大黄鱼皮肤溃烂病病原的研究

从福建省厦门西海域某渔排养殖大黄鱼(Pseudosciaena cyogga)患皮肤溃烂病的肝脏、肌肉分离到2株优势菌。人工感染试验证实其中1株为致病菌。该菌的主要生理生化特性为革兰氏阴性，短杆状，极生单鞭毛，发酵葡萄糖，氧化酶阳性，赖氨酸脱羧酶、鸟氨酸脱羧酶阳性，精氨酸脱羧酶、水杨素、枸橼酸盐阴性，不产生H2S，利用葡萄糖、甘露醇产酸，对O／129(10μg)不敏感。在含0％，10％NaCl的普通培养基上不生长；在含2％，4％，6％，8％NaCl的普通培养基中生长；在温度30，35，40℃下生长良好，但4℃不生长。初步鉴定该病原菌为副溶血弧菌(Vibrio parahaemolyticus)。药敏试验表明，该菌对卡那霉素、复方新诺明、链霉素等10种药物敏感，对青霉素G、氨苄青霉素、利福平等6种药物不敏感。     

大黄鱼倍性检测研究

于2001—2004年对人工诱导大黄鱼的不同发育阶段和不同组织进行倍性检测，胚胎眼泡期采用染色体计数法测定，鱼苗和成鱼阶段用流式细胞法，结果表明，胚胎染色体数目2n=48条为二倍体，3n=72条为三倍体．采用流式细胞法测定鱼苗和成鱼个体时，若受测样品的DNA相对含量值与已知对照组二倍体的比值约为1．5：1，其个体为三倍体；比值约为1：1，则为二倍体，无论用肌肉或尾鳍组织，皆可检测出其个体的倍性。     

大黄鱼(Pseudosciaena crocea)转铁蛋白(Tf)基因克隆及序列分析

提要应用RNeasy animal mini试剂盒抽提大黄鱼total RNA,逆转录合成模板cDNA;同时综合分析从GenBank数据库查询到已报道的转铁蛋白(Tf)基因序列,先设计并合成扩增引物扩增出大黄鱼转铁蛋白部分基因序列,再应用RACE技术,克隆出大黄鱼转铁蛋白全长cDNA基因,全长2461个核苷酸,编码661个氨基酸。应用WCG软件包,采用DNADIST和NEIGHBOR分析方法,构建了转铁蛋白系统进化树。树图显示与传统的分类地位大致相符,可以看出转铁蛋白在海水鱼和淡水鱼的进化上的差异,同时也显示大黄鱼Tf与真鲷的同源性最高。     

大黄鱼病原弧菌拮抗放线菌的筛选与人工诱变

从厦门集美海滩底泥分离得到38株放线菌，用琼脂块法测定它们对大黄鱼病原弧菌-溶藻弧菌和副溶血弧菌的拮抗效果，选择两株拮抗效果较好的放线菌进行诱变，测定诱变后各菌株的抗菌效果，并选择抗菌效果较好的菌株进行第二次诱变，如此反复诱变3次，共得到97株放线菌，结果表明；38株放线菌中的22株对两株病原菌有一定的拮抗作用；用紫外线照射可以获得少量对两株病原菌拮抗作用加强的菌株。     

Diagnosis of iridovirus in large yellow croaker by PCR

A rapid and sensitive PCR-based method for the detection of the large yellow croaker iridovirus(LYCIV) is described,which involves the amplification of a 295 bp fragment of the LYCIV ATPase gene from DNA isolated from naturally infected fish spleen.Sequencing of LYCIV ATPase gene fragment showed it shared 100% nucleotide sequence homology with the corresponding region of the ATPase gene of red sea bream iridovirus(RSIV) and sea bass iridovirus(SBIV),suggesting that LYCIV was homologous with RSIV and SBIV at least in part of the gemone.The specificity and sensitivity of the PCR procedure were tested on the iridovirus-infected fishes,the expected fragment was detected from spleen DNA samples of infected fishes,whereas no fragments were amplified from healthy fish spleen DNA,white spot syndrome baculoviruses(WSBV) DNA and pseudorabies virus(PRV) DNA.Detection limit of this method was 10^-7 ng positive plasmid DNA containing target sequence,equal to about 100 virions.In the infected experiment,first positive detection(1/4) appeared at Day 3 post-infection,all fish(4/4) tested positive at Day 7,however obvious symptoms were observed at Day 8,so LYCIV infection could be detected prior to the appearance of obvious symptoms.These results indicate that this PCR method could be used for early,rapid and specific detection of LYCIV infection.     

免疫添加物对大黄鱼血液白细胞数量及其吞噬功能的影响

采用低聚糖益力素、稳定型维生素C、渔用多维等免疫、营养添加物定期投喂大黄鱼，以粒细胞、淋巴细胞、单核细胞、吞噬百分率、吞噬指数等为指标，跟踪检测试验大黄鱼血液中白细胞组成数量和吞噬细胞吞噬能力的变化。研究结果表明，免疫添加物可以提高大黄鱼血液中白细胞数量和吞噬细胞的吞噬能力，增强大黄鱼的非特异性免疫功能，促进大黄鱼的健康生长。     

小黄鱼(Pseudosciaena polyactis)形态性状与体质量的相关性及通径分析

为了研究野生小黄鱼(Pseudosciaena polyactis)繁殖后代的形态性状对体质量的影响作用,以随机采集的729尾4.5月龄小黄鱼为研究对象,对每尾个体的体质量(g)和8个形态性状(cm):全长(X1)、体长(X2)、头长(X3)、躯干长(X4)、尾部长(X5)、尾柄长(X6)、尾柄高(X7)和体高(X8)进行准确测定,运用相关分析、多元回归分析和通径分析方法,分别计算性状间的相关系数、构建形态性状与体质量的多元回归方程,获得了通径系数和决定系数,进行了养殖小黄鱼的形态性状对体质量的影响研究。结果显示,8个形态性状与体质量的相关系数均达到极显著水平(P0.01);通径分析中,4个形态性状对体质量的通径系数达到极显著水平(P0.01),它们是影响体质量的重要指标,其中体长(通径系数:0.589)对体质量的直接影响最大。应用逐步多元回归分析建立了以体质量为因变量(Y),体长(X2)、躯干长(X4)、尾柄高(X7)和体高(X8)为自变量的回归方程:Y=–32.377+3.064X2–1.511X4+14.285X7+4.438X8。模型等式中形态性状与体质量的相关指数R2=0.927,说明所选性状是影响体质量的主要形态性状。以上分析结果为小黄鱼的选育测量指标的确定提供了理论依据。     

大黄鱼头肾免疫细胞研究

对大黄鱼（Pseudosciaena，crocea）头肾印迹片、光镜片和电镜片进行观察。结果表明，大黄鱼头肾包括淋巴细胞聚集区和颗粒细胞聚集区，但无肾小球结构。头肾免疫细胞丰富，包括淋巴细胞、颗粒细胞（Ⅰ型和Ⅱ型）、单核细胞和巨噬细胞，分别占57．94％、36．43％、5．63％，表明头肾是大黄鱼免疫细胞的生发中心，在免疫防御系统中具重要作用。     

大黄鱼病原副溶血弧菌拮抗菌的筛选

用点种法从大黄鱼肠道优势菌群中筛选出104株病原性副溶血弧菌的拮抗菌,进一步研究拮抗效果最好的11株拮抗菌的抗菌谱和生长曲线,其中NAC平板上筛选的4株拮抗菌的抗菌范围较广,锰营养琼脂培养基上筛选的3株拮抗菌具有生长优势。用BIOLOG板测定了抗菌谱较广、有一定生长优势3株拮抗菌(R22,Y58和J312)的碳源利用情况,结果表明,3株拮抗菌的碳源谱都比较广,都能利用糖类、有机酸和氨基酸等,而且与鱼类营养的竞争较小。16S rRNA基因同源性分析的结果显示与R22,Y58,J312同源性达99%以上的菌株分别来自弧菌属、芽孢杆菌属和假单胞菌属。进一步对R22,J312和Y58三株拮抗菌进行生化鉴定,结果显示R22为溶藻弧菌,J312为荧光假单胞菌,但Y58的种属未能得到确认。所得到的3株拮抗菌R22,Y58,J312在拮抗病原菌、抗菌谱、生长特性和营养利用等方面都初步满足益生菌的筛选条件,有待进一步检测其安全性和实用效果以便能够成为能实用的益生菌。     

GENETIC DIVERSITY OF THE WILD AND REARED PSEUDOSCIAENA CROCEA

The genetic diversity of both wild and reared Pseudosciaena crocea (Richardson) col-lected from Guan-Jing-Yang in Ningde, China in May 1999 was investigated by random amplified poly-morphic DNA (RAPD) in the present study. The polymorphism and mean difference of the wild popula-tion as revealed by RAPD were 18.9% and 0.0960 respectively, and those of the reared stocks were rel-atively lower, with 16.7% in polymorphism and 0.0747 in mean difference. The genetic distance be-tween the two stocks was 0.0041. From the comprehensive investigation, the main reasons for the loss of genetic diversity were probably overilshing, small number of parents as broodstocks and the debatable arti-ficial ranching. Results from this study also showed that the large yellow croaker populations distributed along Fujian coastal waters including Guan-Jing-Yang still potentially wide genetic variability. It is sug-gested that genetic management and prevention should be scientifically conducted in order to maintain and improve the genetic diversity of the P. crocea population.