Probiotic treatment restores protection against lethal fungal infection lost during amphibian captivity.

Host-associated microbiomes perform many beneficial functions including resisting pathogens and training the immune system. Here, we show that amphibians developing in captivity lose substantial skin bacterial diversity, primarily due to reduced ongoing input from environmental sources. We combined studies of wild and captive amphibians with a database of over 1 000 strains that allows us to examine antifungal function of the skin microbiome. We tracked skin bacterial communities of 62 endangered boreal toads, Anaxyrus boreas, across 18 time points, four probiotic treatments, and two exposures to the lethal fungal pathogen Batrachochytrium dendrobatidis (Bd) in captivity, and compared these to 33 samples collected from wild populations at the same life stage. As the amphibians in captivity lost the Bd-inhibitory bacteria through time, the proportion of individuals exposed to Bd that became infected rose from 33% to 100% in subsequent exposures. Inoculations of the Bd-inhibitory probiotic Janthinobacterium lividum resulted in a 40% increase in survival during the second Bd challenge, indicating that the effect of microbiome depletion was reversible by restoring Bd-inhibitory bacteria. Taken together, this study highlights the functional role of ongoing environmental inputs of skin-associated bacteria in mitigating a devastating amphibian pathogen, and that long-term captivity decreases this defensive function.

Gut bacterial communities across tadpole ecomorphs in two diverse tropical anuran faunas.

Animal-associated microbial communities can play major roles in the physiology, development, ecology, and evolution of their hosts, but the study of their diversity has yet focused on a limited number of host species. In this study, we used high-throughput sequencing of partial sequences of the bacterial 16S rRNA gene to assess the diversity of the gut-inhabiting bacterial communities of 212 specimens of tropical anuran amphibians from Brazil and Madagascar. The core gut-associated bacterial communities among tadpoles from two different continents strongly overlapped, with eight highly represented operational taxonomic units (OTUs) in common. In contrast, the core communities of adults and tadpoles from Brazil were less similar with only one shared OTU. This suggests a community turnover at metamorphosis. Bacterial diversity was higher in tadpoles compared to adults. Distinct differences in composition and diversity occurred among gut bacterial communities of conspecific tadpoles from different water bodies and after experimental fasting for 8 days, demonstrating the influence of both environmental factors and food on the community structure. Communities from syntopic tadpoles clustered by host species both in Madagascar and Brazil, and the Malagasy tadpoles also had species-specific isotope signatures. We recommend future studies to analyze the turnover of anuran gut bacterial communities at metamorphosis, compare the tadpole core communities with those of other aquatic organisms, and assess the possible function of the gut microbiota as a reservoir for protective bacteria on the amphibian skin.

The amphibian skin-associated microbiome across species, space and life history stages.

Skin-associated bacteria of amphibians are increasingly recognized for their role in defence against pathogens, yet we have little understanding of their basic ecology. Here, we use high-throughput 16S rRNA gene sequencing to examine the host and environmental influences on the skin microbiota of the cohabiting amphibian species Anaxyrus boreas, Pseudacris regilla, Taricha torosa and Lithobates catesbeianus from the Central Valley in California. We also studied populations of Rana cascadae over a large geographic range in the Klamath Mountain range of Northern California, and across developmental stages within a single site. Dominant bacterial phylotypes on amphibian skin included taxa from Bacteroidetes, Gammaproteobacteria, Alphaproteobacteria, Firmicutes, Sphingobacteria and Actinobacteria. Amphibian species identity was the strongest predictor of microbial community composition. Secondarily, within a given amphibian species, wetland site explained significant variation. Amphibian-associated microbiota differed systematically from microbial assemblages in their environments. Rana cascadae tadpoles have skin bacterial communities distinct from postmetamorphic conspecifics, indicating a strong developmental shift in the skin microbes following metamorphosis. Establishing patterns observed in the skin microbiota of wild amphibians and environmental factors that underlie them is necessary to understand skin symbiont community assembly, and ultimately, the role skin microbiota play in the extended host phenotype including disease resistance.

Inhibitory bacteria reduce fungi on early life stages of endangered Colorado boreal toads (Anaxyrus boreas).

Increasingly, host-associated microbiota are recognized to mediate pathogen establishment, providing new ecological perspectives on health and disease. Amphibian skin-associated microbiota interact with the fungal pathogen, Batrachochytrium dendrobatidis (Bd), but little is known about microbial turnover during host development and associations with host immune function. We surveyed skin microbiota of Colorado's endangered boreal toads (Anaxyrus boreas), sampling 181 toads across four life stages (tadpoles, metamorphs, subadults and adults). Our goals were to (1) understand variation in microbial community structure among individuals and sites, (2) characterize shifts in communities during development and (3) examine the prevalence and abundance of known Bd-inhibitory bacteria. We used high-throughput 16S and 18S rRNA gene sequencing (Illumina MiSeq) to characterize bacteria and microeukaryotes, respectively. Life stage had the largest effect on the toad skin microbial community, and site and Bd presence also contributed. Proteobacteria dominated tadpole microbial communities, but were later replaced by Actinobacteria. Microeukaryotes on tadpoles were dominated by the classes Alveolata and Stramenopiles, while fungal groups replaced these groups after metamorphosis. Using a novel database of Bd-inhibitory bacteria, we found fewer Bd-inhibitory bacteria in post-metamorphic stages correlated with increased skin fungi, suggesting that bacteria have a strong role in early developmental stages and reduce skin-associated fungi.

Deconstructing the Bat Skin Microbiome: Influences of the Host and the Environment.

Bats are geographically widespread and play an important role in many ecosystems, but relatively little is known about the ecology of their associated microbial communities and the role microbial taxa play in bat health, development, and evolution. Moreover, few vertebrate animal skin microbiomes have been comprehensively assessed, and thus characterizing the bat skin microbiome will yield valuable insight into the variability of vertebrate skin microbiomes as a whole. The recent emergence of the skin fungal disease white-nose syndrome highlights the potentially important role bat skin microbial communities could play in bat health. Understanding the determinant of bat skin microbial communities could provide insight into important factors allowing individuals to persist with disease. We collected skin swabs from a total of 11 bat species from the eastern United States (n = 45) and Colorado (n = 119), as well as environmental samples (n = 38) from a subset of sites, and used 16S rRNA marker gene sequencing to observe bacterial communities. In addition, we conducted a literature survey to compare the skin microbiome across vertebrate groups, including the bats presented in this study. Host species, region, and site were all significant predictors of the variability across bat skin bacterial communities. Many bacterial taxa were found both on bats and in the environment. However, some bacterial taxa had consistently greater relative abundances on bat skin relative to their environments. Bats shared many of their abundant taxa with other vertebrates, but also hosted unique bacterial lineages such as the class Thermoleophilia (Actinobacteria). A strong effect of site on the bat skin microbiome indicates that the environment very strongly influences what bacteria are present on bat skin. Bat skin microbiomes are largely composed of site-specific microbiota, but there do appear to be important host-specific taxa. How this translates to differences in host-microbial interactions and bat health remains an important knowledge gap, but this work suggests that habitat variability is very important. We identify some bacterial groups that are more consistent on bats despite site differences, and these may be important ones to study in terms of their function as potential core microbiome members.