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.     

Effects of different dietary carbohydrate‐to‐lipid ratios on growth,plasma biochemical indexes,digestive and immune enzymes activities of juvenile orange‐spotted grouper Epinephelus coioides

An 8‐week feeding trial was conducted to investigate the effects of dietary carbohydrate‐to‐lipid (CHO:L) ratios on growth, liver and muscle glycogen content, haematological indices, and liver and intestinal enzyme activity of juvenile grouper (Epinephelus coioides). Five isonitrogenous (496.0 g/kg protein) and isoenergetic (21.6 KJ/g gross energy) diets with varying CHO:L ratios of 0.65 (D1), 1.31 (D2), 2.33 (D3), 4.24 (D4) and 8.51 (D5), respectively, were fed to triplicate groups of 20 fish (average 10.02 ± 0.1 g) for 8 weeks. Results showed that the weight gain rate, specific growth rate and protein efficiency ratio (PER) of juvenile grouper increased first and then decreased with the increase in CHO:L ratio, reaching a maximum value in the D4 (CHO:L = 4.24) diet. The trend for the feed conversion ratio was opposite to the PER. Along with the diet CHO:L ratios, the apparent digestibility coefficients (ADC) of crude lipid and energy for the juvenile groupers decreased gradually, while the ADC of dry matter, liver and muscle glycogen level increased gradually. Total protein, triglycerides and cholesterol in serum were all maximized in the D4 diet and glucose in the D5 (CHO:L = 8.51) diet. Digestive enzyme activity in the intestine was significantly affected by dietary CHO:L ratio. Liver hexokinase, phosphofructokinase and glucose‐6‐phosphate dehydrogenase activity increased significantly as CHO:L ratio increased. Liver lysozyme and acid phosphatase activity in the groupers fed the D3 (CHO:L = 2.33) diet was significantly higher than that of other diets. Liver fructose‐1,6‐bisphosphatase and alkaline phosphatase activity reached a maximum value in the D4 diet and was significantly higher than that in the D1 diet. Taking the above results together, it can be concluded that an optimal dietary CHO:L ratio of 2.33 is suitable for grouper culture concerning growth performance and health.     

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.     

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.     

Gill damage to juvenile orange‐spotted grouper Epinephelus coioides (Hamilton, 1822) following exposure to suspended sediments

Juvenile Epinephelus coioides were exposed to three nominal concentrations (8, 32, 128 mg wet sediments L^?1) of suspended sediments (SS) from Port Shelter (PS), Mirs Bay (MB) and Victoria Harbour (VH) for 10 and 30 days using semi‐static system. Sediments from VH contained higher concentrations of Cu and polycyclic aromatic hydrocarbons (PAHs) than sediments from PS and MB. Gill damages including lamellar blood sinus dilation and vascular congestion were prevalent after just 10 days of exposure to SS. Fish exposed to SS at the highest concentration of 128 mg L^?1 showed higher incidence of lamellar aneurism. Hyperplasia in the base of lamellae was recorded in fish that had been exposed to contaminated SS from VH. Significant increases in the density of chloride cells and mucous cells were found on the gills of fish that had been exposed to 128 mg L^?1 of SS from PS. Clogging of gills by SS produced hypoxic‐like responses in fish. Polluted sediments from VH produced addictive or synergistic effects between SS and chemical contaminants on fish.     

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.     

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.     

Iron bioavailability of different sources in juvenile grouper Epinephelus coioides

A 70‐day experiment was implemented to study the iron (Fe) bioavailability of three Fe sources in Epinephelus coioides (initial weight, 12.19 ± 0.14 g). Treatments consisted of 0, 50, 90, 130, 170 and 210 mg supplemental Fe/kg from iron sulphate (FeSO₄), ferric citrate (Fe‐citrate) and hydroxy methionine analogue iron (Fe‐MHA). The results showed that the growth performance was not affected by Fe level regardless of Fe sources. Hepatic catalase activity (CAT), the haematocrit value (Hct), haemoglobin (Hb), mean corpuscular volume (MCV) and Fe content in whole body or liver firstly increased significantly and then plateaued as Fe level increased regardless of the Fe source, except significant decreasing of Hct in 210 mg Fe/kg group from FeSO₄. Manganese (Mn) content decreased significantly and then kept stable in the whole body and liver whatever the source was. The slope‐ratio method using FeSO₄ as the reference and Hb content, CAT activity, body and hepatic Fe concentration as the response parameter showed the average relative bioavailability of Fe‐citrate and Fe‐MHA was 103.25 ± 4.35 (n = 4) and 134.5 ± 15.67 (n = 4), respectively, with the maximum value of Fe‐MHA in any case. In conclusion, Fe‐MHA was the most effective to meet preferable haematological index, CAT activity and mineral deposition, with little difference between Fe‐citrate and FeSO₄, athough Fe levels with different sources failed to influence the growth in 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 live or heat‐inactivated autochthonous Bacillus pumilus SE5 on growth performance,immune responses and immune gene expression in grouper Epinephelus coioides

To evaluate the possible dietary application of live and heat‐inactivated probiotic Bacillus pumilus SE5 in grouper Epinephelus coioides, juveniles (14.6 ± 0.2 g) were fed either a basal control diet (without probiotic) or the basal diet supplemented with 1.0 × 10⁸ CFU g^?1 live (T1) and heat‐inactivated B. pumilus SE5 (T2). The heat‐inactivated probiotic significantly improved the final weight, weight gain (WG) and specific growth rate (SGR) at day 60 and significantly decreased the feed conversion ratio (FCR) at day 30 and 60, while the viable probiotic significantly decreased the FCR at day 60 (P < 0.05). Phagocytic activity, serum complement C3 and IgM levels as well as SOD activity elevated significantly in fish fed the heat‐inactivated probiotic for 60 days (P < 0.05). Furthermore, the heat‐inactivated probiotic remarkably up‐regulated expression of TLR2 and pro‐inflammatory cytokines (IL‐8 and IL‐1β) in head kidney (P < 0.05), but the viable probiotic failed to do so. These results indicated that heat‐inactivated B. pumilus SE5 can effectively improve the growth performance and immune responses of E. coioides.     

Investigations on zinc bioavailability of different sources and dietary zinc requirement in juvenile grouper Epinephelus coioides

An experiment was implemented to study the effect of dietary zinc levels from three zinc sources on growth performance, immunity, and tissue mineral elements deposition on juvenile grouper. Based on the zinc free feeds, dietary zinc levels of 0, 10, 20, 40, 80, and 160 mg/kg were added from zinc sulphate (ZnSO₄), tetrabasic zinc chloride (TBZC), and 2‐hydroxy‐4‐(methylthio) butyrate zinc (Zn‐HMB). The results of the 8‐week study indicated that the weight gain rate (WGR) and specific growth rate (SGR) at level 10 mg/kg were significantly higher than the control fed and the reached a plateau until level 80 mg/kg, when the sources were ZnSO₄ and Zn‐HMB. WGR and SGR decreased significantly at level 160 mg/kg compared to 80 mg/kg in group TBZC. Feed coefficient (FCR) of all zinc source groups showed trend of decrease before rising. The alkaline phosphatase (AKP) in serum, catalase (CAT), total superoxide dismutase (SOD), copper‐zinc superoxide dismutase (Cu‐Zn SOD) in liver and zinc concentrations in whole body and vertebrae increased significantly than those in control group and then kept steadily in all treatments. Regardless of the sources, the manganic content of whole body or vertebra showed a downtrend. Based on the WGR, the dietary zinc requirements in juvenile grouper were 52.94 mg/kg from ZnSO₄. The average relative bioavailability (RBV) of TBZC and Zn‐HMB was 169.33% and 200.33%, respectively, compared to ZnSO₄.     

The effects of the tank colour on growth performance and physiological responses in fingerling grouper,Epinephelus coioides

In this study, the effects of different background colours on growth performance and physiological parameters of Epinephelus coioides were investigated. One hundred eighty E. coioides (average weight 18.16 ± 0.07 g) were distributed in black, white and blue 300‐L tanks and fed to satiation for 60 days. The tank colour had no significant effect on the weight gain, condition factor, hepatosomatic index and specific growth rate (p > .05). The lowest amount of viscerosomatic index and the highest carcass protein were recorded in the white tank (p < .05). Daily food intake, feed conversion ratio and plasma glucose were significantly higher in the blue tank (p < .05). The highest levels of haematocrit and plasma triglyceride were observed in the white tank, while the total protein and albumin were higher in the black tank compared to the other tanks. The tank colour had no significant effects on the plasma cortisol level (p > .05). Our findings showed that colours used in this study did not evoke stress and thus had no effect on growth, but some metabolic adaptation occurred in each colour to achieve optimum growth.     

PCR‐DGGE analysis of the autochthonous gut microbiota of grouper Epinephelus coioides following probiotic Bacillus clausii administration

The autochthonous microbiota in the foregut, midgut and hindgut of juvenile grouper Epinephelus coioides following the dietary administration of probiotic Bacillus clausii for 60 days were assessed using polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE). A complex and generally similar bacterial composition along the digestive tract of E. coioides was detected in the DGGE profiles, while several bacteria showed regional specialization. Similarity dendrogram revealed that the bacterial composition of the foregut was more similar to the midgut than the hindgut. Samples collected from the probiotic group and the control group showed generally similar DGGE patterns, while no significant difference in the total number of bands and Shannon index were observed between the probiotic group and the control group, suggested that probiotic B. clausii exerted no significant effect on the gut microbiota of E. coioides. However, various potentially beneficial bacteria, such as Enterococcus sp.‐like and Bacillus pumilus‐like bacterium were selectively stimulated by probiotic B. clausii, while some potential harmful species, like Staphylococcus sp.‐like and Vibrio ponticus‐like bacterium were depressed. These indicated that the gut microbiota was modified to some degree by probiotic B. clausii. Sequences analysis showed that the autochthonous gut bacteria of E. coioides could be classified into four groups, i.e. Proteobacteria, Firmicutes, Actinobacteria and unclassified bacteria.     

The protein requirement of grouper Epinephelus coioides at grow‐out stage

The present study was conducted to investigate the protein requirement of grouper Epinephelus coioides at grow‐out stage (initial weight of 102.8 ± 1.02 g). Six iso‐lipidic diets were formulated using white fish meal and casein as protein sources with graded levels of protein (350, 400, 450, 500, 550 and 600 g/kg). Grouper was hand‐fed twice daily to apparent satiation in triplicates for 8 weeks. The results showed that lack of protein will lead to the declined of weight gain and specific growth rate and suitable protein not only improved growth, but also reduced feed coefficient rate. In addition, high‐protein level diet significantly decreased the morphological index of grouper. For the body proximate composition analysis, the high‐protein diets (500, 550, 600 g/kg) significantly increased the protein contents in the whole body and muscle which was contrary to as observed for the lipid content. High‐protein diets significantly improved the total protein levels of plasma which were contrast to as observed in triglyceride contents. Moreso, the cholesterol content was observed to be significantly decreased after high‐protein diet supplementation. The intestinal protease activity was observed to increase significantly with increasing protein supplementation whereas a decreasing trend was observed for the lipase and amylase activity with the highest going for the groups fed diets containing 350 g/kg protein as compared to the others. The liver alkaline phosphatase, superoxide dismutase and lysozyme activity increased with increasing protein level which later decreased. On the other hand, the acid phosphatase activity showed a significant downward trend. Based on SGR, the broken‐line regression analysis showed that the optimum dietary protein level and digestible protein level of the grouper Epinephelus coioides at grow‐out stage were 466.65 and 395.79 g/kg, respectively.     

Seasonal patterns in the community of gill monogeneans on wild versus cultured orange‐spotted grouper,Epinephelus coioides Hamilton, 1822 in Daya Bay,South China Sea

Epinephelus is among the most important fish genera in the Southeast Asian Pacific coastal systems. In Chinese coast, the orange‐spotted grouper Epinephelus coioides is one of the most significant species based on culture volume, and it is consumed all over the world. Very little information exists on this species’ parasitofauna. Composition and temporal variation in the community structure of the gill monogeneans on wild versus cultured E. coioides from Daya Bay (23°25′N, 117°2′E), South China Sea were determined using seasonal samples taken between April 2008 and January 2009. Eight species of monogeneans of three families and four genera on the gills of E. coioides were found: Neobenedenia melleni, Haliotrema cromileptis, Diplectanum grouperi, Pseudorhabdosynochus justinei, P. lantauensis, P. coioidesis, P. serrani and P. shenzhenensis. Eight of the monogenean species exhibited seasonal variation in their infection dynamics associated with environmental changes during seasons. The variations in the infection dynamics generated changes in the community structure during the sampling periods. Moreover, in the three different host living environments (wild, monocultured and polycultured), the monogenean communities exhibited the different seasonal patterns.     

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.     

Supplementation of heat‐inactivated Bacillus clausii DE5 in diets for grouper,Epinephelus coioides,improves feed utilization,intestinal and systemic immune responses and not growth performance

In recent years, more and more attentions have been paid to the development and application of probiotics in aquaculture, and viable probiotics have been extensively studied, while rare information was available about inactivated probiotics in aquaculture. Therefore, in this study, a feeding trial was designed to investigate the effect of dietary supplementation of heat‐inactivated probiotic Bacillus clausii DE5 on growth performance, immune response and key immune genes expression in head kidney and intestine in grouper Epinephelus coioides. Fish were fed for 60 days with control diet (C) and two experimental diets containing 1.0 × 10⁸ CFU/g live (T1) and heat‐inactivated (T2) B. clausii DE5, respectively. The probiotic treatments did not affect the final weight, weight gain (WG) and specific growth rate (SGR) of E. coioides at days 30 and 60 (p > .05), while both heat‐inactivated and live B. clausii DE5 significantly decreased the feed intake and feed conversion ratio (FCR) at day 60 (p < .05). Serum lysozyme activity and complement C3 level in the two probiotic treatments were significantly higher than those in the control (p < .05). The lysozyme activity and complement C3 level at day 60 were significantly higher than those at day 30 (p < .05), while no significant interaction effect between diet and administration date was observed. Moreover, the heat‐inactivated B. clausii DE5 significantly improved the expression of TLR5, pro‐inflammatory cytokines (IL‐8 and IL‐1β) and TGF‐β1 in head kidney and intestine (p < .05), while the live probiotic did not show any significant effect on the expression of these key immune‐related genes in head kidney and intestine. These results indicate that dietary supplementation of heat‐inactivated B. clausii DE5 effectively improved feed utilization and both the local and systemic immune responses of E. coioides.