A novel heart‐cell line from brown‐marbled grouper Epinephelus fuscoguttatus and its susceptibility to iridovirus

A novel cell line (bmGH) was established from the heart of brown‐marbled grouper Epinephelus fuscoguttatus and its viral susceptibility was evaluated. The bmGH cells have been subcultured to passage 65 in Dulbecco's modified eagle medium:Ham's nutrient mixture F‐12 (1:1) medium (DMEM/F12) which was further supplemented with foetal bovine serum (FBS), carboxymethyl‐chitosan, basic fibroblast growth factor (bFGF) and insulin‐like growth factor‐I (IGF‐I) at 24° C. The heart cells have a fibroblastic morphology and proliferated to confluence 14 days later. The cells grew at a steady rate during subsequent subculture and had a population doubling time of 40·3 h at passage 60. Karyotype analysis showed that these cells exhibited chromosomal aneuploidy with a modal chromosome number of 48. The results of viral susceptibility characterization revealed that cytopathic effects (CPE) of bmGH cells appeared after infection by two iridoviruses, turbot reddish body iridovirus (TRBIV) and lymphocystis disease virus (LCDV). A large number of TRBIV and LCDV particles were also observed in the infected bmGH cells by electron microscope examination. All of these facts indicate that the bmGH cell line established here may serve as a valuable tool for studies of cell‐virus interactions and has potential applications in fish virus isolation, propagation and vaccine development.     

石斑鱼垂体腺苷酸环化酶激活多肽和生长激素释放激素基因的cDNA克隆及表达分析

垂体腺苷酸环化酶激活多肽 (PACAP)和生长激素释放激素 (GHRH)均属于血管活性肠肽家族成员 ,且两者前体基因在脊椎动物的鸟类、两栖类、鱼类中由同一基因编码 ,而哺乳动物是由两个不同基因编码。已有几例关于鱼类编码PACAP和GHRH基因克隆的报道 ,而关于重要海水养殖鱼类石斑鱼的PACAP和GHRH基因未见报道。克隆了PACAP GHRH前体cDNA序列 ,该前体有两种剪接方式 ,包括一个长序列和一个短序列 ,其中长序列编码PACAP和GHRH ,短序列缺失 10 5个碱基 ,仅编码PACAP而缺失编码GHRH的外显子区 ,同样情况在虹鳟和沟鲶中也有报道。通过半定量RT PCR方法对石斑鱼PACAP GHRH前体mRNA在胚胎发育和发育早期以及各部位的表达情况进行了分析。胚胎发育分析结果表明 ,从神经胚期开始 ,PACAP GHRH前体mRNA大量表达 ,提示该蛋白质在神经发育或神经营养方面具有重要作用。PACAP GHRH前体基因在中枢系统的表达量远高于外周组织。在鱼类的眼和鳃发现PACAP GHRH前体分布。     

Dietary sodium alginate administration affects fingerling growth and resistance to Streptococcus sp. and iridovirus, and juvenile non-specific immune responses of the orange-spotted grouper, Epinephelus coioides

The percent weight gain (PWG) and feeding efficiency (FE) of fingerling orange-spotted grouper, Epinephelus coioides, fed diets containing sodium alginate at 1.0 and 2.0 g kg(-1) were calculated on the 2nd, 4th, 6th, and 8th weeks after feeding. Survival rates of the fingerling grouper against Streptococcus sp. and an iridovirus, and non-specific immune parameters such as alternative complement activity (ACH50), lysozyme activity, natural haemagglutination activity, respiratory bursts, superoxide dismutase (SOD) activity, and phagocytic activity of juvenile grouper were also determined when the fish were fed diets containing sodium alginate at 0.5, 1.0, or 2.0 g kg(-1). The PWG and FE of fish were better when the fish were fed diets containing sodium alginate at 1.0, and 1.0 and 2.0 g kg(-1), respectively. The PWG and FE of fish fed the 0, 1.0 and 2.0 g kg(-1) sodium alginate-containing diets after 8 weeks were 271.0%, 454.4% and 327.8%, and 0.61, 0.72 and 0.68, respectively. Fish fed a diet containing sodium alginate at the level of 2.0 g kg(-1) had a significantly higher survival rate than those fed the control diet after challenge with Streptococcus sp. and an iridovirus causing an increase of survival rate by 25.0% and 16.7%, respectively, compared to the control group. The ACH(50) level of fish fed the sodium alginate-containing diets at 2.0 g kg(-1) was significantly higher than those fed the 1.0 g kg(-1) sodium alginate diet and control diet after 12 days, and had increased to 1.9-fold, compared to those fed the control diet. The lysozyme activity, phagocytic activity, respiratory bursts, and SOD level of fish fed the sodium alginate-containing diets at 1.0 and 2.0 g kg(-1) were significantly higher than those fed the control diet after 12 days, and had increased to 1.97- and 1.68-fold, 1.35- and 1.50-fold, 1.63- and 1.81-fold, and 1.23- and 1.31-fold, respectively, compared to those fed the control diet. We therefore recommend dietary sodium alginate administration at 1.0 and 2.0 g kg(-1), respectively, to promote growth and enhance immunity and resistance against Streptococcus sp. and an iridovirus.     

性逆转石斑鱼性腺差异表达基因的克隆和筛选

以 17α 甲基睾丸酮 (17α MT)饲喂 2～ 4龄赤点石斑鱼 (Epinephelusakaara) ,成功地促使其性转变为具有生殖功能的雄鱼 .应用抑制性差减杂交 (SSH)技术构建了石斑鱼性反转前后性腺组织的SMARTcDNA文库及其cDNA差减文库 ,从中随机挑取 12 0 0个克隆进行了PCR和斑点杂交筛选 ,得到 12 0个差异表达cDNA片段 .挑选 71个cDNA克隆测序 ,将所测序列经GenBank检索和生物信息学比较 ,发现有 5 1个cDNA片段序列无明显的同源性 ,2 0个片段与报道的基因有较高同源性 .在这 2 0个具同源性的片段中有 3个片段可能是与性别分化密切相关的重要功能基因 ,它们是钙调蛋白基因、活性蛋白激酶C受体基因和一氧化氮合酶蛋白抑制剂基因 .这 3个基因被分别命名为鱼钙调蛋白基因 (GenBankaccession :AY2 8136 3)、鱼活性蛋白激酶C受体基因 (GenBankaccession :AY2 8136 4)和鱼一氧化氮合酶蛋白抑制剂基因 (GenBankaccession :AY2 8136 5 ) .     

Age and growth of the dusky grouper Epinephelus marginatus (Lowe 1834) in an exploited population of the western Mediterranean Sea

The age and growth of the dusky grouper, Epinephelus marginatus , in the Balearic Islands (western Mediterranean) were studied by otolith analysis from a sample of 358 specimens ranging in total length ( L _T) from 6·6 to 105·6 cm. The specimens came from commercial artisanal and recreational spear fisheries between 1999 and 2003. Otoliths grew asymmetrically throughout the range of L _T studied, showing a clear pattern of alternating translucent and opaque bands. Marginal increment analysis of specimens up to 8 years-old indicated that a single opaque band was formed each year during spring and summer. Whole otoliths allowed ageing specimens up to 10 years old, but above that age whole otoliths yielded lower age estimates than sectioned otoliths. The maximum estimated age was 61 years, which significantly extends the estimated life span of the species from a maximum of 36 years in a previous study. The von Bertalanffy growth parameters were estimated as L _∞= 95·5 cm L _T, K = 0·087 and T _O=−1·12. The study revealed differences in mean L _T at age and age structure between the shallow- and deep-water samples which may be attributed to different fishing pressure and environmental conditions.     

Influence of habitat on grouper abundance in the Florida Keys, U.S.A.

There was a significant inshore to offshore pattern in grouper species composition and density on Florida Keys coral reefs. Red grouper Epinephelus morio and black grouper Mycteroperca bonaci were most abundant on inshore patch reefs. Graysby Cephalopholis cruentata was most abundant on offshore reef types. Density among sites ranged from 0 to 0·43, 0 to 0·26 and 0 to 1·23 100 m⁻² for red grouper, black grouper, and graysby, respectively. Grouper density exhibited no consistency among sites within similar reef types. The densities of red and black grouper were correlated significantly with either physical or biotic habitat parameters. Graysby were observed in microhabitats with whose benthic features differed significantly from the surrounding habitat. This species was observed in high-relief areas with significantly less octocoral species and colony densities. Larger graysby were observed in areas of higher relief and less octocorals than smaller graysby. Habitat was one of several important factors influencing the distribution of groupers among coral reefs types in the Florida Keys.     

青石斑鱼海水循环水养殖水体的细菌群落特征北大核心CSCD

【目的】为揭示工厂化循环水青石斑鱼养殖水体的细菌群落特征,比较患病养殖池与健康养殖池的细菌群落结构差异,探讨细菌群落结构与青石斑鱼病害相关的相关性。【方法】采用Illumina Miseq高通量测序方法,分析比较了患病和健康养殖水体细菌群落结构、α-多样性指数(包括多度、均一度和系统发育多样性);并结合传统方法从患病青石斑鱼病灶部位分离疑似病原菌。【结果】患病和健康养殖水体中细菌群落的α-多样性并无太大差异,但主坐标分析与热图样本聚类分析表明细菌群落结构明显不同。二者的优势细菌门均为Proteobacteria、Verrucomicrobia和Bacteroidetes,但它们的相对丰度差异显著。患病养殖水体中α-Proteobacteria(25.07%)和γ-Protebacteria(22.74%)丰度相当,而健康养殖水体中γ-Protebacteria(40.49%)显著高于α-Proteobacteria(10.87%)。患病水体的Verrucomicrobia丰度(26.4%)远高于健康水体(10.9%);而Bacteroidetes的相对丰度则相反(12.3%vs 20.9%)。主要的差异类群包括α-Proteobacteria的Rhodobacteraceae和Rhodospirillaceae,γ-Proteobacteria的Alteromonadaceae、HTCC2188和Oceanospirillaceae,Verrucomicrobia的Verrucomicrobiaceae和Bacteroidetes的Cryomorphaceae。更表现在核心微生物类群的差异,健康养殖池水体以Glaciecola、HTCC、Sediminicola、Prevotella等对于养殖动物有益或无害的属为核心微生物;而患病养殖池水体则以Vibrio、Rubritalea、Oleibacter等病原菌或对养殖动物不利的属为核心微生物。从患病青石斑鱼的皮肤、肝脏和脾脏共分离得到弧菌20株,Acinetobacter haemolyticus 1株。【结论】患病的青石斑鱼循环水养殖水体中的细菌群落明显不同于健康养殖水体,特别是核心微生物的差异,其以Vibrio等病原菌或对养殖动物不利的属为主。该结果为青石斑鱼循环水养殖系统的管理、病害的诊断和监测提供理论与实验基础。     

Socially controlled male-to-female sex reversal in the protogynous orange-spotted grouper,Epinephelus coioides

Socially controlled sex change in teleosts is a dramatic example of adaptive reproductive plasticity. In many cases, the occurrence of sex change is triggered by a change in the social context, such as the disappearance of the dominant individual. The orange-spotted grouper Epinephelus coioides is a typical protogynous hermaphrodite fish that changes sex from female to male and remains male throughout its life span. In this study, male-to-female sex reversal in male Epinephelus coioides was successfully induced by social isolation. The body length and mass, gonadal change, serum sex steroid hormone levels and sex-related gene expression patterns during the process of socially controlled male-to-female sex reversal in E. coioides were systematically examined. This report investigates the physiological mechanisms of the socially controlled male-to-female sex reversal process in a protogynous hermaphrodite grouper species. The results enable us to study the physiological control of sex change, not only from female to male, but also from male to female.     

Molecular cloning and expression analysis of interferon-gamma-inducible-lysosomal thiol reductase gene in orange-spotted grouper, Epinephelus coioides

In mammals, interferon-gamma-inducible-lysosomal thiol reductase (GILT) has been demonstrated to play a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. In this study a cDNA containing the orange-spotted grouper GILT (OsgGILT) coding sequence has been cloned and its complete sequence determined. The full-length cDNA of OsgGILT gene is 1066 bp nucleotides (nt) encoding a protein of 260 amino acids (aa), with a putative molecular weight of 28.7 kDa. The deduced OsgGILT possesses the typical structural feature of mammalian GILT, including an active-site CXXC motif, a GILT signature sequence CQHGX(2)ECX(2)NX(4)C, and 10 conserved cysteines. The result of real-time PCR showed that OsgGILT mRNA was expressed in heart, liver, brain, gill, kidney and muscle and more highly expressed in spleen. The OsgGILT expression is obviously up-regulated in spleen and kidney after induction with LPS, these results suggest that OsgGILT may be involved in the immune response to LPS challenge in orange-spotted grouper.