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

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol) / The guttural toad, Amietophrynus gutturalis, Power 1927, is a common toad with a broad geographic range through much of temperate, sub-tropical and tropical southern and central Africa. Introduced to the islands of Mauritius and Reunion in the 1960’s, and subsequently to Cape Town in the 1990’s, the species has become invasive in its extra-limital ranges. Determining the invasion history of a species provides valuable information for conservation biologists and managers and it is fundamentally important for improving our understanding of the underlying processes of biological invasions. This study aimed to determine the source populations of the extra-limital populations from Mauritius and Cape Town. Furthermore, studies investigating genetic diversity and demographics of African Bufonidae are largely absent from the literature. Understanding the evolutionary history of the species may also assist with determining their invasive ability and identifying similar features in other bufonids such as Amietophrynus regularis and A. xeros. Using mtDNA sequence data from the 16S and ND2 markers four geographically distinct clades were identified through Bayesian phylogenies and haplotype networks. However, a spatial analysis of molecular variance (SAMOVA) indicated a grouping structure of three clades. A total of 16 haplotypes were identified from 53 samples for the 16S marker and 22 haplotypes were identified from 43 samples for the ND2 marker. Both the Mauritius and Cape Town invasive populations were found to have originated from the eastern clade. However, they matched the common haplotype from this region which was found across a vast area that spans the KwaZulu-Natal province and into the Mpumulanga and Limpopo provinces. This did not allow for identifying a more precise region for the origin of the founder populations. The presence of haplotypes unique to the Cape Town invasive population, which group with the eastern clade, indicates that there has potentially been more than one introduction event. Demographic analysis revealed a recent population expansion in both the northern (Fs = -2.92) and the eastern clades (Fs = -5.03). Significant genetic variation was found among groups (93.92%), with low variation among populations and among populations within groups. Population pairwise differences were found to be significantly different between all clades except between the central and the southern clade. There was a negligible difference in the genetic diversity of the invasive populations when compared to the eastern clade. The eastern clades’ genetic diversity was low compared to the two other clades and demographic analysis revealed that this region has undergone the most recent population expansion. The negligible difference between the eastern clades’ genetic diversity and both invasive populations indicate that founder effects and genetic bottlenecking should have no impact on the invasive populations. / National Research Foundation (NRF)

Genetically modified maize expressing the Bt-protein Cry1Ab (Bt maize) is planted widely in South Africa. Crop residues of Bt maize often end up in aquatic ecosystems where aquatic organisms are exposed to Cry1Ab protein. The effect of this protein on non-target aquatic organisms has not yet been studied in South Africa. The aim of this study was to evaluate the possible effect of exposure to Bt maize on morphological development of Xenopus laevis and Amietophrynus gutturalis tadpoles. Three experiments were conducted with each of X. laevis and A. gutturalis. Five of these were conducted in the bio-secure Amphibian Biology laboratory and one with A. gutturalis in a shade-house facility where microcosms were exposed to natural conditions. In the first experiment of X. laevis and A. gutturalis, which was replicated three times, large portions of maize leaves were placed in the bottoms of microcosms. X. laevis received supplementary pulverised leaves in suspension while A. gutturalis tadpoles fed on provided leaves. For both control and experimental groups microcosms were divided in three groups receiving respectively 15, 30 and 45 g of maize leaves. In the second and third experiment tadpoles only received pulverised Bt maize leaves in suspension. Each replicate (microcosm) contained 50 one-day old tadpoles. Experiment two was conducted to determine whether the Bt-protein has adverse effects on A. gutturalis tadpoles when tadpoles are exposed to the protein in the water but not feeding on the plant material. A total of 100 tadpoles were used during the experiment and tadpoles were placed individually in 250 ml plastic cups that were filled with 100 ml water witch contained an extract of either Bt and non-Bt maize leaves. Tadpoles were fed twice a week with TetraTabimin bottom-feeding fish pellets in suspension. Experiment three was conducted to determine whether the Bt-protein will have adverse effects on A. gutturalis tadpoles when tadpoles feed on Bt maize leaves. Tadpoles were divided into a treatment in which 50 tadpoles were fed Bt maize leaves and a control treatment in which 50 tadpoles were fed non-Bt maize leaves. Tadpoles were placed individually in 250 ml plastic cups that were each filled with 100 ml borehole water. On a weekly basis 10 randomly selected tadpoles were collected, measured and staged for morphological development, using the Nieuwkoop and Faber Normal Table for X. laevis and Gosner stages for A. gutturalis tadpoles. The significant effects observed in some life history parameters of tadpoles exposed to Cry1Ab protein cannot be ascribed to the effect of the protein. Poor husbandry turned out to be the single most important confounding factor. Before follow-up studies are conducted husbandry practices should be optimized. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.

Genetically modified maize expressing the Bt-protein Cry1Ab (Bt maize) is planted widely in South Africa. Crop residues of Bt maize often end up in aquatic ecosystems where aquatic organisms are exposed to Cry1Ab protein. The effect of this protein on non-target aquatic organisms has not yet been studied in South Africa. The aim of this study was to evaluate the possible effect of exposure to Bt maize on morphological development of Xenopus laevis and Amietophrynus gutturalis tadpoles. Three experiments were conducted with each of X. laevis and A. gutturalis. Five of these were conducted in the bio-secure Amphibian Biology laboratory and one with A. gutturalis in a shade-house facility where microcosms were exposed to natural conditions. In the first experiment of X. laevis and A. gutturalis, which was replicated three times, large portions of maize leaves were placed in the bottoms of microcosms. X. laevis received supplementary pulverised leaves in suspension while A. gutturalis tadpoles fed on provided leaves. For both control and experimental groups microcosms were divided in three groups receiving respectively 15, 30 and 45 g of maize leaves. In the second and third experiment tadpoles only received pulverised Bt maize leaves in suspension. Each replicate (microcosm) contained 50 one-day old tadpoles. Experiment two was conducted to determine whether the Bt-protein has adverse effects on A. gutturalis tadpoles when tadpoles are exposed to the protein in the water but not feeding on the plant material. A total of 100 tadpoles were used during the experiment and tadpoles were placed individually in 250 ml plastic cups that were filled with 100 ml water witch contained an extract of either Bt and non-Bt maize leaves. Tadpoles were fed twice a week with TetraTabimin bottom-feeding fish pellets in suspension. Experiment three was conducted to determine whether the Bt-protein will have adverse effects on A. gutturalis tadpoles when tadpoles feed on Bt maize leaves. Tadpoles were divided into a treatment in which 50 tadpoles were fed Bt maize leaves and a control treatment in which 50 tadpoles were fed non-Bt maize leaves. Tadpoles were placed individually in 250 ml plastic cups that were each filled with 100 ml borehole water. On a weekly basis 10 randomly selected tadpoles were collected, measured and staged for morphological development, using the Nieuwkoop and Faber Normal Table for X. laevis and Gosner stages for A. gutturalis tadpoles. The significant effects observed in some life history parameters of tadpoles exposed to Cry1Ab protein cannot be ascribed to the effect of the protein. Poor husbandry turned out to be the single most important confounding factor. Before follow-up studies are conducted husbandry practices should be optimized. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.