The Sierra Nevada yellow-legged frog, Rana sierrae, has been driven close to extinction in part by the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd). Prior studies have shown that skin-associated microbes can inhibit Bd and that differences in microbial community structure may result in distinct disease outcomes for the host. Recent efforts to reintroduce captive-reared R. sierrae to the field have involved experimentally infecting frogs with Bd and clearing infection with the anti-fungal Itraconazole before release, which is thought to reduce future infection intensity. The objective of this project was to investigate the causal impacts of this experimental Bd infection and Itraconazole treatment on the R. sierrae skin microbiome. The skin microbiome was sampled before, during, and after Bd infection and anti-fungal treatment for experimentally infected animals and control animals that were treated with the anti-fungal despite not undergoing Bd infection. Illumina sequencing was done of 16S rRNA gene amplicons and metagenomes for a subset of samples.
Results/Conclusions
Analysis of the 16S data showed that the microbiome was significantly altered by both Bd infection and Itraconazole treatment in terms of community structure and taxonomic composition. Groups of taxa that were dominant on pre-treatment frogs dropped to low relative levels when infected with Bd (e.g. Rubritaleaceae) while other groups increased (e.g. Burkholderiaceae). After Itraconazole treatment, community composition did not recover to pre-treatment composition and was consistently altered in both Bd-infected and uninfected control animals. One dominant amplicon sequence variant (ASV), Frog01, that could not be assigned taxonomy below family (Burkholderiaceae) increased in relative abundance between healthy, infected, and treated status. Shotgun metagenomic data was used to bin and assemble 15 high and medium quality metagenome assembled genomes (MAGs), including a high-quality genome for which the 16S rRNA gene was identical to Frog01, as well as representatives from other taxonomic groups present in 16S rRNA gene sequence data. These MAGs will be used to investigate functional potential, looking especially for interactions with Bd. These results reveal that infection and anti-fungal treatment greatly impact the skin microbiome, which may have lasting effects on host health after reintroduction to the field.