Captivity, Reintroductions, and the Rewilding of Amphibian-associated Bacterial Communities.

Many studies have noted differences in microbes associated with animals reared in captivity compared to their wild counterparts, but few studies have examined how microbes change when animals are reintroduced to the wild after captive rearing. As captive assurance populations and reintroduction programs increase, a better understanding of how microbial symbionts respond during animal translocations is critical. We examined changes in microbes associated with boreal toads (Anaxyrus boreas), a threatened amphibian, after reintroduction to the wild following captive rearing. Previous studies demonstrate that developmental life stage is an important factor in amphibian microbiomes. We collected 16S marker-gene sequencing datasets to investigate: (i) comparisons of the skin, mouth, and fecal bacteria of boreal toads across four developmental life stages in captivity and the wild, (ii) tadpole skin bacteria before and after reintroduction to the wild, and (iii) adult skin bacteria during reintroduction to the wild. We demonstrated that differences occur across skin, fecal, and mouth bacterial communities in captive versus wild boreal toads, and that the degree of difference depends on developmental stage. Skin bacterial communities from captive tadpoles were more similar to their wild counterparts than captive post-metamorphic individuals were to their wild counterparts. When captive-reared tadpoles were introduced to a wild site, their skin bacteria changed rapidly to resemble wild tadpoles. Similarly, the skin bacterial communities of reintroduced adult boreal toads also shifted to resemble those of wild toads. Our results indicate that a clear microbial signature of captivity in amphibians does not persist after release into natural habitat.

Together or Alone: Evaluating the Pathogen Inhibition Potential of Bacterial Cocktails against an Amphibian Pathogen.

The amphibian fungal skin disease Batrachochytrium dendrobatidis (Bd) has caused major biodiversity losses globally. Several experimental trials have tested the use of Janthinobacterium lividum to reduce mortality due to Bd infections, usually in single-strain amendments. It is well-characterized in terms of its anti-Bd activity mechanisms. However, there are many other microbes that inhibit Bd in vitro, and not all experiments have demonstrated consistent results with J. lividum. We used a series of in vitro assays involving bacterial coculture with Bd lawns, bacterial growth tests in liquid broth, and Bd grown in bacterial cell-free supernatant (CFS) to determine: (i) which skin bacteria isolated from a locally endangered amphibian, namely, the Colorado boreal toad (Anaxyrus boreas boreas), are able to inhibit Bd growth; (ii) whether multistrain combinations are more effective than single-strains; and (iii) the mechanism behind microbe-microbe interactions. Our results indicate that there are some single strain and multistrain probiotics (especially including strains from Pseudomonas, Chryseobacterium, and Microbacterium) that are potentially more Bd-inhibitive than is J. lividum alone and that some combinations may lead to a loss of inhibition, potentially through antagonistic metabolite effects. Additionally, if J. lividum continues being developed as a wild boreal toad probiotic, we should investigate it in combination with Curvibacter CW54D, as they inhibited Bd additively and grew at a higher rate when combined than did either alone. This highlights the fact that combinations of probiotics function in variable and unpredictable ways as well as the importance of considering the potential for interactions among naturally resident host microbiota and probiotic additions. IMPORTANCE Batrachochytrium dendrobatidis (Bd) is a pathogen that infects amphibians globally and is causing a biodiversity crisis. Our research group studies one of the species affected by Bd, namely, the Colorado boreal toad (Anaxyrus boreas boreas). Many researchers focus their studies on one probiotic bacterial isolate called Janthinobacterium lividum, which slows Bd growth in lab cultures and is currently being field tested in Colorado boreal toads. Although promising, J. lividum is not consistently effective across all amphibian individuals or species. For Colorado boreal toads, we addressed whether there are other bacterial strains that also inhibit Bd (potentially better than does J. lividum) and whether we can create two-strain probiotics that function better than do single-strain probiotics. In addition, we evaluate which types of interactions occur between two-strain combinations and what these results mean in the context of adding a probiotic to an existing amphibian skin microbiome.

Mr. Toad's Wild Fungi: Fungal Isolate Diversity on Colorado Boreal Toads and their Capacity for Pathogen Inhibition.

The amphibian skin pathogen Batrachochytrium dendrobatidis (Bd) has caused an ongoing biodiversity crisis, including in the locally endangered Colorado boreal toad (Anaxyrus boreas boreas). Although researchers have investigated the bacteria living on amphibian skin and how they interact with Bd, there is less information about fungal community members. This study describes (1) the diversity of culturable fungi from boreal toad skin, (2) which subset of these isolates is Bd-inhibitory, and (3) how Bd affects these isolates' growth and morphology. Most isolates were from the orders Capnodiales, Helotiales, and Pleosporales. Of 16 isolates tested for Bd-inhibition, two from the genus Neobulgaria and three from Pseudeurotium inhibited Bd. Fungal growth in co-culture with Bd varied with weak statistical support for Neobulgaria sp. (isolate BTF_36) and cf Psychrophila (isolate BTF_60) (p-values = 0.076 and 0.092, respectively). Fungal morphology remained unchanged in co-culture with Bd, however, these results could be attributed to low replication per isolate. Nonetheless, two fungal isolates' growth may have been affected by Bd, implying that fungal growth changes in Bd co-culture could be a variable worth measuring in the future (with higher replication). These findings add to the sparse but growing literature on amphibian-associated fungi and suggest further study may uncover the relevance of fungi to amphibian health and Bd infection.

Bacterial Biofilm Thickness and Fungal Inhibitory Bacterial Richness Both Prevent Establishment of the Amphibian Fungal Pathogen Batrachochytrium dendrobatidis.

Host-associated microbial biofilms can provide protection against pathogen establishment. In many host-microbe symbioses (including, but not limited to humans, plants, insects, and amphibians), there is a correlation between host-associated microbial diversity and pathogen infection risk. Diversity may prevent infection by pathogens through sampling effects and niche complementarity, but an alternative hypothesis may be that microbial biomass is confounded with diversity and that host-associated biofilms are deterring pathogen establishment through space preemption. In this study, we use the amphibian system as a model for host-microbe-pathogen interactions to ask two questions: (i) is bacterial richness confounded with biofilm thickness or cell density, and (ii) to what extent do biofilm thickness, cell density, and bacterial richness each deter the establishment of the amphibian fungal pathogen Batrachochytrium dendrobatidis? To answer these questions, we built a custom biofilm microcosm that mimics the host-environment interface by allowing nutrients to diffuse out of a fine-pore biofilm scaffolding. This created a competitive environment in which bacteria and the fungal pathogen compete for colonization space. We then challenged bacterial biofilms ranging in community richness, biofilm thickness, bacterial cell density, and B. dendrobatidis (also known as Bd)-inhibitory metabolite production with live B. dendrobatidis zoospores to determine how B. dendrobatidis establishment success on membranes varies. We found that biofilm thickness and B. dendrobatidis-inhibitory isolate richness work in complement to reduce B. dendrobatidis establishment success. This work underscores that physical aspects of biofilm communities can play a large role in pathogen inhibition, and in many studies, these traits are not studied. IMPORTANCE Our finding highlights the fact that diversity, as measured through 16S rRNA gene sequencing, may obscure the true mechanisms behind microbe-mediated pathogen defense and that physical space occupation by biofilm-forming symbionts may significantly contribute to pathogen protection. These findings have implications across a wide range of host-microbe systems since 16S rRNA gene sequencing is a standard tool used across many microbial systems. Further, our results are potentially relevant to many host-pathogen systems since host-associated bacterial biofilms are ubiquitous.

Predicting fungal infection rate and severity with skin-associated microbial communities on amphibians.

Pathogen success (risk and severity) is influenced by host-associated microbiota, but the degree to which variation in microbial community traits predict future infection presence/absence (risk) and load (severity) for the host is unknown. We conducted a time-series experiment by sampling the skin-associated bacterial communities of five amphibian species before and after exposure to the fungal pathogen, Batrachochytrium dendrobaditis (Bd). We sought to determine whether microbial community traits are predictors of, or are affected by, Bd infection risk and intensity. Our results show that richness of putative Bd-inhibitory bacteria strongly predicts infection risk, while the proportion of putative Bd-inhibitory bacteria predicts future infection intensity. Variation in microbial community composition is high across time and individual, and bacterial prevalence is low. Our findings demonstrate how ecological community traits of host-associated microbiota may be used to predict infection risk by pathogenic microbes.

Identifying fungal-host associations in an amphibian host system.

Host-associated microbes can interact with macro-organisms in a number of ways that affect host health. Few studies of host-associated microbiomes, however, focus on fungi. In addition, it is difficult to discern whether a fungal organism found in or on an ectotherm host is associating with it in a durable, symbiotic interaction versus a transient one, and to what extent the habitat and host share microbes. We seek to identify these host-microbe interactions on an amphibian, the Colorado boreal toad (Anaxyrus boreas boreas). We sequenced the ITS1 region of the fungal community on the skin of wild toads (n = 124) from four sites in the Colorado Rocky Mountains, across its physiologically dynamic developmental life stages. We also sampled the common habitats used by boreal toads: water from their natal wetland and aquatic pond sediment. We then examined diversity patterns within different life stages, between host and habitat, and identified fungal taxa that could be putatively host-associated with toads by using an indicator species analysis on toad versus environmental samples. Host and habitat were strikingly similar, with the exception of toad eggs. Post-hatching toad life stages were distinct in their various fungal diversity measures. We identified eight fungal taxa that were significantly associated with eggs, but no other fungal taxa were associated with other toad life stages compared with their environmental habitat. This suggests that although pre- and post-metamorphic toad life stages differ from each other, the habitat and host fungal communities are so similar that identifying obligate host symbionts is difficult with the techniques used here. This approach does, however, leverage sequence data from host and habitat samples to predict which microbial taxa are host-associated versus transient microbes, thereby condensing a large set of sequence data into a smaller list of potential targets for further consideration.

Comparative Analyses of Vertebrate Gut Microbiomes Reveal Convergence between Birds and Bats.

Diet and host phylogeny drive the taxonomic and functional contents of the gut microbiome in mammals, yet it is unknown whether these patterns hold across all vertebrate lineages. Here, we assessed gut microbiomes from ∼900 vertebrate species, including 315 mammals and 491 birds, assessing contributions of diet, phylogeny, and physiology to structuring gut microbiomes. In most nonflying mammals, strong correlations exist between microbial community similarity, host diet, and host phylogenetic distance up to the host order level. In birds, by contrast, gut microbiomes are only very weakly correlated to diet or host phylogeny. Furthermore, while most microbes resident in mammalian guts are present in only a restricted taxonomic range of hosts, most microbes recovered from birds show little evidence of host specificity. Notably, among the mammals, bats host especially bird-like gut microbiomes, with little evidence for correlation to host diet or phylogeny. This suggests that host-gut microbiome phylosymbiosis depends on factors convergently absent in birds and bats, potentially associated with physiological adaptations to flight. Our findings expose major variations in the behavior of these important symbioses in endothermic vertebrates and may signal fundamental evolutionary shifts in the cost/benefit framework of the gut microbiome.IMPORTANCE In this comprehensive survey of microbiomes of >900 species, including 315 mammals and 491 birds, we find a striking convergence of the microbiomes of birds and animals that fly. In nonflying mammals, diet and short-term evolutionary relatedness drive the microbiome, and many microbial species are specific to a particular kind of mammal, but flying mammals and birds break this pattern with many microbes shared across different species, with little correlation either with diet or with relatedness of the hosts. This finding suggests that adaptation to flight breaks long-held relationships between hosts and their microbes.

Is there convergence of gut microbes in blood-feeding vertebrates?

Animal microbiomes play an important role in dietary adaptation, yet the extent to which microbiome changes exhibit parallel evolution is unclear. Of particular interest is an adaptation to extreme diets, such as blood, which poses special challenges in its content of proteins and lack of essential nutrients. In this study, we assessed taxonomic signatures (by 16S rRNA amplicon profiling) and potential functional signatures (inferred by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt)) of haematophagy in birds and bats. Our goal was to test three alternative hypotheses: no convergence of microbiomes, convergence in taxonomy and convergence in function. We find a statistically significant effect of haematophagy in terms of microbial taxonomic convergence across the blood-feeding bats and birds, although this effect is small compared to the differences found between haematophagous and non-haematophagous species within the two host clades. We also find some evidence of convergence at the predicted functional level, although it is possible that the lack of metagenomic data and the poor representation of microbial lineages adapted to haematophagy in genome databases limit the power of this approach. The results provide a paradigm for exploring convergent microbiome evolution replicated with independent contrasts in different host lineages. This article is part of the theme issue 'Convergent evolution in the genomics era: new insights and directions'.

Community richness of amphibian skin bacteria correlates with bioclimate at the global scale.

Animal-associated microbiomes are integral to host health, yet key biotic and abiotic factors that shape host-associated microbial communities at the global scale remain poorly understood. We investigated global patterns in amphibian skin bacterial communities, incorporating samples from 2,349 individuals representing 205 amphibian species across a broad biogeographic range. We analysed how biotic and abiotic factors correlate with skin microbial communities using multiple statistical approaches. Global amphibian skin bacterial richness was consistently correlated with temperature-associated factors. We found more diverse skin microbiomes in environments with colder winters and less stable thermal conditions compared with environments with warm winters and less annual temperature variation. We used bioinformatically predicted bacterial growth rates, dormancy genes and antibiotic synthesis genes, as well as inferred bacterial thermal growth optima to propose mechanistic hypotheses that may explain the observed patterns. We conclude that temporal and spatial characteristics of the host's macro-environment mediate microbial diversity.

Experimental habitat fragmentation disrupts nematode infections in Australian skinks.

Habitat conversion and fragmentation threaten biodiversity and disrupt species interactions. While parasites are recognized as ecologically important, the impacts of fragmentation on parasitism are poorly understood relative to other species interactions. This lack of understanding is in part due to confounding landscape factors that accompany fragmentation. Fragmentation experiments provide the opportunity to fill this knowledge gap by mechanistically testing how fragmentation affects parasitism while controlling landscape factors. In a large-scale, long-term experiment, we asked how fragmentation affects a host-parasite interaction between a skink and a parasitic nematode, which is trophically transmitted via a terrestrial amphipod intermediate host. We expected that previously observed amphipod declines resulting from fragmentation would result in decreased transmission of nematodes to skinks. In agreement, we found that nematodes were absent among skinks in the cleared matrix and that infections in fragments were about one quarter of those in continuous forest. Amphipods found in gut contents of skinks and collected from pitfall traps mirrored this pattern. A structural equation model supported the expectation that fragmentation disrupted this interaction by altering the abundance of amphipods and suggested that other variables are likely also important in mediating this effect. These findings advance understanding of how landscape change affects parasitism.

Assessment of Bacterial Communities Associated With the Skin of Costa Rican Amphibians at La Selva Biological Station.

Amphibian skin is a suitable environment for rich communities of microorganisms, both beneficial and detrimental to the host. The amphibian cutaneous microbiota has been hypothesized to play an important role as symbionts, protecting their hosts against disease. Costa Rica has one of the most diverse assemblages of amphibians in the world and we know very little about the microbiota of these tropical animals. For comparison with other studies, we explore the diversity of the skin bacterial communities employing16S rRNA amplicon sequencing of swab samples from twelve species of frogs at La Selva Biological Station in Sarapiquí, Heredia province. The predominant phylum detected in our studies was Proteobacteria, followed by Bacteroidetes and Actinobacteria, with these three phyla representing 89.9% of the total bacterial taxa. At the family level, Sphingobacteriaceae and Comamonadaceae were highly represented among samples. Our results suggest that host species and host family are significant predictors of the variation in microbiota composition. This study helps set the foundation for future research about microbiota composition and resilience to unfavorable conditions, leading to improvement in managing strategies for endangered amphibian species.

Host-associated bacterial community succession during amphibian development.

Amphibians undergo significant developmental changes during their life cycle, as they typically move from a primarily aquatic environment to a more terrestrial one. Amphibian skin is a mucosal tissue that assembles communities of symbiotic microbiota. However, it is currently not well understood as to where amphibians acquire their skin symbionts, and whether the sources of microbial symbionts change throughout development. In this study, we utilized data collected from four wild boreal toad populations (Anaxyrus boreas); specifically, we sampled the skin bacterial communities during toad development, including eggs, tadpoles, subadults and adults as well as environmental sources of bacteria (water, aquatic sediment and soil). Using 16S rRNA marker gene profiling coupled with SourceTracker, we show that while primary environmental sources remained constant throughout the life cycle, secondary sources of boreal toad symbionts significantly changed with development. We found that toad skin communities changed predictably across development and that two developmental disturbance events (egg hatching and metamorphosis) dictated major changes. Toad skin communities assembled to alternative stable states following each of these developmental disturbances. Using the predicted average rRNA operon copy number of the communities at each life stage, we showed how the skin bacterial communities undergo a successional pattern whereby "fast-growing" (copiotroph) generalist bacteria dominate first before "slow-growing" (oligotroph) specialized bacteria take over. Our study highlights how host-associated bacterial community assembly is tightly coupled to host development and that host-associated communities demonstrate successional patterns akin to those observed in free-living bacteria as well as macrofaunal communities.

Probiotics as a tool for disease mitigation in wildlife: insights from food production and medicine.

The use of beneficial microbes to improve host attributes, referred to as probiotic therapy, has been increasingly applied to industries, including aquaculture, agriculture, and human medicine, and is emerging in the field of wildlife medicine. However, there is a general lack of shared knowledge regarding successful practices as well as ecological processes that underlie host-microbe interactions. Presently, probiotics are being developed specifically for preventing and treating particular infectious diseases as an alternative to antibiotic treatments and chemotherapy. We review research on probiotics developed for mitigation of infectious disease in the aforementioned industries to gain insight into how probiotics may be effective in reducing wildlife disease risk. We examine the trends of successful in vivo probiotic applications for disease systems and identify common objectives to reduce intestinal pathogens and sexually transmitted and respiratory diseases, inhibit skin pathogens, and serve as environmental prophylaxis to reduce pathogen loads in the environment. We conclude by highlighting the frontier of wildlife probiotics research and identifying knowledge gaps where research is needed.

Composition of Micro-eukaryotes on the Skin of the Cascades Frog (Rana cascadae) and Patterns of Correlation between Skin Microbes and Batrachochytrium dendrobatidis.

Global amphibian decline linked to fungal pathogens has galvanized research on applied amphibian conservation. Skin-associated bacterial communities of amphibians have been shown to mediate fungal skin infections and the development of probiotic treatments with antifungal bacteria has become an emergent area of research. While exploring the role of protective bacteria has been a primary focus for amphibian conservation, we aim to expand and study the other microbes present in amphibian skin communities including fungi and other micro-eukaryotes. Here, we characterize skin-associated bacteria and micro-eukaryotic diversity found across life stages of Cascades frog (Rana cascadae) and their associated aquatic environments using culture independent 16S and 18S rRNA marker-gene sequencing. Individuals of various life stages of Cascades frogs were sampled from a population located in the Trinity Alps in Northern California during an epidemic of the chytrid fungus, Batrachochytrium dendrobatidis. We filtered the bacterial sequences against a published database of bacteria known to inhibit B. dendrobatidis in co-culture to estimate the proportion of the skin bacterial community that is likely to provide defense against B. dendrobatidis. Tadpoles had a significantly higher proportion of B. dendrobatidis-inhibitory bacterial sequence matches relative to subadult and adult Cascades frogs. We applied a network analysis to examine patterns of correlation between bacterial taxa and B. dendrobatidis, as well as micro-eukaryotic taxa and B. dendrobatidis. Combined with the published database of bacteria known to inhibit B. dendrobatidis, we used the network analysis to identify bacteria that negatively correlated with B. dendrobatidis and thus could be good probiotic candidates in the Cascades frog system.

The Effects of Captivity on the Mammalian Gut Microbiome.

Recent studies increasingly note the effect of captivity or the built environment on the microbiome of humans and other animals. As symbiotic microbes are essential to many aspects of biology (e.g., digestive and immune functions), it is important to understand how lifestyle differences can impact the microbiome, and, consequently, the health of hosts. Animals living in captivity experience a range of changes that may influence the gut bacteria, such as diet changes, treatments, and reduced contact with other individuals, species and variable environmental substrates that act as sources of bacterial diversity. Thus far, initial results from previous studies point to a pattern of decreased bacterial diversity in captive animals. However, these studies are relatively limited in the scope of species that have been examined. Here we present a dataset that includes paired wild and captive samples from mammalian taxa across six Orders to investigate generalizable patterns of the effects captivity on mammalian gut bacteria. In comparing the wild to the captive condition, our results indicate that alpha diversity of the gut bacteria remains consistent in some mammalian hosts (bovids, giraffes, anteaters, and aardvarks), declines in the captive condition in some hosts (canids, primates, and equids), and increases in the captive condition in one host taxon (rhinoceros). Differences in gut bacterial beta diversity between the captive and wild state were observed for most of the taxa surveyed, except the even-toed ungulates (bovids and giraffes). Additionally, beta diversity variation was also strongly influenced by host taxonomic group, diet type, and gut fermentation physiology. Bacterial taxa that demonstrated larger shifts in relative abundance between the captive and wild states included members of the Firmicutes and Bacteroidetes. Overall, the patterns that we observe will inform a range of disciplines from veterinary practice to captive breeding efforts for biological conservation. Furthermore, bacterial taxa that persist in the captive state provide unique insight into symbiotic relationships with the host.

Host Ecology Rather Than Host Phylogeny Drives Amphibian Skin Microbial Community Structure in the Biodiversity Hotspot of Madagascar.

Host-associated microbiotas of vertebrates are diverse and complex communities that contribute to host health. In particular, for amphibians, cutaneous microbial communities likely play a significant role in pathogen defense; however, our ecological understanding of these communities is still in its infancy. Here, we take advantage of the fully endemic and locally species-rich amphibian fauna of Madagascar to investigate the factors structuring amphibian skin microbiota on a large scale. Using amplicon-based sequencing, we evaluate how multiple host species traits and site factors affect host bacterial diversity and community structure. Madagascar is home to over 400 native frog species, all of which are endemic to the island; more than 100 different species are known to occur in sympatry within multiple rainforest sites. We intensively sampled frog skin bacterial communities, from over 800 amphibians from 89 species across 30 sites in Madagascar during three field visits, and found that skin bacterial communities differed strongly from those of the surrounding environment. Richness of bacterial operational taxonomic units (OTUs) and phylogenetic diversity differed among host ecomorphs, with arboreal frogs exhibiting lower richness and diversity than terrestrial and aquatic frogs. Host ecomorphology was the strongest factor influencing microbial community structure, with host phylogeny and site parameters (latitude and elevation) explaining less but significant portions of the observed variation. Correlation analysis and topological congruency analyses revealed little to no phylosymbiosis for amphibian skin microbiota. Despite the observed geographic variation and low phylosymbiosis, we found particular OTUs that were differentially abundant between particular ecomorphs. For example, the genus Pigmentiphaga (Alcaligenaceae) was significantly enriched on arboreal frogs, Methylotenera (Methylophilaceae) was enriched on aquatic frogs, and Agrobacterium (Rhizobiaceae) was enriched on terrestrial frogs. The presence of shared bacterial OTUs across geographic regions for selected host genera suggests the presence of core microbial communities which in Madagascar, might be driven more strongly by a species' preference for specific microhabitats than by the physical, physiological or biochemical properties of their skin. These results corroborate that both host and environmental factors are driving community assembly of amphibian cutaneous microbial communities, and provide an improved foundation for elucidating their role in disease resistance.

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.

The Oral and Skin Microbiomes of Captive Komodo Dragons Are Significantly Shared with Their Habitat.

Examining the way in which animals, including those in captivity, interact with their environment is extremely important for studying ecological processes and developing sophisticated animal husbandry. Here we use the Komodo dragon (Varanus komodoensis) to quantify the degree of sharing of salivary, skin, and fecal microbiota with their environment in captivity. Both species richness and microbial community composition of most surfaces in the Komodo dragon's environment are similar to the Komodo dragon's salivary and skin microbiota but less similar to the stool-associated microbiota. We additionally compared host-environment microbiome sharing between captive Komodo dragons and their enclosures, humans and pets and their homes, and wild amphibians and their environments. We observed similar host-environment microbiome sharing patterns among humans and their pets and Komodo dragons, with high levels of human/pet- and Komodo dragon-associated microbes on home and enclosure surfaces. In contrast, only small amounts of amphibian-associated microbes were detected in the animals' environments. We suggest that the degree of sharing between the Komodo dragon microbiota and its enclosure surfaces has important implications for animal health. These animals evolved in the context of constant exposure to a complex environmental microbiota, which likely shaped their physiological development; in captivity, these animals will not receive significant exposure to microbes not already in their enclosure, with unknown consequences for their health. IMPORTANCE Animals, including humans, have evolved in the context of exposure to a variety of microbial organisms present in the environment. Only recently have humans, and some animals, begun to spend a significant amount of time in enclosed artificial environments, rather than in the more natural spaces in which most of evolution took place. The consequences of this radical change in lifestyle likely extend to the microbes residing in and on our bodies and may have important implications for health and disease. A full characterization of host-microbe sharing in both closed and open environments will provide crucial information that may enable the improvement of health in humans and in captive animals, both of which experience a greater incidence of disease (including chronic illness) than counterparts living under more ecologically natural conditions.

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.