Jordan G. Kueneman

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.

Antifungal isolates database of amphibian skin-associated bacteria and function against emerging fungal pathogens

Microbial symbionts of vertebrate skin have an important function in defense of the host against pathogens. In particular, the emerging chytrid fungus Batrachochytrium dendrobatidis, causes widespread disease in amphibians but can be inhibited via secondary metabolites produced by many different skin-associated bacteria. Similarly, the fungal pathogens of terrestrial salamander eggs Mariannaea elegans and Rhizomucor variabilis are also inhibited by a variety of skin-associated bacteria. Indeed, probiotic therapy against fungal diseases is a recent approach in conservation medicine with growing experimental support. We present a comprehensive Antifungal Isolates Database of amphibian skin-associated bacteria that have been cultured, isolated, and tested for antifungal properties. At the start, this database includes nearly 2000 cultured bacterial isolates from 37 amphibian host species across 18 studies on five continents: Africa, Oceania, Europe, and North and South America. As the research community gathers information on additional isolates, the database will be updated periodically. The resulting database can serve as a conservation tool for amphibians and other organisms, and provides empirical data for comparative and bioinformatic studies. The database consists of a FASTA file containing 16S rRNA gene sequences of the bacterial isolates, and a metadata file containing information on the host species, life-stage, geographic region, and antifungal capacity and taxonomic identity of the isolate.

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.

Using "Omics" and Integrated Multi-Omics Approaches to Guide Probiotic Selection to Mitigate Chytridiomycosis and Other Emerging Infectious Diseases

Emerging infectious diseases in wildlife are responsible for massive population declines. In amphibians, chytridiomycosis caused by Batrachochytrium dendrobatidis, Bd, has severely affected many amphibian populations and species around the world. One promising management strategy is probiotic bioaugmentation of antifungal bacteria on amphibian skin. In vivo experimental trials using bioaugmentation strategies have had mixed results, and therefore a more informed strategy is needed to select successful probiotic candidates. Metagenomic, transcriptomic, and metabolomic methods, colloquially called “omics,” are approaches that can better inform probiotic selection and optimize selection protocols. The integration of multiple omic data using bioinformatic and statistical tools and in silico models that link bacterial community structure with bacterial defensive function can allow the identification of species involved in pathogen inhibition. We recommend using 16S rRNA gene amplicon sequencing and methods such as indicator species analysis, the Kolmogorov–Smirnov Measure, and co-occurrence networks to identify bacteria that are associated with pathogen resistance in field surveys and experimental trials. In addition to 16S amplicon sequencing, we recommend approaches that give insight into symbiont function such as shotgun metagenomics, metatranscriptomics, or metabolomics to maximize the probability of finding effective probiotic candidates, which can then be isolated in culture and tested in persistence and clinical trials. An effective mitigation strategy to ameliorate chytridiomycosis and other emerging infectious diseases is necessary; the advancement of omic methods and the integration of multiple omic data provide a promising avenue toward conservation of imperiled species.

Taming wildlife disease: bridging the gap between science and management

Summary Parasites and pathogens of wildlife can threaten biodiversity, infect humans and domestic animals, and cause significant economic losses, providing incentives to manage wildlife diseases. Recent insights from disease ecology have helped transform our understanding of infectious disease dynamics and yielded new strategies to better manage wildlife diseases. Simultaneously, wildlife disease management (WDM) presents opportunities for large‐scale empirical tests of disease ecology theory in diverse natural systems. To assess whether the potential complementarity between WDM and disease ecology theory has been realized, we evaluate the extent to which specific concepts in disease ecology theory have been explicitly applied in peer‐reviewed WDM literature. While only half of WDM articles published in the past decade incorporated disease ecology theory, theory has been incorporated with increasing frequency over the past 40 years. Contrary to expectations, articles authored by academics were no more likely to apply disease ecology theory, but articles that explain unsuccessful management often do so in terms of theory. Some theoretical concepts such as density‐dependent transmission have been commonly applied, whereas emerging concepts such as pathogen evolutionary responses to management, biodiversity–disease relationships and within‐host parasite interactions have not yet been fully integrated as management considerations. Synthesis and applications. Theory‐based disease management can meet the needs of both academics and managers by testing disease ecology theory and improving disease interventions. Theoretical concepts that have received limited attention to date in wildlife disease management could provide a basis for improving management and advancing disease ecology in the future.

Managing Amphibian Disease with Skin Microbiota

The contribution of emerging amphibian diseases to the sixth mass extinction is driving innovative wildlife management strategies, including the use of probiotics. Bioaugmentation of the skin mucosome, a dynamic environment including host and microbial components, may not provide a generalized solution. Multi-omics technologies and ecological context underlie effective implementation.The contribution of emerging amphibian diseases to the sixth mass extinction is driving innovative wildlife management strategies, including the use of probiotics. Bioaugmentation of the skin mucosome, a dynamic environment including host and microbial components, may not provide a generalized solution. Multi-omics technologies and ecological context underlie effective implementation.

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.

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

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. ABSTRACT 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.

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.

Identification of Bufadienolides from the Boreal Toad, Anaxyrus boreas, Active Against a Fungal Pathogen

Amphibian granular glands provide a wide range of compounds on the skin that defend against pathogens and predators. We identified three bufadienolides—the steroid-like compounds arenobufagin, gamabufotalin, and telocinobufagin—from the boreal toad, Anaxyrus boreas, through liquid chromatography mass spectrometry (LC/MS). Compounds were detected both after inducing skin gland secretions and in constitutive mucosal rinses from toads. We described the antimicrobial properties of each bufadienolide against Batrachochytrium dendrobatidis (Bd), an amphibian fungal pathogen linked with boreal toad population declines. All three bufadienolides were found to inhibit Bd growth at similar levels. The maximum Bd inhibition produced by arenobufagin, gamabufotalin, and telocinobufagin were approximately 50%, in contrast to the complete Bd inhibition shown by antimicrobial skin peptides produced by some amphibian species. In addition, skin mucus samples significantly reduced Bd viability, and bufadienolides were detected in 15 of 62 samples. Bufadienolides also appeared to enhance growth of the anti-Bd bacterium Janthinobacterium lividum, and thus may be involved in regulation of the skin microbiome. Here, we localized skin bacteria within the mucus layer and granular glands of toads with fluorescent in situ hybridization. Overall, our results suggest that bufadienolides can function in antifungal defense on amphibian skin and their production is a potentially convergent trait similar to antimicrobial peptide defenses found on the skin of other species. Further studies investigating bufadienolide expression across toad populations, their regulation, and interactions with other components of the skin mucosome will contribute to understanding the complexities of amphibian immune defense.