The waterborne pathogen Batrachochytrium dendrobatidis (Bd), amphibian chytrid, is implicated as being the causative agent for global amphibian declines. The fungus attacks the keratinized skin of adult and postmetamorphic animals and the keratinized mouthparts of tadpoles. Postmetamorphic animals seem to be more susceptible to Bd than tadpoles and adult frogs. Hypotheses exist that the origin of the fungus is in Africa. During the study different aspects of Bd infections in South African frogs were examined including the distribution of Bd, cultivation of Bd, preservation of cultures, the morphology of Bd as an infection as well as in culture and finally differences in host defense. Positive and negative localities for Bd were identified through surveys conducted in South Africa. These data will be contributed to the Bd Mapping Project and the African Bd Database in order to determine whether chytrid has any environmental preferences. Cultures obtained from the positive localities were maintained and cryopreserved for use in numerous experiments. In a future study, DNA extractions from the cultures will be analyzed using multilocus sequence typing in order to determine the sequence type of South African strains in comparison with global strains. This will provide important epidemiological information concerning the origin and control of Bd. The morphology of Bd was also examined using scanning electron microscopy and laser scanning confocal microscopy. Damage due to Bd infections was more severe on the larval mouthparts of Amietia vertebralis than that of Hadromophryne natalensis. The adverse effect of Bd is therefore not limited to postmetamorphic animals. Confocal microscopy uses fluorescent stains and lasers to examine specific structures within organisms. An especially effective stain used during confocal microscopy on Bd is Calcofluor White M2R. Due to its specificity this stain can be used as an effective screening tool for Bd in tissue. The role of antimicrobial skin peptides as a defense against Bd was also examined. A. vertebralis experiences die-offs due to chytrid, while H. natalensis does not experience the same effect in the presence of Bd. H. natalensis possess more antimicrobial skin peptides against Bd with a higher effectiveness than peptides extracted from A. vertebralis. This may explain the observed susceptibility of A. vertebralis to Bd. The relevance of this study is in order to identify areas in South Africa in which the probability of finding Bd is high. This will help in the surveillance of Bd and in the identification of susceptible species to be monitored and protected against the fungus. The effect of Bd on frog species can also be determined by means of exposure experiment using cultures isolated during this study. Through the identification of peptides effective against Bd, predictions can be made with regard to the susceptibility of different frogs to Bd, improving our ability to protect the amphibian biodiversity in South Africa. With the use of confocal microscopy in the examination of Bd, we became the first group to use the method. By the identification of a stain with a high potential as a screening tool, we also contributed to the more efficient identification of Bd in tissue. Keywords: Batrachochytrium dendrobatidis, Bd, amphibian chytrid, distribution, cultivation, antimicrobial skin peptides, laser scanning confocal microscopy, Amietia vertebralis, Hadromophryne natalensis, South Africa / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2009.

abstract: Infectious diseases have emerged as a significant threat to wildlife. Environmental change is often implicated as an underlying factor driving this emergence. With this recent rise in disease emergence and the acceleration of environmental change, it is important to identify the environmental factors that alter host-pathogen dynamics and their underlying mechanisms. The emerging pathogen Batrachochytrium dendrobatidis (Bd) is a clear example of the negative effects infectious diseases can have on wildlife. Bd is linked to global declines in amphibian diversity and abundance. However, there is considerable variation in population-level responses to Bd, with some hosts experiencing marked declines while others persist. Environmental factors may play a role in this variation. This research used populations of pond-breeding chorus frogs (Pseudacris maculata) in Arizona to test if three rapidly changing environmental factors nitrogen (N), phosphorus (P), and temperature influence the presence, prevalence, and severity of Bd infections. I evaluated the reliability of a new technique for detecting Bd in water samples and combined this technique with animal sampling to monitor Bd in wild chorus frogs. Monitoring from 20 frog populations found high Bd presence and prevalence during breeding. A laboratory experiment found 85% adult mortality as a result of Bd infection; however, estimated chorus frog densities in wild populations increased significantly over two years of sampling despite high Bd prevalence. Presence, prevalence, and severity of Bd infections were not correlated with aqueous concentrations of N or P. There was, however, support for an annual temperature-induced reduction in Bd prevalence in newly metamorphosed larvae. A simple mathematical model suggests that this annual temperature-induced reduction of Bd infections in larvae in combination with rapid host maturation may help chorus frog populations persist despite high adult mortality. These results demonstrate that Bd can persist across a wide range of environmental conditions, providing little support for the influence of N and P on Bd dynamics, and show that water temperature may play an important role in altering Bd dynamics, enabling chorus frogs to persist with this pathogen. These findings demonstrate the importance of environmental context and host life history for the outcome of host-pathogen interactions. / Dissertation/Thesis / Ph.D. Biology 2012

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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.