Amphibians are key health indicator species and important sentinels for ecosystem health. With 32% of amphibians threatened with extinction, and an additional 25% without enough data on record to fully understand their status, amphibians are facing what is being called the Amphibian Extinction Crisis. Of caudate amphibians (salamanders/newts), 49.8% are threatened or endangered, making them currently the most threatened vertebrate taxa. As a result of the Amphibian Extinction Crisis, it is imperative that captive breeding facilities be established as a hedge against future amphibian extinctions. Unfortunately, captive assurance colonies face frequent failure due to lack of natural stimuli, which are necessary for amphibian breeding. Therefore, assisted reproductive technologies (ART) have been employed to reproduce endangered amphibians, increasing the success of captive assurance colonies worldwide. Over the last decade, a great deal of attention has been given to restoring anuran (frog/toad) populations through captive breeding, but little attention has been given to preservation of caudate species. Caudate amphibians are notoriously difficult to breed in captivity, thus it is crucial that ART be developed to help preserve endangered caudates sooner, rather than later. The following studies highlight some of the first attempts at developing ART protocols in caudate amphibians. Protocols were developed for: hormone induction of gamete production; cryopreservation of salamander sperm; and in vitro fertilization. These protocols were developed for the common, model species Ambystoma tigrinum in order to assure safety and efficiency for later application in endangered species.

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.

For over three decades, amphibian populations have been declining across the globe. Emerging infectious diseases are responsible for some of these declines. Ranaviruses have caused die-offs in wild amphibian populations on 4 continents, in 5 Canadian provinces, and in over 25 U.S. states. In order to understand host-pathogen dynamics, it is critical to establish baseline information on species susceptibility and the effects of natural stressors. The goal of my thesis research was to quantify the effects of anuran development and exposure to invertebrate predators on species-specific susceptibility to ranavirus. My experiments were designed in factorial arrangements, and consisted of exposure to ranavirus during different developmental stages or with and without predator cues in a controlled environment. I found that exposure to invertebrate predator cues did not increase susceptibility to ranavirus for 4 anuran species tested. Susceptibility differed among embryo, hatchling, larval and metamorph stages, but trends differed among species and did not follow predictions based on Xenopus laevis immune function. Low susceptibility during the embryo stage was the only consistent development result among species, perhaps owing to protective qualities of the vitelline membrane or mucoidal capsules surrounding the embryo. Across 7 anuran species tested, mean mortality rates ranged from 5 – 100%, with Lithobates sylvaticus and Scaphiopus holbrookii most susceptible. I found that infection rates and viral load were correlated with mortality rates, thus these variables are good indicators of susceptibility to ranavirus. My results indicate that ranaviruses can cause catastrophic natural mortality in some anuran species, and likely play a significant role in local population dynamics. For highly susceptible species, ranaviruses could cause local extirpations that lead to species declines. More information is needed on the role of natural (e.g., co-infection, competition) and anthropogenic stressors in driving ranavirus epizootic events. I encourage natural resource agencies to initiate ranavirus surveillance programs, especially for rare species and fragmented populations. Future studies should take an immunogenetic approach to identifying mechanisms driving susceptibility. Identifying mechanisms associated with ranavirus emergence is fundamental to developing science-based conservation strategies.

The conservation of aquatic-breeding amphibian populations and their habitats is increasingly challenged by urban and rural development, which is occurring more intensively and more rapidly than ever before. Some species are now impacted by development throughout their range. This has forced a re-evaluation of the potential of developed landscapes for providing habitat and contributing to regional conservation strategies. For many amphibians, little is known about the criteria necessary for persistence in a developed landscape. Considerable variation in the physiology, habitat requirements, and movement behaviour of amphibians suggests that responses to habitat loss, alteration, and fragmentation are species-specific. In this thesis, I investigate species-habitat relationships for the northern red-legged frog (Rana aurora) and the Pacific chorus frog (Pseudacris regilla) in a mixed urban-rural landscape in southwestern British Columbia to evaluate the potential for species persistence despite urban and rural development throughout their range. I used repeat auditory surveys of the species’ breeding chorus to determine presence or absence at potential breeding wetlands. I then related species occurrence and abundance to characteristics of the aquatic and terrestrial environment measured at multiple spatial scales. Both species were found to use rural and urban wetlands, though R. aurora were rarely detected while P. regilla were common. Occurrence was best explained by characteristics of the terrestrial environment, rather than within-wetland characteristics, though influential terrestrial characteristics and their scale of impact differed between species. Within the context of the developed landscape, I identify species-specific positive and negative habitat associations and suggest the spatial scales at which management of these habitat characteristics will be most effective. These criteria may help to explain the species’ current distribution, prioritize management strategies, predict the effectiveness of habitat conservation and restoration projects, and inform development in municipalities seeking to maintain or enhance amphibian diversity. / Graduate / 2019-09-11

Please read the abstract in the section, 00front, of this document / Dissertation (MSc)--University of Pretoria, 2008. / Zoology and Entomology / unrestricted

Ndumo Game Reserve is the only officially protected area within the Phongolo Floodplain; an area in the northern parts of KwaZulu-­‐Natal known to boast a rich diversity of amphibians, thus becoming one of the focal areas for this study. The study’s aim was to monitor and record amphibian diversity, as well as associated blood parasite biodiversity. For the purpose of monitoring, a number of active and passive techniques were employed. Habitat preferences for the expected species were divided into five types, namely endorheic, lacustrine, palustrine, riverine and terrestrial. Endorheic habitats were found to harbour the highest diversity (70%) of frog species. A permanent song meter was used to passively record calling activity of frog species associated with endorheic systems. This call data indicated peak breeding season, preferred calling times and intensities of the different species. Historical records from the same area were used as a basis to which this study’s data were compared. In the case of the polychromatic Argus Reed Frog Hyperolius argus Peters, 1854, questions were raised concerning the major colour changes during development of the apparent sub-­‐adult to adult life stages, an observation which was has caused some confusion as to whether these forms represented a single species or multiple cryptic species. These issues were clarified using techniques such as DNA extraction and polymerase chain reaction (PCR). Furthermore, a blood parasite survey was conducted. Thin blood smears for morphometrics and whole blood for molecular work, were collected from 29 species and 436 individual frogs. For the majority of the recorded parasites, techniques such as light microscopy were utilized for the morphological description and classification of these parasites. Among the recorded frog blood parasites observed, 20% of the frog specimens were infected with at least one blood parasite group. Hepatozoon and Trypanosoma species accounted for most of the infections; the former demonstrated significant differences in intensity of infection across species, families and habitat types (P = 0.028; P = 0.006; P = 0.007 respectively). Methods, such as transmission electron microscopy, examining the ultrastructure, as well as parasite DNA extraction and 18S rDNA gene sequences for the molecular and phylogenetic characterization, were reserved for Hepatozoon species infecting common toad species (Amietophrynus). Parasite stages observed were measured and compared to each other, as well as to other described African bufonid haemogregarines. Resulting sequences were compared with each other and to comparative haemogregarine sequences selected from GenBank. In the current study a number of important aspects with regards to monitoring and assessment of amphibians in their natural environment were explored, including looking at and determining diversity and prevalence of blood parasites. Furthermore, important data on gaining a better understanding of amphibians and their behavioural activities were also gathered, which should be able to assist in conservation actions to effectively protect South African anurans and their required habitat types. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Ndumo Game Reserve is the only officially protected area within the Phongolo Floodplain; an area in the northern parts of KwaZulu-­‐Natal known to boast a rich diversity of amphibians, thus becoming one of the focal areas for this study. The study’s aim was to monitor and record amphibian diversity, as well as associated blood parasite biodiversity. For the purpose of monitoring, a number of active and passive techniques were employed. Habitat preferences for the expected species were divided into five types, namely endorheic, lacustrine, palustrine, riverine and terrestrial. Endorheic habitats were found to harbour the highest diversity (70%) of frog species. A permanent song meter was used to passively record calling activity of frog species associated with endorheic systems. This call data indicated peak breeding season, preferred calling times and intensities of the different species. Historical records from the same area were used as a basis to which this study’s data were compared. In the case of the polychromatic Argus Reed Frog Hyperolius argus Peters, 1854, questions were raised concerning the major colour changes during development of the apparent sub-­‐adult to adult life stages, an observation which was has caused some confusion as to whether these forms represented a single species or multiple cryptic species. These issues were clarified using techniques such as DNA extraction and polymerase chain reaction (PCR). Furthermore, a blood parasite survey was conducted. Thin blood smears for morphometrics and whole blood for molecular work, were collected from 29 species and 436 individual frogs. For the majority of the recorded parasites, techniques such as light microscopy were utilized for the morphological description and classification of these parasites. Among the recorded frog blood parasites observed, 20% of the frog specimens were infected with at least one blood parasite group. Hepatozoon and Trypanosoma species accounted for most of the infections; the former demonstrated significant differences in intensity of infection across species, families and habitat types (P = 0.028; P = 0.006; P = 0.007 respectively). Methods, such as transmission electron microscopy, examining the ultrastructure, as well as parasite DNA extraction and 18S rDNA gene sequences for the molecular and phylogenetic characterization, were reserved for Hepatozoon species infecting common toad species (Amietophrynus). Parasite stages observed were measured and compared to each other, as well as to other described African bufonid haemogregarines. Resulting sequences were compared with each other and to comparative haemogregarine sequences selected from GenBank. In the current study a number of important aspects with regards to monitoring and assessment of amphibians in their natural environment were explored, including looking at and determining diversity and prevalence of blood parasites. Furthermore, important data on gaining a better understanding of amphibians and their behavioural activities were also gathered, which should be able to assist in conservation actions to effectively protect South African anurans and their required habitat types. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

In this study evidence is presented that suggests transdisciplinary art practices and participatory biology programs may successfully increase public understanding of ecological phenomenon. As today’s environmental issues are often complex and large-scale, finding effective strategies that encourage public awareness and stewardship are paramount for long-term conservation of species and ecosystems. Although artists and biologists tend to stay confined to their professional boundaries, and their discourses largely remain inaccessible to larger audiences, arguments here are presented for a combined approach, which may disseminate knowledge about ecology to non-specialists through novel art-science participatory research and exhibitions. Moreover, historically several scientists utilized varied creative art forms to disseminate scientific insights to a larger populace of non-specialists, such strategies as engaging writings and visually provocative artworks may still be effective to captivate contemporary audiences. In addition such historic hybrid science-art practitioners may have laid a conceptual terrain for some of today’s transdisciplinary art and citizen science practices. Furthermore, seminal ecological artworks from the 20th Century by Joseph Beuys, Patricia Johanson and Hans Haacke utilized novel strategies to reach audiences with a message of wetland conservation, blurring boundaries between art, ecology and activism. More recently artists like Cornelia Hesse-Honegger, Helen and Newton Harrison and others have integrated biological research into their art practices, which resulted in new scientific discoveries. Through my own transdisciplinary artwork about frogs, data suggests that the visual strategies I employ were effective to increase non-specialist understanding of the ecological phenomenon of amphibian declines and deformations. In addition through my participatory biology programs, Public Bio-Art Laboratories and Eco-Actions, evidence suggests that non-specialists achieved an increased awareness of the challenges amphibians and ecosystems currently face. Likewise, that through such participatory citizen science research new scientific insights about the proximate causes for deformities in anuran amphibians at select localities in middle England and Quebec were achieved. Here laboratory and field evidence, generated with the aid of public volunteers, found that non-lethal predatory injury to tadpoles from odonate nymphs and some fishes resulted in permanent limb deformities in post-metamorphic anurans. From an environmental-education and larger conservation standpoint, these findings are very relevant as they offer novel strategies for experientially engaging non-specialist audiences while generating important insights into biological communities and wetland ecosystems.

For nearly a century, amphibian metamorphosis has served as an important model of how thyroid hormones regulate vertebrate development. Consequently metamorphosis has been studied in a number of ways including: morphologically, developmentally, ecologically, and from an endocrine perspective. Over the last two decades, much has been learned about the molecular basis of anuran (frog) metamorphosis. However, very little is known about the molecular underpinnings of urodele (salamander) metamorphosis. Using the axolotl and axolotl hybrids as models, I present some of the first studies on the gene expression changes that occur during urodele metamorphosis. In Chapter 1, the motivation for the research described in the subsequent chapters is presented and the literature is briefly reviewed. In Chapter 2, the first microarray analysis of urodele metamorphosis is presented. This analysis shows that hundreds of genes are differentially expressed during thyroid hormone-induced metamorphic skin remodeling. Chapter 3 extends the analysis presented in Chapter 2 by showing that the transcriptional patterns associated with metamorphic skin remodeling are robust even when the concentration of thyroid hormone used to induce metamorphosis is varied by an order of magnitude. Chapter 4 makes use of the differentially expressed genes identified in Chapters 2 and 3 to articulate the first model of urodele metamorphosis to integrate changes in morphology, gene expression, and histology. In addition, Chapter 4 outlines a novel application for piecewise linear regression. In turn, Chapter 5 makes use of the model presented in Chapter 4 to demonstrate that full siblings segregating profound variation in metamorphic timing begin to diverge in phenotype early during larval development. In Chapter 6 the conclusions drawn from the research are summarized and future directions are suggested.

This dissertation addresses the question of how leaf litter from trees affects animals that live in aquatic environments, with an emphasis on the effect of Chinese tallow (Triadica sebifera) leaf litter on anuran larvae (i.e., frog tadpoles). This question is important to our understanding of how allochthonous inputs to aquatic habitats drive biodiversity in wetlands. It also addresses a timely conservation concern in southeastern Louisiana where invasion by Chinese tallow trees (Triadica sebifera) is displacing native trees. The invasion process is homogenizing forest composition and changing the quantity and quality of litter inputs to ponds from those produced by a mixture of native species to that of a single invasive species. This change in litter quality may have important effects on aquatic animals because leaf litter that falls into ponds is an important source of nutrients and energy in wetland foodwebs. Leaf litter also affects water quality via effects on dissolved oxygen and leaching of defensive compounds, which may subsequently affect the diversity and performance of aquatic animals. Herein I address these issues by presenting a series of studies in which tadpole and aquatic invertebrate responses were tested using leaf litter from Chinese tallow leaves and three native tree species. The major findings of this research are: (1) Leaf litter has a direct effect on water quality (2) Chinese tallow can cause differential survival and performance of tadpoles (3) Differences in water quality due to leaf litter can cause changes in tadpole behavior (4) Chinese tallow leaf litter breaks down much faster than litter from native trees (5) Difference in litter breakdown rates influence aquatic community composition.