M.Sc. / The tigerfish, Hydrocynus vittatus, is one of the most sought-after game fish in Southern Africa, and a vital source of protein and income in contiguous communities. Unfortunately, this species has been placed on the protected species list, alongside the infamous great white shark (Carcharodon carcharias). The loss of this species will not only be disadvantageous to local communities, but will also have a damaging impact on the Southern African tourism industry. After reviewing all available literature on tigerfish, it was noted that studies on the genetics, morphology, distribution, age, growth and maturity were severely lacking. Research on the Pongolapoort Dam and Phongolo River tigerfish was also practically nonexistent. The aims of this study were therefore to: (1) analyse the genetic variation and diversity of five Southern African tigerfish populations and comment on the use of the various populations as possible candidates for restocking; (2) assess whether the genetic composition and caudal colour can be linked to the biogeography of the species; (3) determine the growth patterns of tigerfish otoliths and establish the best ageing technique for this species; and (4) determine the age structure of tigerfish from the Pongolapoort Dam in KwaZulu-Natal, South Africa. Before analysing the genetic composition of various tigerfish populations, it was hypothesised that individuals from the Okavango and Phongolo systems would possess the highest and lowest heterozygosity, respectively. Tigerfish from the Olifants River would be the best choice of brood stock, should population restocking become necessary. The muscle and liver tissues of 117 tigerfish from the Upper Zambezi, Okavango Delta, Olifants and Phongolo Rivers and Pongolapoort Dam, were assessed by starch gel electrophoresis.

A (Na+,K+)-ATPase presente no tecido branquial dos crustáceos osmorreguladores é um componente essencial do sistema de regulação iônica e osmótica e de excreção ativa de NH4 +. Dessa forma, a caracterização cinética da (Na+,K+)-ATPase branquial de Clibanarius vittatus pode fornecer dados importantes para a compreensão dos mecanismos bioquímicos desses processos. Nesse sentido, foram realizados ensaios cinéticos com a fração microsomal obtida do tecido branquial de C. vittatus. Os dados obtidos revelaram a presença de um único pico com atividade ATPase. Os dados obtidos a partir da hidrólise do substrato ATP revelaram a presença de dois sítios, um de alta afinidade que apresenta interação sítio-sítio (nH=1,9; K0,5= 63,8 ± 2,9 nM e V= 19,1 ± 0,8 U/mg) e outro de baixa afinidade, com características Michaelianas (nH=1,0; KM= 44,1 ± 2,6 mM e VM= 123,5 ± 6,1 U/mg). Além disso, foi observada interação do tipo sítio-sítio (nH= 1,8) para estimulação dos íons magnésio (V= 132,0 ± 5,3 U/mg e K0,5= 0,36 ± 0,02 mM). Por outro lado, as interações dos íons sódio (K0,5= 7,4 ± 0,4 mM), potássio (K0,5= 1,51 ± 0,05 mM) e amônio (K0,5= 4,5 ± 0,2 mM) obedeceram a uma cinética Michaeliana. Os dados obtidos mostraram que em condições saturantes de íons potássio, a atividade enzimática aumenta de maneira concentração-dependente quando na presença de íons amônio (V= 290,8 ± 14,4 U/mg e KM= 2,9 ± 0,1 mM). A presença da ouabaína no meio reacional acarretou 66% de inibição (KI= 117,3 ± 3,5 mM), indicando a presença de outras ATPases. A atividade K+-fosfatase, medida usando o substrato sintético PNFF revelou a presença de um único sítio de hidrólise (V= 47,4 ± 1,9 U/mg e KM= 1,0 ± 0,04 mM) com características Michaelianas (nH= 1,1). Entretanto, para os íons Mg2+ (nH= 2,6 e K0,5= 0,6 ± 0,1 mM), K+ (nH= 1,4 e K0,5= 3,7 ± 0,1 mM) e NH4 + (nH= 1,9 e K0,5= 19,3 ± 0,7 mM) foram observadas interações sítio-sítio. Em conjunto, os resultados cinéticos obtidos para a (Na+,K+)-ATPase branquial de C. vittatus, reforçam a possibilidade de que essa enzima contribui eficientemente com o mecanismo adaptativo desencadeado em resposta à exposição a altas concentrações externas de amônia. É importante ressaltar que o presente trabalho representa o primeiro estudo de caracterização cinética da enzima (Na+,K+)- ATPase no ermitão C. vittatus, um animal inserido em um microambiente constituído pela concha. Finalmente, este trabalho agrega informações relevantes relacionadas ao grupo dos Decapoda, em relação ao papel da (Na+,K+)-ATPase nos processos metabólicos e de osmorregulação nos crustáceos. / (Na+,K+)-ATPase is an essential component of osmotic and iono-regulatory system of crustacean gill and plays a relevant role in NH4 + excretion. Thus, the kinetic characterization of Clibanarius vittatus gill microsomal (Na+,K+)-ATPase, can provide crucial data to the understanding of osmoregulating and excretion processes of these animals in different environments, especially the shell microenvironment. In this way, kinetic assays were performed with gill microsomal fraction. The results showed a single protein peak with ATPase activity. The results from ATP hydrolysis revealed the presence of two different sites. One of them is related to the high-affinity with site-site interaction (nH=1.9; K0.5= 63.8 ± 2.9 nM and V= 19.1 ± 0.8 U/mg) and the other shows low-affinity, obeying Michaelis-Menten kinetics (nH=1.0; KM= 44.1 ± 2.6 mM and VM= 123.5 ± 6.1 U/mg). Moreover, site-site interaction (nH= 1.8) was observed to the magnesium ions stimulation (V= 132.0 ± 5.3 U/mg and K0.5= 0.36 ± 0.02 mM). On the other hand, the stimulations of Na+ (K0.5= 7.4 ± 0.4 mM), K+ (K0.5= 1.51 ± 0.05 mM) and NH4 + (K0.5= 4.5 ± 0.2 mM), followed Michaelis-Menten kinetics. At saturating concentrations of potassium ions, the enzyme activity was increased in a dependent-concentration manner with increasing ammonium ions concentration (V= 290.8 ± 14.4 U/mg and KM= 2.9 ± 0.1 mM). Ouabain inhibited 66% of (Na+,K+)-ATPase activity (KI= 117.3 ± 3.5 mM) of the gill microsomal enzyme, indicating the presence of other ATPase activities. The K+-fosfatase activity was also measured using PNPP, a synthetic substrate. PNPP hydrolysis resulted in single site hydrolysis site, obeying the Michaelis-Menten kinetics (nH= 1.1). However, it was observed site-site interactions for Mg2+ (nH= 2.6; K0.5= 0.6 ± 0.1 mM), K+ (nH= 1.4; K0.5= 3.7 ± 0.1 mM) and NH4 + (nH= 1.9; K0.5= 19.3 ± 0.7 mM) ion estimulations. Taken together, these results provide important informations on the kinetic properties of gill (Na+,K+)-ATPase of C. vittatus, emphasizing the synergiistic stimulation induced by K+ and NH4 +, which appear to contribute to the efficient adaptive mechanism triggered by exposure of high external ammonia concentrations. It is important to emphasize that this is the first study on the kinetic properties of (Na+,K+)-ATPase enzyme of the hermit crab C. vittatus. Finally, this study provide information concerning the (Na+,K+)-ATPase role in the metabolic and osmoregulating mechanisms of this Decapoda group

A (Na+,K+)-ATPase presente no tecido branquial dos crustáceos osmorreguladores é um componente essencial do sistema de regulação iônica e osmótica e de excreção ativa de NH4 +. Dessa forma, a caracterização cinética da (Na+,K+)-ATPase branquial de Clibanarius vittatus pode fornecer dados importantes para a compreensão dos mecanismos bioquímicos desses processos. Nesse sentido, foram realizados ensaios cinéticos com a fração microsomal obtida do tecido branquial de C. vittatus. Os dados obtidos revelaram a presença de um único pico com atividade ATPase. Os dados obtidos a partir da hidrólise do substrato ATP revelaram a presença de dois sítios, um de alta afinidade que apresenta interação sítio-sítio (nH=1,9; K0,5= 63,8 ± 2,9 nM e V= 19,1 ± 0,8 U/mg) e outro de baixa afinidade, com características Michaelianas (nH=1,0; KM= 44,1 ± 2,6 mM e VM= 123,5 ± 6,1 U/mg). Além disso, foi observada interação do tipo sítio-sítio (nH= 1,8) para estimulação dos íons magnésio (V= 132,0 ± 5,3 U/mg e K0,5= 0,36 ± 0,02 mM). Por outro lado, as interações dos íons sódio (K0,5= 7,4 ± 0,4 mM), potássio (K0,5= 1,51 ± 0,05 mM) e amônio (K0,5= 4,5 ± 0,2 mM) obedeceram a uma cinética Michaeliana. Os dados obtidos mostraram que em condições saturantes de íons potássio, a atividade enzimática aumenta de maneira concentração-dependente quando na presença de íons amônio (V= 290,8 ± 14,4 U/mg e KM= 2,9 ± 0,1 mM). A presença da ouabaína no meio reacional acarretou 66% de inibição (KI= 117,3 ± 3,5 mM), indicando a presença de outras ATPases. A atividade K+-fosfatase, medida usando o substrato sintético PNFF revelou a presença de um único sítio de hidrólise (V= 47,4 ± 1,9 U/mg e KM= 1,0 ± 0,04 mM) com características Michaelianas (nH= 1,1). Entretanto, para os íons Mg2+ (nH= 2,6 e K0,5= 0,6 ± 0,1 mM), K+ (nH= 1,4 e K0,5= 3,7 ± 0,1 mM) e NH4 + (nH= 1,9 e K0,5= 19,3 ± 0,7 mM) foram observadas interações sítio-sítio. Em conjunto, os resultados cinéticos obtidos para a (Na+,K+)-ATPase branquial de C. vittatus, reforçam a possibilidade de que essa enzima contribui eficientemente com o mecanismo adaptativo desencadeado em resposta à exposição a altas concentrações externas de amônia. É importante ressaltar que o presente trabalho representa o primeiro estudo de caracterização cinética da enzima (Na+,K+)- ATPase no ermitão C. vittatus, um animal inserido em um microambiente constituído pela concha. Finalmente, este trabalho agrega informações relevantes relacionadas ao grupo dos Decapoda, em relação ao papel da (Na+,K+)-ATPase nos processos metabólicos e de osmorregulação nos crustáceos. / (Na+,K+)-ATPase is an essential component of osmotic and iono-regulatory system of crustacean gill and plays a relevant role in NH4 + excretion. Thus, the kinetic characterization of Clibanarius vittatus gill microsomal (Na+,K+)-ATPase, can provide crucial data to the understanding of osmoregulating and excretion processes of these animals in different environments, especially the shell microenvironment. In this way, kinetic assays were performed with gill microsomal fraction. The results showed a single protein peak with ATPase activity. The results from ATP hydrolysis revealed the presence of two different sites. One of them is related to the high-affinity with site-site interaction (nH=1.9; K0.5= 63.8 ± 2.9 nM and V= 19.1 ± 0.8 U/mg) and the other shows low-affinity, obeying Michaelis-Menten kinetics (nH=1.0; KM= 44.1 ± 2.6 mM and VM= 123.5 ± 6.1 U/mg). Moreover, site-site interaction (nH= 1.8) was observed to the magnesium ions stimulation (V= 132.0 ± 5.3 U/mg and K0.5= 0.36 ± 0.02 mM). On the other hand, the stimulations of Na+ (K0.5= 7.4 ± 0.4 mM), K+ (K0.5= 1.51 ± 0.05 mM) and NH4 + (K0.5= 4.5 ± 0.2 mM), followed Michaelis-Menten kinetics. At saturating concentrations of potassium ions, the enzyme activity was increased in a dependent-concentration manner with increasing ammonium ions concentration (V= 290.8 ± 14.4 U/mg and KM= 2.9 ± 0.1 mM). Ouabain inhibited 66% of (Na+,K+)-ATPase activity (KI= 117.3 ± 3.5 mM) of the gill microsomal enzyme, indicating the presence of other ATPase activities. The K+-fosfatase activity was also measured using PNPP, a synthetic substrate. PNPP hydrolysis resulted in single site hydrolysis site, obeying the Michaelis-Menten kinetics (nH= 1.1). However, it was observed site-site interactions for Mg2+ (nH= 2.6; K0.5= 0.6 ± 0.1 mM), K+ (nH= 1.4; K0.5= 3.7 ± 0.1 mM) and NH4 + (nH= 1.9; K0.5= 19.3 ± 0.7 mM) ion estimulations. Taken together, these results provide important informations on the kinetic properties of gill (Na+,K+)-ATPase of C. vittatus, emphasizing the synergiistic stimulation induced by K+ and NH4 +, which appear to contribute to the efficient adaptive mechanism triggered by exposure of high external ammonia concentrations. It is important to emphasize that this is the first study on the kinetic properties of (Na+,K+)-ATPase enzyme of the hermit crab C. vittatus. Finally, this study provide information concerning the (Na+,K+)-ATPase role in the metabolic and osmoregulating mechanisms of this Decapoda group

Tadpoles are known to behaviorally respond to visual, auditory or chemical cues. I examined the influence of group size and light conditions on the activity of Phyllobates vittatus. I performed laboratory experiments to measure locomotor activity. Activity levels of tadpoles were higher in the groups. Light conditions did not influence activity level of tadpoles. My results suggest that the group size is one of the main factors influencing tadpole locomotor activity of Phyllobates vittatus.

M.Sc. / The tigerfish, Hydrocynus vittatus member of the Alestidae, are ferocious and fierce predators capable of consuming prey up to 40% of their size. In South Africa H. vittatus can be found in the major east-flowing rivers from the Limpopo River to the Phongolo River flowing into the Pongolapoort Dam making up the southernmost population. The Pongolapoort Dam also known as Lake Jozini is located in the high-rainfall subtropical region of northern KwaZulu-Natal. The Pongolapoort Dam wall was completed in 1973 to make it the fifth largest dam in South Africa with a total surface area of 2 445.9 x 106 m2. It was primarily built for the irrigation of sugar-cane in the surrounding regions. The Fish Health Assessment Index is used as a quantitative index that identifies morphological alterations and assigns them a score for statistical comparison. The main purpose of the macroscopic Fish Health Assessment Index is to detect gross changes in the health of fish populations through necropsy early enough for remedial actions to be put into place. Histopathology is used as an important diagnostic tool for detecting infectious and non-infectious diseases and can therefore be used to assess the health of fish populations by analysing selected target organs. Catch and release (C&R) angling has become a very widely promoted conservation tool as sport angling continues to increase. Anglers release their fish under the assumption that the fish will survive; however, the end results have been shown to vary greatly, from death of the fish to sub-lethal end points such as exhaustive exercise, injury and/or air exposure which will affect the fitness of the fish. The first aim of the study was to determine the health status of H. vittatus and C. gariepinus from the Pongolapoort Dam by means of a histology-based fish health assessment protocol applied to a total of 45 H. vittatus specimens collected in February 2009 (n = 30) and July 2009 (n = 15) and a total of 19 C. gariepinus specimens collected in October 2009 (n = 10) and April 2010 (n = 9). In the H. vittatus specimens DDT was found in the axial muscle; the concentrations of these in February 2009 (5 403.9 ng∙g-1 lipid) and July 2009 (5 537.4 ng∙g-1 lipid). The Mean Index values showed that the Kidney Index (IK), Gill Index (IG) and Fish Index (IFISH) were higher in fish from the February survey while the Liver Index (IL) was higher in those collected during July. Liver alterations identified included intercellular oedema, granular degeneration, vacuolation, nuclear pleomorphism and lymphocyte infiltration. Kidney alterations included dilation of the glomerulus capillaries, vacuolation and hyaline droplet degeneration. Gill alterations identified included telangiectasia and hyperplasia of the secondary lamella, congestion, and rupture of pillar cells.

M.Sc. (Environmental Management) / The Kruger National Park (KNP) is a world renowned wildlife reserve and a source of South African ecotourism benefiting the economy. The Olifants River is the largest river running through the KNP and it is known to be one of South Africa’s most polluted rivers. In the winters of 2008 and 2009 Nile crocodile carcasses were found in the Olifants River gorge in the KNP. In a very short period nearly the entire population of Nile crocodile was lost, the cause of which was later identified as pansteatitis. Pansteatitis is caused by lipid peroxidation known to be nutritionally mediated, as it is associated with diets high in polyunsaturated fats, often of fish origin and insufficient antioxidants specifically vitamin E, which is solely synthesized by plants and must therefore be obtained through diet. The hypothesis of this study is therefore that the fish inhabiting the Olifants River are antioxidant deficient, more specifically, vitamin E deficient, causing the wildlife e.g. crocodiles and predatory fish species, that feed on the fish, to become vitamin E deficient as well, and subsequently develop pansteatitis. If the hypothesis is true, the top fish predator in this aquatic system, the tigerfish Hydrocynus vittatus, should therefore also exhibit signs of dietary vitamin E deficiency. The aim of this study was to determine if tigerfish from the Olifants River in the KNP are exhibiting any signs of antioxidant deficiency, specifically vitamin E deficiency. The objectives to accomplish this aim were firstly to analyse the total plasma antioxidant activity using an ELISA assay, and secondly, to perform a histology-based fish health assessment on the target organs of the tigerfish to identify any histological alterations, specifically those known to be associated with vitamin E deficiency. The results were compared to the results from fish of the same species from two reference sites where there have been no signs of vitamin E deficiency, such as pansteatitis-related wildlife deaths. The results of the plasma analysis as well as the histological assessment showed no conclusive signs of vitamin E deficiency in tigerfish from the Olifants River. It is therefore unlikely that the pansteatitis in the affected wildlife is caused by dietary vitamin E deficiency in the aquatic system, but rather by vitamin E depleting mechanisms such as the presence of high amounts of toxicants with pro-oxidant properties, which may be causing an imbalance of pro-oxidants and antioxidants within the body. Hence the rate of vitamin E regeneration cannot keep up with the rate of oxidation. This is occurring only in certain species due to their specific behaviour or eating habits.

M.Sc. / Pollutants present in minute concentrations in aquatic environments and which possess long residence times may be accumulated by aquatic organism such as fish, resulting in adverse affects. Bioaccumulation and biomarker responses are often used to qualify and quantify pollutant exposure and effect, and for this reason form a major part of many environmental assessments. To interpret bioaccumulation and biomarker responses the physico-chemical parameters of the environment should be known. This study aimed to spatially and temporally assess the environmental partitioning of heavy metals in three South African freshwater systems, namely the Pongolapoort Dam, Olifants and Luvuvhu Rivers, and to relate these concentrations to bioaccumulation and biomarker responses in tigerfish, Hydrocynus vittatus. This is because there is relatively little known about the bioaccumulation potential and stress responses of tigerfish to pollutants and they have recently become listed as a protected species. Result from this study showed that there were few differences between seasons in terms of metal bioaccumulation in the Pongolapoort Dam with the exception of Se, Zn and Fe. Selenium and Fe concentrations were linked to concentrations found in the environment, whereas Zn was attributed to a disruption in homeostasis within the fish. Increases in MT were found during the winter months and were attributed to increased metal concentrations at this time, namely Zn and Se, whereas decreases in CEA and PC were observed at this time and were linked to depleted energy reserves, stress and a reduction in the presence of pesticides as a result of decreased runoff during the winter months. It was found in the Olifants and Luvuvhu Rivers that there were no distinct decreases in metal concentrations as the rivers flowed through the KNP, and processes such as rainfall, remobilization of sediments, distance of the study area from the source and geology played a great role in the distribution of metals. Metal concentrations in the Olifants River water, sediment and fish were, for the most part, found to be lower than previous studies, possibly due to improvement in management strategies or increased buffering of this river. Only Al and As were significantly higher in tigerfish from the Olifants River, and this was reflected in high MT concentrations. It was suggested that tigerfish from the Olifants River have developed effective mechanisms for the excretion and detoxification of heavy metals that they are exposed to as a result of extended exposure. Concentrations of AChE were also significantly inhibited in tigerfish from the Olifants River which is indicative of greater concentrations of organophosphates and carbamate pesticides than the other sites. Tigerfish from the Pongolapoort Dam had signifcantly higher levels of MT and significantly inhibited concentrations of AChE in comparison to tigerfish from the Luvuvhu River. The tigerfish from the Luvuvhu River had significantly higher concentrations of Se in muscle tissue. Tigerfish from the Luvuvhu River, also experienced stress as a result of pollution as was apparent from significantly depleted energy reserves in comparison to the other sites under study, and higher concentrations of PC and CYP1A which are typical biomarkers responding to halogenated and aromatic pesticides, such as deltamethrin and endosulfan. It was recommended that further studies be done to assess the presence of pesticides within these systems to determine the contribution of these pollutants to the state of tigerfish

Following the construction of the Pongolapoort Dam in 1974, the potential impacts on the lower Phongolo River and floodplain due to alterations in the natural flow regime have caused concerns. Fish communities and population structures are directly influenced by these alterations. The only protected section of the Phongolo River is a 15 km reach and its associated pans that flow through the Ndumo Game Reserve. Historic data shows that the community and population structure of the fish in the Phongolo Floodplain has changed as a result of irregular flood releases. The Ndumo Game Reserve plays an important role in the conservation of many ecologically and economically important species as the pans inside the reserve serve as a refuge area in which these species can breed to replenish the fish numbers in the Phongolo River. The fish diversity inside the Ndumo Game Reserve is also higher when compared to outside. During the high flow period fish move into the floodplain pans as a result of the higher water level. Oreochromis mossambicus, the most common species found in this area, utilize Nyamiti Pan for breeding but the health of this species is under pressure due to severe infestations of Lernaea cyprinacaea and nematode parasites. Lernaea cyprinacaea is a parasitic copepod associated with the introduced exotic fish species Cyprinus carpio which is found in large numbers in various pans inside Ndumo Game Reserve. The presence of this alien species in the refuge area raises concerns as it competes for the same resources as the economically important native fish species. Nyamiti Pan is largely populated by adult cichlid species between the ages of six and ten years old. The importance of flood releases which simulate natural flow regime is emphasised by the negative impacts irregular floods have on fish health, community and population structure. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Following the construction of the Pongolapoort Dam in 1974, the potential impacts on the lower Phongolo River and floodplain due to alterations in the natural flow regime have caused concerns. Fish communities and population structures are directly influenced by these alterations. The only protected section of the Phongolo River is a 15 km reach and its associated pans that flow through the Ndumo Game Reserve. Historic data shows that the community and population structure of the fish in the Phongolo Floodplain has changed as a result of irregular flood releases. The Ndumo Game Reserve plays an important role in the conservation of many ecologically and economically important species as the pans inside the reserve serve as a refuge area in which these species can breed to replenish the fish numbers in the Phongolo River. The fish diversity inside the Ndumo Game Reserve is also higher when compared to outside. During the high flow period fish move into the floodplain pans as a result of the higher water level. Oreochromis mossambicus, the most common species found in this area, utilize Nyamiti Pan for breeding but the health of this species is under pressure due to severe infestations of Lernaea cyprinacaea and nematode parasites. Lernaea cyprinacaea is a parasitic copepod associated with the introduced exotic fish species Cyprinus carpio which is found in large numbers in various pans inside Ndumo Game Reserve. The presence of this alien species in the refuge area raises concerns as it competes for the same resources as the economically important native fish species. Nyamiti Pan is largely populated by adult cichlid species between the ages of six and ten years old. The importance of flood releases which simulate natural flow regime is emphasised by the negative impacts irregular floods have on fish health, community and population structure. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015