Isolation and characterization of a regulator of G protein signaling (RGS16) gene homologue in yellow grouper Epinephelus awoara

Regulators of G protein signaling (RGS) are GTPase-activating proteins (GAP) which act as modulators of G protein-coupled receptors. We isolated a RGS16 homologue in yellow grouper (Epinephelus awoara) spleen using suppression subtractive hybridization and RACE-PCR. The nucleotide sequence of yellow grouper RGS16 full-length cDNA was 700 bp and contained an open reading frame of 537 bp, encoding a putative protein of 178 amino acids. The encoded protein shows 47–61% identities to other homologues. RT-PCR analysis demonstrated that RGS16 was expressed in yellow grouper spleen and up-regulated in kidney heart, liver, and anterior kidney by lipopolysaccharide. This study will help towards validating the specific function of RGS in marine fish.     

河流弧菌对石斑鱼体表黏液的黏附作用

采用^3H-TdR同位素示踪法研究了河流弧菌（Vibrio fluvialis）TS-1对青石斑鱼（Epinephelus awoara）表皮黏液的黏附作用。研究结果显示，经TSB培养的河流弧菌对青石斑鱼表皮黏液的黏附作用极显著高于经TSA培养的河流弧菌（P〈0．01）；河流弧菌经TSB培养后能很好地黏附于青石斑鱼表皮黏液，其黏附量随菌浓度升高而增大并符合饱和黏附动力学特征；不同生长阶段河流弧菌的黏附能力不同，在培养初期阶段细菌的黏附量先是随着培养时间的延长而增大，并在培养24h后黏附量达到最大，而后随培养时间的延长其黏附量急剧下降。本实验研究结果表明，河流弧菌对青石斑鱼表皮黏液有较强的黏附作用，其黏附作用受细菌培养条件、营养状况等自身因素的显著影响。本研究结果有助于了解河流弧菌的流行病学和致病机理。[中国水产科学，2008，15（2）：347—351]     

青石斑鱼微卫星标记的筛选及群体多态性分析

微卫星DNA,又称短串联重复序列(STR)或简单序列重复(SSR),是广泛存在于真核生物基因组中的简单重复DNA片段,一般每个重复单位仅1～6个碱基,重复数为10～20次,其中动物体中以双核苷酸(CA/GT)n最为常见。根据微卫星DNA核心序列两端的保守序列设计引物     

封闭式循环水饲养青石斑鱼试验

在封闭式循环水养殖系统中以全人工配合饲料制成软颗粒饲料投喂青石斑鱼幼鱼,经过33d的饲养,青石斑鱼尾相对增重率为64.6%,平均饲料系数为1.81,养殖成活率95.89%,平均丰满度为1.66。封闭式循环水养殖系统的养殖排出水回收率达到85%以上,养殖单产、饲料效率、生长速度都达到了生产要求。     

网箱养殖青石斑鱼的溃疡病病原

2002年夏季厦门同安湾一带气候炎热,降雨量少,海水盐度偏高,同安湾刘五店的网箱养殖石斑鱼大面积暴发溃疡病.本研究调查了石斑鱼溃疡病暴发的特征和症状,主要表现为肌肉溃疡坏死、眼球脱落、鱼骨暴露等.从病鱼体表及内脏分离出优势菌群命名为TS-628,经回归感染证实TS-628就是引发本次石斑鱼溃疡病的病原菌.对病原菌进行鉴定发现,该菌革兰氏染色呈阴性,电镜下观察菌体呈短杆状,极端单鞭毛,综合研究该菌在形态、生理生化、16S rDNA同源性及药物敏感性等方面的特性,基本确认分离到的病原菌为哈维氏弧菌(Vibrio harveyi),该菌对氯霉素、壮观霉素等多种抗生素敏感,对万古霉素、青霉素G等抗生素不敏感.哈维氏弧菌是水产养殖病害常见病原菌,但作为养殖石斑鱼的病原菌在国内属首次报道.     

Establishment of cell lines from a tropical grouper,Epinephelus awoara (Temminck & Schlegel), and their susceptibility to grouper irido- and nodaviruses

Four tropical marine fish cell lines have been established from the eye, fin, heart and swim bladder of grouper, Epinephelus awoara (Temminck & Schlegel). Optimum media and temperature conditions for maximum growth were standardized. The eye and swim bladder cells were mostly epithelial, but the fin and heart cells were mostly fibroblastic. The viability of cells was 95% after 1 year of storage in liquid nitrogen (-196 degrees C). Besides these four cell lines, previously established grouper brain, kidney and liver cell lines were also used for a viral susceptibility study which showed that all the cell lines were sensitive to grouper iridovirus, whereas only brain, fin and liver cell lines were susceptible to the yellow grouper nervous necrosis virus (a nodavirus). Electron microscopy studies of the grouper irido- and nodaviruses in ultrathin sections of infected cells showed an abundance of viral particles in the cytoplasm of the virus-infected cells indicating the effective replication of these two viruses. It is suggested that these cell lines can be used for the isolation of putative fish specific viruses and provide a valuable tool to study the mechanisms of host-pathogen interactions. Furthermore, these cell lines upon transfection, using pEGFP-C1 and pEGFP-aMT2.5 (ayu metallothionein promoter), produced significant fluorescent signals indicating their utility for exogenous studies.     

In vitro neutralization by monoclonal antibodies against yellow grouper nervous necrosis virus (YGNNV) and immunolocalization of virus infection in yellow grouper, Epinephelus awoara (Temminck & Schlegel)

Mouse monoclonal antibodies (MAbs) were produced by using yellow grouper nervous necrosis virus (YGNNV) as an immunogen, isolated from infected yellow grouper, Epinephelus awoara (Temminck & Schlegel), and propagated in GB cells. In enzyme linked immunosorbent assay (ELISA), 43 hybridoma clones secreting MAbs strongly reacted with the purified virus. Ten of them showed a higher neutralization index (NI) value between 6.5 and 4.5 (log₁₀ NI) than the other 33 MAbs against YGNNV infection in cell culture. All 10 MAbs belonged to the IgG isotype with a κ light chain and recognized the 42 kDa coat protein of YGNNV by Western blot analysis. Immunohistochemical results demonstrated that the viral signals co-located with pathological lesions observed in retina, brain and spinal cord. These results indicate that the MAbs are useful for confirmative diagnosis of YGNNV infection.     

网箱养殖青石斑鱼Epinephelus awoara鳃及体表粘附菌群的PCR-DGGE比较分析

采用免培养的16S rDNA梯度凝胶电泳技术（denaturing gradient gel electrophoresis,DGGE）对海水网箱养殖青石斑鱼Epinephelus awoara鳃和体表粘附菌群结构进行了比较分析。结果表明青石斑鱼鳃粘附菌群结构相对简单,存在绝对优势种群,体表粘附菌群结构较为复杂,无绝对优势种群,聚类分析表明青石斑鱼鳃与体表粘附菌群结构存在较大差异性（相似度52%）,而个体间鳃或体表的粘附菌群结构相似性较好。测序结果表明青石斑鱼鳃与体表粘附菌群以未培养菌为主,鳃的绝对优势菌为Panntoeasp.,体表相对优势菌为Meio-thermussp.、UnculturedAcinetobactersp.、Wautersiella falsenii与未培养菌,提示在生产实践中采用复合菌制剂似乎更为可行。PCR-DGGE技术能区别青石斑鱼鳃与体表粘附菌群的多样性,在可定植益生菌筛选上具指导意义。     

点带石斑鱼对盐度的适应性研究

对均重7．20g的点带石斑鱼进行了兑淡养殖实验，30‰、14‰和9‰三个盐度的生长实验结果表明，盐度越低，生长速度越快。当水体盐度从30‰突变至5‰时，点带石斑鱼完全可以很快适应这种大幅度的盐度变化。其结果对石斑鱼养殖产业远离海岸，深入到广大内陆地区生产有积极的指导意义。     

青石斑鱼肠道菌群研究

以中国沿海重要经济鱼类青石斑为研究对象,分别以营养琼脂和脑心浸液琼脂培养基检测其肠道中总的好气和兼性厌氧菌及专性厌氧菌的数量,以TCBS(Thiosulfate Citrate Bile Salts Su-crose)、麦康凯、NAC(Nalidixic acid-cetri mide)、锰营养盐、TPY(Tryptone-phytone-yeast extract)和SL(Rogosa SL.)琼脂等6种选择性培养基对青石斑肠道菌群进行计数和选择性分离,并对分离的优势菌株进行16S rRNA基因同源性分析。结果表明,青石斑鱼肠道内好气及兼性厌氧菌的总量可达5.3×105CFU/g。在6种选择性培养基中TCBS培养基上生长的菌落数量最多,约占总好气菌及兼性厌氧菌的36%,优势菌与牙鲆弧菌(Vibrio olivaceussp.nov.)同源性最高;麦康凯琼脂平板中生长的菌落数量次之,约占总好气菌及兼性厌氧菌的24%,优势菌与阿氏肠杆菌(E.asburiae)同源性最高;而NAC和锰营养盐琼脂平板中生长的菌落数量较少,优势菌分别与假单胞菌属(Pseudomonassp.)和枯草芽孢杆菌(Bacillus subtilis)的同源性最高。厌氧菌的数量很少,约为总好气和兼性厌氧菌数量的1%,其中TPY培养基中生长的菌落数量约占总厌氧菌数量的53%,优势菌与拟杆菌(Bacteroidessp.)同源性最高;SL培养基中没有检测到细菌。