The amphibian skin microbiome is recognized for its role in defence against pathogens, including the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd). Yet, we have little understanding of evolutionary and ecological processes that structure these communities, especially for salamanders and closely related species. We investigated patterns in the distribution of bacterial communities on Plethodon salamander skin across host species and environments. Quantifying salamander skin microbiome structure contributes to our understanding of how host-associated bacteria are distributed across the landscape, among host species, and their putative relationship with disease. We characterized skin microbiome structure (alpha-diversity, beta-diversity and bacterial operational taxonomic unit [OTU] abundances) using 16S rRNA gene sequencing for co-occurring Plethodon salamander species (35 Plethodon cinereus, 17 Plethodon glutinosus, 10 Plethodon cylindraceus) at three localities to differentiate the effects of host species from environmental factors on the microbiome. We sampled the microbiome of P. cinereus along an elevational gradient (n = 50, 700-1,000 m a.s.l.) at one locality to determine whether elevation predicts microbiome structure. Finally, we quantified prevalence and abundance of putatively anti-Bd bacteria to determine if Bd-inhibitory bacteria are dominant microbiome members. Co-occurring salamanders had similar microbiome structure, but among sites salamanders had dissimilar microbiome structure for beta-diversity and abundance of 28 bacterial OTUs. We found that alpha-diversity increased with elevation, beta-diversity and the abundance of 17 bacterial OTUs changed with elevation (16 OTUs decreasing, 1 OTU increasing). We detected 11 putatively anti-Bd bacterial OTUs that were present on 90% of salamanders and made up an average relative abundance of 83% (SD ± 8.5) per salamander. All salamanders tested negative for Bd. We conclude that environment is more influential in shaping skin microbiome structure than host differences in these congeneric species, and suggest that environmental characteristics that covary with elevation influence microbiome structure. High prevalence and abundance of anti-Bd bacteria may contribute to low Bd levels in these populations of Plethodon salamanders.

Symbiotic bacteria may dampen the impacts of infectious diseases on hosts by inhibiting pathogen growth. However, our understanding of the generality of pathogen inhibition by different bacterial taxa across pathogen genotypes and environmental conditions is limited. Bacterial inhibitory properties are of particular interest for the amphibian-killing fungal pathogens (Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans), for which probiotic applications as conservation strategies have been proposed. We quantified the inhibition strength of five putatively B. dendrobatidis-inhibitory bacteria isolated from woodland salamander skin against six Batrachochytrium genotypes at two temperatures (12 and 18°C). We selected six genotypes from across the Batrachochytrium phylogeny: B. salamandrivorans, B. dendrobatidis-Brazil and four genotypes of the B. dendrobatidis Global Panzootic Lineage (GPL1: JEL647, JEL404; GPL2: SRS810, JEL423). We performed 96-well plate challenge assays in a full factorial design. We detected a Batrachochytrium genotype by temperature interaction on bacterial inhibition score for all bacteria, indicating that bacteria vary in ability to inhibit Batrachochytrium depending on pathogen genotype and temperature. Acinetobacter rhizosphaerae moderately inhibited B. salamandrivorans at both temperatures (μ = 46–53%), but not any B. dendrobatidis genotypes. Chryseobacterium sp. inhibited three Batrachochytrium genotypes at both temperatures (μ = 5–71%). Pseudomonas sp. strain 1 inhibited all Batrachochytrium genotypes at 12°C and four Batrachochytrium genotypes at 18°C (μ = 5–100%). Pseudomonas sp. strain 2 and Stenotrophomonas sp. moderately to strongly inhibited all six Batrachochytrium genotypes at both temperatures (μ = 57–100%). All bacteria consistently inhibited B. salamandrivorans. Using cluster analysis of inhibition scores, we found that more closely related Batrachochytrium genotypes grouped together, suggesting that bacterial inhibition strength may be predictable based on Batrachochytrium relatedness. We conclude that bacterial inhibition capabilities change among bacterial strains, Batrachochytrium genotypes and temperatures. A comprehensive understanding of bacterial inhibitory function, across pathogen genotypes and temperatures, is needed to better predict the role of bacterial symbionts in amphibian disease ecology. For targeted conservation applications, we recommend using bacterial strains identified as strongly inhibitory as they are most likely to produce broad-spectrum antimicrobial agents at a range of temperatures.

Pathogens compete with host microbiomes for space and resources. Their shared environment impacts pathogen-microbiome-host interactions, which can lead to variation in disease outcome. The skin microbiome of red-backed salamanders (Plethodon cinereus) can reduce infection by the pathogen Batrachochytrium dendrobatidis (Bd) at moderate infection loads, with high species richness and high abundance of competitors as putative mechanisms. However, it is unclear if the skin microbiome can reduce epizootic Bd loads across temperatures. We conducted a laboratory experiment to quantify skin microbiome and host responses (P. cinereus: n = 87) to Bd at mimicked epizootic loads across temperatures (13, 17 and 21°C). We quantified skin microbiomes using 16S rRNA gene metabarcoding and identified operational taxonomic units (OTUs) taxonomically similar to culturable bacteria known to kill Bd (anti-Bd OTUs). Prior to pathogen exposure, temperature changed the microbiome (OTU richness decreased by 12% and the abundance of anti-Bd OTUs increased by 18% per degree increase in temperature), but these changes were not predictive of disease outcome. After exposure, Bd changed the microbiome (OTU richness decreased by 0.1% and the abundance of anti-Bd OTUs increased by 0.2% per 1% increase in Bd load) and caused high host mortality across temperatures (35/45: 78%). Temperature indirectly impacted microbiome change and mortality through its direct effect on pathogen load. We did not find support for the microbiome impacting Bd load or host survival. Our research reveals complex host, pathogen, microbiome and environmental interactions to demonstrate that during epizootic events the microbiome will be unlikely to reduce pathogen invasion, even for putatively Bd-resistant species. K E Y W O R D Santifungal, Batrachochytrium dendrobatidis, climate, epidemic, invasion, microbiota, symbiosis

Diverse bacteria inhabit amphibian skin; some of those bacteria inhibit growth of the fungal pathogen Batrachochytrium dendrobatidis. Yet there has been no systematic survey of anti-B. dendrobatidis bacteria across localities, species, and elevations. This is important given geographic and taxonomic variations in amphibian susceptibility to B. dendrobatidis. Our collection sites were at locations within the Appalachian Mountains where previous sampling had indicated low B. dendrobatidis prevalence. We determined the numbers and identities of anti-B. dendrobatidis bacteria on 61 Plethodon salamanders (37 P. cinereus, 15 P. glutinosus, 9 P. cylindraceus) via culturing methods and 16S rRNA gene sequencing. We sampled co-occurring species at three localities and sampled P. cinereus along an elevational gradient (700 to 1,000 meters above sea level [masl]) at one locality. We identified 50 anti-B. dendrobatidis bacterial operational taxonomic units (OTUs) and found that the degree of B. dendrobatidis inhibition was not correlated with relatedness. Five anti-B. dendrobatidis bacterial strains occurred on multiple amphibian species at multiple localities, but none were shared among all species and localities. The prevalence of anti-B. dendrobatidis bacteria was higher at Shenandoah National Park (NP), VA, with 96% (25/26) of salamanders hosting at least one anti-B. dendrobatidis bacterial species compared to 50% (7/14) at Catoctin Mountain Park (MP), MD, and 38% (8/21) at Mt. Rogers National Recreation Area (NRA), VA. At the individual level, salamanders at Shenandoah NP had more anti-B. dendrobatidis bacteria per individual ( ϭ 3.3) than those at Catoctin MP ( ϭ 0.8) and at Mt. Rogers NRA ( ϭ 0.4). All salamanders tested negative for B. dendrobatidis. Anti-B. dendrobatidis bacterial species are diverse in central Appalachian Plethodon salamanders, and their distribution varied geographically. The antifungal bacterial species that we identified may play a protective role for these salamanders.IMPORTANCE Amphibians harbor skin bacteria that can kill an amphibian fungal pathogen, Batrachochytrium dendrobatidis. Some amphibians die from B. dendrobatidis infection, whereas others do not. The bacteria that can kill B. dendrobatidis, called anti-B. dendrobatidis bacteria, are thought to influence the B. dendrobatidis infection outcome for the amphibian. Yet how anti-B. dendrobatidis bacterial species vary among amphibian species and populations is unknown. We determined the distribution of anti-B. dendrobatidis bacterial species among three salamander species (n ϭ 61) sampled at three localities. We identified 50 unique anti-B. dendrobatidis bacterial species and found that all of the tested salamanders were negative for B. dendrobatidis. Five anti-B. dendrobatidis bacterial species were commonly detected, suggesting a stable, functional association with these salamanders. The number of

Life processes of ectothermic vertebrates are intimately linked to the temperature of their environment, influencing their metabolism, reproduction, behaviour and immune responses. In amphibians infected by the generalist chytrid pathogen Batrachochytrium dendrobatidis (Bd), host survival, infection prevalence and infection intensity are often temperature‐ and/or seasonally dependent. However, the transcriptional underpinnings of thermal differences in infection responses remain unknown. Measuring the impact of temperature on host responses to infection is a key component for understanding climatic influences on chytrid disease dynamics. The Bd‐responsive gene pathways in frogs are well documented, but our understanding of salamander immune expression profiles during infection with chytrids remains limited. Here we characterize the transcriptomic responses of Plethodon cinereus using RNA sequencing by comparing skin and splenic gene expression of individuals uninfected, succumbing to Bd infection and naturally cleared of Bd infection at three temperatures. We suggest that amphibian temperature‐dependent susceptibility to Bd is probably driven by shifts in expression of the innate and adaptive immune axes. Our study shows increased expression of transcripts associated with inflammation at lower temperatures and a shift towards increased expression of adaptive immune genes, including MHC (major histocompatibility complex), at higher temperatures. In the face of climate change, and as concerns for the spread of emergent chytrid pathogens increase, our results provide important functional genomic resources to help understand how these pathogenic fungi may continue to affect amphibian communities globally in the future.

Captive management of many wildlife species can be challenging, with individuals displaying health disorders that are not generally described in the wild population. Retrospective studies have identified gastrointestinal (GI) diseases, in particular inflammatory bowel disease (IBD), as the second leading cause of captive adult red wolf ( Canis rufus) mortality. Recent molecular studies show that imbalanced gut microbial composition is tightly linked to IBD in the domestic dog. The goal of the present study was to address two main questions: (1) how do red wolf gut microbiomes differ between animals with loose stool consistency, indicative of GI issues, and those with normal stool consistency and (2) how does dietary type relate to stool consistency and red wolf gut microbiomes? Fresh fecal samples were collected from 48 captive wolves housed in eight facilities in the United States and from two wild wolves living in Alligator River National Wildlife Refuge, NC, United States. For each individual, the stool consistency was categorized as loose or normal using a standardized protocol and their diet was categorized as either wild, whole meat, a mix of whole meat and kibble or kibble. We characterized gut microbiome structure using 16S rRNA gene amplicon sequencing. We found that red wolves with a loose stool consistency differed in composition than wolves with normal stool consistency, suggesting a link between GI health and microbiome composition. Diet was not related to stool consistency but did significantly impact gut microbiome composition; gut microbiome composition of wolves fed a kibble diet were significantly different than the gut microbiome composition of wolves fed a mixed, whole meat and wild diet. Findings from this study increase the understanding of the interplay between diet and GI health in the red wolf, a critical piece of information needed to maintain a healthy red wolf population ex situ .

We engaged pet salamander owners in the United States to screen their animals for two amphibian chytrid fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). We provided pet owners with a sampling kit and instructional video to swab the skin of their animals. We received 639 salamander samples from 65 species by mail, and tested them for Bd and Bsal using qPCR. We detected Bd on 1.3% of salamanders (95% CI 0.0053–0.0267) and did not detect Bsal (95% CI 0.0000–0.0071). If Bsal is present in the U.S. population of pet salamanders, it occurs at a very low prevalence. The United States Fish and Wildlife Service listed 201 species of salamanders as “injurious wildlife” under the Lacey Act (18 U.S.C. § 42) on January 28, 2016, a precautionary action to prevent the introduction of Bsal to the U.S. through the importation of salamanders. This action reduced the number of salamanders imported to the U.S. from 2015 to 2016 by 98.4%. Our results indicate that continued precautions should be taken to prevent the introduction and establishment of Bsal in the U.S., which is a hotspot of salamander biodiversity.

Many parts of the animal body harbor microbial communities, known as animal-associated microbiomes, that affect the regulation of physiological functions. Studies in human and animal models have demonstrated that the reproductive biology and such microbiomes also interact. However, this concept is poorly studied in wild animal species and little is known about the implications to fertility, parental/offspring health, and survival in natural habitats. The objective of this review is to (1) specify the interactions between animals’ reproductive biology, including reproductive signaling, pregnancy, and offspring development, and their microbiomes, with an emphasis on wild species and (2) identify important research gaps as well as areas for further studies. While microbiomes present in the reproductive tract play the most direct role, other bodily microbiomes may also contribute to facilitating reproduction. In fish, amphibians, reptiles, birds, and mammals, endogenous processes related to the host physiology and behavior (visual and olfactory reproductive signals, copulation) can both influence and be influenced by the structure and function of microbial communities. In addition, exposures to maternal microbiomes in mammals (through vagina, skin, and milk) shape the offspring microbiomes, which, in turn, affects health later in life. Importantly, for all wild animal species, host-associated microbiomes are also influenced by environmental variations. There is still limited literature on wild animals compared to the large body of research on model species and humans. However, the few studies in wild species clearly highlight the necessity of increased research in rare and endangered animals to optimize conservation efforts in situ and ex situ. Thus, the link between microbiomes and reproduction is an emerging and critical component in wild animal conservation.