Dissertations / Theses on the topic '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.

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.

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.

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.

Aquaculture is an important protein source for Malaysia. However, the susceptibility of fish to disease leads to heavy losses and hinders production. A potential solution is the supplementation of diets with prebiotics: non-digestible carbohydrates which improve growth and health by modulating gut microbiota to favour beneficial bacteria. Resistant starch (RS) is a prebiotic commonly fed to terrestrial animals, but little work has been performed on aquatic animals. Therefore, this study investigated the use of RS isolated from underutilized crops as a prebiotic in fish. Six species of underutilised legumes (adzuki beans, mung beans, black-eyed peas, pigeon peas, Bambara groundnuts and red lentils) were used for starch isolation via alkaline steeping, followed by processing involving enzyme or acid hydrolysis, and lastly gelatinisation and retrogradation to increase RS yield. Starch was isolated with yields of 25 – 40%, while enzyme hydrolysis pre-treatment was more effective and improved RS content up to 18.06% in most legumes. The starch and RS samples were then supplemented at 5% (w/v) in nutrient broth to investigate their prebiotic effect on fish gastrointestinal lactic acid bacteria. Enzyme pre-treatment improved the growth of W. cibaria, L. garvieae and E. gilvus by up to 43.9% for most legumes tested. Red lentil and adzuki bean enzyme-RS showed highest prebiotic potential and was applied in the zebrafish growth trial, while only red lentil was used in the Asian sea bass growth trial. Supplementation of diets with RS from legumes provided no significant difference in the growth and performance parameters measured in both zebrafish and Asian sea bass when supplemented at 2.5% and fed for five and six weeks respectively. Further studies involving analysis of immune parameters is necessary to identify prebiotic potential on targeted fish. Nevertheless, this study contributed to promote future work in using underutilised legumes as prebiotic source to improve the fish health under commercial conditions of rearing.

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.

A tahiti, le lates calcarifer presente une saison de reproduction annuelle et les animaux males peuvent s'inverser apres cette saison. Les caracteristiques histologiques de l'inversion sont, la degenerescence du tissu germinal male, l'apparition du tissu degenerescent male, et enfin la proliferation du tissu germinal femelle avec persistance du tissu germinal femelle. Ce processus d'inversion peut etre acheve au moins en 17 jours, et necessite une profonde reorganisation morphologique des gonades. Au niveau ultrastructural, nous avons identifie de nombreuses cellules germinales primordiales durant la phase de proliferation du tissu germinal femelle. Ces cellules aux caracteristiques indifferenciees pourraient jouer un role important dans l'ontogenese du tissu germinal femelle. Nous avons aussi mesure les concentrations des steroides sexuels plasmatiques et gonadiques, et effectue des etudes de metabolisme in vitro par des gonades a differents stades de maturation sexuelle et d'inversion. Les principaux resultats de ces etudes sont la mise en evidence d'un steroide de type strogene synthetise in vitro en grande quantite par des gonades en fin d'inversion, et par la detection de fortes concentrations gonadiques dans les gonades des le debut de l'inversion. Ceci suggere un role important des strogenes dans la regulation endocrienne de l'inversion sexuelle du lates calcarifer. Concernant le compose de type strogene, ses caracteristiques biochimiques sont identiques a celles d'un 3-ester d'stradiol. Ce steroide pourrait servir de reserve tissulaire d'stradiol 17

The issue of sustainability has greatest significance in the midst of unilateral bio-socioeconomic degradation resulting from intense and increasing societal pressures placed on the unified global ecology. In such an environment, sustainable development seeks to manage natural resources within a free market economy, aiming to meet the needs of today's population, and to protect and enhance current resource quality and abundance. In this light, techniques of integrated sustainable primary production and wastewater management are the subject matters of this applied research. There are many researchable issues which could be addressed within the subject matter. The first focus in the research scope was driven by the most severe sustainability issue facing Central Queensland (Australia) in 2000: the depletion and degradation of freshwater supplies. Central Queensland (CQ) is an arid sub-tropical region that has suffered from a marked reduction in rainfall and increase in temperature over the last 100 years, {Miles, 2004 #172}, and by the year 2000, conditions had been exacerbated by eight years of severe drought and warmer than average temperatures and resulted in widespread animal and crop failures due to freshwater shortages. Such a problem required a multi-faceted ecological, social, and economic approach. Hence, research centred on investigating the science of integrating regional water-related industries and agribusiness, and biodiverse ecosystems to achieve water and wastewater reuse applications, and associated eco-socioeconomic benefits. Specifically, this research investigates the integration of (a) electrical power station wastewater (b) barramundi culture, (c) red claw culture, (d) constructed wetlands (for water quality management and habitat creation), and (e) hydroponic flower culture. This research produced outcomes of integrated water and wastewater reuse and recycling, marketable agriproducts production (fish, crayfish, and flowers), water and wastewater reuse and conservation, wetland primary production, carbon dioxide sequestration, aquatic pollution control, and biodiversity creation and support. Successful design and management, experimental trialing and evaluation of system components and subjects, and the development of a knowledge base including static and dynamic system models, represent advances in respective research areas, and underpin the emerging discipline of integrated systems approaches to eco-socioeconomic development. Additionally, several gaps in the current body of knowledge regarding integrated systems were filled, and interactive management tools were developed. Apart from this study, the integration of technologies (as described above) has not, to this author's knowledge, been accomplished.

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
國立屏東科技大學
水產養殖系
92
In recent years, the infection disease caused by neuron necrosis virus (NNV) and grouper iridovirus (GIV) dozen of important aquaculture industries in the world. The species affected include fish, amphibian and reptile, and the habitat covered from temperate zone to tropical zone and from seawater to freshwater. To prevent and treat this virus infections are therefore top urgent issues right now. In this study, we prepared formalin inactivated vaccines for yellow grouper neuron necrosis virus (YGNNV) and grouper iridovirus (GIV). The result showed, after injection in combinations of various vaccines treatments injected GIV, YGNNV, and YGNNV+GIV vaccines, respectively the specific immunoreaction of treated seaperch’s tissue fluid were detected by the enzyme-linked immunosorbent assay (ELISA). Soaking in inoculated vaccine three times, the tested fry exhibited non-specific immune response. While soaking in same vaccine twice and injecting it one, the fry showed obvious specific immune response (p<0.05). After three months of inoculated vaccine, the fry injected with YGNNV+GIV treatment has the best survival rate during YGNNV and GIV virus challenge test. This fact implies that mixing vaccines (YGNNV+GIV) possess not only the specific immunoreaction but non-specific protective effects. For the accumulative mortality in field trials, we found that inoculated vaccine injection has a better protective and higher safety effects than soaking one.

碩士
國立中山大學
海洋科學系研究所
107
Fasting is a common process in aquaculture industry. Fasting was thought to influence physiological performance and homeostasis of fishes. To clarify and evaluate physiological changes through peroids of time in fish subjected to fasting is crucial for aquacultural practice. In this study, the Asian seabass (Lates calcarifer) were fasted for 1, 4 and 7 days to evaluate the physiological responses of fish. The secondary stress responses were examined, including hepatosomatic index (HSI), liver glycogen, serum glucose/total protein, serum osmolarity, concentrations of sodium and chloride ions and the expression of heat shock protein 70 (HSP70). The results showed that HSI, liver glycogen content and HSP70 expression, and serum total protein decreased significantly after fasting for 4 and 7 days (p<0.05). However, no change in serum glucose, [Na+] and [Cl-] was found. Our results revealed that fasting stress in 7 days disturbs multiple physiological responses of Asian seabass and provide related information for evaluating the effect of fasting stress on aquaculture fish species.

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.

碩士
國立高雄海洋科技大學
水產食品科學研究所
99
The purpose of this study is to establish the process conditions for preparing collagen peptide from seaperch scale and to study its functionality on calcium-binding (Ca-B) activity. Powder samples from fish scale of seaperch were treated with alcalase, flavourzyme, papain, trypsin, and protamex. The collagen peptides obtained were examined and compared with their Ca-B activity. Among them, peptides from fish scales treated with alcalase, flavourzyme, papain showed higher Ca-B activity, and were selected in the following study. The seaperch scale was then treated with two enzymes system, including alcalase plus flavourzyme and papain plus flavourzyme and alcalase plus papain. Collagen peptide from fish scale treated with alcalase plus flavourzyme showed higher Ca-B activity. The optimal conditions in two enzymes system were listed as follows : concentration of fish scale, 6%; temperature 50 oC; pH 7.5; reaction time, 2 h; alcalase used, 8%, flavourzyme, 6%. Collagen peptides obtained from the above two enzymes system were purified with hydroxyapatite column separation and Q FF column chromatography. Two fractions named Hpt-1, Hpt-2 were obtained from hydroxyapatite column separation, Hpt-2 showed higher Ca-B activity than that of Hpt-1. Hpt-2 was then purified twice with Q FF column chromatography. Three fractions of QFF-1, QFF-2, QFF-3 were found in the chromatogram. Among them, QFF-2 showed the highest Ca-B activity. The QFF-2 was examined with HPLC and showed only one peak in the chromatogram. Analysis on the sequence of amino acid in QFF-2 is being investigated.

博士
國立嘉義大學
農業科學博士學位學程
106
Although tricaine methanesulfonate (MS-222) is often used to tranquilize fish, the guidelines for its use in sea bass, a brackish species, have not been established. The aim of the study reported here was to establish the tranquilizing concentration of MS-222, based on the time required for MS-222 residue elimination and withdrawal. Thirty-six fish (6/group) were immersed in different concentrations of MS-222 (0, 30, 50, 60, 70 and 90 μg/mL) to evaluate the fish physiological behavior. Optimal sedation concentration is 30 μg/mL, and optimal anesthetic concentration is 90 μg/mL. After 200 fish were anesthetized at 90 μg/mL, the fish achieved a healthy recovery within 72 h after the administration of saline. The 10 fish in the control group were subject to the same treatment without anesthesia, 3 out of 10 died. After 108 fish (54/group) were immersed in 30 or 60 μg/mL of MS-222, the sedated fish were healthy during and after the 8 h of transport. However, all the 10 fish in the control group died within 3 days. By high performance liquid chromatography, the residue of MS-222 in sea bass was assessed. In the skinned muscle and liver, the elimination half-life was 5.54 and 5.27 h (30 μg/mL) and 8.72 and 7.15 (60 μg/mL), respectively, and the withdrawal time was at least 4.5 days at 30 μg/mL and 7.5 days at 60 μg/mL. By liquid chromatography tandem-mass spectrometry, the residue of MS-222 in grouper was assessed. In the 30 μg/mL group, the skinned muscle not detect at fifth day, and the liver at day 21 detected 0.005 µg/g. In the 60 μg/mL group, the skinned muscle not detected at day 14, and the liver at day 21 detected 0.007 µg/g.

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.

碩士
國立高雄海洋科技大學
水產食品科學研究所
105
The objective of present work is to study the process conditions for preparing peptidechelated calciumcomplexes (Peptide-Ca)from collagen peptide of perch scale. Collagen peptides were first obtained from perch scale treated with alcalase plus flavourzyme at 50 oC, pH 7.5 for 2 hrs.The optimal conditions for preparing Peptide-Cawere listed as follows: reaction temperature 50 oC at pH 7.5 for 2 hrs.; ratio of peptide to calcium chloride, 2: 1 and calcium chelating activity was 68.4 ± 0.41%. The obtained Peptide-Ca showed good resistance to the treatment of artificial digestive fluid.Methanol and ethanol showed good extraction efficiency in the purification of Peptide-Ca. Main chemical bond inPeptide-Ca was found to be COO− and calcium ion which was identified by Fourier Transform Infrared Spectrometer (FTIR). Among the amino acids of scale collagen pepdide, proline showed the highest efficiency in chelating calcium ion, followed by glycine and hydroxyproline. Hdrolysates from perch scale, tilapia scale and horseshoe shell, all showed good calcium ion chelating activity, the highest was that from horseshoe shell, followed by perch scale and tilapia scale. Moreover,hydrolysates of perch scale also exhibited other metal ion chelating activity, the highest one was Zn2+, followed by Fe2+, Ca2+, and Cr3+, Mg2+.

Increasing coastal eutrophication and rising global temperatures are placing substantial pressure on wild fish populations. In the tropics, routinely high temperatures close to the equator have the combined effects of reducing the solubility of O₂ in water, increasing metabolic rates and enhancing thermal stratification. Such conditions may be particularly pronounced in lentic freshwater environments, and have led to a suite of adaptations by tropical fish species. Inter-specific diversity in hypoxia tolerance is widely acknowledged, however, many species exist not as a single, continuous population, but rather as multiple populations that may be distributed over broad latitudinal gradients, potentially spanning thousands of kilometres. In the absence of horizontal migration and genetic mixing between populations, and with differences in prevailing environmental conditions (such as temperature and dissolved O₂), such populations may become phenotypically and even genotypically divergent over time. Performance of fish populations have been extensively investigated in the context of temperature, particularly with regard to projected temperature increases from climate change models. Despite the intrinsic relationship between temperature, O₂ and metabolism in fish, population differences in hypoxia tolerance have received little research attention to date. To address this knowledge gap, the hypoxia tolerance of geographically and genetically divergent populations of juvenile barramundi from across the distribution in northern Australia was assessed (Chapter 2). Juvenile barramundi were collected from five hatcheries across northern Australia, and were assessed for their resting O₂ consumption rate (ṀO₂) and critical O₂ level (O₂CRIT) using intermittent-flow respirometry. Measurements for all five populations were made at temperatures considered benign for this species across its distribution in Australia (26°C) and at temperatures that may be encountered during the pre-wet season (36°C). A posteriori comparisons revealed significant temperature effects for both resting ṀO₂ and O₂CRIT, but no conclusive evidence for population differences in either measure. The results from this study indicate a similar capacity for barramundi populations to regulate metabolism in response to hypoxia at typical and warm temperatures. The magnitude of temperature effects on O₂CRIT for barramundi was lower than for many other tropical and temperate fish species, indicating that barramundi retain a high capacity to regulate metabolism in hypoxic environments at high temperatures. Over long time scales (tens to hundreds of thousands of years), populations that are genetically divergent may become either locally adapted to a specified range of conditions (if conditions are constrained within a narrow range), or they may retain a large degree of physiological plasticity (if the environments they inhabit are highly variable). Further, dissolved O₂ may display high spatial and temporal variability in coastal freshwater and estuarine systems that is often overlooked in empirical studies. To assess the contribution of population-of-origin or physiological plasticity to hypoxia tolerance, juvenile barramundi (one tropical and one sub-tropical population) were exposed to daily fluctuations in dissolved O₂ (>85% to <10% saturation) for 0 (control), 8 or 16 d (Chapter 3). Fish (separate cohorts) were then assessed for either resting ṀO₂ and O₂CRIT, or haematological parameters. No changes in any parameters were detected after 8 d, however after 16 d a reduction in O₂CRIT, and increases in both haematocrit and haemoglobin were observed. No population differences were detected for any measured parameter. This study demonstrates that barramundi populations are capable of acclimating to diel-cycling hypoxic conditions following repeated exposure, and that such changes are accompanied by improvements to blood-O₂ carrying capacity. Inter-specific diversity in hypoxia tolerance of teleost fish is widely acknowledged, however the extent of intra-specific diversity in hypoxia tolerance is less well understood, due in part to the logistical and temporal constraints of measuring performance across a large number of individuals. Further, the temporal repeatability and hence the reliability of hypoxia tolerance measures have received virtually no attention in the broader scientific literature. To address these knowledge gaps, ~800 juvenile barramundi were first separated into hypoxia tolerance categories (Chapter 4) based on time to loss of equilibrium (LOE) tests: sensitive, intermediate and tolerant. Following a recovery period, fish were then assessed for growth performance, metabolic regulation under hypoxia (O₂CRIT) and repeatability of time to LOE. Further, the relationship between ṀO₂ and DO during O₂CRIT tests was assessed using non-linear regression and broken-stick regression techniques, which represents a novel approach to handling such a large empirical data set. Fish were reliably separated into different hypoxia tolerance categories, yet there were no significant category effects for any of the subsequently measured variables: growth rate, feed conversion ratio, standard metabolic rate or O₂CRIT. Non-linear regression was more robust in describing the relationship between ṀO₂ and DO than the more commonly used broken-stick regression method. Surprisingly, there was no significant relationship between two independent measures of hypoxia tolerance: time to LOE and O₂CRIT. The time to LOE test was broadly repeatable after ~100 d. This study highlights the extent of intra-specific diversity in hypoxia tolerance that can exist within fish populations. All previous assessments of hypoxia tolerance were necessarily conducted on unfed individuals to eliminate the influence of specific dynamic action (SDA) on metabolic responses. In the wild, and on commercial fish farms, metabolism of fish may be elevated above resting levels due to feeding behaviour. Digestive responses are potentially influenced by dissolved O₂ conditions, however, few studies have assessed this possibility in tropical fish. Further, no studies have assessed the effect of hypoxia tolerance phenotype on digestive metabolic responses. Therefore, barramundi were separated into hypoxia tolerance phenotypes (sensitive or tolerant) based on time to LOE tests (Chapter 5). Fish were then fed a restricted ration (2.5% of their body-mass), before being assessed for ṀO₂ under normoxic (>85% saturation) and chronic hypoxic (~35% saturation) conditions. There was no effect of hypoxia tolerance phenotype for any measured parameter, and negligible differences in a range of digestive responses (SDA magnitude, SDA duration, SDA coefficient) under normoxic or hypoxic conditions. The results from this study suggest that barramundi are capable of maintaining maximal digestive capacity even when O₂ drops to 35% saturation. The results from this thesis demonstrate that barramundi are extremely resilient to bouts of environmental hypoxia, and retain a strong tolerance to hypoxic conditions at extremely warm temperatures. The lack of population differences in hypoxia tolerance may be explained by the retention of a high degree of physiological plasticity as a strategy for responding to environmental hypoxia. The extent of phenotypic diversity in hypoxia tolerance is impressive, and the influence of this diversity on performance is still poorly understood. The homogeneity of performance between hypoxia tolerance phenotypes across two experiments and a number of measured parameters suggests that hypoxia tolerance is unrelated to several other performance metrics under either normoxic or hypoxic conditions. Future research should be directed at further exploring the relationship between phenotypic diversity in hypoxia tolerance and fitness, with an objective to more clearly elucidate the ecological consequences of trait variability.

碩士
嘉南藥理科技大學
生物科技系暨研究所
96
The effects of unmethylated CpG oligodexynucleotides (ODNs) on the mammalian immune system are relatively well studied. CpG ODNs have been shown to induce a variety of different immune responses in fish including IFN ??? induction along with antiviral activity, macrophage activation, cytokine production and activation of other immune-related genes and cell proliferation. CpG ODNs can indirectly inducing cellular migration in vitro and enhancing serum lysozyme activity in vivo. Sea bass (Lates calcarifer), a common economical aquaculture species in south Taiwan, was chosen as the objective in this study to investigate cytokines induced by CpG ODNs. Previous studies showed that CpG ODNs can induce trout head kidney cells to produce proliferation inducing factors (PIFs), and then result in cell proliferation. However, B cells depletion did not affect the responses of trout head kidney cells to CpG ODNs stimulation. These results indicate that neither the source of the proliferation-inducing factors or the proliferating responder cells are B cells. The aims of our study are to find the source of PIFs and their target cells. Furthermore, we would try to purify and identify of the proliferation-inducing factors. Our results showed PIFs were proteins secreted by CpG ODNs stimulated phagocytes, and PIFs induced a subpopulation of lymphocyte to proliferate. Besides, phagocytes were supposed to act as accessory cells in this response.

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
國立高雄海洋科技大學
水產養殖研究所
102
Currently, Streptococcus iniae (S. iniae) has become global aquaculture pathogen. The pathogen will infect fish and cause serious death and economy cost. This is a big threat to aquaculture. Those infected fish would have following symptoms: erratic swimming, darkening of the skin, exphthalmia, cornel opacity, ascites, peritonitiss, enlargement of spleen, petchiae around the anus etc. This study is mainly for prepare Streptococcus iniae killed bacterial vaccine to injective and oral immune for Lates Calcarifer. Injection of immune are S. iniae killed bacteria vaccine, intraperitoneal injection at an average weight of 100g of Lates calcarife. Injection concentrations are 1×107, 1×108, 1×109 CFU/250μl/fish for three times. Oral immune as dead bacteria mixes granular feed, concentrations are 1×108, 1×109, 1×1010 CFU/ml/g, continuous feeding of 4 weeks. After immunization use 1×103 CFU/250μl/fish LD50 to challenge. Pre-immune, immune 2-week, 4-week, after challenge and 18-week take blood , applied ELISA, SDS-PAGE and Western Blot to antibody reaction.The results show for injection of immune concentrations in 1×108, 1×109 CFU/250μl RPS are 100%, in 1×107 is 80%, and oral immune RPS ranged from 20% to 40%. This means these two immunizations are effective. Although they both have been tested the existence of antibody, the consequence shows that injective immunization is working better. The results of ELISA reaction reaches peak in the third time of enhancing immunization. After use SDS-PAGE, with Lates calcarife serum speculate 72kDa and 25kDa. Thus, we could speculate 7、26、40、45、55 and 85kDa are important source of immune in western blot. Based on this experiment, we could use Streptococcus iniae killed bacterial vaccine on injective immune to be a direction of future development.

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.

碩士
國立嘉義大學
水生生物科學系研究所
97
Peroxisome proliferator activated receptors (PPARs) are a family of hormone receptors implicated in a collection of fundamental biological processes, mainly related to lipid metabolism regulation. Three subtypes of PPAR, termed α, β and γ have been identified in several vertebrate species. The aim of present study was to �� clone the PPARα gene and �� examine the gene expression of PPARα in various tissues and the related with body composition in different body size of Asian seabass (Lates calcarifer). �� A 256-bp cDNA fragment of PPARα was cloned from Asian seabass by reverse transcription polymerase chain reaction (RT-PCR) using specific oligonucleotide primers which were designed according to known evolutionary conserved sequences of certain PPARα domains, available at NCBI. A comparison between the correspondent PPARα cDNA sequences of the Japanese seaperch (Lateolabrax japonicus), red seabream (Pagrus major), Japanese pufferfish (Takifugu rubripes), house mouse (Mus musculus) and human (Homo sapiens) revealed 93%, 92%, 87%, 79% and 77% identity, respectively. �� Expression of PPARα gene in various tissues and different body size ranged from 3.34 g to 8.03 g and 618 g of Asian seabass were analyzed. Expression of PPARα gene in 3.34 g to 8.03 g of juvenile Asian seabass was detected in the liver, spleen, heart, kidney, brain, pyloric caeca, intestine, dorsal muscle and gill. It showed abundant in the pyloric caeca and intestine, and at a barely detectable level in spleen and gill. Expression of PPARα gene in marketing size (618 g) of Asian seabass was detected in the liver, spleen, heart, head kidney, trunk kidney, brain, pyloric caeca, intestine, dorsal muscle, ventral muscle and gill. It showed abundant in trunk kidney, spleen, heart and brain, and at a lower level in liver, dorsal muscle, ventral muscle and gill. Proximate analyses showed that a negative correlation between the crude lipid level, hepatosomatic index and body weight was found (p<0.05). In conclusion, the 256 bp cDNA fragment of PPARα in Lates calcarifer has been cloned. The highest expression of PPARα gene was found in pyloric caeca and intestine in juvenile Lates calcarifer with body weight of 3.34 g to 8.03 g. It showed abundant in spleen, heart, trunk kidney and brain in marketing size (618 g) of Lates calcarifer.

碩士
國立高雄海洋科技大學
漁業生產與管理研究所
99
This study aims to discuss the current Taiwan sea bass aquaculture production costs and market specifications of management efficiency, and carry out the hypothetical analysis to investigate the market specification as 0.6 kg/individual , 1.2 kg/individual and 1.8 kg/individual of selling points in order to provide farmers more practical farming operation concept.The research in 2009-2010 found that production cost increase significant was in feed expenditure, other costs (cost of water and electricity, personnel, fish breeding cost) were no different. Statistical results on operating in market specifications, the cost variance can be classified into three points as follows: 1.Results of cost imputs analysis: feed and the personnel cost will be due to feeding 1.8 kg/individual specification and has significantly differences, but remaining cost is no significantly differences (p>0.05). It explains that culture process in the cost of water and electricity and the fish breeding costs, 0.6 kg/individual, 1.2 kg/individual and 1.8 kg/individual of specifications has no significantly differences, and has not show different whatever in culture time elongated of process. 2. Results of the profit ratio analysis: market specification as 0.6 kg/individual and 1.8 kg/individual o seedlings have significant differences in fish breeding cost, the remaining personnel, feed, and water and electricity of benefits have no significant difference (p>0.05). 3. Results of productivity analysis: fish breeding productivity has significant different in market specifications (p<0.05).In the other words the market specifications on the fish breeding production sold in which 1.8 kg/individual is higher than that of 1.2 kg/individual and 0.6 kg/individual, and that of 1.2 kg/individual is higher than of 0.6 kg/individual while there is no significant difference in personnel, feed, and water and electricity productivity (p >0.05). Secondly on the analysis of the hypothetical, when 1.8 kg/individual fixed profit at $ 81.25/kg, we discuss the fish breeding profit ratio, total expenditure ratio and net profit, the results of the analysis are as follows: 1. In fish breeding profit ratio results , the 0.6 kg/individual and 1.2 kg/individual could be sold $ 98 and $ 115.5, respectively and has no difference in price statistics. 2. In total expenditure ratio results, the 0.6 kg/individual and 1.2 kg/individual could be sold $ 82 and $ 85.2, respectively and has no difference in price statistics. 3 .In net profir results, the 0.6 kg/individual and 1.2 kg/individual could be sold $ 103.3 and $ 126.4, respectively and has no difference in price statistics.

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.

碩士
國立高雄海洋科技大學
水產養殖研究所
101
The study was aimed to find out analysis the Lates calcarifer growth rate applied replacement of fish meal by various fermented soybean meal. We are going to divide into two stages of experiments and use the fermentation feed which are fermented soybean meal 1, fermented soybean meal 2 and fermented soybean meal 3 to replacement of fish meal. The first phase were divided into A, B and control groups, a total of three groups, four repeat, control group feed commercial formula, and A, B group fermented soybean meal 1 and fermented soybean meal 2 replace 20% fishmeal in commercial formulations. The second phase were divided into groups A, B, C and control group, a total of four groups, three repeated,where A, B and control group renewal of the first stage of the experiment, C group fish out by the control component and replaced 20% fermented soybean meal 3 of the commercial formulations fishmeal feeding trials. Feeding time at every stage of each six weeks, and random sampling once every two weeks, weighed weight, feed for six weeks, the experimental data, we also use the SPSS software to be the experimental data. According to the statistical, the result display the A, B group compare with control group, gain weight, feed intake and feed conversion rate haven’t significantly different(p>0.05). However, compare with A and control group, the specific growth rate are significantly different (p<0.05), C group haven’t significantly different too. The second stage, the result display the A, B, C group compare with control group, gain weight and feed conversion rate haven’t significantly different(p>0.05). But A, B and C group compare with control group, those specific growth rate and feed intake are significantly different(p<0.05). After testing, in measuring Viscerosomatic index and the length of the intestinal microvillus. Viscerosomatic index A, B, Cgroup compare with control group have significantly different (p<0.05), and A, B, C group between have significantly different, index B group> C group> A group> control group; the length of the intestinal microvillus especially the preceding part of m icrovillus bigger than posterior segment microvillus, preceding part of microvillus A, B, C group compare with control group have significantly different (p<0.05), and A, B, C group between have significantly different, length of B group> A group> C group> control group, posterior segment microvillus A, B, C group compare with control group haven’t significantly different(p>0.05). Spine bone analysis, zinc and phosphorus content is normal in the Lates calcarifer . The above results feed fermented soybean meal to replace 20% fishmeal is no effect on the growth of Lates calcarifer.

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
國立臺灣大學
獸醫學研究所
99
The sea water used for fish culture instead of fresh water in the west-south sea shore of Taiwan was very common. Owing to the land fish pond was not wide, the aquaculture in high density caused the high risks for suffering several diseases. In order to prevent the diseases of fish, not only improving the cultivating environments and techniques, but also tried to use various chemical therapy drugs in clinical use. Taiwan is located in the subtropical area, the water temperature was so often warm that the fish always suffered from viral, bacterial infected diseases, others such as parasite, protozoa, alga and mould were also infestation in fish. An experimental trial was performed to determine the half life of oxolinic acid in sea fish Trachinotus blochii and Lates calcarifer of pharmacokinetic and residue studies. A single dose pharmacokinetic study of oxolinic acid was applied by oral administration at the dose of 60 mg/kg b.w./day. A high performance liquid chromatographic method based on fluorescence detection was developed for determination of oxolinic acid in fish blood, tissue samples. Oxolinic acid in sea fish Trachinotus blochii of peak concentration (Cmax) was obtained directly in the concentration time profile; the residue levels in blood, muscle, liver, and kidney samples after 0.5 h depletion were at the maximum concentration. The Cmax in blood was determined to be 0.43 mg/mL and the half life was calculated to be 50.58 h. The Cmax in muscle was determined to be 1.43 mg/g and the half life was calculated to be 25.76 h. The Cmax in liver was determined to be 1.42 mg/g and the half life was calculated to be 45 h. The Cmax in kidney was determined to be 3.14 mg/g and the half life was calculated to be 25.76 h; Oxolinic acid in sea fish Lates calcarifer of Cmax was obtained directly in the concentration time profile; the residue levels in blood, muscle, liver, and kidney samples after 1 h depletion were at the maximum concentration. The Cmax in blood was determined to be 1.55 mg/mL and the half life was calculated to be 170.69 h. The Cmax in muscle was determined to be 2.55 mg/g and the half life was calculated to be 84.51 h. The Cmax in liver was determined to be 8.18 mg/g and the half life was calculated to be 106.61 h. The Cmax in kidney was determined to be 4.72 mg/g and the half life was calculated to be 111.77 h. An experimental trial was performed to establish an adequate depletion period of oxolinic acid in sea fish Trachinotus blochii and Lates calcarifer of pharmacokinetic　and residue studies. The pharmacokinetics of oxolinic acid was applied by oral administration at the dose of 60 mg/kg b.w./day, and was given at a daily dose for five consecutive days. A high performance liquid chromatographic method based on fluorescence detection was developed for determination of oxolinic acid in fish blood, muscle, liver and kidney. Data showed that the average residue levels in sea fish Trachinotus blochii of blood, muscle and kidney decreased to 2.74 ng/mL, 13.82 ng/g, and 21.53 ng/g after 24 days post dosing, respectively; with one exception at 195.01 ng/g in liver tissue. The present study provided data on the depletion of oxolinic acid residues in sea fish Trachinotus blochii after oral administration of five doses of the drug. The residue levels after 24 days depletion decreased to below the maximum residue limits (MRLs) after last oral treatment. At the dosage of 60 mg/kg b.w./day, we suggested that the recommended withdraw period was 36 days; Data also showed that the average residue levels in Lates calcarifer of blood muscle, liver and kidney decreased to MRLs after 2, 12, 36, and 36 days post dosing, respectively. The present study provided data on the depletion of oxolinic acid residues in Lates calcarifer after oral administration of five doses of the drug. The residue levels after 12 days depletion decreased to below the MRLs after last oral treatment. At the dosage of 60 mg/kg b.w./day, we suggested that the recommended withdraw period was 18 days.

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.

碩士
國立高雄海洋科技大學
水產養殖研究所
102
This study assessed the influence of adding commercially available nucleotides and various ratios of soybean meal to replace fishmeal as feed on the growth rates and survival of Lates calcarifer. The experiment involved 6 groups, which included fish that were fed basic feed, feed that comprised soybean meal that replaced 40% or 80% of fishmeal, feed that was supplemented with commercially available nucleotide 0.2% Vannagen, 0.1% Vannagen, or 0.05% Nutrafito Plus, or generic, commercially available sea bass feed. The addition of appropriate nucleotide amounts was expected to reduce the amount of fishmeal necessary, thereby lowering feed costs. The results indicated that the addition of soybean meal and commercially available nucleotides did not substantially increase the rates of specific growth and weight gain. However, the superoxide dismutase immunoreactivity was higher in the feed that included commercially available nucleotides than those that did not (p <0 .05). This result showed that the fish that were given feed supplemented with nucleotides demonstrated an improved ability to remove free radicals, thereby enhancing the immunoreactivity and increasing the survival rate of sea bass. Numerous studies have indicated that adding nucleotide to feed can elevate the immunoreactivity of fishery products, and this study presented similar results. Among the feeds used in the experiment, the costs of the feeds without nucleotides were the lowest. However, because the prices of fishmeal are increasing, adding nucleotides to feeds can reduce costs in the future. Nevertheless, the appropriate amount of nucleotides to be added must be further verified by conducting further experiments.

碩士
國立屏東科技大學
水產養殖系
92
This research discusses the acclimation responses and physiological change, focused on the energetics, of the giant seaperch, Lates calcarifer, under different thermal stress and remnant chlorine concentration environment. The tolerance and adaptive response under this dual adverse circumstance, becomes the basis of environmental impact evaluation. In gentle warming pressure, giant seaperch dies in the water temperature 42~42.4 ℃; In sublethal tolerance experiment, giant seaperch is really resistant to the hypochlorous acid pressure below the water temperature 34 ℃. High survival rate was obtained under chlorine concentration up to 2 mg/ l, but the rate obviously decline when the water temperature is 38 ℃. In blood biochemistry analysis, there are no significant difference of blood glucose change physiologically under water temperature 25~34 ℃. For 38 ℃ group, it is extremely obvious high in blood glucose concentration. The blood lactic acid quantity has rises under the different water temperature, also degree of the rise is becomes the relevance change scope with the temperature. Water temperature and chlorine residue, both contribute the stress for giant seaperch but in different mechanism. The water temperature has created a physiological reaction hyperglycemia, but the chlorine residue actually has the suppression effect on energy metabolism, also its suppression is positive proportional to the remnant chlorine concentration. Changes in blood lactic acid concentration appears is the early time of stress, also proportional to the intensity of stimuli, including thermal elevation and chlorine residue increasing. The concentration of blood lactic acid is suitable for being stress response index as its sensitivity to stressors. In muscle, difference and change of glucose and glycogen content did not show any significant varience within short time. But the lactic acid accumulation in muscle, reveal the physilogical stress caused by the existence of chlorine residue, which indicated energy supply by anaerobic metabolism while giant seaperch is in urgency or burst muscular motion. In liver, however, nearly contains no lactic acid and it is not feasible for monitoring the physiological response of stress reaction. Liver glycogen content demonstrated the chlorine residue suppression on energy metabolism, but under the identical chlorine concentration, the glycogen content change did not reflect the different thermal treatments, no statistic significance obtained. Consumption of energy for physiological regulation resulted in glucose concentration declined and immediately recovered in liver, explained the energy source comes from glycolysis or fatty acid oxidation but glycogenolysis, which changed the glucose content.

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
98
Nocardia seriolae is the causative agent of nocardiosis in cultured fish and causes high mortality in Taiwan and Japan. In this study, N. seriolae was isolated from 14 species of fish in Taiwan and Japan during 1970 to 2009, and Fourty-one N. seriolae strains were identified by 16Sr DNA gene, hsp gene and rpoβ gene analysis. The variations of 16S rDNA, hsp and rpoβ gene of N. seriolae were 0.2% ( 5 Taiwan strains ), 0.2~0.7% ( 6 Taiwan strains, 2 Japanese strains)、0.2~0.7% (6 Taiwan strains, 3 Japanese strains) isolated from ten fish species from 1986 to 2007, respectively. Further study evaluates the in vitro activity of antimicrobial agents against 89 strains of N. seriolae (67 Taiwan strains and 22 Japanese strains). The 50 and 90% MICs values for 89 strains of N. seriolae of the antimicrobial agents used in this study, florfenicol, lincomycin, doxycyclin, oxytetracycline, and kitasamycin were the most active antimicrobial agents against the isolates tested, followed by erythromycin and spiramycin. Furthermore, in an attempt to develop a vaccine against N. seriolae 961113, microfluidizer antigen (MF), and microfluidizer antigen mixed with ISA 763A adjuvant were injected into Asian seabass, Lates calcarifer. Fish were challenged with N. seriolae 961113 and 980401-2 after immunized 4 weeks and 14 week, respectively. RPS values of MF and MF-ISA 763A group challenged by N. seriolae 961113 at 4 weeks after immunization were at 67% and 83%, respectively. However, RPS values of MF and MF-ISA 763A group challenged by 980401-2 strain at 14 week after immunization were at 0% and 67%. A immunostimulatory but not protective in Asian seabass was observed in immunized group challenged by N. seriolae 980401-2 and 961113 strain at 4 weeks and 14 weeks, respectively. These results suggest that cross protective immune responses against N. seriolae are induced in Asian seabass.

Capsalid monogeneans are harmful skin ectoparasites of ornamental and farmed fishes in tropical and subtropical marine environments. Within this group, Neobenedenia includes of particularly virulent species that exhibit low host specificity, a direct life cycle, high fecundity, environmentally resilient eggs, and have been associated with mass mortalities in aquaculture. There is a paucity of information on the interaction between Neobenedenia spp. and their fish hosts. Examination of Neobenedenia spp. infection and invasion behaviour, associated pathology and the effect on host immune responses can enable a deeper understanding of the potential impact of parasites on fish health and the complexity of parasite-host interactions. This study examined the interaction between Neobenedenia sp. and barramundi, Lates calcarifer, an important finfish species in commercial fisheries and aquaculture. Neobenedenia spp. are cryptic in nature, which makes infection success and invasion routes challenging to elucidate. Larval recruitment and microhabitat preference was examined through time (Chapter 2) by using Neobenedenia sp. oncomiracidia (larvae) labelled with a fluorescent marker. Parasites were tracked on the body surface of the host with an epifluorescence stereomicroscope at 10 time intervals post exposure (15, 30, 60, 120 min, 24, 48 h, four, eight, 12, and 16 days). Parasites retained the fluorescent signal throughout the experiment. Neobenedenia sp. larvae settled opportunistically on the fish and then migrated to preferred microhabitats. Once recruitment had ceased (48 h), preferred microhabitats included the eyes, fins, and dorsal and ventral extremities on the main body. Reproduction could be an important factor for Neobenedenia sp. distribution, indicated by parasites aggregating on the fins within 24 h of attaining sexual maturity. Interestingly, some parasites attached beneath the scales of host fish, which may enable the parasite to be almost entirely secluded from the environment and could reduce the efficiency of current parasite management methods (e.g. chemical and freshwater bathing) in aquaculture. High infection intensities of Neobenedenia species are well-known to cause pathology, however, the damage associated with mechanical attachment of the main attachment organ, the haptor, has not been examined. The pathology associated with haptor attachment of Neobenedenia sp. to L. calcarifer was examined through prepared histopathology sections at the haptor-host interface (Chapter 3). Fish were infected with Neobenedenia sp., and skin samples with attached parasites were collected from the eyes, mandible, operculum, middle body, ventral body and caudal fins 20 days post-infection. Histological slides were prepared by embedding, sectioning and staining tissue samples from the site of parasite attachment to the skin of host fish. Epithelial thickness and mucous cell abundance were measured in samples from uninfected and infected fish. Infected fish had lower mucous cell abundance, and the middle and ventral body surfaces had thinner epidermis compared to uninfected fish. Infected fish presented signs of dermal inflammation, epithelial loss, loss of intraepithelial attachment, and vacuolated epidermis compared to uninfected (control) fish. The antibody response and acquired resistance of L. calcarifer to Neobenedenia sp. infections was examined following consecutive experimental infections (Chapter 4). Twenty fish were infected with Neobenedenia sp. oncomiracidia for 10 days with recovery periods (two weeks) between four consecutive exposure events. Before and after each exposure event, each fish was weighed, measured, and blood and mucous samples were collected for ELISA. After each infection the parasites were collected from each fish to analyse infection success, parasite size and reproductive status. Results showed that infected fish had significantly lower feed conversion efficiency than uninfected fish, parasites were significantly smaller on previously exposed fish and Neobenedenia infection success was significantly lower following three exposure events. There was no difference in infection success between the first, second and fourth exposure events. No differences in blood and mucous IgM levels between uninfected and infected fish could be detected by ELISA. This thesis provided an innovative and rigorous approach to standard scientific methodologies to gain new information on the interactions between harmful monogenean parasites and host fish. Fluorescent labelling enabled rapid assessment of infection success and invasion routes of Neobenedenia sp. and revealed intriguing parasite behaviours that could aid parasite survival and reproductive success. Careful precision with histopathology at the haptor-host interface showed morphological differences on the epithelium of L. calcarifer when infected with Neobenedenia sp.. Nevertheless, parasite attachment did not stimulate an immune response to consecutive infections.