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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. 

The field of ecological immunology, or ecoimmunology, has provided valuable insights on the immune responses of diverse host organisms threatened by infectious diseases in many different environments. One infectious disease that has been particularly notable for its impacts on host populations is amphibian chytridiomycosis, which has been linked with amphibian declines around the world. Amphibian immune responses to the pathogen that causes chytridiomycosis (Batrachochytriym dendrobatidis) are not well understood but thought to involve innate immune factors, including the complement system. In this study, we tested the ability of complement proteins to inhibitB. dendrobatidisinin vitrochallenge assays. We found that complement proteins from amphibian plasma that were not heat inactivated reduced the viability and growth ofB. dendrobatidis.The inhibitory efficacy was similar to effects onPseudomonas fluorescens, a bacterium that is known to be inhibited by complement protein activation. These findings suggest inhibition ofB. dendrobatidisthat is consistent with the involvement of the complement system. In addition, we provide methods for standardizing pathogen killing assays, and set a foundation for further investigations on the amphibian complement system and other immune responses to amphibian chytridiomycosis. 

Synopsis Antimicrobial peptides (AMPs) play a fundamental role in the innate defense against microbial pathogens, as well as other immune and non-immune functions. Their role in amphibian skin defense against the pathogenic fungus Batrachochytrium dendrobatidis (Bd) is exemplified by experiments in which depletion of host’s stored AMPs increases mortality from infection. Yet, the question remains whether there are generalizable patterns of negative or positive correlations between stored AMP defenses and the probability of infection or infection intensity across populations and species. This study aims to expand on prior field studies of AMP quantities and compositions by correlating stored defenses with an estimated risk of Bd exposure (prevalence and mean infection intensity in each survey) in five locations across the United States and a total of three species. In all locations, known AMPs correlated with the ability of recovered secretions to inhibit Bd in vitro. We found that stored AMP defenses were generally unrelated to Bd infection except in one location where the relative intensity of known AMPs was lower in secretions from infected frogs. In all other locations, known AMP relative intensities were higher in infected frogs. Stored peptide quantity was either positively or negatively correlated with Bd exposure risk. Thus, future experiments coupled with organismal modeling can elucidate whether Bd infection affects secretion/synthesis and will provide insight into how to interpret amphibian ecoimmunology studies of AMPs. We also demonstrate that future AMP isolating and sequencing studies can focus efforts by correlating mass spectrometry peaks to inhibitory capacity using linear decomposition modeling. 

Abstract To combat the loss of species due to emerging infectious diseases, scientists must incorporate ecological parameters, such as temperature and humidity, to understand how the environment affects host–pathogen interactions. The fungal disease chytridiomycosis is a compelling case study to investigate the role of both temperature and humidity on infectious disease, as both the fungal pathogen (Batrachochytrium dendrobatidis, Bd) and the host (amphibians) are heavily influenced by these abiotic factors. We performed two experiments to investigate the importance of relative humidity and temperature on frog immunity (production of antimicrobial skin secretions) and disease development in captive golden frogs (Atelopus zeteki) of Panama. We found that the quantity of skin secretions significantly decreased over time in frogs moved from low to medium and high relative humidity treatments. FollowingBdexposure, frogs in high temperature (26–27 °C) and high relative humidity (80–90%) had lower pathogen loads and survived significantly longer than frogs kept in all other treatment conditions, including high temperature and low relative humidity. These results suggest that high relative humidity may be an important, although less understood, mediator ofBdinfection and the survival of golden frogs. Because the environment can drastically alter disease dynamics, understanding how temperature and humidity influence chytridiomycosis outcomes in golden frogs may be essential for the success of the reintroduction of captive frogs. 

The immune equilibrium model suggests that exposure to microbes during early life primes immune responses for pathogen exposure later in life. While recent studies using a range of gnotobiotic (germ-free) model organisms offer support for this theory, we currently lack a tractable model system for investigating the influence of the microbiome on immune system development. Here, we used an amphibian species ( Xenopus laevis ) to investigate the importance of the microbiome in larval development and susceptibility to infectious disease later in life. We found that experimental reductions of the microbiome during embryonic and larval stages effectively reduced microbial richness, diversity and altered community composition in tadpoles prior to metamorphosis. In addition, our antimicrobial treatments resulted in few negative effects on larval development, body condition, or survival to metamorphosis. However, contrary to our predictions, our antimicrobial treatments did not alter susceptibility to the lethal fungal pathogen Batrachochytrium dendrobatidis ( Bd ) in the adult life stage. While our treatments to reduce the microbiome during early development did not play a critical role in determining susceptibility to disease caused by Bd in X. laevis , they nevertheless indicate that developing a gnotobiotic amphibian model system may be highly useful for future immunological investigations. This article is part of the theme issue ‘Amphibian immunity: stress, disease and ecoimmunology’. 

ABSTRACT Studying declining and rare species is inherently challenging, particularly when the cause of rarity is emerging infectious diseases (EIDs). Tracking changes in the distribution of pathogens that cause EIDs, and the species made scarce by them, is necessary for conservation efforts, but it is often a time and resource intensive task. Here, we demonstrate how using environmental DNA (eDNA) to detect rare species—and the pathogens that threaten them—can be a powerful tool to understand disease dynamics and develop effective conservation strategies. Amphibian populations around the world have undergone rapid declines and extinctions due to the emerging fungal pathogen,Batrachochytrium dendrobatidis(Bd). We developed and validated a qPCR assay using eDNA sampling methods for some of the most imperiled amphibian species, harlequin frogs (Atelopus varius,Atelopus zeteki,andAtelopus chiriquiensis), and applied this assay in concert with a standard qPCR assay forBdin rainforest streams of Panamá. We confirmed the presence ofAtelopusat sampling locations across three regions. In addition, we used genomic analysis of eDNA samples to show thatBdin Panamá falls within the Global Panzootic Lineage, a lineage associated with disease‐induced declines. We detectedBdDNA in most of our historic sites, and its concentration in water samples correlated with stream characteristics and the pathogen load of the local amphibian community. These results suggest that some populations ofAtelopuspersist in their historic localities. They also show how eDNA analysis can be effectively used for monitoring species presence, pathogen concentrations, and the distribution and spread of pathogen lineages. EIDs are a growing threat to endangered species around the world. Simultaneous detection of rare and declining host species and their pathogens with eDNA will help to provide key insights for effective conservation management. 

Temperature is a critically important factor in many infectious disease systems, because it can regulate responses in both the host and the pathogen. White-nose syndrome (WNS) in bats is a severe infectious disease caused by the temperature-sensitive fungus, Pseudogymnoascus destructans ( Pd ). One feature of WNS is an increase in the frequency of arousal bouts (i.e. when bat body temperatures are elevated) in Pd -infected bats during hibernation. While several studies have proposed that increased frequency of arousals may play a role in the pathophysiology of WNS, it is unknown if the temperature fluctuations might mediate Pd growth. We hypothesized that exposure to a high frequency of elevated temperatures would reduce Pd growth due to thermal constraints on the pathogen. We simulated the thermal conditions for arousal bouts of uninfected and infected bats during hibernation (fluctuating from 8 to 25°C at two different rates) and quantified Pd growth in vitro . We found that increased exposure to high temperatures significantly reduced Pd growth. Because temperature is one of the most critical abiotic factors mediating host–pathogen interactions, resolving how Pd responds to fluctuating temperatures will provide insights for understanding WNS in bats and other fungal diseases. 

Abstract To combat the threat of emerging infectious diseases in wildlife, ecoimmunologists seek to understand the complex interactions among pathogens, their hosts, and their shared environments. The cutaneous fungal pathogen Batrachochytrium dendrobatidis (Bd), has led to the decline of innumerable amphibian species, including the Panamanian golden frog (Atelopus zeteki). Given that Bd can evade or dampen the acquired immune responses of some amphibians, nonspecific immune defenses are thought to be especially important for amphibian defenses against Bd. In particular, skin secretions constitute a vital component of amphibian innate immunity against skin infections, but their role in protecting A. zeteki from Bd is unknown. We investigated the importance of this innate immune component by reducing the skin secretions from A. zeteki and evaluating their effectiveness against Bd in vitro and in vivo. Following exposure to Bd in a controlled inoculation experiment, we compared key disease characteristics (e.g., changes in body condition, prevalence, pathogen loads, and survival) among groups of frogs that had their skin secretions reduced and control frogs that maintained their skin secretions. Surprisingly, we found that the skin secretions collected from A. zeteki increased Bd growth in vitro. This finding was further supported by infection and survival patterns in the in vivo experiment where frogs with reduced skin secretions tended to have lower pathogen loads and survive longer compared to frogs that maintained their secretions. These results suggest that the skin secretions of A. zeteki are not only ineffective at inhibiting Bd but may enhance Bd growth, possibly leading to greater severity of disease and higher mortality in this highly vulnerable species. These results differ from those of previous studies in other amphibian host species that suggest that skin secretions are a key defense in protecting amphibians from developing severe chytridiomycosis. Therefore, we suggest that the importance of immune components cannot be generalized across all amphibian species or over time. Moreover, the finding that skin secretions may be enhancing Bd growth emphasizes the importance of investigating these immune components in detail, especially for species that are a conservation priority.