Comparative antioxidant enzyme study in freshwater fish with different types of feeding behaviour

Comparative studies were made of the activities of Cu,Zn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase in organ homogenates from two herbivorous (grass carp and silver carp) and three omnivorous fish (barbel, crucian carp and common carp). The protein contents and lipid peroxidation of the organ homogenates were also compared. These comparative measurements provided control values for subsequent toxicological examinations. For the herbivorous fish, the highest total superoxide dismutase activity was observed in the kidney, followed in turn by the liver or spleen. In contrast, the highest total superoxide dismutase activity in the barbel was found in the roe, in the crucian carp in the liver, and in the common carp in the liver too. The quantitative distributions of the two peroxide metabolism enzymes glutathione peroxidase and catalase in the organ homogenates present very varied pictures. The results of the lipid peroxidation are similarly difficult to fit into one framework, even for fish following the same diet. It appears that the enzyme activities mentioned in points 4 and 5, together with the lipid peroxidation, depend not on the species, but on the variety.     

Comparative antioxidant enzyme study in freshwater fishes. II. Distribution of antioxidant enzymes and lipid peroxidation in omnivorous fish organs

Comparative studies were performed on the activities of superoxide dismutases, catalase and glutathione peroxidase in organ homogenates from three omnivorous fishes, the barbel, crucian carp and common carp. The lipid peroxidation and protein contents of organ homogenates were also compared. These comparative measurements primarily provide control values for subsequent toxicological examinations. The highest total superoxide dismutase activities were found in the liver or roe, kidney, heart and spleen in every cases. The antioxidant enzyme activities and other studied parameters of the organ homogenates partly appear to depend on the feeding mode, but are rather characteristic of the fish variety.     

The activity of pro- and antioxidant systems in the liver of freshwater fishes during different seasons

The content of lipid peroxidation products--diene conjugates, lipid hydroperoxides, thiobarbituric acid reactive substances (TBARS), vitamins A, E and carotenoids and the activity of antioxidant enzymes--superoxide dismutase, glutathione peroxidase and catalase in the liver of freshwater fishes of different species (silver carp, grass carp and common carp) in different seasons have been studied. It was established the activity of antioxidant defence system in the liver of fish depends significantly on the season and fish species. In particular, the content of lipid peroxidation products in the liver of freshwater fishes at the beginning of winter and spring was significantly higher compared to their content at the beginning of summer and autumn. The superoxide dismutase and glutathione peroxidase activities in the liver of these fish species at the beginning of winter and spring were significantly lower than at the beginning of summer and autumn while the seasonal changes of catalase activity in the liver of fish are expressed insignificantly. The content of vitamins E, A1, A2 and carotenoids in the liver of fishes of different species at the beginning of winter and spring was significantly lower than at the beginning of summer and autumn. The content of lipid peroxidation products and vitamins E, A1 and A2 in the liver of common carp is significantly lower than in the liver of silver carp and grass carp and species differences in antioxidant enzymes activity are insignificant.     

淡水鱼类微囊藻毒素去毒酶基因的克隆

微囊藻毒素去毒酶在鱼类微囊藻毒素去毒过程中起着关键作用,研究成功克隆鲢鱼、鳙鱼、草鱼、鲫鱼、鳜鱼、罗非鱼等淡水鱼类微囊藻毒素去毒酶基因cDNA核心片段而首次获得这些淡水鱼类微囊藻毒素去毒酶氨基酸序列。鲢鱼、鳙鱼、草鱼、鲫鱼、鳜鱼、罗非鱼微囊藻毒素去毒酶基因与人、小鼠、大鼠、牛、猪、羊的谷胱甘肽S-转移酶基因氨基酸同源性为60%左右,表明淡水鱼类微囊藻毒素去毒酶基因在进化上变异性较大,与其承担微囊藻毒素去毒代谢之特殊功能相适应。     

Molecular cloning and characterization of alpha-class glutathione S-transferase gene from the liver of silver carp, bighead carp, and other major Chinese freshwater fishes

Two full-length cDNAs encoding glutathione S-transferase (GST) were cloned and sequenced from the hepatopancreas of planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). The silver carp and bighead carp GST cDNA were 920 and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from the liver of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratu), mud carp (Cirrhinus molitorella), and tilapia (Oreochromis nilotica). All these GSTs could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using a computer program, and was found to fit the classical two-domain GST structure. Using the genome walker method, a 875-bp 5'-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish might be regulated by LPS, released from the toxic blue-green algae producing microcystins. To compare the constitutive expression level of the liver GST gene among the six freshwater fishes with completely different tolerance to microcystins, beta-actin was used as control and the ratio GST/beta-actin mRNA (%) was determined as 130.7 +/- 6.6 (grass carp), 103.1 +/- 8.9 (bighead carp), 92.6 +/- 15.0 (crucian carp), 72.3 +/- 7.8 (mud carp), 58.8 +/- 11.5 (silver carp), and 33.6 +/- 13.7 (tilapia). The constitutive expression level of the liver GST gene clearly shows that all the six freshwater fishes had a negative relationship with their tolerance to microcystins: high-resistant fishes (phytoplanktivorous silver carp and tilapia) had the lowest tolerance to microcystins and the high-sensitive fish (herbivorous grass carp) had the highest tolerance to microcystins. Taken together with the reciprocal relationship of constitutive and inducible liver GST expression level in some of the tested fish species to microcystin exposure, a molecular mechanism for different microcystin detoxification abilities of the warm freshwater fishes was discussed.     

不同发育阶段草鱼肾脏蛋白质组差异的初步分析

为了研究草鱼在不同发育阶段抗病能力差异的发育遗传学原因,以1龄草鱼和3龄草鱼的免疫器官肾脏为材料研究其在蛋白质组水平的差异。提取1龄草鱼和3龄草鱼肾脏的全蛋白,用二维聚丙烯酰胺凝胶电泳进行蛋白质的分离,染色后,扫描成像,经PDQUST软件分析,分别检测到大约900个蛋白质点。这些蛋白质点主要分布在pH4.5-7之间,其分子量大部分在66.2kDa以下。在3龄草鱼中检测到了3个在1龄草鱼中没有的差异蛋白质点,和20个上调、下调的差异点。经MALDI-TOF质谱分析和数据库搜索,证明在3龄草鱼肾脏中上调的差异点中有4个是与免疫或肾脏发育相关的蛋白质。这些初步的研究结果提示草鱼在不同发育阶段抗病能力的差异可能具有其发育遗传学基础。       

Molecular characterization of heat shock protein 70 genes in the liver of three warm freshwater fishes with differential tolerance to microcystin-LR

Heat shock protein 70 (HSP70) protect cell from oxidative stress by preventing the irreversible loss of vital proteins and facilitating their subsequent regeneration. Silver carp (Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idellus), and Nile tilapia (Oreochromis nilotica) are three warm freshwater fishes with differential tolerance to microcystin-LR (MC-LR). Full-length cDNAs encoding the HSP70 were cloned from the livers of the three fishes. The HSP70 cDNAs of silver carp, grass carp, and Nile tilapia were 2356, 2348, and 2242 bp in length and contained an open-reading frame of 1950 bp (encoding a polypeptide of 649 amino acids), 1950 bp (649 amino acids), and 1917 bp (638 amino acids), respectively. Like mammalian HSP70, the HSP70 of the three fish was also composed of an ATPase domain from residues 1 to 383 (44 kDa), substrate peptide binding domain from residues 384 to 544 (18 kDa), and a C-terminus domain from residues 545 to 649 (10 kDa). The relatively high conservation of HSP70 sequences among different vertebrates is consistent with their important role in fundamental cellular processes. Using beta-actin as an external control, RT-PCR within the exponential phase was conducted to determine the constitutive and inducible expression level of HSP70 gene among the three fishes (6-12 g) intraperitoneally injected with MC-LR (50 μg kg(-1) body weight). Both constitutive and inducible liver mRNA levels of the fish HSP70 genes showed positive relationships with their tolerance to MC-LR: highest in Nile tilapia, followed by silver carp, and lowest in grass carp. The differential expression pattern of liver HSP70 genes in the three fish indicated a potential role of HSP70 in the detoxification process of MC-LR.     

Structural conservation and food habit-related liver expression of uncoupling protein 2 gene in five major Chinese carps

The full-length cDNA of grass carp (Ctenopharyngodon idellus) and silver carp (Hypophthalmichthys molitrix) uncoupling protein 2 (UCP2) was obtained from liver. The grass carp UCP2 cDNA was determined to be 1152 bp in length with an open reading frame that encodes 310 amino acids. Five introns (Intron 3, 4, 5, 6 and 7) in the translated region, and partial sequence of Intron 2 in the untranslated region of grass carp UCP2 gene were also obtained. Gene structure comparison between grass carp and mammalian (human and mouse) UCP2 gene shows that, the UCP2 gene structure of grass carp is much similar to that of human and mouse. Partial UCP2 cDNA sequences of bighead carp (Aristichthys nobilis) and mud carp (Cirrhinus molitorella), were further determined. Together with the common carp (Cyprinus carpio) UCP2 sequence from GenBank (AJ243486), multiple alignment result shows that the nucleotide and amino acid sequences of the UCP2 gene, were highly conserved among the five major Chinese carps that belong to four subfamilies. Using beta-actin as control, the ratio UCP2/beta-actin mRNA (%) was determined to be 149.4 +/- 15.6 (common carp), 127.4 +/- 22.1(mud carp), 96.7 +/- 12.7 (silver carp), 94.1 +/- 26.8 (bighead carp) and 63.7 +/- 16.2 (grass carp). The relative liver UCP2 expression of the five major Chinese carps, shows a close relationship with their food habit: benthos and detritus-eating fish (common carp and mud carp) > planktivorious fish (silver carp and bighead carp) > herbivorous fish (grass carp). We suggest that liver UCP2 might be important for Chinese carps to detoxify cyanotoxins and bacteria in debris and plankton food.     

Experimental induction of Sphaerospora renicola (Myxosporea) infection in common carp (Cyprinus carpio) by transmission of SB-protozoans

The authors have experimental evidence that the protozoa causing the swimbladder inflammation (SBI) of the common carp (Cyprinus carpio) are indentical with presporogonic stages of Sphaerospora renicola Dyková et Lorn, 1982 parasitizing the renal tubules. Homogenates prepared from the thickened and inflamed swimbladder of naturally infected common carp, when injected into the abdominal cavity of fish, produced renal sphaerosporosis in the infection-free common carp if the homogenates contained the parasites described by Kovács -Gayer et al. (8). By intraperitoneal injection, the Unidentified Blood Organisms (UBOs) living in the blood of the common carp were transmissible to common carp, from the blood of which they were demonstrable for a long time. However, they were not transformed into Sphaerospora. To other cyprinids (gibel carp, silver carp, grass carp, tench, roach) neither the blood stages nor the swimbladder stages were transmissible from the common carp.     

长江水系钱草鱼遗传结构及变异性的RAPD研究

采用随机扩增多态性DNA（RAPD）技术对长江中游的湖北嘉鱼与江西瑞昌两个地理群体和中游的汉江和湘江两大水系的鲢和草鱼的群体进行了遗传学研究。发现长江水系链的遗传变异要高于草鱼,与现今生物量成反比的反常现象。鲢遗传多样性从大到小的分布是嘉鱼→湘江→瑞昌→汉江。草鱼遗传多样性从大到小的分布是瑞昌→汉江→湘江→嘉鱼,鲢和草鱼的遗传多样性地理分布并不一致。遗传分化指数Gst分别为12.3%和17.5%,表明鲢和草鱼的四个地理群体的遗传分化度较低,可能与地理较近和基因交流频繁有关。     

二龄草鱼脾脏、肝脏组织高表达甘露糖结合凝集素mRNA

Innate immunity is expected to be very important in fish. Mannose-bingding lectin (MBL) participates in the innate immune system as an activator of the complement system and as an opsonin after binding to certain carbohydrate structures on microorganisms. In this experiment, total mRNA was isolated from spleen, liver, gills, thymus, head kidney and kidney of adult and immature grass carp Ctenopharygodon idllus. The cDNA of MBL was obtained by RT-PCR using total mRNA from the spleen of carp as template. Such cDNA was labled with ^32p and used as probe for Northern analysis, and autoradiographic signals were quantified by densitometry analysis. The results showed that MBL was high expressed in the spleen and liver and low in gills, thymus, head kidney and kidney of adult grass carp, and MBL was much lower expressed in spleen and liver of immature grass carp than those of adult grass carp. The results might partially explain why immature grass carp are vulnerable to grass carp hemorrhage virus (GCHV) whereas adult grass carp are not.This suggested that MBL mav be an imoortant anti-GCHV factor [Acta Zoologica Sinica 50 (1): 137 - 140. 2004].     

草鱼与鲢鱼肥胖基因克隆与序列分析

肥胖基因产物leptin是调节哺乳动物摄食、能量代谢等生命活动的重要细胞因子。 应用RT-PCR和RACE法获得了草鱼(Ctenopharyngodon idellus)和鲢鱼(Hypophthalmichthys molitrix) leptin基因的全长cDNA序列分别为1 096 bp和1 176 bp,编码173和172个氨基酸。氨基酸序列同源性分析表明,草鱼和鲢鱼的leptin序列与其它鲤科鱼类leptin的同源性较高,而与其他鱼类的leptin同源性很低,但所有鱼类的leptin均含有用于形成二硫键的高度保守的半胱氨酸。系统进化树分析显示,草鱼和鲢鱼leptin与其他鱼类leptin聚于一进化分支。应用PCR和Genome Walker方法,进一步获得了草鱼和鲢鱼leptin基因的内含子和5′侧翼区序列。结果表明,获得的草鱼和鲢鱼leptin基因长度分别为2 129 bp和2 192 bp,含有与其他脊椎动物leptin相似的基因结构（含三个外显子和两个内含子）。本研究为深入研究鱼类肥胖基因结构功能关系与鱼类抗肥胖品系定向遗传选育奠定了良好的基础。     

氨氮胁迫下不同食性鱼类仔鱼抗氧化和非特异性免疫的差异响应

为探究氨氮胁迫对不同食性鱼类的影响,研究选取不同食性的4种鱼类(鲢Hypophthalmichthys molitrix、草鱼Ctenopharyngodon idella、团头鲂Megalobrama amblycephala和黄颡鱼Pelteobagrus fulvidraco)仔鱼为研究对象,探讨不同浓度氨氮(0、1、2和3 mg/L)短期胁迫(96h)对其生长、抗氧化和非特异性免疫响应的影响及其分子机制。结果显示:(1)氨氮胁迫导致4种仔鱼的体长生长速度呈现剂量依赖性减缓;(2)不同浓度氨氮导致4种仔鱼体内T-AOC、CAT和GPx含量显著降低(P<0.05), 2和3 mg/L氨氮暴露显著降低了草鱼、团头鲂和黄颡鱼仔鱼体内SOD活力(P<0.05),仅检测到黄颡鱼gpx及鲢sod转录水平出现显著性下调(P<0.05);(3)在不同浓度氨氮胁迫下, 4种仔鱼相关免疫基因转录水平均呈现一定的上调,仅鲢il1β转录水平显著下降(P<0.05),相对地草鱼仔鱼LYZ含量显著性下降,黄颡鱼仔鱼LYZ含量在2 mg/L氨氮组显著性上升(P<0.05)。双因素...     

草鱼出血病病毒对其它鱼的感染性研究

用草鱼出血病病鱼分离出的草鱼出血病病毒(Grass carp hemorrhage virus,GCHV)感染其它常见鱼并用ELISA方法检查感染鱼组织提取液,结果表明:青鱼、鲢鱼、布氏鳌条对GCHV抗体呈阳性反应;鲤鱼、鳙鱼、鲫鱼、团头鲂、泥鳅则呈阴性反应。综合感染鱼发病症状及死亡特征,初步认为:青鱼对GCHV是易感的,GCHV能在鲢鱼、布氏蟹条体内增值,但毒力较低,鳙鱼、鲫鱼、团头鲂、鲤鱼、泥鳅能抗GCHV感染。     

基于Miseq测序技术分析黄颡鱼不同养殖模式下池塘微生物群落结构多样性

利用Miseq测序技术分析了黄颡鱼Pelteobagrus fulvidraco (Richardson)三种养殖模式[(黄颡鱼单养、黄颡鱼混养草鱼(Ctenopharyngodon idellus, Valenciennes)和黄颡鱼混养草鱼、鳙(Aristichthys nobilis, Richardson)、鲢(Hypophthalmichthys molitrix, Valenciennes)]下养殖水体及底泥微生物群落结构多样性。结果显示, 12个样本共获得了655100条合格16S rDNA序列, 每个样本获得的有效序列数目为41256—77508, 可归纳为608—3686个分类操作单元。α多样性指数结果表明, 在三种养殖模式下末期水体的微生物群落丰度和多样性均高于养殖初期, 黄颡鱼混养草鱼模式下水体的群落丰度最高(Chao1指数: 2199.25, ACE指数: 2374.60), 黄颡鱼混养草鱼、鲢、鳙模式下水体微生物群落多样性最高(Shannon指数: 4.88, Simpson指数: 0.021)。样本聚类分析及PcoA主坐标分析结果表明, 黄颡鱼单养及黄颡鱼、草鱼混养模式下的水体和底泥的菌群组成及群落结构相似性较高, 黄颡鱼、草鱼、鲢、鳙混养模式下样本单独形成一类。从门水平的比较分析发现, 所有样本检测到细菌有8门, 其中变形菌门(Proteobacteria)的物种丰度在所有样本中占绝对优势地位。放线菌门(Actinobacteria)在养殖末期升高成为水体中占比第二的优势门, 尤其是黄颡鱼混养草鱼模式下(37.6%)。黄颡鱼在单养模式下, 养殖末期水体拟杆菌门(Bacteroidetes)细菌含量均明显增加(1.95%到10.98%)。从属水平的比较分析发现, 各样本之间的优势属组成有较大差异, 其中黄颡鱼在单养模式下水体样本中气单胞菌属(Aeromonas)在养殖末期丰度占优势(5.81%)。从微生物群落结构和多样性的角度考虑, 黄颡鱼、草鱼、鲢、鳙混养是一种值得推荐的养殖模式。     

葛洲坝水利枢纽兴建后对青、草、鲢、鳙繁殖生态效应的研究

本文分析了葛洲坝枢纽截流后,大坝上、下游草、青、鲢、鳙的繁殖条件,产卵场位置,产卵规模,鱼卵、鱼苗成色以及4种鱼的群体组成等。指出除原宜昌产卵场发生变化外,其余产卵场基本存在,并在上游新发现了几处产卵场。讨论了大坝对4种鱼的影响程度和上游存在草鱼地方性群体,论证了4种鱼不必过坝产卵的理由。       

草鱼线粒体型超氧化物歧化酶的生化遗传特性

超氧化物歧化酶 (SOD)是一种对生物细胞保护至关重要、在进化上比较保守的酶。因此 ,超氧化物歧化酶作为分子钟或分子标记已被广泛应用于生物进化研究、群体遗传结构分析以及品系鉴定。但鱼类SOD的生物化学和遗传学特性都尚未进行过系统和深入的研究。为使这一重要的分子标记能更好地应用于鱼类遗传育种、种质资源保护以及进化研究 ,本实验采用聚丙烯酰胺梯度凝胶垂直电泳法 ,研究了草鱼线粒体型超氧化物歧化酶 (fm SOD)的同功酶形式 ,生化遗传表型、亚基组成以及金属类型。实验结果表明 ,草鱼fm SOD有三种不同的同功酶形式 ;按从正极到负极的排列分别命名为fm SOD 1 ,fm SOD 2 ,fm SOD 3。这三种不同的fm SOD在草鱼群体中可构成 3种不同的生化遗传学表型 :表型 1个体只含有迁移率最快的fm SOD 1同功酶 ;表型3个体只含有迁移率最慢的fm SOD 3同功酶 ;而表型 2个体中含有所有三种不同形式的同功酶。在野生草鱼群体中 ,存在所有三种表现型 ;而在基因纯合型的雌核发育草鱼群体中只检测到表型 1和表型 3。野生草鱼群体中三种表现型的个体数之比符合一对等位基因分离的 1∶2∶1孟德尔遗传分离比例。由这些实验结果得出以下结论 :(1 )草鱼fm SOD是由细胞核DNA上的基因所编码而不是由线粒体DNA上的基因所编码的     

Study of the haemoglobin content of a single erythrocyte (M index) in various cultured fish species

Investigations of the erythrocyte count, haemoglobin content and M index of 7 fish species were performed under equivalent environmental conditions in the same period of the year. The species were: bighead Hypophthalmichthys nobilis (Richardson), carp Cyprinus carpio L., silver carp Hypophthalmichthys molitrix (Val.), grass carp Ctenopharyngodon idella (Val.), sheat-fish Silurus glanis L. , pike Esox Indus L., pike-perch Lucioperca lucioperca (L.). Bighead and carp are carnivorous and omnivorous, silver carp and grass carp are herbivorous and sheat-fish, pike and pike-perch are predatory. Significant differences were demonstrable between M values of fish living on different type of food, where the M index is: It is concluded that M index values are useful as one parameter differentiating between fish with distinctly different food habits.