This thesis consists of three sections, all of which are linked to the ecology of infectious disease and the decline of amphibians caused by chytridiomycosis. This thesis represents a detailed analysis of factors that allow the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), to persist in the Peruvian Andes. Collectively, these three sections elaborate on the current knowledge of the disease, how it persists in an area, as well as recommendations for future disease mitigation. Chapter one is a review of environmental and biological factors that contribute to the persistence of Bd. Here we address the use of biotic and abiotic reservoirs by the pathogen that allow Bd to persist. Biological reservoirs include both amphibian and non-amphibian hosts, thus allowing Bd to persist and proliferate when susceptible host densities recover. More than 40% of all amphibian populations are in decline and more than a third are at risk of extinction making it important to develop conservation measures for endangered and diminishing biodiversity. By understanding disease dynamics between reservoirs and other susceptible hosts, mitigation strategies can be developed to save amphibian populations from loss of genetic diversity or extinction. Chapter two addresses the current distribution and disease status of Bd in the Kosñipata Valley in southeastern Peru. We investigated the post-epizootic distribution and prevalence of Bd by examining trends in Bd prevalence across multiple sample periods. We modeled infection prevalence using logistic regression as a function of species, sample period, reproductive mode, life stage, and elevation and their two-way interactions. The most parsimonious model of prevalence contained two-way interactions between sample period and elevation, and reproductive mode and life stage. Overall we observed that prevalence varied over time; prevalence was higher in 2012-2015 than it was in 2008-2009. The interaction between elevation and sample period revealed that while prevalence decreased with elevation during the wet season, it generally increased with elevation during the dry season. We concluded that the prevalence of Bd in the Kosñipata Valley appears to have stabilized over time, indicating enzootic Bd. Long-term monitoring of infection in hosts is important because temporal patterns in prevalence and infection intensity are associated with changes in population abundance which could lead to future changes in the disease state. Chapter three examines Hypsiboas gladiator as a proposed reservoir species for Bd. We conducted field surveys to determined prevalence and calculated Bd loads across life stages, and calculated zoospore shedding rates of H. gladiator and sympatric species. Results showed that H. gladiator had the highest prevalence and Bd load, followed by Pristimantis toftae and then P. platydactylus. We tried to determine if prevalence and Bd load varied with distance from streams but found no direct evidence to support our claims. We also conducted susceptibility trials of H. gladiator to Bd. Results from our susceptibility trials are inconclusive. Shedding rates were higher in 2014 than 2013 for all species and varied by year. At this time we cannot determine if H. gladiator is a reservoir species for Bd.

No description available.

Chytridiomycosis is a fungal skin disease in amphibians that is primarily caused by Batrachochytrium dendrobatidis (Bd). We analyzed whole genome sequences of 40 bacterial isolates that had been previously cultured from the skin of four amphibian species from Virginia, USA, and tested for their ability to inhibit Bd growth via an in vitro challenge assay. These 40 isolates spanned 11 families and 13 genera. The aim of this study was to identify genomic differences among the amphibian skin bacterial isolates and generate hypotheses about possible differences that could contribute to variation in their ability to inhibit the growth of Bd. We identified sixty-five gene families that were present in all 40 isolates. We also looked for the presence of biosynthetic gene clusters. While this set of isolates contained a wide variety of biosynthetic gene clusters, the two most abundant clusters with potential anti-fungal activity were siderophores (N=17) and Type III polyketide synthases (N=20). We then analyzed the isolates belonging to the phylum Proteobacteria in more detail. We identified 197 gene families that were present in all 22 Proteobacteria. We examined various subsets of the Proteobacteria for genes for specific compounds with known activity against fungi, including chitinase and violacein. We identified a difference in the number, as well as amino acid sequences, of predicted chitinases found in two isolates belonging to the genus Agrobacterium that varied in their inhibition of Bd. After examining the annotated genomes, we identified a predicted chitinase in a Sphingomonas isolate that inhibited the growth of Bd that was absent from the five Sphingomonas isolates that did not inhibit Bd growth. The genes vioA, vioB, vioC, vioD and vioE are necessary to produce violacein, a compound which inhibits the growth of Bd. Differences in these genes were identified in three out of the four Janthinobacterium isolates. Of these three isolates, two showed strong inhibition of Bd growth, while the third inhibited Bd growth to a lesser extent. Using comparative genomics, we generated several testable hypotheses about differences among bacterial isolates that could contribute to variation in ability to inhibit Bd growth. Further work is necessary to test the various mechanisms utilized by amphibian skin bacterial isolates to inhibit Bd. / Master of Science / Many amphibian population declines around the world have been caused by chytridiomycosis, a skin disease. This disease is caused by the fungus Batrachochytrium dendrobatidis (Bd). The skin of amphibians is also home to many bacteria that can provide important functions for the amphibian host, like preventing infection by Bd. To understand how these bacteria might provide protection, we examined the entire genomes of 40 bacterial isolates that reside on the skin of four amphibian species from Virginia, USA. These bacteria were previously tested for their ability to prevent Bd growth and 40 of them were chosen for sequencing based on selecting closely related isolates that varied in their ability to inhibit Bd growth. This allowed us to compare their genomes and generate hypotheses about possible genomic differences that could contribute to the variation in Bd growth inhibition. We identified sixty-five gene families that were present in all 40 bacteria. We also looked for sets of genes (biosynthetic gene clusters) that are known to produce secondary metabolites, which are compounds that can include antifungals. The two most abundant clusters we identified that had the potential to produce compounds that inhibit fungal growth were siderophores and Type III polyketide synthases. We then looked for genes that were not part of biosynthetic gene clusters that could produce specific compounds that can inhibit Bd growth, such as chitinase and violacein. We found variation in chitinase genes in several isolates that seemed to be associated with the ability to inhibit Bd growth. In addition, there were some differences in violacein genes that should be examined more in future studies. Overall, we suggest that using comparative genomic approaches can be valuable for identifying key bacterial functions in the microbiome.

Depuis plusieurs décennies, un déclin important de la biodiversité est observé à l'échelle mondiale. Les amphibiens constituent aujourd'hui le groupe le plus vulnérable sur la planète. Près d'un tiers des espèces recensées dans le monde est à ce jour menacé d'extinction. Dans le cadre de ce doctorat, nous nous sommes intéressés dans un premier temps à l'émergence d'une maladie infectieuse des amphibiens, la chytridiomycose, provoquée par le champignon pathogène Batrachochytrium dendrobatidis (Bd). Nous avons étudié la répartition actuelle de ce champignon en France, démontré son impact sur la batrachofaune locale et mis en évidence le rôle de la Grenouille taureau (Lithobates catesbeianus) comme vecteur de transmission du pathogène. Afin de limiter la dissémination de Bd, nous avons également élaboré deux protocoles d'hygiène à mettre en oeuvre lors d'interventions sur le terrain ou lors d'utilisations d'amphibiens en laboratoire. Dans une seconde partie, nous avons développé une nouvelle méthode d'inventaire de la biodiversité basée sur la détection de l'ADN environnemental (ADNe). Nous avons mis en évidence que la persistance de l'ADNe dans un écosystème d'eau douce était d'environ 15 jours et que cette méthode innovante permettait d'améliorer la détection d'espèces exotiques envahissantes, comme la Grenouille taureau. Nous avons ensuite développé cette approche pour le suivi d'autres groupes taxonomiques (poissons, macro-invertébrés, chiroptères, etc.), dans des milieux différents et en utilisant notamment les technologies de séquençage nouvelle génération. / Since several decades, a significant decline in biodiversity is observed worldwide. Amphibians are now the most vulnerable group on the planet. Nearly a third of known species in the world is today threatened of extinction. Among many causes, diseases appear as an emerging threat worldwide. As part of this PhD, we were interested at first to the emergence of an infectious disease of amphibians, chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). We studied the current distribution of this fungus mostly in France, showed the impact on local batrachofauna and highlighted the role of the Bullfrog (Lithobates catesbeianus) as a vector for transmission of the pathogen. To limit the spread of Bd, we also developed two hygiene protocols to implement during field trips or use of amphibians in the laboratory. In the second part of this thesis, we have developed a new method of biodiversity inventory based on the detection of environmental DNA (eDNA). We have shown that the persistence of vertebrates (fish and amphibian) eDNA in freshwater ecosystems was about 15 days and that this innovative method greatly improves the detection of invasive alien species, such as Bullfrog. We are then developed this approach for monitoring other taxonomic groups (fish, macro-invertebrates, bats, etc...), in various environments, taking advantage of bio-technological developments such as next generation DNA sequencing.

Orientador: Luís Felipe de Toledo Ramos Pereira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T11:16:08Z (GMT). No. of bitstreams: 1 Oliveira_IgorSoaresde_D.pdf: 4104401 bytes, checksum: 76046f01be0657270cbc44223cd762b9 (MD5) Previous issue date: 2015 / Resumo: As mudanças climáticas possuem potencial para alterar o funcionamento dos ecossistemas através da extinção de espécies e das conexões entre a biota e o ambiente, alterando os padrões aos quais estamos acostumados e exigindo adaptação. As alterações climáticas terão como uma de suas consequências a elevação oceânica, que pode redesenhar as costas de todos os continentes terrestres e afetar a biota associada. Nesse contexto, sobressaem-se os anfíbios como um grupo sensível que sofre diversas pressões e apresenta declínios populacionais, se destacando como vertebrados mais ameaçados da atualidade, com diversas espécies pouco conhecidas. Sendo assim, nesse trabalho analisamos múltiplos efeitos que dificultam a conservação dos anfíbios. Avaliamos os potenciais efeitos das mudanças climáticas e da elevação oceânica sobre os anfíbios com distribuição costeira. Também desenvolvemos um índice para classificar espécies com dados insuficientes com a finalidade de oferecer uma alternativa para a busca de informações para esses taxa e mudar sua classificação atual. Por fim, analisamos a disponibilidade histórica de clima adequado para uma linhagem endêmica do fungo quitrídio em busca de padrões que pudessem explicar sua distribuição e raridade atuais. Utilizamos ferramentas como modelos de distribuição de espécies e sistema de informação geográfica em busca de respostas às nossas questões. Nossos resultados corroboram as mudanças climáticas como um fenômeno com potencial devastador e alertam para potenciais perigos da elevação oceânica. Além disso, nosso índice para espécies deficientes em dados pode auxiliar a direcionar esforços em busca de novas informações. Por fim, nossas análises com relação ao fungo quitrídio corroboram a hipótese de endemismo para a linhagem Bd-Brazil e também evidenciam hábitat adequado passado para o Bd-GPL. Além disso, verificamos baixa sobreposição de nicho climático entre essas duas linhagens, indicando possibilidade de competição. Assim, esperamos que nossos resultados tenham contribuído para o conhecimento de múltiplas ameaças à conservação dos anfíbios e sirvam para direcionar futuros estudos / Abstract: Climate change has the potential to change ecosystem functioning through species extinction and disrupting connection between biota and environment, thus changing natural patterns and requiring adaptation. One certain consequence of climate change is the sea level rise, which is expected to redraw coastal shorelines worldwide and broadly affect coastal-associated biota. In this context, amphibians represent a sensitive group under several current pressures, exhibiting population decline, highlighted as the most current threaten vertebrates on Earth, and also, with several "data deficient" species. Thus, herein we analyzed multiple effects that hamper amphibian conservation. We evaluated potential climate change effects and sea level rise on amphibians with coastal distribution. Also, we developed an index to classify data deficient species in order to offer an alternative further research of such species to gather sufficient information to change their current status. Finally, we analyzed historical availability of suitable habitat for and endemic lineage of the chytrid fungus searching for patterns that explain its current distribution and rarity. We used tools as species distribution models and geographic information system to answer our questions. Our results corroborate climate change as a potentially devastating phenomena and we stress potential threatens derived from sea level rise. Moreover, the index we developed for data deficient species seems to work properly and may drive further effort in searching for further information for those species. Finally, our analyzes related to the chytrid fungus supported the hypothesis of endemism of Bd-Brazil, and also showed past environmental suitability for Bd-GPL. In addition, we verified low environmental niche overlap these two lineages, indicating possibility of competition. Thus, we expect our results may improve current knowledge about multiple threats to amphibian conservation, as well as, they may be used to guide further research / Doutorado / Ecologia / Doutor em Ecologia

Master of Science / Department of Clinical Sciences / James W. Carpenter / In many species, temporary increases in glucocorticoids (GC) can be used to identify changes in adrenal activity in response to acute stressors. For this research, GC metabolites were identified in fecal extracts from various Atelopus species. The objectives were to identify possible correlates between GCs and health status, assess the impact of husbandry protocols on adrenal activity, and evaluate the sub-lethal effects of antifungal bacteria used for protection of frogs against the chytrid fungus (Batrachochytrium dendrobatidis; Bd). The first study examined whether fecal GC concentrations can be correlated with animal health and behavior changes in a captive setting. Atelopus zeteki with varying degrees of dermatitis were categorized based on the severity of their skin abnormalities and GC metabolite concentrations were analyzed to detect correlations between severity of disease and GC metabolite concentrations. Similarly, behaviors that may indicate elevated stress levels (e.g., time spent in hide) were analyzed to detect correlation between disease and behavior changes. There was no correlation between fecal GC metabolites and health status of the animal or between health status and amount of time spent in hide. The second study established ex situ colonies of two Panamanian frog species, Atelopus certus and Atelopus glyphus, to determine how male group size affects behavior and GC levels. When housed in groups of eight, animals initially had elevated GC concentrations and interacted aggressively, but these instances declined substantially in the first 2 weeks of being housed together. Thus, captive Atelopus populations can be housed in same-sex enclosures without causing sub-lethal stress on the individuals involved. The third study examined the ability of antifungal bacterium from Central America to propagate on Atelopus skin as a preventative treatment for Bd and the sub-lethal effects of each bacteria species on adrenal function based on GC analysis. Four species of bacteria (Pseudomonas sp., Pseudomonas putida, Chryseobacterium indolgenes, and Stenotrophomonas maltophili) were found to be successful Bd inhibitors in vitro. There were no detectable effects of bacterial exposure with GC metabolite concentrations over time for any of the treatments assessed.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol) / There have been many cases reported of amphibian populations declining. These are often due to anthropogenic factors such as habitat destruction and pollution. However, some declines have not had an obvious cause and many of these have been investigated and found to be due to pathogenic disease. Batrachochytrium dendrobatidis is a recently described pathogen of frogs. The population declines that have been associated with chytridiomycosis have occurred in relatively undisturbed areas such as national parks. The declines tend to occur at higher altitudes or in colder climates. This is thought to be because of the frog immune system being slower at lower temperatures. Chyt1id fungus has been found in frog populations throughout the world. Little research has been carried out in Africa although, chytridiomycosis has already been seen in Kenya and South Africa. In this project frogs were sampled from selected transects in the Western Cape and three sites in the Northern Cape. The effect of altitude on the occurrence of infection was tested in the Western Cape. It was found that 18 frogs were infected in the Western Cape and the effect of altitude was not significant. Large numbers of dead and dying frogs were found in two of the Northern Cape sites and the incidence of chytridiomycosis was high in these populations. Chytrid was found in two Bufogariepensis from the Eastern Cape and in Xenopus petersii from Kasanka National Park, Zambia. More research is needed on chytridiomycosis in these populations. The frogs in the Western Cape seem to survive with chytrid fungus infection whereas, the frogs in the Northern Cape are dying. This suggests another factor acting on the Northern Cape frog populations.

acase@tulane.edu

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.

Amphibians are the most threatened taxon on the planet. Declines have been associated with over-exploitation, habitat loss, pollution, and pathogenic diseases, but of these factors, pollution and disease have been relatively under-studied. Here, I investigated: 1) the impacts of commonly used pesticides on aquatic communities, 2) the effect of these pesticides on amphibian susceptibility to the pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd), and 3) whether there are non-amphibian hosts of Bd and 4) how to best quantify the survival of Bd through ontogeny of the host. In my first research chapter, I quantified the effects of environmentally relevant concentrations of the mot commonly used synthetic fungicide in the US, chlorothalonil, on 34 species-, 2 community- and 11 ecosystem-level responses in a multitrophic-level system. Chlorothalonil increased mortality of amphibians, gastropods, zooplankton, algae, and a macrophyte (reducing taxonomic richness), reduced decomposition and water clarity, and elevated dissolved oxygen and net primary productivity. These ecosystem effects were indirect but were predictable based on changes in taxonomic richness. A path analysis suggests that chlorothalonil-induced reductions in biodiversity and top-down and bottom-up effects facilitated algal blooms that shifted ecosystem functions. In my second chapter, I investigated how a wide range of ecologically relevant concentrations of chlorothalonil affected four species of amphibians (Osteopilus septentrionalis, Rana sphenocephala, Hyla squirella and H. cinerea). I also evaluated the effects of chlorothalonil on liver tissue, immune cell density, and the stress hormone, corticosterone. Chlorothalonil killed nearly every amphibian at the expected environmental concentration (EEC) and, at concentrations to which humans are commonly exposed (up to the EEC), it was associated with elevated corticosterone levels and changes in immune cells. Three species (O. septentrionalis, R. sphenocephala, and H. cinerea) showed a non-monotonic dose-response, with low and high concentrations causing significantly greater mortality than intermediate concentrations and controls. Corticosterone exhibited a similar non-monotonic dose response and chlorothalonil concentration was inversely associated with liver tissue and immune cell densities. These studies on chlorothalonil emphasize the need to re-evaluate its safety and to further link anthropogenic-induced changes in biodiversity to altered ecosystem functions. In my third research chapter, I investigated the effects of chlorothalonil and atrazine, one of the most commonly used herbicides in the US, on amphibian susceptibility to Bd, a leading cause of amphibian extinctions. Relative to controls, atrazine monotonically reduced Bd growth in culture and on tadpoles. In contrast, chlorothalonil non-monotonically reduced Bd growth in culture and on tadpoles, with low and high concentrations causing significantly greater mortality than intermediate concentrations and controls. This study is one of only a handful of studies to document a non-monotonic dose response of an invertebrate (Bd) to a pesticide. Although both pesticides reduced Bd growth on tadpoles and in culture, neither eliminated Bd entirely, and because we know little about the long-term effects of the pesticides on hosts (e.g., immunosuppression), I do not recommend using these chemicals to control Bd. In my fourth research chapter, I investigated whether there are non-amphibian hosts for Bd. Non-amphibian hosts could explain how Bd is able to persist in the environment after amphibians are extirpated, and the extreme virulence and distribution of Bd. In laboratory and field studies, I found that crayfish, but not mosquitofish, were hosts for Bd. I found that crayfish could be infected with Bd, could maintain that infection long term (at least 3 months) and could transfer that infection to susceptible amphibians. I also revealed that exposure to water that previously held Bd caused significant crayfish mortality and gill recession, suggesting that Bd releases a chemical that can cause host pathology in the absence of infection. Most efforts to conserve and restore amphibian populations challenged by Bd have been unsuccessful, but managing alternative hosts offers a new and potentially more effective approach to managing Bd. Likewise, identifying the specific pathology-inducing chemical released by Bd might facilitate the development of new strategies to reduce the risk posed by this pathogen. The fifth and sixth research chapters are aimed to improve the quality and efficiency of Bd research. During amphibian development, Bd infections transition from the mouthparts of tadpoles to the skin of post-metamorphic frogs but this transition has never been quantified and thus researchers might be sampling the wrong parts of amphibian bodies to detect Bd. I showed that Bd abundance in O. septentrionalis mouthparts declined from Gosner stages 35-42 and increased on epidermis from Gosner stages 38-46. Assuming our findings are general across species, I recommend sampling mouthparts of amphibians less than Gosner stage 41 and hind limbs of amphibians greater than Gosner stage 41. This should provide researchers with guidance on where to sample to maximize detection of Bd. I also investigated whether Trypan blue dye could be used to determine the viability of Bd. I showed that the proportion of zoospores stained with Trypan blue dye matched the proportion of known dead zoospores added to cultures. In contrast, all of the zoosporangia stage (including known dead zoosporangia) of Bd stained blue. These results demonstrate that Trypan blue can be used to determine the viability of Bd zoospores but not zoosporangia. I recommend using Trypan blue to report the number of live zoospores to which hosts are exposed and to help determine whether factors have lethal or sublethal effects on Bd. My work demonstrates that managing exposure to contaminants and biological reservoirs for Bd might provide new hope for imperiled amphibians. Further exploring how pesticides and pathogens are contributing to amphibian declines will allow us to formulate crucial management and conservation plans to begin remediation.