The decline of amphibian populations around the world is a well documented phenomenon. The Baw Baw Frog Philoria frosti belongs to a group of high-elevation, mountain-top amphibians in Australia that have undergone recent population declines, but an understanding of the responsible agents is deficient or absent for most species. The inability to diagnose agents of decline has mostly been attributed to a paucity of knowledge on the natural history of these species. / The discipline of conservation biology provided a scientific basis for commencing investigation into the decline of P. frosti. This thesis examines the pattern and extent of decline, and the autoecology and demography of the species, in order to provide a basis for evaluating conceivable decline-agents, and to establish a platform to commence diagnosis of the decline. (For complete abstract open document)

Amphibian populations are declining on a global scale and although disease outbreaks are a commonly accepted hypothesis they are not the only one. My aims for my thesis were to study the home range of Leiopelma hamiltoni, to determine whether a photographic database could be used to individual identified them and monitor the health status of the population. Habitat loss is a possible cause. For this reason monitoring an animals' home range is a possible method to detect early impacts the population is facing. By tracking 12 L. hamiltoni within a 12 m x 6 m grid on Maud Island, it was shown that the home range size can vary from 0.5 m2 to 25 m2 based on the minimum convex polygon method. However, to track multiple individuals it is important to be able to distinguish among frogs. The commonly used methods of identification, such as toe clipping, pose potentially detrimental effects. Therefore, non-invasive methods based on natural markings need to be established. Through the use of the dark pigmented patterns found on the skin of L. hamiltoni individuals can be identified on recapture with a mean accuracy of 93%. By developing a database to maintain the photographs used for individual identification, the database can also be used to monitor the status of the population. During 2003 numerous L. hamiltoni were observed with denuded patches predominantly on the facial region. By monitoring five individuals within the captive facility at the University of Canterbury it was discovered that frogs appear to be able to cure themselves. Through researching the home range requirements and developing a photographic database to monitor the population status of L. hamiltoni, it will aid in the management of ensuring the long-term survival of this archaic species of frog.

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.

Amphibians are declining at alarming rates globally. Multiple factors contribute to these declines, including chemical contaminants and emergent diseases. In recent years, agrochemical use, especially fungicide applications, has increased considerably. Previous studies have demonstrated that these agrochemicals leave application sites and enter wetlands via runoff and have detrimental effects on non-target organisms. For example, exposure to contaminants can have multifarious effects on amphibians, such as reducing their ability to deal with a secondary stressor, such as disease. A pathogen that is found concomitant with chemical contaminants in aquatic systems is Batrachochytrium dendrobatidis (Bd). Bd has decimated amphibian populations worldwide. Susceptibility to this pathogen varies across amphibian life stages, and is greater in adults than larvae. Consequently, it is important to examine the effects of simultaneous and serial Bd and agrochemical exposure throughout amphibian development. I assessed the combined effects of 3 different fungicides and Bd on two amphibian species: Cuban tree frogs (Osteopilus septentrionalis) and grey tree frogs (Hyla versicolor), both simultaneously and across life stages. To elucidate the complexities of these interactions, I conducted two experiments, one in the laboratory and another in outdoor mesocosms. Frogs were exposed to most of the possible combinations of fungicides and Bd as tadpoles and metamorphs. The presence of fungicides during the tadpole stage caused no difference in timing to metamorphosis and therefore no extension of time animals were exposed to the pathogen. Fungicides did not reduce fungal growth; in fact, tadpoles exposed simultaneously to a fungicide and Bd, regardless of the specific fungicide, had increased fungal loads compared to acetone controls. Additionally, animals exposed to both stressors simultaneously had higher mortality compared to controls or any of the stressors singly. Lastly, the fungicide had persistent effects on amphibian health by affecting susceptibility to Bd later in ontogeny. Frogs exposed to any of the three fungicides as a tadpole had higher Bd prevalence, Bd abundance, and Bd-induced mortality when challenged with Bd after metamorphosis, an average of 71 days after their last fungicide exposure. In conclusion, I found no benefits of fungicides for amphibians. In fact, results indicate both immediate and delayed negative effects of exposure to fungicides and Bd. These findings highlight the importance of studying multiple potential contributors to amphibian declines, simultaneously and sequentially, to understand net effects of stressors on amphibian performance.

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.

Amphibian populations are declining on a global scale and although disease outbreaks are a commonly accepted hypothesis they are not the only one. My aims for my thesis were to study the home range of Leiopelma hamiltoni, to determine whether a photographic database could be used to individual identified them and monitor the health status of the population. Habitat loss is a possible cause. For this reason monitoring an animals' home range is a possible method to detect early impacts the population is facing. By tracking 12 L. hamiltoni within a 12 m x 6 m grid on Maud Island, it was shown that the home range size can vary from 0.5 m2 to 25 m2 based on the minimum convex polygon method. However, to track multiple individuals it is important to be able to distinguish among frogs. The commonly used methods of identification, such as toe clipping, pose potentially detrimental effects. Therefore, non-invasive methods based on natural markings need to be established. Through the use of the dark pigmented patterns found on the skin of L. hamiltoni individuals can be identified on recapture with a mean accuracy of 93%. By developing a database to maintain the photographs used for individual identification, the database can also be used to monitor the status of the population. During 2003 numerous L. hamiltoni were observed with denuded patches predominantly on the facial region. By monitoring five individuals within the captive facility at the University of Canterbury it was discovered that frogs appear to be able to cure themselves. Through researching the home range requirements and developing a photographic database to monitor the population status of L. hamiltoni, it will aid in the management of ensuring the long-term survival of this archaic species of frog.

Amphibian populations worldwide are currently in decline. One approach to preventing extinction of some of the affected species is to create assurance colonies. These sustainable captive populations might some day be used to reestablish wild populations. One issue with creating assurance colonies is successful breeding; often difficulties arise when attempting to breed exotic animals in captivity. Sexual conditioning, a form of Pavlovian conditioning, has been shown to improve breeding behavior. In this project the efficacy of sexual conditioning to improve breeding behavior in the dyeing dart frog (Dendrobates tinctorius) was tested. The frogs were trained with a stimulus that was either predictive of or independent of exposure to a member of the opposite sex. The group trained with the predictive stimulus showed shorter latencies to a variety of breeding behaviors and produced more eggs than the control groups. The sexual conditioning procedure also increased expression of various breeding behaviors allowing for careful examination of calls and visual signaling within this species.

Fundação de Amparo a Pesquisa no Estado do Rio Grande do Sul / On the last decades, amphibian populations have been suffering a global decline, and different factors can be related to this process. Parallel observations between the decrease of stratospheric ozone concentration in the late 1970s and amphibian decline in early 1980s aroused interest to study the possible association among increased incidence of ultraviolet radiation (UV) and amphibian decline. The UV radiation has important biological impact on living organisms due to their ability to induce lesions in the DNA molecule, which may result in the induction of mutations or cell death. Photoreactivation is an important DNA repair mechanism that promotes the reversal of UV-induced DNA damage by photolyase enzymes after the absorption of UVA and visible light photons. In this study, we evaluated the biolocial effects of UVB and UVA through morphological, histological, locomotor and survival of Hypsiboas pulchellus (Anura: Hylidae) tadpoles. Tadpoles kept in the laboratory were submitted to acute and chronic treatments, composed of different doses of UVB and UVA exposures. The results of survival curves indicate that the H. pulchellus is very sensitive to UVB radiation comparative to UVA. However, despite the high tadpole survival after UVA, they showed serious malformations after metamorphosis. Tadpoles body weight also suffered influence of different UV radiation doses, however the body length was bit affected. The locomotor capacity (horizontal and vertical speed) of UVB-exposed tadpoles has been significantly changed. In addition, UVB radiation had severe impact on the skin (stratum corneum) and mouth (rows of teeth and jaw sheaths) keratin structures of tadpoles, indicating that these should be important impacts of solar UV radiation in reducing the performance of these organisms. In parallel, it was also possible to observe the extreme importance of activation of photolyases for the repair of DNA damage after treatment with UVB but not UVA. Therefore, in this study, we present a wide characterization of the biological effects of solar UVB and UVA on the performance of H. pulchellus tadpoles. / Durante as últimas décadas, as populações de anfíbios vêm sofrendo um declínio generalizado e diferentes fatores são sugeridos como responsáveis por esse efeito. Observações paralelas entre o rápido decréscimo da concentração do ozônio estratosférico no final da década de 1970, com o declínio de anfíbios no início da década de 1980, despertaram grande interesse para se estudar as possíveis associações entre o aumento da incidência de radiação ultravioleta (UV) na superfície e a redução do número de espécies de anfíbios. Sabe-se que a radiação UV solar tem importante impacto biológico na vida dos organismos devido a sua grande capacidade de induzir lesões na molécula de DNA, que podem levar a indução de mutações ou morte celular. Um dos principais mecanismos de reparo de DNA é a fotorreativação que ocorre por meio das enzimas fotoliases, as quais promovem reversão dessas lesões ao absorver fótons de luz UVA e luz visível. Neste projeto, avaliou-se a ação biológica da radiação UVB e UVA a partir de dados morfológicos, histológicos, locomotores e de sobrevivência de girinos da espécie Hypsiboas pulchellus (Anura: Hylidae). Girinos coletados e mantidos no laboratório foram submetidos a tratamentos agudos e crônicos, compostos por exposições a diferentes doses de luz UVB e UVA. Os resultados de sobrevivência indicam que os girinos de H. pulchellus são muito sensíveis à radiação UVB em relação à UVA. Entretanto, apesar da alta sobrevivência dos indivíduos submetidos à radiação UVA, estes apresentaram sérias malformações em longo prazo. A massa corporal dos girinos também sofreu influência das diferentes doses de radiação UV aplicadas, entretanto o comprimento total foi pouco influenciado. A capacidade locomotora (vertical e horizontal) dos organismos expostos à luz UV foi bastante alterada, onde se destaca a grave redução da atividade locomotora dos indivíduos tratados com radiação UVB. Adicionalmente, a radiação UVB teve impacto severo nas estruturas de queratina da pele (estrato córneo) e da boca (queratodontes e mandíbula) dos girinos, indicando que esses devem ser impactos principais da radiação UV solar na redução da performance desses organismos. Em paralelo, também foi possível observar a extrema importância da ativação das enzimas fotoliases para a reparação dos danos de DNA, assim como os seus efeitos após os tratamentos com radiação UVB, mas não com UVA. Portanto, no presente trabalho, foi possível apresentar uma ampla caracterização dos efeitos biológicos da radiação UVB e UVA solar na performance de girinos de H. pulchellus.

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.

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