Dissertations / Theses on the topic 'Barramundi (Lates calcarifer)'

Disease is a major impediment to world aquaculture, amplified by the increase of the intensity of aquaculture relieving pressure from over depleted wild stocks, but with intensity brings disease and particularly disease of the fragile gill organ, exposed directly to the water environment. There is little literature on barramundi biology and the various forms of culture impacting on health, particularly the gill and much research is required in gaining a further understanding of this popular eating fish. The light microscope is a pivotal tool with cytology and histology mandatory in assessing gill health. The gill biopsy should be considered part of a clinical examination as the water medium surrounding the gill and on the gill contains often fragile organisms that would otherwise be lost in fixation for histology alone, but easily viewed with cytology. Barramundi are easily anaesthetised and recovered like many terrestrials and gill re-growth is rapid, healing within days. Biopsies should be viewed unstained with and without phase contrast and then stained and reviewed, recognizing some ectoparasites maybe lost with anaesthetic agents and stains. The sacrificing of the fish after a live gill biopsy is necessary with histology and microbiology our major tools for diagnostics, with no other non invasive methods readily available as for terrestrials. Every year many new water organisms related to aquaculture are described in the literature and the finding of novel and new organisms makes the veterinary examination of the live fish exciting yet imperative. A major concern is the gill pathogens found in wild barramundi were similar to those found in culture. For example the prevalence of the parasite Henneguya a Myxosporidean was 90% in sea cages 60 km offshore from Darwin in the Bathurst Island river system and 66% for ponded fish with water drawn from the Darwin Elizabeth river, compared to 33% infected in the wild habitat of the Mary river system close to Darwin by road. However the bacterial disease Epitheliocystis had a prevalence of 66% in the sea cages and 18% of similarly sized fish in the Mary river system, yet nil found in the pond farm, but in this case sample numbers were restricted. Consequently the surveillance for new fish pathogens and monitoring for existing pathogens in the wild ecosystems and aquaculture facilities is necessary and must include the macro and micro flora and fauna surrounding such facilities as they are potentially affected from aquaculture waste streams. The sustainability of aquaculture in open water culture must be considered with great concern for many reasons, but disease by its nature could overwhelm a species and other aquatic life quickly disseminated in a dynamic water medium. Freshwater culture of barramundi has problems with off flavour and disease, particularly recirculating aquaculture systems due to undercapitalization and possibly at this stage with existing type farms not suited for the culture of barramundi with one farm having all fish sampled diagnosed with systemic bacteraemia and gill Epitheliocystis. Commonly fish sampled from freshwater culture had suffered pathological changes to the gill, particularly hyperplasia indicating the fish are continually affected by issues of water quality and disease. Pond culture appeared to control gill disease issues by affording lower stocking rates, high water exchanges from a river within metres, fallow and the flavour of the fish similar to wild catch or sea cage culture, when purged in brackish water. The decreased environmental and ecosystem risks, coupled with the pond farmer reporting good profits with a simple form of culture, also suitable for intensification is a success story for barramundi production for today and the future.

This study characterises various aspects of barramundi (Lates calcarifer) humoral immunity, including ontogeny, temperature modulation and kinetics following challenge with Streptococcus iniae. It was discovered that Staphylococcal protein A (SpA) was able to efficiently isolate antibody from serum, and that all barramundi Ig found in serum is tetrameric with a weight of approximately 800 kDa. This tetramer is composed of 8 heavy chains (72 kDa) and 8 light chains (28 kDa). Denaturing, non-reducing electrophoresis demonstrated differential disulfide polymerization (redox forms) of the tetrameric Ig which was consistent with those observed with other species. Polyclonal and monoclonal antibodies were produced against the protein A purified barramundi Ig, and various ELISA formats were developed. These serological tools were used to investigate aspects of barramundi humoral immunity. Examination of ontogeny of humoral immunity, revealed that barramundi possess minimal maternal antibody (<10 μg/ml wet weight) post-hatch, which is depleted rapidly (within 3 days). By day 8 systemic Ig is able to be detected, which continues to increase over the following months. However, it is not until seven week post-hatch that barramundi fingerlings are able to mount a prolonged immune response following vaccination with S. iniae. Environmental temperature was also found to significantly impact the ability of barramundi to respond to vaccination with S. iniae. Barramundi maintained at low temperatures (<230C) displayed a diminished, delayed and highly variable humoral immune response following vaccination, with many of the experimental animals failing to respond to primary vaccination. These responses could be mediated by either administering a booster vaccine or by elevating the environmental temperature. This study also demonstrated that there was a relationship with specific serum antibody and protection against S. iniae, with fish possessing high levels of specific Ig being protected from lethal challenge, while those with low titres being more susceptible to disease. Specific antibody in barramundi could be generated through natural exposure to the bacterium from the environment or through vaccination. Thus bath vaccination of fish (50,000) held at two facilities resulted in elevated systemic antibody levels and lower observed mortality, when compared to the unvaccinated control fish. Infections due to S. iniae were determined to be associated with elevated water temperatures. Laboratory trials and field data indicated that water temperatures between 24 and 280C resulted in the highest barramundi mortality. A weak association was also determined with low pH and mortality, with fish exposed to low pH’s (<6.0) being more susceptible to infection. No association was observed with mortality and salinity. Four monoclonal antibodies (Mab’s) were also generated against a 21 kDa protein from cell wall of S. iniae. The Mab’s displayed a high level of specificity for S. iniae, including those from Australia, Israel and America, and minimal cross-reactivity with other bacterial species tested. The Mab’s were used in an immunohistochemical study that confirmed the neurotropic nature of S. iniae infections, as well as demonstrating the presence of the bacterium in the intestine of infected fish.

This study characterises various aspects of barramundi (Lates calcarifer) humoral immunity, including ontogeny, temperature modulation and kinetics following challenge with Streptococcus iniae. It was discovered that Staphylococcal protein A (SpA) was able to efficiently isolate antibody from serum, and that all barramundi Ig found in serum is tetrameric with a weight of approximately 800 kDa. This tetramer is composed of 8 heavy chains (72 kDa) and 8 light chains (28 kDa). Denaturing, non-reducing electrophoresis demonstrated differential disulfide polymerization (redox forms) of the tetrameric Ig which was consistent with those observed with other species. Polyclonal and monoclonal antibodies were produced against the protein A purified barramundi Ig, and various ELISA formats were developed. These serological tools were used to investigate aspects of barramundi humoral immunity. Examination of ontogeny of humoral immunity, revealed that barramundi possess minimal maternal antibody (<10 μg/ml wet weight) post-hatch, which is depleted rapidly (within 3 days). By day 8 systemic Ig is able to be detected, which continues to increase over the following months. However, it is not until seven week post-hatch that barramundi fingerlings are able to mount a prolonged immune response following vaccination with S. iniae. Environmental temperature was also found to significantly impact the ability of barramundi to respond to vaccination with S. iniae. Barramundi maintained at low temperatures (<230C) displayed a diminished, delayed and highly variable humoral immune response following vaccination, with many of the experimental animals failing to respond to primary vaccination. These responses could be mediated by either administering a booster vaccine or by elevating the environmental temperature. This study also demonstrated that there was a relationship with specific serum antibody and protection against S. iniae, with fish possessing high levels of specific Ig being protected from lethal challenge, while those with low titres being more susceptible to disease. Specific antibody in barramundi could be generated through natural exposure to the bacterium from the environment or through vaccination. Thus bath vaccination of fish (50,000) held at two facilities resulted in elevated systemic antibody levels and lower observed mortality, when compared to the unvaccinated control fish. Infections due to S. iniae were determined to be associated with elevated water temperatures. Laboratory trials and field data indicated that water temperatures between 24 and 280C resulted in the highest barramundi mortality. A weak association was also determined with low pH and mortality, with fish exposed to low pH’s (<6.0) being more susceptible to infection. No association was observed with mortality and salinity. Four monoclonal antibodies (Mab’s) were also generated against a 21 kDa protein from cell wall of S. iniae. The Mab’s displayed a high level of specificity for S. iniae, including those from Australia, Israel and America, and minimal cross-reactivity with other bacterial species tested. The Mab’s were used in an immunohistochemical study that confirmed the neurotropic nature of S. iniae infections, as well as demonstrating the presence of the bacterium in the intestine of infected fish.

Other than the study by Griffiths (2009) on gill diseases, there has been no comprehensive study and report on the major diseases of Asian seabass (or barramundi) Lates calcarifer Bloch. It is a food fish species of growing importance in Asia and Australia. This study investigates some of the major diseases encountered in the various stages of the culture of L. calcarifer, at the histopathological, ultrastructural and molecular levels. Culture practices can have significant impacts on fish health. Disease outbreaks are influenced by factors involving the host, environment and pathogen. Current knowledge on diseases of L. calcarifer, and these factors which may influence disease outbreaks are discussed in Chapter 1. This is the first report of an intestinal Eimeria infection in L. calcarifer. The Eimeria infection was associated with severe pathology and significant mortality in the absence of other pathogens. It was detected in diseased L. calcarifer in all five nurseries in Ca Mau, Vietnam. Although these were small scale nurseries which stocked an average of 3000 to 5000 fish at any one time, a mortality rate of up to 30% was reported and is the cause of significant economic losses for these nurseries. Moderate to heavy Eimeria infestation were observed in greater than 80% of diseased fish examined. This high rate of Eimeria infestation is suspected to be linked to the low daily water exchange rates practised in these nurseries. However, the examination of only diseased fish does not allow the determination of prevalence. A systemic iridovirus infection was concurrently observed in some of the fishes but was not consistently present when compared to the Eimeria infection. Molecular analysis showed that the Eimeria of L. calcarifer from Vietnam formed clades with the Eimeria detected in L. calcarifer cultured in Australia, but clustered separately from other known Eimeria species. Although Cryptosporidium was detected in these L. calcarifer tissues, it could not be demonstrated histologically or ultrastructurally, suggesting a low grade infestation or perhaps an environmental contaminant in fish tissues tested. In situ hybridization using labeled PCR products showed that labeled DNA probes generated from 18S PCR products could not be used to distinguish between closely related genera such as Cryptosporidium and Eimeria. Future investigation to determine the origin, transmission and risk factors associated with this Eimeria infestation in L. calcarifer are needed. ‘Scale drop syndrome’ is a novel disease first reported in L. calcarifer in Penang, Malaysia in 1992. Cases with similar gross and clinical presentations were observed in Singapore in 2002, 2006 and 2009. Affected fish have loose scales, which dropped off easily when handled. The disease was initially observed in 100-300g fish, and later in larger fish up to 5kg bodyweight. Cumulative mortalities of 40 to 50% were reported by farms, posing significant economic losses of larger more valuable fish. This investigation forms the first pathological description of ‘scale drop syndrome’ (SDS) in L. calcarifer. To aid recognition of new cases for study, a case definition was developed for ‘scale drop syndrome’ in L. calcarifer as a systemic vasculitis associated with tissue necrosis in all major organs including the skin, with apparent targeting of cells of epithelial origin. Attempts to isolate or detect the causative agent(s) by cell culture, PCR and immunohistochemistry have proven unsuccessful. Further studies to elucidate the definitive aetiology, isolate the causal agent(s) and reproduce the disease will help better understanding and control of SDS. Although systemic iridoviral disease has been previously reported in many freshwater and marine fish species, this study forms the first report of this disease in L. calcarifer. Systemic iridoviral disease was observed in 5 to 20g L. calcarifer usually 2 to 3 weeks post-transfer into sea cages at two farms. Inclusion bodies suggestive of a systemic iridovirus infection were observed in clinically healthy L. calcarifer from the land-based nursery of one of these two farm; the presence of an iridovirus infection was supported by positive PCR results using Red Sea bream iridovirus (RSIV) primer 1. The presence of inclusions was not accompanied by any tissue necrosis in these clinically healthy fish. This finding suggested that the systemic iridovirus infection occurred before stocking at sea, and did not originate from wild fish or older fish in adjacent sea cages as initially suspected by this farm. Immunohistochemistry on tissues of clinical cases of systemic iridovirus gave positive results using the Red Sea bream iridovirus monoclonal antibody (RSIV M10), although intensity varied between tissues, possibly related to varying exposure of different tissues to fixation chemicals. Inclusion bodies in clinically healthy fish from the same farm did not show positive reaction with RSIV M10. This may be due to a lack of antigenic expression by the viral infected cells at this early stage of infection. Viral nervous necrosis (VNN) is a serious disease of hatchery reared L. calcarifer fry in this study. Mortalities of 50 to 100% were reported in 3wo fry. VNN can be difficult to diagnose in older fry, where it can be associated with few vacuolations or an absence of viral inclusions ‘Pot belly disease’ (PBD) was previously reported in L. calcarifer fry less than 1g, in association with an intracellular coccobacillus infection and mortalities of 80 to 100%. In this study, PBD was observed in 120g L. calcarifer at two sea cage farms, in association with significant granulomatous enteritis. The extent of the granulomatous enteritis is likely to have an effect on affected fish. It was observed concurrently with systemic iridoviral disease at one farm and nocardiosis at another farm. Diagnosis by histopathology and the lack of other confirmatory tests for PBD may result in underdiagnosis of this disease. The epidemiology of PBD needs further study to establish origin and modes of transmission, to facilitate better disease control. Diseases associated with infections by ubiquitous bacteria such as Vibrio, Tenacibaculum were commonly observed in L. calcarifer post-handling. Tenacibaculosis and vibriosis often occurred concurrently with other diseases such as streptococcosis, systemic iridviral disease or PBD. Streptococcosis can affect fish up to 3kg bodyweight, resulting in significant mortalities greater than 40 to 50%. Like SDS, because streptococcosis can affect up to market size fish, they can cause considerable economic losses. Although vaccines against Streptococcosis are available, conflicting views are held on the efficacy of Streptococcus vaccines by various research groups. Overall, the South-east Asian L. calcarifer farms which practiced vaccination against Streptococcus iniae reported a reduction of mortality, especially in fish greater than 1 to 1.5kg bodyweight. Nocardiosis has been reported as an emerging disease in marine food fish species caused by acid fast filamentous branching bacterium. Although nocardiosis was observed histopathologically in L. calcarifer at two sea cage farms, the numbers of samples examined were small and no other tests were attempted due to lack of suitable samples. More intensive and extensive study is needed to determine the significance of nocardiosis in L. calcarifer. Chronic granulomatous enteritis was not uncommon in the cases submitted to the Fish Health Laboratory in Perth. Although the peritonitis was associated with heavy bacteria infection, it is unclear if these are secondary invaders. Schipps, Bosmans & Humphreys (2009) reported that Vibrio harveyi and Photobacterium damsela damsela vaccinations appeared to be not efficacious, suggesting that these bacteria were not the primary cause of the disease. It is well recognized that disease outbreaks in farmed fish are influenced by the interaction between host, the environment and pathogens. While serious diseases are often reported in association with specific aquatic pathogens, not much is known about the risk factors which trigger fish disease outbreaks. Disease outbreaks often occur after stressful events such as net transfers, recent handling or poor water quality. In fact, diseases are often caused by ubiquitous pathogens that are commonly present in the culture environment. Although further research is necessary to gather more information to improve diagnosis and management of specific diseases, general health management strategies can be applied at the various stages in the culture of L. calcarifer to minimize disease outbreaks. This is discussed for L. calcarifer in Chapter 6. Observations of types of disease agents may be influenced by site conditions or the types of tests or materials examined. For example, some parasites may be more prevalent in certain sites where intermediate hosts abound, or loosely attached ectoparasites may be lost unless wet mount microscopic examinations of fresh tissues were carried out. The study of emerging diseases such as scale drop syndrome (SDS) or pot belly disease (PBD) in L. calcarifer has been hampered by lack of confirmatory diagnostic tools and inadequate knowledge on critical epidemiological factors such as mode of transmission or potential reservoirs. While ideally identification and isolation of the causal agent will help fulfil Koch’s postulates, it may be possible to improve the understanding of disease via cohabitation or infectivity trials using tissue homogenates from diseased fish when pure isolates are not available. There is a need to conduct research to not only establish a definitive aetiology, but also to identify risk factors to facilitate successful disease control. The successful management of disease in aquaculture does not lie in any one strategy but an integrated management of all risks encountered during the culture cycle against disease occurrence or incursions.

Barramundi (Lates calcarifer) is a centropomid teleost with a wide distribution across the Indo Pacific. In Australia, barramundi are native to the tropical zone from Exmouth Gulf in Western Australia, across the northern part of the continent, to the Mary River in Queensland. Barramundi are protandrous hermaphrodites, and are euryhaline, with a catadromous life history. Barramundi are a valuable Australian resource, with important commercial and recreational fisheries and aquaculture production to the value of $11 million dollars per year. Recent declines in the availability of the fish in some rivers has led to an interest in the possibility of restocking rivers with barramundi from other areas. Determining the genetic structure of barramundi populations in Australia is important for understanding biogeographic history, and appropriate management practices for both aquaculture and recreational and commercial fishing. Previous studies have concentrated on the east coast of Australia, and have largely ignored the western populations. In this study, I obtained DNA data from barramundi populations across the Australian range of the species, as well as populations from Papua New Guinea and Indonesia. The aims of this study were to use the genetic data to determine: 1. if populations in Western Australia show genetic differences between geographic regions 2. if these populations show an ancestral split from populations in the east of Australia and 3. the ancestral origins of Australian barramundi. Previous studies of DNA data from barramundi have discovered an east/west split occurring at the Torres Strait that was assumed to be caused by the closing of the strait during lowered sea levels. However, these studies suffered from a bias in sampling area, concentrating either on the eastern half of the range of barramundi, or on the western tip of the range. Data from these studies were combined and reanalyzed. Two major clades were discovered, with considerable biogeographic structuring, but their geographic locations did not coincide with the reported vicariance event at the Torres Strait. Instead,historical divisions among freshwater drainage systems appeared to have driven the evolutionary history of barramundi in Australia. In order to investigate these historical divisions further, a 290 bp section of the mitochondrial DNA control region was sequenced in 284 barramundi from seven populations across the Australian geographic range of the species and from one population in Papua New Guinea and one population in Indonesia. Analyses of molecular variance within and among populations showed significant geographic structuring, based on biogeographical provinces and drainage divisions. Nested clade analyses indicated that these geographical associations were the result of restricted gene flow, range expansion, and past fragmentation events. I hypothesise that the Ord River area in the west of the continent was the ancestral source population for the rest of the species' range across Australia, with Indonesia being the most likely origin of this source. Populations of barramundi from the Pilbara region are genetically distinct and geographically isolated, with strong evidence of an ancestral divide along geographical barriers to dispersal. There is a strong association between Papua New Guinea and Australia, although further investigations using the cytochrome b region of mitochondrial DNA indicated a more ancestral divide between the two than is currently evident, which could reflect an ancient geographical divide between the two, or could be evidence of a secondary migration route to Australia. For a more detailed study of evolutionary processes acting on populations of barramundi in Western Australia, allelic diversity was examined at five microsatellite loci. All loci were polymorphic and genotypic frequencies conformed to Hardy-Weinberg expectations, with no significant linkage between loci evident in any population. Measures of within population diversity were significantly related to latitude, suggesting southerly migration from a northern source population. The Ord River was the most genetically diverse population, and the most likely ancestral migration source to the area, with diversity decreasing down the west coast. Although there were significant differences among populations, the nuclear microsatellite data do not indicate the same degree of genetic structuring as is evident in the mitochondrial data. This may be a consequence of rapid evolutionary change at microsatellite loci, with past separations or population differences masked by recombination and back mutation of the microsatellite alleles. However, the nature of nuclear and mitochondrial inheritance may also indicate life history differences between the sexes, where significant genetic contribution to gene flow by males and limited female gene flow may lead to preservation of maternally inherited population substructure. The principal findings from this study are: * There is no genetic evidence for an east/west division of barramundi populations in Australia, as suggested by previous research. * Despite barramundi's catadromous life history, and ability to disperse through marine waters, the present genetic structure indicates a division principally among river drainages. From a population genetic viewpoint, the species can be regarded as freshwater, rather than marine. * The most likely origin of barramundi in Australia is the Ord River region, with Indonesia as the route of migration. * Differences in the population structure demonstrated by nuclear and mitochondrial data indicate possible life history differences between the sexes. * Barramundi populations in different biogeographical provinces may have been substantially isolated over a long period of time, and may therefore represent independently evolving populations. This has important implications for fishery management and translocation issues for restocking rivers.

Barramundi (Lates calcarifer) is a centropomid teleost with a wide distribution across the Indo Pacific. In Australia, barramundi are native to the tropical zone from Exmouth Gulf in Western Australia, across the northern part of the continent, to the Mary River in Queensland. Barramundi are protandrous hermaphrodites, and are euryhaline, with a catadromous life history. Barramundi are a valuable Australian resource, with important commercial and recreational fisheries and aquaculture production to the value of $11 million dollars per year. Recent declines in the availability of the fish in some rivers has led to an interest in the possibility of restocking rivers with barramundi from other areas. Determining the genetic structure of barramundi populations in Australia is important for understanding biogeographic history, and appropriate management practices for both aquaculture and recreational and commercial fishing. Previous studies have concentrated on the east coast of Australia, and have largely ignored the western populations. In this study, I obtained DNA data from barramundi populations across the Australian range of the species, as well as populations from Papua New Guinea and Indonesia. The aims of this study were to use the genetic data to determine: 1. if populations in Western Australia show genetic differences between geographic regions 2. if these populations show an ancestral split from populations in the east of Australia and 3. the ancestral origins of Australian barramundi. Previous studies of DNA data from barramundi have discovered an east/west split occurring at the Torres Strait that was assumed to be caused by the closing of the strait during lowered sea levels. However, these studies suffered from a bias in sampling area, concentrating either on the eastern half of the range of barramundi, or on the western tip of the range. Data from these studies were combined and reanalyzed. Two major clades were discovered, with considerable biogeographic structuring, but their geographic locations did not coincide with the reported vicariance event at the Torres Strait. Instead,historical divisions among freshwater drainage systems appeared to have driven the evolutionary history of barramundi in Australia. In order to investigate these historical divisions further, a 290 bp section of the mitochondrial DNA control region was sequenced in 284 barramundi from seven populations across the Australian geographic range of the species and from one population in Papua New Guinea and one population in Indonesia. Analyses of molecular variance within and among populations showed significant geographic structuring, based on biogeographical provinces and drainage divisions. Nested clade analyses indicated that these geographical associations were the result of restricted gene flow, range expansion, and past fragmentation events. I hypothesise that the Ord River area in the west of the continent was the ancestral source population for the rest of the species' range across Australia, with Indonesia being the most likely origin of this source. Populations of barramundi from the Pilbara region are genetically distinct and geographically isolated, with strong evidence of an ancestral divide along geographical barriers to dispersal. There is a strong association between Papua New Guinea and Australia, although further investigations using the cytochrome b region of mitochondrial DNA indicated a more ancestral divide between the two than is currently evident, which could reflect an ancient geographical divide between the two, or could be evidence of a secondary migration route to Australia. For a more detailed study of evolutionary processes acting on populations of barramundi in Western Australia, allelic diversity was examined at five microsatellite loci. All loci were polymorphic and genotypic frequencies conformed to Hardy-Weinberg expectations, with no significant linkage between loci evident in any population. Measures of within population diversity were significantly related to latitude, suggesting southerly migration from a northern source population. The Ord River was the most genetically diverse population, and the most likely ancestral migration source to the area, with diversity decreasing down the west coast. Although there were significant differences among populations, the nuclear microsatellite data do not indicate the same degree of genetic structuring as is evident in the mitochondrial data. This may be a consequence of rapid evolutionary change at microsatellite loci, with past separations or population differences masked by recombination and back mutation of the microsatellite alleles. However, the nature of nuclear and mitochondrial inheritance may also indicate life history differences between the sexes, where significant genetic contribution to gene flow by males and limited female gene flow may lead to preservation of maternally inherited population substructure. The principal findings from this study are: * There is no genetic evidence for an east/west division of barramundi populations in Australia, as suggested by previous research. * Despite barramundi's catadromous life history, and ability to disperse through marine waters, the present genetic structure indicates a division principally among river drainages. From a population genetic viewpoint, the species can be regarded as freshwater, rather than marine. * The most likely origin of barramundi in Australia is the Ord River region, with Indonesia as the route of migration. * Differences in the population structure demonstrated by nuclear and mitochondrial data indicate possible life history differences between the sexes. * Barramundi populations in different biogeographical provinces may have been substantially isolated over a long period of time, and may therefore represent independently evolving populations. This has important implications for fishery management and translocation issues for restocking rivers.

Barramundi (Lates calcarifer) is a centropomid teleost with a wide distribution across the Indo Pacific. In Australia, barramundi are native to the tropical zone from Exmouth Gulf in Western Australia, across the northern part of the continent, to the Mary River in Queensland. Barramundi are protandrous hermaphrodites, and are euryhaline, with a catadromous life history. Barramundi are a valuable Australian resource, with important commercial and recreational fisheries and aquaculture production to the value of $11 million dollars per year. Recent declines in the availability of the fish in some rivers has led to an interest in the possibility of restocking rivers with barramundi from other areas. Determining the genetic structure of barramundi populations in Australia is important for understanding biogeographic history, and appropriate management practices for both aquaculture and recreational and commercial fishing. Previous studies have concentrated on the east coast of Australia, and have largely ignored the western populations. In this study, I obtained DNA data from barramundi populations across the Australian range of the species, as well as populations from Papua New Guinea and Indonesia. The aims of this study were to use the genetic data to determine: 1. if populations in Western Australia show genetic differences between geographic regions 2. if these populations show an ancestral split from populations in the east of Australia and 3. the ancestral origins of Australian barramundi. Previous studies of DNA data from barramundi have discovered an east/west split occurring at the Torres Strait that was assumed to be caused by the closing of the strait during lowered sea levels. However, these studies suffered from a bias in sampling area, concentrating either on the eastern half of the range of barramundi, or on the western tip of the range. Data from these studies were combined and reanalyzed. Two major clades were discovered, with considerable biogeographic structuring, but their geographic locations did not coincide with the reported vicariance event at the Torres Strait. Instead, historical divisions among freshwater drainage systems appeared to have driven the evolutionary history of barramundi in Australia. In order to investigate these historical divisions further, a 290 bp section of the mitochondrial DNA control region was sequenced in 284 barramundi from seven populations across the Australian geographic range of the species and from one population in Papua New Guinea and one population in Indonesia. Analyses of molecular variance within and among populations showed significant geographic structuring, based on biogeographical provinces and drainage divisions. Nested clade analyses indicated that these geographical associations were the result of restricted gene flow, range expansion, and past fragmentation events. I hypothesise that the Ord River area in the west of the continent was the ancestral source population for the rest of the species’ range across Australia, with Indonesia being the most likely origin of this source. Populations of barramundi from the Pilbara region are genetically distinct and geographically isolated, with strong evidence of an ancestral divide along geographical barriers to dispersal. There is a strong association between Papua New Guinea and Australia, although further investigations using the cytochrome b region of mitochondrial DNA indicated a more ancestral divide between the two than is currently evident, which could reflect an ancient geographical divide between the two, or could be evidence of a secondary migration route to Australia. For a more detailed study of evolutionary processes acting on populations of barramundi in Western Australia, allelic diversity was examined at five microsatellite loci. All loci were polymorphic and genotypic frequencies conformed to Hardy-Weinberg expectations, with no significant linkage between loci evident in any population. Measures of within population diversity were significantly related to latitude, suggesting southerly migration from a northern source population. The Ord River was the most genetically diverse population, and the most likely ancestral migration source to the area, with diversity decreasing down the west coast. Although there were significant differences among populations, the nuclear microsatellite data do not indicate the same degree of genetic structuring as is evident in the mitochondrial data. This may be a consequence of rapid evolutionary change at microsatellite loci, with past separations or population differences masked by recombination and back mutation of the microsatellite alleles. However, the nature of nuclear and mitochondrial inheritance may also indicate life history differences between the sexes, where significant genetic contribution to gene flow by males and limited female gene flow may lead to preservation of maternally inherited population substructure. The principal findings from this study are: • There is no genetic evidence for an east/west division of barramundi populations in Australia, as suggested by previous research. • Despite barramundi’s catadromous life history, and ability to disperse through marine waters, the present genetic structure indicates a division principally among river drainages. From a population genetic viewpoint, the species can be regarded as freshwater, rather than marine. • The most likely origin of barramundi in Australia is the Ord River region, with Indonesia as the route of migration. • Differences in the population structure demonstrated by nuclear and mitochondrial data indicate possible life history differences between the sexes. • Barramundi populations in different biogeographical provinces may have been substantially isolated over a long period of time, and may therefore represent independently evolving populations. This has important implications for fishery management and translocation issues for restocking rivers.

The research investigated the effectiveness of proteins from tuna hydrolysate (TH) and poultry by product meal (PBM), as fishmeal (FM) protein replacements. The results demonstrated that replacement of 10% FM with TH improved growth, immunity, intestinal health and disease resistance in juvenile barramundi. The addition of 10% TH in bioprocessed PBM not only improved the physiology of the fish but also increased the fish growth when 100% fishmeal protein was replaced by PBM protein.

The effects of pre-freezing treatments such as time-temperature abuse, use of ice, freeze–thaw cycles, use of polyphosphate and post freezing treatments such as glazing and packaging were assessed on the microbiological and physiochemical properties of barramundi (Lates calcarifer) fillets. The results indicated that the use of sodium tripolyphosphate, constant temperature and use of ice in the form of slurry can improve the shelf life of barramundi fillets.

The Barramundi (Lates calcarifer Bloch) is a large, percoid fish, highly valued as a commercial and recreational species. In Queensland, it is distributed in estuaries, coastal habitats and freshwater areas accessible to the sea north from about the Noosa River. This study reports on a three year investigation of the movements, reproduction and growth of barramundi at 15 sites along the east Queensland coast. Of the 524 adult and sub-adult barramundi tagged in coastal areas and estuaries of eastern Queensland between 1981 and 1984, 136 (26%) were recaptured. Most recaptures (75%) occurred within a year of the fish being tagged and 32% were recaptured within three months of release. Movements of tagged fish were usually less than five kilometres, with 25 km regarded as rare. While most fish were recaptured at or near the location where they were released (usually an estuary), in the Burdekin delta area there were movements along coastal foreshores and into adjacent streams. Unlike other parts of Australia and Papua New Guinea, barramundi in eastern Queensland are generally not catadromous. The large proportion of short and ephemeral rivers and an increasing number of barriers across the larger river systems have restricted the freshwater habitat available for barramundi. In eastern Queensland, peak spawning occurs from November to February although some spawnings do occur as early as September and as late as April. Gametogenesis commences in August/September and is apparently initiated by a seasonal increase in water temperature and photoperiod. Only weak evidence was found supporting multiple spawning and only one modal size class of developing eggs was generally present in ovaries. Fecundity was high and was found to be exponentially related to length. Barramundi mature as males and later, between about 900 and 1000 mm total length, change sex to females. Length-weight relationships, for both sexes, in all areas were strongly linear. In most areas there were significant differences between male and female length-weight regressions. For each area, estimates of the von Bertalanffy growth parameters K, L00 and t 0 ranged from 0.23 to 0.25, 1189 mm to 1274 mm and -0.44 to -0.49 years respectively. Growth rates were initially faster than those established for barramundi in the Northern Territory, Gulf of Carpentaria and Papua New Guinea, and this as considered to be a possible response to heavy exploitation or environmental conditions.

This research evaluated nutritional compositions of the selected and bioprocessed plant ingredients and their potential to replace fishmeal in barramundi (Lates calcarifer) diets. Bioprocessing including fermentation, germination and enzymatic treatments improved the nutritional profiles of the plant ingredients that were reflected in the enhanced growth and physiology of the barramundi fed the test diets with less fishmeal based protein source. Further, the inclusion of the test diets improved the water quality of the rearing environment.

The research investigated the complete replacement of fishmeal (FM) with a mixture of multiple protein ingredients including poultry by-product meal (PBM), black soldier fly, Hermetia illucens (HI) larvae meal, and fish protein hydrolysates (FPHs) originating from recycled food wastes. The results showed that supplementation of HI and FPH separately or concurrently with PBM can replace FM completely from the juvenile barramundi diet with an improvement in physiological and immunological responses and final product quality.

Research Doctorate - Doctor of Philosophy (PhD)
High pressure processing (HPP) is well-known as an innovative processing technology that can extend shelf-life and improve physicochemical properties of fish muscle during storage. However, research for all potential HPP applications on barramundi fish has not been done before. In this study, HPP was applied prior to conventional freezing to improve physicochemical properties of barramundi muscle and to abate the adverse effects of freezing on barramundi muscle during frozen storage. HPP was also used with or without the combination of chitosan coating to extend the shelf-life of barramundi during chilled storage. HPP was also employed to produce barramundi gels, especially, to reduce salt concentration in these gels. For raw barramundi muscle stored in frozen condition at - 18 °C, application of HPP at 150 MPa and initial temperature of 4 °C for 3 min prior to freezing resulted in barramundi fillet with higher hardness and springiness, lower drip loss and stable pH compared to non-pressurised samples during frozen storage for up to 18 weeks. HPP under these conditions also did not induce cooked appearance and lipid oxidation of barramundi fillet, two major drawbacks in pressurised fish muscle. Thus, HPP prior to freezing may be a good option for barramundi processors. Before chilled storage at 4 ± 1 °C, raw barramundi muscle was treated under 3 different conditions including pressurisation (HPP), chitosan coating and pressurisation (HC) and chitosan coating (CHI). Barramundi samples were investigated on day 1, 8, 15 and 23 during chilled storage. HPP at 200 MPa and 4 ± 1 °C for 3 min significantly improved texture profile, pH, drip loss and TVB-N of barramundi muscle as compared to CHI and control treatment. However, HPP did not delay microbial growth compared to CHI and control treatment. Application of HC treatment significantly hampered the development of TVB-N, microbial growth and improved texture and drip loss of barramundi muscle compared to control, HPP and CHI treatment. However, HC treatment resulted in a cooked appearance and acceleration of lipid oxidation, two major drawbacks in pressurised fish muscle. Pressurisation significantly decreased the activity of protease, but did not reduce the activity of LOX. Microscopic images also showed that the microstructures of pressurised samples were better preserved than the cracked microstructure of unpressurised samples. In this study, HPP was found to be the most favourable treatment for improving the quality of barramundi muscles stored in chilled condition for up to 23 days. For the application of HPP to produce barramundi gels, barramundi minced muscles with 1% and 2% added salt were pressurised at 300, 400 and 500 MPa at ≤ 10 °C and 50 °C for 10 min. Pressure induced barramundi gels (PG) exhibited a lower gel strength and poorer texture such as hardness and springiness as compared to conventional heat induced gels. Comparable water holding capacity to heat induced gels was only obtained at a salt concentration of 2% and at pressures ≥ 400 MPa. SEM images showed a compact network with smoother surface of barramundi minced muscle with 2% added salt and HPP at ≥ 400 MPa as compared to conventional heat induced gels. Gelling properties of barramundi minced muscle with 1.5% and 2 % added salt were assessed after HPP at 300, 400 and 500 MPa at 4 °C (initial temperature) for 10 min and subsequent cooking at 90 °C for 30 min. Whiteness, gel forming ability, water holding capacity, hardness and springiness of the barramundi gels increased as applied pressure and salt concentration increased. At 2% salt concentration, HPP resulted in barramundi gels with higher gel strength and smoother texture as compared to conventional heat induced gels (0.1 MPa, 90 °C for 30). At a reduced salt concentration (1.5%) and HPP at ≥ 400 MPa, the quality (gel strength, water holding capacity, hardness and springiness) of pressurised, cooked gels are comparable to those heat-induced gels with 2% added salt, but the microstructure is smoother. Scanning electron microscope images of pressurised, cooked gels showed a compact network with smoother surface than those of heat-only induced gels. Thus, application of HPP prior to cooking could be an effective method to enable reduced salt concentration in barramundi gels. Barramundi minced muscle with 1% and 2% added salt was gelled by different combinations of pressurisation (300, 400 and 500 MPa at 4 °C for 10 min), cooking (0.1 MPa, 90°C for 30 min) and setting (0.1 MPa, 50 °C for 2 h) to improve their mechanical properties and lower the amount of salt added to barramundi gels. At the low salt concentration of 1%, pressurisation prior to cooking (P - C) treatment resulted in barramundi gels with comparable mechanical properties and water holding capacity to those of conventional heat induced (HI) gels with 2% added salt. At a salt concentration of 2%, pressurisation prior to setting (P - S) and P - C gels exhibited higher mechanical properties and water holding capacity than HI gels. Scanning electron microscope images showed a smooth and dense microstructure of P - C and P - S gels, whereas the microstructure of HI gels is rough and less compact. P - S and P - C treatment can result in higher mechanical and functional properties of barramundi gels at conventional salt concentration (2%) compared to HI gels. P - C treatment can lower the salt concentration added to barramundi gels to 1% which is very significant for health-conscious consumers. To produce a shelf-stable barramundi product by high pressure thermal sterilisation (HPTS), barramundi muscle in a brine was treated at 600 MPa and three different temperatures (90, 110 and 120 °C) for 5 min. Barramundi muscle retorted at F_o = 3.38 was used as control. HPTS at 600 MPa and 110 °C and 120 °C, for 5 min, respectively, produced stable barramundi products, which were stored at room temperature and tested for up to 1 year. Hardness and springiness, of HPTS sterilised barramundi samples were enhanced (i.e. increased) compared to retorted samples. Gumminess, chewiness and cohesiveness of HPTS sterilised barramundi muscle were also similar to retorted samples and did not decrease as processing temperature increased. TBA and pH was also similar in all treatments, except for a significant increase of pH of samples treated at 600 MPa and 90 °C for 5 min after 3 months of storage. In general, HPTS could be a feasible option to produce a sterilised barramundi product with better overall quality and is recommended for more research on other HPTS barramundi products.

碩士
輔仁大學
生命科學系碩士班
92
The first case of suspected viral infection of the giant seaperch (Lates calcarifer, Bloch ) fry, which caused 50-90% mortality, was reported in Thailand in 1970, and cases of viral infections of the cultured giant seaperch followed in Australia and Southeastern Asia. The first virus infection in farmed giant seaperch was reported in Taiwan in 1993 and cases occurred continuously. In 2001, the farmed giant seaperch in south Taiwan appeared poor appetite, ataxia and death. We sampled and dissected some ill fish and found extensive hemorrhagic areas and swollen on the anterior liver, which contained lots of cavities histologically. Under the transmission electronic microscope, there were 160-200 nm hexahedral viral particles, whose electronic compact center was 100-120 nm, and 10 to 20 particles aggregated in tissues. We homogenized the hepatic tissue of ill fish, removed the pellet by centrifugation, filtrated the supernatant through 0.22 m filters, and obtained the fish tissue extract. As we injected the tissue extract into healthy giant seaperch, cavities in hepatic tissues were observed. We tried to replicated the giant seaperch virus from the established grouper cell lines, however, they were not susceptible to the virus containing hepatic extract, and we had to establish giant seaperch cell lines for virus replication. We cultured cells from brain, heart, liver, kidney, spleen and muscle, and obtained four types of giant seaperch brain (sp b) cells, three flat and one spindle, and kidney (giant seaperch kidney, sp k) cells, swim bladder (giant seaperch swim bladder, sp b) cells, as well as muscle (giant seaperch muscle, sp m) cells, which appeared spindle. Characterization of cells revealed that cell lines sp b-7 and sp b-2 showed the best growth rate as cultured in the medium containing 15% fetal bovine serum and incubated at 36 °C. Chromosome analysis of cell line sp sb-2 showed that its chromosome number was 40 to 46. The susceptibility study of giant seaperch cell lines to virus from the liver extract of ill fish showed that sp sb cells appeared cytopathic effect (CPE) as incubated with 101- to 103-fold dilutions of virus. However, no significant CPE observed in other giant seaperch cell lines. To detect virus from the tissue extracts of ill fish and cell extracts which showed CPE by PCR, we designed primers referred to the frog virus 3 (FV3) major capsid protein, however, no PCR product was amplified. It is suggested that this giant seaperch virus is not FV3 related or the virus amount in the tissue extract is not sufficient for PCR amplification.

A knowledge of the factors which regulate insulin-like growth factors (IGFs) in teleost fish is important for understanding the physiological process of growth and thus devising strategies to improve growth in culture systems. In mammals, two IGF molecules have been identified. Whilst IGF-I is predominantly regulated by growth hormone (GH) and the nutritional status of the animal, GH does not appear to regulate IGF-II which is thought to be important for fetal development. Although the IGF system has been well characterised in mammals, there is a paucity of data on IGFs in teleost fish. The detection of IGFs in teleost species has generally been determined using non-homologous competitive binding assays, often without complete assay validation. In addition, there are few studies which investigate the mechanisms of IGF regulation. The aim of the present study was to determine the effect of nutritional status and water temperature on the growth and regulation of IGFs in juvenile barramundi, Lates calcarifer. Following acidic size exclusion chromatography, circulating IGF-I and IGF-II were detected in the serum of juvenile barramundi using type I and type II radioreceptor (RRA) assays, respectively. Both RRA were rigorously validated using the recommended protocol for the measurement of IGFs in biological fluids (Bang et al., 1994). Peaks containing the IGF molecules were serially diluted and demonstrated parallelism to human IGF-I and IGF-II standard reference curves, in the type I and type II RRA, respectively. IGF binding proteins were identified as a false peak of immunoreactivity, ranging in molecular size from 12.3 - 66 kDa. The IGF binding proteins (IGFBPs) were further characterised using an IGF binding protein assay with subsequent neutral size exclusion chromatography and Western ligand blots. High quantitative recovery was demonstrated in the type I (99 ± 2.7 %) and type II (97 -± 3.4 %) RRA by the addition of unlabelled IGF-I or IGF-II respectively to the serum prior to analysis. Infra- and inter-coefficients of variation were within acceptable literature ranges being 1.7 ± 0.3 % and 8.2 ± 2.1 % respectively for the type I RRA and 3.9 ± 0.6 % and 8.3 ± 2.8 % respectively for the type II RRA. These findings satisfied the requirements of IGF assay validation and thus provided a method for detecting both IGF-I and IGF-II in the serum of juvenile barramundi. The present study demonstrated that ration size regulates the growth, level of circulating IGF-I and expression of hepatic IGF-I mRNA in juvenile barramundi. In contrast, the expression of IGF-I mRNA in the brain, the ratio of the alternatively spliced Ea-4 : Ea-2 IGF-I mRNA transcripts and the concentration of circulating IGF-II were not significantly affected by ration size. As hepatic IGF-I mRNA and circulating levels of IGF-I were reduced during starvation and there was an accompanying decreased growth, it is likely that systemic IGF-I of hepatic origin is important for somatic growth in this species. The response of IGF-I, both pre- (hepatic only) and post-translational, but not IGF-II, to ration size in juvenile barramundi is similar to findings in gilthead seabream, thereby providing further evidence for the general principle of regulation of the GH:IGF-I axis in fish by nutritional status. Dietary protein and energy regulated the growth and level of circulating IGF-I in juvenile barramundi, providing support for the theory of nutritional regulation of IGF-I, but not IGF-II, in this species. Since decreased dietary protein and energy caused a reduction in the concentration of circulating IGF-I, which was accompanied by decreased growth, it is likely that systemic IGF-I is affected by protein and energy restriction in this species. Although the mechanisms of this regulation remain unknown, results from studies conducted in other teleost fish demonstrate that protein and energy restriction result in an insensitivity of the liver to GH. Although the role of IGF-II remains somewhat uncertain in teleost fish, it is reasonable to suggest that IGF-II in fish, as in mammals, is not directly influenced by nutrition or GH. Although water temperature affected growth in both trials there was no clear effect of temperature on circulating IGF-I or IGF-II. The results of this study provide a technique for detecting changes in circulating IGF levels and indicate that environmental parameters, including ration size, dietary protein and energy content and water temperature affect the growth and the synthesis of insulin-like growth factor-I in juvenile barramundi, Lates calcarifer.

Alyssa Budd studied the epigenetic effects of temperature on sex change in barramundi, revealing the first evidence for temperature-induced epigenetic changes in the gonads of a sequential hermaphrodite. Alyssa’s results further our understanding of sex change in fish and highlight the potential for temperature as a sustainable method for sex control in aquaculture.

Fish reared in aquaculture must be provided with an adequate diet for growth and reproduction. Although there is a paucity of literature on broodstock nutrition generally, particularly for male fish, it is known that nutrition has profound effects on reproductive physiology. Most of the available information documents dietary effects on direct reproductive outcomes, such as fecundity, spawning performance, egg and larval quality. Information on the mechanisms by which these processes are regulated is rare and this lack of understanding is one of the major constraints to improved broodstock management. Barramundi, Lates calcanfer (Bloch), also known as Asian sea bass or giant perch, is a highly valued food fish forming the basis of major fishery and aquaculture industries in Australia and south-east Asia. However, hatchery production of barramundi continues to be problematic, with unpredictable male fish performance in hatcheries. There is no published literature on the relationship between nutrition and reproduction in barramundi and brood fish are generally fed trash fish supplemented with vitamins. In order to contribute to our understanding of brood stock nutrition, particularly of male barramundi, this study has investigated the effect of three major nutritional regimes - feeding frequency (food deprivation), dietary protein:energy ratio and dietary fatty acid profile - on standard nutritional indices such as growth and body composition and on plasma steroid concentrations, GSI and histological stage of the gonads of male barramundi. To test the effect of feeding frequency, groups of relatively small (700 ± 25 g) or relatively large (1000 ± 70 g) male barramundi were fed daily (D), every three days (3D) or every seven days (7D) for 24 weeks. A fourth group of each size was starved for 12 weeks and refed for a further 12 weeks. Starvation resulted in loss of body weight and tissue nutrients, while nutrients were regained and compensatory growth occurred during the refeeding period. Low feeding frequency (7D) resulted in reduced or no growth in fish, whilst fish fed more frequently (D or 3D) showed higher growth. These findings were also reflected in improved body composition of fish fed more frequently. In general, gonads were found to be cycling with a range of stages of development including development of gonia cells and appearance of spermatocytes and spermatids apparent in any particular population (treatment group) of fish. However, a larger proportion of testes from barramundi that were starved were immature compared with those of fish in other treatments. Feeding regime affected the hormone production in the smaller and larger fish differently. Although starvation or low nutrient intake did not influence the low Oestradio1-17β (E₂) levels in small fish, the relatively higher E2 level in larger fish was clearly reduced by starvation. In contrast, relatively high Testosterone (T) level of small fish was reduced by starvation but low T level was not influenced by starvation in large fish. In the less extreme treatments, the effects were not as clear. The large fish in D or 3D regimes increased plasma T level although not significantly and plasma E₂ level significantly decreased at week 18. A period of refeeding after starvation clearly influenced the concentration of plasma steroid hormones in both size groups. To test the effect of protein:energy ratio, groups of male barramundi were presented with diets containing 50% protein and either 15 MJ.kg⁻¹, 18 Mike, 21 M.J.kg⁻¹ or 24 MJ.kg⁻¹ by varying dietary lipid for a 24 week period. Comparable growth was observed in all animals with low energy diets being consumed at a greater rate to compensate for the lack of energy. Body composition analysis showed that high dietary energy led to greater fat storage. As with the starved animals, gonads of fish fed the lowest energy diet were consistently found to be at an early developmental stage with tightly packed gonia cells and gonads of fish in other treatments showed evidence of normal cycling. Dietary energy level did not significantly affect the plasma hormone levels in male barramundi. Plasma E2 was relatively low throughout and did not show differences between treatments. Plasma T level reduced with time and similarly did not vary between treatments. Plasma 1 1keto Testosterone (11kT) did not show any particular trend with dietary protein:energy ratio, even in the lowest energy diet. To test the effect of dietary fatty acid levels, groups of fish were fed with one of four diets (50 % protein, 21 M.J.kg⁻¹) containing either linseed oil (enhanced levels of 18:3 n-3), soybean oil (enhanced levels of 18:2 n-6), fish oil (enhanced levels of 20:5 n-3 and 22:6 n-3) or Aquagrow (enhanced levels of 20:4 n-6) for 18 weeks. Fish fed a diet high in short chain n-3 fatty acids derived largely from linseed oil had lower growth than those of other treatments. Fish fed short chain n-6 and long chain n-3 fatty acids had intermediate growth and fish fed long chain n-6 had the highest growth. Tissue fatty acid profiles were highly correlated with the dietary fatty acid profile, but evidence was also obtained that barramundi preferentially accumulate long chain HUFA into the gonad. No apparent effect on the stage of gonadal development as a result of dietary fatty acid profile was observed. Nor did dietary fatty acid profile affect the circulating concentration of plasma T or E₂. Plasma E₂ level was low at week 18 and plasma T level showed similar changes in all treatments. Plasma 11kT level clearly declined with time and it is suggested that this may have been in response to the high dietary fatty acid levels negatively affecting hormone production. Alternatively, high dietary fatty acid levels used in this study may have inhibited plasma T production with subsequent decreases in production of 11kT from its precursor T. The principle conclusion from this study is that extreme cases of nutrition (starvation, refeeding, low dietary energy) impact on male barramundi reproductive development, but under less extreme conditions, there appeared to be little effect. This is in agreement with some of the data in the literature, which indicates that male fish expend less energy on reproduction and so are less affected by moderate changes in nutritional conditions. Conclusions regarding the effects of the moderate treatments are constrained since there appeared to be other circumstances impacting upon the experiments. Difficulties experienced with poor quality feeds and resulting long acclimation periods, disease events and the fact that relatively small gonad sizes observed in all experiments, even in the presence of gametogenesis indicate that the conditions may not have been ideal for reproductive development. Thus, this study must be considered a preliminary investigation. It does nevertheless provide a significant platform for future work regarding the effects of nutrition on male barramundi broodstock development and teleost reproduction in general.

碩士
國立宜蘭大學
生物技術研究所碩士班
95
Lates calcarifer (bloch) belongs to the Lates genus, Centropomidae family, Perciformes order and Osteichthyes class. This tropical and sub-tropical fish is located in the coastal brackish water throughout Okinawa to the Indian Ocean. Lates calcarifer is massively cultured in Southeastern Asia and Southern Taiwan. Nervous Necrosis Virus (NNV), a member of the Betanodaviridae, is an icosahedral, non-enveloped RNA virus 25-30 nm in diameter. NNV mainly infects juveniles, destroying the nerves of the brain, leading to abnormal swimming and high mortality of infected fish, causing devastating economic impact to the aquaculture industry. Since the larvae of Lates calcarifier are highly susceptible to NNV, this research study developed primary and continuous cell lines from the liver, kidney, and the gas bladder of bloch. The liver and kidney cells were propagated over 90 passages while the gas bladder cell line was propagated over 150 passages, maintained in 5% FBS, L-15 medium, and grew quickly at both 28°C and 32°C. Epinephelus lanceolatus NNV was first used to test these different cell lines for susceptibility and results showed that Lates calcarifer gas bladder (LCGB) was highly susceptible. Then, wild type Plectropomus leopardus Nervous Necrosis Virus (PLNNV) was replicated in LCGB and the viral titer was 107 TCID50/mL. Large quantities of PLNNV was grown in LCGB, collected and purified, serving as the antigen for anti-NNV monoclonal antibodies production in BALB/c mice. After limiting dilution and screening, 3 different hybridomas secreting monoclonal antibodies against NNV were successfully produced. The 3 monoclonal antibodies were confirmed by Western blot and IPMA against purified NNV. Subsequently, cloning of PLNNV capsid gene and expression of recombinant proteins in E. coli was done in pQE30 vector and M15 host. A Western blot of the monoclonal antibodies against the recombinant protein proved all 3 monoclonal antibodies produced in this study were against the NNV capsid protein. These monoclonal antibodies are important tools to have for further researches on Nervous Necrosis Virus.

Canola meal (CM) is one of many potential plant ingredients for fishmeal replacement in fish diets. Many fish species have performed good growth when fed with dietary CM. However, there is limited information for using this ingredient in barramundi. In order to use this ingredient for aquaculture feeds, the information such as nutritional value, nutrient digestibility and ingredient utilisation have to be provided. Therefore, the present study described in this thesis was carried out to: (1) characterise nutritional composition and determine nutrient and energy digestibility of four Australian CMs with respect to different origin and processing method; (2) assess effects of diets with serial inclusion levels of two different CMs regarding different processing methods (expeller and solvent extraction) on growth performance and feed utilisation; (3) examine effects of CMs on changes in plasma chemistry, histology of digestive, metabolic organs and hepatic gene expression. To achieve the above objectives, two experiments were undertaken. The first experiment (digestibility experiment) was designed with six diets (four CMs: three solvent extracted (SE) CMs from Newcastle, Footscray, Numurkah and one expeller extracted (EX) CM from Pinjarra), a diet with fishmeal (FM) as the sole protein and a diet based on lupin kernel meal (LM) were included as reference diets. Each CM test diet and LM diet were made by incorporation of 30 % of test ingredient and 70 % of basal mash (FM reference). Dry matter, protein, energy, amino acid and yttrium content of the diets, ingredients and faeces were analysed to enable the determination of the apparent digestibility of corresponding parameters. The second experiment (growth experiment) included eight dietary treatments each with three replicates, one FM reference diet (sole protein as fishmeal) (FM), one lupin (LM) diet (300 g/kg LM) and the CM diets (100, 200, 300 g/kg as either SE CM or EX CM). Performance indices such as feed intake, weight gain, DGC, FCR, protein and energy retention were determined. Following, an examination of the health effects and molecular responses of fish fed the CM containing diets compared to the FM and LM diets were also carried out. Plasma samples were analysed for biochemical parameters. The liver, kidney, caeca, distal intestine and stomach were used for histological analysis. For molecular expression, genes involved in fatty acid metabolism (FAS, SCD and FXR) and energy production pathways (CS and PDK) and others involved in detoxification (CYP1A1, CYP3A, CYP2N, GST, GHGPx and GPx) were examined using RT-qPCR. The relative expression level of each gene in each sample was determined by normalising the cycle threshold values for each gene to Ef1-α. Compositional analysis of the ingredients showed that the protein content of the SE CMs (370 to 423 g/kg DM) was higher than that of the EX CM (348 g/kg DM), but the lipid content was lower than that of the EX CM. Among the SE CMs, the protein digestibility of the CMs from Numurkah and Newcastle was similar (84.1 % and 86.6 % respectively), corresponding to that of the LM but significantly higher than that of the CM Footscray (74.5 %). The protein digestibility was the lowest (63.1 %) for the EX CM. The energy digestibility of the CMs (43.1 % to 52.5 %) was similar to that of the LM (54.8 %) except for the lower of the SE CM Footscray (32.4 %). The SE CMs provide 276 to 366 g/kg DM of digestible protein while that of the EX CM is only 220 g/kg DM. The digestible energy content of the SE CM Footscray (6.5 MJ/kg) was significantly lower than that of other CMs (8.7 to 10.6 MJ/kg DM). After an eight week culture period the feed intake, growth performance, and protein retention efficiency of fish fed with dietary CM levels were similar or even higher to those of fish fed the FM and the LM diets. The FCR is also similar or better than the control diets. The exception to this was for fish fed with the 300 g/kg EX CM diet. The diet containing 300 g/kg EX CM depressed growth performance, feed intake, and increased FCR. In general, the SE CM can be used up to 300 g/kg diet without negative growth effects while 200 g/kg is the maximum acceptable level of the EX CM for barramundi. Plasma biochemistry parameters were fairly similar among each of the dietary treatments. There were no modifications in the morphology of the liver, kidney, caeca, distal intestine or stomach of fish caused by any of the experimental diets. The expression of genes involved in fatty acid metabolism and TCA cycle was not influenced by fish fed with CM containing diets relative to the FM control and LM diets. However, fish fed with the diet containing 300 g/kg EX CM were shown to downregulate the expression of some genes acting in detoxification pathways (Lc CYP1A1, Lc CYP3A, Lc CYP2N and Lc GST), but not Lc GPx, Lc PHGPx and Lc GR. Overall, this study demonstrates that CM is a promising plant ingredient for FM replacement in barramundi based on determined digestible values and feed utilisation. However, implications regarding different origin and processing method importantly affect CM utilisation for barramundi.

Asian seabass, Lates calcarifer, is a protandrous catadromous fish species cultured worldwide for commercial aquaculture. Farmed Asian seabass exhibit precocious sex inversion before two years of age and this phenomenon is the major impediment to maintaining broodstock in a hatchery. In the wild, age, seasonal temperatures and movement of the fish to a brackish or saline environment apparently affect spawning and sex inversion of Asian seabass. This series of experiments investigated the role and relationships between age and the hatchery conditions which appear to induce sex inversion in captivity. Asian seabass grown in freshwater under natural conditions were transported to the research facility at James Cook University, Australia to conduct four experiments as follows. In the first experiment investigating age effects, Asian seabass of four different sizes grown in freshwater were transferred to salinewater (32 g L⁻¹) at 28 °C and 14: 10 L:D photoperiod and fed to satiety twice daily. Eight groups of Asian seabass (n=8/group, two groups per each size) according to their body weight were allocated to 3000 L tanks in a single enclosed room. In the rest of the experiments, 14 month old seabass grown in freshwater under natural temperature in the same farm were transferred to the research facility and held in freshwater at 28 °C until acclimatized to the experimental conditions at different salinities (0 g L⁻¹, 20 g L⁻¹or 30 g L⁻¹ salinity), different temperatures (22 °C, 25 °C, 28 °C, 31 °C and 34 °C) and both salinity versus temperature (24 °C, 29 °C or 34 °C at each of 0 g L⁻¹ or 30 -32 g L⁻¹) in experiments two, three and four, respectively. Each experiment additional rearing conditions were similar, except the variable environmental parameter tested. Fish were anaesthetized to collect the blood samples for plasma steroids assays at the beginning and at the end of the experimental period. Upon collection of blood, the fish were sacrificed; brain and gonad were removed aseptically, and labeled vials were placed in liquid N₂ at ⁻80 °C for aromatase assay for all experimental fish, while histological analyses were conducted for last two experiments. Brain aromatase activity appeared to respond to age/size rather than environmental conditions, while gonadal aromatase was detectable only in the 700 – 1000 g fish group, plasma T increased in response to the environmental change in fish groups of 300 – 500 g and 700 – 1000 g while the 50 – 100 g and 2.5 – 4 kg fish had no increases (P˃0.05). Plasma E₂ increased significantly in all groups of fish in experiment one, while 11 KT was detected in the 700 – 1000 g and 2.5 – 4 kg fish and was significantly different (P˂0.05). Results indicated that the hormonal conditions are pre-requisite for inducing sex change in captive Asian seabass from 435 ± 27 g body weight. The results of the second experiment indicated that there were no differences (p˃0.05) between the aromatase activities in the brains of fish held at 0 g L⁻¹, 20 g L⁻¹ or 30 g L⁻¹ salinity, while no differences (p ˃ 0.05) between the gonadal aromatase activities were also observed in any of the treatment groups except for fish held in 10 g L⁻¹ and 30 g L⁻¹ salinities, respectively. The highest gonadal aromatase level was recorded in fish held in 0 g L⁻¹and 20 g L⁻¹. Plasma T concentration in fish in all treatments at the end were not different (P ˃ 0.5), while the highest E₂ level was recorded in fish held at 0 g L⁻¹ followed by fish held at 10 g L⁻¹ and 30 g L⁻¹, respectively. However, no measurable amount of 11- KT was detected in any salinity group of fish in this experiment. Results of the third experiment which examined the effects of temperature on sex change indicated that there was an increase in plasma E₂ levels with increasing temperature from 25 °C, while no significant difference was observed among all treatment temperatures except at 25 °C. However, fish held at 22 °C expressed higher E₂ level than at either 25 °C and 28 °C. Significantly higher plasma T levels were detected in fish held at 31 °C and 34 °C, while a reducing trend was observed towards lower temperature regimes. Fish held at 22 °C had significantly lower plasma T than all others as well as those sampled at the beginning. The plasma 11- KT was at non-detectable levels in all experimental temperatures as shown in the initial fish sampled. The average aromatase activity in the brain was highest at 28 °C among all temperatures, but no significant differences were observed. The average aromatase activity in the gonad was higher at 31 °C, followed by 34 °C and 28 °C. No or very low levels of gonad aromatase activity was recorded in fish sacrificed prior to treatment. The aromatase activity was greater in brain than in gonad suggesting that the initial responses to changes in environmental temperatures occur in aromatase produced in the brain. The results of the final experiment indicated that there was an increase in plasma E₂ level with temperature in fish held at 34 °C, whereas no significant difference was observed at 24 °C and 29 °C, although the highest plasma T level was detected in fish at 34 °C which, except for those fish held at 24 °C in freshwater, had significantly lower levels than at the beginning. Plasma 11- KT was significantly greater in fish held at 24 °C compared with 29 °C or 34 °C, which was opposite to that of E₂. Aromatase activity in the brain was higher at 29 °C than at either 24 °C or 34 °C, whereas gonadal aromatase was recorded the highest at 34 °C. It is apparent from the data presented in the final experiment that there is a relationship between culture water temperature, independent of salinity, and induction of sex change as demonstrated by histological staging and measured through changes in the concentrations of aromatase and reproductive hormones.

Formulated ‘artificial’ diets have the potential to overcome inherent nutritional and financial drawbacks associated with live foods used for larvae fish culture. Artificial formulations also provide a vehicle for accurate manipulation of nutritional constituents, enabling investigations into the nutrient requirements of fish larvae. In order to develop species-specific larval food formulations, optimal macronutrient requirements and appropriate nutrient sources must be established. The nutritional value of a food ingredient is determined by its nutritional profile and nutrient availability, and therefore nutrient digestibility is an essential part of the evaluation of novel food ingredients. Since the proteolytic capacity of barramundi larvae is limited, optimal sources of dietary protein must be established to optimise both their amino acid profile, and protein digestibility. An integrated approach was therefore adopted in this study to evaluate protein sources for barramundi larvae in terms of their amino acid profile and digestibility and in their capacity to support optimal growth and survival when incorporated into food formulations. Furthermore, the endocrinal mediation of the nutritional control of growth is investigated through thyroid hormone analysis, and nutritional effects on digestive physiology are examined through pepsin development. The thyrotropic hormone system is a major regulatory mechanism for the control of growth in teleosts. Thyroid hormones (triiodothyronine, T3 and L-thyroxine, T4) mediate extrinsic processes, such as nutrition, to regulate growth in juvenile and adult fish, and are regulated by nutritional quality and quantity. While thyroid hormones regulate growth, survival, development and metamorphosis in fish larvae, data are lacking on an endocrine-nutrition link at the larval stage. By applying an endocrinal approach to the nutritional control of growth, we may achieve a better understanding of the underlying processes governing the physiological status of fish. The research described in this thesis was therefore designed to clarify the quantitative and qualitative protein requirement of barramundi, Lates calcarifer, larvae, and to investigate possible nutritional links to thyroid hormone concentration. Dietary protein and energy contents were initially manipulated in larval food formulations to determine baseline macronutrient inclusion levels. Barramundi larvae (14 days after hatch, DAH) were fed microbound diets (MBD), varying in gross dietary protein (45, 50 and 55%) and energy (18 and 21 MJ.kg-1) for a period of 14 days. All fish were then sacrificed, measured for total length and a sub-sample taken for dry weight analysis. Carcass T3 and T4 were measured by radioimmunoassay, following chloroform/NH3OH extraction. In following experiments, marine animal meals (fish meal, squid powder, Artemia meal, mussel meal, prawn meal and krill meal) were evaluated for their suitability for inclusion into MBD for barramundi larvae. Each of these meals was included in dietary formulations to a total of 50% gross protein. These formulations were evaluated in terms of growth and survival of barramundi larvae, amino acid composition of protein sources, protein digestibility and resulting carcass thyroid hormone levels. To improve the limited digestibility of fish meal, subsequent experiments incorporated fish meal hydrolysates and acid-denatured fish meal into MBD for evaluation in growth trials with barramundi larvae. An optimal diet was found to contain at least 21 MJ.kg-1 dietary energy and derived its protein from a combination of fish meal and squid powder (9:1 ratio). The limited digestibility of fish meal was improved approximately two-fold by acid denaturation, and the moderate inclusion of denatured fish meal into food formulations improved larval growth significantly, while the entire replacement of untreated fish meal with denatured fish meal did not improve growth above that of diets containing intact fish meal. The reasons for this are unclear, though carcass pepsin level was depressed for larvae fed the formulation containing no intact fish meal, indicating that larvae may adapt to less digestible protein sources. The high leaching rates typically attributed to MBD are assumed to be responsible for the poor growth and/or survival of larvae fed diets containing autolysates and hydrolysates in this study. Thyroid hormone levels had no direct correlation to dietary energy level, protein source or protein inclusion level, though T4 correlated with growth independently of these nutritional manipulations. This finding indicates that T4 is important in the growth process of barramundi larvae, but is not directly mediated by specific nutritional inputs. This study developed a microbound diet which supported up to 58% survival and significant growth in barramundi larvae from 14-28 DAH. The diet that supported the best rates of growth and survival contained 21 MJ.kg-1 utilisable dietary energy and at least 50% dietary protein, comprised of a 9:1 ratio of fish meal to squid powder. This study utilised integrated methodology to improve diet composition to increase growth and survival in barramundi larvae fed MBD, and to investigate the underlying mechanisms behind growth promotion. Growth trials remain the most conclusive way to determine optimal nutrient requirements for formulated foods, however, biochemical composition and physical properties can be used to narrow-down the wide range of nutrient sources available. Digestibility is of critical importance to the study of protein sources in food formulations for fish larvae, and a major area for the improvement of native animal meal protein sources. The diet developed in this study is a critical step in the development of species-specific weaning and larval diets for barramundi. Amino acid profiles for optimal growth have been refined, and the digestibility of fish meal has been increased to improve larval growth, thus potentiating early weaning protocols and diminishing Artemia requirements. The development of co-feeding and weaning protocols with this diet is expected to increase growth through optimised amino acid profile and increased energy and fatty acid availability, while reducing costs in barramundi hatcheries through reduced Artemia requirements.

The purpose of this research was to study polybrominated diphenyl ethers (PBDEs) and the effect they have in North Queensland, Australia, specifically in reference to a commercially important fish species, barramundi (Lates calcarifer). This thesis is separated into four main sections: determination of PBDE levels in Ross Creek, Townsville, QLD; toxicokinetics of PBDE-47 in barramundi; optimization of an enzyme-linked immunosorbent assay (ELISA) for detection of vitellogenin (Vtg) in barramundi; and assessing the (anti-)estrogenic effect of PBDE-47 in barramundi. Levels of two common PBDE congeners, PBDE-47 and PBDE-209 were measured in sediments at three sites along Ross Creek in Townsville, QLD. Levels were found to range from below detection (0.2 μg kg-1 dw) to 0.35±0.2 μg kg-1 (dw) for PBDE-47 and from below detection (0.2 μg kg-1 dw) to 0.85±0.07 μg kg-1 (dw) for PBDE-209. Male juvenile barramundi were injected with either a low (1 mg kg-1 bw) or a high (10 mg kg-1 bw) dose of PBDE-47 and then sampled over the course of 14 days in order to determine the depuration rate of PBDE-47 in barramundi. PBDE-47 was found to depurate at a rate of 0.041- 0.069 day-1, a rate which falls well within the range of the literature for depuration of PBDE-47 in fish. An optimal ELISA for the detection of Vtg production in barramundi was determined after comparing the component reagents of a pre-existing ELISA with component reagents developed during this study. Two commercially available Vtg standards, a lipophylised Rainbow Trout Vtg standard (RT Vtg standard) and a lipophylised Atlantic Salmon Vtg standard (Salmon Vtg standard) (both from Caymen Chemical Co), were compared to a purified barramundi Vtg fraction obtained after size exclusion chromatography of plasma from barramundi in which Vtg production was induced by repeated injection of large doses of 17β-estradiol (E2). In addition, a commercially available monoclonal mouse anti-striped bass Vtg primary antibody (ND-3G2, Biosense) was compared with two polyclonal sheep anti-barramundi Vtg antibodies (Sh-0404JCU and Sh-0404-SJCU) created by inoculating sheep with one of the size exclusion chromatography purified Vtg fractions. The optimal ELISA was determined to be the preexisting ELISA using ND-3G2 as the primary antibody and RT Vtg standard for quantification, although promising results obtained with the purified barramundi Vtg fractions, Sh-0404JCU and Sh-0404-SJCU suggest that further purification could lead to a better barramundi specific ELISA in the future. Finally, male, juvenile barramundi were exposed to PBDE-47 in two separate experiments to study whether PBDE-47 has an estrogenic or anti-estrogenic effect, with Vtg production measured by ELISA as the endpoint for estrogenic behaviour. In the first experiment barramundi were given either a low (1 mg kg-1 bw) or a high (10 mg kg-1 bw) dose of PBDE-47 by intraperitoneal (i.p.) injection, and then sampled over the course of 14 days to determine the time course induction of Vtg production. Vtg levels in samples were not quantifiable but the qualitative data allowed for assessment of trends and patterns. Two interesting conclusions were apparent from the data. The first is that male barramundi appear to produce Vtg without exposure to xeno-estrogens, a hypothesis that is supported by literature that has found low natural levels of E2 production in males of many fish species. The second is that the high dose of PBDE-47 suppressed Vtg production between days 7 and 14 with Vtg levels rising much slower in the high dosed fish than in either the control or low dosed fish. In the second experiment barramundi were given either a single low (1 mg kg-1 bw) or a high (10 mg kg-1 bw) dose of PBDE-47 by i.p. injection then sampled 3 and 6 days after injection, or were given two low (1 mg kg-1 bw) or a high (10 mg kg-1 bw) doses of PBDE-47 by i.p. injection, with three days between injections, then sampled 3 and 6 days after the second injection. This was done to determine whether a repeated dose of PBDE-47 had more of an effect on Vtg production than a single dose. The Vtg levels in these samples was quantifiable and the results showed that a double injection of PBDE-47 significantly suppressed the production of Vtg (P<0.0001) at both a low and high dose. In addition, at 6 days post final injection there was a small, but significant difference (P=0.0355) between the fish that received a single low dose and a single high dose, confirming that a single high dose of PBDE-47 can suppress Vtg production as well.

Adrien Marc studied the fertility of the iconic Australian barramundi. He optimized advanced reproductive techniques to assess sperm quality, developed reliable sperm storage procedures, and explored the influence of sperm quality on larval development. The Australian aquaculture industry is using his results to improve breeding processes.

碩士
國立宜蘭大學
生物技術研究所碩士班
96
This study demonstares the induction of apoptosis in BM (barramundi muscle) and BSB (barramundi swim bladder) cell lines, by grouper iridovirus (GIV) infection. The apoptosis was detected and confirmed through various methods. At MOI of 10, cytopathic effect (CPE) characterized as cell shrinkage and rounding was first observed in both GIV-infected BM and BSB cells at as early as 45 min pi (post-infection). GIV infection appeared to progress more rapidly in BM cells than in BSB cell as suggested by the faster development of CPE in the infected BM cells. Cell viability assay also showed a stronger inhibitory effect on the BM (26.6%) and BSB (54.8%) cells by GIV at 4 hpi. The DNA fragmentation, Annexin V and Hoechst 33258 assays were performed at 45 min pi. The chromatin nick could be observed by TUNEL assay at 2 hpi. The above results demonstrate that BM and BSB cell lines can be triggered apoptosis by GIV. The heat-inactivated GIV and UV-inactivated GIV were used to infect cells. The results showed only UV-inactivated can induce apoptosis. Caspase-3, -8, and -9 activities in the BM- and BSB-infected cells were early activated at 30 min pi., and as the infection goes along, caspase-3, -9 had the maxima activities at 45 min pi. However, caspase-8 had the maxima activity in comparison to the mock-infected cells at 1 hpi. GIV-induce apoptosis was inhibited by a pan-caspase inhibitor, z-VAD-fmk, indicating a caspase-activation pathway. The above results showed that GIV can induce BM and BSB cells apoptosis and the apoptosis pathway is caspase-activation dependent pathway.

碩士
國立宜蘭大學
生物技術研究所碩士班
96
By definition, viruses are unable to replicate within a host cell. Virus infection can trigger events that lead to apoptosis, however some viruses are known to encode gene products or induce host gene products that block apoptosis and use the host-cell macro-molecular machinery and energy supplies to replicate. In previous studies, it was suggested that GIV could induced apoptosis in barramundi muscle cells via caspase-3, -8, -9 activation pathway. In this study, a proteomic method was applied to identify alterations in protein expression profile in barramundi muscle cells and being an indicator of apoptosis after GIV infection. From this screening, 7 novel proteins were identfied, including Initiation factor 4A 1B （eIF4A1B）, Proteasome 20S, ADP-ribosylation factor 1 （Arf-1）, Ribose 5 phosphate isomerase（RPIA）, Tyrosine Hydroxylase（TH）, RAN binding protein 1, and Phosphoglycerate mutase 1（PGAM1）, were overexpressed in GIV-infected barramundi muscle cells. In addition, we also determined these genes expression levels by real-time PCR. The results showed that except Initiation factor 4A 1B （eIF4A1B）but, ADP-ribosylation factor 1（Arf-1）, Ribose 5 phosphate isomerase （RPIA）and Tyrosine Hydroxylase （TH）were up regulated conpared to the mock-infected cells. However, the Phosphoglycerate mutase 1（PGAM1） expression only rises slightly is heightened in comparison to the mock-infected cells. Summarized our results indicated that GIV could induce host genes expression level, by GIV infection and supports viral, and replication virus-infected cells of premature death. Our results provide an information for understanding of GIV viral replcation in the host cells and possible mechenisms apoptosis regulatory systems.

Australian barramundi (Lates calcarifer), are distributed over much of the northern and northeastern coast where they inhabit rivers, estuaries and near coastal waters spanning some 16 degrees of latitude (10ºS - 26ºS). Over their distribution, populations of barramundi experience differences in thermal environmental conditions that vary from warmer and more consistent tropical conditions at northern latitudes (mean yearly range of 23.2 – 32 ºC), to cooler and more variable conditions at southern latitudes (mean yearly range of 18.5 – 27.7 ºC). Australian barramundi populations show strong genetic structuring and this, coupled with exposure to varying thermal environments, may have led to temperature tolerance differences among these populations indicative of local adaptation. As barramundi are currently cultured to some degree within all mainland states of Australia, the replication of optimal culture conditions, at significant cost to farmers, is necessary to ensure the success of primary breeding objectives. Identifying the underlying genetic mechanisms contributing towards peak growth and survival under a range of culture temperatures in barramundi would therefore be of significant benefit to the aquaculture industry. Research described in this thesis aimed to identify whether or not barramundi populations at the extreme of their Australian distribution exhibited evidence for local phenotypic thermal adaptation and, if so, whether underlying genetic differences could be established. To demonstrate evidence of local adaptation, initial experiments aimed to differentiate between two genetically distinct populations of barramundi based upon their tolerance to increased water temperatures. To determine this, loss of swimming equilibrium (LOSE) was used as a predictor of upper thermal tolerance in fish from a warm-water adapted (Darwin, Northern Territory) and cool-water adapted (Gladstone, Queensland) population. Barramundi from both populations were simultaneously exposed to an increase in water temperature from 28 ºC to 40 ºC at the rate of 2 ºC/h. LOSE was recorded as the time taken for individual fish to demonstrate loss of swimming equilibrium after water heating began. Significant differences in upper thermal tolerance, suggestive of local adaptation, were evident between the two populations with warm adapted, northern barramundi demonstrating a significantly longer time until LOSE at 40 ºC than cool adapted, southern barramundi (518.5 ± 8.0 min and 452.8 ± 8.0 min respectively, ANOVA; F₁, ₂₂ = 7.86, P≤0.01). However, as LOSE challenge tests are not practical to the identification of commercial broodstock; the response to temperature was also evaluated within dissociated caudal fin cells as a means of providing a sensitive and non-invasive method with which to determine upper thermal tolerance in whole animals. Prior to measurements of LOSE, small fin clips were taken from each fish and enzymatically digested to produce 'free' caudal fin cells. Cells were incubated at 40 º C for 1 h as a thermal stress, before cell staining with Propidium Iodide (stains dead cells) and Calcein AM (stains live cells) was used, allowing for the determination of a dead/live cell ratio. Thermal tolerance results generated from dissociated caudal fin cells strongly correlated cell viability with LOSE measurements (average r = 0.69), confirming that this method can be used to discriminate between populations with different thermal tolerances without having to directly thermally challenge valuable broodfish. In doing so, these results provide strong evidence that thermal tolerance differences amongst barramundi populations arise due to significant contribution from differences at the genetic level. Having demonstrated that divergent populations of barramundi show strong evidence for genetic adaptation to temperature, the expression of a group of genes likely to be involved in this species' response to an acute heat stress was examined. The acute heat shock response, as indicated by the expression of genes within the cellular stress (Hsp90α, Hsp90β, Hsc70, Hsp70), metabolic (CiSy, CcoII, Ldh) and growth (Igf1, Mstn1) related pathways, was examined following an increase in water temperature from 28 ºC to 36 ºC over 30 min. Barramundi were maintained at the acute stress temperature of 36 ºC for 1 hr before being returned to 28 ºC and allowed to recover at this temperature for a further 2 weeks. Muscle tissue sampling over the experimental period allowed for the expression quantification of stress, metabolic and growth related genes via real time quantitative PCR (RT-qPCR), where a robust and reliable normalization approach identified both α-tub and Rpl8 as appropriate genes for the analysis of gene expression in response to an acute heat stress. Hsp90α and Hsp70 of the inducible heatshock response pathway showed a massive up-regulation of gene expression in response to heat stress, whilst the constitutive heat shock genes Hsp90β and Hsc70 showed no change over the course of the experiment and a small increase after 2 weeks of recovery respectively. Of the three genes representing the metabolic pathway (CiSy, CcoІІ and Ldh) only CcoІІ changed significantly showing a decrease in gene expression which may suggest a small suppression of aerobic metabolism. Igf1 of the growth pathway showed no significant differences in response to an acute heat stress, whilst Mstn1 increased at the beginning of the heat stress, but returned to basal levels soon after. Overall, the results demonstrate that an acute heat stress in L. calcarifer caused a significant increase in the expression of genes from the cellular stress response pathway along with a potential decrease in aerobic metabolism and only relatively minor changes to the growth pathway. These results highlight the hardy nature of L. calcarifer and demonstrate the importance of an adaptive gene expression response in coping with the sudden temperature changes routinely encountered on a daily basis within its natural environment. Having identified key genes from temperature responsive pathways, differences in the phenotypic performance of barramundi populations to temperature (as highlighted in Chapter 2), were interpreted using gene expression data. Following on from the analysis of gene expression in response to an acute heat stress, key genes from the cellular stress, metabolic and growth pathways were analysed via RT-qPCR in both a warm (Darwin, Northern Territory) and cool (Gladstone, central Queensland) water adapted barramundi population reared at either hot (36 ºC), control (28 ºC) or cool (22 ºC) temperatures for 106 days. Growth indicators were periodically measured during the growth trial and white muscle tissue was also sampled at day 0 (T=0), day 3 (T=3), day 9 (T=9) and day 106 (T=106) for gene expression analysis. At a rearing temperature of 22 ºC, a higher final weight in cool adapted barramundi over warm adapted barramundi (145.9 ± 11.1 g and 89.9 ± 3.5 g, respectively) was underpinned by a significantly faster induction of the cellular stress response and greater expression of Hsp90α. Conversely, no changes in heat shock protein (Hsp) gene expression were observed in barramundi reared at either 36 ºC or 28 ºC. Genetically regulated adaptation to cool temperatures in barramundi therefore seems to be correlated with changes to the cellular stress response pathway. Regulation of metabolic and growth associated genes to temperature were consistent between populations and were not affected by a control (28 ºC) or a cool (22 ºC) rearing temperature. At 36 ºC, both warm and cool adapted barramundi exhibited a significant decrease in CcoII expression consistent with expectations associated with alterations in aerobic capacity, however, CiSy expression remained unchanged. The impaired growth of both populations reared at 36 ºC was accompanied by a decrease in the expression of Igf1 and an increase in the expression of Mstn1. As such the expression of both genes can be reliably used to indicate the growth status of barramundi at high temperatures, however, long term control of growth at cool temperatures seems to be under the control of alternate gene pathways, as no significant differences in the expression of these two growth related genes was observed. To further investigate population differences, the underlying transcriptome profile of barramundi reared over a long term period was examined via Illumina mRNA deep sequencing as a means of determining the major contributing gene categories giving rise to the phenotypic differences in population growth. White muscle tissue from warm and cool adapted barramundi reared for 106 days was sampled and used for pathway expression analysis in conjunction with the phenotypic data collected previously. Gene ontology (GO) analysis revealed enrichment in categories relating to the regulation of peptidase activity as well as microtubule, cytoplasmic and cellular metabolic based processes. Further analysis of the GO category "microtubule based process" with associated genes from the "response to stress" category revealed an apparent reorganisation of cytoskeletal elements in response to an induced cold stress in northern barramundi reared at 22 ºC, when compared with northern barramundi reared at 36 ºC. Between southern barramundi and northern barramundi reared at 36 ºC, an analysis of the "endopeptidase inhibitor activity" GO category, in conjunction with stress genes, indicated a suppression of the immune complement system in southern barramundi, along with an increase in the cellular stress response. As southern populations of barramundi from a cooler environment grew significantly faster at 22 ºC than northern barramundi populations from a warm environment; the results of the present study show that southern populations of barramundi exhibit underlying molecular adaptation to cooler water temperatures, but still retain a tolerance for warm water temperatures. Furthermore, GO profiling has revealed groups of genes that underlie population differences in temperature tolerance as a means to prioritize the analysis of differential gene expression in studies of local adaptation in the future. The results of this thesis demonstrate the occurrence of local adaptation to environmental temperature amongst Australian populations of barramundi from significantly different environments. Both phenotypic and genetic indicators reveal the hardy and adaptable nature of barramundi to adverse temperatures in accordance with the variable characteristics of estuarine environments. Specifically, barramundi from the northern end of the species distribution range show a greater tolerance to short, but significant spikes, in water temperature compared with barramundi from southern populations. However, gene expression analysis and growth data reveal that southern populations of barramundi show adaptive traits leading to better growth at cooler temperatures when compared with northern populations. The performance of southern barramundi at cool temperatures was accompanied by significant differences in the expression of heat shock genes and other associated stress responsive genes, suggesting that faster induction and higher expression of heat shock genes aids the continuation of growth at cooler temperatures. The expression of nominated growth related genes was only affected at high rearing temperatures and it is therefore likely that growth at cooler temperatures is under the influence of alternate mechanisms, which could prove interesting for future research.

碩士
國立屏東科技大學
水產養殖系所
103
Abstract: The study was to evaluate the effects of water temperature on growth, feed intake and body composition of juvenile barramundi (Lates calcarifer). Triplicate groups of fish (initial wt:10 g) were reared in recirculating system with five temperature (23 °C, 26 °C, 29 °C, 32 °C and 35 °C) for 30 days. Survival (100%) were the same (p > 0.05) among all treatments. Feed intake and specific growth rate (SGR) were the highest (p < 0.05) in 29 °C and 32 °C rearing group, followed by 26 °C and 35 °C rearing group, and the lowest in 23 °C rearing group. Fish reared in 29 °C and 32 °C show lower feed conversion ratio than other treatments. When the water temperature were 29 °C and 32 °C, body lipid content was lower but protein content was higher than other treatments. The results suggest that the adequate rearing temperature are 29 °C and 32 °C.

Fish farming with Recirculating Aquaculture Systems (RAS) is becoming widespread to fill the demand gap due to diminishing wild caught sea foods. Barramundi fish has a high demand as a premium Australian seafood, and is grown as an RAS farmed-fish. However, the accumulation of ‘earthy’ or ‘muddy’ off-flavours due to taint accumulation as geosmin (GSM) or 2-methylisoborneol (MIB) in the fish-flesh of is a major concern. Inconsistent quality of farmed barramundi has been identified as a major issue in buyer resistance. Established predictive models for chemical taint in fish-flesh have been based on steady-state assumptions. However, it was thought debatable as to whether a steady-state assumption could be upheld i.e. there was no evidence that the net chemicals exchange is zero across the fish body and RAS water phase. Against this background, an original, new and quantitative model that predicts the time dependent concentration of taste-taint chemicals as GSM and MIB in harvested fish-flesh was developed (Hathurusingha & Davey, 2013; Hathurusingha & Davey, 2014; Davey & Hathurusingha, 2014). This model is based on conservation of mass and energy, and thermodynamic processes established in (bio)chemical engineering with chemical uptake and elimination routes into and from the fish considered. The model was simulated for two RAS species, barramundi (Lates calcarifer) and rainbow trout (Onchorhynchus mykiss) with independent data (n ≥ 14) and showed good agreement with experimental observations. A major benefit of this new model is that simulations can be used to investigate a range of growth protocols in RAS farming to minimize taint in fish-flesh. An advantage is that it can readily be simulated in standard spread-sheeting tools by users with a range of sophistication. Extensive experimental testing of the new model was carried out in both pilot- and commercial-scale plants using low concentrations (≤ 10 mg L⁻¹) of hydrogen peroxide (H₂O₂) as a benign biocide to limit natural occurring taste-taint chemicals in the RAS growth water, and subsequently into the fish-flesh. A dedicated methodology and new dosing apparatus (ProMinent Fluid Control Pty Ltd, Germany) for controlled H₂O₂ dosing was developed. The analyses of taste-taint chemicals as GSM and MIB in water and fish-flesh was carried out with Solid-Phase Micro-Extraction (SPME) followed by Gas Chromatography Mass spectroscopy (GC-MS) (skills training was obtained at both the University of Laval and University of Waterloo, Canada). Preliminary investigations with a low concentration of H₂O₂ (5 mg L⁻¹) in pilot-scale (2,500 L) studies with barramundi fish demonstrated its potential to mitigate development of GSM and MIB in RAS water. It was found that controlled dosing of low concentrations of H₂O₂ did not impact the pH level in growth waters and was not detrimental to the health and well-being of the fish as fingerlings (0.01 kg) and until harvest at 240 days (0.8 kg). Additional benefits of H₂O₂ as benign biocide include a fish product of whiter colour, an increased dissolved oxygen concentration (Cₒₓ) in the growth water, a reduction in the number of gill flukes, and improved particles distribution with increased C:N ratio, and; improved availability of organic carbon in the growth water. Based on these preliminary investigations H₂O₂ was ‘optimised’ at a (low) concentration of 2.5 mg L⁻¹ as a benign biocide. This was investigated in commercial-scale studies (conducted at Barra Fresh Farm, South Australia) for a typical growth of 240 day for barramundi as the selected RAS fish. The emerging risk methodology of Davey and co-workers (e.g. Chandrakash et al., 2015) was applied for the first time to investigate quantitatively the impact of naturally occurring fluctuations in taste-taint chemicals in the RAS water and their accumulation in the fish-flesh. This predictive approach was justified because of the prohibitively expensive time and analytical costs that experimental studies would have necessitated. A Refined Monte Carlo (with Latin Hypercube) simulation of GSM and MIB in the growth water (Cᴡ), water temperature (T) and growth time (t) was used to simulate typical RAS farmed barramundi. It was found in RAS farming of barramundi it would be expected some 10.10 % of all 240 day harvests, averaged over the long term, would result in fish with taste-taint as GSM above the desired consumer rejection threshold concentration (0.74 μg kg⁻¹) due to natural fluctuations in an uncontrolled RAS environment. For MIB this predicted failure rate was 10.56 % (Hathurusingha & Davey, 2016). The vulnerability to taste-taint failure as GSM and MIB was shown to be principally controlled by the time to fish harvest, and to a lesser extent by concentration and fluctuation of these taint chemicals in the RAS water. This work was of practical benefit because growth time can be readily controlled by farmers. The methodology appears generalizable and therefore is applicable to a range of RAS farmed fish (and possible crustaceans e.g. prawns- Macrobrachium sp.). In extensive commercial-scale RAS studies with barramundi and controlled H2O2 dosing, fish grown from fingerlings to harvest at 240 day was investigated. This was to observe an entire production cycle. Results from a H₂O₂ ‘treated’ growth tank (30,000 L) were compared directly with those obtained from an identical ‘control’ tank (30,000 L). Increased organic matter (three (3) to four (4) times pilot-scale findings) reduced H₂O₂ efficacy through inhibiting generation of reactive oxygen species (ROSs). This is thought to be a consequence of the need to scale (48 times volume) the pilot-scale studies for in-tank mixing. Analyses of fish-flesh (n ≥ 167) showed (moderate) predicted exponential correlation between taste-taint concentrations in the fish-flesh and the growth-mass of the fish for both GSM and MIB as predicted. In addition, the research findings highlighted that accumulation of taste-taint compounds was mainly governed by the combined effect of mass of the fish (mᵳ) and taste-taint concentrations in the growth water (Cᴡ). Comparisons between the model predictions and experimental observations showed good agreement over the range of low taste-taint concentration (0 to 2, μg kg⁻¹), especially below the consumer rejection threshold (~ 0.7 μg kg⁻¹). However, a minor anomaly was an over-prediction for greater concentrations (2 to 11, μg kg⁻¹). Current predictions are therefore conservative or ‘safe’ by about 20 %. Possible reasons for over prediction might be attributed to rapid fluctuation of taste-taint concentration in growth water with growth time and different (exponential) growth constants shown by larger and smaller fish, and; errors in obtaining representative samples from fish-flesh. Model predictions and experiments further highlighted that the new model could be meaningfully applied to RAS systems with lower variations and/or lower taste-taint concentrations in RAS growth water. These theoretical and experimental results are the first for RAS farmed fish covering an entire production period to harvest. Approval for this research was gained from both The University of Adelaide Animal Ethics Committee Science and, Australian Pesticides and Veterinary Medicines Authority (see Appendices F and G). Research findings will be of immediate benefit to RAS farmers, fish processors and risk analysts in foods processing.
Thesis (Ph.D.) -- University of Adelaide, School of Chemical Engineering, 2016.

Growth has traditionally been the most important trait amongst the many targeted to boost the productivity and profitability of aquaculture farms. Several genetic approaches have been investigated and employed in recent years to enhance the growth rate of aquatic species. Of these, the use of a single gene approach has recently gained popularity amongst aquaculture scientists looking at the improvement of productivity. The adoption of a single gene approach has spiked the characterization and study of a number of candidate genes of major effect on growth and one such gene is that of myostatin (Mstn). MSTN is a protein that inhibits muscle growth and that in beef cattle is responsible for inducing "double muscling", a phenotype with increased muscle mass. A number of studies have shown that genes similar to that of the cattle Mstn are present in the whole animal kingdom and in many species have a significant association with growth functions. Because of its central role in the regulation of growth, Mstn is therefore a very interesting candidate gene for the improvement of aquaculture productivity. Detailed evolutionary analyses have revealed that Mstn comprises with its closest relative growth and differentiation factor-11 (Gdf-11) a small gene family that evolved from multiple events of gene or genome duplication. The structure of the Mstn/Gdf11 family resembles the "one-to-four" model seen for many genes isolated from species encompassing the diversity of the animal kingdom. In the one-to-four gene model invertebrates possess a single gene homologue (i.e. Mstn/Gdf11), higher vertebrates like mammals possess two paralog genes (i.e. Mstn and Gdf11) and at least four paralogs are present in teleost fish (i.e. Mstn-1 and Mstn-2). Gene duplication events may have engendered the origin of new functions compared to those initially exerted by the Mstn/Gdf11 ancestor. While some of these new physiological roles arisen following duplication are well studied and understood in mammalian model species, the history of functional evolution of the Mstn/Gdf11 family in species of high relevance for aquaculture production is still largely unclear. This thesis addresses key issues important for providing significant advances in the understanding of Mstn-like genes in aquaculture species using barramundi (Lates calcarifer) and the black tiger prawn (Penaeus monodon) as model species. As at least two Mstn paralogs (Mstn-1 and Mstn-2) are present in fish like L. calcarifer, it is important for the purpose of growth improvement to understand whether subfunctionalization occurred following the event of genome duplication. In this thesis the Mstn-2 gene paralog, including its upstream region, was isolated and characterized from the L. calcarifer. Through a detailed analysis of gene expression this thesis provides preliminary evidence of differentiation of MSTN paralogs. Firstly, differential regulation as well as specific tissueresponses in the muscle, liver, gill and brain of L. calcarifer was observed after nutritional deprivation. In particular, the LcMstn-1 expression increased in liver (~4 fold) and muscle (~3 fold) and diminished in brain (~0.5 fold) and gill (~0.5 fold), while that of LcMstn-2 remained stable in brain and muscle and was up regulated in gill (~2.5 fold) and liver (~2 fold). In addition, it also suggested that Mstn genes in fish may regulate different growth and developmental processes by revealing an independent regulation of each paralog throughout the embryonic development with the Mstn-2 generally more abundant than the Mstn-1 and a diametrically opposite correlation of their expression with muscle hypertrophy in juvenile fish. Analyses of relationship between Mstn transcript abundance and muscle hypertrophy showed in fact that the expression of LcMstn-1 was only marginally associated with fiber size (r = 0.384, p = 0.064) while that of LcMstn-2 showed a highly significant negative correlation (r = -0.691, p < 0.0001). Differential regulation of Mstn paralogs was supported by in silico analyses of regulatory motifs that revealed, at least in the immediate region upstream the genes, a differentiation between Mstn-1 and Mstn-2. The Mstn-1 in particular showed a significantly higher conservation of regulatory sites among teleost species compared to its paralog indicating that this gene might have a highly conserved function in the taxon. Conversely, invertebrates possess a single ortholog of the MSTN/GDF11 family. In this thesis the Mstn/Gdf11 gene ortholog was identified and characterized in the Penaeid shrimp, Penaeus monodon. The overall protein sequence and specific functional sites were highly conserved with other members of the MSTN/GDF11 family. Gene transcripts of pmMstn/Gdf11, assessed by real-time PCR, were detected in a variety of tissue types and were actively regulated in muscle across the moult cycle. To assess phenotypic function in shrimp, the pmMstn/Gdf11 gene expression was down-regulated by tail-muscle injection of sequence-specific double-stranded RNA. Shrimp with reduced levels of pmMstn/Gdf11 transcripts displayed a dramatic slowing in growth rate compared with that of control groups. Findings from this study place the MSTN/GDF11 gene at the centre of growth regulation in shrimp suggesting that this gene has an opposite role in invertebrates compared to higher vertebrates. In the former, levels of gene expression may positively regulate growth. The outcomes of this thesis have provided significant advances in the understanding of Mstnlike genes in aquaculture species such as barramundi (L. calcarifer) and the black tiger prawn (P. monodon), towards the development of MSTN-based technologies for the enhancement of growth. In particular, this research emphasized that significant differences exist between Mstn-1 and Mstn-2 in fish whereby an efficient enhancement of growth may arise from specific targeting of one paralog only. It also revealed for the first time that the invertebrate Mstn/Gdf11 does not inhibit growth. This last finding in particular will cause an inversion of tendency, whereby research aiming to improve growth of invertebrates like crustaceans should investigate strategies to enhance the activity of MSTN/GDF11 and not reduce it, such as the case of vertebrate MSTN.

Barramundi, or Asian seabass (Lates calcarifer), is considered one of the most important commercial tropical aquaculture species in Southeast Asia and Australia. Despite its importance, efforts to improve barramundi production via selective breeding have been challenging, primarily due to difficulties in controlling sex change. Barramundi is a protandrous hermaphrodite, where fish first mature as male and then, a few years later, sex change into female. The gonadal development of barramundi provides a good model to study the genetic mechanisms underlying natural sex change in fish; however, this natural sex change process poses significant challenges for barramundi broodstock management as high quality male broodfish change sex, which requires the constant recruitment of new male broodstock. Alternatively, and relevant to selective breeding programs, the sequential sex change of barramundi results in an age difference between the sexes in spawning groups, where females are one generation older than the males. As the rate of genetic gain is contingent on the generation interval, this inter-generation breeding thereby halves the annualised rate of genetic progress that could otherwise be made through a single-generation selection program. Therefore, sex control of barramundi in hatcheries through either inducing precocious females, or preventing natural sex change from male to female, is of great importance to the industry. Sex control for fishes (including teleosts) is feasible as the course of gonadal development is a flexible process and may be subject to modification by extrinsic factors, especially during early gonadal development when animals are most sensitive to environmental stimuli (widely referred to as the labile period). Manipulation of environmental factors (e.g. addition of sex steroids, changing water temperature or pH) during early gonadal development may override genetically pre-programmed sex determination mechanisms, thus altering the resultant phenotypic sex of the fish. Yet, the process of testicular development in barramundi is unknown. In particular, there is no information on when and how testicular differentiation commences in the species and what happens when exogenous steroids are subsequently administered. To address these knowledge gaps, the research that is reported in this thesis was conducted in three steps. Firstly, the timing of sexual development and differentiation in the protandrous barramundi was determined through tracking morphological differentiation and gene expression of key sex genes from early embryogenesis through to spermatogenesis. This provided an understanding of the timing of initial gonadal differentiation into the male testis state and an indication of the likely labile period whereby sex could be manipulated through the addition of exogenous factors such as hormones. Once the timing and process of testicular differentiation was established, the research then went on to examine the effect of applying exogenous steroid and non-steroidal hormones for inducing precocious feminisation and/or retaining masculinisation. This was examined through a feeding trial of exogenous steroid to juveniles and use of implants with estrogen and fadrozole in male barramundi. The labile period of sex determination in barramundi was identified by examining the gonadal morphology of fish from newly hatched larvae till 9 months post-hatch, when barramundi gonads were previously reported as differentiated testes. As primordial germ cells (PGCs) of male fish remain morphologically unchanged for longer periods and are differentiated as the final step of testicular differentiation, early histological indicators for testicular differentiation, such as the slit-like lumen in stromal tissue, appearance of blood vessels, and invagination of the epithelial tissues into lobular structures, are often used. Results showed that the barramundi proto-testes originates as a strip of cells attached to the dorsal coelemic cavity at 4 days post hatch (dph), with the formation of a slit-like lumen by 44 dph (fish total length (TL) 25.8 ± 3.3 mm), which is the first morphological indication of testicular differentiation in the species. This slit-like lumen later develops into the testes efferent duct. Other major indicators of testicular development were invagination among epithelial cells and the formation of capillary vessels at 60 dph, the organization of primary germ cells into lobules at 90 dph and differentiation of the somatic cells into Sertoli cells at 120 dph. Final testicular differentiation was histologically discernible at 140 dph when the somatic cells had differentiated into Sertoli cells and germ cells into spermatocytes. Noticeably, the male-associated gene dmrt1 (double-sex and Mab-2 related transcription factor 1) was detected exclusively to be expressed in Sertoli cells by in situ hybridisation using an mRNA probe. Additionally, gene expression of the important male-related genes, dmrt1 and cyp11b (cytochrome P450 11β-hydroxylase gene), were examined using RT-qPCR. Cyp11b and dmrt1 were expressed highest in the gonad at 90 and 120 dph, respectively, with dmrt1 expression observed to rapidly increase from 70 to 120 dph. The beginning of sex differentiation to the finalisation of testicular development in barramundi was determined in this study to occur from 44 to 140 dph (TL 169.4 ± 40.3 mm). The next steps in this study were to assess the feminising effect of 17 β-estradiol (E₂) and 17 α-ethinylestradiol (EE₂) on barramundi during their determined labile period. Fish were fed pellets containing 10 mg E₂ kg⁻¹ food, 20 mg E₂ kg⁻¹ food, 5 mg EE₂ kg⁻¹ food and 10 mg EE₂ kg⁻¹ food from 30 to 160 dph, which covers the period of initial male gonad differentiation identified in the first study. The effect of E₂ and EE₂ treatment on gonadal morphology and dmrt1 and cyp19a1a gene expression was analysed by histology and RT-qPCR. Results showed that orally delivered E₂ and EE₂ induced observable changes in gonad morphology. Previtellogenic oocytes (PO) were observed in 33% and 50% of the fish fed with 20 mg E₂ kg⁻¹ food at 160 dph and 12 months post hatch (mph), respectively, while PO were not observed in control fish. The 10 mg E₂ kg⁻¹ food treatment did not induce feminisation, but significantly suppressed testicular development. Treatment with EE₂ resulted in fibrosis within gonad tissues at a dose-dependent rate. E₂ administration resulted in upregulation of the gene cyp19a1a and down-regulation of dmrt1. EE₂ significantly suppressed expression of dmrt1 at 160 dph and 12 mph; while cyp19a1a was not significantly different at 160 dph and was significantly downregulated at 12 mph. The result of this study showed that E₂ is a more suitable hormone than EE₂ to induce precocious feminisation of barramundi, and when delivered through the feed it has a dose-dependent feminising effect. Whilst feeding of estrogen steroids to juvenile barramundi results in sex change of some individuals in a selective breeding program, from a consumer awareness perspective it may be better to only administer hormone to males selected for use as female broodstock to allay any fears about hormone treatment of fish that may be destined for human consumption. Because of this, it was deemed vital to assess if there is an alternative route to sex change males. The literature shows that sex plasticity of some teleost species remains after sexual differentiation, therefore a trial was conducted to produce precocious female barramundi after fish had differentiated as males. Here, the efficacy of two dosages of E₂ delivered via implants to induce the early feminisation of barramundi were evaluated. Six-month-old male barramundi (405 ± 50 g body weight (BW)) were given a single cholesterol-based pellet implant containing either 0 mg E₂ kg⁻¹ BW (untreated control), 4 mg E₂ kg⁻¹ BW ('low dose'), or 8 mg E₂ kg⁻¹ BW ('high dose'). At 4 and 9 weeks post-implantation, gonads were histologically assessed for morphological changes, liver condition histologically examined and expression profiles of key male (dmrt1, cyp11b and esr1) and female (cyp19a1a and foxl2) sex-related genes examined using RT-qPCR. At 9 weeks post-implantation, significant gonadal morphological changes were observed in E₂-treated fish, while all control fish remained as male. In the 'high dose' E₂ treatment group, 77.8 % (7/9) of fish sex-changed completely to female, signified by gonads containing oocytes (20-30 μm) and no observed residual sperm. Comparably, 44.4 % (4/9) of fish in the 'low dose' E₂ treatment group had sex-changed, while remaining fish showed complete testicular regression with gonads containing only undifferentiated germ cells. In the 'high dose' E₂ treatment, increased expression of female-biased genes (cyp19a1a and foxl2) was observed, while downregulation of male-biased genes (dmrt1, cyp11b and esr1) was induced. The success of artificially-induced sex change in barramundi provides an important tool that is critical to improving selective breeding of this species. Efforts to control sex of barramundi also means having the ability to maintain high quality broodfish as males. The results from the feeding and implant trials illustrated the important role of the cyp19a1a aromatase gene in barramundi feminisation, suggesting a possible way to block natural sex change in barramundi may be to reduce aromatase activity (estrogens are synthesised from androgens through a reaction catalysed by cytochrome P450 aromatase). Accordingly, a final trial was conducted to assess the impact of a non-steroidal aromatase inhibitor, Fadrozole (FAD), administered solely or with E₂ cholesterol pellet implants, on the gonadal development of mature male barramundi. Fifteen-month-old male barramundi (2102 ± 126 g BW) were divided into four groups of 12 individuals each. Fish in the first group were implanted with a cholesterol pellet without FAD or E₂ as control (n = 12). The three treated groups of fish were implanted with 8 mg E₂ kg⁻¹ (n = 12), 8 mg FAD kg⁻¹ (n = 12), or 8 mg E₂ and 8 mg FAD kg⁻¹ were both implanted into individual fish (n = 12). Survival rates were 100% for all groups during the experiment with no damage observed in liver and kidney of any fish. At the final sampling 9 weeks after treatment, 8 % (1/12) of the untreated control fish were found in early stages of sex reversion (stage T1), whereas 100% of FAD-treated fish were male and 100% of E₂-treated fish were female. Binary treatment with FAD and E₂ resulted in 42 % (5/12) males, 42 % (5/12) females and 16% (2/12) transitional fish. In the FAD-treated fish, expression of male genes (dmrt1 and cyp11b) were significantly upregulated, whilst female-biased gene (foxl2) downregulated. Exposure to E₂ resulted in significantly higher expression of cyp19a1a and foxl2; and lower expression of dmrt1, cyp11b and esr1 than control fish. FAD neutralised the effects of E₂, in terms of gonad morphology and gene expression. In summary, this PhD research has revealed the important process of sex differentiation of barramundi, a sequential hermaphroditic teleost species. It unveiled the morphological changes at cellular levels of barramundi gonads from undifferentiated to maturity of testes, while revealing the expression patterns of the important sex-related genes during the process. The timing of this process provided the time window for testing the responsiveness of barramundi gonads to the exogenous steroids for sex control purposes. This research has determined a suitable hormone treatment for barramundi feminisation, including hormone types and doses, timing and administration route. It also filled our knowledge gap in gene expression of the important male- and female-biased genes in barramundi gonad exposed to exogenous steroids and non-steroid hormones. Although the effect of masculine fadrozole was investigated in this thesis, it is necessary to optimise the dose for better results. The process of obtaining young females by means of hormonal treatment in this study also require further research of their reproductive potential, as the ultimate goal is to produce dams for selective breeding programs.

The ability of tropical fish to adapt and/or acclimate to their native thermal habitat is of biological interest, especially in the face of climate change. Evidence for this can be sought at the phenotypic, genotypic, transcript abundance and/or deduced protein sequence level. In this thesis a multi-level approach is employed to examine the question of thermal adaptation in a tropical fish. I use the Australian barramundi (Lates calcarifer) as a target species because genetically distinct populations occur throughout their tropical distribution range. The phenotype of L. calcarifer from two such divergent populations was investigated for a response to two ecologically relevant stressors, namely temperature and swimming challenge. Subsequently investigation of genotype, transcript abundance and deduced protein sequence of the candidate gene lactate dehydrogenase-B (ldh-b) in two discrete Australian L. calcarifer populations were carried out. This candidate gene was chosen because of its known metabolic (aerobic) function and previous association with thermal adaptation and swimming performance in the temperate fish Fundulus heteroclitus. This thesis presents novel evidence for local adaptation of tropical Australian L. calcarifer to their native thermal habitats using a comparative and integrative multi-level approach, as follows: Chapter 2 investigated at the organismal level whether juveniles (n = approx. 300) from two genetically discrete Australian L. calcarifer populations (Darwin, NT: 12° S, 130° E and Bowen, QLD: 20° S, 148° E; herein referred to as northern and southern, respectively) possess differences in mortality rate, growth rate and exhaustive swimming capacity following 28 day acclimation to both cold- (20°C) and heat-stress (35°C) conditions. This chapter uses swimming challenge as an ecologically relevant aerobic stress, applied in the presence and absence of thermal stress, to establish physiological or metabolic differences between these discrete L. calcarifer populations. Among-population differences were consistent with local thermal adaptation, with southern juveniles from the cooler climatic region exhibiting significantly higher performance than their northern counterparts under cold-stress (20°C) conditions (F₁, ₁₂ = 18.023; p = 0.001). Conversely, northern populations from the warmer climatic region performed significantly better than their southern counterparts under heat-stress (35°C) conditions (F₁, ₁₂ = 13.948; p = 0.003). Mortality rates differed significantly from expectation at the two temperature stress conditions, with cold-stress resulting in significantly reduced mortality (1.4 %) for the southern population (p < 0.05) and heat-stress resulting in significantly increased mortality for both populations (53.2 and 17.6 % respectively), but particularly for the southern population (p < 0.02). These phenotypic differences indicate that discrete L. calcarifer populations may be locally adapted to their native thermal regime across their tropical Australian distribution and provides the basis for subsequent investigations of genotype, transcript abundance and deduced amino acid sequences for the candidate gene lactate dehydrogenase-b (ldh-b). Chapter 3 investigates the ldh-b genotype in Lates calcarifer and congeneric Lates niloticus and compares the nucleotide (exons and introns) and deduced amino acid sequence of the ldh-b locus among these two tropical fishes. Ldh-b was 5,004 and 3,527 bp in length in L. calcarifer and L. niloticus, respectively, with coding regions comprising 1,005 bp in both species. A high level of sequence homology existed between species for both coding and non-coding regions (> 97% homology), corresponding to a 98.5% amino acid sequence homology. All six known functional sites within the encoded protein sequence (LDH-B) were conserved between the two Lates species. This chapter also identifies putative regulatory motifs and elements embedded within non-coding (intron) regions including 10 simple sequence repeat (SSR) motifs and 30 putative microRNA elements (miRNAs).. Five single nucleotide polymorphisms (SNPs) were also identified within putative miRNA regions. This ldh-b characterization provided valuable sequence for the subsequent population genetics and transcript abundance studies. Chapter 4 uses a population approach to assess the level of nucleotide (exons and introns) and deduced amino acid sequence variation in the ldh-b locus both among and within Australian L. calcarifer populations from different thermal regimes (n = 8). We identified a high homology of nucleotide and amino acid sequences among discrete populations lending further support to a hypothesis for selective constraint acting on the ldh-b locus in this tropical fish (Chapter 6). Interestingly, we found that the southernmost population sampled (Gladstone, Queensland, 23°S 151°E) differed significantly (p < 0.05) from all the other six populations screened with F(ST) values ranging from 0.12 to 0.30, making it the ideal target population for future research on this thermally sensitive species. This chapter also reveals the presence of several intronic SNPs which may impact several overlapping putative miRNA elements and may ultimately influence the expression of the ldh-b locus itself or other loci of the transcriptome. Chapter 5 investigates transcript abundance by quantifying the magnitude of variation in hepatic ldh-b transcripts among the two sampled populations (Darwin, NT and Bowen, QLD) following 28 day acclimations to cold-stress (20°C), heat-stress (35°C) and native control temperature (25 or 30°C) treatments. Ldh-b transcript abundance was also quantified in individuals subjected to swimming stress in control temperatures and in the presence of cold- or heat-stress, following acclimation to these temperatures. This chapter uses L. calcarifer-specific qRT-PCR primers and Sybr GreenER fluorescence based assays to quantify ldh-b transcript abundance. Fish from the southern and northern populations exhibited a significant increase in hepatic ldh-b transcript abundance (F₅, ₄₁ = 6.459; p < 0.001 and F₅, ₃₉ = 3.866; p = 0.006 respectively) in response to swimming stress at their respective native culturing temperatures (25°C and 30°C respectively) (i.e. in the absence of thermal stress). Fish from both populations exhibited a significant increase in hepatic ldh-b transcript abundance following heat-stress (35°C) acclimation compared to controls. Fish from the southern, but not the northern, population possessed significantly higher abundance of hepatic ldh-b transcripts following cold-stress (20°C) acclimation compared to controls. These observations suggest that the southern population possesses a unique transcriptional response to that of the northern population and that southern fish may be evolutionarily accustomed to greater variance in seasonal temperatures. Chapter 6 uses an analytical approach, in lieu of empirical protein data, to identify evidence of selective constraint acting on ldh-b in two tropical congeneric Lates (L. calcarifer and L. niloticus) and Plectropomus (P. leopardus and P. laevis) species compared to one temperate congeneric species pair (Fundulus). In regard to both coding nucleotide (ldh-b) and amino acid (LDH-B) sequences, pairwise and phylogenetic comparisons identify that the two tropical species pairs are more homologous to one another (94 – 96%) than either is to the temperate species pair (90 – 92%). Observed pairwise species differences between coding and deduced amino acid sequences do not occur at known functional residues. In summary, this analytical approach identifies that selective constraint appears to be acting on both coding nucleotide and deduced amino acid sequences of this candidate gene. Chapter 7 synthesizes the combined results of the comparative and integrative multi-level approach used in this thesis and presents novel ideas for future studies which would expand on the biological insight generated by this thesis and could provided further evidence for local adaptation of Australian L. calcarifer to their native tropical habitats.

The research covered in this thesis concentrated primarily on improving production protocols for juvenile barramundi Lates calcarifer through studies on diet and feeding habits, pond rearing techniques, effects of photoperiod on growth, and weaning strategies. Juvenile barramundi are produced in northern Australia for two reasons; to supply seed for the aquaculture industry, and to supply fingerlings for recreational fisheries enhancement programs. Within this context, two related studies were undertaken: firstly, an analysis of the proposal to introduce Nile perch to Australia, which preceded the barramundi production studies; and secondly, an evaluation of the use of circulus patterns on scales for discriminating wild and hatchery-produced barramundi. A review of the historical and present distribution of barramundi (a catadromous species) in Queensland indicated that barriers (barrages, weirs and dams) built in river systems, coupled with the inability to negotiate even minor stream barriers, have restricted access of this fish to much of its original, natural habitat. Further, while the construction of dams has created vast new aquatic habitats (potentially at least 100 000 ha in Queensland), these have also been inaccessible to barramundi via its normal life-cycle movements. To fill the 'niche' made available by the decreasing distribution of barramundi, it was proposed that a congener of barramundi, the Nile perch Lates niloticus, be introduced to establish sport fisheries in tropical impoundments. The principal rationale for this introduction was that, unlike barramundi, the Nile perch reproduces in fresh waters and, hence, once established would be capable of sustaining breeding populations. Contrarily, however, three lines of evidence suggested that the introduction of the Nile perch would have negatively impacted upon Australian aquatic fauna. The lower temperature tolerance of the species and analysis of water temperatures in rivers in eastern Australia indicated that its range would have extended to temperate regions, thus endangering established fisheries for native species in those areas. The introduction of the Nile perch, an opportunistic predator, to Lakes Victoria and Kyoga in eastern Africa caused a drastic decrease in species diversity and fish biomass. L. niloticus is not restricted to lacustrine habitats, and known features of its biology indicate that it could have colonised and adversely affected the fauna in a broad range of freshwater habitats in Australia. The risks associated with the proposed introduction were considered to outweigh the potential benefits, and hence it was abandoned. As an alternative, attention was given to hatchery production of barramundi as a means of supplying fingerlings for stocking fresh waters for enhancement of recreational fisheries in northern Australia. Hatchery-reared barramundi fry were studied to determine feeding behaviour, diel feeding patterns, stomach evacuation rates, daily food consumption and growth rates. At 16-18 mm total length (TL), the feeding behaviour of the fry changed abruptly from a roving zooplanktivore to that of a lurking predator. A distinct change in pigmentation accompanied the change in feeding behaviour. Fry reared in hatchery ponds were obligate zooplanktivores between 10 and about 17 mm. Between about 17 and 50 mm the diet changed progressively from zooplankton to insect larvae to small vertebrates. The fry were visual feeders, taking food throughout the day, and showed a peak in feeding activity at dusk. Feeding continued at a reduced level under moonlight conditions, but ceased in total darkness. Stomach evacuation rates for 16 mm fry under continuous feeding and non-feeding conditions were 47 and 210 minutes, respectively; for 37 mm fry the rates were 73 and 108 minutes, respectively. The daily rations for these two size groups were 19-86% and 38-56%, respectively. Specific growth rates were 13-16% body weight/day for fry reared in ponds. Laboratory-based experiments were conducted to determine the vulnerability of different sized barramundi fry to predation by nymphs of the dragonfly Pantala flavescens. Mortality of 10 mm mean TL fry was significantly greater in the presence of dragonfly nymphs, whereas 20 mm mean TL fry exhibited comparatively minor levels of mortalities. The results accorded with feeding behaviour patterns of the different sized fry and the development of an escape response in barramundi fry at 16-18 mm TL. An examination of the growth rates of P. flavescens in newly-filled ponds, the development of the pond fauna on which the barramundi fry feed, and the growth rates of fry, indicated a rearing strategy to optimise survival of barramundi fry reared in freshwater ponds. An experiment was conducted to determine the effect of extended periods of light on the growth, survival, feeding pattern and daily food consumption of barramundi fry reared in a freshwater hatchery. There was no significant difference in growth or survival of fry, initially 11-12 mm TL, in either 12, 18 or 24 hours light. Fish exposed to 12L/12D photoperiod fed continuously during daylight, and ceased feeding in darkness. Under continuous daylight conditions, fish fed throughout the normal daytime period, but ceased feeding at a time corresponding to the normal onset of darkness; feeding started again near midnight. Daily food consumption for 34 mm fish was approximately 40% more in continuous light than in 12L/12D photoperiod. The results clearly showed that there was no advantage to be gained by rearing barramundi fry in extended light regimes. An experiment was conducted to determine if survival during weaning was affected by the size of the fry at the initiation of weaning. At the outset of the trials, feeding of live zooplankton was discontinued and a commercially available salmon starter crumble was dispensed by automatic feeders every hour for the 12 hours of daylight (photoperiod 12L/12D). Four trials were undertaken using fish initially 12.8, 13.6, 16.7 and 19.6 mm TL. Survival through the 10-day weaning period averaged 39, 58, 97 and 92%, respectively. An asymptotic curve described the relationship between initial size and survival, and indicated that survival of greater than 90% could be expected with fry greater than 16 mm TL at the time of weaning. This is the size at which barramundi fry change their feeding habit from that of a roving zooplanktivore to a lurking predator. Cost-benefit analyses indicated a considerable economic saving in delaying weaning until the fry are 16 mm TL. A study was conducted to determine if hatchery-reared and wild barramundi could be distinguished by the patterns of circulus spacing on the scales. Proprietary software and digitising equipment was used to obtain measurements of circulus spacing within one millimetre of the focus of the scales. Discriminant analyses separated the groups with up to 83% accuracy. As the technique utilises innate tags laid down in response to the rearing environment, it has considerable potential for evaluating the efficacy of large-scale enhancement programs. However, because scales from fish larger than 350 mm TL were too thick and heavily pigmented to be reliably read, the applicability of the technique with barramundi is limited to fish smaller than 350 mm TL.

Scope As is the case overseas, the wild fisheries of Australia are under increasing threat from the pressures of over-fishing, habitat destruction and water quality degradation. In addition, inshore fisheries that are dependant on freshwater flows to provide nutrient pulses and nursery habitats are also affected by changes in natural flow regimes as a result of water impoundment and extraction (Robins, Halliday et al. 2005). The barramundi (Lates calcarifer) is an important commercial fish species in Australia worth $8.8 million in 2004/05 (ABARE 2006), and supports valuable tourism and recreational fishing industries. Commercial catch displays a high degree of inter-annual variation; a characteristic that many fishers believe is the result of climate variability. However, apart from rainfall and freshwater flow, previous studies of the barramundi have not examined the impacts from climate in any detail, and existing management strategies do not consider natural climate variability or climate change. This study examined the effects from long-term (biannual to decadal) and short-term (inter-annual) climate variability, extreme and threshold climate events, and anthropogenic climate, change on the commercial catch of wild barramundi in north-east Queensland. The possibility of incorporating climate parameters into the management of the fishery was also examined. Methods A life cycle model of the barramundi was developed to link climate parameters with the complex developmental stages of the species from spawning in the estuary through maturation in freshwater rivers. Fisheries and climate data were extracted from a variety of sources and compiled for analysis. A gamma distributed logarithm link function model was constructed to calculate total freshwater flow for those years when records were incomplete. Correlation analysis identified significant relationships between climate parameters and catch, and forward stepwise ridge regression was used to develop a model of barramundi catch using climate parameters as predictors. The impact of threshold events was determined by non-linear analysis and the effects of extreme events on barramundi habitat were qualified against MODIS satellite imagery. A selection of climate change scenarios from a range of global climate models (GCMs), were run through the predictive model developed to determine the likely impacts of future anthropogenic climate change on the fishery. Results In the near-pristine Princess Charlotte Bay area, warm sea surface temperatures, high rainfall, increased freshwater flow and low evaporation (all measures of an extensive and productive nursery habitat) were significantly correlated with barramundi catch two years later as recorded by the CFISH logbook system. These results suggest that early barramundi survival is enhanced in these conditions. Catchability was significantly increased after high freshwater flow and rainfall events in the year of catch, a result that reinforced previous observations that mature fish in freshwater habitats are flushed into the commercial estuarine fishery. October – December rainfall and April – June flow showed non-linear asymptotic relationships and annual evaporation a quadratic relationship, with commercial catch two years later. Curves peaked at approximately 325 mm, 245 000 Ml and 2 000 mm respectively, a result that demonstrated that once these hydro-meteorological threshold events occurred, the response from the fishery was reversed and subsequent commercial barramundi catch reduced. A comparative analysis of data from the Fitzroy River area, a catchment and near shore area that has been highly modified by human intervention, showed only increased freshwater flow prior to the wet season enhanced subsequent barramundi catch. This result indicated that the anthropogenic changes to habitat either affected or masked the relationship between other climate variables and barramundi catch in the area. Total long-term barramundi landings as recorded by the Queensland Fish Board for six regions along the north-east coast of Queensland showed a near decadal cycle. Correlation analyses returned significant relationships between catch and the January – March average L-index (a measure of the latitude of the subtropical ridge) two, three and four years prior to catch, and the Quasi-biennial Oscillation (QBO) three and four years prior to catch. These results suggest that each of these cycles affects climate in the north-east Queensland region and subsequent survival of barramundi in the early life cycle stages, and provides an opportunity to estimate catch a number of years in advance. A forward stepwise ridge regression model was built to predict commercial barramundi catch in Princess Charlotte Bay. The model contained July – September rainfall and annual evaporation two years prior to catch and explained 62% of the variance in catch and had a cross validated predictive R2 of 59%. A second model also contained April – June flow in the year of catch (a measure of catchability). This second model explained 69% of the variance in catch and had a cross validated R2 of 61%, however, the improvement was not statistically significant. Using the nine global climate models in the OZCLIM program set for three initiating TAR SRES markers (A1B, A2 and B1), a suite of twelve climate change scenarios was generated for the years 2030 and 2070 for Princess Charlotte Bay. These scenarios were then run through the predictive barramundi model developed. Results indicated that due to a likely increase in annual evaporation, barramundi catch in the area will decrease for all future climate scenarios including those that show an increase in July – September rainfall. An analysis to calculate future sea surface temperatures using REEFCLIM indicated that, depending on the availability of suitable habitats, it is possible that the range of the species will extend further south by up to 800 km by the year 2070 as temperatures increase. Conclusions Results from this study indicate that a significant proportion of the variability seen in commercial barramundi catch in north-east Queensland is driven by variability in climate. Climate signals are significant at both short and long-term time frames and for some variables the impact is non-linear beyond a defined threshold. Anthropogenic changes to the fishery habitat alter or mask the relationship between climate and barramundi catch, and possibly affect the reproductive success of the species. The likely impact of future anthropogenic climate change will be a reduction in barramundi catch in areas where an increase in evaporation results in a subsequent decrease in shallow wetland habitats essential for early life cycle survival. This thesis provides supporting evidence for policy makers to improve significantly both the prediction of future barramundi catch and the sustainable management of the species by considering the impacts of climate variability and climate change on the species, and by incorporating climate variables into catch models.

Barramundi (Lates calcarifer), also known as Asian seabass, is an emergent aquaculture fish species possessing excellent farming attributes. As a result of its euryhaline life-history, culture of barramundi is extremely flexible and the species displays fast growth in freshwater, brackish, and/or marine environments, where it can be farmed in ponds, cages, or intensive recirculation tank-based systems. Barramundi has been farmed for over 40 years in tropical waters of the Indo-West Pacific region and has recently been introduced into Europe and North America due to increasing consumer demand and high market value, and global production is rapidly increasing. Whereas a wealth of knowledge on the biology and culture of the species have contributed to a developing industry, barramundi farmers still rely on unimproved stocks and often experience inconsistent production as a result of high variability in fish growth and survival between batches. However, for the barramundi industry to meet its full potential in the future it will need to rapidly move towards the instigation of well-informed genetic improvement programs. In order to assist with the design and implementation of a selective breeding program for barramundi, this research aimed to address key knowledge gaps related to the genetic diversity of farmed populations and to the underlying genetic basis of commercially important growth traits. In addition, this research investigated larval progeny testing as a means to rapidly estimate breeding values (EBV) of broodstock currently held at commercial hatcheries, or broodstock to be incorporated into a breeding program. One significant challenge for genetic studies of farmed barramundi relies on the fact that the species is a strict mass spawner and therefore the number and size of families cannot be fully manipulated by strip spawning and artificial fertilization. Such reproductive behavior has limited the knowledge on the genetic diversity of farmed cohorts, as pedigrees can only be reconstructed through DNA parentage analysis. Therefore, exploring how many families may be produced through mass spawning and, most importantly, retrieved at the end of the production cycle is critical for the development of breeding programs for barramundi. To investigate the fate of genetic diversity over the entire culture cycle, offspring from three commercially produced batches were genotyped with microsatellite markers at 18 days post hatch (dph) and then later at harvest (273-469 dph) for inference of familial relationships. Results demonstrated that it is possible to produce and retrieve at harvest over 100 families from a single spawning event using large numbers of broodstock (e.g. 33 fish). In addition, genetic diversity within generations was found much more resilient than previously thought. Despite significant changes in relative family frequencies over the culture period (P < 0.05), family lines identified at the larval stage were still available for selection by the time of harvest. The stability of several indices to gauge possible changes across the two sampling periods (i.e. effective population sizes, inbreeding rates, number of alleles, allelic richness, observed and expected heterozygosity and Rxy relatedness coefficient) also confirmed no subsequent loss of genetic diversity throughout the culture cycle. Therefore, early demographic census would provide an accurate representation of genetic diversity available for selection at harvest. Moreover, a cost-effective genotyping sample scheme targeting to reduce the overall costs of breeding programs of barramundi was also investigated. Here, examination of family contributions and mean family body weights at harvest showed that genotyping the top 1.5% of the population (or 750 individuals > 2.17 S.D. heavier than the population mean) will capture > 75% of family-specific genetic diversity present, whereas continued sampling after this was ineffective. This study suggests that genetic diversity in a barramundi breeding nucleus can be boosted and maintained by using several large breeding groups per generation. However, before efficient selective breeding programs can be implemented, it is also fundamental to have a comprehensive understanding of key genetic parameters of growthrelated traits, such as heritabilities (h²), genetic correlations (r(g)) and genotype by environment interactions (G x E). This knowledge is necessary to predict genetic gains and the efficiency of a breeding program, as well as to determine if genotypes selected under a particular environment will express the same growth performance when reared in a different environment. Heritability estimates for three separate batches measured and genotyped at harvest were consistently high for fish weight (mean W h² ~ 0.40), standard length (mean Ls h² ~ 0.37) and body depth (mean BD h² ~ 0.40). As additive genetic effects play a significant role in barramundi body size, selection of heavier (or longer/deeper) fish at harvest is expected to greatly improve fish growth rates. Lower heritability values were found for Fultons condition factor (0.00 ≤ K h² ≤ 0.20) and body shape (0.00 ≤ H h² ≤ 0.12), however, positive genetic correlations with fish weight (0.36 ≤ r(g) ≤ 0.41) indicate that selection for heavier fish may also improve fish condition and shape. Also of importance, very high genetic correlations were found for the same growth trait (W, Ls or BD) in fish reared in either fresh vs. sea water cages (r(g) ≥ 0.97), or commercially reared in an intensive tank system vs. a semi-intensive pond (r(g) ~ 0.99). The lack of G x E interactions suggest that the offspring of breeding candidates selected in a breeding nucleus, or in a particular farm, will express superior growth rates in a range of farming conditions which are representative for the barramundi industry. The lack of G x E interactions coupled with high heritability for growth are encouraging outcomes for the wider barramundi industry and the shared investment in a breeding program should return high genetic gains for all stakeholders alike. Nevertheless, the benefits from a future barramundi breeding program would only be capitalized after a few generations, i.e. a decade or longer. Therefore, alternative means to rapidly ascertain the genetic merit for growth rate of existing broodstock would be highly desirable. In this study, several larval progeny traits strongly associated with the individual growth capacity were investigated as potential early predictors of long-term family growth. High genetic correlations between early and late growth traits would indicate the feasibility of larval progeny testing as a method to infer EBVs of parental broodstock. Firstly, a protocol to measure larval size (Ls), total RNA, total DNA, total protein, RNA/DNA, protein/DNA and the proportion of cells dividing within a single larva was developed. Secondly, the heritability of these traits was estimated from a dataset of ca. 400 18 dph larvae with known pedigrees. Thirdly, the genetic correlations between heritable larval traits and fish harvest size (weight (W) and Ls)) were determined by combining the larval dataset with that of ca. 2000 harvested fish with known pedigrees. All larval traits exhibited moderate to high heritability at 18 dph (0.19 ≤ h² ≤ 0.51), indicating that their expression is under additive genetic control and therefore that they could have predictive power to estimate parental EBV for long term growth. This was confirmed by positive genetic correlation between all larval traits (except protein/DNA) and fish harvest W and Ls (r(g) ≥ 0.60). In particular, larval RNA/DNA, total RNA and the proportion of cells dividing had the highest predictive power to determine genetic differences in growth potential among barramundi broodstock shortly after spawning (0.81 ≤ r(g) ≤ 0.88). Results indicate that testing cellular traits in larval progeny may allow hatchery managers to immediately re-spawn the highest EBV ranked broodstock for stocking grow-out systems, thereby avoiding costs associated with rearing slow-growing families. Furthermore, early progeny testing may allow fish breeders working with long-lived and highly fecund multiple spawners like barramundi to cull introduction of broodstock with inferior genes for growth. In summary, this research bridges several missing links in the knowledge of genetic diversity and parameters of farmed barramundi necessary for the establishment of a selective breeding program for the species. It has been shown for the first time that despite the mass spawning nature of the species, sufficient genetic diversity can be generated and maintained in a breeding nucleus through to harvest and that growth rates can greatly be improved through selection for increased body weight at harvest. Furthermore, this research unveiled the existence of significant genetic correlations between cellular larval traits and fish harvest size which may be explored as a novel progeny testing tool to fast-track the improvement of fish growth. In summary, this research bridges several missing links in the knowledge of genetic diversity and parameters of farmed barramundi necessary for the establishment of a selective breeding program for the species. It has been shown for the first time that despite the mass spawning nature of the species, sufficient genetic diversity can be generated and maintained in a breeding nucleus through to harvest and that growth rates can greatly be improved through selection for increased body weight at harvest. Furthermore, this research unveiled the existence of significant genetic correlations between cellular larval traits and fish harvest size which may be explored as a novel progeny testing tool to fast-track the improvement of fish growth.