Outbreaks of pathogens that threaten both human and nature have increased in recent years. Infectious and transmittable diseases, such as chytridiomycosis, which is caused by the emerging pathogen Batrachochytrium dendrobatidis, has been identified as one of the most important drivers of the current declines in amphibian numbers. This pathogen has spread globally and is not only responsible for the declines in amphibian population numbers, but also for the extinction of species in several countries. As part of the Amphibian Conservation Action Plan, the IUCN recommended ex situ breeding of amphibian species to try and stem the global loss of amphibian species. Due to chytridiomycosis being one of the most eminent threats for amphibians, it poses an additional threat for the ex situ breeding plan. There is thus a need for safe and effective measures to treat chytridiomycosis, especially in breeding programs for endangered species. F10 (Health and Hygiene) is a veterinary antiseptic that has shown to be 100% effective in killing B. Dendrobatidis in vitro. Before any chemical treatment can be applied the efficacy and toxicity of F10 has to be determined to establish if F10 can be effectively applied across different amphibian species and across different life stages. We propose to develop a treatment protocol for F10 for the effective treatment of amphibian chytridiomycosis by challenging juveniles of Amietophrynus gutturalis with B. dendrobatidis and subsequently treating the infection with a proposed concentration of F10. The survival of B. dendrobatidis zoospores was also determined in the presence of F10. The results obtained showed survival of tadpoles at a 1:10,000 concentration of F10 for 30min, and juveniles at a concentration of 1:2000 for 15 min. Furthermore the in vitro tests showed that the B. dendrobatidis zoospores died after 10 min at a 1:10,000 concentration and 30 min at a 1:15,000 concentration. The successful treatment of tadpoles as well as juveniles will increase any species chance for survival, especially when treating tadpoles as the pathogen will then be eradicated before the tadpole metamorphoses and reaches the disease-susceptible life stage. By establishing a partnership between the industry, academic and zoo/wildlife communities we hope to maximise the likelihood of implementing this program in the future and thus ensuring long term sustainability. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Outbreaks of pathogens that threaten both human and nature have increased in recent years. Infectious and transmittable diseases, such as chytridiomycosis, which is caused by the emerging pathogen Batrachochytrium dendrobatidis, has been identified as one of the most important drivers of the current declines in amphibian numbers. This pathogen has spread globally and is not only responsible for the declines in amphibian population numbers, but also for the extinction of species in several countries. As part of the Amphibian Conservation Action Plan, the IUCN recommended ex situ breeding of amphibian species to try and stem the global loss of amphibian species. Due to chytridiomycosis being one of the most eminent threats for amphibians, it poses an additional threat for the ex situ breeding plan. There is thus a need for safe and effective measures to treat chytridiomycosis, especially in breeding programs for endangered species. F10 (Health and Hygiene) is a veterinary antiseptic that has shown to be 100% effective in killing B. Dendrobatidis in vitro. Before any chemical treatment can be applied the efficacy and toxicity of F10 has to be determined to establish if F10 can be effectively applied across different amphibian species and across different life stages. We propose to develop a treatment protocol for F10 for the effective treatment of amphibian chytridiomycosis by challenging juveniles of Amietophrynus gutturalis with B. dendrobatidis and subsequently treating the infection with a proposed concentration of F10. The survival of B. dendrobatidis zoospores was also determined in the presence of F10. The results obtained showed survival of tadpoles at a 1:10,000 concentration of F10 for 30min, and juveniles at a concentration of 1:2000 for 15 min. Furthermore the in vitro tests showed that the B. dendrobatidis zoospores died after 10 min at a 1:10,000 concentration and 30 min at a 1:15,000 concentration. The successful treatment of tadpoles as well as juveniles will increase any species chance for survival, especially when treating tadpoles as the pathogen will then be eradicated before the tadpole metamorphoses and reaches the disease-susceptible life stage. By establishing a partnership between the industry, academic and zoo/wildlife communities we hope to maximise the likelihood of implementing this program in the future and thus ensuring long term sustainability. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

<p>Per-and  polyfluoroalkyl  substances  (PFAS)  are  a  large  group  of  emerging  contaminants  that include astrong carbon-flourine bond that makes the compounds resistant to physical, chemical and  biological  degradation.  They  are  found  in  drinking  water  supplies,  daily  human  products, manufacturing  facilities,  and  in  areas  where  aqueous  film-forming  foam  (AFFF)was  used  to extinguish fires. Toxicity levels of these chemicals can vary depending on the characteristics of the specific chemical; longer carbon chain has shown to be more bioaccumulative and toxic than shorter chain length PFAS. Many studies have recognized perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) to be a substantial concern due to their known toxicity to wildlife. For example, studies show strong evidence that PFOA and PFOS suppress the antibody response from animals. Due to adverse health effects and public concern, the U.S stopped perfluorooctanoic acid (PFOA) manufacturing and switched to the production of an alternative fluorinated compound known  as  hexafluoropropylene  oxide  (HFPO)  dimer  acid  or  GenX,  which  is  thought  to  beless bioaccumulative and therefore, potentially less toxic. These anthropogenic pollutants are one of many stressors acting on aquatic organisms like anurans. Natural stressors such as the devastating fungal  pathogen Batrachocytrium  dendrobatidis(Bd)  is  another  stressor  impacting  amphibian populations.  Despite the co-occurrence of these stressors, no studies have examined interactive effects of the fungal pathogen Bd and PFAS, or whether PFAS effects carry over into the terrestrial environment aftera larvae  exposure. This study  tested the growth and developmental effects of PFOS, PFOA, and GenX, on gray treefrog (Hyla versicolor) tadpoles, followed by a Bd challenge in metamorphs. Our results demonstrate that a PFAS larval exposure interacted witha terrestrial Bdchallenge to influence growth and development. Bdexposed animals were significantly shorter (smaller snout vent length)  and had a significant increase in body condition and mass. This is the first study to report effects on amphibian terrestrial life stages after larval exposure to PFAS and to report an increased sensitivity to Bd. The environmentally relevant concentrations tested in this study  (<10  parts  per  billion)  lend  ecological  significance  to  these  results  however,  additional studies are needed to understand the mechanisms behind these effects.</p>

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Emerging infectious diseases are a threat to wildlife populations and conservation efforts. One example of this is the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis and has been linked to amphibian populations declines worldwide. There have been numerous attempts to mitigate the effects of Bd on amphibians, all with mixed results. Two factors that have previously been found to correlate with Bd infection intensity and prevalence are the amphibian skin bacterial communities and environmental temperatures. Some naturally occurring bacteria on the skin of amphibians and warmer temperatures can limit Bd infection. For my dissertation research, I aimed to 1) assess the amphibian skin bacterial communities across species, developmental stage, infection status, and different local environments, and 2) understand and predict the effect of a natural, varying temperature regime on the growth of Bd from constant temperature data. In Chapter 1, I reviewed the amphibian chytrid fungus and the effects of varying temperature on organisms' performance or trait rates. In Chapter 2, I sampled bacterial communities on ranid tadpoles and three ranid frog species at Mianus River Gorge Preserve in Bedford, New York, USA. I found that tadpoles had significantly different bacterial alpha diversity measurements than adult frogs, with higher Faith's phylogenetic diversity, Shannon diversity, and amplicon sequence variant (ASV) richness. Bacterial communities between the three different adult frogs species were not different. Additionally, infected frogs did not have significantly different bacterial communities than uninfected frogs. In Chapter 3, I predicted Bd growth in three varying temperature environments with Bayesian hierarchical models assuming different thermal performance curves. My predictions overestimated the growth of Bd in varying temperature environments, and the choice of thermal performance curve used in the models strongly impacted the predictions by altering the implied relationship between Bd's growth rate and temperature. In Chapter 4, I aimed to improve modeling methods for predicting in vitro Bd growth in varying temperature environments by adding additional features to the model based on observed biological phenomena, specifically a temperature-dependent delay period for Bd development. However, the model parameters were unidentifiable with this added complexity when only optical density data are available to quantify growth, highlighting the need to match the appropriate data to the complexity of the model. In Chapter 5, I created a mechanistic model that was parameterized by a combination of optical density, MTT assays (a metabolic assay), and zoospore count data to learn more about Bd growth dynamics. I also examined how many days of zoospore count data are needed to fit the mechanistic model. By combining these three data sources, I increased the ability to estimate most model parameters. My dissertation added to both the amphibian skin bacterial community literature, supporting differences between tadpoles and adult frog bacterial communities, and added new data from a previously unsurveyed area. Attempts are being made to use bacterial communities to limit diseases in many wildlife populations, through a probiotic. To use skin bacterial communities, factors that shape these communities need to be understood to ensure the successful application of a probiotic. My dissertation also added to the thermal ecology literature, showing that current methods and my optical density Bayesian hierarchical model do not accurately predict performance in varying temperature environments. As temperatures are changing around the world and temperature variability is expected to increase in many places, predicting how organisms will perform in new thermal environments is becoming increasingly important. / Doctor of Philosophy / Infectious diseases around the world have led to wildlife population declines. Chytridiomycosis is a disease in amphibians caused by the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). Bd infects the skin of amphibians and can cause death. The composition of amphibian skin bacterial communities, bacteria that live on the skin of amphibians, can limit the growth of Bd on amphibians and reduce disease. Due to some species of bacteria inhibiting Bd growth, attempts have been made to try to use bacteria to limit disease in amphibians. But, we still do not know to what extent some host and environmental factors influence host bacterial communities, and how this might influence disease in amphibians. Warmer environmental temperatures have also been associated with reduced chytridiomycosis in amphibians. However, the effect of temperature is often studied at constant temperatures instead of natural, varying temperatures. The impact of varying temperature on Bd growth dynamics is still not fully understood. My dissertation research examined 1) differences in amphibian bacterial communities in different species and at different developmental stages (tadpoles vs. frogs), and 2) whether I can accurately predict Bd growth in varying temperature environments. First, I examined skin bacterial communities of three frog species at Mianus River Gorge, in Bedford, NY. I found that tadpoles had more diverse bacterial communities than adult frogs and that adults from the three species had similar bacterial communities, and that Bd infection status did not correlate with skin bacterial community composition. Second, I examined how temperature impacts the growth of Bd and whether we can predict how Bd grows in natural, fluctuating temperature conditions. Specifically, I used data from lab experiments in which I grew Bd at constant temperatures to fit a model and then predict how Bd grew in temperatures that fluctuate over the day as they would in nature. I found that current methods that use constant temperature data to predict how Bd grows in natural temperature scenarios are not accurate. Third, I attempted to improve modeling methods to predict Bd growth in natural temperature scenarios by specifying that Bd development is dependent on temperature. I found that the increasing model complexity without the correct type or amount of data leads to not being able to fit the model. Lastly, I combined three different types of Bd growth data to fit a new model that describes Bd growth. Fitting this new model with three data sources, I learned more about Bd growth and was more certain about the values of the parameters in the model. Additionally, this model has parameters and model components directly related to Bd growth, unlike in the previous Chapters' models. Using this model will allow us to examine how temperature influences specific Bd growth stages in future studies. My dissertation examined host and environmental factors that influence skin bacterial communities. Determine how these factors shape and change host bacterial communities will allow scientists to successfully use bacteria to reduce disease in amphibians and other wildlife. Additionally, I examined methods in the literature and built my own model to predict Bd growth in varying temperature environments. I found that taking constant temperature data from the lab to predict Bd growth in more natural varying temperature environments is not accurate and future studies need to improve these methods. Developing these methods is becoming more important as temperatures change around the world and organisms are exposed to new temperatures. Improving these methods would allow more accurate predictions about organisms' performance in new environmental conditions.

Chytridiomycosis is an emerging cutaneous fungal disease that contributes to recent global declines and extinction of amphibian species, caused by infection of the skin with a fungus known as Batrachochytrium dendrobatidis (Bd). Many species of frogs secrete antimicrobial peptides onto their skin that are capable of killing Bd. This thesis is an investigation of the effect of corticosteroids on cutaneous innate immunity in frogs, in the context of infection with Bd. The general hypothesis was that injections of glucocorticoids would impair the cutaneous synthesis of these antimicrobial peptides, thereby increasing susceptibility to Bd infection. The objective of the first experiment was to measure and compare gene expression levels of cutaneous AMP’s in frogs treated with glucocorticoids with sham-treated controls. Wild-caught Lithobates pipiens were acclimatized and administered either the corticosteroid methylprednisolone or saline every 48 hours. Norepinephrine-elicited cutaneous secretions were collected prior to the first injection of corticosteroid or saline, and then every 8 days for 40 days. Gene expression of the AMP’s brevinin and ranatuerin in the cutaneous secretions was quantified relative to the reference genes EF1-α and RPL8 using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Corticosteroid treatment was associated with a significant (P<0.027) increase in brevinin gene expression, which was most notable at 24-40 days of corticosteroid administration. Ranatuerin expression followed a similar but nonsignificant trend. The second experiment was a pilot study intended to establish a Bd challenge protocol in L. pipiens. Frogs were immersed in water containing 0, 104, 105 or 106 zoospores of Bd strain JEL 423. Cutaneous swabs were collected prior to challenge and tested for Bd by qPCR; unexpectedly, some tested positive, indicating pre-challenge infection. The analysis was complicated by an identified cross-reactivity of the assay with other fungi. The findings of the first experiment refuted the hypothesis, and suggested that corticosteroids promote rather than impair AMP gene expression in the skin of L. pipiens, under these experimental conditions. Further, the second study demonstrated that none of the frogs showed clinical abnormalities or died, despite exposure to Bd zoospores and despite molecular and histologic evidence of cutaneous Bd infection in some frogs. / NSERC Discovery Grant