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Anthropogenic disturbances can alter the abiotic composition of freshwater systems. These compositional changes can act as physiological stressors towards system inhabitants. However, little is known about how these altered abiotic factors influence stress hormones (corticosterone) in larval amphibians. Throughout the following chapters, I examined the effects of several abiotic factors on baseline and stress-induced corticosterone levels in the larvae of four amphibian species: Jefferson salamander (Ambystoma jeffersonianum), spotted salamander (A. maculatum), wood frog (Rana sylvatica), and grey treefrog (Hyla versicolor). Chapter II examined corticosterone level differences throughout development in A. jeffersonianum and R. sylvatica larvae under field, mesocosm, and laboratory venues. Baseline corticosterone levels in R. sylvatica increased near metamorphic climax in all venues, but not in A. jeffersonianum. Rather, baseline corticosterone levels differed with respect to venue throughout development in A. jeffersonianum. Chapter III examined corticosterone level differences among free-living A. jeffersonianum populations and possible abiotic factors underlying these hormone differences. Corticosterone levels significantly differed across populations. Increased baseline corticosterone levels significantly correlated to low pH. There was also a trend for increased baseline corticosterone levels to be positively correlated with chloride levels and negatively correlated with conductivity. Chapter IV examined the effects of laboratory manipulated pH on corticosterone levels in A. jeffersonianum, A. maculatum, R. sylvatica, and H. versicolor. There was a significant correlation between increased baseline corticosterone levels to low pH in all four species. Prey consumption (in both Ambystoma species) and survival (in A. jeffersonianum, A. maculatum, and R. sylvatica) were also negatively correlated to low pH. Chapter V examined the effects of increased conductivity on corticosterone levels in A. jeffersonianum, R. sylvatica, and H. versicolor. Increased conductivity exposure significantly correlated to increased baseline corticosterone levels in A. jeffersonianum and R. sylvatica. Prey consumption in A. jeffersonianum was also negatively correlated to increased conductivity. My dissertation shows that abiotic factors, such as pH and conductivity, can influence corticosterone levels in larval amphibians. These results suggest that corticosterone levels in larval amphibians may be a suitable biomarker reflective of altered freshwater habitat quality. However, my results also suggest that one should use a high degree of caution when using corticosterone levels in larval amphibians as a means to infer the health status of a population. / Ph. D.

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. / Ph. D. / In light of the global losses of amphibian diversity due to, in part, the skin disease chytridiomycosis (caused by the fungus Batrachochytrium dendrobatidis [Bd]); the discovery that some amphibian-skin bacteria can inhibit Bd growth provides hope for amphibian conservation via their use as probiotics to control Bd infections. However, experiments testing these bacteria have yielded inconsistent results, suggesting a limited understanding about the factors influencing the diversity of amphibian-skin microbes and their ability to inhibit Bd. Also, efforts to identify effective candidates for probiotic therapy are still premature. Thus, my dissertation had an ecological emphasis and focused on complementing conservation efforts focused on probiotics. First, I assessed whether environmental conditions influence bacteriallyproduced products, which can have antifungal properties. Specifically, I surveyed low and highelevation populations of an amphibian species to assess the skin-bacteria and their products. I determined that, while skin bacterial communities were similar across an environmental gradient, their products differed, suggesting potential different antifungal properties. Second, I assessed the ability of different culture media types (low vs high nutrient concentrations) to grow a high portion and most representative fraction of the amphibian-skin bacteria. I found that culture media with low nutrient concentrations allowed the growth of a higher diversity of the bacteria occurring on the amphibian-skin, including the abundant members, and also determined that including a large number of amphibians is the best way to improve culture collections. Third, I assessed the fungal diversity occurring in the skin of different amphibian species and how it might response to Bd infections, and examined whether skin-fungi interact with co-occurring bacteria. I found that the amphibian species was the most important driver of the fungal diversity, and that Bd infection did not influence the diversity of these communities. Moreover, I identified the most diverse fungal phyla occurring in the amphibian-skin and determined that these fungi might interact with co-occurring bacteria. My dissertation contributes to our understanding about the influence of the environmental conditions in the amphibian-skin bacteria, expands our limited knowledge on the amphibian-skin fungi, and complement current amphibian conservation efforts.

The purpose of this research is, first, to determine by laboratory methods, that species specificity does not exist in closely allied taxonomic animals; second, to determine a unit of activity for the gonadotropic hormone. For this purpose a quantitative method for determining potency is necessary to ascertain the seasonal production of the gonadotropic factor in fish. A further aim in this investigation is to demonstrate that the diversity of the gonadotropic factor, in relation to phylogenetic variations, is not as ineffectual as previously reported.

Global amphibian declines have been attributed to numerous and often synergistic causes, such as invasive species, pathogens, and ultraviolet-B (UV-B) radiation. The effects of these stressors are context dependent and can vary with location, species, and populations. As sensitivity to UV-B has shown inconsistencies across amphibian taxa, it can be expected that variation also occurs between populations of a single species. High elevation populations of the long-toed salamander (Ambystoma macrodactylum) face exponentially higher UV-B radiation levels relative to low elevation populations and these levels are predicted to increase in conjunction with continued ozone depletion. We hypothesized that breeding long-toed salamander females at high elevations have modified oviposition behavior to better protect embryos from UV-B induced damage. In addition, we hypothesized that long-toed salamander embryos at high elevation would exhibit elevated photolyase activity, a photo-reactivating enzyme that repairs UV-B radiation-induced damage to DNA. We predicted that this behavioral defense strategy would be employed together with an elevated physiological response as a correlated defense response to increased levels of UV-B radiation in high elevation populations. We surveyed high and low elevation long-toed salamander breeding sites throughout Oregon to quantify oviposition site characteristics and associated UV-B profiles. We simultaneously collected embryos for quantification of photolyase activity in a bacterial transformation assay. We found significant differences in oviposition behavior across elevations, with high elevation breeding females ovipositing in deeper water and using UV-B protective refugia. Oviposition sites at low elevations, however, were most often found in UV-B exposed microhabitats located at the surface of the water. This population difference in oviposition behavior resulted in a standardization of UV-B and temperature conditions for long-toed salamander embryos across elevation. In contrast, we found no population differentiation in photolyase activity between high and low elevation breeding sites. This indicates that behavioral selection for UV-B protected oviposition substrates may either be negating the need for increased photolyase activity in long-toed salamander embryos, or that populations lack the capacity to adapt a heightened physiological response to UV-B at high elevations. Together, these results show how trade-offs in physiology and behavior are a unique adaptation to a significant environmental stressor. Further research into the susceptibility of amphibian species to changing environmental conditions may help to demonstrate the effectiveness of correlated trait responses and plasticity in behavior, and species persistence under changing climate regimes. / Graduation date: 2012

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Amphibians are regarded as sensitive indicators of environmental change and are therefore excellent subjects for use in ecotoxicology. The Phongolo River floodplain is South Africa’s most diverse natural floodplain system and hosts more than 40 frog species. It is also a malaria endemic region and is subjected to active spraying with Dichlorodiphenyltrichloroethane (DDT) through means of indoor residual spraying over the summer months. The upper Phongolo River runs through agricultural landscape and is subjected to runoff from forest plantations, orchards and sugar cane plantations. In this study residue levels of 22 different organochlorine pesticides (OCPs) were analysed in selected amphibian species from in and around the Ndumo Nature Reserve coupled with 12 different biomarker response assays to determine environmental exposure levels and possible sublethal effects in amphibians from the lower Phongolo River floodplain. Seasonal change, direct influence of anthropogenic activity and the influence of species’ aquatic preference in habitat selection were all factors considered during this assessment. Stable Isotope analyses were performed on 11 different food web components In order to determine the food web structure pertaining to Xenopus muelleri (Müller's platanna). Samples were collected during both high and low flow seasons from inside and outside Ndumo Nature Reserve. Organochlorine pesticide bioaccumulation was analysed in whole frog samples using a GC-μECD. Results indicated significant seasonal variation in OCP levels and exposure composition. Significant differences between inside and outside sites were also noted. Dichlorodiphenyltrichloroethane in its different isomer forms and their metabolites along with the hexachlorocyclohexane (HCH) isomers was the two main contributing OCP groups detected. Total OCP levels from all sample sets ranged between 8.71 ng/g lipid and 21,399.03 ng/g lipid. An increase in OCP accumulation was observed for X. muelleri over a period of one year. Organochlorine pesticides are known to have neurotoxic effects causing imbalances in Na+, K+, and Ca+ ion exchange. Hyperactivity has been reported in Rana temporaria (European Common frog) tadpoles exposed to p,p-DDT concentrations above 110,000 ng/g lipid. Despite OCP levels measured in frogs from this study being lower than reported toxic levels, the biomarker response assays indicated definite oxidative stress responses correlating to OCP bioaccumulation, with other minor responses shown. Cellular energy allocation showed a shift in the main energy source type from proteins to lipids correlating to increased OCP bioaccumulation. A slight inhibition response was noted in the hepato-somatic index correlating to γ-HCH bioaccumulation. Stable isotope analyses indicated food web structure differences between inside and outside the reserve, with outside showing less clear distinction between trophic groups and nitrogen enrichment of primary producers. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Amphibians are regarded as sensitive indicators of environmental change and are therefore excellent subjects for use in ecotoxicology. The Phongolo River floodplain is South Africa’s most diverse natural floodplain system and hosts more than 40 frog species. It is also a malaria endemic region and is subjected to active spraying with Dichlorodiphenyltrichloroethane (DDT) through means of indoor residual spraying over the summer months. The upper Phongolo River runs through agricultural landscape and is subjected to runoff from forest plantations, orchards and sugar cane plantations. In this study residue levels of 22 different organochlorine pesticides (OCPs) were analysed in selected amphibian species from in and around the Ndumo Nature Reserve coupled with 12 different biomarker response assays to determine environmental exposure levels and possible sublethal effects in amphibians from the lower Phongolo River floodplain. Seasonal change, direct influence of anthropogenic activity and the influence of species’ aquatic preference in habitat selection were all factors considered during this assessment. Stable Isotope analyses were performed on 11 different food web components In order to determine the food web structure pertaining to Xenopus muelleri (Müller's platanna). Samples were collected during both high and low flow seasons from inside and outside Ndumo Nature Reserve. Organochlorine pesticide bioaccumulation was analysed in whole frog samples using a GC-μECD. Results indicated significant seasonal variation in OCP levels and exposure composition. Significant differences between inside and outside sites were also noted. Dichlorodiphenyltrichloroethane in its different isomer forms and their metabolites along with the hexachlorocyclohexane (HCH) isomers was the two main contributing OCP groups detected. Total OCP levels from all sample sets ranged between 8.71 ng/g lipid and 21,399.03 ng/g lipid. An increase in OCP accumulation was observed for X. muelleri over a period of one year. Organochlorine pesticides are known to have neurotoxic effects causing imbalances in Na+, K+, and Ca+ ion exchange. Hyperactivity has been reported in Rana temporaria (European Common frog) tadpoles exposed to p,p-DDT concentrations above 110,000 ng/g lipid. Despite OCP levels measured in frogs from this study being lower than reported toxic levels, the biomarker response assays indicated definite oxidative stress responses correlating to OCP bioaccumulation, with other minor responses shown. Cellular energy allocation showed a shift in the main energy source type from proteins to lipids correlating to increased OCP bioaccumulation. A slight inhibition response was noted in the hepato-somatic index correlating to γ-HCH bioaccumulation. Stable isotope analyses indicated food web structure differences between inside and outside the reserve, with outside showing less clear distinction between trophic groups and nitrogen enrichment of primary producers. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015