Characterization of immune genes in an urban Plethodon cinereus population

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Nina Naghshineh, Graduate School of Arts and Sciences - Biology, Fordham University, Bronx, NY and J.D. Lewis, Graduate School of Arts and Sciences - Biology, Fordham University, Armonk, NY

Background/Question/Methods
Amphibians are at risk of extinction globally due to emerging infectious diseases and habitat degradation. Chytridiomycosis, a cutaneous infection caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is a main driver of these declines. Interestingly, the cutaneous microbial community of eastern redback salamanders, Plethodon cinereus, provides defense against Bd. Yet, few studies have focused on the role of amphibian immune responses in mediating infection. Here, we characterized five immune genes that may be integral in amphibian response to Bd. We sampled liver tissue of P. cinereus individuals at an urban woodland in New York City. DNA was extracted and primers were developed to isolate five genes implicated in the amphibian immune response (i.e., cathepsin C, protein phosphatase 2 catalytic subunit alpha, CXCR3, fibroblast activation protein alpha, interferon regulatory factor 3). Sanger sequencing was performed and we used Geneious software to compare gene sequences to Xenopus laevis, a closely related species with known sequence information for these genes. We also compared gene sequences among P. cinereus individuals. We hypothesized that homologs of all genes of interest would be present in P. cinereus individuals. Further, there would be minimal sequence variation among P. cinereus individuals as similar environmental stressors impact this population.

Results/Conclusions
In support of our hypotheses, all five genes were present in the Plethodon cinereus individuals that we sampled. Additionally, a non-significant (p > 0.01) number of SNPs were found among the individuals tested. The population that we sampled is known to be affected by Bd. Thus, these results suggest that the inflammation-related, innate immune genes that we assayed for may be involved in mediating infection by Bd. As the target sequences were located in exons, the non-significant number of SNPs we observed indicate that, at a genetic level, similar physiological stressors may be eliciting comparable responses in P. cinereus individuals. Nonetheless, the minor genetic variation that we discovered could be due to differences in Bd load across generations. In sum, this evidence paves the way for the characterization of immune genes, their expression, and roles in different P. cinereus populations along a gradient of land use. Future work, linking individual genetic variation to Bd load will further illuminate the role of the amphibian immune system in mediating infection by Bd. Understanding the interplay of microbial community composition, disease prevalence, and the host immune system in amphibian susceptibility to Bd is integral to the advancement of conservation strategies globally.