328, 329 heat shock response, 161 Helcogramma medium, 53 Hemichromis bimaculatus, 31 Hemichromis letourneuxi, 31 Hemiodidae, 34 Hemiscyllium ocellatum, 332 Hemitripterus americanus, 220, 221, 274 hemoglobin, 258–85 allosteric regulation at subcellular level, 278 Bohr eVect, 215, 259, 263–5, 271, 280 buVering power of surface histidine components, 263 Haldane eVect, 281 Hb–O2 binding aYnity, 207, 258, 498 phosphate regulation, 276–80 mean cell Hb concentration (MCHC), 258 multiple forms of Hb, 268–73 NO binding, 283–4 oxygen equilibrium curves, 258–62, 260 (fig.), 262(fig.) P50 value, 215 role as O2 ‐sensing mechism, 286 Root eVect, 259, 263–5, 271, 277, 280 translational modification of function, 284–5 Hepsetus odoe, 55 herring (Clupea harengus), 47, 62 schooling, 50, 62 Heteropneustes fossilis, 229, 230, 237, 389 hexokinase (HK), activity, 340–1 high‐motility group proteins HMG‐Y and HMG2a, 89 Hill’s coeYcient, 281 Hill’s n‐value, 261, 268 Hippoglossoides dubius, 377 Hoplerythrinus unitaeniatus, 42, 198, 203, 209, 214, 305 Hoplias lacerdae, 198, 209, 211, 229, 306, 307, 310 Hoplias malabaricus, 198, 211, 221, 229, 305, 306, 307, 310 Hoplias microlepis, 335, 344 Hoplosternum littorale, 203, 277 Hoplosternum thoracatum, 61 hormonal disruption, 115–17 5‐HT see serotonin H2S levels, plasma, 234, 321 Hybognathus hankinsoni, 33 hydrogen sulphide (H2S) levels in plasma, 234, 321 as sensor or transducer of O2 234, 321 hydrothermal vents, 270 hydroxysteroid dehydrogenases (HSDs), 85–6 hypercapnia, 6 Hyphessobrycon pulchripinnis, 30 Hypostomus plecostomus, 203 Hypostomus regani, 198, 203, 214 hypothalamus—pituitary—gonad (HPG) axis, 83–5 hypophysiotropic factors, 387 hypoxia, 487–505 515 INDEX acclimation or acclimatization, 499 defining, 488–95 and ecological interactions, 57–64 eVects on growth, appetite and assimilation, 376–81, 377(fig.) environmental hypoxia, 489–91 exposure time, 498–500 functional hypoxia, 493–8 future research considerations, 501–2 oscillating, plant respiration, 500 hypoxia tolerance, stable cellular ATP, 447–8 hypoxia‐induced death, mechanism, 447 hypoxia‐inducible factors, 450–1, 467–75 activation in thermal acclimation, 159–60 isoforms, 469–71 HIF‐1 and HIF‐2 122–4 HIF‐1alpha, role in hypoxia resistance, 285–6 HIF‐1alpha, mRNA expression, 476 HIF‐1beta (ARNT), 123, 467, 473 phylogeny, 471(fig.), 474(fig.) structural analysis of HIF‐1alpha amino acid sequence, 473(fig.) regulation by O2 468(fig.), 475–6 relation between function and hypoxia tolerance, 477–8 hypoxia‐inducible genes, 122–4 hypoxic chemotransduction, 232–5 hypoxic ventilatory response, 193–256 oxygen‐sensing mechanisms, 220–32 physiological significance, 215–19 Hypseleotris sp., 33 J Japanese eel (Anguilla japonica), 196, 211, 306, 307, 319 Japanese mudskipper (Periophthalmus modestus), male parental care, 56 jeju (Hoplerythrinus unitaeniatus), 42, 198, 203, 209, 214 hypoxic bradycardia, 305 jellyfish, sea nettle, 60 jellyfish (Aurelia aurita), 60 Jordanella floridae, 55 K Katsuwonus pelamis, 40, 50, 168, 199 Kattegat, shift in dominance from demersal to pelagic fish, 82 kawakawa (Euthynnus aYnis), 268 killifish, see also Gulf killifish (Fundulus grandis) killifish (Fundulus heteroclitus) glycogen stores in brain, liver, and muscle, 457(tab.) pyruvate dehydrogenase kinase, 456 killifish, mangrove (Kryptolebias [Rivulus] marmoratus), 5 Kryptolebias (Rivulus) marmoratus, 54 Krytopterus bicirrhus, 35 KB (TASK‐like) channel, 234 regulation by hypoxia, 235 I L ice and snow cover, 2 icefish (Chaenocephalus aceratus), 305 icefish (Chionodraco hamatus), 284 Ictalurus melas, 34, 229 Ictalurus nebulosus, 229, 230 Ictalurus punctatus, 87, 198, 222, 229, 230, 284, 366, 370 insulin‐like growth factor binding protein (IGFBP)–1 114 insulin‐like growth factors, aVecting ovarian growth, 87–8 invertebrate prey, interactions, 62–3 Labeo bicolor, 33 Labeo capensis, 272 Labrochromis ishmaeli, 31 labyrinthine fishes (Anabantoids), spontaneous emersion, 53 lactate dehydrogenase, 343, 402, 450–1 lactate production, turtles, 403 Lake Nabugabo, 58 Lake Victoria basin, structural and low‐ oxygen refugia, 57 Lamna nasus, 268 516 INDEX Lampetra fluviatilis, 195, 283 Lampetra japonica, 230 largemouth bass (Micropterus salmoides), fluctuating O2 levels for growth, 382 Lates calcarifer, 270, 278 Lates niloticus, 30, 57–8 Leiopotherapon unicolor, 211, 308 Leiostomus xanthurus, 369 Lepidonotothen kempi, 172 Lepidosiren, 304 Lepidosiren paradoxa, 202 Lepisosteus, 42 Lepisosteus oculatus, 61, 203, 305 Lepisosteus osseus, 61, 203, 229, 230 Lepisosteus platyrhincus, 43, 214, 221, 229 Lepomis gulosus, 30 Lepomis marginatus, 30 Limanda ferruginea, 308 Limanda limanda, 368, 377 Liposarcus pardalis, 305, 333 Liza aurata, 34, 38, 61–2 locomotor activity, change in response to hypoxia, 45, 52 Loligo forbesi, 177 longjaw mudsucker (Gillichthys mirabilis), 89, 288, 330, 451, 452(tab.) myoglobin, 334 S‐adenoylmethionine synthase and cystathione synthease, 458 Lota lota, 158 Louisiana, Atchafalaya River, 83 lungfish, African (Protopterus aethiopicus), 40, 42, 202, 215 glycogen stores, in brain, liver, and muscle, 457(tab.) inositol phosphates, 276 nest construction, 55 lungfish, African (Protopterus annectens), 229 lungfish (Protopterus amphibius), 280 M Macropodus opercularis, 54, 390 Macrourus holotrachys, 271 Macrozoarces americanus, 274 malformations, fish larvae, 110–15 maneuverability, 62 mangrove killifish (Kryptolebias [Rivulus] marmoratus), emergence response, 54 MAP kinase phosphatase, 1 (KP‐1), 89 MAP‐kinase phosphates, 465 marlin, striped (Tetrapterus audax), 268 Mastacembelus circumcinctus, 34 medaka , marine (Oryzia melanostigma) localization of omTERT mRNA in testes, 124(fig.) sex ratio, and sex diVerentiation, 125–6 medaka (Oryzias latipes), 87, 108 gene expression patterns, 89 neurotransmitters, 91–2 sex reversal, 87 sex‐determining gene DMY, 119 Megalops atlanticus, 203, 214 Megalops cyprinoides, 203, 270, 278, 280 Menidia beryllina, 49 metabolic responses to environmental hypoxia, 490(fig.) metabolism, 361–90, 443–88 aerobic scope and growth, 371–3 depression freshwater burbot, 158 shrimp (Palaemon), 158–9 during hypoxia exposure, 443–88 metabolic and molecular responses, 451–66 coordination of these responses, 466–78 metabolic rates RMR and SMR, 362–4, 371 suppression, 459–68 and oxygen consumption (VO2 max), 364 (fig.) specific dynamic action of food (SDA), 373 time to acclimate and acclimatize, 450–1 Metynnis, 30 Micropogonias undulatus, 90, 91, 93–5, 96, 99, 123, 272 Micropterus dolomieui, 105, 229, 306, 307 Micropterus salmoides, 382 Misgurnus anguillicaudatus, 203 mitochondria anaerobiosis, 144 densities, 287 densities and functions, 154–5 KATP channels, 331 mitochondrial electron transport chain, ATP production, 446 mitochondrial enzymes, 335 517 INDEX mitochondrial hypothesis, oxygen‐sensing mechanisms, 233 mitochondrial membrane, cholesterol import, 85 Mnierpes macrocephalus, 53 Modiolus barbatus, 161 molecular responses to hypoxia, 444–88 coordination of these responses, 466–78 mollies, 29, 56 moray, brown (Gymnothorax unicolor), Hb components, 269 moray (Muraena helena), Hb components, 269 Morone saxatilis, 36, 282, 368, 382–3 mosquitofish (Gambusia holbrooki), 35, 59 eVect of oxygen on mating behavior, 100 (fig.) sexual fitness of hypoxia‐acclimated, 262 mudfish (Labeo capensis), 272 mudminnow (Umbra limi) air‐breathing, synchronous, 214 foraging behaviour, 62 mudskipper, Japanese (Periophthalmus modestus), male parental care, 56 mudsucker (Gillichthys mirabilis), 89, 288, 330, 451, 452(tab.), 458 Mugil cephalus, 34, 37, 38, 39, 199, 212, 213 mullet (Liza aurata), 34, 38, 61–2 mullet (Mugil cephalus), 37, 38, 39, 199, 212, 213 Muraena helena, 269 muscle contraction, genes, 465 mussels, Modiolus barbatus and Mytilus galloprovincialis, 161 Mustelus, 47 Mustelus norrisi, 51 myoglobin, 273–5 better maintenance of ATP levels and oxygen consumption, 333–4 oxygen transport, 454–5 red muscle, vs white muscle, 52 Myoxocephalus octodecimspinosus, 274 Myoxocephalus scorpius, 199, 337 Mytilus galloprovincialis, 161 Myxine glutinosa, 278, 312 Naþ/Hþ exchanger, 156, 281 Naþ/Kþ ATPase, 171, 172, 331, 400, 430 nase (Chondrostoma nasus) developmental defects, 110 embryos, 105, 107 Neoceratodus, 304 Neoceratodus forsteri, 204 Neochromis nigricans, 31 Neolamprologis tretocephalus, 31 neuroepithelial cells (NECs), 207, 218(fig.), 219(fig.), 228–30, 236(fig.) oxygen chemotransduction, 235–7 proposed model for oxygen sensing, 236(fig.) neuroglobin, 275–6 neuropeptide Y, identified in gill neuroepithelial cells, 230(tab.) neurotransmitters, 85, 91–2, 237–9 identified in gill neuroepithelial cells by immunohistochemistry, 229(tab.) and neuromodulators, 415–17 response to hypoxia, 91 Nile perch (Lates niloticus), predation on cichlids, 57–8 nitric oxide (NO) SarcKATP channels, 331 vasodilatation of vascular smooth muscle, 283 nitric oxide synthase, identified in gill neuroepithelial cells nitrogen, limiting nutrient (SW), 6 Nocomis biguttatus, 33 North Sea, warming‐induced reduction of cod recruitment, 177 Northward geographical shifts, 176 Notothenia angustata, 270 Nototheniodei Hb polymorphisms, 270, 271 Pagothenia borchgrevinki, 270, 279, 305, 332 Notropis spp., 33 Noturus gyrinus, 34 nucleus lateralis tuberis, 386 nucleus preopticus, 386 N O naked goby (Gobiosoma bosc), 60 Nannoperca australis, 35 ocean pout (Macrozoarces americanus), 274 oceanic oxygen minimum zones (OMZs), 4 518 oceanic oxygen minimum zones (OMZs), (continued ) continental margins, 4 global distribution (map), 5 oceans, anthropogenic CO2 accumulation, 175 Odax pullus, 267 Odontamblyopus lacepedii, 41, 204 Oligocottus maculosus, 29, 38, 41, 53 Oligocottus snyderi, 53 oligotrophic lakes, development of hypoxia, 4 Oncorhynchus kisutsch, 382 Oncorhynchus mykiss see rainbow trout Oncorhynchus nerka, 152, 369 Oncorhynchus tshawytscha, 87, 105, 107, 108 oogenesis, 84–5 opercular displays, 64 Ophiocephalus (Channa) striatus, 388, 389 Ophiodon elongatus, 35, 200 Opsanus beta, 200, 216 Oreochromis esculentis, 31 Oreochromis microlepidotus, 210 Oreochromis mossambicus, 229, 230, 457, 461, 462 Oreochromis niloticus, 31, 200, 210, 367, 388 Orthodon microlepidotus, 200 Oryzia latipes, 87, 89, 91–2, 108, 119, 124 Oryzia melanostigma, 124 oscar (Astronotus ocellatus), 28, 31, 37, 59, 212, 213 glycogen stores in brain, liver, and muscle, 457(tab.) hypoxia, protein synthesis, 333 suppression of protein synthesis, 463 Oslofjord, Norway, 6 Osteoglossum, 36 ovarian growth, insulin‐like growth factors, 87–8 oxidative stress, in response to hypoxemia, 155–6 oxygen arterial O2 transfer factor [TaO2], 489 enhanced utilization under hypoxia, 333–4 Hb–O2 binding aYnity, 207, 258, 498 see also dissolved oxygen (DO) oxygen consumption (VO2 max), and metabolism, 364(fig.) oxygen equilibrium curves (OECs), 258–62, 260(fig.), 262(fig.) INDEX oxygen partial pressure thresholds (kPa), 258–60 aquatic surface respiration (ASR), 1–10(tab.) species changing spontaneous locomotor activity in response to hypoxia, 11–12(tab.) thresholds for regulation of routine metabolic rate (Pcrit, in kPa), 1–10(tab.) oxygen secretion mechanisms, 257, 263–5 oxygen tension critical (POcrit), 1–10(tab.), 444–5, 487, 498 acclimatization, 499 oxygen transport, 257–88, 454–5 capacity limitation concept, 144–6 chemotransduction in fish gill NECs, 235–7 concentration, and biomass (kg wet wt. h‐1 trawling), 82(fig.) digestive organs and hypoxia, 384–6 eVect on mating behavior of male mosquito fish, 100(fig.) myoglobin, 273–5, 333–4, 454–5 O2‐carrying capacity responses, 259–62 partial endothermy, 267–8 saturations, and temperatures, 48, 146–52 sensor or transducer, role of hydrogen sulphide (H2S), 234, 321 whole organism oxygen demand, and ventilation, 150 oxygen‐sensing mechanisms, 217–31 branchial vs extra‐branchial chemoreceptors, 222–4 cellular models of sensing, 232–4 chemoreceptor plasticity, 231 chemosome hypothesis, 235 extra‐branchial site of O2 sensing, 223 hypoxic chemotransduction, 232–5 internally vs externally oriented chemoreceptorss, 220–2 membrane hypothesis vs mitochondrial, 232–3 three distinct populations of receptors, 221 P PACAP, 27 and, 38, identified in gill neuroepithelial cells, 230(tab.) INDEX Pachycara brachycephalum, 147, 152, 153, 172, 285 Pacific salmon (Oncorhynchus nerka), temperature window of aerobic scope for spawning, 152 pacu (Piaractus brachypomus) embryonic development, 105 glycogen stores, in brain, liver, and muscle, 457(tab.) hormonal disruption, 93–4(tab.) mortality of embryo and larvae, 107(tab.) oxygen uptake (MO2 ) and ventilation during exposure to hypoxia, 209(fig.) pacu (Piaractus mesopotamicus), 37, 200, 211, 212, 310 Pagothenia borchgrevinki, 270, 279, 305, 332 Pagrus major, 60, 113–14 Palaemon spp., 158–9 Pangasius hypothalamus, 230 Paracheirodon axelrodi, 30 Paracheirodon innesi, 30 paradise fish (Macropodus opercularis), 390 Paralichthys lethostigma, 369, 380, 382 parental care, 54–6 fanning, 55 mouth brooding, 54 ovoviviparity, 54 Parophrys vetulus, 200 PAS domains, 467 pelagic fish, and demersal fish, shift in dominance, 82 peptide hormones, ghrelin, 386 Perca flavescens, 35, 58 Perca fluviatilis, 35, 59, 229 perch, climbing, (Anabas testudineus), 53 perch, fw (Perca flavescens), 35, 58 perch, Nile (Lates niloticus), predation on cichlids, 57–8 perch, redfin (Perca fluviatilis), 35, 59, 229 perch, sea see sea bass (Dicentrarchus labrax) perch, spangled (Leiopotherapon unicolor), 211, 308 perch, yellow (Perca flavescens), 35, 58 perciformes, Hb polymorphisms, 271(fig.) Percina maculata, 35 Periophthalmodon schlosseri, 40 Periophthalmus modestus, 56 Petrocephalus catostoma, 34 519 phosphate regulation, hemoglobin–O2 binding aYnity, 276–80 phosphatidylethanolamine, 431, 433 phosphofructokinase (PFK), 342 phosphofructokinase‐2 (PFK‐2), 466 phosphorus anthropogenic, 6 limiting nutrient (FW), 6 phylogenetic analysis, isoforms of HIF beta, 471(fig.), 474(fig.) phylogenetic relationships, 500 Piabucina festae, 42, 61, 204 Piaractus brachypomus, 93–4, 105, 107, 209, 457 Piaractus mesopotamicus, 37, 200, 211, 212, 310 pike (Esox lucius), 33, 59 Pimelodella picta, 35 Pimephales promelas, 29, 33, 58, 62 piranha (Serrasalmus rhombius), 277 plaice (Platessa platessa), 269, 368, 374 hypoxia‐related reduction in growth, 377 specific growth rates at diVerent durations of hypoxia, 380(tab.) Platessa platessa, 269, 368, 374, 377, 380 Platichthys flesus, 200, 211, 269, 335–7 Platichthys stellatus, 200 Pleuragramma antarcticum, 270 Pleuronectes americanus, 308, 309, 313, 366 Pleuronectes platessa, 201 pleuronectiformes, Hb polymorphisms, 271(fig.) Poecilia latipinna, 56, 261 Poecilia reticulata, 29 Poecilia sphenops, 35 polar fishes see Antarctic; Arctic Polypterus, 42 Pomacentrus amboinensis, 96 Pomatoschistus microps, 55 Pomatoschistus minutus, 49, 55 porbeagle shark (Lamna nasus), 268 preconditioning, 348, 349(fig.) predator‐prey relationships, 57, 59 hypoxic refugia, 57–9 prey vulnerability under hypoxia stress, 52 pregnenolone, 85 Prochilodus scrofa, 201 Prognathochromis perrieri, 31 Prognathochromis venator, 32 prolyl hydroxylase (PHD), 468(fig.), 475 520 INDEX protein functional capacity, influence by pH, 157 protein synthesis, 171–4 Protopterus aethiopicus, 202, 215, 276, 337 Protopterus amphibius, 280 Protopterus annectens, 202, 229 Protopterus dolloi, 202 Protopterus spp., 40, 42, 55, 276, 287, 304 Pseudocrenilabrus multicolor victoriae, 32, 56 Pterogoplichthys spp., 276, 287 Pterophyllum, 36 Pungitus pungitus, 34 pyruvate dehydrogenase kinase, 456 pyruvate kinase (PyK), 343 R rainbow trout (Oncorhynchus mykiss), 87, 105, 106, 119, 156, 168–70, 172–3, 199 adenosine, decrease in heart rate, 332 allosteric regulation of Hb at subcellular level, 278 anoxia in, 400 apoptotic early diplotene oocytes, 119 beta‐adrenergic receptors of erythrocytes, 283 blood and heart glucose levels and heart lactate levels, 337(tab.) Ca2þ influx cold‐induced compensation, 169 no change with temperature in atrial myocytes, 170 cold acclimation, 156, 172–3 exercise‐induced functional hypoxia, 282 exogenous catecholamines, eVects on on ventilation volume, frequency, and amplitude, 225–6(tab.) fH‐water PO2 relationships, 306, 307 glycogen stores, 423(tab.) in brain, liver, and muscle, 457(tab.) Hb components, 269 heart rate, stroke volume and venous tone, 315(fig.) hydrogen sulphide (H2S), 234, 321 hyperventilation in response to acute anemia, 221 hypoxia and central regulation of appetite, 387 interactive eVects of temperature and severe hypoxia, 345, 346(fig.) maximum recorded decreases in metabolic rate, 462(tab.) mean circulatory filling pressure (MCFP), 314, 316(fig.) metabolic rate suppression, 460–1 Naþ/Hþ exchanger, 281 neuroepithelial cells (NECs), containing neurosecretory synaptic vesicles, 237 oxygen‐carrying capacity, 259–62 PO2 of peak ventilation, 211(tab.) ryanodine‐induced impairment in cardiac performance, 168 SarcKATP channels, 331 specific growth rates, 367(tab.) and food intake, 379(fig.) SR Ca2þ‐release channel in beat‐to‐beat regulation of cardiac contractility, 168 ram ventilation, 51, 206 Rasboro taeniata, 33 rat, glycogen stores, 423(tab.) ray (Torpedo marmorata), 195 red muscle vs white muscle, 52 see also myoglobin red sea bream (Pagrus major) blastula and gastrula stages, 113–14 larvae, predation by jellyfish, 60 redfin perch (Perca fluviatilis), 35, 59, 229 release (CICR)‐induced Ca2þCa2 reproduction, 79–144 eVects of hypoxia on HPG axis, steroidogenesis, and sex hormones, 88–92 gene expression profile, 87–9 hypothalamus—pituitary—gonad (HPG) axis, 83–5 impairment and population decline, 125–6 other vertebrates, 120–2 reproductive behaviors, 96–8 sex determination and diVerentiation, 87–9, 118–20, 125–6 steroidogenesis, 85–7 research into hypoxia, future, 501–2 reservoirs, 4 respiration, TCA cycle, mRNA coding for genes involved, 456 521 INDEX Rhamdia quelen, 366, 457 Rhinelepis strigosa, 201, 204, 214 Rhinichthys atratulus, 33 Rhinichthys cataractae, 33 Rhinogobius, 55 Rhodeus amarus, 401 Rivulus hartii, 33 Roeboides guatemalensis, 30, 33 ryanodine, cold‐induced sensitivity, 168–9 S Sacramento blackfish (Oreochromis microlepidotus), 210 sailfin molly (Poecilia latipinna), 56 chronic exposure to extreme hypoxia, 261 salinity, temperature, and growth, 382–3 Salminus maxillosus, 201, 306, 307 Salmo gairdneri see rainbow trout (Oncorhynchus mykiss) Salmo salar, 106–7, 146, 284 Salmo trutta, 87, 92, 105, 107, 108–10, 229, 230 salmon catfish (Arius leptaspis), 280 salmonids DO as limiting factor, 3 Hb polymorphisms, 271 insulin‐like growth factors, aVecting ovarian growth, 87–8 see also Atlantic salmon; brown trout; Chinook; Coho; Pacific salmon; rainbow trout; sockeye Salvelinus alpinus, 282, 310 Salvelinus fontinalis, 382 sand eels, 52–3 sand goby (Pomatoschistus minutus), male parental care, 49, 55 sandbar shark (Carcharhinus plumbeus), 282–3 saratoga (Scleropages jardinii), 270, 278 scalloped hammerhead shark (Sphyrna lewini), 284 Scartelaos histophorus, 41 schooling, Atlantic herring, induction of hypoxia along the axis of motion, 50, 62 Scleropages jardinii, 270, 278 Scomberomorus niphonius, 60 scombroids, Hb polymorphisms, 271(fig.) Scophthalmus maximus, 201, 211, 261, 271, 280, 366, 368, 372 Scorpaena guttata, 279 sculpin, buValo (Enophrys bison), 275 sculpin (Cottus gobio), predation on alevins, 110(fig.) sculpin (Myoxocephalus octodecimspinosus), 274 sculpin, short‐horned (Myoxocephalus scorpius), 337, 340 sculpins (Cottidae), 29, 38, 41, 53, 333 Scyliorhinus canicula, 105, 107, 108, 195, 309, 320 Scyliorhinus stellaris, 195, 229 SDA (specific dynamic action of food), 373–5, 387, 390 sea bass (Dicentrarchus labrax) acclimation studies, 280 fluctuating O2 levels for growth, 382 gastrointestinal blood flow during hypoxia, 384, 385(fig.) no response by using ASR, 62 oxygen‐carrying capacity, 261 specific growth rates (SGR), 367–8(tab.) sea nettle jellyfish (Chrysaora quinquecirrha), 60 sea raven (Hemitripterus americanus), 221, 222 myoglobin, 274 Sebastolobus alascanus, 279 selachiformes, Hb polymorphisms, 271(fig.) Semotilus atromaculatus, 33 Semotilus margarita, 33 serotonin (5‐HT), 85, 91, 237–8 identified in gill neuroepithelial cells, 229(tab.) paracrine role in rat carotid body, 231 Serrasalmus rhombius, 277 sex determination, 87–9, 118–20 sex diVerentiation, 117–19 imposex, 125 and sex ratio, zebrafish and medaka, 125–6 sex reversal, medaka, 87 sex steroid hormones, 117 sex‐determining gene, DMY, 119 shark, epaulette (Hemiscyllium ocellatum), 332 shark, Florida smoothhound (Mustelus norrisi), 51 shark, porbeagle (Lamna nasus), 268 shark, sandbar (Carcharhinus plumbeus), 282–3 522 shark, scalloped hammerhead (Sphyrna lewini), 284 shrimp (Palaemon), metabolic depression, 158–9 Siamese fighting fish (Betta splendens), 54 reduced opercular displays, 64 signaling, adenosine accumulation, 155 Silurus glanis, 201, 266 silver bream (Sparus sarba), 367(tab.) hypoxia‐related reduction in growth, 376, 377(fig.) silver catfish (Rhamdia quelen), 366(tab.) glycogen stores in brain, liver, and muscle, 457(tab.) silverside (Menidia beryllina), 49 skipjack tuna (Katsuwonus pelamis), 47, 50, 168, 199 small‐mouth bass (Micropterus dolomieui), 105, 229 fH—water PO2 relationships, 306, 307 larval survival times, 105 snakehead fish (Channa striatus), 388, 389 (fig.) social behavior, in response to aquatic oxygen availability, 62 sockeye salmon (Oncorhyncus nerka), 369 thermal optimum of growth performance, 149 Solea solea, 49 Southern flounder (Paralichthys lethostigma), 369, 380, 382 Spanish mackerel (Scomberomorus niphonius), 60 Sparus auratus, 273 Sparus sarba, 367 species interactions between fish and their invertebrate prey, 62–3 predator‐prey relationships, 57, 59 specific dynamic action of food (SDA), 373 specific growth rate (SGR), 365 sperm motility, predictor for sperm quality, 102–3 spermatogenesis, 84–5 Sphyrna lewini, 284 Sphyrna tiburo, 47, 51, 195 spleen, role, 286 spontaneous emersion, 53 spontaneous locomotor activity, change in response to hypoxia, 45 INDEX spontaneous swimming activity, 44–52 spot (Leiostomus xanthurus), 369 Squalus acanthias, 195, 226, 284 squid (Loligo forbesi), migratory movements, 177 steroidogenesis, 85–7 disruption of sex steroid hormones by hypoxia, 91 enzymes, 91 schematic pathway, 86(fig.) steroidogenic acute regulatory protein (StAR), 85 stickleback (Culaea inconstans), 62, 63 stress hormones, catecholamines and corticosteroids, 155–6 striped bass (Morone saxatilis), 282, 368, 382, 383(fig.) sturgeon, Adriatic (Acipenser naccarii), 47, 50, 196 eVects of exogenous catecholamines on ventilation volume, frequency, and amplitude, 226(tab.) sturgeon, Amur (Acipenser shrenckii), 113 sturgeon, green (Acipenser medirostris), 281 sturgeon, Siberian (Acipenser baeri), 195 ventilatory responses to hypoxia, 206(fig.) sturgeon, white (Acipenser transmontanus), 196, 211, 368, 382 sturgeons Hb polymorphisms, 271 systemic vascular resistance, 320 swamp eel (Synbranchus marmoratus), 42, 204, 305, 314 swim bladder, evolution, 257 swimming distance, Atlantic cod, diVerent oxygen saturationsand temperatures, 48 swimming speed, 47 Symenchelis parasitica, 269 Symphysodon aequifasciatus, 457 Synbranchus marmoratus, 42, 204, 305, 314 Synodontis nigrita, 34 Synodontis nigriventris, 28 T tambaqui (Colossoma macropomum), 28, 29, 197, 222, 223 INDEX eVects of exogenous catecholamines on ventilation volume, frequency, and amplitude, 226(tab.) induction of bradycardia, 308 O2‐carrying capacity, 261 O2 receptors, 305 tarpon (Megalops atlanticus), 203, 214 tarpon, ox‐eye (Megalops cyprinoides), 270, 278, 280 Taurulus (Cottus) bubalis, 53 emergence from hypoxic tidepools, 53 temperate, vs polar fishes, energy‐saving lifestyles, 151 temperature interaction with severe hypoxia (anoxia), and cardiac function, 345–6 and oxygen solubility, 266 salinity and hypoxia, 382–3 see also thermal tolerance temperature adaptation cellular mechanisms, 162–75 role of hypoxemia, 153–62 systemic signaling and oxidative stress, 159–62 thermal windows in climate sensitivity, 175–8 tench (Tinca tinca), 201, 222, 265 teratogens, 110–15 testosterone, 85, 86(fig.), 87 estradiol, and triiodothyronine, disruption in carp (Cyprinus carpio), 93 testosterone/estradiol (T/E2), 116 Tetrapterus audax, 268 thermal tolerance, 143–94 cellular mechanisms of temperature adaptation, 162–75 energy budget, turnover, and allocation, 170–5 key cellular parameters (I—IV), 174(fig.) temperature adaptation, role of hypoxemia, 153–62 thermal windows in climate sensitivity, 175–8 thermally induced hypoxemia, 144–53 see also temperature thermal windows climate sensitivity, 175–8 climate‐induced shifts at ecosystem level, 177–8 523 Thermarces cerberus, 270 Thunnus albacores, 50, 201, 221 Thunnus maccoyii, 268 Thunnus obesus, 201, 268 Thunnus thynnus, 168, 268 tidepool sculpin Oligocottus maculosus, 29, 38, 41, 53 tilapia (Oreochromis mossambicus), 229 glycogen stores in brain, liver, and muscle, 457(tab.) maximum recorded decreases in metabolic rate, 462(tab.) metabolic rate suppression, 460–1 tilapia (Oreochromis niloticus), 31, 200, 210 assimilation eYciency, 388 specific growth rates (SGR), 367(tab.) tilapiines, moving young to oxic habitat, 55 Tinca tinca, 201, 222, 265 Torpedo marmorata, 195, 230 traira (Hoplias malabaricus), 198, 211, 221, 222, 229, 305, 306, 307, 310 trairao (Hoplias lacerdae), 198, 209, 211, 229, 306, 307, 310 Trematomus bernachii, 305 Trichogaster leeri, 61 Trichogaster trichopterus, 43, 54, 204 triiodothyronine (T3), 116 Tripterygiidae, 270 Tropheus moorii, 32 tropical habitats, coral reef crevices, 6 trout see brown; rainbow ‐ tryptophan hydroxylase, 91 tuna, bigeye tuna (Thunnus obesus), 268 tuna, bluefin tuna (Thunnus thynnus), 168, 268, 274 tuna, skipjack, (Katsuwonus pelamis), 47, 50, 168, 199 tuna, southern bluefin (Thunnus maccoyii), 268 tuna, yellowfin tuna (Thunnus albacores), 50 myoglobin, 275 O2 chemoreceptors, 221 temperature‐insensitive oxygen binding, 267 ventilatory responses to hypoxia, 206(fig.) turbot (Scophthalmus maximus), 201, 211 acclimation studies, 280 Hb polymorphisms, 271 524 INDEX turbot (Scophthalmus maximus), (continued ) oxygen uptake, metabolic scope and feeding ratio, 372(fig.) oxygen‐carrying capacity, 261 specific growth rates (SGRs), 366, 368(tab.) at diVerent durations of hypoxia, 380 (tab.), 381(fig.) turtles brain activity during anoxia, 411–15 glycogen stores, 423(tab.) lactate production, 403 metabolic rate suppression, 461 tyrosine hydroxylase, identified in gill neuroepithelial cells, 230(tab.), 238 U Umbra limi, 21, 62, 204 Umbra—cyprinid prey assemblages, 59 upwelling areas, development of hypoxia, 4 urotensin, 387 W Wadden Sea, Germany, 176 water column stratification, 6 white sturgeon (Acipenser transmontanus) see sturgeon winter flounder (Pleuronectes americanus), 308 acclimation temperature and heart rate, 309(fig.) specific growth rates (SGR), 366(tab.) water PO2 and various cardiac parameters, 313(fig.) Wisconsin lakes, centrarchid‐Esox predator assemblage, 59 wolYsh (Anarhichas lupus), 307, 312 water PO2 and various cardiac parameters, 313(fig.) wolYsh, spotted (Anarhichas minor), 366(tab.) specific growth rates at diVerent durations of hypoxia, 380(tab.) X V Xiphophorus helleri, 35 van’t HoV relationship, 266 vascular endothelial growth factor (VEGF), 115 HIF, 123 vascular smooth muscle, vasodilatation with NO, 283 vasoactive intestinal peptide, identified in gill neuroepithelial cells, 230(tab.) Venn diagram, up‐ and down‐regulated genes in response to hypoxia exposure, 479(fig.) ventilation ram, 51, 206 whole organism oxygen demand, 150 see also gill ventilation ventilatory responses to hypoxia, 193–256 visual acuity, 265–6 vitellogenin (VTG), 117 voltage‐dependent anion channels (VDACs), 342 vomiting response to hypoxia, 386 von Hippel‐Lindau protein (VHL), 469 Y Y chromosome, sex‐determining gene DMY, 119 yellow perch (Perca flavescens), 35, 58 yellowtail flounder (Limanda ferruginea), 308 Yssichromis argens, 32 Z zebrafish (Danio rerio), 64, 89–92, 94, 101, 108, 197 3beta‐HSD, CYP11A, and CYP19B, 92 acute hypoxic exposure, eVect on heart rate, 311(fig.) altering aggression, 64 branchial O2 chemoreceptors, 228–9 INDEX cardiac performance, 325(fig.) increased cardiac myocyte density, 328 chemoreceptor plasticity, 231–2 chronic hypoxia, eVect on cardiovascular development, 322–4 developmental defects, 110–15 disruption in apoptosis, 112 malformations, 111(fig.) eVects of acute graded hypoxia on ventilation frequency, 217(fig.) gene expression patterns, 89–90 GnRH and GtH, 90–1 hormonal levels, 94(tab.) lack of a hypoxia‐induced bradycardia in early larval stages, 310 maximum recorded decreases in metabolic rate, 462(tab.) metabolic rate suppression, 460–1 molecular responses to hypoxia, 452(tab.) mRNA coding for genes involved in TCA cycle, 456 neuroepithelial cells (NECs), 228–30 containing neurosecretory synaptic vesicles, 237 525 early larval stages, 231 proposed model for oxygen sensing, 236(fig.) whole‐cell voltage‐clamp recording, 219(fig.) neuroglobin, 275–6 normoxia vs hypoxia embryos, 108 oogonia, 101(fig.) spermatogonia, 101(fig.) O2 chemotransduction in fish gill NECs, 235–7 PO2 of peak ventilation, 211(tab.) sex determination and diVerentiation, 87 sex ratio and sex diVerentiation, 125–6 ventilatory responses to hypoxia, 206 viability of developmental stages in anoxia, 106(tab.) VTG levels, 118(fig.) Zoarces viviparus, 110, 145, 155–6, 159–60, 285, 328, 334–5 Zoarcidae (eelpouts), 270, 285 OTHER VOLUMES IN THE FISH PHYSIOLOGY SERIES VOLUME 1 Excretion, Ionic Regulation, and Metabolism Edited by W. S. Hoar and D. J. Randall VOLUME 2 The Endocrine System Edited by W. S. Hoar and D. J. Randall VOLUME 3 Reproduction and Growth: Bioluminescence, Pigments, and Poisons Edited by W. S. Hoar and D. J. Randall VOLUME 4 The Nervous System, Circulation, and Respiration Edited by W. S. Hoar and D. J. Randall VOLUME 5 Sensory Systems and Electric Organs VOLUME 6 Environmental Relations and Behavior Edited by W. S. Hoar and D. J. Randall Edited by W. S. Hoar and D. J. Randall VOLUME 7 Locomotion Edited by W. S. Hoar and D. J. Randall VOLUME 8 Bioenergetics and Growth Edited by W. S. Hoar, D. J. Randall, and J. R. Brett VOLUME 9A Reproduction: Endocrine Tissues and Hormones Edited by W. S. Hoar, D. J. Randall, and E. M. Donaldson VOLUME 9B Reproduction: Behavior and Fertility Control Edited by W. S. Hoar, D. J. Randall, and E. M. Donaldson VOLUME 10A Gills: Anatomy, Gas Transfer, and Acid-Base Regulation Edited by W. S. Hoar and D. J. Randall VOLUME 10B Gills: Ion and Water Transfer Edited by W. S. Hoar and D. J. Randall VOLUME 11A The Physiology of Developing Fish: Eggs and Larvae Edited by W. S. Hoar and D. J. Randall 527 528 VOLUME 11B OTHER VOLUMES IN THIS SERIES The Physiology of Developing Fish: Viviparity and Posthatching Juveniles Edited by W. S. Hoar and D. J. Randall VOLUME 12A The Cardiovascular System VOLUME 12B The Cardiovascular System Edited by W. S. Hoar, D. J. Randall, and A. P. Farrell Edited by W. S. Hoar, D. J. Randall, and A. P. Farrell VOLUME 13 Molecular Endocrinology of Fish Edited by N. M. Sherwood and C. L. Hew VOLUME 14 Cellular and Molecular Approaches to Fish Ionic Regulation Edited by Chris M. Wood and Trevor J. Shuttleworth VOLUME 15 The Fish Immune System: Organism, Pathogen, and Environment VOLUME 16 Deep Sea Fishes Edited by George Iwama and Teruyuki Nakanishi Edited by D. J. Randall and A. P. Farrell VOLUME 17 Fish Respiration Edited by Steve F. Perry and Bruce Tufts VOLUME 18 Muscle Growth and Development VOLUME 19 Tuna: Physiology, Ecology, and Evolution Edited by Ian A. Johnson Edited by Barbara A. Block and E. Donald Stevens VOLUME 20 Nitrogen Excretion Edited by Patricia A. Wright and Paul M. Anderson VOLUME 21 The Physiology of Tropical Fishes Edited by Adalberto L. Val, Vera Maria F. De Almeida-Val, and David J. Randall VOLUME 22 The Physiology of Polar Fishes Edited by Anthony P. Farrell and John F. SteVensen VOLUME 23 Fish Biomechanics Edited by Robert E. Shadwick and George V. Lauder VOLUME 24 Behaviour and Physiology of Fish VOLUME 25 Sensory Systems Neuroscience Edited by Katherine A. Sloman, Rod W. Wilson, and Sigal Balshine Edited by Toshiaki J. Hara and Barbara S. Zielinski VOLUME 26 Primitive Fishes Edited by David J. McKenzie, Anthony P. Farrell, and Colin J. Brauner VOLUME 27 Hypoxia Edited by Jeffrey G. Richards, Anthony P. Farrell, and Colin J. Brauner