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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. 

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. 

The emerging fungal pathogenBatrachochytrium salamandrivorans(Bsal) threatens the diversity of amphibians, particularly in North America where it is projected to invade. Amphibian skin defenses include a mucosal layer containing microorganisms that can potentially modulate host response to pathogens such asBsal. In this study, we focused on the composition of the skin microbiome across life stages of spotted salamanders (Ambystoma maculatum). We also evaluated the stress hormone corticosterone and skin microbiome response to inoculations withBsaland probiotics at both the larval and juvenile developmental stages, and the response to different environmental conditions. Results indicated that both bacterial and fungal communities found on the skin significantly differed in structure and diversity between life stages ofA. maculatum. Exposure to three different probiotics (Bacillus thuringiensis,Chryseobacterium rhizoplanae, andPenicilliumsp.) andBsalevoked shifts in the microbiome of larvae and juveniles, and the metabolite profile of the larval mucosal layer ofA. maculatum. Despite changes in the microbiome, all tested probiotics andBsalwere unable to persist on the skin. Larval bacterial microbiomes shifted in response toBsalandB. thuringiensiswith no significant impacts on antifungal function or bacteria richness, however fungi strongly responded toBsalandB. thuringiensisapplication. This indicates that developmental shifts in the microbiome can be initiated by microbial applications such asB. thuringiensis, a widely used mosquito larvicide. Overall, experimental results indicate that life stage, growth and development, and environmental conditions appeared to be the main factors driving changes in the amphibian skin microbiome and potential anti-Batrachochytriumfunction. 

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. 

Bats are widespread mammals that play key roles in ecosystems as pollinators and insectivores. However, there is a paucity of information about bat-associated microbes, in particular their fungal communities, despite the important role microbes play in host health and overall host function. The emerging fungal disease, white-nose syndrome, presents a potential challenge to the bat microbiome and understanding healthy bat-associated taxa will provide valuable information about potential microbiome-pathogen interactions. To address this knowledge gap, we collected 174 bat fur/skin swabs from 14 species of bats captured in five locations in New Mexico and Arizona and used high-throughput sequencing of the fungal internal transcribed (ITS) region to characterize bat-associated fungal communities. Our results revealed a highly heterogeneous bat mycobiome that was structured by geography and bat species. Furthermore, our data suggest that bat-associated fungal communities are affected by bat foraging, indicating the bat skin microbiota is dynamic on short time scales. Finally, despite the strong effects of site and species, we found widespread and abundant taxa from several taxonomic groups including 

Abstract Variable harlequin frogsAtelopus variushave declined significantly throughout their range as a result of infection with the fungal pathogenBatrachochytrium dendrobatidis(Bd). The Panama Amphibian Rescue and Conservation Project maintains an ex situ population of this Critically Endangered species. We conducted a release trial with surplus captive-bredA. variusindividuals to improve our ability to monitor frog populations post-release, observe dispersal patterns after freeing them into the wild and learn about threats to released frogs, as well as to determine whether natural skin toxin defences of frogs could be restored inside mesocosms in the wild and to compare Bd dynamics in natural amphibian communities at the release site vs a non-release site. The 458 released frogs dispersed rapidly and were difficult to re-encounter unless they carried a radio transmitter. No frog was seen after 36 days following release. Thirty frogs were fitted with radio transmitters and only half were trackable by day 10. Tetrodotoxin was not detected in the skins of the frogs inside mesocosms for up to 79 days. Bd loads in other species present at sites were high prior to release and decreased over time in a pattern probably driven by weather. No differences were observed in Bd prevalence between the release and non-release sites. This trial showed that refinements of our methods and approaches are required to study captiveAtelopusfrogs released into wild conditions. We recommend continuing release trials of captive-bred frogs with post-release monitoring methods, using an adaptive management framework to advance the field of amphibian reintroduction ecology. 

The amphibian chytrid fungus, Batrachochytrium salamandrivorans ( Bsal ) threatens salamander biodiversity. The factors underlying Bsal susceptibility may include glucocorticoid hormones (GCs). The effects of GCs on immunity and disease susceptibility are well studied in mammals, but less is known in other groups, including salamanders. We used Notophthalmus viridescens (eastern newts) to test the hypothesis that GCs modulate salamander immunity. We first determined the dose required to elevate corticosterone (CORT; primary GC in amphibians) to physiologically relevant levels. We then measured immunity (neutrophil lymphocyte ratios, plasma bacterial killing ability (BKA), skin microbiome, splenocytes, melanomacrophage centres (MMCs)) and overall health in newts following treatment with CORT or an oil vehicle control. Treatments were repeated for a short (two treatments over 5 days) or long (18 treatments over 26 days) time period. Contrary to our predictions, most immune and health parameters were similar for CORT and oil-treated newts. Surprisingly, differences in BKA, skin microbiome and MMCs were observed between newts subjected to short- and long-term treatments, regardless of treatment type (CORT, oil vehicle). Taken together, CORT does not appear to be a major factor contributing to immunity in eastern newts, although more studies examining additional immune factors are necessary. This article is part of the theme issue ‘Amphibian immunity: stress, disease and ecoimmunology’.