Advances in aquaculture technologies are being investigated to support the replenishment of local fisheries, develop marine food fish farming opportunities and to increase seafood production globally. In order to promote the expansion and development of aquaculture technologies required to raise new finfish species, a number of key bottlenecks restricting commercial-scale culture need to be addressed, including the ability to control fish reproduction in captivity and to produce high quality seeds. One candidate species for large-scale production, and the focus of this work, is common snook. Prized as a food fish in Mexico, Central and South America and as a popular game fish along the Gulf coast of the United States; common snook are economically important having both a high market value and recreational demand. Despite recent advances in captive spawning, a number of reproductive bottlenecks still need to be addressed such as lack of spontaneous spawning in captivity, poor fertilization rates and inconsistent production of high quality eggs and larvae. Therefore, the overall aim of this thesis was to better understand the reproductive biology of common snook in order to develop protocols to improve the reliability of captive spawning in closed recirculating aquaculture systems and the quality of eggs produced as a basis for commercial scale cultivation. First, this PhD project described oocyte development in common snook and validated a non-invasive method for assessing reproductive condition in wild and captive stocks (Chapter 2). This was done by using a tiered and adaptable staging scheme to compare the wet mount technique with histological preparations of ovarian biopsies. When compared with histology, the wet mount provided an immediate and precise method for determining whether female broodstock were candidates for hormonal induction. In fishery biology, an understanding of fish reproductive success and population reproductive potential is critical for designing and implementing effective fisheries management strategies. The wet mount technique provides a tool for non-lethal, low-cost determination of reproductive status in wild fish stocks. The next research chapter focused on spawning induction of captive snook populations. The first trial compared the effects of slow and regular release GnRHa implants whereas the second trial investigated the effects of GnRHa, alone or in combination with the dopamine antagonist, pimozide (PIM), on milt characteristics and plasma steroid levels in captive male common snook broodstock (Chapter 3). In an effort to better enable reliable control of reproduction under captive conditions, the annual plasma sex steroid profile of captive male and female broodstock maintained under natural photo-thermal conditions was also examined. When possible, milt samples were collected pre and post implantation; sperm density, sperm motility and spermatocrit were documented among individual males. The assigned treatments appeared to have no or little effects on milt production in male broodstock although plasma steroid levels were found to be significantly elevated in individuals treated with GnRHa in combination with the dopamine antagonist, pimozide. At the time this work was performed, no data on spawning dynamics, including individual spawning performance, had been reported for common snook in captivity. Mass spawning tanks are complex systems where fish are left to spawn naturally and fertilized eggs are collected with little or no control over the mating of the animals. Therefore, the third part of this thesis explored the potential of DNA profiling for monitoring mating outcomes in captive broodstock by employing eight microsatellite markers to detect and quantify individual parental contributions for 2,154 larvae obtained from the three broodstock tanks (Chapter 4). The panel of loci was generally robust and allowed unambiguous assignment of 89% of larvae to a single family. Overall, spawn contribution data 1) provided a confirmation of GnRHa treatment efficacy in female snook with a minimum stage of oogenesis (late secondary growth-SGl) required for successful spawning, 2) identified a potential impact of handling on maturation and spawning of captive broodstock and 3) confirmed that, through photothermal conditioning, captive broodstock can spawn over consecutive days and several times per year including outside of their natural spawning season. The exogenous cues that tropical species use to synchronize key life events like reproduction remain largely unstudied, therefore, my PhD project also investigated the influence of tidal cycle on reproductive activity in common snook (Chapter 5). Real-time quantitative RT-PCR assays were developed and validated to measure the temporal expression patterns of gonadotropin genes (fshβ and lhβ) during the reproductive cycle in males and females. These were evaluated in relation to sex steroid production, LH blood plasma levels, gonadal development and tidal cycle. The phylogenetic analysis of the deduced amino acid sequence of common snook for fshβ and lhβ revealed strong identity with other teleosts (75-90%). Additionally, the mRNA profiles of fshβ and lhβ in the pituitary of females displayed a clear pattern of expression concomitant with histological changes in oocyte development. Histological observations of gonads suggested a circa-tidal rhythm of follicular development. The findings, as a whole, provided new information supporting the role of tidal cycle on the entrainment of gametogenesis allowing for a better understanding of the environmental control of reproduction in common snook. Although the primary research emphasis in this PhD was on broodstock spawning and gamete quality, the final chapter focuses on larval ontogeny. The goal of this research was to gain improve understanding of the early life history characteristics of common snook in order to improve larval culture technologies. To do so, a combination of digital photography and histological techniques were used to document the embryonic and early larval development (0 to 14 days post hatch-DPH) of hatchery-reared individuals (Chapter 6). Larvae hatched 15 h after fertilization at 28°C, lacked pigmentation, had a rudimentary digestive tract and undeveloped visual system. Development was rapid and by 3 DPH larvae had almost doubled in length, the yolk sac was nearly exhausted, the mouth was open and eyes were pigmented with a well-structured retinal layer. The alimentary canal was differentiated into three distinct sections including the foregut, midgut and hindgut. Food was observed in the gut (rotifers) and structural epithelium organelles, such as the nucleus, mitochondria, and dark vesicles, were all present in high numbers. The swim bladder was formed and inflated. In summary, understanding early ontogenetic development in common snook can help provide information needed to address key bottlenecks seen in captive cultivation, such as the high incidence of larval mortality observed during the transition from endogenous to exogenous feeding. Overall, this doctoral work 1) validated molecular and endocrine analytical tools for future studies of common snook reproductive physiology, 2) provided a better understanding of both broodfish requirements in tank systems as well as the endocrine control of reproduction and spawning at the level of the brain-pituitary-gonadal axis, 3) increased our knowledge in genetic management of captive broodstock, in terms of parentage assignment and 4) offered new insight into wild population reproductive strategy as well as how reproduction is entrained through environmental cues and the pathways leading to oocyte recruitment and maturation. The new information presented here can be used to conserve wild snook stocks through production of farm raised individuals as a sustainable source of seafood and for fisheries enhancement.

M.Sc. (Zoology) / Please refer to full text to view abstract

The research assessed the potential of developing a selective breeding programme for the UK rainbow trout industry. Levels of genetic variation at 12 microsatellite loci were first compared in seven different commercial strains. The Observed heterozygosity ranged from Ho = 48.1% in a gold rainbow trout strain (GTR) to Ho = 66.4% in a newly derived broodstock population constructed from a number of different sources (GIT). The Expected Heterozygosity (He) was highest in GIM1 (He= 79.5%) and lowest in the GTR strain (He = 56.9%). The Effective number of alleles (Mae) showed that the GIM1, GIM2, GIM3, and GIT strain (5.4; 5.2; 4.8; 4.2) were significantly more variable than the other strains and that GTR strain had the lowest value (2.5). There appears to be substantial genetic variability within the commercial United Kingdom rainbow trout strains surveyed in this study. This appears to be the case despite very different management histories and levels of record keeping. The strains appear to be genetically distinct (based on population genetic analyses), though the reasons for this remain unclear (and possibly unanswerable given the poor records kept by the different companies). The Glenwyllin farm strains (GIM) were chosen to form the base population for the project because of their high genetic variability, disease free status and because the farm produced around 20 million ova per year, so any genetic gains would have a widespread impact. The farm has an early (Strain A) and a late spawning (Strain B) and these were mated in a partial factorial design, 20 females and 20 neomales per strain (A & B) were chosen on the basis of maturity and gamete quality in November 2002 so that each male was crossed to 4 females (2 in the same strain and 2 in the other), a total of 160 families were created. All broodstock were biopsied to enable them to be genotyped. The families were reared separately up to the eyed stage at which point the eggs from each family were divided into three to generate three communal replicate populations. One of these was sent to a fingerling producer (Iwerne Spring) for ongrowing to fingerling size and formed the basis of a commercial production trial at Test Valley Trout farm (TVT) in Hampshire. When the fish reached an average weight of 5 g they were transferred from Iwerne Spring to TVT and 1500 were randomly selected, PIT tagged and biopsied to enable them to be assigned to their family using 11 multiplexed microsatellite loci. Parental assignment was based on exclusion (FAP) but the results were compared with another parental assignment based on likelihood (PAPA). Of the 1500 offspring (OIM) PIT tagged 1242 82.8% could be assigned to a single family utilizing different combinations of more than 6 loci (6 to 11). The growth of the 1500 OIM fish was tracked throughout the grow out period before they were finally harvested and fully processed. The results of OIM strain at the end of the trial period were mean weight of 415.5 g, and a mean length of 314.5 mm. The visual measurement of colour gave a mean flesh colour values of 26.01 on the 20-34 scale (SalmoFan™), and 11.0 with the colotimetry evaluation of colour (a*). The heritability results for the IOM strain were 43 ± 9% for weight, 42 ± 9% for gutted, and 28 ± 8% for length. The heritability estimates for the visual colour variables were 19 ± 7% and when using the colorimeter, the red chromaticity (a*) heritability was 14 ± 6%. Therefore, the heritability results of the IOM strain indicate that there are opportunities of substantial and rapid improvement of the growth rate and flesh colour traits. Also no line effects were observed or indications of non-additive genetic variation. In contrast to these last results, the overall survival of the GIM strain from the time of the physical tagging with PIT until harvest was 52.8%, and survival heritability was extremely low, 3 ± 2%, hardly significant.

Arapaima gigas is the largest scaled freshwater fish in the world reaching over 250 kg. With growth rates of 10 kg+ within 12 months, A. gigas is considered as a promising candidate species for aquaculture development in South America. However, the lack of reproductive control in captivity is hindering the industry expansion. The work carried out in this doctoral thesis therefore aimed to better understand the species’ reproductive physiology, develop tools to identify gender and monitor gonad development, test hormonal therapies to induce ovulation and spawning and characterise the cephalic secretion for its potential roles in pheromone release and during parental care. Initially, a genomic study investigated the overall extent of polymorphism in A. gigas, which was found to be surprisingly low, with only 2.3 % of identified RAD-tags (135 bases long) containing SNPs. Then, a panel with 293 single nucleotide polymorphism (SNP) was used to characterise the genetic diversity and structure of a range of Amazon populations. Results revealed populations from the Amazon and Solimões appeared to be genetically different from the Araguaia population, while Tocantins population comprised individuals from both stocks. This data provided a tool for broodstock identification and future management. The PhD then aimed to evaluate the effects of slow-release mGnRH implants and different broodstock size pairings on maturation and spawning. Results showed that the implants stimulated the brain-pituitary-gonad axis resulting in increased plasma levels of testosterone (females) and 11-ketotestosterone in males, respectively regardless of pairing sizes. However, no spawning was observed. Results also showed the release of sex steroids with potential pheromonal action through the cephalic secretion, a biological fluid released from the adult head along the reproductive period. Thereafter, a non-surgical field endoscopy method was developed and validated for ovarian assessment and gender identification. The method was then used to describe the female gonopore and obtain biopsy of the ovary through cannulation which allowed the description of oogenesis in A. gigas. Importantly, oocytes obtained by cannulation confirmed that adult females under investigation were maturing with oocytes in final maturation stage but failed to ovulate/spawn. Another hormonal induction trial was therefore performed in which a combination of GnRHa (mGnRHa/sGnRHa) was used by injection to induce ovulation and spawning in selected maturing females with effects on oocyte maturation monitored post-induction through biopsy. However, this trial appeared to not be successful at inducing ovulation or spawning. Finally, the peptidome and proteome of the cephalic secretion was further characterised through the comparison between parental and non-parental fish. Results highlighted the complex role of this biological fluid including potential roles on the developing offspring during the parental care period. Overall, this doctoral thesis provided new basic and applied data on A. gigas reproduction and tools that can be used in future studies to better understand the environmental and hormonal control of oogenesis and spawning.

Cleaner fish, including ballan wrasse (Labrus bergylta) have been proposed as a sustainable solution to sea lice infestations affecting farmed Atlantic salmon (Salmo salar) globally. However, in order to become sustainable, ballan wrasse need to be farmed. This thesis investigated the establishment of captive broodstock and protocols to optimise hatchery performance and productivity of ballan wrasse. High throughput sequencing was used to develop a panel of novel single nucleotide polymorphic markers (SNPs). These SNPs were used to investigate the phylogeographic structuring of ballan wrasse populations within northern geographic ranges including the UK and Norway. Results indicated fine scale population structuring within the UK suggesting that founder broodstock should be sourced locally to minimise the risk of genetic introgression with wild ballan wrasse. Secondly, captive breeding was benchmarked from harems to determine total egg production over the spawning season. Data quantified the spawning periodicity and seasonal changes in egg quality parameters. In addition, microsatellite markers identified the parental contribution to each spawning event of captive broodstock. Results confirmed, for the first time, the repeat-batch spawning behaviour and suggested that spawning events were single-paired matings. Furthermore, bottlenecks in commercial production were investigated including the benthic adhesive eggs and complex spawning behaviours of ballan wrasse within broodstock tanks. Experiments were conducted to optimise the spawning dynamics and egg productivity using fragmented spawning zones and coloured substrates. Finally, an effective bath treatment for removal of the adhesive gum layer of eggs using the proteolytic enzyme alcalase® was found to assist in egg disinfection and incubation. Overall, this research provides important baseline data on the management of broodstock and the optimisation of hatchery protocols to improve the commercial productivity and performance of ballan wrasse for use as a biological control of sea lice of farmed Atlantic salmon.

This study assessed the importance of overwash and breaching events on the ichthyofaunal community structure in the medium-sized temporarily open/closed Mpekweni Estuary located on the southeast coastline of southern Africa. The fish in the littoral zone of the estuary were sampled using a 5m seine net while the channel region was sampled using two nets, a smaller meshed 30m seine net to target the estuarine spawning species and the juvenile estuarine-dependant marine spawners, and a larger meshed 50m seine net to target the larger marine and freshwater spawning species. Intensive monthly sampling over two years provided data on selected physico-chemical and biological parameters. During the two year sampling period from November 2005 to October 2007 the estuary breached in late July 2006 and remained open till the sandbar re-formed across the mouth in late December 2006. Thus, sampling encompassed three open/closed phases 1) initial closed period, 2) open period and 3) re-closed period after the berm was re-formed. The open period was divided into two phases 1) the out-flow phase and 2) the tidal phase. A total of 36 fish species representing 19 families were sampled using the various seine nets employed during the investigation. In the littoral zone, the estuarine spawners (Estuarine Utilisation Category, [EUC] I), mainly the Gobiidae, Glossogobius callidus, dominated the samples numerically and by biomass. The smaller estuarine spawning species sampled in the channel were numerically dominated by Gilchristella aestuaria in conjunction with two other EUC I species, Atherina breviceps and G. callidus. The estuarine-dependant marine spawners (EUC II), however, dominated the ichthyofaunal biomass of the channel. The abundance and biomass of the larger species targeted were dominated by estuarine-dependant marine spawning species (EUC II), principally Rhabdosargus holubi. During the closed periods of the estuary, total fish abundance and biomass displayed weak seasonal patterns. The breaching event and subsequent open period was associated with a decrease in the total abundances of fish in the littoral zone and channel of the estuary, reflecting the out-flow of estuarine biomass-rich water into the marine environment. The breaching event coincided with a shift in the community composition of the ichthyofauna, reflecting the recruitment of marine spawning species into the estuary. Numerical analysis identified two distinct spatial fish communities within the estuary, a community associated with the mouth region and one comprising the rest of the estuary. The absence of any further spatial patterns in the ichthyofaunal community structure within the Mpekweni Estuary appear to be ascribed to the virtual absence of horizontal patterns in physico-chemical and biological parameters recorded in the system. Cohort analyses were employed to determine possible recruitment events for selected estuarine and marine spawning species. The estuarine spawning species displayed continuous recruitment patterns throughout the study, which appeared to be unaffected by the breaching event. Conversely, the larger marine spawning species displayed multiple cohorts, indicating non-continuous recruitment. Subsequent retrospective analysis of cohorts for the different species identified summer recruitment peaks that coincided with the breaching event and open period. Minor recruitment of marine spawning species also occurred during overwash events. The recruitment of ichthyofauna into the estuary was quantified during three distinct hydrological events: overwash, out-flow phase immediately after breaching and tidal phase during the period when the mouth was open. Estimates of fish recruitment were highest during the outflow phase immediately after the estuary breached and declined as the estuary became tidally inundated with marine water. Although not as high as the outflow and tidal phases, recruitment was evident during overwash events. Results of the current study highlight the importance of both breaching and overwashing events in structuring the ichthyofaunal community composition in a medium-sized southern African temporarily open/closed estuary. These results are broadly in agreement with similar studies conducted both locally and in other regions of the world.

M.Sc. (Aquatic Health) / This study took place in the Groot Marico River catchment situated in the North West Province of South Africa. The catchment holds many rivers and associated tributaries that are home to a variety fish species with some being endemic to the catchment. A small minnow species, the Marico barb Barbus motebensis was chosen as the study species due to it being endemic to the catchment with limited information available on them with regards to their reproductive status in terms of their reproductive timing and number of potential offspring. Two tributaries from the upper Groot Marico River were chosen as sampling sites for the collection of B. motebensis across the four seasons. These two tributaries presented two genetically different populations, which allowed for a comparison of the findings to ascertain whether any differences in reproductive aspects occurred between the two tributary populations during the different seasons. The Marico barb is under threat from predation pressure posed by the largemouth bass Micropterus salmoides introduced into the system. Micropterus salmoides is a known alien invasive fish that eats any animal food item it encounters, especially fish. Due to B. motebensis and M. salmoides sharing the same habitat preferences, B. motebensis stands little chance of survival in the same water inhabited by the predatory M. salmoides. Many studies have shown bass to drive other fish species to the point of extirpation from a system through predation pressure. Currently B. motebensis is classified as vulnerable on the Red List of Threatened Species, although this status may be further compromised with the presence of M. salmoides...