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This research studied the dietary effect of arachidonic acid (ARA) on growth performance and immune responses in juvenile grouper (Epinephelus coioides). The test diets used casein as protein source, and contained 9 g/100g lipid. Cod liver oil and arachidonic acid oil were used to adjust the ARA levels to 0, 0.2, 0.4, 0.6, 0.6, 0.8, and 1.0 g/100 g diet in the 5 test diets, respectively. Result of 12-week feeding trial show that the ARA supplementation had no significantly effect on the weight gain of the grouper, although 0.6 % and 0.8% groups showed higher weight gains than other groups. Liver fatty acid composition of the groupers was affected by dietary ARA. Liver ARA concentration, ARA/EPA and ARA/DHA increased with increasing dietary ARA levels. The immune responses was affected significantly by the dietary treatments. Dietary supplementation of ARA significantly enhanced plasma lysozyme activity. Lysozyme activity was increased with increasing dietary ARA levels. Dietary ARA significantly also enhanced head-kidney leucocytes respiratory burst activity. The respiratory burst activity of the 0.6 % ARA group was significantly higher than the other groups. Dietary ARA significantly enhanced head-kidney leucocyte proliferation and the dosages of 0.6 % and 0.8 % ARA were significantly more effective than the other groups. The blood phagocytic activity of the 0.6 % ARA was significantly higher than the other groups. The blood cytokine IL-1£] and TNF-£\ was significantly higher in the 0.8 % ARA group than the other groups. This research shows that feeding the grouper diets cointaining ARA at an optimal level could significantly increase immune responses of the fish.

In recent years, the skin and muscle hemorrhagic ulcer occurred in grouper hatchery, suggesting that the bacteria extracellular protease was involved. In this study I surveyed the extracellular protease activity of marine bacteria and investigated the characteristics of the pathogen isolated from diseased grouper. The sampling sites of background survey were located along the coast of Taitung. The cultured media were MB2216 and PPESII. The extracellular protease activity was measured using milk seawater medium (MS) and dye-modified proteins of azocasein and azoalbumin. The extracellular protease activities of the marine bacteria were 1~5 U/mg. The average protease activities of the bacteria from MB2216 media were higher than those from PPESII media. The extracellular protease activity assay using azocasein and azoalbumin had similar tendency but the result by the clear zone measurement on the MS medium was of high variations. The FI-02 isolated from the diseased grouper skin was identified as Aeromonas hydrophila by the biochemicial characteristics and 16S rDNA sequence analysis. FI-02 was able to grow on the LB broth with 0~4% NaCl. The FI-02 was resistant to ampillicin, cefazolin, erythomycin, sulfisoxazole, whereas it was sensitive to streptomycin, doxycline, norfloxacin, cefotazime, oxolinic acid, and tetracycline. When FI-02 growing in the four media, the extracellular protease activities were higher than 4 U/mg. The highest protease activity was 6.08 U/mg when the bacteria grew on the LB medium. After heating on 27, 37, 42 and 56¢J for 20 mins, the protease activities remained the same. In the investigation of the fish immunity, owing to the complex compositions of the fish serum, no conclusion can be drawn on the specificity of the antibody against the bacterium.

The grouper is a high-value fish in Taiwan. However, nervous necrosis virus, which contains two single strand RNAs and doesn¡¦t have an envelope, causes groupers die in early stages of development. It economically impacts on the aquaculture of this marine fish. Vaccination is one of the best methods to prevent viral diseases. Virus-like particles (VLPs) were used for studying the ability and efficiency of producing antibodies against dragon grouper nervous necrosis virus. Different injection dosages and injection frequency of VLPs were performed on Epinephelus lanceolatus. The anti-sera of vaccinated fish were acquired by microvoluminal phlebotomization method and analyzed by antigen-capture dot blotting with ECL detections, which are the best choices for qualitative and quantitative assays. The signal of antibodies in the vaccinated fish was detected every week after primary immunization. The antibody signal reached 8.4x106 U in one month when the dragon groupers were injected with 10 µg of VLPs, but giving additional injection of VLPs didn¡¦t increase production of antibodies after one month. Moreover, dragon groupers that were injected with 50 µg and 100 µg of VLPs generated antibody signals up to 7.2x106 U and 6.7x106 U, respectively. The antibody signal can remain at a higher level in the presence of high dosage injection for at least three weeks. Furthermore, a polyvalent vaccine that included killed bacteria was applied in field to test dragon grouper immune response. The results not only support the data that dragon grouper boots antibody production within a short period after immunization, but also demostrates that the production of antibody by dragon grouper against virus and bacteria can be monitored by the microvoluminal phlebotomization method.

This research determined the influence of dietary vitamin E levels on the growth, body composition and various health indices of juvenile Epinephelus malabaricus. Five experimental diets were formulated by adding all-rac-a-tocopherol in 0, 200, 400, 1000 and 3000mg/kg feed to the basal feed that used casein as the sole protein source. The juveniles, having an average weight of 30g, were cultivated in an indoor closed recirculation system, with 6 fishes in each tank, and 3 tanks for each experimental feed. After the 17-week feeding trial, weight gain of the fish (170-250%) was not significantly affected by the vitamin E treatments. The supplemental level of vitamin E was positively related to the lipid levels in liver, but was negatively related to the protein levels in liver. Total w-3 HUFA of polar lipids in body muscle were decreased with the increasing supply of vitamin E. It was observed on the 11th week and beyond that the fish fed with the vitamin-free diet were prone to fail in taking in feed particles although their responses to feeding were of no difference to the supplemental groups. But once the supplemental levels of vitamin E were higher than 400mg/kg no difference in feeding failure was detected, indicating deficiency of vitamin E detrimentally affected fish feeding. Plasma AST activities in the 0 and 3000mg/kg groups were significantly higher than those of the other groups. Histological sections of fish liver indicated that liver cells in low vitamin E groups showed signs of vacuolization. TBARS (thiobarbituric acid reactive substances) decreased with the increasing vitamin E levels in the feed. Vitamin E showed limited enhancement on the immune parameters studied. Except that agglutination titter was significantly higher in fish fed the high vitamin E diet (3000mg/kg) and serum lysozyme and erythrocyte SOD showed highest activity in 200 and 400mg/kg groups, intracellular super oxide anion of macrophages and serum alternative complement pathway activities were not significantly affected by vitamin E supplementation.

The Betanodaviruses caused nerve necrosis in several fishes. There are two RNAs in the Betanodaviruses. RNA1 encodes RNA dependent RNA polymerase and RNA2 encodes capsid protein. I analyzed the RNA2 sequences of the viruses isolated from two species of grouper, Epinephelus malabaricus (MGNNV) and E. lanceolatus (DGNNV). The similarity of two grouper viruses (GNNV) was 99%. Their similarities to DlEV and SJNNV were 87.8% and 78.6%, respectively. The capsid protein was successful expressed and assembled to virus-like particles. Deleting N- and C-termini revealed different impacts on VLP formation. Deletion of 35 or 52 residues at the N-terminus completely ruined the VLP assembly, presumably due to removal of positively charged residues for binding RNAs. When deletions were restricted to 4, 16, or 25 N-terminal residues, the assembly of VLPs remained. The ability of VLP formation diminished when 4 to 11 C-terminal residues were deleted. The termini that can be deleted without seriously destructing the VLPs are 25 and 3 residues at N- and C-termini, respectively. Expression the ORF of RNA2 in E. coli formed virus-like particles, indistinguishable from native virus particles in appearance, whereas a mutant of Asp-75 expressed no VLPs. The emergence of a trimer band in mutant D75N as wild type suggested that the Asp-75 mutation could halt the packaging process in the trimer stage, not proceeding to assemble intact VLPs. The D54N mutant remained the ability of VLP formation but lost packaging high molecular weight of RNAs. Another Asp mutant at C-terminus, D335A, lost the ability of VLP assembly. The D335 may play an important role on the instability of VLP structure.

This research determined the in vitro, intraperitoneal injection and dietary immunostimulatory effects on the grouper Epinephelus malabaricus of chitosan and its derivatives with different molecular weight, chitosan, polyglucosamine and N-acetyl-chitooligosaccharides. Respiratory burst activity of head-kidney phagocytes isolated from the grouper incubated in vitro with the chitosans at a range of concentrations was studied. Respiratory burst activity generally decreased with increasing dosage of chitosan products. N-acetyl-chitooligosaccharides were significantly more potent in enhancing respirtatory burst activity than the other two chitosans. Respiratory burst activity of head-kidney phagocytes of the grouper injected with three kinds of chitosans at 4 dosages was assayed. N-acetyl-chitooligosaccharides caused significantly higher respiratory burst activity than the other two chitosans. N-acetyl-chitooligosaccharides at the dosage of 10 µg/g was found to enhance the highest respiratory burst activity among treatments. In the time series assay with intraperitoneal injection by N-acetyl-chitooligosaccharides at a dosage of 10 µg/g, it was found that enhancement of NBT reduction occurred early in the time course of the study and is similar to the time series response of the glucan treatment. When the groupers (120g) were fed with diets containing 5 concentrations of N-acetyl-chitooligosaccharides including 0, 0.5, 1, 1.5, and 2 g/100g and stocked in indoor closed recirculation systems for 7 weeks, weight gain of the fish was not significantly affected by the dietary treatments. The immune status measured by respiratory burst activity, alternative complement pathway, agglutination titer, lysozyme activity and superoxide dismutase activity was not significantly affected by N-acetyl-chitooligosaccharides supplement. But feeding the grouper with N-acetyl-chitooligosaccharides at 1 g/100g diet seems to lower the immunity of the fish, although the effects were statistically insignificant.

Iridovirus infections have led to serious economic loss in the aquaculture industry in Kaohsiung County as well as the whole Southern Taiwan region. Identified susceptible host species in this region includes hybrid grouper (Epinephelus hybrid), giant seaperch (Lates calcarifer), largemouth bass (Micropterum salmoides), king grouper (Epinephelus lanceolatus), spotted butter fish (Scatophagus argus), yellow-wax pomfrat (Trachinotus blochii), goldlined seabream (Sparus sarba), humpback grouper (Chromileptes altivelis), Mangrove red snapper (Lutjanus argentimaculatus). In this study, a diagnostic PCR primer pair CY15n-F/CY15nR, and its nested primer pair RY16-F/RY16-R, were designed and applied to amplify virus-specific products of 1339 bp and 305 bp, respectively. This primer set did not amplify products from lymphocystis disease virus, largemouth bass virus, or healthy control fish DNA. This sensitive technique can detect the presence of 50 fg plasmid with viral DNA insert in the presence of 100 ng/£gl host DNA, or 0.05 fg DNA from infected fish. Comparing sequences of CY15 fragment, ATPase gene, predicted major capsid protein and partial DNA polymerase genes among iridoviruses, it is suggested that the viruses found in this area should be classified as the Megalocytivirus of Iridoviridae. These viruses are clearly different from the Ranavirus, another fish-pathogenic iridovirus. Those iridoviruses can be classified into two genotypes: the CY630 type, which is the Taiwan grouper iridovirus; and the CY113 type, which is similar to red seabream iridovirus (RSIV). The identity in CY15 fragment sequences is about 91%. Microscopically, enlarged cells can be found in organs of infected fishes. They appear in the spleen, head kidney, and trunk kidney in infected groupers. The enlarged cells may be relocated from other organs. In giant seaperch, the enlarged cells appeared in the above mentioned organs, and also in the digestive tract and the heart. Two kinds of the enlarged cells in grouper can be distinguished by their H&E staining properties: the basophilic and the eosinophilic enlarged cells. The result from in situ hybridization and electron microscopy suggest that the viruses only appear in the basophilic enlarged cells. Both nucleus and cell volume increase in basophilic enlarged cells, while only the cell volume increases in the eosinophilic enlarged cells. The viruses appeared first in the nuclei of the basophilic enlarged cells, after the mid-phase of the infection they distributed into the whole cells. Judging from the results of phagocytosis, acid phosphatase activity and the ultrastructure of infected cells, it is suggested that this target cell is macrophage or monocyte. The viral capsid is assembled in the viromatrix, and the virogenic stroma can be either ring-shped or disc-shaped. The diameter of mature virus is 120-130 nm from side to side, or 160-170 nm from apex to apex. The electron-lucent space between the capsid and the envelope is about 20-50 nm. The virus particles can be found in (1) lysosome-like vesicles in the cytoplasm, if the host cell still has its nucleus; or (2) the viromatrix, When the host nucleus is dissolved or only has some vestige nuclear membrane left.

Two experiments were undertaken to assess the dietary effects of microbial phytase on growth performance, mineral utilization, and immune responses in groupers, Epinephelus coioides. Basal diet contained fish meal and soybean meal as protein source and no phosphorus supplement. In experiment I, test diets containing 0 (0.2% sodium phosphate was supplemented; 0+P), 200, 400, 600, 800 FTU phytase/ kg were assigned to triplicate tanks and were fed for 12wk. Fish fed diet containing 0+P or 400 FTU phytase/kg showed better weight gains. Supplementation of phytase had no effect on feed efficiency, body tissue proximate composition, vertebral ash, and vertebra and whole-body phosphorus concentrations. Phosphorus utilization was improved and excretion tended to reduce for fish fed diets containing 400 and 600 FTU phytase/kg than the other treatments. Experiment II evaluated the effects of phytase supplementation on utilization of phosphorus, zinc and iron, and relative immune responses of the groupers. Three diets were formulated base on the results of experiment 1 to contain 0, 0+P or 400 FTU phytase/kg. The results of the 8-wk feeding trial indicated that phosphorus, zinc and iron utilizations were higher for fish fed diet containing inorganic phosphorus (0+P) than the phytase-containing diet. Phytase supplementation did not significantly affect immune responses. The present results indicated that phytase at the dosage of 400 FTU/kg is a suitable level in grouper diets, which would increase utilization and lessen excretion of dietary phosphorus.

The purpose of this study was for building the data on blood and serum chemistry of grouper ‚ and expect these data can be helpful to know the fish condition in early stages‚ in order to remedy as soon as possible and decrease the loss that caused of fish disease. Three experiments were conducted to establish the data on blood and serum chemistry of grouper. In the first experiment‚ the clinical chemistry including red and white blood cell counts‚ Glutamic oxalacetic transaminase (GOT)‚ Glutamic pyruvic transaminase (GPT)‚ total protein‚ albumin and blood ion of Na‚ K and Ca were built in different size of healthy orange spotted grouper¡]Epinephelus coioides¡^. The results showed that the white blood cell counts was divided into two major parts 70-90&#x00B4;103 cell/ml and 110-140&#x00B4;103 cell/ml. Red blood cell counts was landed on 1.85-3.21&#x00B4;106 cell/ml. The range of GOT and GPT were 0-45 IU/L and 0-350 IU/L. Total protein and albumin were 3.8-5.6 and 0.7-1.6 g/dL. In the second experiment‚ the clinical chemistry of orange spotted grouper after infected by dactylogurus‚ trichodinla‚ nervous necrosis virus (NNV) and NNV with Amyloodinium ocellatum were collected and compared with normal orange spotted grouper. The white blood cell counts and the concentration of K+ in the infected NNV and dactylogurus of fish were significantly higher (P<0.05) than normal fish‚ while the granulocyte in infected fish was significantly lower (P<0.05) than normal fish. In the dactylogurus and trichodinla infections of fish‚ the concentration of Ca2+ was significantly higher (P<0.05) than normal fish. In the third experiment‚ the size of monocytes and neutrophils of brown marbled grouper¡]E. fuscoguttatus¡^ were grown in 8 hours and 16 hours‚ respectively‚ after challenged with Photobacterium damselae subsp. damselae‚ and the eosinophils size of challenge group fish bigger (P<0.05) than control group fish after challenged for 16 hours.