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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. 

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. 

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. 

Abstract Understanding host immune function and ecoimmunology is increasingly important at a time when emerging infectious diseases (EIDs) threaten wildlife. One EID that has emerged and spread widely in recent years is chytridiomycosis, caused by the fungal pathogenBatrachochytrium dendrobatidis(Bd), which is implicated unprecedented amphibian declines around the world. The impacts ofBdhave been severe for many amphibian species, but some populations have exhibited signs of persistence, and even recovery, in some regions. Many mechanisms may underpin this pattern and amphibian immune responses are likely one key component. Although we have made great strides in understanding amphibian immunity, the complement system remains poorly understood. The complement system is a nonspecific, innate immune defense that is known to enhance other immune responses. Complement activation can occur by three different biochemical pathways and result in protective mechanisms, such as inflammation, opsonization, and pathogen lysis, thereby providing protection to the host. We currently lack an understanding of complement pathway activation for chytridiomycosis, but several studies have suggested that it may be a key part of an early and robust immune response that confers host resistance. Here, we review the available research on the complement system in general as well as amphibian complement responses toBdinfection. Additionally, we propose future research directions that will increase our understanding of the amphibian complement system and other immune responses toBd. Finally, we suggest how a deeper understanding of amphibian immunity could enhance the conservation and management of amphibian species that are threatened by chytridiomycosis. 

Abstract Avoiding extinction in a rapidly changing environment often relies on a species’ ability to quickly adapt in the face of extreme selective pressures. In Panamá, two closely related harlequin frog species (Atelopus variusandAtelopus zeteki) are threatened with extinction due to the fungal pathogenBatrachochytrium dendrobatidis(Bd). Once thought to be nearly extirpated from Panamá,A. variushave recently been rediscovered in multiple localities across their historical range; however,A. zetekiare possibly extinct in the wild. By leveraging a unique collection of 186Atelopustissue samples collected before and after theBdoutbreak in Panama, we describe the genetics of persistence for these species on the brink of extinction. We sequenced the transcriptome and developed an exome‐capture assay to sequence the coding regions of theAtelopusgenome. Using these genetic data, we evaluate the population genetic structure of historicalA. variusandA. zetekipopulations, describe changes in genetic diversity over time, assess the relationship between contemporary and historical individuals, and test the hypothesis that someA. variuspopulations have rapidly evolved to resist or tolerateBdinfection. We found a significant decrease in genetic diversity in contemporary (compared to historical)A. variuspopulations. We did not find strong evidence of directional allele frequency change or selection forBdresistance genes, but we uncovered a set of candidate genes that warrant further study. Additionally, we found preliminary evidence of recent migration and gene flow in one of the largest persistingA. variuspopulations in Panamá, suggesting the potential for genetic rescue in this system. Finally, we propose that previous conservation units should be modified, as clear genetic breaks do not exist beyond the local population level. Our data lay the groundwork for genetically informed conservation and advance our understanding of how imperiled species might be rescued from extinction. 

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. 

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’.