牙鲆鱼肠道弧菌感染症及病原特性研究

对一起养殖牙鲆Paralichthys olivaceus病害进行了发病情况、临床表现、病理变化及病原学方面的检验,结果表明为细菌性败血感染症。经过对10尾病(死)牙鲆做病变肝组织及腹水中细菌的染色检查,取其中6尾的肝、肾、腹水为材料做细菌分离与纯培养后进行形态及理化特性测定,选择代表菌株进行16S rRNA基因序列测定及系统发育学分析,结果表明分离做纯培养的12株菌(HQ010223-1至HQ010223-12)为同种细菌———弧菌属Vibrio的鱼肠道弧菌V.ichthyoenteri。经以代表菌株(HQ010223-1)对健康牙鲆做人工感染试验,表明该菌在所检牙鲆病例的相应病原学意义;用37种抗菌类药物对6株菌所做的药敏试验,结果显示在不同菌株间无明显的敏感与耐药性差异。     

First isolation and characterization of a new species of Vibrio from diseased flounder (Paralichthys olivaceus Temminck et Schlegel)

Appropriate pathogenic bacteria were isolated from diseased (or dead) flounder (Paralichthys olivaceus L) occurring in a mariculture farms of Qinhuangdao, which all diseased fishes expressed bacterial septicaemia. The phenotypic information of the 12 pure cultures was studied extensively, including morphological characteristics, colony characteristics in different media, physiological and biochemical characteristics, and the mole fraction G C ratio of the DNA for representative strain. The results show that the isolates belong to a new species of Vibrio, and are designated as Vibrio qinhuangdaora sp.nov, by the area from which the diseased flounders are collected (Qinhuangdao) based on its biological properties following Rules of International Code of Nomenclature of Bacteria. At the same time,studies on the serum homology ofisolates, and pathogenicity of isolates were conducted by experimental infection. The results show that all the isolates are of serologic similarity, and the isolates have strong pathogenicity to flounder. The studies can extensively reflect the main biological properties of this new species of Vibrio.     

大菱鲆不同家系生长性能的比较

为培育优良的大菱鲆养殖新品种,收集了4个不同地理群体的大菱鲆作亲本,根据巢式设计,采用人工受精技术,在群体间和群体内构建了全同胞和半同胞家系,对其不同生长时期(生长阶段)全同胞家系的生长性能进行了比较.综合不同生长时期家系的体质量差异分析和不同生长阶段家系的生长速度分析,结果表明:来自英国和法国的群体间和群体内家系的生...     

微卫星标记在大菱鲆(Scophthalmus maximus L.)家系系谱印证中的应用研究

用8个微卫星标记对大菱鲆7个人工选育家系进行了系谱鉴别和遗传多样性研究。8个微卫星位点的平均多态信息含量为0.6721,平均杂合度为0.7206。8个微卫星位点在7个家系分析中,共检测到40个等位基因,每个位点的等位基因数在38个之间,每个位点平均有5个等位基因。根据已知亲本及子代基因型,成功的推断出了7个家系中缺失亲本的基因型。在双亲未知的情况下8个微卫星位点累积排除概率是97.07%,已知单亲时累积排除概率达99.63%。用UPG-MA法对7个家系的137个子代个体进行了聚类分析,90.5%同一家系的子代个体能完全聚类到一起,分类结果与系谱来源基本一致。微卫星标记可以为大菱鲆混养家系进行亲权鉴定和系谱构建提供技术支持。     

Food and feeding habits of juvenile flounder Platichthys flesus (L.), abd turbot Scophthalmus maximus L. in the åland archipelago, northern Baltic Sea

The food choice of juvenile flounder (Platichthys flesus) and turbot (Scophthalmus maximus) was studied in the northern Baltic Sea during the years 1988, 1989, 1994 and 1995. The diet included organisms from 30 species/taxa in flounder (n = 306) and 10 species/taxa in turbot (n = 41). Flounder 45 mm mainly consumed meiofauna (dominating taxon: Harpacticoida, Copepoda) and larger fish (46–101 mm) consumed macrofauna (dominating taxa: Oligochaeta, Amphipoda and Chironomidae). In terms of biomass, macrofauna dominated for all sizes of flounders, and meiofauna was important only for the smallest fish. A strong seasonal variation could be detected in the diet. In spring, macrofauna dominated for all size classes of fish (only fish > 30 mm were caught in spring), while in summer and autumn meiofauna dominated the diets for fish 45 mm in size. Juvenile turbot (22–88 mm) consumed macrofauna and small fish. Turbot 30 mm consumed mainly amphipods, while > 30 mm turbot consumed mysid shrimps, amphipods and fish.The ontogenetic shift from meio- to macrofauna-sized prey in flounders occurs at a larger fish size in the northern Baltic Sea than reported in other areas, possibly depending on the increased relative importance of meiofauna in the northern Baltic. The seasonal variation in the diet could be due to seasonally changing abundances in the zoobenthos, or for the small fish (1-group, spring), to switching from meio- to macrofauna in order to optimize their energy gain. The 0-group flounders consumed meiofauna for a long period, possibly due to a learning-process or simply due to easy availability of meiofauna. Turbot has a much larger mouth gap than flounders, thus allowing them to consume macrofauna from the beginning of their benthic life.     

人工诱导雌核发育牙鲆的染色体及核型证明

于1994-1996年,分别在威海海洋渔业捕捞公司（石岛）和鸿洋实业总公司龙须岛育苗场采集人工培育的3－5龄牙鲆亲鱼,采用紫外线照射法使牙鲆精子遗传物质失活,并用冷休克法抑制受精卵第二极体释放,从而获得雌核发育二倍体牙好。原肠期采用空气干燥法、Giemsa染色,得到雌核发育二倍体、正常二倍体及单倍体的染色体制片,进行染色体和核型的分析。结果表明,牙鲆的雌核发育二倍体和正常二倍体的染色体数均为2n=48,核型为48t,即48条端部着色点染色体,臂数NF=48,两者的核型设有明显差异;单倍体为24条端部着丝点染色体;在3个组别中,第一号染色体上都有一明显的次缢痕。雌核发育二倍体牙鲆的诱导率为98％。     

Rearing in intermediate salinity enhances immunity and disease-resistance of turbot (Scophthalmus maximus L.)

Studies were conducted to investigate the non-specific immune responses and disease-resistance of juvenile turbot Scophthalmus maximus,cultured at four different salinities(8,20,32 and 40) . Three concentrations(3.75 × 10 7,3.75 × 10 8 and 3.75 × 10 9 CFU/ml) of Vibrio anguillarum suspension were employed at each salinity to determine the 4-day LD 50 . The serum lysozyme activity,the alternative complement pathway activity(ACH50) and the phagocytosis percentage of head kidney in turbot were tested at 24,48 and 72 h post-challenge of V. anguillarum(1.1 × 10 8 CFU/ml,0.1 ml) ,respectively,to evaluate the non-specific immune responses at the selected rearing salinities. Fish reared at salinity 20 had the lowest mortality,namely,the highest 4-day LD 50 value(8.88 ± 0.17) . Besides,the lysozyme activity,ACH50 and the phagocytosis of turbot were the highest at the salinity 20,but with the lowest at the salinity 40 treatment regardless of sampling time. In addition,the non-specific immune activities kept increasing within 72 h post-challenge of V. anguillarum,except that the lysozyme activity increased from 24 to 48 h,and then decreased from 48 to 72 h at 40 significantly. These results together indicate that rearing in intermediate salinity(20) was able to enhance the immunity and disease-resistance of turbot.     

大菱鲆体重和形态性状的遗传动态分析

In order to elucidate the genetic mechanism of growth traits in turbot during ontogeny, developmental genetic analysis of the body weights, total lengths, standard lengths and body heights of turbots was conducted by mixed genetic models with additive-dominance effects, based on complete diallel crosses with four different strains of Scophthalmus maximus from Denmark, Norway, Britain, and France. Unconditional genetic analysis revealed that the unconditional additive effects for the four traits were more significant than unconditional dominance effects, meanwhile, the alternative expressions were also observed between the additive and dominant effects for body weights, total lengths and standard lengths. Conditional analysis showed that the developmental periods with active gene expression for body weights, total lengths, standard lengths and body heights were 15–18, 15 and 21–24, 15 and 24, and 21 and 27 months of age, respectively. The proportions of unconditional/conditional variances indicated that the narrow-sense heritabilities of body weights, total lengths and standard lengths were all increased systematically. The accumulative effects of genes controlling the four quantitative traits were mainly additive effects, suggesting that the selection is more efficient for the genetic improvement of turbots. The conditional genetic procedure is a useful tool to understand the expression of genes controlling developmental quantitative traits at a specific developmental period(t-1→t) during ontogeny. It is also important to determine the appropriate developmental period(t-1→t) for trait measurement in developmental quantitative genetic analysis in fish.     

半滑舌鳎病原鳗利斯顿氏菌表型及分子特征研究

2008年12月,江苏赣榆某渔场养殖半滑舌鳎出现大量死亡,症状主要表现为:头部、鳃盖及鳍基出血,尾鳍腐烂,腹腔膨胀并积有大量腹水,部分病鱼肠管脱出肛外。从病鱼肝脏、腹水中分离出大量优势生长的细菌,人工感染试验证明其对半滑舌鳎有较强的致病性。对3株分离菌进行了形态特征、理化特性、胞外酶及溶血素活性等表型生物学性状检验;测定了代表菌株的16S rRNA和gyrB基因序列,分析了16S rRNA和gyrB两种基因序列的同源性,并构建了系统发生树,比较了两种基因对分离菌的鉴定能力;结果表明gyrB基因用于细菌种间鉴定更具优越性;基于16S rRNA和gyrB基因的系统发育学分析表明分离菌与鳗利斯顿氏菌具有高同源性,根据分离菌的表型及分子特征,判定分离菌为利斯顿氏菌属(Listonella MacDonell and Colwell 1986)的鳗利斯顿氏菌[Listonella anguillarum (Bergeman 1909) MacDonell and Colwell 1986]。胞外酶活性及溶血活性检测表明3株分离菌均具有淀粉酶、蛋白酶、卵磷脂酶等胞外酶活性,在含7%家兔脱纤血液营养琼脂培养基上呈β型溶血,PCR检测3株供试菌均可扩增出大小约493 bp的溶血素基因和大小约248bp的金属蛋白酶基因。分离菌的耐药谱测定结果显示,对供试49种抗菌药物中的青霉素G等13种药物耐药,对羧苄青霉素等6种药物存在敏感与耐药的株间差异。     

养殖水体中氨氮对大菱鲆的急性毒性效应研究

为了评估大菱鲆(Scophthalmus maximus)耐氨氮性状的遗传参数和为循环水养殖大菱鲆提供科学指导,作者研究了养殖海水中氨氮对4月龄和8月龄大菱鲆的急性毒性效应,同时对比了氨氮对白化和正常个体、雌性和雄性个体的急性毒性效应差别。结果表明:非离子氨对4月龄大菱鲆的24、48、72、96 h LC50分别为2.19、1.94、1.80、1.72 mg/L,对8月龄大菱鲆的24、48、72、96 h LC50分别为3.64、3.02、2.93、2.86 mg/L,非离子氨对4月龄和8月龄大菱鲆的安全质量浓度分别为0.17和0.29 mg/L;氨氮对白化和正常大菱鲆以及雌性和雄性大菱鲆的急性毒性效应均没有显著性差异,研究结果为进一步选育大菱鲆的耐氨氮品系提供科学依据。     

三倍体牙鲆的核型证明

于1990，1993 - 1994年以捕自青岛近海的牙鲆亲鱼，采用冷休克法（0－4°C）进行多倍体的人工诱导实验，以期掌握牙鲆细胞遗传学特征和研究遗传育种方法。通过实验得到了三倍体牙鲆并进行培育。在原肠胚期采用空气干燥法、Giemsa染色获得正常二倍体、三倍体的染色体制片。镜检得，牙鲆二倍体染色体以2n=48，均为端部着丝点染色体，核型为24t，其管数NF=48；三倍体的染色体3n=72，也都为端部着丝点染色体，其臂数NF=72，并依据二倍体的枝型得出了三倍体的核型图。     

大菱鲆营养需求与饲料研究进展

大菱鲆是欧洲沿海的特有名贵鱼类之一，于1992年引入我国，目前已发展成为我国北方的一个主养品种，取得了巨大的经济和社会效益，被视为我国海水养殖的一个新的经济增长点。本文中总结了近年来国内外有关大菱鲆营养与饲料方面的研究报道，结果表明，大菱鲆对蛋白质有很高的营养需求，饲料中蛋白质的含量对大菱鲆幼鱼的生长起着重要的作用，对鱼体的增重方面影响极显著，对配合饲料的转换率影响显著。脂肪可以促进大菱鲆蛋白质的转化利用，从而达到节约饲料蛋白质的目的。饲料中的脂肪酸对大菱鲆特别是其仔稚鱼的生长很重要。目前国际上大菱鲆的饲料配制水平差别较大，研究认为大菱鲆配合饲料中适宜的蛋白质含量为42％以上，脂肪含量为12％以上。随着大菱鲆对各种营养参数的研究完善后，必将会提高饲料的配制水平。展望了大菱鲆营养需求与配合饲料的研究前景和发展方向。     

大菱鲆(Scophthalmus maximus)家系选育 F2 早期选择反应和现实遗传力估计

为了评估大菱鲆选育F2的早期育种效果,采用3种方法,即,Ⅰ:比较F2选择系和对照系的最小二乘均值,Ⅱ:比较连续2代(F1和F2)选择系目标性状间的育种值,Ⅲ:比较F2选择系和对照系的育种值,估测了6月龄大菱鲆体重的选择反应,并进而分析了现实遗传力和遗传增益。结果表明,3种方法的选择反应估测值存在一定差异,其相应估测的现实遗传力和遗传增益也不相同;3种方法估测选择反应、现实遗传力和遗传获得的均值分别为3.1983±0.5880、0.2941±0.0531和8.70±1.60,显示出足够大的早期选择反应、中等的现实遗传力和较低的遗传获得,预示利用家系选育对大菱鲆进行遗传改良能够取得良好的育种成效,同时,这些群体还有很大的遗传改良潜力。     

四株鳖源致病性嗜水气单胞菌(Aeromonas hydrophila)的表型、分子鉴定及其毒力基因检测

采用生态毒理学、表观分类学及分子生物学方法,对具白底板病典型症状濒死中华鳖内脏中分离获得的4株病原菌开展了以致病性、表型分析、分子鉴定及毒力基因检测为内容的实验研究,结果表明:(1)4株病原菌均具致病性,致死力由大到小依次为ZHYYZ-2、ZHYYZ-4、ZHYYZ-1、ZHYYZ-3;(2)4株病原菌均为呈短杆状、具溶血活性的革兰氏阴性细菌,VITEK2型全自动细菌鉴定与药敏系统和ATBExpression型细菌鉴定与药敏智能系统均显示为嗜水气单胞菌;(3)ZHYYZ-1、ZHYYZ-2、ZHYYZ-3、ZHYYZ-4的16SrDNA序列长度分别为1460、1464、1466、1461,经Blast同源性检索表明它们所扩增的16SrDNA序列与GenBank数据库中登记的71株嗜水气单胞菌的相似性均为99%;(4)经PCR特异性检测,各实验菌均含有Aha、AHH、AerA和OMP。根据4株实验菌的表型和分子生物学特征,判定它们均为气单胞菌属的致病性嗜水气单胞菌。     

大菱鲆低温生长遗传参数及相关的估计

The objectives of this present research were to assess the heritability of growth traits under low temperature conditions in turbot(Scophthalmus maximus L.), and to analyze the correlation between body weight(BW) and body length(BL). There were 536 individuals from 25 full- and half-sib families involved in this study. During the entire 90-day period, which was initiated at 233 dph(day old) and ended at 323 dph, the individuals' BW and BL were weighed consecutively six times every 18 days. The heritability of BW and BL and the correlation between these two traits were estimated based on an individual animal model with the derivative-free restricted maximum likelihood(DFREML) method. These results showed that the specific growth rates(SGR) of 25 families was from 0.75±0.11 to 1.05±0.14 under water temperature of 10.5–12°C. In addition, the heritability of BW and BL estimated under low-temperature were 0.32±0.04 and 0.47±0.06, respectively. The BW had a medium heritability(0.2–0.4), and the BL had a high heritability(0.45), which suggested that selection for increased weight and length was feasible. Moreover, there was potential for mass selection on growth. The genetic and phenotypic correlations between BW and BL were 0.95±0.01 and 0.91±0.01(P 0.01), respectively. A significant correlation between BW and BL showed that BL could be instead of BW for indirect selection, which could be effectively implemented in the breeding program.     

牙鲆(Paralichthys olivaceus)细菌败血感染症及病原检验与分析

采用调查统计、剖检病变、病原分离鉴定与人工感染健康鱼致病作用试验的方法，对自然发生的养殖牙鲆细菌败血感染症病例，进行了发病情况、临床表现及病理变化等方面的检验，同时取病(死)牙鲆的肝、腹水、腐烂肌肉组织为材料进行细菌分离，对分离于10尾鱼20株纯培养菌的形态特征、理化特性等进行鉴定。结果表明，分离出来的两种细菌，一种是弧菌属(Vibrio Pacini 1854)细菌的一个新种(sp．nov．)并定名为牙鲆弧菌(Vibrio olivaceus sp．nov．)；另一种是气单胞菌属(Aeromonas Kluyver and van Niel 1936)的嗜水气单胞菌(A．hydrophila)。通过人工感染试验等表明该两种分离菌为相应感染症的致病菌。     

饥饿对大菱鲆(Scophthalmus maximus)雌核发育二倍体仔鱼前期生长的影响

针对饥饿对大菱鲆雌核发育二倍体仔鱼前期生长发育的影响进行研究,并对仔鱼的饥饿不可逆点进行了测定。结果表明,水温14.5—16℃的培育条件下,大菱鲆雌核发育饥饿仔鱼卵黄于5—6日龄消耗殆尽,油球6—7日龄消耗殆尽;饥饿仔鱼最高初次摄食率出现在4日龄,为85.3%;仔鱼的饥饿不可逆点出现在8日龄;饥饿仔鱼全长生长速度显著慢于投喂仔鱼,6日龄后全长呈负增长,并且出现胸角突出、体色暗黑、消化道萎缩等较为明显的饥饿特征。本实验同时对大菱鲆常规二倍体仔鱼的饥饿耐受情况对照研究,表明,常规二倍体饥饿仔鱼的卵黄和油球消耗规律、初次摄食率变化、PNR点及全长生长与雌核发育仔鱼无显著差异。     

凡纳滨对虾(Litopenaeus vannamei)病原副溶血弧菌(Vibrio parahaemolyticus)的表型及分子特征

采用常规的形态及生理生化特性检验及分子生物学等方法,对引起凡纳滨对虾幼虾发生毁灭性死亡的病原菌进行了致病性、表型生物学及分子生物学的系统研究。结果表明,分离菌为弧菌属(Vibrio Pacini 1954)的副溶血弧菌(Vibrio parahaemolyticus),对凡纳滨对虾、中国对虾及日本对虾仔虾均有强致病性。分离鉴定的4株副溶血弧菌具有淀粉酶、明胶酶、卵磷脂酶活性,但不具有蛋白酶、脂肪酶活性,KP溶血均呈阴性,且均具有K抗原;代表菌株(JGB080708-1株)的16S rRNA基因序列(长度为1456bp,GenBank登录号为GQ205448)和gyrB基因序列(长度为1195bp,GenBank登录号为GQ205453)与副溶血弧菌的两种基因序列相似性均可达98%以上。病原菌的耐药性检测结果显示,对供试49种抗菌药物中的林可霉素等6种药物耐药。     

用显微注射方法分析牙鲆肌肉发育调节基因MyoD, Myf5的启动子序列

克隆了牙鲆(Paralichthys olivaceus)肌肉发育调节基因MyoD和Myf5的启动子序列,分别为608 bp和1 073 bp。对序列进行分析发现MyoD和Myf5的启动子含有参与肌肉发育调控的基因的结合位点。将MyoD和Myf5的启动子连接到GFP载体上,构建了重组质粒MyoDP-GFP和Myf5P-GFP,并注射到一细胞或两细胞的斑马鱼胚胎中,对这两个基因的启动子进行了瞬时表达研究。结果表明,牙鲆MyoD和Myf5的启动子可以驱动绿色荧光蛋白特异地在斑马鱼肌肉纤维中表达,因此牙鲆608 bpMyoD和1073 bpMyf5的启动子包含着可以驱动MyoD和Myf5正常表达的必需元件,它们是肌肉特异的,并且可以跨物种行使其功能。     

牙鲆幼鱼对n-3高度不饱和脂肪酸的吸收及与生长关系的初步研究

于1996年9-10月在中国水产科学研究院黄海水产研究所小麦岛实验基地,采用人工投喂的方法,给牙鲆幼鱼投喂含不同含量n-3高度不饱和脂肪酸（n-3HUFA）的人工饵料,研究牙鲆幼鱼对饵料中n-3HUFA的消化、吸收以及n-3HUFA的含量与日增重率、饵料效率的关系。结果表明,在饵料中添加0.60％—2.40％的n-3HUFA可有效提高牙鲆幼鱼的日增重率和饵料效率;肝脏中n-3HUFA的含量与饵料中n-3HUFA（尤其是DHA）的含量成正比;牙鲆幼鱼对饵料中n-3HUFA的表观消化吸收率随其含量的增加呈下降趋势。