Rapid detection of SNPs in candidate genes regulating the growth of orange‐spotted grouper,Epinephelus coioides (Hamilton, 1822), using semiconductor sequencing

Orange‐spotted grouper (Epinephelus coioides) is one of the most important marine food species in Southeast Asia and China and has been cultured for decades. In this study, we fully utilized the limited capacity of semiconductor sequencing, the high efficiency of long‐range PCR for target enrichment and a non‐indexed pooling strategy to screen single‐nucleotide polymorphisms (SNPs) in a breeding population of orange‐spotted grouper. Forty‐one genomic DNA fragments, with a total length of approximately 180 kb, including 22 candidate genes that control growth, and from a DNA pool of 20 heaviest and 22 lightest individuals of the sampled population were successfully sequenced using an Ion Torrent Personal Genome Machine. 3 503 466 clear reads were produced with a length of 192 ± 56 bp, 86.8% of which were mapped to the reference with an average coverage depth of 2567‐fold and physical coverage of 98.8%. Finally, 1623 high‐quality SNPs were adopted. Compared with Sanger sequencing of three random common regions, the sensitivity and specificity of our approach were 39.4% and 100.0% respectively. A mutation located at the third position of the previously labelled start codon of growth hormone receptor type 1 invalidated the start codon. Furthermore, comparison of the frequencies of genotypes and alleles of this site between the two extreme groups, prediction of signal peptide and identification of conservative mRNA sequences suggested that the functional start codon is likely located at the position of another downstream in‐frame ATG in the mutant. These detected SNP markers will provide important tools for the selective breeding of orange‐spotted grouper.     

What causes cannibalization‐associated suffocation in cultured brown‐marbled grouper,Epinephelus fuscoguttatus (Forsskål, 1775)?

During culture of grouper, cannibalism is a frequent phenomenon that usually causes economic loss. Grouper culture often requires grading to prevent size‐dependent cannibalism. In comparison with orange‐spotted (Epinephelus coioides) and giant grouper (E. lanceolatus), failure to swallow prey during cannibalism is frequently observed in brown‐marbled grouper (E. fuscoguttatus). The cannibal cannot engulf the entire prey and the two fish ultimately end up dying together. Herein, we attempted to compare morphometric differences among orange‐spotted, giant and brown‐marbled grouper. We established a cannibal–prey total length (TL) relationship for brown‐marbled grouper: TL_prey=0.71 TL_cannibal−1.75. According to the equation, a difference of approximately 50% in TL is a threshold to use for grading the grouper. This value is much larger than that used (30%) in orange‐spotted and giant grouper, and this size difference results in a higher incidence of failed cannibalism attempts in brown‐marbled grouper. It is our belief that the standard grading method will fail to produce as good an effect in brown‐marbled grouper as is seen in orange‐spotted and giant grouper. Therefore, in addition to grading, other manipulations such as satiation feeding, nutritional supplementation and optimal stocking densities should be applied to reduce cannibalism of brown‐marbled grouper.     

Effects of dietary supplementation with coconut oil on the growth,fatty acid profiles and some lipid metabolism relative gene expressions of orange‐spotted grouper Epinephelus coioides

This study investigated the effects of coconut oil as a dietary supplement on the growth, lipid metabolism and related gene expressions of juvenile orange‐spotted grouper Epinephelus coioides. Coconut oil at concentrations of 0, 10, 30 and 50 g/kg was used to replace dietary lipids in a basal diet containing 150 g/kg lipids. The four experimental diets were, respectively, fed to triplicate groups of juvenile groupers (initial weight: 8.53 ± 0.13 g) in a recirculating system for 8 weeks. Fish fed the diet containing 50 g/kg coconut oil exhibited lower (p < .05) weight gain than did fish fed the diet containing 30 g/kg coconut oil; however, no significant differences in weight gain were observed between fish fed diets containing 0 and 10 g/kg coconut oil. Hepatic carnitine palmitoyltransferase‐1, fatty acid synthase, fatty acid elongase, fatty acid desaturase and peroxisome proliferator‐activated receptor gamma gene expressions were all the highest in fish fed the diet containing 10 g/kg coconut oil. Fish fed the coconut oil‐free basal diet demonstrated upregulated gene expression of neuropeptide Y. The results suggest that dietary supplementation with 10 g/kg coconut oil exerted beneficial effects on lipid metabolism by E. coioides.     

Design and performance of recirculating aquaculture system for marine finfish broodstock development

Tropical and subtropical climatic conditions in India present an ideal and unique opportunity for being the leader in tropical marine finfish aquaculture. However, the problem persist due to non-availability of marine finfish seed for the culture. In response to this problem, broodstock development of different tropical marine finfishes for seed production was started. The present study was undertaken to design a recirculating aquaculture system (RAS) and studying their performance in managing the various water quality required for the marine finfish broodstock development and breeding. The design of RAS, developed in the present study, included a broodstock tank, egg collection chamber, electrical pump, rapid sand filter, venturi type protein skimmer and biological filter. Two RAS were designed, one was stocked with a demersal fish species, orange spotted grouper (Epinephelus coioides) and the other was stocked with a pelagic fish species, Indian pompano (Trachinotus mookalee) at the rate 1 and 0.5 kg/m³ with a sex ratio of 1:1 and 1:2 (female: male) respectively. Various physio-chemical parameters, viz, total ammonia nitrogen (TAN), nitrite, nitrate, pH, alkalinity, temperature, free carbon dioxide (CO₂) and dissolved oxygen (DO) of both tank water were analyzed to assess the performance of recirculating aquaculture system in maintaining the water quality. Gonadal development of the fishes was assessed and the spawning performance was recorded and finally, economic performance of the system was also evaluated. During the entire experimental period, mean monthly total ammonia nitrogen was less than 0.07 and 0.06 mg L⁻¹ and mean monthly nitrite was less than 0.02 and 0.01 mg L⁻¹ in orange spotted grouper and Indian pompano RAS tanks respectively. The pH (7.8–8.2), DO (>4 mg/L) and alkalinity (100–120 mg/L) were found to be in optimum range in both recirculating aquaculture systems. Carbon dioxide was found to be nil during the entire experimental period in both the systems. In fact these levels were comparable or less than that is reported as the permissible limits for broodstock development. Indian pompano and Orange spotted grouper matured and spawning was obtained with production of fertilized eggs round the year. Economic evaluation showed the price of 10,000 fertilized eggs of orange spotted grouper to be US $ 1.33. The design of RAS devised in the present study is efficient in controlling and maintaining optimum water quality for broodstock development of both demersal and pelagic finfishes. The fishes stocked in RAS attained final maturation and round the year spawning was obtained.     

Using Bacillus subtilis E20‐fermented soybean meal as replacement for fish meal in the diet of orange‐spotted grouper (Epinephelus coioides,Hamilton)

The potential of Bacillus subtilis E20‐fermented soybean meal (FSBM) as a partial alternative component of fish meal (FM) in fed diets of orange‐spotted grouper (Epinephelus coioides) was evaluated in this study. An FM‐based diet and seven diets containing 10%, 20% and 30% and 10%, 20%, 30% and 40% of FM replaced by soybean meal (SBM) and FSBM, respectively, were fed to grouper for 84 days to evaluate possible substitution levels of FM by tracking growth performance, digestive enzyme activity and morphological changes in the liver and distal intestine. No significant differences in survival and muscle composition of grouper were found between controls and treatments. Growth performance and feed efficiency of fish fed diets with FM replaced by FSBM up to 30% were not significantly different from controls, whereas significantly decreased growth performance and feed efficiency occurred with diets containing >20% of SBM. Based on the feed efficiency, the maximum substituted levels of FM by SBM and FSBM in grouper diets were 18.36% and 29.32%, respectively, based on broken‐line analyses. Histopathological changes in the liver and distal intestine, and significantly lower activity levels of digestive enzymes, including pepsin in the stomach and trypsin, chymotrypsin, amylase and lipase in the distal intestine, were found in fish fed a diet containing 30% of FM replaced by SBM. However, these parameters were improved by the substitution of FSBM. It is therefore believed that FSBM has great potential to be used as a protein source in grouper diets in partial replacement of FM.     

Effects of dietary docosahexaenoic acid sources,microalgae meal and oil,on growth,fatty acid composition and docosahexaenoic acid retention of orange‐spotted grouper,Epinephelus coioides

This study evaluated the effects of Aurantiochytrium spp. microalgae meal and oil as dietary docosahexaenoic acid (DHA) sources on the growth, fatty acid composition and DHA retention of orange‐spotted grouper, Epinephelus coioides. Dietary fish oil was replaced with microalgae meal or oil to provide an equal amount of DHA as a fish oil‐containing basal diet. In total, three experimental diets were fed to triplicate groups of fish (initial wt: 8.48 ± 0.06 g) in a recirculating system for 8 weeks. The weight gain and feed efficiency of the fish did not differ significantly among the experimental diets. The fatty acid composition of the whole body of the fish generally reflected the composition of their diet. The concentration of eicosapentaenoic acid in the whole body was higher in the fish fed the fish meal control diet than in those fed the two experimental diets The fish fed the control diet and those fed the diet containing microalgae oil exhibited higher DHA concentrations than did the fish fed the diet containing microalgae meal. The whole‐body DHA retention was the highest in the fish fed the diet with microalgae oil, followed by the fish fed the control diet. The lowest whole‐body DHA retention was observed in the fish fed the diet containing microalgae meal. The results suggested that the oil from Aurantiochytrium spp. microalgae can be used as DHA source for the grouper. DHA utilization by the fish was higher when the diet was supplemented with microalgae oil than with dry microalgae meal.     

GnRHa‐induced Multiple Spawns and Volition Spawning of Captive Spotted Rose Snapper,Lutjanus guttatus,at Mazatlan,Mexico

Sexual maturation and induced spawning treatments were carried out with captive spotted rose snapper, Lutjanus guttatus. A total of 3013 × 10⁶ eggs (64.7% were floating) were produced from eight treated females in 42 spawns induced with GnRHa implants during the course of the present study. GnRHa ethylene‐vinyl acetate copolymer effective doses were 204 ± 11 µg/kg in June 2005, and 224 ± 13 µg/kg in July 2005. General fertilization was 50.9 ± 34.5% and 12–14 h after spawning, viability of floating eggs was 90.4 ± 12.4%. Mean incubation period at 29–31 C was 18–20 h, and mean hatching was 94.4 ± 8.2% (73–100%). Newly hatched larvae were 2.18 ± 0.15 mm in total length (TL). One month after the last hormone experiment, previously GnRHa‐treated and untreated fish began spawning voluntarily. Hormone‐treated breeders had higher fecundity than untreated fish, producing 72.5 million eggs versus 13.9 million eggs for the untreated fish, over the following 11 mo. Combined data of volitional spawning for total egg fertilization, viability, hatching, and larval TL were 77.7 ± 1.8%, 90.3 ± 1.3%, 87.9 ± 2%, and 2.50 ± 0.12 mm, respectively. These results can ensure the sustainability of a commercial hatchery.     

Environmentally conditioned,year‐round volitional spawning of cobia (Rachycentron canadum) in broodstock maturation systems

Year‐round control of the spawning cycle of cobia (Rachycentron canadum) has been established by using water temperature manipulation. To compare the effectiveness of using this method to induce volitional spawning in cobia, two 80 m³ recirculating aquaculture systems (RAS) were used. Temperatures in one of the maturation tanks (‘Mat 1’) were maintained between 27 and 29°C for 12 months of the 15.5‐month study period. Temperatures in the second maturation tank (‘Mat 2’) were allowed to fluctuate naturally throughout the year and ranged from 20 to 32°C. A total of 101 spawning events occurred in the tanks between the spring of 2008 and the summer of 2009 (3 April 2008 to 17 June 2009). Of the 38 total spawning events in Mat 1, 17 of them (44.7% of all Mat 1 spawning events) occurred during the off‐season (fall and winter). The egg viability rates did not differ significantly (P > 0.05) between on‐ and off‐season spawns in Mat 1. Conversely, cobia broodstock exposed to natural water temperatures (no environmental manipulation) in Mat 2 followed the natural pattern of warm water (>26°C) dependence, limiting egg production to spring and summer seasons. This method of water temperature manipulation allows for effective control of the cobia reproductive cycle without compromising egg viability.     

Evaluation of Stocking Density during Second‐Year Growth of Largemouth Bass,Micropterus salmoides,Raised Indoors in a Recirculating Aquaculture System

Largemouth bass (LMB), Micropterus salmoides, are a highly desirable food fish especially among Asian populations in large cities throughout North America. The primary production method for food‐size LMB (>500 g) has been outdoor ponds that require two growing seasons (18 mo). Indoor, controlled‐environment production using recirculating aquaculture system (RAS) technologies could potentially reduce the growout period by maintaining ideal temperatures year‐round. Researchers conducted a 26‐wk study to evaluate optimal stocking densities for growout of second‐year LMB to food‐fish size in an indoor RAS. LMB fingerlings (112.0 ± 38.0 g) were randomly stocked into nine 900‐L tanks to achieve densities of 30, 60, or 120 fish/m³ with three replicate tanks per density. The RAS consisted of a 3000‐L sump, ¼ hp pump, bead filter for solids removal, mixed‐moving‐bed biofilter for nitrification, and a 400‐watt ultraviolet light for sterilization. Fish were fed a commercially available floating diet (45% protein and 16% lipid) once daily to apparent satiation. At harvest, all fish were counted, individually weighed, and measured. Total biomass densities significantly increased (P ≤ 0.05) with stocking rate achieving 6.2, 13.2, and 22.9 kg/m³ for fish stocked at 20, 60, and 120 fish/m³, respectively. The stocking densities evaluated had no significant impact (P > 0.05) on survival, average harvest weight, or feed conversion ratio which averaged 92.9 ± 5.8%, 294.5 ± 21.1 g, and 1.8 ± 0.3, respectively. After approximately 6 mo of culture, LMB did not attain target weights of >500 g. Observed competition among fish likely resulted in large size variability and overall poor growth compared to second‐year growth in ponds. Additional research is needed to better assess the suitability of LMB for culture in RAS.     

Enhanced immune responses and effectiveness of refined outer membrane protein vaccines against Vibrio harveyi in orange‐spotted grouper (Epinephelus coioides)

Vibriosis is a severe infection occurring in many commercially important marine fish species. In this study, vaccines containing Vibrio harveyi recombinant outer membrane protein K (rOmpK), outer membrane protein U (rOmpU) and rOmpK‐OmpU fusion protein in addition to the metabolizable Montanide^TM ISA 763 A VG adjuvant were developed and evaluated in the orange‐spotted grouper. The results indicate that recombinant V. harveyi protein‐based vaccines resulted in a remarkably higher expression of IL‐1β and IL‐8 at 24 hr, and greater antibody production, as early as 2 weeks postimmunization. Notably, enhanced immune responses and significant protective efficacy against V. harveyi infections were observed in the fusion protein vaccine‐injected fishes with relative per cent survival value of 81.8%. Additionally, the rOmpK‐OmpU antisera presented a high bactericidal effect on not only V. harveyi, but also Vibrio parahaermolyticus and Vibrio alginolyticus. Our results demonstrated that the fusion protein rOmpK‐OmpU was an effective vaccine candidate that exhibited potentially great versatility for controlling vibrio infections.     

The optimal dietary protein level of large‐size grouper Epinephelus coioides

An 8‐week feeding trial was conducted to determine the requirement of protein for large‐size grouper Epinephelus coioides (initial body weight: 275.07 ± 1.56 g). Six iso‐lipidic (124 g/kg) diets were formulated containing graded levels of protein (350, 400, 450, 500, 550 and 600 g/kg). Grouper was hand‐fed twice daily to apparent satiation with triplicate. The results showed that significantly high weight gain, specific growth rate and significantly low feed conversion ratio were observed in fish fed 450 g/kg protein group. High‐protein level diets significantly increased protein content and significantly decreased lipid content of fish body and muscle. Total protein and cholesterol content in serum of 600 g/kg group were significantly higher than those of 350 g/kg group. However, serum glucose and triglyceride contents of fish fed low‐protein diets were significantly higher than those of fish fed high‐protein diets. Meanwhile, liver glutamic‐pyruvic transaminase and glutamic‐oxaloacetic transaminase in high‐protein diet groups were significantly higher than those of low‐protein diet groups. The intestinal protease activity in high‐protein diet groups was significantly higher that of low‐protein diet groups, but lipase and amylase showed opposite trend. With the increasing of dietary protein level, the activities of alkaline phosphatase, superoxide dismutase and lysozyme in liver of grouper increased significantly compared with 350 g/kg group, while the activities of acid phosphatase decreased significantly. With specific growth rate as the evaluation index, the optimum dietary protein level of large‐size grouper Epinephelus coioides was 438.39 g/kg by fitting the broken‐line regression analysis.     

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.     

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.     

Induced maturation and fertilized egg production of the red‐spotted grouper,Epinephelus akaara,using adaptive physiology of photoperiod and water temperature

As a preliminary investigation into the effects of environmental factors on gonadal development using adaptive physiological characteristics, we examined the involvement of photoperiod and water temperature in the ovarian development of the red‐spotted grouper, Epinephelus akaara. During October 2014 (non‐breeding season), the photoperiod and water temperature were adjusted to 12L:12D and 18°C respectively. Every 3 weeks, daylight was increased as follows a 13L:11D and 14L:10D in the photo‐thermal treatment group, and the control group was maintained under natural conditions. After 9 weeks, water temperature was increased to 23°C in the photo‐thermal treatment group. In photo‐thermal treatment group, GSI increased significantly after 9 weeks (1.36 ± 0.19) and 12 weeks (3.31 ± 0.20) compared with the control group. At 12 weeks after photo‐thermal treatment, all fishes matured into ovaries containing late vitellogenic oocytes. The expression of FSHβ and LHβ mRNAs also increased significantly. However, in the control group, the ovarian development was maintained at an immature stage. Based on these results, we demonstrate that exposure to long photoperiod and high water temperature can induce the sexual maturation of immature red‐spotted grouper during the non‐breeding season.     

Digestibility,growth and pigmentation of astaxanthin,canthaxanthin or lutein diets in Lake Kurumoi rainbowfish,Melanotaenia parva (Allen) cultured species

New cultured ornamental fish namely Lake Kurumoi rainbowfish Melanotaenia parva (Allen) run into reduced of colour performances when reared in the aquaria, consequently, fish feed must be added with carotenoids as a pigment source. The aim of this study was to evaluate the digestibility, growth and pigmentation of astaxanthin, canthaxanthin and lutein in diet. Apparent digestibility coefficients (ADC) of dry matter, lipid, protein, carotenoids, growth and pigmentation were studied in twenty fish after 14 and 56 days of observation. The single‐dose supplementation of 100 mg/kg of astaxanthin, canthaxanthin, or lutein diets on fish was fed by apparent satiation. The basal diet without carotenoids was used as control. The result showed that the ADC of carotenoids of test diets was higher compared to control. Fish fed astaxanthin diet had higher survival rate (96.67 ± 2.89%), colour measurements of lightness (57.60 ± 7.46%), a*‐values (4.66 ± 1.20), total carotenoids content in skin (33.75 ± 5.02 mg/kg) and muscle (2.16 ± 0.74 mg/kg). Astaxanthin also increased the growth after 14 days (2.00% ± 0.19%/days) but there was no significantly different at the end of experiment. The yellowish‐orange colour performance was more rapidly achieved by fish fed astaxanthin diet after 28 days experimentation. These values suggested that dietary carotenoids were required and astaxanthin diet was superior to other diets for skin pigmentation of Lake Kurumoi rainbowfish.     

Growth performance and disease resistance against Vibrio vulnificus infection of novel hybrid grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus)

Groupers are economically important for aquaculture in Thailand. A novel hybrid grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus) has been successful cross‐bred; therefore, the present work aimed to assess the hybrid's traits. The growth performance, strength and tolerance to a pathogenic bacterial infection of this hybrid were compared with its parent species, tiger grouper and giant grouper. The results of all measured growth parameters indicated that the hybrid strain grew fastest followed by giant and tiger grouper respectively. The expressions of the growth‐related genes, insulin‐like growth factor (IGF) I and II, were also analysed in fish muscle and liver which are the main target organs in fish growth regulation. Among tested species, similar expression patterns of IGF‐I and IGF‐II were detected in both organs. The levels of these genes in liver and muscle of hybrid and giant grouper were higher than those of tiger grouper comparable with the growth manner. After challenge with Vibrio vulnificus, the immunological parameters, clearance time of Vibrio in haemolymph and survival was measured to verify the fish immunity. Leucocyte number, lysozyme activity and the ability to eliminate the pathogen were very high in hybrid and giant grouper while these parameters were lower in tiger grouper. Correspondingly, the mortality rate of tiger grouper was higher than others and % survival at the end of observation time (15 days post challenge) was lowest in infected tiger grouper. Altogether, the results suggested that the hybrid grouper has desirable traits that will improve cultured grouper.     

Gonadal recrudescence and induced spawning in Barbus altianalis

Gonadal recrudescence in Barbus altianalis from River Nile, Jinja (33°05E; 0°45N) Uganda was studied prior to induced spawning. Oocytes in B. altianalis were found to develop in cohorts suggesting batch spawning throughout the year with pronounced spawning activities coinciding with rainfall peaks of April and September. There was a strong positive correlation between rainfall and gonadosomatic index (r_S = 0.75, P = 0.008). B. altianalis had low fecundity (6.0 ± 2.0 per gram) and large‐sized eggs (2.97 ± 0.1 mm). Induced spawning was successful with exogenous hormones and water current. Results from this study suggested that total striping may not be appropriate, but larger eggs would confer high survival of offsprings under optimal conditions. The success registered in induced spawning of B. altianalis provides a breakthrough in seed production for multiplication and culture of this economically important fish species.     

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.     

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.