Bacterial communities of the threatened Western Pond Turtle may be impacted by land use.

As semi-aquatic species that use both terrestrial and aquatic habitats, freshwater turtles and their microbial communities are especially sensitive to the impacts of habitat disturbance. In this study, we use 16S rRNA amplicon sequencing to characterize the shell and cloacal bacterial communities of turtles in the San Francisco Bay Area. We captured western pond turtles (Actinemys/Emys marmorata) across eight sites located in urban and rural environments, along with invasive red-eared sliders (Trachemys scripta elegans). We assessed differences in western pond turtle bacterial communities diversity/composition between shell and cloacal samples and evaluated how alpha/beta diversity metrics were influenced by habitat quality. We found phylum-level bacterial taxonomic turnover in the bacterial communities of western pond turtles relative to the host tissue substrate samples. Our findings indicate that location identity elicits a high degree of lower-level (i.e. species/genus) bacterial taxonomic turnover. Further, we found that samples originating from good quality habitat had poorer shell bacterial communities but more diverse cloacal ones. The shell bacterial communities of red-eared sliders overlapped with those western pond turtles suggesting the existence of microbial dispersal between these two species. Our results add to our current understanding of turtle symbiont microbial ecology by establishing patterns of bacterial symbiont variation in an urban to rural gradient.

Wildfires disturb the natural skin microbiota of terrestrial salamanders.

Environmental change can disturb natural associations between wildlife and microbial symbionts, in many cases to the detriment of host health. We used a North American terrestrial salamander system to assess how the skin microbiota of amphibians responds to wildfires. In northern California's redwood/oak forests, we assessed how recent wildfires affected the skin microbiota of three different salamander species (Taricha sp., Batrachoseps attenuatus, and Ensatina eschscholtzii) over two different sampling seasons in 2018 and 2021. We found species-specific responses to wildfire disturbance on the alpha diversity of the skin microbiota of terrestrial salamanders, although burning in general altered the composition of the skin microbiota. The effect of burning on alpha diversities and body condition indices varied by sampling season, suggesting an additional effect of annual climatic conditions on body condition and skin microbiota response. We tested all salamanders for Batrachochytrium dendrobatidis and found four infected individuals in 2018 and none in 2021. Our study documents correlations in the skin microbiota response to an increasing source of disturbance in western North American ecosystems. In addition, our results highlight the need to consider the effects of increased wildfire regimes/intensities and longitudinal effects on wildlife-associated microbiota and animal health.

From the organismal to biosphere levels: environmental impacts on the amphibian microbiota.

This review summarizes the role of environmental factors on amphibian microbiotas at the organismal, population, community, ecosystem, and biosphere levels. At the organismal-level, tissue source, disease status, and experimental manipulations were the strongest predictors of variation in amphibian microbiotas. At the population-level, habitat quality, disease status, and ancestry were commonly documented as drivers of microbiota turnover. At the community-level, studies focused on how species' niche influence microbiota structure and function. At the ecosystem-level, abiotic and biotic reservoirs were important contributors to microbiota structure. At the biosphere-level, databases, sample banks, and seminatural experiments were commonly used to describe microbiota assembly mechanisms among temperate and tropical amphibians. Collectively, our review demonstrates that environmental factors can influence microbiotas through diverse mechanisms at all biological scales. Importantly, while environmental mechanisms occurring at each of the different scales can interact to shape microbiotas, the past 10 years of research have mostly been characterized by targeted approaches at individual scales. Looking forward, efforts considering how environmental factors at multiple organizational levels interact to shape microbiota diversity and function are paramount. Generating opportunities for meaningful cross-disciplinary interactions and supporting infrastructure for research that spans biological scales are imperative to addressing this gap.

Life stage and proximity to roads shape the skin microbiota of eastern newts (Notophthalmus viridescens).

Host-associated microbiomes play an essential role in the health of organisms, including immune system activation, metabolism and energy uptake. It is well established that microbial communities differ depending on the life stage and natural history of the organism. However, the effects of life stage and natural history on microbial communities may also be influenced by human activities. We investigated the effects of amphibian life stage (terrestrial eft vs. aquatic adult) and proximity to roadways on newt skin bacterial communities. We found that the eft and adult life stages differed in bacterial community composition; however, the effects of roads on community composition were more evident in the terrestrial eft stage compared to the aquatic adult stage. Terrestrial efts sampled close to roads possessed richer communities than those living further away from the influence of roads. When accounting for amplicon sequence variants with predicted antifungal capabilities, in the adult life stage, we observed a decrease in anti-fungal bacteria with distance to roads. In contrast, in the eft stage, we found an increase in anti-fungal bacteria with distance to roads. Our results highlight the need to consider the effects of human activities when evaluating how host-associated microbiomes differ across life stages of wildlife.

Skin bacterial metacommunities of San Francisco Bay Area salamanders are structured by host genus and habitat quality.

Host-associated microbial communities can influence physiological processes of macroorganisms, including contributing to infectious disease resistance. For instance, some bacteria that live on amphibian skin produce antifungal compounds that inhibit two lethal fungal pathogens, Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). Therefore, differences in microbiome composition among host species or populations within a species can contribute to variation in susceptibility to Bd/Bsal. This study applies 16S rRNA sequencing to characterize the skin bacterial microbiomes of three widespread terrestrial salamander genera native to the western United States. Using a metacommunity structure analysis, we identified dispersal barriers for these influential bacteria between salamander families and localities. We also analysed the effects of habitat characteristics such as percent natural cover and temperature seasonality on the microbiome. We found that certain environmental variables may influence the skin microbial communities of some salamander genera more strongly than others. Each salamander family had a somewhat distinct community of putative anti-Bd skin bacteria, suggesting that salamanders may select for a functional assembly of cutaneous symbionts that could differ in its ability to protect these amphibians from disease. Our observations raise the need to consider host identity and environmental heterogeneity during the selection of probiotics to treat wildlife diseases.

Prevalence of Batrachochytrium dendrobatidis in immature eastern hellbenders Cryptobranchus alleganiensis from North Carolina, USA.

Batrachochytrium dendrobatidis (Bd) has been detected in wild hellbender Cryptobranchus alleganiensis populations, with rare instances of chytridiomycosis and Bd-induced mortality. To date, Bd surveillance in hellbender populations has been disproportionately focused on adult age classes. A lingering question is whether Bd might contribute to hellbender declines through disproportionate negative effects on immature age classes. The objective of this study was to quantify Bd prevalence and load in immature hellbenders in western North Carolina, USA. We conducted field surveys during 2018 and 2019 and collected 88 skin swabs from 84 hellbenders spanning 3 age classes. Bd was detected on 11% of individuals, including 8 larvae and 1 juvenile. We did not detect symptoms of chytridiomycosis or a decline in body condition in Bd-positive hellbenders. Load varied from approximately 1-153 zoospore equivalents for the 9 Bd-positive hellbenders and was not associated with size class of the individual. While hellbenders appeared to be abundant in each survey reach, more work is needed to determine whether Bd may increase the vulnerability of immature hellbenders to anthropogenic stressors.

A novel bioaugmentation technique effectively increases the skin-associated microbial diversity of captive eastern hellbenders.

Captive environments are maintained in hygienic ways that lack free-flowing microbes found in animals' natural environments. As a result, captive animals often have depauperate host-associated microbial communities compared to conspecifics in the wild and may have increased disease susceptibility and reduced immune function. Eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) have suffered precipitous population declines over the past few decades. To bolster populations, eastern hellbenders are reared in captivity before being translocated to the wild. However, the absence of natural microbial reservoirs within the captive environment diminishes the diversity of skin-associated bacteria on hellbender skin and may negatively influence their ability to defend against pathogenic species once they are released into the wild. To prepare hellbenders for natural bacteria found in riverine environments, we devised a novel bioaugmentation method to increase the diversity of skin microbial communities within a captive setting. We exposed juvenile hellbenders to increasing amounts of river water over 5 weeks before translocating them to the river. We genetically identified and phylogenetically compared bacteria collected from skin swabs and river water for alpha (community richness) and beta (community composition) diversity estimates. We found that hellbenders exposed to undiluted river water in captivity had higher alpha diversity and distinct differentiation in the community composition on their skin, compared to hellbenders only exposed to well water. We also found strong evidence that hellbender skin microbiota is host-specific rather than environmentally driven and is colonized by rare environmental operational taxonomic units in river water. This technique may increase hellbender translocation success as increasing microbial diversity is often correlated with elevated disease resistance. Future work is necessary to refine our methods, investigate the relationship between microbial diversity and hellbender health and understand how this bioaugmentation technique influences hellbenders' survival following translocation from captivity into the wild.

Comment on "Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity".

Scheele et al (Reports, 29 March 2019, p. 1459) bring needed attention to the effects of amphibian infectious disease. However, the data and methods implicating the disease chytridiomycosis in 501 amphibian species declines are deficient. Which species are affected, and how many, remains a critical unanswered question. Amphibians are imperiled; protective actions require public support and robust science.

Invasive vegetation affects amphibian skin microbiota and body condition.

Invasive plants are major drivers of habitat modification and the scale of their impact is increasing globally as anthropogenic activities facilitate their spread. In California, an invasive plant genus of great concern is Eucalyptus. Eucalyptus leaves can alter soil chemistry and negatively affect underground macro- and microbial communities. Amphibians serve as excellent models to evaluate the effect of Eucalyptus invasion on ground-dwelling species as they predate on soil arthropods and incorporate soil microbes into their microbiotas. The skin microbiota is particularly important to amphibian health, suggesting that invasive plant species could ultimately affect amphibian populations. To investigate the potential for invasive vegetation to induce changes in microbial communities, we sampled microbial communities in the soil and on the skin of local amphibians. Specifically, we compared Batrachoseps attenuatus skin microbiomes in both Eucalyptus globulus (Myrtaceae) and native Quercus agriflolia (Fagaceae) dominated forests in the San Francisco Bay Area. We determined whether changes in microbial diversity and composition in both soil and Batrachoseps attenuatus skin were associated with dominant vegetation type. To evaluate animal health across vegetation types, we compared Batrachoseps attenuatus body condition and the presence/absence of the amphibian skin pathogen Batrachochytrium dendrobatidis. We found that Eucalyptus invasion had no measurable effect on soil microbial community diversity and a relatively small effect (compared to the effect of site identity) on community structure in the microhabitats sampled. In contrast, our results show that Batrachoseps attenuatus skin microbiota diversity was greater in Quercus dominated habitats. One amplicon sequence variant identified in the family Chlamydiaceae was observed in higher relative abundance among salamanders sampled in Eucalyptus dominated habitats. We also observed that Batrachoseps attenuatus body condition was higher in Quercus dominated habitats. Incidence of Batrachochytrium dendrobatidis across all individuals was very low (only one Batrachochytrium dendrobatidis positive individual). The effect on body condition demonstrates that although Eucalyptus may not always decrease amphibian abundance or diversity, it can potentially have cryptic negative effects. Our findings prompt further work to determine the mechanisms that lead to changes in the health and microbiome of native species post-plant invasion.

Amphibian Host and Skin Microbiota Response to a Common Agricultural Antimicrobial and Internal Parasite.

Holistic approaches that simultaneously characterize responses of both microbial symbionts and their hosts to environmental shifts are imperative to understanding the role of microbiotas on host health. Using the northern leopard frog (Lithobates pipiens) as our model, we investigated the effects of a common trematode (family Echinostomatidae), a common agricultural antimicrobial (Sulfadimethoxine; SDM), and their interaction on amphibian skin microbiota and amphibian health (growth metrics and susceptibility to parasites). In the trematode-exposed individuals, we noted an increase in alpha diversity and a shift in microbial communities. In the SDM-treated individuals, we found a change in the composition of the skin microbiota similar to those induced by the trematode treatment. Groups treated with SDM, echinostomes, or a combination of SDM and echinostomes, had higher relative abundances of OTUs assigned to Flavobacterium and Acinetobacter. Both of these genera have been associated with infectious disease in amphibians and the production of anti-pathogen metabolites. Similar changes in microbial community composition between SDM and trematode exposed individuals may have resulted from stress-related disruption of host immunity. Despite changes in the microbiota, we found no effect of echinostomes and SDM on host health. Given the current disease- and pollution-related threats facing amphibians, our study highlights the need to continue to evaluate the influence of natural and anthropogenic stressors on host-associated microbial communities.

Local adaptation of the MHC class IIß gene in populations of wood frogs (Lithobates sylvaticus) correlates with proximity to agriculture.

Major histocompatibility complex (MHC) genes code for membrane-embedded proteins that are involved in parasite/pathogen recognition. The link between the MHC and immunity makes these genes important genetic markers to evaluate in systems where infectious disease is associated with population declines. As human impacts on wildlife populations continue to increase, it is also essential to evaluate the role of MHC and immunity in the context of anthropogenic change. Amphibians are an ideal model to test the role of the MHC in infectious disease resistance, as parasites and anthropogenic disturbances currently threaten populations worldwide. We characterized the diversity of MHC class IIß peptide binding region alleles, 13 microsatellite loci, and population-level trematode resistance in 14 populations of wood frogs (Lithobates sylvaticus) in northwestern Pennsylvania with varying geographic distances to agriculture. To assess local adaptation in the MHC IIß, we compared genetic differentiation of MHC IIß and microsatellite markers (FST). We also tested for an effect of isolation by distance on genetic differentiation of MHC IIß and microsatellite markers. In addition, we evaluated whether population-level MHC IIß diversity and common allele frequencies correlate with distance to agriculture and trematode resistance. We found no evidence for genetic structure based on microsatellite analysis nor an effect of isolation by distance on neutral and immunogenetic markers. However, we did detect structure based on the MHC IIß locus, suggesting that it is under local selection. The MHC IIß allele Lisy-DAB*1 was more common in populations living near agricultural sites. Populations with higher MHC IIß diversity showed increased resistance to trematodes. Our results suggest that wood frog populations experience immunogenetic differences at a small scale. In addition, agriculture may disturb natural associations between hosts and parasites through its influence on immunocompetence, underscoring the importance of examining the effects of environmental context on host-parasite interactions.

Captivity-Induced Changes in the Skin Microbial Communities of Hellbenders (Cryptobranchus alleganiensis).

Variation in environmental conditions can result in disparate associations between hosts and microbial symbionts. As such, it is imperative to evaluate how environmental variables (e.g., habitat quality) can influence host-associated microbiome composition. Within wildlife conservation programs, captive conditions can negatively influence the establishment and maintenance of "wild-type" microbiotas within a host. Alternative microbial communities can result in the proliferation of disease among captive stock or upon reintroduction. Hellbenders (Cryptobranchus alleganiensis) are a threatened salamander for which extensive captive management is currently employed. Using metabarcoding, we characterized the skin microbiota of wild and captive hellbenders from two subspecies in the state of Missouri, the eastern (C. a. alleganiensis) and the Ozark hellbender (C. a. bishopi). Both subspecies in our study included wild adults and captive juveniles that were collected from the wild as eggs. Our objectives were to investigate differences in the skin microbial communities' richness/diversity, composition, and functional profiles of microbes between wild and captive individuals. Captive eastern hellbenders possessed richer communities than wild cohorts, whereas the opposite pattern was observed within the Ozark subspecies. We found significant microbial community structure between wild and captive populations of both subspecies. Microbiota structure translated into differences in the predicted metagenome of wild and captive individuals as well. As such, we can expect captive hellbenders to experience alternative microbial structure and function upon reintroduction into the wild. Our study provides a baseline for the effect of captivity on the skin microbial communities of hellbenders, and highlights the need to incorporate microbiota management in current captive-rearing programs.

Influence of immunogenetics, sex and body condition on the cutaneous microbial communities of two giant salamanders.

The complex association between hosts and microbial symbionts requires the implementation of multiple approaches to evaluate variation in host physiology. Within amphibians, heterogeneity in immunogenetic traits and cutaneous microbiota is associated with variation in disease resistance. Ozark (Cryptobranchus alleganiensis bishopi) and eastern hellbenders (C. a. alleganiensis) provide a model system to assess variation in host traits and microbial communities. Ozark hellbenders have experienced declines throughout their range, are federally endangered and experience wound retardation that is absent in the eastern subspecies. Previous microbial investigations indicate differentiation in the composition of the skin microbiota of both hellbender subspecies, but it is not clear whether these patterns are concurrent with diversity in the major histocompatibility complex (MHC) genes. We characterized the MHC IIB and the skin microbiota of hellbenders in Missouri, where both subspecies co-occur though not sympatric. We compared the microbiota composition and MHC diversity between both subspecies and investigated whether individual-level MHC diversity, sex and body condition were associated with microbiota composition. Overall, MHC IIB diversity was lower in Ozark hellbenders compared to the eastern subspecies. Multivariate statistical comparisons identified microbiota differentiation between Ozark and eastern hellbenders. MHC IIB allele presence/absence, allele divergence, body composition and sex defined grouping of hellbender microbiotas within populations. Differentiation of the cutaneous microbiotas and MHC IIB genes between eastern and Ozark hellbenders suggests that differences exist in immunity between the two subspecies. This study demonstrates how simultaneous assessments of host genetic traits and microbiotas can inform patterns of microbial community structure in natural systems.

Cutaneous Microbial Community Variation across Populations of Eastern Hellbenders (Cryptobranchus alleganiensis alleganiensis).

Multicellular hosts maintain complex associations with microbial communities. While microbial communities often serve important functional roles for their hosts, our understanding of the local and regional processes that structure these communities remains limited. Metacommunity analyses provide a promising tool for investigating mechanisms shaping microbiome heterogeneity, which is essential for predicting functional variation between hosts. Using a metacommunity framework, we examined heterogeneity in the skin microbiome of the eastern hellbender (Cryptobranchus alleganiensis alleganiensis). Hellbenders are broadly distributed throughout river systems in the eastern United States, but are present in specific environmental locations throughout their range. The large range of the species and history of population fragmentation suggest that local and regional processes contribute to the distribution of cutaneous symbiont diversity. Therefore, we characterized the skin and environmental bacterial communities at eight rivers throughout the range of the species. We observed variation among hellbender populations in skin microbial community diversity and proportion of shared operational taxonomic units (OTUs) between animal and river water communities. Among populations sampled, we noted significant clumped OTU turnover (i.e., Clementsian structure) resulting in unique cutaneous communities. In addition, we observed a significant positive correlation between skin community divergence and hellbender population genetic divergence. Host-population skin community dissimilarity did not correlate strongly with distance between sampling locations, indicating a weak spatial effect on the distribution of symbionts. These results suggest that species sorting mechanisms (i.e., local processes) structure local skin microbial communities in hellbenders. The variation in skin community composition observed among host populations foreshadows a similar pattern in important functional characteristics (e.g., resistance to dysbiosis). Future work should focus on investigating forces shaping microbiome structure in eastern hellbenders, examining functional variation among populations, and evaluating effectiveness of microbiome management recommendations.

Characterization of the Cutaneous Bacterial Communities of Two Giant Salamander Subspecies.

Pathogens currently threaten the existence of many amphibian species. In efforts to combat global declines, researchers have characterized the amphibian cutaneous microbiome as a resource for disease management. Characterization of microbial communities has become useful in studying the links between organismal health and the host microbiome. Hellbender salamanders (Cryptobranchus alleganiensis) provide an ideal system to explore the cutaneous microbiome as this species requires extensive conservation management across its range. In addition, the Ozark hellbender subspecies (Cryptobranchus alleganiensis bishopi) exhibits chronic wounds hypothesized to be caused by bacterial infections, whereas the eastern hellbender (Cryptobranchus alleganiensis alleganiensis) does not. We assessed the cutaneous bacterial microbiome of both subspecies at two locations in the state of Missouri, USA. Through 16S rRNA gene-based amplicon sequencing, we detected more than 1000 distinct operational taxonomic units (OTUs) in the cutaneous and environmental bacterial microbiome. Phylogenetic and abundance-based dissimilarity matrices identified differences in the bacterial communities between the two subspecies, but only the abundance-based dissimilarity matrix identified differences between wounds and healthy skin on Ozark hellbenders. The higher abundance of OTUs on Ozark wounds suggests that commensal bacteria present on the skin and environment may be opportunistically colonizing the wounds. This brief exploration of the hellbender cutaneous bacterial microbiome provides foundational support for future studies seeking to understand the hellbender cutaneous bacterial microbiome and the role of the bacterial microbiota on chronic wounds of Ozark hellbenders.