Dietary manganese requirement of juvenile hybrid grouper,Epinephelus lanceolatus × E. fuscoguttatus

An 8‐week feeding trial was conducted to estimate the optimum dietary manganese (Mn) requirement for juvenile hybrid grouper, Epinephelus lanceolatus × E. fuscoguttatus. The basal diet was formulated to contain 520 g/kg crude protein from casein and fishmeal. Manganese methionine was added to the basal diet at 0 (control group), 2.5, 5, 10, 20 and 40 mg Mn/kg diet providing 7.48, 10.34, 13.76, 19.72, 31.00 and 53.91 mg Mn/kg diet, respectively. Each diet was randomly fed to triplicate groups of juveniles, and each tank was stocked with 20 fish (initial weight, 60.06 ± 0.68 g). The manganese content in rearing water was monitored and kept below 0.01 mg/L. Results showed that the weight gain ratio (WGR), protein efficiency ratio (PER), specific growth rate (SGR), Mn contents in whole body, liver and vertebra, and activities of hepatic Mn superoxide dismutase (Mn‐SOD), total SOD (T‐SOD) and glutathione peroxidase (GSH‐PX) were significantly improved by dietary Mn supplementation (p < .05). However, dietary Mn did not affect arginase (DArg) activity. The highest feed conversion ratio (FCR) was observed in fish fed the basal diet (p < .05). No significant differences were found on the Cu and Zn contents in whole body by supplementing dietary Mn. Supplemented Mn in diets had significantly effect on liver and vertebral trace element deposition (p < .05). Fish fed the basal diet had the highest Fe and Zn contents in vertebra (p < .05). There were no significant differences on hepatic pyruvate decarboxylase (PDC) activity with supplemented Mn levels below 13.76 mg/kg. As biomarker of oxidative stress, malondialdehyde (MDA) content in liver was significantly higher in fish fed the basal diet (p < .05). Using the broken‐line models based on SGR, dietary Mn requirement of the juvenile hybrid grouper was estimated to be 12.70 mg/kg diet.     

The optimum dietary isoleucine requirement of juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂)

A six‐week growth trial was conducted to evaluate the optimum dietary isoleucine requirement of juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂). Seven isoenergetic (3,400 kcal/kg of dry matter), isoproteic (496 g/kg of dry matter) and isolipidic (70 g/kg of dry matter) diets were formulated to contain graded Ile levels (7.3, 11.3, 15.7, 19.6, 23.5, 26.8 and 30.8 g/kg, dry‐matter basis). Each experimental diet was fed to triplicate groups of 12 hybrid grouper juveniles (average initial body weight: 6.00 ± 0.01 g/fish). Experimental fish were randomly distributed into 21 glass tanks (L 60 × W 45 × H 50 cm) connected to mechanical and biological water filters as a recycling system. Fish were fed twice daily (08:00 and 16:00) to apparent satiation. After the sampling of the growth trial, the remaining fish in each group were fed their corresponding diets for 2 d and then exposed to 4 mg Cu²⁺ · L^?1 water for 24 hr. Results showed that growth performance and feed utilization were significantly affected by different dietary Ile levels (p < .05). Weight gain percentage (WG%), protein productive value (PPV), protein efficiency ratio (PER) and feed efficiency (FE) were increased as dietary Ile level increased, reaching a peak value at 19.6 g/kg dietary Ile, and thereafter, these four parameters declined as dietary Ile level continued to increase. Daily feed intake (DFI) showed an opposite tendency of variations as FE. The quadratic regression analysis of WG%, PPV, PER and FE against dietary Ile levels indicated that the optimum dietary Ile requirement for hybrid grouper was estimated to be 19.8, 20.8, 19.4 and 19.1 g/kg dry matter, respectively. Among all experimental treatments, fish fed 19.6 g/kg dietary Ile had the highest expression of growth and protein synthesis‐related genes, including growth hormone (GH) in pituitary, insulin‐like growth factor 1 (IGF‐1), growth hormone receptor 1 (GHR1), target of rapamycin (TOR) and S6‐kinase 1 (S6K1) in liver. Gut micromorphology was significantly influenced by dietary Ile levels. After the exposure to 4 mg Cu²⁺ · L^?1 water for 24 hr, fish fed 19.6 g/kg dietary Ile had the highest survival and the best immunologic manifestation among all experimental treatments. Generally, the optimum dietary Ile requirement for maximum growth of hybrid grouper was estimated to be 19.8 g/kg dry matter, corresponding to 39.9 g/kg dietary protein.     

Quantitative trait locus mapping of growth‐related traits in inter‐specific F1 hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus) in a tropical climate

Growth‐related traits are the main target of genetic breeding programmes in grouper aquaculture. We constructed genetic linkage maps for tiger grouper (Epinephelus fuscoguttatus) and giant grouper (E. lanceolatus) using 399 simple sequence repeat markers and performed a quantitative trait locus (QTL) analysis to identify the genomic regions responsible for growth‐related traits in F₁ hybrid grouper (E. fuscoguttatus × E. lanceolatus). The tiger grouper (female) linkage map contained 330 markers assigned to 24 linkage groups (LGs) and spanned 1,202.0 cM. The giant grouper (male) linkage map contained 231 markers distributed in 24 LGs and spanned 953.7 cM. Six QTLs affecting growth‐related traits with 5% genome‐wide significance were detected on different LGs. Four QTLs were identified for total length and body weight on Efu_LG8, 10, 13 and 19 on the tiger grouper map, which explained 6.6%–12.0% of the phenotypic variance. An epistatic QTL with a reciprocal association was observed between Efu_LG8 and 10. Two QTLs were identified for body weight on Ela_LG3 and 10 on the giant grouper map, which explained 6.9% of the phenotypic variance. Two‐way analysis of variance indicated that the QTL on Efu_LG13 interacts with the QTLs on Ela_LG3 and 10 with large effects on body weight. Furthermore, these six QTLs showed different features among the winter, summer and rainy seasons, suggesting that environmental factors and fish age affected these QTLs. These findings will be useful to understand the genetic structure of growth and conduct genetic breeding in grouper species.     

The high prevalence of pathogenic Vibrio harveyi with multiple antibiotic resistance in scale drop and muscle necrosis disease of the hybrid grouper,Epinephelus fuscoguttatus (♀) × E. lanceolatus (♂), in China

Scale drop and muscle necrosis disease with high mortality widely occurred recently in the hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), a crucial cultured marine fish species in China. In this study, 30 Harveyi clade isolates of 27 Vibrio harveyi strains were isolated from diseased hybrid groupers in the south‐east and north‐east coastal areas of China. A total of 22 V. harveyi strains were determined to be pathogenic, and most challenged fish died within 2 days of infection; surviving individuals exhibited scale drop and deep dermal lesions as naturally diseased fish. Although five typical virulence genes, including luxR, toxR_Vh, chiA, serine protease and vhh widely existed in V. harveyi, no obvious correlation was established between virulent strains and virulence genes harboured in them. Furthermore, multiple antibiotic resistance was widely exhibited in Harveyi clade strains, particularly for penicillins, polypeptides, lincomycins, acetylspiramycin, streptomycin, metronidazole and bacitracin. And the multiple antibiotic resistance indices were gradually decreased from southern to northern areas of China. This study demonstrated that the pathogenic V. harveyi with multiple antibiotic resistance is highly prevalent in hybrid grouper in China, which requires particular attention.     

Soy protein concentrate as an alternative in replacement of fish meal in the feeds of hybrid grouper,brown‐marbled grouper (Epinephelus fuscoguttatus) × giant grouper (E. lanceolatus) juvenile

Hybrid grouper juveniles (body weight, 6.1 ± 0.7 g) (brown‐marbled grouper, Epinephelus fuscoguttatus × giant grouper, E. lanceolatus) were fed with six isoproteic (50% crude protein) and isolipidic (12% crude lipid) feeds containing different levels of soy protein concentrate (SPC) in replacement of fish meal (SPC at 20%, 30%, 40%, 50% and 60% protein) and control feed (SPC0) for 6 weeks. Hybrid grouper juveniles were cultured in 100‐L fibreglass tank equipped with flow‐through water system and fed twice a day to apparent satiation level. The highest and lowest growth was recorded in fish fed SPC20 and SPC60 respectively. However, growth of SPC20 was not significantly higher than those fed SPC0, SPC30, SPC40 and SPC50 (p > .05). A decreasing growth trend was observed with the increasing level of SPC from feed SPC40 to SPC60. A noticeable better feed utilization was also observed in fish fed SPC0, SPC20, SPC30, SPC40 and SPC50 compared to fish fed SPC60 (p < .05). The fish condition factor, hepatosomatic index, viscerosomatic index and whole body proximate content of the fish were not affected by the graded levels of SPC. However, the body lipid content was significantly lower in fish fed SPC40 to SPC60 (p < .05). The apparent digestibility coefficient (ADC) of protein and lipid was significantly higher in fish fed SPC0 and SPC20 compared to other dietary treatments (p < .05). Based on the regression analysis on specific growth rate, the study suggests that the hybrid grouper grow best at 21.4% and can utilize up to 50% inclusion level of SPC in protein without significantly affect their growth and its body condition.     

Molecular cloning,characterization, tissue expression and nutritional regulation of O‐GlcNAc transferase gene in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂)

O‐GlcNAc transferase gene (OGT) was considered as the sole rate‐limiting enzyme in the O‐GlcNAc modification. In the present study, the OGT gene of hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) was cloned and characterized, and its expression in response to dietary carbohydrate level and acute glucose treatment was investigated. The full‐length of OGT (GenBank accession no. KY656469 ) was 4,063 bp, including a 302 bp 5′untranslated terminal region (UTR), a 3,165 bp coding region that encoded 1,054 amino acids residues and a 596 bp 3′ UTR. The highly conservation of OGT gene between fish and mammals was also observed through multiple sequences alignment and phylogenetic analysis. O‐GlcNAc transferase gene was ubiquitously expressed in all detected tissues with highest expressions in brain and liver, to a lesser degree, in eye, heart, kidney and intestine. The increasing dietary carbohydrate from 8.02% to 16.08% had no significant effect on the mRNA expression of OGT. However, the expression of OGT was slightly elevated at 6 hr post‐glucose injection, and the elevation became significant at 24 hr time‐point. These data may enhance our understanding on the nutritional regulation of OGT and O‐GlcNAc modification in fish species.     

Isolation,identification and pathogenicity of Vibrio harveyi,the causal agent of skin ulcer disease in juvenile hybrid groupers Epinephelus fuscoguttatus × Epinephelus lanceolatus

The hybrid grouper, Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂), is a newly bred cultivated marine fish species of high economic value. However, a skin ulcer disease with high mortality has occurred, and the responsible pathogen remains unknown. In this study, we summarized the epidemic status and external signs of this disease. We screened potential pathogens and finally isolated one bacterial strain ML01 from affected fish. We subjected healthy juvenile hybrid groupers to bacterial challenge tests with the isolate by immersion, immersion after dermal abrasion and intraperitoneal injection, respectively. Within 14 days post‐infection, the isolate ML01 caused mass mortality of juveniles infected via immersion after dermal abrasion or intraperitoneal injection. Diseased juveniles displayed obvious signs of skin ulcers. The median lethal dose of ML01 by intraperitoneal injection was 1.10 × 10⁵ colony‐forming units. ML01 was identified as Vibrio harveyi by bacterial morphology, analytical profile index identification, 16S rDNA sequencing and multilocus sequence analysis. Antibiotic susceptibility tests showed that ML01 was sensitive to ceftriaxone, doxycycline and minocycline. The results of this study suggest that V. harveyi is the causal agent of skin ulcer disease in juvenile hybrid groupers, thus providing a basis for effective control and prevention of this disease.     

Effects of dietary supplementation of probiotics on the growth,activities of digestive and non‐specific immune enzymes in hybrid grouper (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀)

Effects of Bacillus cereus BC‐01, Lactobacillus acidophilus LAG01, Clostridium butyricum CBG01 and their combinations as supplementation on the growth performance, digestive enzyme activities and serum non‐specific immunity of hybrid grouper (Epinephelus lanceolatus ♂×E. fuscoguttatus ♀) were assessed. Seven different diets, that is one control diet (basal feed without any probiotics, CT) and six treatment diets containing single B. cereus (Bs), L. acidophilus (Ls) and C. butyricum (Cs) at 1.0 × 10⁹ cfu/kg feed, and also their combinations in equal proportion at 1.0, 2.0 and 3.0 × 10⁹ cfu/kg feed (BLC1, BLC2 and BLC3) were prepared respectively. After 60‐day feeding trial, the final weight, specific growth rate,food consumption, food conversion efficiency and apparent digestibility coefficient of fish in Ls and BLC3 were significantly higher compared with the control (p < .05). The activities of pepsin and trypsin in the intestine of fish for Ls and BLC3 were significantly higher relative to the control (p < .05). Relative to controls, significantly enhanced amylase and lipase activities in proximal intestine except for Cs and BLC1 and lipase activities in distal intestine except for Cs were observed (p < .05). Meanwhile, activities of superoxide dismutase in the serum of fish for all treatments, lysozyme and catalase in Ls and BLC3, and glutathione peroxidase except for Cs were significantly enhanced (p < .05). Based on the above, dietary supplementation of single L. acidophilus at 1.0 × 10⁹ cfu/kg or combination of three strains at 3.0 × 10⁹ cfu/kg for hybrid grouper is recommended.     

Embryonic and larval development of a hybrid between kelp grouper Epinephelus moara ♀ × giant grouper E. lanceolatus ♂ using cryopreserved sperm

Hybridization was used to take advantage of desirable traits in offspring. In the present study, we applied the cryopreserved Epinephelus lanceolatus sperm into interspecific hybridization with E. moara. Successful hybridization between these two species was achieved and cultured in 23–24°C seawater (34‰). There was no difference in survival rate between hybrid (E. moara ♀ × E. lanceolatus ♂) and non‐hybrid (E. moara ♀ × E. moara ♂) at 24 hrs post hatch (HPH), but less hybrid (14.35 ± 8.02%) hatched than non‐hybrid (93.60 ± 1.65%), which might be due to irregularity cell cleavage and skeletal deformities from the formation of embryonic body to the later embryonic development. Similar phenomenon was found in hybrid embryos from fertilization with fresh sperm, indicating that species variations between parents, rather than cryopreserved sperm, resulted in deformities in embryos. Mean hatch time of the hybrid was 1 hr faster than that of E. moara. The hybrid was 1.95 ± 0.06 mm in total length when newly hatched and reached 38.00 mm at 58 days post hatch (DPH), which showed faster growth than E. moara (recorded in the previous study). Considering its faster growth, E. moara × E. lanceolatus hybrid was a potential breeding production in aquaculture. Over 212,000,000 larvae have been produced and launched in the market since 2015. The results of this study also shed some lights on further comparative studies in grouper hybrid performance.     

Vibrio harveyi (VirB11) recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides)

Despite significant improvements in aquaculture to compensate wild catch, disease organisms have thrived in limiting its national and global potential. Using antibiotics, in a bid to remedy the havoc, has given rise to complications, attracting attention to disease prevention by immune enhancement against diseases. Grouper production has been inhibited for the threats of bacterial infection, particularly of Vibrio origin. Considering the rise in vibriosis cases, improved vaccines are necessary; moreover, recombinant vaccines, the choice for trial in the present experiment have been effective and more specific in improving immunity. The current work deals with grouper immune system enhancement with a recombinant vaccine developed from VirB11 gene in Vibrio harveyi. VirB11 was cloned in V. harveyi for recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides). As indicated by the results, recombinant VirB11 protein showed effectiveness in conferring protection against vibriosis with observable specific antibody response in enzyme‐linked immunosorbent assay (ELISA) analysis; a significant increase (p < 0.05) in antibody levels was observed after a week and after 8 weeks post‐vaccination. From the weeks post‐vaccination, log2 (antibody titres) in the sera of vaccinated groups reached a peak of 14.2 at week 5 in the vaccinated group in comparison with a peak of approximately 5 and 2 in adjuvant and PBS controls. As indicated by the challenge results, 90% relative survival was observed in vaccinated group and 13% relative survival in control group I (adjuvant control). The cumulative performance of protein concludes VirB11 commendable for recombinant vaccine development.     

Use of response surface methodology to study the combined effects of temperature and salinity on hatching and deformity of the hybrid grouper,Epinephelus fuscoguttatus (♀) × Epinephelus polyphekadion (♂)

This study seeks to identify the optimal combination of temperature and salinity for the embryo development of the hybrid groupers, Epinephelus fuscoguttatus (♀) × Epinephelus polyphekadion (♂), using the central composite design and response surface methodology. Results have shown that the low hatching rates of fertilized eggs and high deformity rates of larvae were the result of lower or higher temperature/salinity levels. The linear effects of temperature and salinity were significant in both hatching rates and deformity rates (p < .01). Similar results were observed in the quadratic effects of temperature and salinity (p < .01). Effects of temperature were positively related to salinity effects (p < .05). Salinity effects were more remarkable than temperature on hatching rates, but not the same on deformity rates. Model equations were established for temperature and salinity effects on hatching rates and deformity rates. A high coefficient of determination (R² > .97) was found for the adequacy and predictive capability of model equations, indicating that these models can be applied to prediction. The optimal combination of temperature and salinity was 27.58°C/30.94, which was derived from the statistical optimization approach. In this condition, the hatching rate was the highest at 85.73%, and the deformity rate was the lowest at 5.96%, with a high desirability function of 0.957. The results of this study can be applied to improve hatching and decrease deformity in future cultures of hybrid groupers.     

Effects of rhythmic temperature change on the growth,body composition and energy budget of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀)

Effects of rhythmic temperature change on the growth, body composition and energy budget of hybrid grouper (Epinephelus lanceolatus ♂ × E. fuscoguttatus ♀) were investigated. Nine groups of fish received repeating cycles of low‐temperature manipulation (22°C) for 1, 2 and 4 days followed by recovery temperature (28°C) for (3, 7 and 11 days), (6, 14 and 22 days) and (12, 28 and 44 days) respectively, designated as L1R3, L1R7, L1R11, L2R6, L2R14, L2R22, L4R12, L4R28 and L4R44 respectively. In the control group (C), fish were reared at 28°C throughout the whole experiment. After 96‐day feeding trail, the final weight, relative weight gain rate, specific growth rate, food conversion efficiency and apparent digestibility coefficient of fish in L4R28 were significantly higher than those of control (p < .05). The crude protein contents of fish in L2R14, L2R22, L4R12, L4R28 and L4R44 were significantly higher compared to the control (p < .05). According to energy budget, fish in L4R28 exhibited significantly higher proportion of food energy assimilated into growth and lower proportion consumed for excretion than the control fish (p < .05). These results indicated the occurrence of complete or over growth compensation in hybrid grouper, and the use of repeated cycles of low temperature (22°C) for 4 days followed by recovery temperature (28°C) for 28 days could obtain enhanced growth, that is overcompensation, with improved apparent digestibility coefficient, food conversion efficiency and energetic efficiency into growth. This technique would be more practically applicable in indoor culture for this species.     

Effects of alternative dietary oils on lipid metabolism and related gene expression in hybrid grouper (♀Epinephelus fuscoguttatus × ♂E. lanceolatu)

An 8‐week growth trial was conducted to evaluate effects of dietary oil sources on growth, enzymes activity and genes expression levels related to lipid metabolism of hybrid grouper (♀Epinephelus fuscoguttatus × ♂E. lanceolatu) juveniles. Seven iso‐lipid (97 g/kg of dry matter) and iso‐protein (503.5 g/kg of dry matter) experimental diets were formulated containing 50 g/kg fish oil (FO; acting as controls) or various vegetable oils (VOs): corn oil (CO), sunflower oil (SO), tea oil (TO), olive oil (OO), rice oil (RO) and mixed oil (MO; comprising equal amounts of these oils). Each diet was fed to triplicate groups of 40 fish for per repetition (15.09 ± 0.01 g) for 56 days. The results show that (a) alternative dietary oils had no significant effects on final weight compared with control group (p > .05); (b) compared with FO group, VOs significantly changed the contents of serum lipoproteins, cholesterol, triglycerides and the activity of liver lipid‐metabolizing enzymes (p < .05); (c) CO group had the least effect on the serum lipoproteins, triglycerides and cholesterol of grouper compared with control; the activity of liver lipid‐metabolizing enzymes in RO and control group was the closest; (d) the mRNA levels of Δ6 Fatty acid desaturase (Δ6Fad), hormone‐sensitive lipase (HSL) and lipoprotein lipase (LPL) were not significantly effected by lipid sources, but CO, TO, OO and MO significantly down‐regulated the expression of fatty acid synthetase (FAS) mRNA level in liver, while RO opposite (p < .05); (e) vegetable oil significantly up‐regulated peroxisome proliferator‐activated receptor α (PPARα) and peroxisome proliferator‐activated receptor β (PPARβ) mRNA levels, while TO and RO down‐regulated peroxisome proliferator‐activated receptor γ (PPARγ) mRNA levels (p < .05); and 6) MO significantly increased the mRNA levels of heart‐type fatty acid‐binding protein (H‐FABP) and adipocyte‐type fatty acid‐binding protein (A‐FABP) (p < .05), while other VOs had no effect on them (p > .05). In conclusion, dietary substitution of FO by VO in diet affected lipid metabolism of grouper, which may be regulated by PPARs.     

Effects of fishmeal replacement by Tenebrio molitor meal on growth performance,antioxidant enzyme activities and disease resistance of the juvenile pearl gentian grouper (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀)

A 50‐day feeding trial was conducted for juvenile pearl gentian grouper (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀) to determine the optimal amount of Tenebrio molitor meal (TM) that could replace fishmeal (FM) in feeds without reducing growth performance, antioxidant enzyme activities and resistance. Six isonitrogenous and isolipidic diets were formulated with TM replacing 0% (T0), 6.25% (T6), 12.5% (T12), 18.75% (T18), 25% (T25), 31.25% (T31) FM respectively. Each diet was randomly divided into triplicate groups with 30 juveniles (initial weight 6.60 ± 0.00 g). After feeding trail, fish were challenged by Vibrio harveyi for 7 days. The results showed that each treatment had better survival rate between 96.67% and 100%. Fish fed with control and T6 diet showed lower final weight and weight gain rate as compared to other diets. The feed conversion ratio of fish fed on T12 and T18 diet had no significant difference compared to control diet. As compared to control diet, other diets had no significant differences on specific growth rate and temperature growth coefficient. Fish fed with T6 diet had highest liver glutathione reductase activities. No significant differences were observed in liver superoxide dismutase activities in fish fed with T6, T12, T25, T31 and control diet. Fish fed on T18 diet had lowest hepatic malondialdehyde contents and a highest relative per cent survival. According to a broken‐line model, the recommended substituted ratio of TM for FM was estimated to be 12.3% for grouper obtaining maximum WGR, corresponding to additive amount of TM is 4.92%.     

Growth and feed performance,digestibility and acute stress response of juvenile grouper (Epinephelus fuscoguttatus) fed diets with hydrolysate from milkfish offal

Nutritional qualities of fish processing by‐products can further be improved through enzymatic hydrolysis. The objective of this study was to elucidate the efficacy of hydrolysed milkfish offal at different inclusion levels when fed to juvenile grouper, Epinephelus fuscoguttatus, with an initial body weight of 2.88 ± 0.06 g. The animals were fed for 56 days with seven diets supplemented with 0 (control), 5%, 15% and 25% of milkfish offal (MO) and milkfish offal hydrolysate (MOH). The diets were formulated to be isonitrogenous (45%) and isolipidic (11%). The diets were assigned to 21 tanks (15 fish per tank) with each diet having three replicates. Results from the experimental trials indicated that feed conversion efficiency, feed intake and weight gain of fish significantly (P < 0.05) improved when fed diets with MOH. No significant differences within the rest of the dietary treatments were observed. Survival rate (>90%) did not differ in all the dietary treatments. Proximate composition (crude protein, crude fat and ash) indicated no significant difference among fish fed from all the dietary treatments. Apparent digestibility of MOH indicated a 95% and 66% digestibility for protein and dry matter respectively. Plasma stress parameters (cortisol and glucose) were not influenced by the dietary treatment when fish were subjected to an acute stressor (5‐min chasing). Liver morphology indicated normal hepatocyte shape and the presence of lipid droplets in fish fed from all the dietary treatments. The results indicated that milkfish offal processed as hydrolysate can be utilized in grouper diets and can promote growth and feed efficiency when supplied at 10–15%.     

Characterization of triploid hybrid groupers from interspecies hybridization (Epinephelus coioides ♀ × Epinephelus lanceolatus ♂)

Interspecies hybridization is widely used in aquaculture as a beneficial strategy. Diploid and triploid hybrids have been detected from the interspecies hybridization of Epinephelus coioides ♀ × Epinephelus lanceolatus ♂. This is the first report of triploidization through hybridization in grouper. Confirmation has been obtained through flow cytometry, karyotyping and erythrocyte nuclei measurement. The chromosome numbers of E. coioides, E. lanceolatus, diploid hybrid grouper are 48 and triploid hybrid grouper are 72. Measurements of erythrocyte nuclei indicate that triploid fish have a larger nuclear surface than the diploid groupers, and the average ratio of triploid to diploid surface area is 1.59. During the first 1.5 years, triploid hybrid groupers grow faster than diploid hybrid groupers or either parent species. The average growth rate of triploid hybrids is 1.61 times greater than that of diploid hybrids at 6 months of age and 1.43 times greater at 18 months of age. The triploid hybrid groupers are inferior in gonadal development, with no primary‐growth‐stage oocytes appearing in the gonads at 18 months of age. Morphological studies indicate that triploid hybrid groupers have distinctive differences in snout length, eye diameter, body trunk shape, and tail shape development compared with diploid hybrid groupers. Triploid hybrid groupers have an advantage in growth ability, and artificial breeding of triploid groupers might be of great potential use in the grouper aquaculture industry.     

Experimental infection of brown‐marbled grouper,Epinephelus fuscoguttatus (Forskal), with Vibrio parahaemolyticus identifies parvalbumin beta‐2 subunit I,alpha‐2‐macroglobulin,nattectin and immunoglobulin light chain,differentially expressed in resistant grouper

The mechanisms through which brown‐marbled grouper accomplishes resistance to infection, particularly against Vibrios, are not yet fully understood. In this study, brown‐marbled grouper fingerlings were experimentally infected with Vibrio parahaemolyticus, to identify disease resistance grouper, and the serum proteome profiles were compared between resistant and susceptible candidates, via two‐dimensional gel electrophoresis (2‐DE). The results showed that putative parvalbumin beta‐2 subunit I, alpha‐2‐macroglobulin, nattectin and immunoglobulin light chain proteins were among proteins that significantly overexpressed in the resistant fish as compared to the susceptible group of fish, whereas apolipoprotein E and immunoglobulin light chain proteins were observed to be differentially overexpressed in the susceptible fish. Further analysis by peptide sequencing revealed that the immunoglobulin light chain proteins identified in the resistant and susceptible groups differed in amino acid composition. Taken together, the results demonstrated for the first time that putative parvalbumin beta‐2 subunit I, alpha‐2‐macroglobulin, nattectin and immunoglobulin light chain are among important proteins participating to effect disease resistance mechanism in fish and were overexpressed to function collectively to resist V. parahaemolyticus infection. Most of these molecules are mediators of immune response.     

Parasites of cultured and wild brown‐marbled grouper Epinephelus fuscoguttatus (Forsskål, 1775) in Lampung Bay,Indonesia

A total of 210 Epinephelus fuscoguttatus (brown‐marbled grouper) was examined for parasites. During three consecutive seasons (two rainy and one dry season from 2002 to 2004), 35 specimens each taken from floating net cages of the National Sea Farming Development Centre (Balai Budidaya Laut) and from wild catches in Lampung Bay, South Sumatra, Indonesia were studied. Twenty‐five (cultured grouper) and 30 (wild grouper) parasite species/taxa were identified, with an infracommunity ranging from one to nine (cultured) and three to 14 parasite species (wild), demonstrating a species‐rich parasite fauna even in the cultured fish. Protozoans (1 species), microsporeans (1), myxozoans (1), digeneans (8), monogeneans (5), cestodes (3), nematodes (8), acanthocephalans (2) and crustaceans (6) were found. The most abundant parasites were the monogeneans Pseudorhabdosynochus epinepheli and Pseudorhabdosynochus lantauensis for both, cultured and wild grouper during all seasons. For the cultured fish, the prevalence of monoxenous ectoparasites (e.g. P. epinepheli, P. lantauensis, Capsalidae gen. et sp. indet., Benedenia epinepheli) was in most cases higher than that of heteroxenous endoparasites. This contrasts the wild grouper, where heteroxenous parasites such as Allopodocotyle epinepheli and Raphidascaris sp. occurred at a similar prevalence compared with the fairly abundant Pseudorhabdosynochus spp. No seasonality of infestation was observed for both cultured and wild fish. The high levels of infestation of potentially pathogenic monogeneans throughout the year could result in significant parasite outbreaks at the locality studied.