Master of Science / Department of Civil Engineering / Steven K. Starrett / Current aquaponic technology ranges from backyard hobbyist to technologically advanced commercial production. A single source for protein (fish) and nutrients/vitamins (vegetables), development of a technologically simplified commercial-scale system is a realistic solution for many impoverished nations. This study develops a simplified aquaponic facility to be implemented in rural northern Uganda. Research objectives were to: (1) identify simplified commercial-scale system design components, (2) establish a water quality baseline, (3) identify plant/tilapia production ratios, (4) identify construction materials available in northern Uganda, (5) integrate culturally familiar elements, (6) complete preliminary facility design, and (7) calculate facility water balance. The study established that a viable simplified design achieves: (1) water circulation with weir gravity flow and one return pump, (2) tank cleaning with strategically sloped floors and manual waste siphoning, and (3) breeding control with raised bottom fishnets. Submerged aeration is critical to optimal fish growth, and cannot be eliminated despite surface aeration’s low energy appeal. Baseline water quality parameter values of DO > 3 mg/L, pH > 5.5, and TAN > 3 mg/L (2 mg/L average) were established for the pilot study configuration and hydraulic retention time (HRT). A plant/tilapia ratio of 2.5 ft[superscript]2/lb was identified for the proposed facility’s design. The simplified design was assessed compatible with concrete block construction local to northern Uganda. Incorporating the following culturally familiar elements will facilitate technology adoption: utilize native fish (tilapia) and vegetable crops identified in community markets, replace commercially produced plant tank raft components with woven matting from locally available natural materials, and identify the unfamiliar proposed tank design with newly adopted raceway culture techniques at a well-known Ugandan national fishery institute. A proposed facility preliminary design represents local materials, identified plant/tilapia ratio, minimum HRT, and simplified design components for tilapia densities ranging from 12 to 3 gal/lb. With the facility supplied by both rainwater and groundwater, corresponding water balances for 12 to 3 gal/lb densities ranged from a 9,735 gal/yr well supply demand to a 10,984 gal/yr rainwater surplus.

Thesis (MScEng (Mechanical and Mechatronic Engineering))--University of Stellenbosch, 2006. / Successful housing and breeding of exotic animals or plants often requires an environment that is quite different to the ambient conditions present. The current study approached the problem of sub-optimal water temperatures experienced by Central African Bream (Tilapia) housed within a South African greenhouse during winter months. A theoretical and experimental study of fundamental heat and mass transfer processes relevant to an aquacultural greenhouse was conducted. Experimental results were generally in agreement with those of previous researchers; while evaporation tests were found to concur particularly well with an analytical equation developed. The experimental results were used to develop a simple glass greenhouse model to evaluate the expected thermal behaviour during the coldest time of the year. Manipulation of the model revealed that water has the ability to absorb large quantities of solar radiation and regulate temperature fluctuations within such a system, and that the appropriate use of thermal insulation during both the night and day can maintain acceptable water temperatures for extended periods of time. With the conclusions drawn from the experimentation and modelling done, an optimised conceptual greenhouse design was presented, along with associated guidelines and principles for attaining the required water temperatures, and consequently providing the exotic fish specie with a healthy environment.

The bacterial pathogens Streptococcus agalactiae and S. iniae have the capacity to infect a wide range of fish species throughout the world, with Nile tilapia (Oreochromis niloticus) being particularly susceptible. Global tilapia aquaculture production was estimated to be 3.5 million tonnes in 2008, and has a significant contribution in the global farmed fish market. Due to their ability to adapt to a wide range of culture systems the commercialisation of tilapia production has occurred in more than 100 countries. However, countries such as China have suffered from severe and extensive outbreaks of streptococcosis in cultured tilapia continuously for many years. Such large-scale outbreaks in China have resulted in a loss of approximately US$0.4 billion in 2011. Fish are permanently exposed to a plethora of pathogens and natural disease outbreaks are complex host-pathogen interactions that seldom involve single pathogen infections. As a consequence, simultaneous infections, alternatively called concurrent or co-infections, are starting to receive interest from aquatic disease researchers. Streptococcus agalactiae and S. iniae infections can both occur in the same geographic area and both S. agalactiae and S. iniae have been found to be present on the same farm in a single disease outbreak. It has been found that a disease outbreak caused by one these pathogens can be followed by another outbreak from the other. These two pathogens have serious effects on the tilapia aquaculture industry yet there is no information regarding S. agalactiae and S. iniae co-infections. Such information would be valuable for understanding epidemiology and the development of improved treatment and control of aquatic streptococcosis infections. The overall aim of this study was to investigate the pathogenesis of S. agalactiae and S. iniae in Nile tilapia. One important aspect of investigating simultaneous infections was to examine if there was any competition or synergy between S. agalactiae and S. iniae in vitro or in vivo. It was found that competition between S. agalactiae and S. iniae in vitro was inconsistent between different experimental systems. Results indicated that there was either no interaction between bacterial species or they coexisted during in vitro competition assays. Whereas, an in vivo model utilising wax moth larvae (Galleria mellonella) suggested that during a simultaneous infection with S. agalactiae and S. iniae the total levels of larval mortality were lower than expected indicating that the pathogens may have interacted with one another in a competitive manner. Investigations were also conducted to identify the expression of virulence factors in vitro for S. agalactiae and S. iniae. Comparisons were then made to ascertain any inter- and intra-species variation. Results demonstrated that both S. agalactiae and S. iniae strains possessed a capsule but varied in their haemolytic activity, blood survival and resistance to complement-mediated killing. These variations suggested that the two bacterial species differed in their mechanisms of pathogenicity where aquatic S. agalactiae strains may initially have a more systemic spread of infection and aquatic S. iniae strains may utilise a more localised spread of infection within the host. This hypothesis was tested through the development of a robust and reliable challenge model for S. agalactiae and S. iniae in Nile tilapia. Through this work it was apparent that fish infected with S. iniae experienced an acute infection with morbidity/mortality occurring 1 – 3 days after exposure. Whereas, the S. agalactiae challenged fish showed a more chronic infection with morbidity/mortality occurring from 1 – 6 days after exposure. Findings clearly demonstrated a more systemic spread of infection during a S. agalactiae challenge with high bacterial loads in all the organs examined. Streptococcus iniae was observed in fewer organs of infected fish and bacterial numbers were substantially lower. Concurrent infections are complex in natural conditions and in experimental studies. As a result a substantial amount of research will be required to fully understand the nature of co-infection with these two streptococci. This study has provided a solid foundation upon which to base future work.

Assignment (MPhil)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Many species of tilapia such as Oreochromis mossambicus are euryhaline, able to adapt to different salinity waters. Their ability to withstand high salinity levels has given rise to the possibility of using tilapia as baitfish for tuna line fishery. The purpose of the study was to determine the survival rate of tilapia O. mossambicus during direct transfer from freshwater to the salinity levels of 0, 15, 20, 22.5, 25, 27.5, 30, 32.5, and 35 ppt. The data was analysed through means of univariate ANOVAand regression analysis. O. mossambicus showed no mortality to all salinity regimes up to 25 ppt. Mortality was observed at 27.5 ppt, with 100% mortality at 35 ppt. LC 50 and LC 90 were found to be 30.5 and 34.2 ppt, respectively. The results indicate that tilapia (0. mossambicus) will survive a direct transfer to salinities up to 25 ppt. acclimation will be required in the event of transfer to salinity levels above 25 ppt, in order to prevent significant levels of mortalities. / AFRIKAANSE OPSOMMING: Meeste van die tilapia spesies soos Oreochremis mossambicus het die vermoë om by water van verskillende soutgehaltes aantepas. Dit is hierdie vermoë om hoë sout vlakke te weerstaan wat die moontlikheid vir gebruik as lewende aas in die tuna langlyn visvangbedryf moontlik maak. Die doel van hierdie studie was om die oorlewingsvlak van tilapia, O. mossambicus te bepaal by die oorplasing van varswater direk na soutwater by vlakke van 0, 15, 20, 22.5, 25, 27.5, 30, 32.5, en 35 dele per duisend. Die data is verwerk deur gebruik te maak van eenvariant ANOVAen regressie analises. O. mossambicus het geen mortaliteite tot gevolg gehad by al die oorplasings van vlakke tot en met 25 dele per duisend sout nie. Mortaliteite is wel gevind vanaf 27.5 dele per duisend, met 100 % mortaliteite by 35 dele per duisend. LC 50 en LC90 was gewees 30.5 en 34.2 dele per duisend onderskeidelik. Die resultate toon aan dat tilapia (0. mossambicus) sal oorleef by direkte oorplasing na soutwater by vlakke van tot en met 25 dele per duisend. Tilapia wat na hoër vlakke as 25 dele per duisend oorgeplaas wil word, sal eers geleidelik moet akklimatiseer om mortaliteite te beperk.

Thesis (PhD (Botany and Zoology))--Stellenbosch University, 2008. / Endocrine disruption is an alteration of the chemical messaging processes in the body. The value of studies‐ and monitoring of endocrine disruption using techniques included in the field of toxicogenomics is undoubtedly supported by scientific literature over the past four decades, as is demonstrated in Chapter 1 where I review relevant literature on the topic. Clearly, well sustained bio‐monitoring will include studies both in vitro and in vivo, and very well on transcriptional and translational levels. Animals are providing good models for in vivo studies to report or monitor endocrine disruption. It is imperative though to first understand such an animal’s biology, especially its endocrine system, and characterize what is considered “normal” for a species before engaging in endocrine disrupting exposures. A multitude of studies report endocrine disruption in relation to reproductive systems, with more recent work illustrating alteration of metabolism related to thyroidogenic disruption within the last decade. It is therefore essential to consider sex determination and ‐differentiation when studying sentinel species. Apart from the obvious academic interest in the matter of sex differentiation, altered patterns of sex differentiation in certain appropriate species provide for a very convincing endpoint in monitoring estrogenic endocrine disruption. As I approach to study a potential sentinel species for the southern African subcontinent, I set forward to study aspects of endocrine disruption influencing the reproductive system in a piece‐meal manner, starting with estrogenic endocrine disruption as this is the best studied facet of the endocrine disruption hypothesis to date. Yet, one learn from vast amounts of literature that in cases where sex is not exclusively determined by the genetic fraction of an individual, a number other characteristics may very well be used to determine estrogenic disruption in ecosystems. Quantitative production of the egg yolk precursor protein (vitellogenin) resides under these characteristics, and in the proposed sentinel, South African tilapiine, Oreochromis mossambicus phenotypic sex can be altered by environmental sex determination. The present study therefore targeted firstly the product most often used in tier I screening processes, vitellogenin (VTG). Specimens of O. mossambicus were cultured for this purpose from wild breeding stock, sampled at 5 day intervals and the transcription levels of vitellogenin gene (vtg) studied in those. Hereby, Chapter 2 describes the cloning of partial vtg gene and subsequent temporal expression of vtg quantitatively in O. mossambicus. To shed light on the state of gonadal differentiation sub‐samples were subjected to histology, illustrated in Chapter 3. In addition the quantitative vtg responses has been described in this study at a transcriptional level, both of adult males and juveniles subjected to low and very high levels of natural estrogens. In addition, a 3 kb 5’ flanking region of vtg was cloned and sequenced, and several putative binding sites identified for transcription factors of vtg, including several estrogen responsive elements (EREs). These indicate the expected regulational process of vtg by estrogens. Subsequently I measured the transcription levels of the only enzyme capable of aromatizing androgens into estrogens, Cytochrome P450 19 (cyp19) as has been characterized in Chapter 3. For stable binding of an estrogen to an ERE, binding of the ligand to its specific nuclear receptor (Estrogen receptor, ESR) is required. Since E2 is known to have different mechanisms of action in vertebrates, the expression levels of the ESRs were evaluated in our sample set after cloning 3 different homologues of ESR in O. mossambicus. The results on this matter is discussed in Chapter 4 and provides in addition to data on vtg and cyp19 a platform of “normal” transcription levels of these candidate genes involved in estrogenic endocrine disruption of O. mossambicus. Ultimately, characterization of those candidate genes involved extensively in phenotypic sex, contribute to our understanding of sex determination and differentiation in this species in a small way.

Tilapia culture in Indonesia was started with the Mozambique Tilapia (Oreochromis mossambicus) in the 1930’s, and the Nile Tilapia (Oreochromis niloticus) the 1960’s. The genetic improvement program of the Nile Tilapia, has led Indonesia to be one of the main tilapia producers in the world. On the other hand, shrimp aquaculture in the country was not started until the 1960’s, it became more popular after the eye ablation technology for broodstock maturation was developed in the early 1980’s. The first experimental study was conducted to investigate the feasibility of low salinity shrimp farming in a polyculture system with tilapia. Polyculture increased the survival for shrimp (77% compared to 62%), but at the same time decreased the survival of tilapia (87% compared to 97%). Together, the data on survival, specific growth rates, and feed conversion ratios showed that the shrimp performed well at low salinity. The second experimental study investigated the feasibility of brackishwater shrimp farming in a polyculture system with tilapia. Polyculture increased the survival for shrimp (82% compared to 65%), and had higher survival for the tilapia (60% compared to 43%). The Red hybrid Tilapia strain used in the study experienced mortalities after one month, suggesting the need for a salt tolerant strain. The presence of tilapia stimulated the growth of microalgae (Chlorella dominance), promoted higher numbers of heterotrophic bacteria in the water, and had lower presumptive vibrios on TCBS agar. A challenge study was conducted by mixing pathogenic luminescent Vibrio harveyi UAZ-651 into shrimp and tilapia feed. The survival of shrimp in monoculture were significantly lower (20%) compared to in polyculture systems (75 - 95%). Mortality was not found in tilapia. Based on 16S rRNA gene sequence, shrimp monoculture water was dominated by marine Vibrio spp., while the polyculture system had Bacillus spp. and Vibrio spp. with high homology to V. cholerae. The presence of Bacillus spp. which produce a lactonase enzyme AiiA, seems to inhibit vibrio growth. While providing advantages, shrimp-tilapia polyculture might also contribute to streptococcosis transmission. Injecting shrimp with Streptococcus iniae and S. agalactiae resulted in mortalities. S. iniae caused higher mortality in the shrimp cultured in 20 ppt (40%) compared to 10 ppt (20%), and no mortality in 5 ppt. S. agalactiae caused higher mortality in 5 ppt (40%) compared to 10 ppt (20%) and 20 ppt (20%). Quorum sensing (QS) is a density dependent cell to cell communication process in bacteria. Based on challenge studies in shrimp, the luminescent Vibrio harveyi BB120 wild-type strain caused 75-90 % mortality through injection of 106 CFU/shrimp. The mortality patterns in the QS mutants suggest that QS defined, when specific virulence genes were expressed or repressed. As QS in V. harveyi consists of three different circuits, further experiments deployed six mutants lacking either a synthase or a receptor for each circuit. The highest survival in the CqsS (a receptor for CAI-1 circuit) mutant group indicates that the CAI-1 circuit is the most crucial for virulence, followed by the AI-2 and HAI-1 cascades. Chitin acquisition and oxygen scavenging may be two reasons for luminescence in V. harveyi evolution and why they infect shrimp.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: A 2010 Food and Agriculture Organization (FAO) report indicated that 85% of the world´s fish stocks overexploited. This highlights the importance of commercial aquaculture as a sustainable alternative to capture fisheries. Growth rates for international aquaculture production are slowing, although an average annual increase of 12.6% occurred in Africa between 1970 and 2008. Sub-Saharan Africa accounted for 0.50% of the total world aquaculture production in 2008. The South African aquafeed industry recognized the need for aquaculture research in alternative feed ingredients to ensure a more sustainable future. With routine use of antibiotics in aquaculture, antibiotic resistance have emerged, and novel strategies are needed. Currently, alternative feed ingredients and practical substitution of antibiotics are two of the major challenges faced in aquaculture. The use of plant proteins is preferred over animal proteins such as fish meal. By developing sustainable aquafeeds, that have both nutritional and health benefits, the gastrointestinal tract (GIT) microbiota of the host should be considered. The fish GIT is an open system and acts as a route of infection for pathogenic bacteria. A comparative study was done to determine the structure of the GIT microbiota in Mozambique tilapia (Oreochromis mossambicus) and rainbow trout (Oncorhynchus mykiss). Tilapia and trout are important freshwater aquaculture species in South Africa. Tilapia is a warmwater herbivorous fish with a long coiled GIT, whereas trout is a coldwater carnivore and its GIT is short and compartmentalized. Tilapia was reared in recirculation systems with lower water pH and trout in flow-through systems with higher water pH. Tilapia diets consisted of plant-based protein, whereas trout diets contained animal-based proteins. To understand the role of the autochthonous bacteria in the GITs of these fish, we determined community structure along the length of the respective GITs. A highly reproducible DNA fingerprinting technique, automated ribosomal intergenic spacer analysis (ARISA), was used that targeted the intergenic spacer region (ITS) in bacteria. Community analysis based on operational taxonomic units (OTUs) was used to statistically compare the microbiota in different segments. The different GIT compartments in trout also showed distinct bacterial community structures. However, the degree of variation between identical segments between different fish was shown, and consequently, individual microbiotas was not seen. ARISA was further used to determine if formulations containing dietary phytogenics will modify the GIT microbiota. The GIT of fish plays a role in food digestion, while the resident GIT bacteria are important contributors to disease control and nutrient utilization. During two feeding trials tilapia was fed 16 and trout six different diets. Microbial diversity was lower in the GIT of tilapia as compared to trout and no significant shifts in community structure were detected in both species. With a high-throughput DNA sequencing technique, we followed a metagenomic approach to classify the autochthonous microbiota of tilapia and trout on a phylogenetic level. Pyrosequencing was used to define a core microbiota that would enable comparative feeding studies to be carried out. The proposed microbiota of tilapia consisted of genera from the Proteobacteria (5), Firmicutes (1), Actinobacteria (1), Fusobacteria (1) and Cyanobacteria (2). The microbiota of trout comprised genera of the Proteobacteria (3), Firmicutes (3) and Actinobacteria (1). Three genera from the Proteobacteria were common in tilapia and trout, confirming the dominance of this group in the GIT of freshwater fish. The variability and distribution of Lactobacillus in the GITs of both species was determined. Culturing of lactic acid bacteria (LAB) from the GIT of tilapia proves to be difficult and this was confirmed by the low-abundance of particularly Lactobacillus. A number of other LAB genera were identified that confirmed LAB as part of the autochthonous microbiota in freshwater cultured fish. The detection of secondary bacterial fish pathogens as part of the resident microbiota was also established. The use of 454 pyrosequencing in this study, gave a deeper insight into the structure of bacterial communities in the GITs of tilapia and trout. This research is novel and based on our knowledge, the first study to determine the core microbiota in two South African aquaculture species. Data based on community diversity and structure can be used as a basis for further studies. One important study would be to determine the effect environmental stressors would have on the core microbiota. / AFRIKAANSE OPSOMMING: Volgens `n 2010-verslag van die FAO (Food and Agriculture Organization) is 85% van die wêreld se visbronne oorbenut. Dit beklemtoon die belangrikheid van kommersiële akwakultuur as `n alternatief tot visvangste. Internasionaal neem die groeitempo van akwakultuur produksie af, alhoewel groei in Afrika tussen 1970 en 2008 jaarliks met `n gemiddeld van 12.6% toegeneem het. In 2008 was lande suid van die Sahara verantwoordelik vir 0.50% van die wêreld se akwakultuur produksie. Die Suid-Afrikaanse akwavoer industrie het die behoefte raakgesien vir akwakultuur navorsing in alternatiewe voerbestanddele om 'n meer volhoubare toekoms te verseker. Die roetine gebruik van antibiotika in visboerderye het bygedra tot die ontstaan van weerstandbiedende organismes, en daarom is nuwe strategieë nodig. Tans is alternatiewe voeding en die praktiese vervanging van antibiotiese stowwe belangrike uitdagings in akwakultuur. Die gebruik van plantaardige proteïne in viskos word verkies bo dierlike proteïene soos vismeel. Deur die ontwikkeling van volhoubare viskos, wat beide voedingswaarde en voordele vir die gesondheid van visse inhou, moet die mikrobiota in die spysverteringskanaal (SVK) van die gasheer oorweeg word. Die SVK van visse is `n oop sisteem en dien sodoende as `n infeksie kanaal vir patogeniese bakterieë. `n Vergelykende studie is uitgevoer om SVK mikrobiota struktuur te bepaal in Mosambiek tilapia (Oreochromis mossambicus) en reënboogforêl (Oncorhynchus mykiss). Tilapia en forêl is twee belangrike varswater spesies in Suid-Afrikaanse akwakultuur. Tilapia is `n warmwater herbivoor met `n lang, gekronkelde SVK. In teenstelling is forêl `n kouewater vis met `n kort SVK, wat uit verskillende segmente bestaan. Tilapia is in hersirkulasie waterstelsels met `n lae pH gekweek terwyl forêl in deurvloei waterstelsels met `n hoër pH gekweek is. Tilapia diëte het plantaardige proteïne bevat tewyl forêl diëte proteïne vanaf dier-afkoms bevat het. Om die rol van SVK bakterieë beter te verstaan is populasie struktuur bepaal langs die lengte van die SVK. Geoutomatiseerde ribosomale intergeniese spasie analise (ARISA) is `n herhaalbare DNS vingerafdruk tegniek wat die intergeniese spasies gebied (ITS) in bakterieë teiken. Analise gebaseer op operasionele taksonomiese eenhede (OTEs) is gebruik om die mikrobiota van SVK segmente statisties te vergelyk. Die SVK kompartemente in forêl het ook duidelike bakteriële populasie strukture getoon. `n Groot variasie tussen identiese segmente van verskillende vise is waargeneem en individuele mikrobiota kon nie uitgesonder word nie. ARISA is gebruik om die effek van plantaardige dieet formulasies op die SVK mikrobiota te toets. Die SVK van visse speel `n rol in voedselvertering, terwyl die natuurlike SVK bakterieë `n belangrike rol speel in siektebeheer en die benutting van voedingstowwe. Twee voedingsproewe is op tilapia en forêl uitgevoer, met onderskeidelik 16 en 6 verskillende diëte,. Mikrobiese diversiteit was laer in die tilapia SVK in vergeleke met die SVK van forêl en geen beduidende verskuiwings in populasie struktuur is in beide visse waargeneem nie. `n Hoë deurvloei DNS-volgorde bepalings tegniek, gekombineer met `n metagenomiese benadering is gebruik om die mikrobiota van tilapia en forêl op `n taksonomiese vlak te bepaal. “Pyrosequencing” is gebruik om `n kern microbe populasie te identifiseer ten einde vergelykende studies te vergemaklik. Die voorgestelde kern mikrobiota in tilapia het bestaan uit genera van die Proteobakterieë (5), Firmikute (1), Aktinobakterieë (1), Fusobakterieë (1) en Sianobakterieë (2). Die voorgestelde kern mikrobiota van forêl het bestaan uit genera van die Proteobakterieë (3), Firmikutes (3) en Aktinobakterieë (1). Drie genera van die Proteobakterieë het algemeen in tilapia en forêl voorgekom, wat die oorheersing van hierdie groep in die SVK van varswatervisse bevestig. Die veranderlikheid en verspreiding van Lactobacillus in die SVKs van beide visse is bepaal. Dit is moelik om melksuurbakterieë (MSB) uit die SVK van tilapia te isoleer en `n lae voorkoms van Lactobacillus is waargeneem. Ander MSB genera is ook geïdentifiseer wat MSB as deel van die plaaslike mikrobiota in varswater gekweekte visse bevestig. Verder is daar ook vasgestel dat sekondêre patogene deel uitmaak van die plaaslike mikrobiota. In hierdie studie het 454 “pyrosequencing” `n dieper insig gegee oor die struktuur van bakterie-populasies in die SVK van tilapia en forêl. Hierdie navorsing is oorspronklik en volgens die literatuur die eerste studie wat `n kern mikrobe populasie in twee Suid-Afrikaanse akwakultuur spesies waargeneem het. Data wat gebasseer is op populasie diversiteit en struktuur kan gebruik word as basis vir verdere studies. Een so `n studie kan die effek van omgewings stresfaktore op die kern mikrobiota in hierdie vise bepaal.

In externally fertilizing fishes, multiple factors of the spawning environment may affect the sperm viability, and thus the fertilization rate. In this thesis, the sperm activation effect of osmolality of non-electrolytes and electrolytes activation media, pH and ion channel inhibitors on Nile tilapia, Oreochromis niloticus, and the effect of environmentally relevant pollutants (cadmium, malathion and rotenone) on sperm fitness (motility and morphology) were investigated. Seminal fluid samples collected from male fishes (200-250g) were subjected to activation treatments, then analyzed for sperm motility using motility score, and motility variables using Hobson sperm tracker for straight line velocity (VSL), beat cross frequency (BCF) and percentage of motile cells (MOT). For the ion channel inhibitors and pollutants, the effect on sperm motility variables of VSL, VCL (curvilinear velocity) and LIN (linearity) were determined. Multivariate analysis was also carried out to determine the effects of ion channel inhibitors and pollutants on sperm subpopulations. The effects of pollutants on sperm morphology were observed using microscopy techniques, namely, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Sperm motility was initiated when the sperm were exposed to hypoosmotic electrolytes and non-electrolytes solution. We also found that sperm show optimal activity at pH range of 6-8 which depicts that the effect of pH on sperm motility is negligible. Lanthanum (calcium channel blocker) and flunarizine (sodium-calcium exchanger pump blocker) were found to inhibit sperm motility at 25 and 5 µM, respectively, suggesting that both ion channels play a significant role in sperm activation in O. niloticus. In contrast amiloride, ouabain and quinine showed no effects on activation, indicating that epithelial sodium channels, sodium-potassium ATPase and voltage gated potassium channels respectively are unlikely to have major roles in sperm activation or motility. The spermatozoa of Oreochromis niloticus were uniflagellate with clearly differentiated oval-shaped head, midpiece and flagellum. Sperm exposed to hypoosmotic shock showed swelling of the midpiece and sleeve structure. The pollutants showed dose- and time-dependent effect on sperm motility of the fast linear sperm subpopulation. Sperm morphology was not affected. Sperm motility was inhibited at 0.44, 0.03 and 0.063 µM, cadmium, malathion and rotenone respectively. Both cadmium and malathion exerted effects very quickly after exposure. The effect of cadmium, which can exert toxicity by calcium antagonism, is consistent with the effects of calcium channel blockes and further supports an important role for calcium in sperm activation and motility. Malathion had effects at relatively low, environmentally relevant concentrations, suggesting the presence of functionally important acetylcholinesterase activity in sperm, and also the presence of activation cytochrome P450 activity. Rotenone, a well known mitochondrial poison, affected motility only after 15 min of pretreatment. The alteration of sperm trajectories in fast linear spermatozoa subpopulation by pollutants at submicromolar concentrations as demonstrated in our study implies potentially serious consequences for fish populations in polluted environments. Furthermore the results indicate that fish sperm motility as assessed by CASA could be an ecologically relevant, sensitive, and ethically acceptable method for toxicity testing in environmental risk assessment.

M.Sc. / South Africa is renowned for its exploitable mineral resources and continues to be a major player in the world’s mineral markets. The country is well known for containing the world’s largest gold and platinum repositories and electroplating industries, which is the major cause for delivering by-products such as cadmium (Cd), chromium (Cr) and nickel (Ni). Environmental pollution caused by active mining and seepage from closed mines, continuously threatens South African water resources. Such pollution can cause a shift in water chemistry and increase the availability of certain metals to the living organisms of such a system. Even at low concentrations metals are amongst the most toxic environmental pollutants. As a result of their persistence and capacity to accumulate in the environment, metals have a lasting detrimental effect on the ecosystem. Although there is progress in the treatment of metallic wastes, the discharge thereof by industries is still a serious water pollution problem. In the past, chemical analysis of water has proven to be of great use for the detection of pollutants within the environment. The value of chemical analysis alone has become limiting, as chemical analysis supplies information on the levels of chemicals at a certain time. Furthermore, the monitoring of water quality variables often does not reflect long-term events that may play a critical role in determining the ecosystem health. It is now generally understood that measurements of only the physical and chemical attributes of water cannot be used as surrogates for assessing the health of an aquatic ecosystem. The new trend is to incorporate biological monitoring into Abstract existing monitoring strategies. Fish are entirely dependent on the aquatic environment for their survival, rendering them a good monitor of water pollution. Macroscopic changes in organs are preceded by changes at the tissue, cellular or molecular level. These changes are the net result of adverse biochemical and physiological changes within an organism. Histological analysis is a therefore very sensitive parameter and a valuable technique in determining cellular changes in target organs as a result of exposure to stressors. Fish histology can thus be used as an indicator of exposure to contaminants and assess the degree of pollution. Because of the subjective nature of morphological studies correlations with other quantitative studies are difficult. However, incorporation of quantitative methods is essential to the continued development of histopathology as a biomarker of pollution exposure, and to the interpretation of histological responses. The aim of this study is to qualitatively and quantitatively describe the toxic induced histological changes in the selected organs of Oreochromis mossambicus after acute exposure to Cd, Cr and Ni. Fish were exposed to 10% (n=20) and 20% (n=20) of the LC50 concentration of Cd, Cr and Ni respectively under controlled conditions (23 ± 1°C) for 96 hours in an environmental room with a control group (n=5) for each exposure.

Seven families of XX and YY homozygous Oreochromis niloticus were produced by mitotic gynogenesis from XY neofemales and their genetic status was verified by multilocus DNA fingerprinting and progeny testing. Two of these gynogenetic families and their corresponding diploid controls were used with 64 AFLP primer combinations in different levels of screening (XX/YY grand pool; XX/YY family pool; XX/YY gynogenetics and XX/XY control individuals) to search for sex-linked or sex-specific markers. Grand pool screening did not reveal any sex-linked markers. Subsequent family pool and individual level screening identified four sex-linked AFLP markers from two primer combinations, three Y-linked (OniY425, OniY382, OniY227) and one X-linked (OniX420). Two of these (OniX420, OniY425) were shown to be allelic. Single locus PCR markers were developed for all of those markers. Linkage analysis of these markers and the sex locus within the source families revealed tight linkage, with estimated map distances of 13cM, 17cM and 20cM for OniY382, OniY227 and OniX420/OniY425 respectively. However, these sex-linked AFLP markers failed to consistently identify sex in unrelated individuals. To develop an effective system for parentage analysis in normal and gynogenetic progeny, AFLPs and multiplexed polymorphic microsatellite loci were investigated. Both were found to be effective, but microsatellites were more appropriate since they are codominant and some loci showed high gene-centromere recombination rates, suitable for discriminating meiotic from mitotic gynogenetics, while AFLPs are dominant markers. Spontaneous diploidization of the maternal chromosome set (SDM) was observed in gynogenetic progeny of one XY neofemale. Maternal inheritance and ploidy status were verified by multilocus DNA fingerprinting and chromosome karyotyping. Close genetic linkage between the red gene and an autosomal sex-reversal gene(s) in gynogenetic progeny and influences of autosomal sex-reversal gene(s) producing males in a fully inbred XX clonal line were previously reported in O. niloticus. To test if the same autosomal sex-reversal locus was responsible in both cases, a series of test crosses was carried out involving XX clonal neomale(s) and homozygous red females. The results indicated the involvement of more than one autosomal sex-reversal locus, one of which is linked to red body colour.