免疫低聚糖对中国对虾的免疫效用研究

为证实免疫低聚糖(D-氨基葡聚寡糖,D-Amino-Oligosacellaride)对中国对虾(Penaeus chinensis)的免疫增强作用,进行了不同剂量的注射及饲料投喂实验.结果表明,注射免疫低聚糖可显著增强中国对虾酸性磷酸酶和溶茵酶等血清免疫指标,添加于饲料中投喂可以提高对虾成活率,降低饵料系数,且饲料中添加量≥3‰的效果较佳.     

莫桑比克鳗鲡对温度、盐度和pH的耐受性试验

就莫桑比克鳗鲡对水温、盐度和pH的耐受性进行了初步研究.试验结果表明,适宜水温为12.0～35.0℃,临界上限为40.0℃.临界下限为0℃;适宜盐度为0～15.临界极限为45;适宜pH为4.0～9.5,临界上限为11.0,临界下限为2.5.     

鲤鱼肝原代细胞的分离与培养方法

本研究在酶消化法的基础上,通过比较不同的肝组织分离和培养条件,获得鲤鱼肝原代细胞培养的最佳培养方法和条件。首先,结合细胞数量和台盼蓝染色所得的细胞存活率,比较了胰蛋白酶消化鲤鱼肝组织的不同反应温度和时间,结果表明:肝组织用0.25%(m/V)胰蛋白酶在25℃消化40 min时细胞分离效果最好。其次,经酶消化得到的肝原代细胞粗制液,设置4种不同的细胞纯化和培养条件:1)将细胞直接重悬于含10%胚牛血清(FBS)的L-15/DMEM培养基中培养;2)以10%鲤鱼血清取代培养基中的胚牛血清重悬细胞并培养;3)将细胞经Percoll液密度梯度离心纯化后,重悬于含10%胚牛血清的L-15/DMEM培养基中培养;4)细胞经Percoll纯化后,重悬于含10%鲤鱼血清的L-15/DMEM培养基中培养。利用光学显微镜观察肝细胞的形态,进而通过HE染色法检测,结果表明经Percoll纯化后,肝细胞纯度显著提高;采用MTS/PMS法检测肝细胞的增殖情况,结果表明鲤鱼血清代替胚牛血清培养细胞,肝细胞活力显著提高。本研究将为建立稳定的毒理学肝原代细胞实验模型奠定基础。     

饥饿和再投喂期间尼罗罗非鱼生长、血清生化指标和肝胰脏生长激素、类胰岛素生长因子-Ⅰ和胰岛素mRNA表达丰度的变化

在室内可控条件下,对尼罗罗非鱼[初始体质量(62.50±3.44)g]进行饥饿28d和随后再投喂21d的处理,于饥饿第0、7、14、21、28天和再投喂第14、21天进行采样分析,研究饥饿和再投喂期间尼罗罗非鱼生长、血清生化指标和肝胰脏生长激素(GH)、类胰岛素生长因子-Ⅰ(IGF-Ⅰ)和胰岛素(IN)mRNA表达丰度的变化。结果显示,与饥饿第0天相比,饥饿超过7d鱼体体质量显著降低(P<0.05),再投喂21d显著增加(P<0.05);肝体比随饥饿时间延长显著降低(P<0.05),恢复投喂后较饥饿时升高,但显著低于饥饿前水平(P<0.05)。在血清指标上,甘油三酯、血糖、碱性磷酸酶、谷草转氨酶和谷丙转氨酶均随饥饿时间延长而逐渐降低,恢复投喂后均有不同程度提高,但转氨酶活性显著低于饥饿前水平(P<0.05);饥饿和再投喂对血清总胆固醇、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇无显著影响(P>0.05)。在激素方面,与饥饿第0天相比,饥饿使血清GH含量及其肝胰脏mRNA表达丰度显著升高,血清IGF-Ⅰ及其肝胰脏mRNA表达丰度降低,恢复投喂后两者均显著升高(P<0.05);INmRNA表达丰度在饥饿7~21d显著升高(P<0.05),饥饿第28天时无显著差异(P>0.05),再投喂后显著降低(P<0.05)。     

First report of Shewanella sp. and Listonella sp. infection in freshwater cultured loach,Misgurnus anguillicaudatus

A novel disease outbreak occurred among cultured loach, Misgurnus anguillicaudatus in China. The diseased fish displayed lethargy, loss of appetite, black discolouration, anasarca and ventral skin hyperaemia. Clinical signs were observed in most of the fish examined, which consisted of discoloured liver, swollen spleen and petechial haemorrhage in the intestine and peritoneal membranes. The causative agent was found to be two species of bacteria by experimental infection, identified as Shewanella sp. and Listonella sp. The result showed that these were likely to be Shewanella putrefaciens and Listonella anguillarum respectively. Bacterial identification consisted of physiological and biochemical tests as well as 16S rRNA sequence analysis. In addition, antibiotic susceptibility testing was performed, revealing that Shewanella putrefaciens and Listonella anguillarum were susceptible to enrofloxacin, ciprofloxacin, levofloxacin, pipemidicacid and norfloxacin. This is the first report of S. putrefaciens and L. anguillarum from cultured loach.     

鲤鱼血清蛋白,血红蛋白,LDH和EST同工酶的电泳分析

采用聚丙烯酰胺凝胶电泳技术,分析了鲤鱼性成熟期雌雄个体的血清蛋白、血清脂蛋白和血红蛋白,结果是雌雄个体血清蛋白电泳图谱有差异,雌性(14条)比雄性(13条)多1条蛋白带;血清脂蛋白和血红蛋白无性别差异。同时,测定了鲤鱼血清、肝胰脏、心脏、背部肌肉和眼晶体的乳酸脱氢酶(LDH)同工酶和酯酶(EST)同工酶,LDH具有组织特异性,除在肝胰脏分离出9条酶带外,在血清中也分离出8条酶带,表明有LDH—c基因的作用,EST同工酶表现出明显的多态性,表明在鲤鱼各组织中有多个基因座位起作用。     

花刺参钙调蛋白cDNA克隆及组织分布

钙调蛋白（ calmodulin,CaM）是一种钙离子（ Ca2＋）结合蛋白,在钙信号传导过程中起重要作用。文章扩增到花刺参（Stichopus monotuberculatus）钙调蛋白（命名为StmCaM）cDNA全长序列。StmCaM cDNA全长1394 bp,其中5＇UTR长138 bp,3＇UTR长806 bp,ORF为450 bp。ORF推导出的StmCaM含149个氨基酸（包含起始蛋白酸）,其预测分子量约为16.7 kDa,理论等电点为4.1。StmCaM氨基酸序列与其他物种的钙调蛋白高度相似,相似度百分比为95.9%~98.0%。采用realtime PCR分析StmCaM基因在花刺参不同组织中的表达,结果显示其在呼吸树中表达量最高,体腔细胞、肠次之,而体壁中表达量最低,几乎检测不到。     

远海梭子蟹人工育苗及中间培育技术研究

2011—2012年在海南琼海进行远海梭子蟹(Portunus pelagicus)亲蟹培育、人工育苗及稚蟹中间培育技术研究。结果表明,亲蟹培育平均成活率和抱卵率分别为94.05%和85.61%。在水温28.5～29.6℃的条件下,从第1期溞状幼体发育变态至仔蟹期约需10～14 d,投放溞状幼体625.29万只,培育出大眼幼体100.12万只,成活率为16.01%,培育成仔蟹苗20.25万只,育苗成活率为3.24%。经过中间培育获得1.0 cm的蟹苗10.46万只,成活率达51.65%。     

Gene Expression in Black Tiger Prawns Following Intramuscular Injection of β-gal Plasmid

Gene expression in black tiger prawns (Penaeus monodon) was studied following intra-muscular injection of CMVGal plasmid into the second abdominal segment. We used an in situ staining technique to detect -gal expression in one- and three-month-old injected prawns. We found that only one of the three-month-old prawns expressed the marker gene (2 days after injection), and the site of expression was confined to the sixth abdominal segment away from the injection site. We repeated the experiment on a new batch of three-month-old prawns, but using fluorometric determination technique. This time we found that -gal expression was detected (6/42) at the site of injection after 2, 7, and 14 days. In two other test samples, transgene expression was detected in the sixth abdominal segment only, further confirming the possibility of injected DNA dispersal. The results of the study also suggest that direct gene transfer is a feasible technique in black tiger prawns.