Animals often form functionally important relationships with bacteria. To understand rules of microbiome assembly and function it is critical to integrate data from multiple disciplines. We used culturing assays, molecular microbiomics and pathogen diagnostics, and proteomics to assess how host-associated bacteria interact with one another, with host-secreted peptides, and with pathogens. We collected skin swabs and skin secretions from 73 individuals of four wild salamander species: red-backed salamander (n = 30), two-lined salamander (n = 12), seal salamander (n = 11), and eastern newt (n = 20) in Virginia. We collected skin swabs and skin secretions from 10 African clawed-frogs from a laboratory colony. We identified bacterial taxa for which we have culturable representatives based on 16S rRNA microbiome data, and those bacteria were grown in co-culture assays to quantify their inhibitory properties. We identified host-secreted peptides via high-resolution mass-spectrometry, and then tested the hypothesis that niche filtering by host-secreted peptides influence which bacteria colonize amphibian skin. Finally, we determined how effective host skin secretions (i.e., the mucosome) were at killing two fungal pathogens, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), and tested the hypothesis that components of bacterial and peptide diversity are predictive of pathogen-killing ability.
Results/Conclusions
We found that the five amphibian species differed in skin microbiome composition and Bd infection levels. In wild salamanders, the average Bd prevalence was 29% (range 3 – 65%), in which eastern newts had the highest prevalence and red-backed salamanders had the lowest. We found 12 bacterial taxa that were shared among the salamander and frog species sampled. All 12 bacterial taxa matched culturable bacteria known to inhibit Bd growth in culture (>99% 16S rRNA sequence similarity). We found that those bacteria, while inhibitory of Bd, did not inhibit the growth of one another. This suggests that some bacteria may be conserved across Amphibia given their inhibitory function against pathogens and their non-inhibitory properties against each other. We found that the wild salamander species differed in their mucosome ability to kill Bsal, but not Bd. Two-lined salamanders and red-backed salamanders had significantly higher Bsal killing ability than eastern newts. We discovered hundreds of peptides from salamander and frog skin via de novo sequencing from mass spectrometry data, and are in the process of data analysis. Understanding how peptide diversity influences bacterial diversity and how their shared diversity is linked to host disease susceptibility is essential to identifying rules of microbiome assembly.