Diverse Relationships between Batrachochytrium Infections and Antimicrobial Peptide Defenses Across Leopard Frog Populations.

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.

Towards the generation of gnotobiotic larvae as a tool to investigate the influence of the microbiome on the development of the amphibian immune system.

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'.

Temperature shifts associated with bat arousals during hibernation inhibit the growth of Pseudogymnoascus destructans.

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.

Once a reservoir, always a reservoir? Seasonality affects the pathogen maintenance potential of amphibian hosts.

Host species that can independently maintain a pathogen in a host community and contribute to infection in other species are important targets for disease management. However, the potential of host species to maintain a pathogen is not fixed over time, and an important challenge is understanding how within- and across-season variability in host maintenance potential affects pathogen persistence over longer time scales relevant for disease management (e.g., years). Here, we sought to understand the causes and consequences of seasonal infection dynamics in leopard frogs (Rana sphenocephala and Rana pipiens) infected with the fungal pathogen Batrachochytrium dendrobatidis (Bd). We addressed three questions broadly applicable to seasonal host-parasite systems. First, to what degree are observed seasonal patterns in infection driven by temperature-dependent infection processes compared to seasonal host demographic processes? Second, how does seasonal variation in maintenance potential affect long-term pathogen persistence in multi-host communities? Third, does high deterministic maintenance potential relate to the long-term stochastic persistence of pathogens in host populations with seasonal infection dynamics? To answer these questions, we used field data collected over 3 years on >1400 amphibians across four geographic locations, laboratory and mesocosm experiments, and a novel mathematical model. We found that the mechanisms that drive seasonal prevalence were different from those driving seasonal infection intensity. Seasonal variation in Bd prevalence was driven primarily by changes in host contact rates associated with breeding migrations to and from aquatic habitat. In contrast, seasonal changes in infection intensity were driven by temperature-induced changes in Bd growth rate. Using our model, we found that the maintenance potential of leopard frogs varied significantly throughout the year and that seasonal troughs in infection prevalence made it unlikely that leopard frogs were responsible for long-term Bd persistence in these seasonal amphibian communities, highlighting the importance of alternative pathogen reservoirs for Bd persistence. Our results have broad implications for management in seasonal host-pathogen systems, showing that seasonal changes in host and pathogen vital rates, rather than the depletion of susceptible hosts, can lead to troughs in pathogen prevalence and stochastic pathogen extirpation.

When Defenses Fail: Atelopus zeteki Skin Secretions Increase Growth of the Pathogen Batrachochytrium dendrobatidis.

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.

Host species is linked to pathogen genotype for the amphibian chytrid fungus (Batrachochytrium dendrobatidis).

Host-pathogen specificity can arise from certain selective environments mediated by both the host and pathogen. Therefore, understanding the degree to which host species identity is correlated with pathogen genotype can help reveal historical host-pathogen dynamics. One animal disease of particular concern is chytridiomycosis, typically caused by the global panzootic lineage of the amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd), termed Bd-GPL. This pathogen lineage has caused devastating declines in amphibian communities around the world. However, the site of origin for the common ancestor of modern Bd-GPL and the fine-scale transmission dynamics of this lineage have remained a mystery. This is especially the case in North America where Bd-GPL is widespread, but disease outbreaks occur sporadically. Herein, we use Bd genetic data collected throughout the United States from amphibian skin swabs and cultured isolate samples to investigate Bd genetic patterns. We highlight two case studies in Pennsylvania and Nevada where Bd-GPL genotypes are strongly correlated with host species identity. Specifically, in some localities bullfrogs (Rana catesbeiana) are infected with Bd-GPL lineages that are distinct from those infecting other sympatric amphibian species. Overall, we reveal a previously unknown association of Bd genotype with host species and identify the eastern United States as a Bd diversity hotspot and potential site of origin for Bd-GPL.

Thermal Performance Curves of Multiple Isolates of Batrachochytrium dendrobatidis, a Lethal Pathogen of Amphibians.

Emerging infectious disease is a key factor in the loss of amphibian diversity. In particular, the disease chytridiomycosis has caused severe declines around the world. The lethal fungal pathogen that causes chytridiomycosis, Batrachochytrium dendrobatidis (Bd), has affected amphibians in many different environments. One primary question for researchers grappling with disease-induced losses of amphibian biodiversity is what abiotic factors drive Bd pathogenicity in different environments. To study environmental influences on Bd pathogenicity, we quantified responses of Bd phenotypic traits (e.g., viability, zoospore densities, growth rates, and carrying capacities) over a range of environmental temperatures to generate thermal performance curves. We selected multiple Bd isolates that belong to a single genetic lineage but that were collected across a latitudinal gradient. For the population viability, we found that the isolates had similar thermal optima at 21°C, but there was considerable variation among the isolates in maximum viability at that temperature. Additionally, we found the densities of infectious zoospores varied among isolates across all temperatures. Our results suggest that temperatures across geographic point of origin (latitude) may explain some of the variation in Bd viability through vertical shifts in maximal performance. However, the same pattern was not evident for other reproductive parameters (zoospore densities, growth rates, fecundity), underscoring the importance of measuring multiple traits to understand variation in pathogen responses to environmental conditions. We suggest that variation among Bd genetic variants due to environmental factors may be an important determinant of disease dynamics for amphibians across a range of diverse environments.

Divergent regional evolutionary histories of a devastating global amphibian pathogen.

Emerging infectious diseases are a pressing threat to global biological diversity. Increased incidence and severity of novel pathogens underscores the need for methodological advances to understand pathogen emergence and spread. Here, we use genetic epidemiology to test, and challenge, key hypotheses about a devastating zoonotic disease impacting amphibians globally. Using an amplicon-based sequencing method and non-invasive samples we retrospectively explore the history of the fungal pathogen Batrachochytrium dendrobatidis (Bd) in two emblematic amphibian systems: the Sierra Nevada of California and Central Panama. The hypothesis in both regions is the hypervirulent Global Panzootic Lineage of Bd (BdGPL) was recently introduced and spread rapidly in a wave-like pattern. Our data challenge this hypothesis by demonstrating similar epizootic signatures can have radically different underlying evolutionary histories. In Central Panama, our genetic data confirm a recent and rapid pathogen spread. However, BdGPL in the Sierra Nevada has remarkable spatial structuring, high genetic diversity and a relatively older history inferred from time-dated phylogenies. Thus, this deadly pathogen lineage may have a longer history in some regions than assumed, providing insights into its origin and spread. Overall, our results highlight the importance of integrating observed wildlife die-offs with genetic data to more accurately reconstruct pathogen outbreaks.

Automated detection of frog calls and choruses by pulse repetition rate.

Anurans (frogs and toads) are among the most globally threatened taxonomic groups. Successful conservation of anurans will rely on improved data on the status and changes in local populations, particularly for rare and threatened species. Automated sensors, such as acoustic recorders, have the potential to provide such data by massively increasing the spatial and temporal scale of population sampling efforts. Analyzing such data sets will require robust and efficient tools that can automatically identify the presence of a species in audio recordings. Like bats and birds, many anuran species produce distinct vocalizations that can be captured by autonomous acoustic recorders and represent excellent candidates for automated recognition. However, in contrast to birds and bats, effective automated acoustic recognition tools for anurans are not yet widely available. An effective automated call-recognition method for anurans must be robust to the challenges of real-world field data and should not require extensive labeled data sets. We devised a vocalization identification tool that classifies anuran vocalizations in audio recordings based on their periodic structure: the repeat interval-based bioacoustic identification tool (RIBBIT). We applied RIBBIT to field recordings to study the boreal chorus frog (Pseudacris maculata) of temperate North American grasslands and the critically endangered variable harlequin frog (Atelopus varius) of tropical Central American rainforests. The tool accurately identified boreal chorus frogs, even when they vocalized in heavily overlapping choruses and identified variable harlequin frog vocalizations at a field site where it had been very rarely encountered in visual surveys. Using a few simple parameters, RIBBIT can detect any vocalization with a periodic structure, including those of many anurans, insects, birds, and mammals. We provide open-source implementations of RIBBIT in Python and R to support its use for other taxa and communities.

Los anuros (ranas y sapos) se encuentran dentro de los grupos taxonómicos más amenazados a nivel mundial. La conservación exitosa de los anuros dependerá de información mejorada sobre el estado y los cambios en las poblaciones locales, particularmente para las especies raras y amenazadas. Los sensores automatizados, como las grabadoras acústicas, tienen el potencial para proporcionar dicha información al incrementar masivamente la escala espacial y temporal de los esfuerzos de muestreo poblacional. El análisis de dicha información requerirá herramientas robustas y eficientes que puedan identificar automáticamente la presencia de una especie en las grabaciones de audio. Como las aves y los murciélagos, muchas especies de anuros producen vocalizaciones distintivas que pueden ser capturadas por las grabadoras acústicas autónomas y también son excelentes candidatas para el reconocimiento automatizado. Sin embargo, a diferencia de las aves y los murciélagos, todavía no se cuenta con una disponibilidad extensa de herramientas para el reconocimiento acústico automatizado de los anuros. Un método efectivo para el reconocimiento automatizado del canto de los anuros debe ser firme ante los retos de los datos reales de campo y no debería requerir conjuntos extensos de datos etiquetados. Diseñamos una herramienta de identificación de las vocalizaciones: la herramienta de identificación bioacústica basada en el intervalo de repetición (RIBBIT), el cual clasifica las vocalizaciones de los anuros en las grabaciones de audio con base en su estructura periódica. Aplicamos la RIBBIT a las grabaciones de campo para estudiar a dos especies: la rana coral boreal (Pseudacris maculata) de los pastizales templados de América del Norte y la rana arlequín variable (Atelopus varius), críticamente en peligro de extinción, de las selvas tropicales de América Central. Mostramos que RIBBIT puede identificar correctamente a las ranas corales boreales, incluso cuando vocalizan en coros con mucha superposición, y puede identificar las vocalizaciones de la rana arlequín variable en un sitio de campo en donde rara vez se le ha visto durante censos visuales. Mediante relativamente unos cuantos parámetros simples, RIBBIT puede detectar cualquier vocalización con una estructura periódica, incluyendo aquellas de muchos anuros, insectos, aves y mamíferos. Proporcionamos implementaciones de fuente abierta de RIBBIT en Python y en R para fomentar su uso para otros taxones y comunidades.

Whole exome sequencing identifies the potential for genetic rescue in iconic and critically endangered Panamanian harlequin frogs.

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 varius and Atelopus zeteki) are threatened with extinction due to the fungal pathogen Batrachochytrium dendrobatidis (Bd). Once thought to be nearly extirpated from Panamá, A. varius have recently been rediscovered in multiple localities across their historical range; however, A. zeteki are possibly extinct in the wild. By leveraging a unique collection of 186 Atelopus tissue samples collected before and after the Bd outbreak 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 the Atelopus genome. Using these genetic data, we evaluate the population genetic structure of historical A. varius and A. zeteki populations, describe changes in genetic diversity over time, assess the relationship between contemporary and historical individuals, and test the hypothesis that some A. varius populations have rapidly evolved to resist or tolerate Bd infection. We found a significant decrease in genetic diversity in contemporary (compared to historical) A. varius populations. We did not find strong evidence of directional allele frequency change or selection for Bd resistance 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 persisting A. varius populations 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 amphibian complement system and chytridiomycosis.

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 pathogen Batrachochytrium dendrobatidis (Bd), which is implicated unprecedented amphibian declines around the world. The impacts of Bd have 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 to Bd infection. Additionally, we propose future research directions that will increase our understanding of the amphibian complement system and other immune responses to Bd. Finally, we suggest how a deeper understanding of amphibian immunity could enhance the conservation and management of amphibian species that are threatened by chytridiomycosis.

Daily fluctuating temperatures decrease growth and reproduction rate of a lethal amphibian fungal pathogen in culture.

BACKGROUND: Emerging infectious diseases (EIDs) are contributing to species die-offs worldwide. We can better understand EIDs by using ecological approaches to study pathogen biology. For example, pathogens are exposed to variable temperatures across daily, seasonal, and annual scales. Exposure to temperature fluctuations may reduce pathogen growth and reproduction, which could affect pathogen virulence, transmission, and environmental persistence with implications for disease. We examined the effect of a variable thermal environment on reproductive life history traits of the fungal pathogen Batrachochytrium dendrobatidis (Bd). Bd causes chytridiomycosis, an emerging infectious disease of amphibians. As a pathogen of ectothermic hosts, Bd can be exposed to large temperature fluctuations in nature. To determine the effect of fluctuating temperatures on Bd growth and reproduction, we collected temperature data from breeding pools of the Yosemite toad (Anaxyrus canorus), a federally threatened species that is susceptible to chytridiomycosis. We cultured Bd under a daily fluctuating temperature regime that simulated Yosemite toad breeding pool temperatures and measured Bd growth, reproduction, fecundity, and viability. RESULTS: We observed decreased Bd growth and reproduction in a diurnally fluctuating thermal environment as compared to cultures grown at constant temperatures within the optimal Bd thermal range. We also found that Bd exhibits temperature-induced trade-offs under constant low and constant high temperature conditions. CONCLUSIONS: Our results provide novel insights on variable responses of Bd to dynamic thermal conditions and highlight the importance of incorporating realistic temperature fluctuations into investigations of pathogen ecology and EIDs.

Response to Comment on "Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity".

Lambert et al question our retrospective and holistic epidemiological assessment of the role of chytridiomycosis in amphibian declines. Their alternative assessment is narrow and provides an incomplete evaluation of evidence. Adopting this approach limits understanding of infectious disease impacts and hampers conservation efforts. We reaffirm that our study provides unambiguous evidence that chytridiomycosis has affected at least 501 amphibian species.

Cryptic diversity of a widespread global pathogen reveals expanded threats to amphibian conservation.

Biodiversity loss is one major outcome of human-mediated ecosystem disturbance. One way that humans have triggered wildlife declines is by transporting disease-causing agents to remote areas of the world. Amphibians have been hit particularly hard by disease due in part to a globally distributed pathogenic chytrid fungus (Batrachochytrium dendrobatidis [Bd]). Prior research has revealed important insights into the biology and distribution of Bd; however, there are still many outstanding questions in this system. Although we know that there are multiple divergent lineages of Bd that differ in pathogenicity, we know little about how these lineages are distributed around the world and where lineages may be coming into contact. Here, we implement a custom genotyping method for a global set of Bd samples. This method is optimized to amplify and sequence degraded DNA from noninvasive skin swab samples. We describe a divergent lineage of Bd, which we call BdASIA3, that appears to be widespread in Southeast Asia. This lineage co-occurs with the global panzootic lineage (BdGPL) in multiple localities. Additionally, we shed light on the global distribution of BdGPL and highlight the expanded range of another lineage, BdCAPE. Finally, we argue that more monitoring needs to take place where Bd lineages are coming into contact and where we know little about Bd lineage diversity. Monitoring need not use expensive or difficult field techniques but can use archived swab samples to further explore the history-and predict the future impacts-of this devastating pathogen.

Quantifying Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans Viability.

The disease chytridiomycosis is responsible for global amphibian declines. Chytridiomycosis is caused by Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), fungal pathogens with stationary and transmissible life stages. Establishing methods that quantify growth and survival of both life stages can facilitate research on the pathophysiology and disease ecology of these pathogens. We tested the efficacy of the MTT assay, a colorimetric test of cell viability, and found it to be a reliable method for quantifying the viability of Bd and Bsal stationary life stages. This method can provide insights into these pathogens' growth and reproduction to improve our understanding of chytridiomycosis.

Disease and the Drying Pond: Examining Possible Links among Drought, Immune Function, and Disease Development in Amphibians.

Drought can heavily impact aquatic ecosystems. For amphibian species that rely on water availability for larval development, drought can have direct and indirect effects on larval survival and postmetamorphic fitness. Some amphibian species can accelerate the timing of metamorphosis to escape drying habitats through developmental plasticity. However, trade-offs associated with premature metamorphosis, such as reduced body size and altered immune function in the recently metamorphosed individual, may have downstream effects on susceptibility to disease. Here, we review the physiological mechanisms driving patterns in larval amphibian development under low water conditions. Specifically, we discuss drought-induced accelerated metamorphosis and how it may alter immune function, predisposing juvenile amphibians to infectious disease. In addition, we consider how these physiological and immunological adjustments could play out in a lethal disease system, amphibian chytridiomycosis. Last, we propose avenues for future research that adopt an ecoimmunological approach to evaluate the combined threats of drought and disease for amphibian populations.

Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity.

Anthropogenic trade and development have broken down dispersal barriers, facilitating the spread of diseases that threaten Earth's biodiversity. We present a global, quantitative assessment of the amphibian chytridiomycosis panzootic, one of the most impactful examples of disease spread, and demonstrate its role in the decline of at least 501 amphibian species over the past half-century, including 90 presumed extinctions. The effects of chytridiomycosis have been greatest in large-bodied, range-restricted anurans in wet climates in the Americas and Australia. Declines peaked in the 1980s, and only 12% of declined species show signs of recovery, whereas 39% are experiencing ongoing decline. There is risk of further chytridiomycosis outbreaks in new areas. The chytridiomycosis panzootic represents the greatest recorded loss of biodiversity attributable to a disease.

Non-lethal isolation of the fungal pathogen Batrachochytrium dendrobatidis (Bd) from amphibians.

The ability to isolate and purify pathogens is important for the study of infectious disease. A protocol for isolating Batrachochytrium dendrobatidis (Bd), a lethal pathogen of amphibians, has been available for over a decade, but the method relies on sacrificing infected animals. We validated a non-lethal protocol for Bd isolation that uses biopsy punches from toe webbing to collect skin samples from live amphibians in remote field locations. We successfully isolated Bd from the Cascades frog Rana cascadae and found a positive association between Bd infection and probability of Bd growth in culture. Recapture rates of sampled animals suggest that our isolation protocol did not affect frog survival. The ability to collect isolates from live animals will facilitate investigations of the biology of Bd and enhance amphibian conservation efforts.

Opening the file drawer: Unexpected insights from a chytrid infection experiment.

Infection experiments are critical for understanding wildlife disease dynamics. Although infection experiments are typically designed to reduce complexity, disease outcomes still result from complex interactions between host, pathogen, and environmental factors. Cryptic variation across factors can lead to decreased repeatability of infection experiments within and between research groups and hinder research progress. Furthermore, studies with unexpected results are often relegated to the "file drawer" and potential insights gained from these experimental outcomes are lost. Here, we report unexpected results from an infection experiment studying the response of two differentially-susceptible but related frogs (American Bullfrog Rana catesbeiana and the Mountain yellow-legged frog Rana muscosa) to the amphibian-killing chytrid fungus (Batrachochytrium dendrobatidis, Bd). Despite well-documented differences in susceptibility between species, we found no evidence for antibody-mediated immune response and no Bd-related mortality in either species. Additionally, during the study, the sham-inoculated R. catesbeiana control group became unexpectedly Bd-positive. We used a custom genotyping assay to demonstrate that the aberrantly-infected R. catesbeiana carried a Bd genotype distinct from the inoculation genotype. Thus R. catesbeiana individuals were acquired with low-intensity infections that could not be detected with qPCR. In the Bd-inoculated R. catesbeiana treatment group, the inoculated genotype appeared to out-compete the cryptic infection. Thus, our results provide insight into Bd coinfection dynamics, a phenomenon that is increasingly relevant as different pathogen strains are moved around the globe. Our experiment highlights how unexpected experimental outcomes can serve as both cautionary tales and opportunities to explore unanswered research questions. We use our results as a case study to highlight common sources of anomalous results for infection experiments. We argue that understanding these factors will aid researchers in the design, execution, and interpretation of experiments to understand wildlife disease processes.