Probiotic colonization of Xenopus laevis skin causes short-term changes in skin microbiomes and gene expression

Probiotic therapies have been suggested for amelioration efforts of wildlife disease such as chytridiomycosis caused by Batrachochytrium spp. in amphibians. However, there is a lack of information on how probiotic application affects resident microbial communities and immune responses. To better understand these interactions, we hypothesized that probiotic application would alter microbial community composition and host immune expression in Xenopus laevis . Accordingly, we applied three amphibian-derived and anti- Batrachochytrium bacteria strains (two Pseudomonas spp. and one Stenotrophomonas sp.) to X. laevis in monoculture and also as a cocktail. We quantified microbial community structure using 16S rRNA gene sequencing. We also quantified genes involved in X. laevis immune responses using quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) and skin transcriptomics over 1 and 3-week periods. All probiotic treatments successfully colonized X. laevis skin for 3 weeks, but with differential amplicon sequence variant (ASV) sequence counts over time. Bacterial community and immune gene effects were most pronounced at week 1 post-probiotic exposure and decreased thereafter. All probiotic treatments caused initial changes to bacterial community alpha and beta diversity, including reduction in diversity from pre-exposure anti- Batrachochytrium bacterial ASV relative abundance. Probiotic colonization by Pseudomonas probiotic strain RSB5.4 reduced expression of regulatory T cell marker ( FOXP3, measured with RT-qPCR) and caused the greatest gene expression changes detected by transcriptomics. Single bacterial strains and mixed cultures, therefore, altered amphibian microbiome-immune interactions. This work will help to improve our understanding of the role of the microbiome-immune interface underlying both disease dynamics and emergent eco-evolutionary processes. IMPORTANCE Amphibian skin microbial communities have an important role in determining disease outcomes, in part through complex yet poorly understood interactions with host immune systems. Here we report that probiotic-induced changes to the Xenopus laevis frog skin microbial communities also result in significant alterations to these animals’ immune gene expression. These findings underscore the interdependence of amphibian skin immune-microbiome interactions.