PS 43-63 - Under the skin: Heterogeneity in immunity-related genes of Plethodon cinereus along an urbanization gradient

Background/Question/Methods

Urbanization and chytridiomycosis, a cutaneous infection caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), jointly pose threats to amphibian populations globally. Interestingly, a salamander endemic to the Northeastern USA, Plethodon cinereus, exhibits relatively low susceptibility to Bd. This is attributed to the presence of putatively Bd-inhibitory microbes in the P. cinereus microbiome. Another factor in disease outcomes is the host immune response. Yet, the functional genetic underpinnings of amphibian responses to infection are largely unknown. Here, we take steps towards connecting the role of the host microbiome with the endogenous host immune response in combating infection. Specifically, we explored genetic heterogeneity in genes related to immune function in nine P. cinereus populations located along a 65-km urbanization gradient originating in New York City. Using custom primers to exons within complement component 1qA chain (CC1qA), tumor necrosis factor receptor-associated factor 3 (TRAF3), and GRB2-associated binding protein (GRB2), we amplified P. cinereus DNA in one-hundred individuals. Sanger sequencing was performed, and we used the Geneious software to assemble contigs, confirm sequence identity, and build phylogenies. We hypothesized that sequence heterogeneity among individuals for each gene studied would group individuals and populations based on their associated land-use classifier (e.g., urban, suburban, exurban).

Results/Conclusions

Nucleotide sequence heterogeneity among Plethodon cinereus individuals averaged at 1% for CC1qA, TRAF3, and GRB2. These single nucleotide polymorphisms (SNPs) resulted in a limited number of amino acid sequence changes, indicating that the exons studied are relatively conserved. This suggests that these genes serve important functions in the P. cinereus immune response. In support of our hypothesis, SNPs and amino acid residue changes grouped by population for all the genes studied. Interestingly, certain amino acid substitutions in CC1qA were more prevalent in urban populations. There was no significant clustering of individuals based on land-use classifier for TRAF3 or GRB2. Nonetheless, these patterns suggest that certain populations along our urbanization gradient may be in the early stages of directional selection; although, more data is needed. Together, our findings are indicative of the unique stressors faced by distinct populations which may be influencing P. cinereus immunity genetics. Future work should delve into the connection between specific stimuli (e.g., chytridiomycosis) and associated genetic variation. Understanding these types of responses from the level of the genome to the microbiome in resilient species like P. cinereus is essential to designing strategies to protect at-risk amphibian populations globally.