Economic analysis of tiger grouper <Emphasis Type="Italic">Epinephelus fuscoguttatus</Emphasis> and humpback grouper <Emphasis Type="Italic">Cromileptes altivelis</Emphasis> commercial cage culture in Indonesia

This study presents an economic analysis of tiger and humpback grouper at different production scales in Indonesia. The results highlight the non-viability of small-scale tiger grouper farming, with a 5-year projected negative cumulative cash flow of −IDR 18,102,650.00 and a negative net present value (NPV) of −IDR 22,059,576.28. An increased production scale of tiger grouper highlights a marginal viability for medium-scale farms (with a 5-year projected cumulative cash flow of IDR 198,320,673.00, a positive NPV of IDR 105,578,440.42; a benefit cost ratio of 1.25; an internal rate of return (IRR) of 88% and a payback period of 0.99 years), and an economically viable large-scale cage culture (with a 5-year projected cumulative cash of IDR 707,746,923.00; a NPV of IDR 406,801,749.07; a benefit cost ratio of 1.33; an internal rate of return of 157%; and a payback period of 0.57 years). The economic analysis of humpback grouper at different production scales highlighted a positive cumulative cash and NPV, a benefit cost ratio over 2, an internal rate of return over 300% and a payback period <1 year. A sensitivity analysis revealed that increased survival rate up to 80% would increase cumulative cash and NPV of small-scale tiger grouper cage culture. Additionally, improved profitability performance was associated with decreasing major production costs, increasing production and price of the product.     

Dietary polysaccharide from <Emphasis Type="Italic">Angelica sinensis</Emphasis> enhanced cellular defence responses and disease resistance of grouper <Emphasis Type="Italic">Epinephelus malabaricus</Emphasis>

The purpose of this study was to determine the effect of Angelica sinensis polysaccharide (ASP) supplemented in diet on the innate cellular immune response and disease resistance in grouper, Epinephelus malabaricus. Fish were fed diets containing different doses of ASP (0, 500 and 3,000 mg kg⁻¹ diet) for 12 weeks. After 12 week feeding, the respiratory burst activity, phagocytic activity, and leukocytes proliferation in head kidney were assayed. The functional immunity in terms of cumulative mortality was also assessed by a challenge with live Edwardsiella tarda. Results showed that the respiratory burst activities in ASP-supplemented groups were increased significantly. The respiratory burst index was the highest in fish-fed 3000 mg kg⁻¹ diet and the lowest in control. The phagocytic activities in ASP-supplemented groups were significantly higher than that of control. No significant difference in phagocytic activity was observed between ASP-supplemented groups. ASP stimulated the head kidney leukocytes proliferation significantly, despite the absence of lipopolysaccharide (LPS) or not. The cumulative mortalities of fish fed with 3000 mg ASP kg⁻¹ diet were significantly lower than those fed with 500 mg ASP kg⁻¹ diet and control diet after 96 h of challenge. In conclusion, dietary ASP enhanced some cellular immune parameters and disease resistance against E. tarda in grouper.     

Establishment and characterization of a heart-derived cell line from goldfish (<Emphasis Type="Italic">Carassius auratus</Emphasis>)

The goldfish Carassius auratus, a freshwater fish in the family Cyprinidae, was one of the earliest fish to be domesticated for ornamental purposes. A cell line was established from goldfish heart (GH) tissue to create a biological monitoring tool for viral diseases. The GH cell line was optimally maintained at 25 °C in M199 medium supplemented with 10–20% fetal bovine serum. A chromosomal analysis indicated that the cell line remained diploid, with a mean chromosomal count of 100. In viral inoculation assays, significant cytopathic effects (CPEs) were caused by epizootic hematopoietic necrosis virus (EHNV), Andrias davidianus iridovirus (ADIV), and Bohle iridovirus (BIV) infections in the fish cells and the viral titers (average value) of EHNV, ADIV, and BIV in GH cells reached 10^5.0, 10^4.5, and 10^5.0 TCID₅₀/0.1 mL, respectively, within 7 days. However, no CPE was observed in the cells infected with viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), or grass carp reovirus (GCRV). These results suggest that the GH cell line is a valuable tool for studying viral pathogenesis.     

Morphological development of larval and juvenile blacktip grouper, <Emphasis Type="Italic">Epinephelus fasciatus</Emphasis>

The morphological development of larval and juvenile blacktip grouper, Epinephelus fasciatus, was examined using a hatchery-reared series. By about 5 mm body length (BL), the larvae developed characteristic pigmentation patterns of groupers, such as melanophores on the dorsal part of the gut, on the tips of the second-dorsal and pelvic fin spines, and on the midpoint of the tail to form a cluster. In addition, characteristic spines of groupers, such as spinelets on the second-dorsal and pelvic fin spines, and the preopercular angle spine developed by about 6 mm BL. The notochord end was in the process of flexion in larvae of 6–7 mm BL, by when major melanophores, spines, and jaw teeth started to appear. After the fin ray counts attained the adult complement at about 14 mm BL, somewhat densely pigmented bands started to appear on the body. The fish had the five distinct transverse bands and attained adult-like attire at about 40 mm BL when the major head spines disappeared and body parts in relation to BL became stable.     

Pyridoxine deficiency of grouper, <Emphasis Type="Italic">Epinephelus coioides</Emphasis>: physiological and biochemical alteration

Pyridoxine is essential for animals. The purpose of this study was to investigate the pyridoxine deficiency symptoms and the fit dose of pyridoxine to keep normal physiological function in grouper, Epinephelus coioides. Graded levels of pyridoxine (0, 1, 2, 4, 8, 16, 32 mg pyridoxine kg⁻¹ diet) were fed to grouper juveniles (14.83±0.31 g) for 8 weeks, and then some physiological and biochemical parameters were measured. Pyridoxine deficiency symptoms such as anorexia, poor survival, convulsion, helically swimming and hyperirritability were observed in fish fed the pyridoxine-deficient diet, and these fish also showed low whole blood haemoglobin level, liver glutamic-pyruvic transaminase (GPT) and superoxide dismutase (SOD) activities. Slight symptoms of pyridoxine deficiency also appeared among fish fed the diet with very low dose (1 mg pyridoxine kg⁻¹ diet) of pyridoxine in 6 weeks. Broken-line regression analysis showed that the optimal pyridoxine levels for these fish to keep normal survival, whole blood haemoglobin level, liver GPT and SOD activities were approximately 1.75, 1.87, 2.22 and 2.05 mg kg⁻¹, respectively.     

Growth and survival of grouper <Emphasis Type="Italic">Epinephelus</Emphasis><Emphasis Type="Italic">coioides</Emphasis> (Hamilton) larvae fed free-living nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> at first feeding

The free-living nematode, Panagrellus redivivus, was tested as live food for grouper Epinephelus coioides larvae during the first feeding stage. A series of experiments were conducted to determine the acceptability of the free-living nematodes in grouper larvae at first feeding, the optimum nematode density and the response of the larvae to nutritionally enriched nematode. All experiments were conducted in 200-L conical tanks filled with 150-L filtered seawater and stocked at 15 larvae L⁻¹. Duration of feeding experiments was up to day 21 (experiment 1) and 14 days (experiment 2 and 3). Brachionus plicatilis and Artemia (experiment 1) and Brachionus plicatilis alone (experiment 2 & 3) was used as the control treatment. Observations indicated that the grouper larvae readily fed on free-living nematodes as early as 3 days posthatching, the start of exogenous feeding. Optimum feeding density for the larvae was 75 nematodes ml⁻¹. The enrichment of cod liver oil or sunflower oil influenced the total lipids and n-3 highly unsaturated fatty acids of P. redivivus, which in turn influenced those of the grouper larvae, however, growth and survival of the larvae were not affected (P > 0.05). The results from this investigation showed that the nematode, P. redivivus, can be used as first live food for grouper larvae from the onset of exogenous feeding until they could feed on Artemia nauplii.     

Establishment of a model for chemoattraction of <Emphasis Type="Italic">Symbiodinium</Emphasis> and characterization of chemotactic compounds in <Emphasis Type="Italic">Acropora tenuis</Emphasis>

Corals harbor symbiotic dinoflagellates, Symbiodinium spp., acquired from surrounding environments. Because Symbiodinium are present at low densities in the water column, corals may attract these symbionts using chemotactic compounds. To examine whether corals contain chemotactic compounds, we established an assay to measure the chemotactic activity for Symbiodinium using an extract of the coral Acropora tenuis, a major reef-building coral in Japan. Our assay revealed that Symbiodinium strain NBRC102920 (clade A), which is taken up by juvenile A. tenuis polyps, is attracted to crude A. tenuis extracts. We found that the chemotactic compounds are water-soluble, heat-labile macromolecules and that the chemotactic activity was inhibited by N-acetyl-d-glucosamine (GlcNAc). We separated the GlcNAc-binding fraction (Fr-ActL) and identified it as the most plausible candidate for the chemoattractant, since the chemotactic activity of the crude A. tenuis extract appeared to be mainly attributable to the activity of Fr-ActL and was also inhibited by the addition of GlcNAc. These results indicate that chemoattraction is mediated via the binding of Symbiodinium to Fr-ActL.     

Screening of enzyme activity for assessing the condition of larvae in the seven-band grouper <Emphasis Type="Italic">Epinephelus septemfasciatus</Emphasis> and devil stinger <Emphasis Type="Italic">Inimicus japonicus</Emphasis>

We conducted screening tests to determine whether enzyme activity is a suitable biomarker for assessing the physiological condition of marine fish larvae. The rearing experiments consisted of three trials, of which two were conducted using the seven-band grouper Epinephelus septemfasciatus for a period of 5 days after hatching (DAH), and one was conducted using the devil stinger Inimicus japonicus for 10 DAH. The trials were conducted under three different rearing-tank environments (shallow tank, intermediate tank, deep tank) in a water volume of 100 l and an aeration rate of 50 ml/min. We determined survival, surface death, growth, and enzyme activities (trypsin, esterase, and alkaline phosphatase). The highest survival rates and lowest surface deaths in both species occurred among the larvae grown in the deep tank. There was a significant and negative correlation between survival at 5 DAH and alkaline phosphatase activity at 0 DAH in the seven-band grouper. The same correlation was found between survival at 10 DAH and trypsin and alkaline phosphatase activity at 1 DAH in the devil stinger. Based on these results, we conclude that the activity of a specific enzyme is a candidate for assessing the physiological condition of marine fish larvae.     

Nucleic acids digestion by enzymes in the stomach of snakehead (<Emphasis Type="Italic">Channa argus</Emphasis>) and banded grouper (<Emphasis Type="Italic">Epinephelus awoara</Emphasis>)

Dietary nucleic acids (NAs) were important nutrients. However, the digestion of NAs in stomach has not been studied. In this study, the digestion of NAs by enzymes from fish stomach was investigated. The snakehead pepsins (SP) which were the main enzymes in stomach were extracted and purified. The purity of SP was evaluated by SDS-PAGE and HPLC. The snakehead pepsin 2 (SP2) which was the main component in the extracts was used for investigating the protein and NAs digestion activity. SP2 could digest NAs, including λ DNA and salmon sperm DNA. Interestingly, the digestion could be inhibited by treatment of alkaline solution at pH 8.0 and pepstatin A, and the digestion could happen either in the presence or absence of hemoglobin (Hb) and BSA as the protein substrates. Similarly, the stomach enzymes of banded grouper also showed the NAs digestion activity. NAs could be digested by the stomach enzymes of snakehead and banded grouper. It may be helpful for understanding both animal nutrition and NAs metabolic pathway.     

Spatial learning-induced <Emphasis Type="Italic">egr</Emphasis>-<Emphasis Type="Italic">1</Emphasis> expression in telencephalon of gold fish <Emphasis Type="Italic">Carassius auratus</Emphasis>

The immediate-early gene (egr-1) expression was used to examine the neuron’s response in telencephalon of goldfish during spatial learning in small space. Fishes were pre-exposed in the experimental apparatus and trained to pick food from the tray in a rectangular-shaped arena. The apparatus was divided into identical compartments comprising three gates to provide different spatial tasks. After the fish learned to pass through the gate one, two more gates were introduced one by one. Fish made more number of attempts and took longer time (P < 0.05) to pass through the first gate than the gate two or three. This active learning induces the expression of egr-1 in telencephalon as established by western blot analysis. Subsequently, the fish learn quickly to cross the similar type of second and third gate and make fewer errors with a corresponding decline in the level of egr-1 expression. As the fish learned to pass through all the three gates, third gate was replaced by modified gate three. Interestingly, the level of egr-1 expression increased again, when the fish exhibit a high exploratory behavior to cross the modified gate three. The present study shows that egr-1 expression is induced in the telencephalon of goldfish while intensively acquiring geometric spatial information to pass through the gates.     

Cloning and characterization of <Emphasis Type="Italic">Bax1</Emphasis> and <Emphasis Type="Italic">Bax2</Emphasis> genes of <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis> and evaluation of transcript expression in response to grass carp reovirus infection

Multidomain proapoptotic Bcl-2-associated X (Bax) protein is an essential effector responsible for mitochondrial outer membrane permeabilization, resulting in cell death via apoptosis. In this study, two Bax genes of grass carp (Ctenopharyngodon idellus), designated as CiBax1 and CiBax2, were isolated and analyzed. The obtained CiBax1 cDNA is 2058 bp long, with a 579 bp open reading frame (ORF) coding a protein of 192 amino acid residues. The full-length cDNA of CiBax2 is 1161 bp, with a 618 bp ORF coding 205 amino acids. Both CiBax1 and CiBax2 are typical members of Bcl-2 family containing conserved Bcl and C-terminal domains, and they share conserved synteny with zebrafish Bax genes despite the grass carp Bax mapping to different linkage groups. Phylogenetic analysis showed that CiBax1 was clustered with Bax from most teleost fish, and CiBax2 was close to Bax2 from teleost fish but far separated from that of Salmo salar. Quantitative real-time PCR analysis revealed broad expression of CiBax1 and CiBax2 in tissues from healthy grass carp, but the relative expression level differed. The mRNA expression of CiBax1 and CiBax2 was both upregulated significantly and peaked in all examined tissues at days 5 or 6 post-infection with grass carp reovirus. Subcellular localization indicated that CiBax1 protein was localized in both nucleus and cytosol, while CiBax2 protein only in cytosol. Moreover, CiBax2, but not CiBax1 was colocalized with mitochondrion under normal condition. Taken together, the findings would be helpful for further understanding of the function of Bax in teleost fish.     

Homogeneity of <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> from farmed amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> in Japan

Streptococcus dysgalactiae strains have been isolated from cultured amberjack Seriola dumerili and yellowtail Seriola quinqueradiata in Japan. To characterize the fish isolates, we performed genetic analysis and compared the biochemical properties of these isolates with those of the S. dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis strains isolated from mammals. The genetic analysis revealed that the fish isolates were genetically very similar to each other with high DNA–DNA relatedness (>95.4%) and sequence homology. Meanwhile, the DNA relatedness between mammalian isolates and the fish isolates was 73.4–82.6%. In biased sinusoidal gel electrophoresis (BSFGE) analysis, the restriction patterns of mammalian isolates were different from those of fish isolates. The fish isolates did not show streptokinase activity in plasminogen obtained from mammals. These characteristics enabled us to distinguish between the fish isolates and the Sdd and Sde strains isolated from mammals. In order to obtain epidemiological information on the fish isolates, BSFGE patterns from 284 S. dysgalactiae strains from fish in Japan were examined. Based on the results of BSFGE analysis, the fish isolates were classified into 16 groups (AP1–AP16) with restriction enzyme ApaI. The dendrogram based on BSFGE analysis indicated that all fish isolates using in this study were closely related.     

Establishment,characterization of a new cell line from heart of half smooth tongue sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

A new cell line was established from the heart of a cultured marine fish, half smooth tongue sole (Cynoglossus semilaevis), designated as CSH (Cynoglossus semilaevis heart cell line). The CSH cells grow over 400 days in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 2 ng/ml basic fibroblast growth factor (bFGF). The suitable temperature for the cell growth was 24–30°C with the optimum growth at 24°C and a reduced growth at 12 and 30°C. FBS and bFGF concentration were the two important components for CSH cells proliferation. Twenty percent FBS in the medium was found to be the optimum concentration and bFGF promoted the growth of CSH cells. The double time of the cells at 24°C was determined to 73.39 h. Chromosome analysis revealed that 44% of the cells maintained a normal diploid chromosome number (2n = 42) in the CSH cells at Passage 58. The fluorescent signals were observed in CSH after the cells were transfected with green fluorescent protein (GFP) reporter plasmids. CSH cells showed the cytopathic effect (CPE) after infection with lymphosystis disease virus (LCDV). Moreover, the LCDV particles can be observed in the cytoplasm of virus-infected cells by electron microscopy, and a segment of MCP gene for major capsid protein of LCDV was found by PCR amplification DNA of virus-infected cells.     

Effect of shelter acclimation on the post-release survival of hatchery-reared black-spot tuskfish <Emphasis Type="Italic">Choerodon schoenleinii</Emphasis>: laboratory experiments using the reef-resident predator white-streaked grouper <Emphasis Type="Italic">Epinephelus ongus</Emphasis>

A critical component of many releasing projects is the identification and subsequent implementation of optimal release strategies that can decrease post-release predation mortalities. We performed laboratory experiments to investigate whether acclimation to shelters affects the post-release survival of hatchery-reared black-spot tuskfish Choerodon schoenleinii in the presence of a reef-resident predator, the white-streaked grouper Epinephelus ongus. Tuskfish were exposed to groupers under three different experimental conditions/treatments: (1) acclimation of fish to shelters prior to their exposure to groupers; (2) no acclimation of fish to shelters, but with shelters available during their exposure to groupers; (3) fish not acclimated to shelters and no shelters available during their exposure to groupers. Tuskfish that were acclimated to shelters utilized shelters more frequently than did non-acclimated fish, and the survival rate of acclimated fish was higher than those of fish in the other treatments. These results suggest that pre-release acclimation to shelters improves the post-release survival of hatchery-reared black-spot tuskfish.