中华绒螯蟹DNA指纹图谱的初步研究

本文运用ＤＩＧ标记的人源小卫星探针３３．６与ＨａｅⅢ消化的中华绒螯基因组ＤＮＡ杂交,建立了中华绒螯非放射性ＤＮＡ指纹图谱,证明上前在缺乏来自中华绒螯蟹基因组ＤＮＡ本身的同源小卫星ＤＮＡ的情况下,运用异源小卫星探针进行中华绒螯蟹ＤＮＡ指纹图谱的研究是可行的;实验表明运用人源小卫星探针３３．６进行中华绒螯蟹ＤＮＡ指纹图谱研究时,该探针可检测到１０个以上位点的遗传变异,每个位点呈现丰富的多态性。     

鳙小卫星DNA的克隆

鳙基因组ＤＮＡ 经内切酶Ｓａｕ３ＡⅠ完全酶切后，与Ｂａｍ ＨⅠ－ ｐＵＣ１８ 质粒连接，转化ＤＨ５α后，获得９３３ 个重组体。用人源小卫星探针３３ ．１５ 对重组体进行原位杂交筛选，获得１４ 个阳性克隆，取其中的１ 个阳性克隆（ 即小卫星ｐＢＣ１７４） 作探针，与５ 尾鳙和４ 尾鲢基因组ＤＮＡ 杂交，能产生具有高度变异性的、个体特异性的ＤＮＡ 指纹图，表明克隆的鳙小卫星ｐＢＣ１７４ 可用于鱼类ＤＮＡ 指纹分析。     

转基因鱼试验湖浮游生物群落DNA指纹与理化因子的关系

对转基因鱼试验湖７个采样点的浮游生物群落进行了ＰＣＲ－ＤＧＧＥ指纹分析，并进而探讨了ＤＮＡ指纹与理化因子的关系。结果表明：（１）ＰＣＲ－ＤＧＧＥ指纹图谱中共含１０４谱带，其中原核谱带５８条，真核谱带４６条，其多态性位点分别为８７．９％和８２．６％。（２）各站点的理化因子中，Ⅰ站的ＴＰ含量最高（０．１０ｍｇ／Ｌ），ＴＮ含量在Ⅲ站最高（０．３４ｍｇ／Ｌ），且各站点波动较大，Ⅴ站的透明度最低（６３．００ｃｍ）；ＮＯ３－Ｎ、ＮＨ４－Ｎ、ｐＨ、ＤＯ、ＣＯＤ及电导率在各站点变化不大。基于原核生物图谱的相似性聚类分析表明，浮游生物群落Ⅵ和Ⅶ、Ⅳ和Ⅴ、Ⅱ和Ⅲ相似度分别较高，Ⅰ站有别于其它各站，单独为１枝；基于真核生物图谱的聚类分析显示，Ⅰ、Ⅱ站聚为１枝，Ⅳ、Ⅴ、Ⅵ和Ⅶ依次相聚后与Ⅲ站聚为另１大枝，Ⅰ、Ⅱ站与Ⅲ站相距较近；理化因子的主成分分析结果与之基本吻合。因此，浮游生物群落ＤＮＡ多态性与环境理化因子是密切相关的，这类资料的积累将为在分子水平上建立水质预测预报体系奠定基础，为评价转基因鱼生态安全性提供依据。     

DNA指纹技术及其在水产上的应用

ＤＮＡ指纹技术及其在水产上的应用ＤＮＡＦＩＮＧＥＲＰＲＩＮＴＩＮＧＴＥＣＨＮＯＬＯＧＹＡＮＤＩＴＳＡＰＰＬＩＣＡＴＩＯＮＩＮＦＩＳＨＥＲＩＥＳ张跃朱新平罗建仁夏仕玲中国水产科学研究院珠江水产研究所,广州５１０３８０）ＺｈａｎｇＹｕｅＺｈｕＸｉｎｐｉｎ...     

两种不同地方种群尼罗罗非鱼遗传差异和分子遗传标记↑（*）

用ＡｐａⅠ，Ｂｃ１Ⅰ，Ｂｇ１Ⅱ，ＤｒａⅠ，ＥｃｏＲⅠ，ＥｃｏＲＶ，ＨｉｎｄⅢ，ＫｐｎⅠ，ＰｓｔⅠ，ＰｖｕⅡ，ＳａｃⅠ，ＸｂａⅠ，ＸｈｏⅠ等１３种限制性核酸内切酶对来自尼罗河上游和下游的两种不同地方种群尼罗罗非鱼的线粒体ＤＮＡ（ｍｔＤＮＡ）进行酶切分析，得到这两种罗非鱼的ｍｔＤＮＡ分子大小为：上游尼罗为１６９０９±９０ｂｐ，下游尼罗为１６９１２±１００ｂｐ。研究中还发现我国养殖的这两种不同地方种群尼罗罗非鱼，每一群体内个体间均存在ｍｔＤＮＡ酶切片段长度多态现象，而且群体间在ｍｔＤＮＡ水平上具有一定差异，依此构建了这两种不同地方种群尼罗罗非鱼ｍｔＤＮＡ限制性内切酶酶切图谱，建立了识别它们的分子遗传标记。     

中国对虾皮下及造血组织坏死杆状病毒（HHNBV）DNA探针的制备及应用

ＨＨＮＢＶ是１９９３年中国对虾暴发性流行病的主要病原之一。从纯化的病毒中提取ＤＮＡ，用ＥｃｏＲｌ限制性内切酶酶切成ＤＮＡ片段，与ＰＵＣ１８体外重组，建立ＨＨＮＢＶＤＮＡ文库。从中筛选出三个重组质粒（５＃、８＃、９＃），它们所插入的片段大小分别为：２．８Ｋｂ、０．８Ｋｂ、１．６Ｋｂ，它们分别用光敏生物素标记成探针，与感染ＨＨＮＢＶ的病虾ＤＮＡ呈阳性反应，以此利用制备的ＨＨＮＢＶＤＮＡ探针的高敏感性和特异性来检测虾病。     

杂交一代（尼罗罗非鱼♀×奥利亚罗非鱼♂）及春亲本基因组ＮＤＡ的比较

采用ＲＡＰＤ技术，用２０个随机引物对尼罗罗非鱼，奥利亚罗非鱼及其杂交一代的精巢ＤＮＡ进行扩增反应，发现引物ＯＰＺ－１４和ＯＰＺ－１８在尼罗罗非鱼和奥利亚鱼之间扩增出有差异的ＤＮＡ片段，作为鉴定两种鱼的分子标记有较高可信度，对这两种鱼的杂交一代基因组ＤＮＡ的ＲＡＰＤ扩增能获得中够的多态性，２０个引物中有８个引物扩增出差异性产物，表明杂交一代基因杂合性增强，这是杂种优势得以形成的重要遗传物质基础之一。     

红罗非鱼在咸淡水中生长快

红罗非鱼在咸淡水中生长快红罗非鱼是世界性养殖的主要鱼类。美国加勒比海洋研究中心对红罗非鱼从淡水到海水的最佳生长盐度和水温进行了测定。试验用红罗非鱼平均体重０．５６～１．２０９，放养在室内４２个０．３３ｍ’的水槽中，密度７５尾／ｍ’。试验水温分别为２２...     

罗非鱼微卫星DNA指纹图谱的构建

采用微卫星DNA指纹图谱技术对奥利亚罗非鱼、尼罗罗非鱼和橙色莫桑比克罗非鱼进行品种鉴定和遗传多样性分析。从103对罗非鱼微卫星引物中筛选出82对多态性高、带型清晰稳定的引物,用这 82对引物检测3种罗非鱼的遗传结构,共检测到605个等位基因,平均等位基因数7.37个,数据经Popgen32计算和EXCEL工具作图,构建3种罗非鱼的DNA指纹数据库,并绘制了DNA指纹图谱模式图。结果显示,奥利亚罗非鱼、尼罗罗非鱼和橙色莫桑比克罗非鱼的平均观测杂合度分别为0.179 6、0.530 1和0.312 2,平均期望杂合度分别为0.203 2、0.678 6和0.291 3,平均多态信息含量分别为0.075 4、0.608 3和0.152 8,由此可见,尼罗罗非鱼群体的遗传多样性水平最高,奥利亚罗非鱼群体遗传多样性最低。同时筛选得到16对具特异性条带的核心引物组合可将3种罗非鱼完全区分,每个位点可在其中一种罗非鱼中扩增出独有的条带,从而将这种罗非鱼与其它两种区分开来,将16对特异引物的图谱数据转化成计算机可以识别的数码指纹,可以方便地应用于罗非鱼及其杂交种的鉴定研究。为解决罗非鱼品种混杂遗传渐渗问题和保护罗非鱼种质资源提供技术支撑。     

鱼类仔鱼期的摄食和生长

鱼类仔鱼期的摄食和生长殷名称（上海水产大学，２０００９０）关键词鱼类，仔鱼期，摄食，生长ＦＥＥＤＩＮＧＡＮＤＧＲＯＷＴＨＯＦＴＨＥＬＡＲＶＡＳＴＡＧＥ．ＯＦＦＩＳＨ￥ＹｉｎＭｉｎｇｃｈｅｎｇ（ＳｈａｎｇｈａｉＦｉｓｈｅｒｉｅｓＵｎｉｖｅｒｓｉｔｙ，２...     

Association between the interannual variation in the oceanic environment and catch rates of bigeye tuna (Thunnus obesus) in the Atlantic Ocean

The environmental processes associated with variability in the catch rates of bigeye tuna in the Atlantic Ocean are largely unexplored. This study used generalized additive models (GAMs) fitted to Taiwanese longline fishery data from 1990 to 2009 and investigated the association between environmental variables and catch rates to identify the processes influencing bigeye tuna distribution in the Atlantic Ocean. The present findings reveal that the year (temporal factor), latitude and longitude (spatial factors), and major regular longline target species of albacore catches are significant for the standardization of bigeye tuna catch rates in the Atlantic Ocean. The standardized catch rates and distribution of bigeye tuna were found to be related to environmental and climatic variation. The model selection processes showed that the selected GAMs explained 70% of the cumulative deviance in the entire Atlantic Ocean. Regarding environmental factors, the depth of the 20 degree isotherm (D20) substantially contributed to the explained deviance; other important factors were sea surface temperature (SST) and sea surface height deviation (SSHD). The potential fishing grounds were observed with SSTs of 22–28°C, a D20 shallower than 150 m and negative SSHDs in the Atlantic Ocean. The higher predicted catch rates were increased in the positive northern tropical Atlantic and negative North Atlantic Oscillation events with a higher SST and shallow D20, suggesting that climatic oscillations affect the population abundance and distribution of bigeye tuna.     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth performance,digestive enzyme activity and immune response of Japanese sea bass,Lateolabrax japonicus fed with fructooligosaccharide

In this experiment, a feeding trial was performed to determine the effects of fructooligosaccharide (FOS) on growth performance, digestive enzyme activity and immune response of Japanese sea bass, Lateolabrax japonicus juveniles (initial weight 38.3 ± 0.5 g), and the fish were examined following feeding with six levels of FOS (0, 0.5, 1, 2, 4 and 6 g/kg) for 28 days. Significant enhancement of weight gain (WG) and specific growth rate (SGR) was found in fish fed 1 g/kg FOS incorporated diets (p < .05), while the feed conversion ratio (FCR) in the 1, 2 g/kg FOS groups reduced significantly compared with the control (p < .05). Besides, the crude lipid in the 4, 6 g/kg FOS groups increased significantly compared with the control (p < .05). On the other hand, the erepsin and lipase activities significantly elevated in intestine of fish fed 2 g/kg FOS (p < .05) and the lysozyme activity in serum of fish fed 2 g/kg FOS were significantly higher than that in the control (p < .05). Moreover, the alkaline phosphatase activities in serum of fish fed 0.5, 1, 2 g/kg FOS were significantly higher than in control (p < .05). Regression analysis showed that the relationships between dietary FOS levels and either SGR, FCR, erepsin or lysozyme activities were best expressed by regression equations, and the optimal inclusion levels are 1.37, 1.80, 3.06, 3.11, 1.93 and 1.80 g/kg for SGR, FCR, erepsin, lipase, lysozyme and total superoxide dismutase activities, respectively. Overall, this study revealed that FOS incorporated diets could beneficial for L. japonicus culture in terms of increasing the growth, digestion and immune activities. Under the present experimental condition, the optimal supplementary level of FOS in the diet of L. japonicus is 1–3 g/kg.     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

The optimal dietary protein level of juvenile silver sillago Sillago sihama at three dietary lipid levels

A 5 × 3 factorial growth trial was conducted to evaluate optimal dietary protein and lipid levels (dietary protein level, DP; dietary lipid level, DL) for juvenile Sillago sihama (S. sihama) (2.0 ± 0.02 g, initial weight). Fish were fed 15 diets containing 5 DPs (350, 400, 450, 500 and 550 g/kg) and 3 DLs (60, 90 and 120 g/kg) for 8 weeks. The interaction between proteins and lipids significantly influenced the feed conversion ratio, condition factor, body composition, antioxidant indices and lipase activity (p < .05). DP 450 g/kg showed the highest average final body weight. DPs 500 and 550 g/kg significantly decreased the protein efficiency ratio (p < .05). DL 120 g/kg showed the highest percentage weight gain. The low feed conversion ratio was found in diets P45L12, P55L9 and P55L12. Diet P45L12 showed high superoxide dismutase activities. DP 450 g/kg showed the lowest average malondialdehyde content. Lipase activity was increased by increasing DP (p < .05) with a fall at DP 550 g/kg. Under the present experimental conditions, the optimal DP for S. sihama was 450 g/kg under the DL 120 g/kg.     

Involvement of gonadal steroids in final oocyte maturation of white perch (Morone americana) and white bass (M. chrysops): in vivo and in vitro studies

Plasma estradiol-17 (E₂), testosterone (T), 17,20-dihydroxy-4-pregnen-3-one (DHP) and 17,20,21-tri-hydroxy-4-pregnen-3-one (20-S) levels were measured by radioimmunoassay (RIA) in white perch (Morone americana) and white bass (M. chrysops) that were induced to undergo final oocyte maturation (FOM) with human chorionic gonadotropin (hCG). Plasma DHP levels increased in females of both species in association with oocyte germinal vesicle migration (GVM) and germinal vesicle breakdown (GVBD) and decreased thereafter. Plasma 20-S levels also increased with oocyte GVM in white bass, but were several-fold lower than DHP levels. Circulating E₂ and T levels were greatest during GVM and GVBD in both species and decreased to low levels during oocyte hydration and ovulation. Follicles from white perch and white bass which received a priming injection of hCG in vivo, produced both DHP and 20-S in vitro after exposure to hCG and their oocytes underwent GVBD. Ovarian incubates from unprimed fish of either species produced only E₂ and T and their oocytes did not complete GVBD. Oocytes from unprimed bass, but not perch, matured when follicles were exposed to hCG in vitro. Both trilostane and cycloheximide blocked in vitro production of DHP and 20-S and oocyte GVBD by white perch follices. DHP and 20-S were equipotent inducers of FOM in the GVBD bioassay. None of several other structurally-related steroids tested were effective within a physiological range of concentrations. These results indicate a role for DHP and 20-S in the control of FOM in white perch and white bass.