The purpose of the present study was to quantify the optimal zinc requirements of juvenile grouper (Epinephelus malabaricus). Basal diet contained 52 % crude protein from derived egg white and mackerel¡]Scomberomorus commerson¡^muscle. This semipurified basal diet which contained 10.0 mg Zn/kg was supplemented with ZnSO4 at levels of 0, 4, 8, 12, 24, 36 or 140 mg Zn/kg, respectively. The test diets were fed to juvenile groupers with an initial weight of about 5.9 g. All treatments were triplicated, each tank had 12 fish. After 18 weeks of rearing, dietary zinc levels have significant effects on the growth of fish. Weight gain and feed efficiency were significantly lower in grouper fed the unsupplemental diet than the supplemental groups. No significant difference among fish groups was found in survival rate, condition factor, muscle composition, ash contents of vertebra and scale, as well as some hematological parameters. Zinc concentrations in serum, muscle, vertebra and scale were affected by the supplemental zinc level. There was no significant difference in nonspecific immunity parameters including phagocytosis of macrophage, alternative complement pathway (ACH50) activity, agglutination titer and lysozyme activity among dietary treatments. The activity of the copper and zinc metalloenzyme superoxide dismutas in red blood cells was also not affected by dietary zinc intake. Broken-line regression analysis of weight-gain, feed efficiency as well as serum-, vertebra- and scale zinc concentrations against dietary zinc intake indicated requirement values of 33.7, 35.6, 32.5, 28.9 and 32.6 mg Zn/kg diet, respectively. Therefore, based on the results of growth performance and tissue zinc concentrations, the dietary zinc requirement of the juvenile groupers was between 28-36 mg Zn/kg diet.

Piscine nodaviruses are members of genus Betanodavirus, which infect more than 30 species of fish and cause massive mortality in larvae and juveniles. The infection causes great economic losses to aquaculture and sea-ranching. To study the dissociation and reassembly of betanodavirus, virus-like particles (VLPs) of dragon grouper nervous necrosis virus (DGNNV) were used. The experiments with calcium-chelating or reducing/oxidizing reagents elicited that the DGNNV VLPs required only calcium for particle assembly. With the recombinant VLPs, site-directed mutagenesis can be employed to investigate the roles of calcium-binding ligands in particle formation. In the mutational analysis of DxxDxD that is putatively involved in the coordination of calcium ions, the results showed that the D133N mutation significantly disrupted the assembly of VLPs while D130N and D135N mutants produced heterogeneous particles with broken shapes. The thermal stability of the VLP-forming fractions demonstrated that VLPs of D135N mutant were stable at a temperature of 85¢XC, which is slightly higher than that for wild-type, whereas VLPs of D130N mutant could not tolerate the thermal effects at a temperature higher than 60¢XC. It is deduced that three aspartate residues of the motif DxxDxD are all important for the efficient formation of DGNNV VLPs and, among them, the DxxD provides a more stable coordinate of calcium-ligand than DxD.

The sustainability of grouper culture as an alternative livelihood option for coastal fishers was investigated in the southern provinces of Thailand. A sequenced approach using qualitative and quantitative research methods was used to explore factors that could undermine the potential for grouper culture to contribute to a sustainable livelihood. A study was initially conducted in 28 villages in 6 provinces in southern Thailand to provide baseline information on grouper aquaculture and fisheries, focusing on culture practice, the actors and institutions involved in grouper culture and beneficiaries. Factors contributing to the variation in levels of uptake of grouper culture between provinces was explored. This initial overview facilitated the description of the grouper culture system in terms of its boundaries, hierarchies, interconnections and processes and the development of a framework for the assessment of the sustainability of the grouper culture system. A case study approach was used to investigate in-depth the role and sustainability of grouper culture at the community and household level. Three communities were selected for study in Trang and Satun provinces. Research was guided by the sustainability assessment framework, which comprised five components: Livelihoods, Institutions, Environment, Markets and Production Systems. Data was collected using qualitative, participatory methods and a quantitative survey. The research found that grouper culture has the potential to contribute to a sustainable livelihood for coastal fishers in southern Thailand, although problems with fish disease and mortality, fluctuations in the availability of seed fish and water quality undermine the ability of most farmers to obtain the maximum benefit from grouper culture. Non-technical factors including motivation and incentive, access to financial capital and levels of social capital were also observed to influence sustainability of grouper culture. Grouper culture was found to be an activity in which members of all wealth groups could participate with institutional support, access to credit, and access to wild sources of seed and feed. It is unlikely to provide a suitable alternative to fishing, as the two activities contribute in different ways to the livelihoods of coastal households, but may support livelihood diversification.

Fish populations are regulated by both external environmental factors, e.g., water quality parameters and habitat, and internal reproductive biology and physiology processes. For many species and populations there is often ample external information, while critical internal, i.e., life-history trait (LHT), information is not available. For this study, I determined LHTs and energy allocation patterns for Epinephelus guttatus (red hind), E. striatus (Nassau grouper), and Mycteroperca venenosa (yellowfin grouper) harvested from The Bahamas. I determined age ranges, and how growth patterns and rates differed among the study species. The maximum ages were: 17, E. guttatus; 22, E. striatus; and 13, M. venenosa. Epinephelus striatus was estimated to have the slowest, while M. venenosa had the fastest growth rate. A gonad histological classification system and the ageing data were used to determine the spawning seasons, sex ratios, size and age of sexual maturation and sex change and gonadosomatic indices (GSIs) for the study species. The peak spawning months were January-February for E. guttatus, December-January for E. striatus and March-April for M. venenosa. The fifty-percent sexual maturity estimates were 235 total length mm (Tlmm) (2.05 year old, yo), 435 Tlmm (4.00 yo), and 561 Tlmm (4.66 yo) for E. guttatus, E. striatus and M. venenosa, respectively. The size and age range of sex change for E. guttatus was between 257-401 Tlmm, ~4-5 years old and between 716-871 Tlmm, ~8-9 yo for M. venenosa. I determined protein and lipid concentrations in muscle and gonad tissues to ascertain energy allocation patterns. For all species and sexes except for female E. guttatus, the proportion of energy delegated to somatic growth declines as a fish grows longer, while reproduction energy allocation increases. The results of each study were compared to previous studies conducted throughout the tropical western Atlantic Ocean, and were related to species-specific ecological and spawning behaviors. The findings of each study highlight that the LHTs of the study species greatly differ and these differences will impact population dynamics and need to be considered for management initiatives. In the final chapter, the effects of fishing on LHTs are reviewed and fishery management options are discussed.

The accurate identification of taxa from mixed assemblages using genetic analysis remains an important field of molecular biology research. The common principle behind the development of numerous documented genetic detection technologies is to exploit specific nucleotide sequences inherent to each taxon. This body of work focuses on practical applications of real-time nucleic acid sequence-based amplification (RT-NASBA) in marine science, and is presented in four case studies. Each study represents novel work in the genetic identification of respective taxa of interest using RT-NASBA. Two case studies documented the development of an assay targeting mitochondrial 16S rRNA to discern legally salable grouper species in the U.S. from fraudulently mislabeled surrogate fish. This technology was first validated using lab-based, benchtop instrumentation, and was then adapted into a complete field detection system. The third study documented an internally controlled RT-NASBA (IC-NASBA) assay for the detection and quantification of the harmful algal bloom-causing dinoflagellate, Karenia mikimotoi, by targeting the ribulose-1, 5-bisphosphate carboxylase-oxygenase (RuBisCO) large-subunit gene (rbcL). The final section of this dissertation details the preliminary development of an IC-NASBA assay targeting large subunit rRNA for the quantification of Enterococcus, which is a genus of bacteria commonly used as an indicator of fecal pollution in recreational marine water. My results show that RT-NASBA provides a suitable format for the accurate identification of target species from these taxa which include prokaryotes, as well as both unicellular and multicellular eukaryotes.

The groupers are vital fish in the market of over 350 million dollars, while grouper nervous necrosis virus (NNV) has caused mass mortality at about 100% in larvae and juveniles, which impacts on economic of marine cultured fish. The monoclonal antibody is one of the best methods to identify the epitopes on the 3D structure. For evaluation, the Balb/c mice were injected with DGNNV and virus-like particles (VLPs) in this study. The results showed that ascite of mAb-cells produced 1200 times higher than the cell secretion in the medium whereas our best clone hAb_VLP8 can only produced 100 times less antibody than the cell secretion. In the meantime before the monoclonal producer is established, the hAb_VLP8 could be used for ascite production to gain high antibody production.

Groupers are an important mariculture fish in Taiwan and Southeast Asian countries. The rapidly growing orange spotted grouper (Epinephelus coioides) has experienced relatively severe bacterial disease problems. The proliferation of pathogens in fish can be suppressed by commensal microbiota. In this context, probiotic seem to offer an attractive alternative. Bacillus subtilisis a probiotic bacteriumthat is administered in diet to suppress proliferation of pathogens. In the present study, E.coioideswere fed for 6 months with diets containing B.subtilis at 0 (control), 0.1 % and 1 %. Percent weight gain and feed efficiency of the 0.1 and 1 % groups were significantlybetter than the control group. The innate cellular response, respiratory burst of the fish fed the 1 % and 0.1 % diet was significantly higher compared to the control group on 10 or 20 days after feeding, and even moresignificanton 30 days.ProbioticBacillus subtilis increased the fish¡¦s intestinal microbial diversity as measured by visible band number and Shannon diversity indexin DGGE analysis. Probiotic Bacillus subtilis also stimulated the population of bacterial species likePaenibacillussp,Lactobacillus oenistrain 59 b, and Methilacidophiluminfernorumstrain V4 that beneficial for Epinephelus coioides. The best dose of probiotic Bacillus subtilis based on growth performances, innate cellular responses and profile of microbiota in fish intestines is 0.1 %, which showed equal efficacy as the 1% diet.

The groupers are vital fish in Taiwan, the market of grouper fry over 300 million dollars. While grouper nervous necrosis virus (NNV) has caused mass mortality, especially 100% in larvae and juveniles, which economically impacts on culture of marine fish. The vaccination is one of the best methods to against viral diseases. The dragon grouper (Epinephelus lanceolatus), malabar grouper (E. malabaricus) and brown-marbled grouper (E. fuscoguttatus) were injected with different dosages and injection frequencies of virus-like particles (VLPs) of DGNNV, which is by the first claimed. The anti-sera of vaccinated fish were analyzed with eight kinds of immunology methods, among which antigen-capture ELISA was the best choice for qualitative and quantitative assays. The signal of antibodies in the vaccinated fish was detected in all groupers in one week after primary immunization, and the antibody titers increased markedly in one month. In dragon grouper, fish was injected with 10 £gg of VLPs, the antibody titer reached 1.05. To given booster injection once, antibody titers were raised to 35.7%. In malabar grouper, after injected twice with 50 £gg of VLPs, the antibody titer raised 33.3% than inoculation once in six weeks. After brown-marbled grouper was injected with 450 £gg of VLPs, the high antibody titer reached to 1.57 at five weeks post primary immunization. Specific antibodies still can be detected after seven months. In the in vitro assay with MGNNV of 103.5 TCID50/mL, neutralizing antibody titer of control fish were all lower than 1:50. The neutralizing antibody titer of anti-serum of dragon grouper was detected at 1:200 at one week, and raised to 1:1600 at four weeks and 1:6400 at eleven weeks after primary vaccination. In malabar grouper and brown-marbled grouper, the neutralizing antibody titers were 1:3200 and 1:400, respectively, in one month. The antibody titer can not increased by Freund¡¦s complete adjuvant. The fish produced high antibody titer and high protection by immunization with VLPs.

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ABSTRACT The mortalities for aquaculture animals in a newly established pond tend to be high. The phenomenon can be described as the ¡§new pond syndrome¡¨. The main reason for the phenomenon is still unclear. However, it is suspected that the accumulation of ammonia due to insufficient nitrifying bacteria in the system may have been the cause. The purposes of this thesis are to test the theory and to rectify the problem by introducing a nitrifying trickling filtration system using E. amblycephalus as the experimental animal. It was found that the ammonia concentrations did increase to the levels detrimental to the newly hatched fries within 6 days. The LD50 of ammonia to the young fries was determined to be 0.034 ppm in 48 hr. The nitrifying filtration system could reduce the ammonia concentrations substantially and raise the breeding rates for the fries. On two separate trials, one with fish tanks in the laboratory and the other in the ponds of a fish farm, 12.3% and 4.2% of the newly hatched fries were still alive after 34 days, respectively, as compared to 0% and 0.035% for the controls. Meanwhile, a pH value of equal to or greater than 8.6 was detrimental to the fries. Therefore, the low breeding rates for the E. amblycephalus fries may have been caused by the ¡§new pond syndrome¡¨ due to the toxic effects of accumulated ammonia in the early stage of the pond usage. Removal of ammonia with the filtration system could increase the survival rates for the fries.