Diagnosis of nervous necrosis virus in orange-spotted grouper,Epinephelus coioides, by a rapid and convenient RT-PCR method

Viral nervous necrosis （VNN） causes high mortality in marine fish, especially in the grouper, worldwide and in China. Since there is no effective vaccine or drug to deal with VNN, early detection and prevention is important to block its outbreak. In this study, a reverse transcription-polymerase chain reaction （RT-PCR） was developed for the rapid, convenient, and sensitive detection of the VNN pathogen, nervous necro- sis virus （NNV）, in the grouper. The whole process was completed within 3.5 h from the RNA extraction to PCR product visualization. The detection limit of this method was 200 copies of NNV RNA standard, which corresponded to 200 copies of virus particles. This RT-PCR method was specific to the NNV detection with no cross-reactivity to other fish viral disease pathogens, such as infectious pancreatic necrosis virus （IPNV）, infectious hematopoietic necrosis virus （IHNV）, spring viraemia of carp virus （SVCV）, epizootic haematopoietic necrosis virus （EHNV）, and large yellow croaker iridovirus （LYC1V）. With this method, the orange-spotted grouper （Epinephelus coioides） fry from hatcheries with or without incidence of the VN- N epidemic in Fujian Province were detected. The results showed that all or 93% of the fry from the two hatcheries with incidence of the epidemic were diagnosed as positive, while 40% or 25% of fry from the t- wo hatcheries without the VNN epidemic were also detected as NNV positive, indicating that this RT-PCR method can be used for rapid, sensitive detection of NNV infection and applied in the VNN epidemic alert.

A Preliminary Study on Fertilization Biology between Epinephelus coioides(♀) and Epinephelus lanceolatus(♂)

Crossbreeding between Epinephelas coioides （ ♀ ） and Epinephelus lanceolatus （♂） was conducted by artificial insemination. Fertilized eggs were col- lected at 0, 30, 90 s, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 min post-insemination respectively, which were fixed by Smith＇s solution, embedded by paraffins and stained by H.E. According to the characteristics of Epinephelus coioides eggs, tissue section method was modified. The results showed that the sperms of Epinephelus lanceolatus rapidly entered the eggs of Epinephelus coioides at 30 s - 1 rain post-insemination. Observation results of tissue sections showed that mature eggs of Epinephelus coioides remained at metaphase of secondary maturation division. The eggs were activated after sperm penetration into the egg. With the develop- ment of secondary maturation division, at 2 rain post-insemination, eggs reached the metaphase of secondary maturation division ; at 3 - 6 rain post-insemination, sperm asters appeared; at 5 min post-insemination, eggs extruded secondary polar body; at 7 -15 rain post-insemination, male pronucleus and female pronucleus moved closer to each other and fused finally, forming a clear junction line; subsequently, zygote nucleus formed and karyotheca became faint; at 15 min pest-insem- ination, first karvokinetic division was develoued.

Identification of Epinephelus malabaricus and Epinephelus coioides using DNA markers

Using multi-molecular marker technologies and based on morphological criteria, the genetic relationship between Epinepheelus malabaricus and E, coioides was examined in the hope of resolving the long-standing issue of identifying these two species. Results showed that： （ 1 ） E. coioides and E. malabaricus should be identified as two species, the consistency of mitochondrial DNA cytochrome b gene sequence between E. malabaricus and E. coioides is 94.4%, and the genetic similarity by AFLP was 0.753 9 ; （2） Hybridization exists between E. malabaricus and E. coioides, the specific RAPD and AFLP fragments are found to be useful in the identification of these two species, and the genetic properties （both with exterior and inheritance） of hybrid is significantly biased to the male parents ; and （3） AFLP was a potentially powerful tool in constructing the genetic linkage map for these two groupers.

Growth performance,fatty acid composition,and lipid metabolism are altered in groupers(Epinephelus coioides)by dietary fish oil replacement with palm oil

In this study,we conducted a 56-d feeding trial to investigate the effects of replacing the fish oil(FO)with palm oil(PO)on the performance,tissue fatty acid(FA)composition,and mRNA levels of genes related to hepatic lipid metabolism in grouper(Epinephelus coioides).Five isolipidic(13%crude lipid)and isonitrogenous(48%CP)diets were formulated by incrementally adding PO to the control diet(25%fish meal and 9%added FO)to replace FO in the control diets.Triplicate groups of 30 groupers(initial weight:12.6±0.1 g)were fed one of the diets twice daily,to apparent satiety.The replacement of FO with 50%PO revealed maximum growth without affecting the performance and whole-body proximate compositions,and replacing FO with 100%PO revealed a comparable(P>0.05)growth with that of the control diet,suggesting PO as a suitable alternative to FO.The analysis of FA profiles in the dorsal muscle and liver though reflected the FA profile of the diet,PO substitutions above 50%could compromise(P<0.05)the FA profile in the liver and flesh of the fish species in comparison with the control diet.Furthermore,the mRNA levels of FAS,G6PD,LPL,PPARА,and D6FAD genes in the liver had positive linear and/or quadratic responses,but the SCD,HSL,ATGL,FABP,SREBP-1C and ELOVL5 had the opposite trend,with increasing dietary PO inclusion levels,whereas the mRNA level of ACC was not affected by dietary treatments.The optimal level of PO substitution for FO was estimated to be 47.1%of the feed,based on the regression analysis of percent weight gains against dietary PO inclusion levels;however,it might affect the FA profile in the liver and flesh of the fish species,and further study is required to investigate whether the changes in tissue FA composition will affect the welfare and market value over a production cycle of grouper.

Immunity,feed,and husbandry in fish health management of cultured Epinephelus fuscoguttatus with reference to Epinephelus coioides

Groupers are dispersed worldwide in the tropical and subtropical waters.They are prized in the live reef fish trade,making them candidates with high market value and consumer demand.In the Asian-Pacific region,the brown-marbled grouper(Epinephelus fuscoguttatus)is widely adapted as an aquaculture species.However,health management remains a major concern in the stressful intensive culture process.The present review discusses techniques and current knowledge in the cultivation of E.fuscoguttatus towards healthy fish growth focusing on aspects of immunity,feed,and husbandry.Understanding how the fish immune system responds during infections provides insights into the intricate ways fish resist pathogens.This information is helpful when developing vaccination strategies or immunostimulant compounds to strengthen fish immunity.Feeds that are formulated according to the needs of the fish ensure optimal growth and using suitable alternative ingredients may lower production cost without compromising fish health.Good husbandry practices contribute to a favourable environment for the fish to grow,while interspecific hybridization may be a convenient approach to generate hardier species.Nevertheless,it has been noted that research in E.fuscoguttatus is relatively scarce in contrast to the closely-related orange-spotted grouper(E.coioides),which is used in the present article as a reference.Ultimately,the identified gaps in knowledge between the two species warrant species-specific research in E.fuscoguttatus to promote fish health and ensure continued success in aquaculture.

两种重要经济石斑鱼肠道微生物群落结构及代谢功能的比较分析

为揭示虎龙杂交斑(Epinephelus fuscoguttatus♀×E.lanceolatus♂)和斜带石斑鱼(Epinephelus coioides)肠道微生物群落的结构及功能差异,利用16S rDNA高通量测序技术对其肠道微生物群落进行分析。结果表明:(1)虎龙杂交斑肠道微生物群落以变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和放线菌门(Actinobacteria)为主,斜带石斑鱼中以变形菌门、梭杆菌门(Fusobacteria)和厚壁菌门为主。(2)在属水平,虎龙杂交斑和斜带石斑鱼肠道中均含有高丰度的弧菌属(Vibrio)。鲸杆菌属(Cetobacterium)、发光杆菌属(Photobacterium)和丙酸菌属(Propionigenium)是斜带石斑鱼肠道中的标志微生物,而虎龙杂交斑肠道中的标志微生物为棒状杆菌属1(Corynebacterium 1)、变形杆菌属(Proteus)、葡萄球菌属(Staphylococcus)和类香菌属(Myroides)。(3)网络分析显示,变形菌门在两种石斑鱼肠道中均占主导地位。(4)功能预测表明,虎龙杂交斑肠道微生物群落中抗坏血酸与糖醛酸代谢、植物-病原体相互作用和胰岛素信号通路显著高于斜带石斑鱼,斜带石斑鱼肠道微生物群落的功能在万古霉素类抗生素和链霉素生物合成、亚油酸代谢、双酚降解以及类黄酮生物合成的相关途径中显著富集。研究结果有望加深对石斑鱼肠道微生物群落的认识,为选择合适的石斑鱼养殖品种,提高石斑鱼养殖产业的经济效益提供科学参考。

Protective immunity of orange-spotted grouper(Epinephelus coioids) against a nervous necrosis virus isolated from China,and determination of the complete sequences of the virus

On the basis of the sequence and analysis of genome from the orange-spotted nervous necrosis virus（ OGNNV）, China strain, a pair of special primers were designed according to the nucleotide sequences of RNA2 from OGNNV. The major capsid protein （ MCP）gene of OGNNV was cloned by means of reverse-transcriptase polymerase chain reaction （RT-PCR） and ligated into the pET32a expression plasmid. The MCP gene of OGNNV was 1 017 bases, encoded a protein of 338 amino acid with a molecular mass of 37.1 kDa. Recombinant protein with a molecular mass of 57.4 kDa was expressed in E. coli BL21 （DE3）. Vaccine was prepared from the recombinant protein expressed in recombinant cells. The juvenile orange-spotted groupers （8 cm in average length） were immunized by intraperitoneal injection. Group A was challenged with infected tissue filtrates 25 d post-vaccination. The mortality in the vaccined group （ A1,30% ） was a little higher than the unvaccined group （ B2, 27.8% ）. Group B was challenged after three vaccine injections. The mortality in the vaccined group （B1, 16.7% ） was lower than the unvaccined group （132, 27.8% ）, And the relative percentage survival （RPS） value of vaccined group, compared with the unvaccined group, was 40%. The anti-recombinant protein sera with a 1 ： 100 dilution were mixed with double volume of infected tissue filtrates and incubated at 4 ℃ for 12 h and then intramuscularly injected into the juvenile orange-spotted grouper. Treatment of infected tissue filtrates with anti-recombinant protein serum resulted in a significantly lower mortality of fish （ Group C1, mortality of 18.18% ）, compared with the fish （ Group C2, mortality of 40% ） which received infected tissue filtrates treated with control serum. Results implied the potential use of the capsid protein in immunization against OGNNV.

斜带石斑鱼mstn对其肌肉细胞增殖分化及相关基因表达的影响

斜带石斑鱼Epinephelus coioides是具有较高经济价值的海水鱼,如何促进石斑鱼生产性状改良一直是科学研究的重点之一。肌肉生长抑制素(mstn,myostatin)是调节肌肉生长的重要转化因子。本研究中,Mstn的亚细胞定位表明Mstn在斜带石斑鱼细胞质中呈点状分布。利用酶标仪测定被Mstn重组蛋白刺激肌肉原代细胞1~5 d后的A值,发现实验组细胞生长明显减缓,随着处理时间的增加,细胞活性差异进一步扩大。利用流式细胞仪测定被Mstn重组蛋白刺激的肌肉原代细胞细胞周期,发现实验组细胞周期受阻。用Mstn重组蛋白对肌肉原代细胞进行刺激,通过RT-qPCR检测Mstn重组蛋白对mstn信号通路及下游相关基因的影响,结果表明随着处理浓度的升高,p21、smad3和mrf4 mRNA的表达量也逐渐而上升,其中,smad3和mrf4表达量的上升不存在显著性差异;当处理浓度为100和1000 nmol/L时,p21存在显著性差异;随着处理浓度的升高,myod和myog mRNA的表达量均下降,但myod mRNA下降趋势不显著,myog mRNA显著下降,当处理浓度为100和1000 nmol/L时存在显著性差异。本研究揭示了Mstn定位于斜带石斑鱼GS细胞质;Mstn重组蛋白上调smad3、mrf4的表达,下调myod、myog的表达,来促进细胞的分化;同时Mstn重组蛋白能上调p21的表达,抑制细胞的增殖。本研究验证了Mstn在肌肉发育过程中的生物学功能,为后续深入开展mstn对鱼类肌肉发育的影响研究奠定了基础。