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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. 

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. 

Environmental temperature is a key factor driving various biological processes, including immune defenses and host-pathogen interactions. Here, we evaluated the effects of environmental temperature on the pathogenicity of the emerging fungal pathogen, Batrachochytrium salamandrivorans ( Bsal ), using controlled laboratory experiments, and measured components of host immune defense to identify regulating mechanisms. We found that adult and juvenile Notophthalmus viridescens died faster due to Bsal chytridiomycosis at 14°C than at 6 and 22°C. Pathogen replication rates, total available proteins on the skin, and microbiome composition likely drove these relationships. Temperature-dependent skin microbiome composition in our laboratory experiments matched seasonal trends in wild N . viridescens , adding validity to these results. We also found that hydrophobic peptide production after two months post-exposure to Bsal was reduced in infected animals compared to controls, perhaps due to peptide release earlier in infection or impaired granular gland function in diseased animals. Using our temperature-dependent susceptibility results, we performed a geographic analysis that revealed N . viridescens populations in the northeastern United States and southeastern Canada are at greatest risk for Bsal invasion, which shifted risk north compared to previous assessments. Our results indicate that environmental temperature will play a key role in the epidemiology of Bsal and provide evidence that temperature manipulations may be a viable disease management strategy. 

Abstract Accurately predicting the impacts of climate change on wildlife health requires a deeper understanding of seasonal rhythms in host–pathogen interactions. The amphibian pathogen,Batrachochytrium dendrobatidis(Bd), exhibits seasonality in incidence; however, the role that biological rhythms in host defences play in defining this pattern remains largely unknown.The aim of this study was to examine whether host immune and microbiome defences againstBdcorrespond with infection risk and seasonal fluctuations in temperature and humidity.Over the course of a year, five populations of Southern leopard frogs (Rana[Lithobates]sphenocephala) in Tennessee, United States, were surveyed for host immunity, microbiome and pathogen dynamics. Frogs were swabbed for pathogen load and skin bacterial diversity and stimulated to release stored antimicrobial peptides (AMPs). Secretions were analysed to estimate total hydrophobic peptide concentrations, presence of known AMPs and effectiveness ofBdgrowth inhibition in vitro. The diversity and proportion of bacterial reads with a 99% match to sequences of isolates known to inhibitBdgrowth in vitro were used as an estimate of predicted anti‐Bdfunction of the skin microbiome.Batrachochytrium dendrobatidisdynamics followed the expected seasonal fluctuations—peaks in cooler months—which coincided with when host mucosal defences were most potent againstBd. Specifically, the concentration and expression of stored AMPs cycled synchronously withBddynamics. Although microbiome changes followed more linear trends over time, the proportion of bacteria that can function to inhibitBdgrowth was greatest when risk ofBdinfection was highest.We interpret the increase in peptide storage in the fall and the shift to a more anti‐Bdmicrobiome over winter as a preparatory response for subsequent infection risk during the colder periods when AMP synthesis and bacterial growth is slow and pathogen pressure from this cool‐adapted fungus is high. Given that a decrease in stored AMP concentrations as temperatures warm in spring likely means greater secretion rates, the subsequent decrease in prevalence suggests seasonality ofBdin this host may be in part regulated by annual immune rhythms, and dominated by the effects of temperature.