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Chytridiomycosis, a cutaneous fungal disease caused by two related fungi, Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), is a major cause of amphibian population declines worldwide. Bd has a broad distribution, covering every continent except for Antarctica. Meanwhile, Bsal is currently restricted to Europe and Asia. Treatment options for both Bd and Bsal remain a major area of concern for wild populations. Recently, implants for long-term, parenteral antifungal drug administration have shown some promise in fungal diseases in wildlife. We investigated the safety and efficacy of an intracoelomic implant of the antifungal drug terbinafine in greater sirens exposed to Bd. While the efficacy results were inconclusive, the implants did not cause harm and led to detectable levels of terbinafine in plasma. The use of such implants in amphibians remains an important area for further investigation. In a separate study, we explored the use of a probiotic bacteria and addition of a known secondary bacterial pathogen in Bsal-infected animals. The objectives of this study were to determine the safety and efficacy of using probiotic bacteria to prevent and treat Bsal chytridiomycosis, and to evaluate bacteremia and septicemia as components of morbidity and mortality in animals that succumb to disease. While the probiotic treatment did not impact mortality rates, there was evidence of bacterial infiltration into the blood and organs in Bsal-exposed animals. Finally, while there is an ever-growing body of research elucidating the acute impacts of Bsal, sublethal effects on population dynamics must be considered as well. We used a two-part study design to investigate the impacts of Bsal exposure on individual reproductive potential, and on reproductive success under breeding conditions. This study provided evidence for terminal investment, increased reproductive effort in the face of pathogen exposure, in eastern newts exposed to Bsal. Overall, the findings of this research provide more information about new avenues for treatment for chytridiomycosis and their potential drawbacks, and expand on the existing knowledge of Bsal pathogenesis. These findings will provide a foundation for future amphibian disease research, informing mitigation efforts for this disease in managed and free-ranging amphibian populations.

Chytridiomycosis is an emerging infectious disease that is significantly reducing global amphibian populations. The disease is caused by Batrachochytrium dendrobatidis (Bd), a fungus that lethally modifies amphibian skin. Recent research has suggested that the cutaneous microbiome of individual amphibians may play a role in susceptibility to the pathogen. In this study, twelve cave salamanders (Eurycea lucifuga) were collected. Cutaneous bacteria from each salamander were isolated and identified using Sanger Sequencing. Additionally, a Bd-challenge assay was performed to determine each isolate’s antifungal activity. Results indicated many microbial isolates possessed inhibitory capabilities against Bd, which may contribute to the cave salamander’s lack of population decline due to chytridiomycosis. This research may contribute to an increased understanding of potential defenses against Bd and ultimately to the development of a treatment to mitigate the chytridiomycosis epidemic.

Chytridiomycosis is a life-threatening disease which infects amphibian species worldwide. Chytrid fungi Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans have been found to be the primary causes of chytridiomycosis. In past research, it has been found that the skin microbiome of certain amphibian species could have fungal inhibition properties, reducing the effects of the chytrid fungi. In this study, 15 eastern red-spotted newts (Notophthalmus viridescens) were collected, to determine if cutaneous bacteria of the newts secrete antifungal compounds. Pure cultures of each bacterium morphotype were produced from mass culture plates, with 188 morphotypes being isolated. The antifungal properties of the skin microbiome were obtained through a Bd assay, which was completed to find comparisons of which bacteria inhibited Bd.

Emergent infectious diseases are a major driver of the accelerated rates of biodiversity loss that are being documented around the world. Global losses of amphibians provide evidence of this, especially those associated with chytridiomycosis, a lethal skin disease caused by the fungus Batrachochytrium dendrobatidis (Bd). Amphibian skin can harbor diverse bacterial communities that, in some cases, can inhibit the growth of Bd. Thus, there is interest in using skin bacteria as probiotics to mitigate Bd infections in amphibians. However, experiments testing this conservation approach have yielded mixed results, suggesting a lack of understanding about the ecology of these microbial communities. My dissertation research aimed to assess basic ecological questions in microbial ecology and to contribute to the development of probiotics using amphibian skin bacteria. First, to assess whether environmental conditions influence the function of amphibian skin bacterial communities, I conducted a field survey across low and high elevation populations of an amphibian host to assess their skin bacterial communities and metabolite profiles. I found that similar bacterial communities produced different metabolites at different locations, implying a potential functional plasticity. Second, since culturing is critical for characterizing bacteria, I aimed to identify the culture media (low vs high nutrient concentration) that recovers the most representative fraction of the amphibian skin bacterial community. I found that media with low nutrient concentrations cultured a higher diversity and recovered a more representative fraction of the diversity occurring on amphibian skin. I also determined that sampling more individuals is critical to maximize culture collections. Third, I assessed the diversity of the amphibian skin fungal community in relation to Bd infection across eight amphibian species. I determined that amphibian species was the most important predictor of fungal diversity and community structure, and that Bd infection did not have a strong impact. My dissertation highlights the importance of environmental conditions in the function of amphibian skin bacteria, expands our knowledge of the understudied fungal component of the amphibian skin microbiome, and complements current efforts in amphibian conservation.

Amphibian populations are declining globally at historically rapid rates, and while a multitude of factors have contributed to amphibian population declines, emerging infectious diseases, such as chytridiomycosis and ranavirus have been linked to a large proportion of the reported amphibian mass mortality events. Distribution and infection prevalence data for chytridiomycosis and ranavirus are lacking, and effective surveillance is crucial. This project aims to describe anuran richness, relative abundance, habitat occupancy, and community structure and to identify the chytridiomycosis and ranavirus infection prevalence rates of the anuran population at Dairymen’s Inc. Wood frogs, spring peepers, boreal chorus frogs, northern leopard frogs, gray treefrogs, American toads, green frogs, mink frogs, and American bullfrogs were the nine anuran species that were recorded in Vilas County, Wisconsin. The infection prevalence rates of Bd and frog virus 3 were 11 ± 5.6 with 95% C.I. and 0%, respectively. This study is the first to report the presence of Bd-infected anurans in Vilas County, however further research is needed to provide reliable frog virus 3 infection rates in this county.

Batrachochytrium dendrobatidis (Bd), a fungal pathogen that causes chytridiomycosis in amphibians, has infected >500 species and caused declines and extinctions in >200 species. Recently, a second deadly fungal pathogen that also causes chytridiomycosis, Batrachochytrium salamandrivorans (Bsal) was discovered. The presence of these lethal pathogens in international trade threatens amphibian diversity. In this dissertation, I use a novel modeling approach to predict disease risk from Bd and/or Bsal to amphibian populations in North America and Asia by incorporating pathogen habitat suitability, host availability, the potential presence of an invasive carrier species, and pathogen invasion history. First I identify Bsal threat to North American salamanders to be greatest in the Southeast US, the West Coast, and highlands of Mexico. I then investigate the compounded risk of Bd and Bsal in North America and find highest relative risk in those same areas and in the Sierra Nevada Mountains and the northern Rocky Mountains. However, when I incorporate the historical context of Bd into the model by treating the eastern US as the native range of Bd, I find invasive Bd risk to be highest in the mountain ranges of the western US and Mexico, which could influence how amphibians in these areas respond to Bsal. I also uncover a pattern that suggests the translocation of the American bullfrog (Rana catesbeiana) to western North America may have facilitated Bd spread. Last, I investigate the threat of Bd in Asia and find that mountain forests in China, Southeast Asia, and the Pacific Islands have the highest relative Bd risk, and the potential presence of R. catesbeiana and amphibian richness may have a strong influence on Bd risk. I also predict that climate change may lead to a 3-23% decrease in total area of Bd risk in Asia by 2070. These findings provide helpful guidance for conservation planning and management by further highlighting the role of R. catesbeiana in Bd spread and identifying areas at highest risk to prioritize for surveillance, monitoring, and species susceptibility studies needed to enhance our understanding of the pathogen invasion history, disease dynamics, and potential interactions of Bd and Bsal.

Fungal diseases have recently caused severe multi-species die-off events in wildlife. In particular, chytridiomycosis, a skin disease caused by the chytrid pathogen Batrachochytrium dendrobatidis (Bd), has provoked extinctions and severe declines in many amphibian populations across the globe. Here, I investigate the combination of environmental, ecological, and genetic factors that determine a species' ability to persist despite infection. In Chapter 1, I review recent literature-including experimental Bd challenges and longitudinal studies-to illustrate how integrating genomic approaches into current research, both for hosts and pathogens, will improve our understanding of factors promoting disease outcome. In Chapter 2, I apply a traitbased approach to investigate how species-specific attributes control the degree of exposure, probability of infection, and pathogen intensity, during and after Bd emergence in a naïve amphibian community at El Copé, Panamá. I find a signature of phylogenetic history in disease responses associated with increased pathogen exposure via shared life history traits such as reproductive mode and habitat use. I identify key hosts at the time of Bd emergence, characterized by a high probability of infection, pathogen load, and abundance. In Chapter 3, I characterize temporal turnover of skin microbial communities and their relation to Bd prevalence and infection intensity in three natural populations of the common coqui (Eleutherodactylus coqui) in Puerto Rico that persist with enzootic Bd infections. I find that natural fluctuations in temperature and precipitation between months can create opportunities for microbial replacement, potentially attenuating pathogen transmission, and thus contributing to host persistence in E. coqui populations. In Chapter 4, I experimentally infect E. coqui frogs under conditions known to confer the survival advantage to the host, or alternatively to the pathogen, by performing inoculations in mesocosms over two seasons. I demonstrate that natural fluctuations in environmental conditions shift the survival advantage among the pathogen, the host, and its microbial community. In summary, my results advance our understanding of multi-host single-pathogen systems by quantifying the relative roles of host heterogeneity and environmental controls at different disease stages after pathogen emergence.

Environmental and anthropogenic stressors negatively affect amphibians in a variety of ways, often increasing their vulnerability to pathogen infection and mortality. Sampling for the pathogenic fungus Batrachochytrium dendrobatidis (Bd) was conducted in order to: 1) determine the presence of chytrid infection in stream-associated plethodontid salamanders of southeastern Kentucky, and 2) evaluate differences in infection intensity between salamanders residing in intact forest streams, timber-harvested streams, and surface-mined streams. During 14 sampling sessions occurring between March, April and May of 2013, DNA samples from 306 individual salamanders within 8 species from the family Plethodontidae were collected; additional amphibians (i.e. frogs, newts) were opportunistically sampled when encountered. Approximately 2.1% of the salamanders and 50% of the frogs sampled from intact streams, 2.3% of the salamanders and 80% of the frogs sampled from the harvested streams, and none of the salamanders and 100% of the frogs sampled from the mined streams tested positive for Bd. No significant differences in occurrence of Bd or infection intensity were detected between the treatment sites (x2 = 0.59; p-value = 0.75), or between individuals of a species between different treatments (see tables). These findings are the first to demonstrate that Batrachochytrium dendrobatidis is present in amphibians of eastern Kentucky.

Recently emerged diseases in natural populations present novel problems for biodiversity conservation. Integrated approaches are needed to better understand disease-related threats, to mitigate these threats, and to assist population recovery. My dissertation research confronts the global amphibian biodiversity crisis. The recently emerged infectious disease chytridiomycosis, caused by the chytrid fungal pathogen Batrachochytrium dendrobatidis (Bd), infects hundreds of species around the world and is a major contributor to amphibian population declines. I use a multi-faceted approach to address critical issues of disease-caused amphibian declines. In this dissertation, I implement a novel methodology to gain insights on variability of host response to Bd (Chapter 2). Next, I analyze the spatial genetic structure of post-decline populations to aid conservation (Chapter 3). Finally, I examine the genetic underpinnings of pathogen attenuation – loss of virulence – using genomic sequencing (Chapter 4).First, I examine differential disease progression and host response in two related species (Chapter 2). Determining how different hosts respond to infection by a widespread pathogen is essential for understanding - and ultimately limiting - the devastating effects of emerging infectious diseases. Previous work demonstrated that susceptibility to chytridiomycosis is variable among species, but the mechanism(s) that underlie the difference between winners and losers remains a mystery. I used an integrative approach to analyze host response to infection in two related toad species that are thought to differ in susceptibility: the invasive Cane Toad (Bufo marinus) and the threatened Boreal Toad (Bufo boreas). With my results, I characterize the nature of differential susceptibility and compare host response using genome-wide gene expression analysis. The susceptible B. boreas exhibited high pathogen loads, loss in body weight, severe changes in the epidermis, and dramatic transcriptomic changes without a robust immune response. Conversely, the resistant B. marinus exhibited low pathogen loads, stable body weight, only mild disruption of the epidermis and relatively few changes in transcriptomic profile. Together our results show intrinsic differences in host response between related species, which are likely to be an important factor in explaining variation in response to a deadly emerging pathogen in wild populations.Second, I conducted a conservation genetics study of an endangered amphibian species in Yosemite National Park (Chapter 3). The most striking example of chytrid-associated population declines in North America is the mountain yellow-legged frog (Rana muscosa and Rana sierrae), including populations in Yosemite National Park. A clear picture of genetic structure and demography of remaining R. sierrae populations is critical to short-term management and conservation. I conducted a study to describe phylogeographic patterns of R. sierrae in Yosemite NP in collaboration with ecologists and park biologists. I utilized a recently developed method for multilocus amplicon sequencing that allows sequence data collection from a vast collection of swabs that contain low quantities of input DNA. My analysis of population genetic structure suggests that three genetic clusters occur in Yosemite NP with a significant signature of isolation by distance. This analysis of population genetic structure adds a critical component to the population recovery plan and will assist management strategies such as translocations, reintroductions, and monitoring.Third, I investigated the genomic changes associated with virulence attenuation in a lab-evolved Bd strain (Chapter 4). Despite recent efforts to characterize the diversity of Bd lineages, there are many questions that remain about the genetic underpinnings of pathogenicity. In a collaborative study, I take advantage of an accidental case of virulence reduction in a Bd strain that was lab passaged over many generations. I analyzed the genomic changes in strain samples cryo-archived before and after virulence attenuation. I found multiple patterns that may be linked to attenuation including decreases in chromosome copy number and mutations in putative virulence genes. These results contribute to the growing body of knowledge of how changes in pathogen genomes occur within a relatively short period of time, which has major implications for host-pathogen dynamics in natural systems.In conclusion, my dissertation provides important new contributions to the study of host-pathogen interactions with specific relevance to the fields of disease biology, conservation genetics, and pathogen evolution. Integrating genomic tools into a variety of experimental methods enabled not only valuable novel insights, but also opened up many new opportunities for further exploration of disease-caused amphibian declines using the generated genomic and computational resources.

Amphibian populations have undergone unprecedented declines in recent decades. Many of these declines are due to the spread of the cutaneous fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis. The Panamanian golden frog (Atelopus zeteki) has not been seen in the wild since Bd spread through western Panama. In response to initial declines, golden frogs were collected from wild populations and placed in captive colonies with the goal of future reintroductions. An understanding of this species' natural defense mechanisms against Bd is needed for reintroduction to be successful. Previous studies indicate that cutaneous bacteria are an important defense mechanism for some amphibians and applying antifungal bacteria to the skin of Bd-susceptible amphibians (probiotic therapy) can prevent chytridiomycosis. Therefore, the goals of my dissertation were to characterize the bacterial community of A. zeteki and determine if probiotic therapy could be used to prevent chytridiomycosis in this species. I initially characterized the bacterial community of wild and captive golden frogs using samples collected prior to the initial declines and after approximately eight years in captivity. I found that the community structure of the microbiota was significantly different between wild and captive frogs; however, the offspring of the original captive frogs still shared 70% of their microbial community with wild frogs. Then, I characterized the Bd-inhibitory properties of 484 bacteria isolated from 11 species of free-living Panamanian amphibians. I found a large proportion of bacteria (75.2%) had the ability to inhibit Bd and this trait was widely distributed among bacterial taxa, although there was also significant variation within bacterial genera in their ability to inhibit Bd growth. I then experimentally tested the ability of four of these isolates to prevent chytridiomycosis in captive golden frogs. None of them successfully prevented infection; however, there were several frogs that cleared infection and this was correlated with composition of the bacteria initially present on their skin. Overall these results demonstrate that the structure of microbial communities of A. zeteki are important to host health and building on this might provide the best hope for reintroducing this iconic species back to its native habitat.

Symbiotic microbial communities (microbiomes) are ubiquitous inhabitants of multicellular organisms and are increasingly recognized to play roles in host health and development. The skin-associated microbiome of amphibians may affect resistance to the potentially lethal disease, chytridiomycosis, which is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). The Sierra Nevada yellow-legged frog (Rana sierrae) is threatened with extinction, and Bd is a major driver of declines in these frogs. However, some R. sierrae populations are able to co-exist with the pathogen, persisting despite Bd infection. Understanding why some R. sierrae populations persist with Bd may provide clues for how to minimize the impact of Bd. However, while preliminary studies highlight the possibility that symbiotic bacteria affect disease resistance, very little is known about natural diversity, stability, or function of the amphibian skin microbiome. I tested the hypothesis that differences in skin-associated bacterial communities (referred to simply as the microbiome for brevity) can account for differences in the outcome of R. sierrae infection, i.e. population extinction versus persistence. I surveyed microbiomes from multiple R. sierrae populations and showed that populations that persisted or declined to extinction due to Bd indeed harbored different skin bacterial communities, consistent with a protective effect of bacteria in persistent populations. At the same time I found evidence that Bd infection itself may drive variation in the microbiome, both at the local scale (among frogs within a given population), and landscape scale (among populations). Correlations between microbiome composition and Bd infection severity among wild frogs may be the result of either microbiome-mediated resistance to disease, in which bacterial assemblages control the degree of Bd infection, or, alternatively could result from Bd-induced disturbance causing changes in the microbiome. Using a laboratory experiment, I demonstrated that Bd infection alters the R. sierrae microbiome, while I found no direct evidence that the microbiome limits the severity of Bd infection, at least within the range of microbiome variability represented in this study. In addition, some of the bacterial taxa that were sensitive to Bd infection might have been predicted to inhibit the growth of Bd based on results in other study systems. These results highlight the importance of considering microbiome stability when assessing the potential for the microbiome to limit pathogen growth. Understanding why microbial communities vary among individuals or populations is an important step toward understanding the diversity and function of the microbiome. I present an experiment that tested the effects of natural variation in the aquatic environment and genetic variation among R. sierrae individuals in shaping the skin microbiome. By integrating controlled experiments with field surveys at multiple spatial scales, these studies reveal new insights into amphibian skin microbiome assembly, function, and sensitivity in the face of infection by an important fungal pathogen, and show the importance of these characteristics of the microbiome in wild amphibian populations.

Declines in Rana muscosa within the last ten years have been so severe that this species is threatened with extinction. The fungal pathogen Batrachochytrium dendrobatidis (Bd) has emerged as a major contributor to declines in the already small and fragmented populations of this species. Adults, juveniles and larvae continue to be found within affected populations from year to year, albeit in smaller numbers. The work presented within this dissertation asked whether immune responses could be enhanced against Bd and examined the effect of physiological factors on Bd prevalence and related mortality in R. muscosa in lab and field caught animals. I studied the possibility of protecting individuals against Bd using immunizations and examined the beneficial effects of sub-clinical Bd infections and survival between life stages. My work also included examining interactions between seasonal changes in endocrine function related to reproduction and prevalence of Bd. In laboratory studies I found that immunizations with killed Bd prior to live exposure in juvenile R. muscosa frogs was not protective. No difference in how quickly animals became infected nor in mortality between groups, was found. However, exposure to Bd during the larval stage, at sub-infectious doses, was protective in those same animals when re-exposed to Bd as sub-adults. This work provides two points for further investigation; infectious dose may be a determining factor in the pathogenesis of Bd and protective responses developed in larvae that are capable of resolving or building a memory response to infection with Bd. Within field populations of R. muscosa I detected seasonal fluctuations in Bd prevalence and the hormones involved in the stress and reproduction, corticosterone and testosterone. I found no association between either hormone and Bd prevalence within and between populations This finding refutes the immunocompetence handicap hypothesis claim that these two hormones are immunosuppressive in all vertebrates and that disease and breeding are positive correlated with each other The strongest predictors of infection prevalence and intensity were month and site of collection or population sampled. This supports work showing Bd is constrained by temperature within the environment but further suggests that factors unique to sites are important in modulating disease.

The conservation of endangered species is best achieved by studies that consider both ecological and evolutionary aspects of population viability. Integrative research of this type is especially important for amphibian species as they have been plagued by rapid, global declines and extinctions in recent decades. The aims of this dissertation were to contribute to the conservation of two such declining amphibians, the Panamanian golden frogs, Atelopus varius and A. zeteki, as well as demonstrate the combined utilities of morphological, ecological, and genetic analyses in providing an integrative framework for informing wildlife conservation initiatives. The results of this research engender several important considerations for the conservation of these critically endangered and culturally important amphibians. In my first study, multivariate phenotypic and mitochondrial DNA analyses were used to verify that previously defined management units are supported by concordant patterns of variation among populations as well as to identify phenotypic traits that may be associated with adaptive divergence. The second study examined the contributions of alternative landscape factors to patterns of genetic variation among golden frog populations, highlighting the importance of maintaining the integrity of low slope areas, such as riparian habitat corridors and mountain ridges, for gene flow. The third and final study demonstrated the importance of genetic and phenotypic variability in allowing golden frog populations to adapt to environmental change by examining the relationship between thermoregulation behavior and susceptibility to a fungal epidemic. Here I demonstrated that the behavioral fever response mounted by one golden frog population is effective in reducing the odds of infection during a chytridiomycosis epidemic. I concluded by summarizing the implications of this work for the management of Panamanian golden frogs and considering the applicability of these findings to the conservation of other endangered species of Atelopus.