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Amphibian and reptile microbiomes are important contributors to host health and ecosystem dynamics but understudied compared to microbiomes of other vertebrates. Through study of amphibian and reptile microbiomes, investigators can advance theory and practice in animal health, ecosystem function, conservation, and more. This special issue ofFrontiers of Amphibian and Reptile Sciencefeatures six publications in the field of amphibian and reptile microbiome research, and here, we highlight the contributions of these and other recent publications to four main areas of recent advancement in the field: 1) expanding “microbiome” beyond bacteria, 2) expanding study of host life-history stages and anatomical microbial habitats, 3) characterizing whole microbial communities, particularly in disease ecology studies, and 4) developing and implementing databases and bioinformatics tools. We also discuss several avenues for future research that would bring amphibians and reptiles to the forefront of microbiome research. With the continued implementation of classic culture-based and sequencing approaches paired with cutting-edge toolsin vivo,in vitro, andin silico, herpetofauna microbiome research is poised for exciting growth in the near future. 

Disruption of habitat connectivity alters host movement patterns and pathogen exposure in wildlife. Changes in exposure dynamics have led to increased research interest in host-associated microbial communities (i.e., microbiomes), particularly in how repeated encounters with pathogens may drive microbial filtering processes that favor the assembly of pathogen-inhibiting microbiomes, a concept known as the adaptive microbiome principle. Understanding how habitat connectivity and pathogen exposure shape adaptive microbiomes remains a key frontier in disease ecology. For widely distributed waterborne pathogens such as the amphibian chytrid fungusBatrachochytrium dendrobatidis(Bd), sustained host exposure in contiguous terrestrial–aquatic habitats may promote selection of microbiomes with enhanced antifungal properties. In contrast, under conditions of high habitat split, where key habitats such as forests and water bodies are spatially disconnected, limited exposure to Bd during the pre-breeding and overwintering seasons may hinder the selection of Bd-inhibitory microbiomes that are critical for host protection during the subsequent breeding season, when pathogen exposure in water bodies is at its peak. Our results demonstrate that habitat split may limit recruitment of putative Bd-inhibitory skin bacteria, while Bd loads increase with habitat split for certain amphibian species. Results from Joint Species Distribution Models also indicate that habitat split is a key driver of reduced skin bacterial diversity, even after accounting for biotic and abiotic metrics. Our study provides evidence that spatial connectivity among natural habitats is essential for maintaining multiple levels of biodiversity, from host species to their associated functional microbiomes, highlighting a critical link between environmental disturbance, microbial defenses, and disease dynamics. 

Abstract Ongoing amphibian population declines are caused by factors such as climate change, habitat destruction, pollution and infectious diseases not limited to chytridiomycosis. Unfortunately, action is taken against these factors once population collapses are underway. To avoid these post hoc responses, wildlife endocrinology aims to analyse physiological mediators that predict future population declines to inform wildlife management. Mediators typically investigated are stress hormones known as glucocorticoids, which are produced by the Hypothalamus—Pituitary—Interrenal axis (HPI axis). The HPI axis is the part of the endocrine system that helps amphibians cope with stress. Chronic increases in glucocorticoids due to stress can lead to immune dysfunction, which makes amphibians more susceptible to infectious diseases. Despite this predictive potential of glucocorticoids, interpretation of glucocorticoid data is confounded by sampling design and type. Glucocorticoid monitoring classically involves blood sampling, which is not widely applicable in amphibians as some are too small or delicate to sample, and repeated samples are often valued. To address this, we tried to validate skin swabbing via corticosterone (CORT) and adrenocorticotropin hormone (ACTH) injections in adults of two amphibian species: Eastern red-spotted newts, Notophthalmus viridescens viridescens, with natural skin infections with Batrachochytrium dendrobatidis (Bd) upon collection in the field, and Northern leopard frogs, Rana (Lithobates) pipiens, raised in captivity and naïve to Bd exposure. Further, we determined the predictive potential of skin glucocorticoids on Bd load in the field via correlations in Eastern red-spotted newts. We found that hormones present in the skin are not related to the HPI axis and poorly predict infection load; however, skin hormone levels strongly predicted survival in captivity. Although skin swabbing is not a valid method to monitor HPI axis function in these species, the hormones present in the skin still play important roles in organismal physiology under stressful conditions relevant to wildlife managers. 

A diverse metabolome exists on amphibian skin that mediates interactions between hosts and skin microbiomes. Tetrodotoxin is one such metabolite that occurs across a variety of taxa, and is particularly well studied in newts of the genusTarichathat are susceptible to infection with chytrid fungi. The interaction of tetrodotoxin with the skin microbiome, including pathogenic fungi, is not well understood, and here we describe these patterns across 12 populations ofTaricha granulosaandT. torosain Washington, Oregon, and California. We found no correlation of TTX andBatrachochytrium dendrobatidis(Bd) infection in eitherT. granulosaorT. torosa, a pattern inconsistent with a previous study. In addition, TTX, but not Bd, was significantly correlated with the skin microbiome composition inT. granulosa. InT. torosa, however, Bd, but not TTX, was correlated with the skin microbiome structure. The relationship between TTX and skin microbiome composition differed between species, with significant correlations observed only inT. granulosa, which exhibited higher TTX concentrations. We also detected significantly higher abundances of bacterial taxa (e.g., Pseudomonadaceae) associated with TTX production in newts with higher skin TTX. These taxa (ASVs matchingAeromonas, Pseudomonas, Shewanella, andSphingopyxis) were associated with all body sites of previously sampledT. granulosa, but not found in soil samples. Our results suggest that toxins can shape the newt skin microbiome and may influence pathogen infection through indirect mechanisms, as TTX showed no direct inhibition of Bd orB. salamandrivoransgrowth. 

Some of the amphibian populations in Panama are demonstrating slow recovery decades after severe declines caused by the invasion of the fungal pathogenBatrachochytrium dendrobatidis(Bd). However, new species remain to be described and assessed for the mechanisms of disease resilience. We identified seven skin defense peptides from a presumably novel leopard frog species in the Tabasará range, at Buäbti (Llano Tugrí), Ngäbe-Buglé Comarca, and Santa Fe, Veraguas, Panama, herein called the Ngäbe-Buglé leopard frog. Two of the peptides were previously known: brevinin-1BLb fromRana (Lithobates) blairiand a previously hypothesized “ancestral” peptide, ranatuerin-2BPa. We hypothesized that the peptides are active againstBdand shape the microbiome such that the skin bacterial communities are more similar to those of other leopard frogs than of co-occurring host species. Natural mixtures of the collected skin peptides showed a minimum inhibitory concentration againstBdof 100 μg/ml, which was similar to that of other leopard frogs that have been tested. All sampled individuals hosted high intensity of infection withBd. We sampled nine other amphibian species in nearby habitats and found lower prevalence and intensities ofBdinfection. In addition to the pathogen load, the skin microbiomes were examined using 16S rRNA gene targeted amplicon sequencing. When compared to nine co-occurring amphibians, the Ngäbe-Buglé leopard frog had similar skin bacterial richness and anti-Bdfunction, but the skin microbiome structure differed significantly among species. The community composition of the bacterial skin communities was strongly associated with theBdinfection load. In contrast, the skin microbiome composition of the Ngäbe-Buglé leopard frog was similar to that of five North American leopard frog populations and the sympatric and congenericRana (Lithobates) warszewitschii, with 29 of the 46 core bacteria all demonstrating anti-Bdactivity in culture. Because of the highBdinfection load and prevalence in the Ngäbe-Buglé leopard frog, we suggest that treatment to reduce theBdload in this species might reduce the chytridiomycosis risk in the co-occurring amphibian community, but could potentially disrupt the evolution of skin defenses that provide a mechanism for disease resilience in this species. 

Abstract Human‐induced climate change, land use changes, and urbanization are predicted to dramatically impact landscape hydrology, which can have devastating impacts on aquatic organisms. For amphibians that rely on aquatic environments to breed and develop, it is essential to understand how the larval environment impacts development, condition, and performance later in life. Two important predicted impacts of climate change, urbanization, and land use changes are reduced hydroperiod and variable larval density. Here, we explored how larval density and hydroperiod affect development, morphology, physiology, and immune defenses at metamorphosis and 35 days post‐metamorphosis in the frogRana pipiens. We found that high‐density larval conditions had a large negative impact on development and morphology, which resulted in longer larval periods, reduced likelihood of metamorphosis, smaller size at metamorphosis, shorter femur to body length ratio, and reduced microbiome species evenness compared with animals that developed in low‐density conditions. However, animals from the high‐density treatment experienced compensatory growth post‐metamorphosis, demonstrating accelerated growth in body size and relative femur length compared with animals from the low‐density treatments, despite not “catching‐up” in size. We also observed an increase in relative gut length and relative liver size in animals that had developed in the high‐density treatment than those in the low‐density treatment, as well as higher bacterial killing ability, and greater jump distances relative to their leg length across different temperatures. Finally, metabolic rate was higher overall but especially at higher test temperatures for animals that developed under high‐density conditions, indicating that these animals may expend more energy in response to acute temperature changes. While the effects of climate change have direct negative effects on larval development and metamorphosis, animals can increase growth rate post‐metamorphosis; however, that compensatory growth might come at a cost and reduce their ability to cope with further environmental change such as increased temperatures. 

The use of museum specimens for research in microbial evolutionary ecology remains an under-utilized investigative dimension with important potential. Despite this potential, there remain barriers in methodology and analysis to the wide-spread adoption of museum specimens for such studies. Here, we hypothesized that there would be significant differences in taxonomic prediction and related diversity among sample type (museum or fresh) and sequencing strategy (medium-depth shotgun metagenomic or 16S rRNA gene). We found dramatically higher predicted diversity from shotgun metagenomics when compared to 16S rRNA gene sequencing in museum and fresh samples, with this differential being larger in museum specimens. Broadly confirming these hypotheses, the highest diversity found in fresh samples was with shotgun sequencing using the Rep200 reference inclusive of viruses and microeukaryotes, followed by the WoL reference database. In museum-specimens, community diversity metrics also differed significantly between sequencing strategies, with the alpha-diversity ACE differential being significantly greater than the same comparisons made for fresh specimens. Beta diversity results were more variable, with significance dependent on reference databases used. Taken together, these findings demonstrate important differences in diversity results and prompt important considerations for future experiments and downstream analyses aiming to incorporate microbiome datasets from museum specimens. 

Most hosts contain few parasites, whereas few hosts contain many. This pattern, known as aggregation, is well-documented in macroparasites where parasite intensity distribution among hosts affects host–parasite dynamics. Infection intensity also drives fungal disease dynamics, but we lack a basic understanding of host–fungal aggregation patterns, how they compare with macroparasites and if they reflect biological processes. To begin addressing these gaps, we characterized aggregation of the fungal pathogenBatrachochytrium dendrobatidis(Bd) in amphibian hosts. Utilizing the slope of Taylor’s Power law, we found Bd intensity distributions were more aggregated than many macroparasites, conforming closely to lognormal distributions. We observed that Bd aggregation patterns are strongly correlated with known biological processes operating in amphibian populations, such as epizoological phase (i.e. invasion, post-invasion and enzootic), and intensity-dependent disease mortality. Using intensity-dependent mathematical models, we found evidence of evolution of host resistance based on aggregation shifts in systems persisting with Bd following disease-induced declines. Our results show that Bd aggregation is highly conserved across disparate systems and contains signatures of potential biological processes of amphibian–Bd systems. Our work can inform future modelling approaches and be extended to other fungal pathogens to elucidate host–fungal interactions and unite host–fungal dynamics under a common theoretical framework.