Conditioning,maturation and year‐round natural spawning of orange‐spotted grouper,Epinephelus coioides (Hamilton, 1822), in recirculating aquaculture system

The present experiment was aimed at studying the conditioning, maturation and natural spawning of orange‐spotted grouper, Epinephelus coioides, in a recirculatory aquaculture system (RAS). Thirty fish (n = 30; 3.35 ± 0.05 kg) were stocked in a circular tank of 125 m³ capacity fitted with an RAS for conditioning and broodstock development. After 15 days, 15 fish were implanted with 17 α methyl testosterone and letrozole at the rate of 5 mg and 0.2 mg/kg body weight, respectively, for conversion from female to male. The gonadal development started after 1 month, and by 90th day, 63.53 ± 3.78% and 2.07 ± 0.84% of the oocytes attained a size of 400–500 μm and 500–600 μm respectively. Natural spawning commenced in the RAS from 4th month onward after stocking and spawning continued round the year. The spawning pair showed courtship behaviour with a typical vertical burst of swimming just before release of gametes. The total number of eggs spawned during 1 year was 47.23 million with spawning frequency varying form 5 to 13 times per month. The association of spawning events with new moon day (lunar cycle) weakened as time progressed. The mean monthly fertilization and hatching rates varied from 77.80 ± 3.34% to 83.70 ± 1.76% and 82.80 ± 4.21% to 88.33 ± 1.39% respectively. The study proved that RAS is an efficient system that provides a stable, controllable and conducive environment for year‐round natural breeding of orange‐spotted grouper.     

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.     

Targeted Sequencing of Myogenic Regulatory Factors and Myostatins Reveals an Association between MSTN‐1 and Interorbital Distance in Orange‐spotted Grouper,Epinephelus coioides (Hamilton, 1822)

The aim of this study was to investigate single nucleotide polymorphisms (SNPs) in genes that are presumed to control muscle growth and to determine their potential association with growth in a cultured population of orange‐spotted grouper, Epinephelus coioides. Seven genes, myogenic regulatory factor 4 (MRF4), Myf5, MSTN‐1, MSTN‐2, MyoD1, MyoD2, and myogenin, were selected for the investigation, covering approximately 26 kbp. First, the two clades for the genes MSTN (myostatin) and MyoD were confirmed in this species using Bayesian inference analysis of the phylogenetic relationships. Then, the seven genes were enriched by polymerase chain reaction and sequenced using an Ion Torrent Personal Genome Machine. A total of 586 SNPs were discovered. Linkage disequilibrium was decayed by 50% within 250 bp based on the combined data, which means that there was high resolution in the association mapping. A mixed linear model considering the population structure and kinship was used to detect the associations between genotypes and phenotypes. Only one site (KR269814.1:g.22T>G) in MSTN‐1 was found to be significantly associated with a measured trait, the interorbital distance (false discovery ratio < 0.05), and it explained 12.4% of the phenotype variation of this trait. This study provides insight on strategies for molecular marker‐assisted breeding in orange‐spotted grouper.     

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 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.     

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.     

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.     

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.     

Comparative analysis of the predominant carotenoids and chemical components in the common and orange‐muscle mutant of Haliotis gigantea

The Xishi abalone (Haliotis gigantea) is an economically significant aquaculture species in southern China. We identified a novel orange‐muscle mutation present in less than 2% of the cultured population. High‐performance liquid chromatography and electrospray ionization mass spectrometry identified zeaxanthin and β‐carotene as the two main pigments present in the orange‐muscle abalone. The concentrations of zeaxanthin and β‐carotene detected in the orange‐muscle abalones were significantly higher than those detected in the common abalones (p < 0.01). Notably, the concentration of zeaxanthin was approximately 16.5 times higher in orange‐muscle abalones than in common abalones, which was also the species with highest carotenoid content reported in shellfish to date. Furthermore, the contents of total amino acids and essential amino acids in the orange‐muscle abalone were higher than that in the common abalone, though not significantly, while the content of non‐essential amino acids was significantly higher in the orange‐muscle abalone than that in the common abalone (p < 0.05). The saturated fatty acid content in the orange‐muscle abalones was slightly lower than that in the common abalones, and the unsaturated fatty acid content was higher in the orange‐muscle abalones than that in the common abalones. These results could provide a basis for characterizing the mechanism by which carotenoids accumulate in abalones and further guide the breeding of abalone with orange‐muscle mutants in the future.     

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.     

Effects of dietary levan on growth performance,nonspecific immunity,pathogen resistance and body composition of orange‐spotted grouper (Epinephelus coioides H.)

This study investigated the effects of levan produced by Bacillus licheniformis FRI MY‐55 on growth performance, intestinal count of viable bacteria, immune status, pathogen resistance and body composition of orange‐spotted grouper (Epinephelus coioides). Orange‐spotted grouper were fed diets supplemented with levan at concentrations of 0 (control), 5.0, 10.0, 25.0 and 50.0 g kg^?1 for 12 weeks. The final weight and per cent weight gain were significantly higher in the 25.0 g kg^?1 levan‐supplemented group than in the control group (p < 0.05). All levan‐supplemented diets significantly decreased the count of total viable aerobic bacteria and Vibrio spp. in the intestines of groupers (p < 0.05). Serum total protein levels, globulin levels, lysozyme activity and survival rate of orange‐spotted grouper after challenge with V. harveyi were significantly higher in the 25.0 g kg^?1 levan‐supplemented group than in the control group (p < 0.05). This study also found that the 10.0, 25.0 and 50.0 g kg^?1 levan‐supplemented diets significantly increased the crude protein level in the body composition of orange‐spotted grouper. Overall, the results of this study indicate that dietary levan (25.0 g kg^?1) could be an effective method for enhancing the growth performance and disease resistance in orange‐spotted grouper.     

Polymorphisms of the growth hormone gene in domesticated red sea bream populations (Pagrus major) based on minisatellite genotypes and nucleotide sequences

The genetic diversity of the growth hormone gene in domesticated red sea bream (pmaGH) was evaluated using a minisatellite DNA marker located in intron 3 (pmaGH22) and nucleotide sequences. The number of alleles of pmaGH22 was largely decreased in domesticated strains of red sea bream, and the possibility of selection pressures was also detected based on the analysis of the Hardy–Weinberg equilibrium in some strains. However, each strain inherited a small number of alleles of pmaGH22, and the entire domesticated population (combining all strains) showed a large number of alleles (n = 17), similar to the allelic richness of the wild population (n = 18.5). Based on nucleotide sequencing analysis, three synonym mutations were found in the coding regions, and also several SNPs and indels were found in the noncoding regions. In addition, four genealogies of growth hormone haplotypes were identified based on principal coordinate analysis, and these genealogies of pmaGH partly reflected allele size ranges of pmaGH22. Several haplotypes shared alleles of pmaGH22, and also fragment size homoplasy in pmaGH22 was suspected. These alleles of pmaGH22 and the haplotypes will be a useful indicator for divergence of pmaGH and for broodstock individual selection with minimum inbreeding effect.     

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%.     

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.     

Microbial communities associated with early stages of intensively reared orange‐spotted grouper (Epinephelus coioides)

The gut microbiota plays key roles in the health and general welfare of fish larvae, the present study characterized the bacterial communities associated with grouper Epinephelus coioides larvae during a period of 22 days post hatch (DPH) in an intensive hatchery using both cultivation‐based and cultivation‐independent approaches. Both approaches confirmed that bacteria were present in the gut of larvae before and after the onset of exogenous feeding, and the number of cultiviable bacteria increased gradually from 2 DPH to 22 DPH. A more complex bacterial profile was present in larvae fed fertilizer oyster eggs for 4 days (8 DPH), probably as a result of the onset of exogenous feeding. Interestingly, similar internal microbiota were observed in larvae fed fertilized oyster eggs for 4 days (8 DPH) and rotifers for 2 weeks (22 DPH), although different microbial communities were present in the two feeds. This might suggest that the gut environment of E. coioides larvae selects for a common microbiota, which is more closely related with the rearing water than the two feeds. Therefore, bacterial community of the rearing water may play a critical role in the establishment of gut microbiota of fish larvae and more attention should be paid to its practical modulation by using probiotics. In addition, some potentially beneficial bacteria, such as Lactococcus spp., were the major components of the microbiota associated with fertilized oyster eggs, while these bacteria were not detected in larvae samples.