Figure 1.
Experimental bacterial infection model in P. dolloi.
A) Delivery of E. ictaluri to P. dolloi intranasally. B) Skin ulcers (black arrows) produced by E. ictaluri in an infected P. dolloi individual. No ulcers were observed in control specimens.
Figure 2.
Lungfish J chain molecular analysis.
A) Alignment of the J chain amino acid sequences of lungfish (JX999962), mouse (AAH06026), human (ABI63362), chicken (BAB83927), Xenopus (AAC05636), bullfrog (ACO52584), coelacanth (ENSLACP00000023629) and shark (AAO24897). “SS” indicates the interchain disulfide bridges and “TP” marks the Cys paired with the secretory tail of IgA or IgM. The conserved N-linked glycosilation site is indicated by “+”. Numbers (1–8) indicate the Cys residues in human sequences. “*” Indicates identical amino acids between all sequences, whilst “.” and “:” show conservative substitutions. Regions 1–3 were identified as in Braathen et al (2007) B) Conservation of Cys residues between lungfish and other vertebrates. C) Phylogenetic tree showing the evolutionary relationship of J chain molecules in vertebrates. The tree was constructed using the Neighbour-Joining method in MEGA 4 and was bootstrapped 10,000 times. GenBank accession numbers are as follows: lungfish (JX999962), mouse (AAH06026), human (ABI63362), chicken (BAB83927), Xenopus (AAC05636), bullfrog (ACO52584), coelacanth (ENSLACP00000023629) and shark (AAO24897).
Figure 3.
Tissue distribution of J chain expression in control Nigerian spotted lungfish (P. dolloi) (n = 6).
The relative expression of each gene was normalized first with the house keeping genes, and then divided by the average expression level in the pre-pyloric spleen, which had the lowest value. Bars represent means ± standard error of four fish. Different letters represent statistically significant groups after Tukey's test (p<0.05).
Figure 4.
Expression of J chain by B cells of Nigerian spotted lungfish (P. dolloi).
A) Mean percentage of IgM+ and IgM+ J chain+ B cells and B) Mean percentage of IgW+ and IgW+ J chain+ B cells in the pre-pyloric spleen and gut of control P. dolloi (n = 3) studied by double FISH staining. Cells were counted from 20 different fields (×60). Differences were statistically significant when p<0.05. n.s: non-significant differences. C–E) Examples of representative FISH staining showing IgM+ J chain+ lymphocytes (white asterisks) from a gut cryosection. C) Cy5-IgM FISH probe D) Cy3-Jchain FISH probe E) DAPI nuclear stain. F–H) Examples of representative FISH staining showing a IgW+ J chain− lymphocyte (yellow asterisk) from a pre-pyloric spleen cryosection. F) Cy5-IgW FISH probe G) Cy3-Jchain FISH probe H) DAPI nuclear stain.
Figure 5.
Fold change of J chain expression in pre-pyloric spleen, post-pyloric spleen, gut, kidney and lung of Nigerian spotted lungfish infected with Edwardsiella ictaluri compared to samples from PBS mock control fish.
Bars represent means ± standard error of four fish. Different letters represent statistically significant groups after Tukey's test (p<0.05).
Figure 6.
P. dolloi gut epithelial cells (GEC) express J chain.
A) P. dolloi gut cryosection stained with the nuclear stain DAPI (blue) showing the characteristic nuclear morphology of epithelial cells B) P. dolloi gut cryosection stained with Cy-3 J chain Stellaris FISH probe. Epithelial cells contained numerous J chain transcripts (dots). Ep: epithelial cells, Lu: lumen, Bm: basal membrane C) Gut epithelial cells were dissected from control P. dolloi gut cryosections using LCM. Total RNA from P. dolloi pre-pyloric spleen, LCM gut epithelial cells (GEC) and muscle was extracted and used as template for RT-PCR using J chain (J), IgM, IgW and cytokeratin 8 (CK-8) specific primers. EF-1α served as cDNA control. “–” indicates J chain negative control. M (marker) indicates the DNA ladder. The sequences of all PCR products were confirmed by cloning and sequencing.