Molecular characterization of nonstructural protein NS38 of grass carp reovirus

Viral nonstructural proteins in both enveloped and non-enveloped viruses play important roles in viral replication. Protein NS38 of Grass carp reovirus (GCRV), has been deduced to be a non-structural protein, and, consistent with other reoviruses, is considered to cooperate with the NS80 protein in viral particle assembly. To investigate the molecular basis of the role of NS38, a complete protein was expressed in E.coli for the first time. It was found that there is a better expression of NS38 induced with IPTG at 28 ℃ rather than 37 ℃. In addition, the antiserum of NS38 prepared with purified fusion protein and injected into rabbit could be used for detecting NS38 protein expression in GCRV infected cell lysate, while there is not any reaction crossed with purified virus particle, confirming NS38 is not a component of the viral structural protein. The result reported in this study will provide evidence for further viral protein-protein and protein-RNA interaction in dsRNA viruses replication.     

Intensive culture of grass carp and hybrid grass carp larvae

Intensive culture of grass carp and hybrid grass carp (Ctenopharyngodon idella x Aristichthys nobilis ) larvae was conducted under Florida (U.S.A.) conditions. The influence of different rearing facilities (indoor tanks, outdoor tanks and cages) and different methods of fish feeding using live zooplankton and artificial food was tested. High survival (86–100%) and satisfactory grass carp growth (47–56 mg) were obtained in the outdoor tanks and cages during the 10–day experiment. It is believed, that the technique described can be used after some improvements for commercial–scale grass carp larvae rearing. Low survival (1–3%) was obtained in the hybrid culture experiment even though satisfactory growth rates (84–212 mg) were obtained after 13 days. High mortality was attributed to genetic abnormalities caused by hybridization. The hybrid does not appear to be a promising fish for culture.     

Expression and identification of inclusion forming-related domain of NS80 nonstructural protein of grass carp reovirus

Grass carp reovirus (GCRV), a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstructural protein NS80, encoded by GCRV segment 4, has a high similarity with uNS in MRV(Mammalian orthoreoviruses), which may be associated with viral factory formation. To understand the function of the uNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region (335.742) was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28oC. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80(335.742) protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody (mouse) and NS80(335-742) specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.     

The relationship between feeding preferences and consumption rates in grass carp and grass carp × bighead carp hybrids

The relationship between food preference hierarchy and consumption rate was examined for the grass carp, Ctenopharygodon idella , and its Fl hybrid (grass carp × bighead carp). Preferences of both types of fish were similar, although hybrids consumed vegetation at a much lower rate. For both, relative preference in feeding trials with mixed plant species was significantly correlated with consumption rate in monospecific feeding experiments. No correlations were found between preference and caloric content, protein content or relative water content. The hypothesis is proposed that preference hierarchies reflect relative handling times of different food types and that these fish maximize 'through-put' by preferentially consuming plant species which can be most rapidly ingested.     

The grass carp for weed control

The agricultural and recreational use of waterways is decreased by a too luxurous growth of various species of aquatic plants. Weed control has to be carried out at least once every year. The old-fashioned hand-cutting has nearly been abandoned, due to shortage of manpower and high costs. For the same reasons mechanical weed control methods are not very popular everywhere. Possibilities for chemical control of aquatic weeds in Dutch waterways are restricted.The grass carp (Ctenopharyngodon idella) could offer an inexpensive biological alternative. Experiments showed, that this fish is an efficient weedcontrol agent under Dutch circumstances. It is presumed, that its impact on various functions of the surface water is less (or at least less rigorous) than that of modern mechanical or chemical methods. Still it is felt, that this impact (side-effects) should be investigated thoroughly before introduction of this exotic species into our aquatic environment. For this reason a Working Party was formed within the framework of the Dutch Agricultural Research Council, section Weed Research.Preliminary results indicate that the grass carp does not eradicate plant-species; in the experiments remnants of the original vegetation remained, so that recovery was possible. Furthermore the macrophytic diversity was only slightly decreased. These observations indicate that the grass carp shows very little selectivity in type of food and in space. From a biological point of view this is rather ideal for any weed control agent. Up till now no clear influence on the composition or quantity of the microflora was found.The quantity of macrofauna and macrobenthos decreased in grass carp plots, for unknown reasons, but the rate of diversity does not differ from the rate of diversity in the control plots. Influence of grass carp stocking on growth, survical and breeding of endemic fishes will be studied in the coming years.     

Cloning of the grass carp growth hormone cDNA

We have constructed a cDNA library in lamda gt11 using mRNA isolated from the pituitary glands of the grass carp (Ctenopharyngodon idellus). Based on the published sequence of the rainbow trout growth hormone cDNA, we synthesized two oligonucleotide probes. One of these hybridized strongly with a specific mRNA fragment from the grass carp pituitary. Using this probe, we have isolated six positive clones carrying an insert of approximately 1.2 Kb. By restriction enzyme digestion, all the clones were determined to be identical. Sequence determination on one of them indicated that it has an open reading frame coding for 210 amino acids. Both the nucleotide and translated amino acid sequence are highly homologous to those of the salmon growth hormone and the common carp. A putative signal peptide consisting of hydrophobic amino acids can be identified at the 5' end of the sequence. A polyadenylation signal, ATTAAA, was also present 12 base upstream from the poly A tail.     

草鱼前体脂肪细胞的原代培养

构建脂肪细胞体外培养体系,研究脂肪细胞分化过程,是探讨动物脂质代谢规律的重要手段.目前,哺乳动物方面,自20世纪60年代起至今,国内外已成功构建了大鼠[1,2]、人[3,4]、牛[5,6]和猪[7,8]的前体脂肪细胞体外培养模型,鱼类方面的工作则开始不久.     

草鱼、鲢和瓦氏黄颡鱼幼鱼感应流速的比较

在21±1℃水温下,使用自行制作的鱼类游泳行为测试槽,采用流速递增的方法测定了草鱼(Ctenopharyngodon idellus)(体长9.64±3.12 cm,体重20.97±16.71 g)、鲢(Hypophthalmichthys molitrix)(体长10.60±3.45 cm,体重24.37±18.61 g)和瓦氏黄颡鱼(Pelteobagrus vachelli)(体长11.54±1.46 cm,体重20.98±6.94 g)幼鱼个体的感应流速.结果表明:草鱼、鲢和瓦氏黄颡鱼幼鱼的感应流速分别是(8.21±0.68)、(6.74±0.84)和(33.11±3.06) cm·s-1,相对感应流速分别是(0.91±0.22)、(0.67±0.15)和(2.9±0.29) BL·s-1;鲢和草鱼幼鱼的感应流速差异不显著(P＞0.05),但瓦氏黄颡鱼比草鱼和鲢幼鱼感应流速大近4倍,差异显著(P＜0.05);因此,草鱼和鲢幼鱼较瓦氏黄颡鱼幼鱼对流速的感应更敏感.本实验结果可为设计鱼道、确定拖网速度等提供一定的科学指导.     

Differential expression of two C‐type lectins in grass carp Ctenopharyngodon idella and their response to grass carp reovirus

The cDNAs of two C‐type lectins in grass carp Ctenopharyngodon idella, galactose‐binding lectin (galbl) and mannose‐binding lectin (mbl), were cloned and analysed in this study. Both of them exhibited the highest expression level in liver, whereas their expression pattern differed in early phase of embryonic development. Following exposure to grass carp reovirus (GCRV), the mRNA expression level of galbl and mbl was significantly up‐regulated in liver and intestine.     

Molecular characterization of glutathione peroxidase gene from the liver of silver carp, bighead carp and grass carp

The cDNAs encoding glutathione peroxidase (GPx) were cloned and sequenced from the liver of three Chinese carps with different tolerance to hepatotoxic microcystins, phytoplanktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis), and herbivorous grass carp (Ctenopharyngodon idellus). Using genome walker method, a 750 bp 5'-flanking region of the silver carp GPx gene was obtained, and several potential regulatory elements were identified in the promoter region of the GPx gene. The silver carp GPx gene was widely expressed in all tissues examined. Despite phylogenetic analysis, assigning this newly described carp GPx to the group of mammalian GPx2, the carp GPx seems more similar to GPx1 from a physiological point of view. The constitutive expression pattern of the three carp liver GPx gene, shows a positive relationship with their tolerance to microcystins.     

Comparison of early developmental stages and adults of grass carp, Ctenopharyngodon idella, and hybrid carp (female grass carp × male bighead Aristichthys nobilis)

Hybrid carp and grass carp from the zygote to the prolarval stage and adults were compared to establish distinguishing characteristics. Zygote diameter, yolk sac and embryo length were not different between the fishes. Body pigmentation patterns showed distinct separation among the prolarvae of the hybrids and grass carp. Total and preanal myomeres were higher in grass carp larvae than in the hybrids. The grass carp and hybrid larvae also differed in 38% of the proportional morphometric characters.
Average meristic counts of adults were significantly higher in hybrids compared with grass carp. Adults of the two fishes showed significant differences in 63% of the proportional morphometric measurements. Gill raker counts showed no overlap and lateral line scales were smaller and more numerous in the hybrid adults.     

Feeding behaviour of yearling and older hybrid grass carp

Hybrid grass carp resulting from the cross of a female grass carp, Ctenopharyngodon idella (Val.), and a male bighead carp, Hypophthalmichthys ( Aristichthys ) nobilis Rich., 12–18 months old ( c . 300 mm T.L.) were studied in a two-part experiment to determine feeding preference and total daily consumption fish^-1 on selected species of aquatic plants. Fish were maintained in circular pools with 6840·8 1 of water inside a temperature-controlled greenhouse. Preference tests were conducted at three temperature ranges; 25–28° C, 17–20° C and 12–15° C. Based on the time to complete consumption or the relative quantity consumed, the most preferred plant was Lemna gibba when in combination with six other species. Chara sp., Najas guadalupensis and Potamogeton peciinatus were readily consumed and considered to be of about equal preference. Ceratophyllum demersum and Myriophyllum brasiliense were least preferred. Hybrid grass carp generally consumed as much plant material species^-1 and in the same order of preference at the 12–15°C range as they did at 25–28° C. In the second part, mean daily consumption (g) fish^-1 at 25·7–31·0° C for five plant species tested separately was as follows: Chara sp. 369·8; Lemna gibba 178·2; Najas guadalupensis 172·6; Hydrilla verticillata 106·4 and Ceratophyllum demersum 8·8.     

Purification and characterization of grass carp mitochondrial aldehyde dehydrogenase

The molecular biology and enzymology of aldehyde dehydrogenase (ALDH) have been extensively investigated. However, most of the studies have been confined to the mammalian forms, while the sub-mammalian vertebrate ALDHs are relatively unexplored. In the present investigation, an ALDH was purified from the hepatopancreas of grass carp (Ctenopharygodon idellus) by affinity chromatographies on α-cyanocinnamate-Sepharose and Affi-gel Blue agarose. The 800-fold purified enzyme had a specific activity of 4.46 U/mg toward the oxidation of acetaldehyde at pH 9.5. It had a subunit molecular weight of 55 000. Isoelectric focusing showed a single band with a pI of 5.3. N-terminal amino acid sequencing of 30 residues revealed a positional identity of ∼70% with mammalian mitochondrial ALDH2. The kinetic properties of grass carp ALDH resembled those of mammalian ALDH2. The optimal pH for the oxidation of acetaldehyde was 9.5. The K_m values for acetaldehyde were 0.36 and 0.31 μM at pH 7.5 and 9.5, respectively. Grass carp ALDH also possessed esterase activity which could be activated in the presence of NAD⁺.     

Purification and characterization of grass carp mitochondrial aldehyde dehydrogenase

The molecular biology and enzymology of aldehyde dehydrogenase (ALDH) have been extensively investigated. However, most of the studies have been confined to the mammalian forms, while the sub-mammalian vertebrate ALDHs are relatively unexplored. In the present investigation, an ALDH was purified from the hepatopancreas of grass carp (Ctenopharygodon idellus) by affinity chromatographies on alpha-cyanocinnamate-Sepharose and Affi-gel Blue agarose. The 800-fold purified enzyme had a specific activity of 4.46 U/mg toward the oxidation of acetaldehyde at pH 9.5. It had a subunit molecular weight of 55000. Isoelectric focusing showed a single band with a pI of 5.3. N-terminal amino acid sequencing of 30 residues revealed a positional identity of approximately 70% with mammalian mitochondrial ALDH2. The kinetic properties of grass carp ALDH resembled those of mammalian ALDH2. The optimal pH for the oxidation of acetaldehyde was 9.5. The K(m) values for acetaldehyde were 0.36 and 0.31 microM at pH 7.5 and 9.5, respectively. Grass carp ALDH also possessed esterase activity which could be activated in the presence of NAD(+).     

寄生在鲤鱼及草(鲩)鱼的鱊头槽绦虫的多样性

研究了寄生于鲤鱼及草(鲩)鱼的鱊头槽绦虫(Bothriocephalus acheiloghathi)的多样性.作者在1986-2000年收集材料,遍及中国19个省及自治区.在江苏等5省只收集到其它寄生虫的材料,故结果未列入附录1和2.研究结果表明,鲤鱼及草鱼的寄生鱊头槽绦虫的地理分布截然不同.寄生于鲤鱼的鱊头槽绦虫分布于黄河水系的西北地区及黑龙江水系的东北各省及长江上游的四川、云南和贵州,南方各省的鲤鱼不感染这种绦虫.寄生于草鱼的鱊头槽绦虫分布于沿珠江水系的广东、广西及闽江水系的福建,感染池塘的幼龄草鱼,天然水体中未发现感染阳性的草鱼.交义感染试验表明,寄生在这两种鲤科鱼类的头槽绦虫有极强的宿主特异性,草鱼感染的头槽绦虫一年内大部分时间排出发育完全的胚胎卵,而鲤鱼感染的头槽绦虫排出的虫卵多为早期分裂卵,甚至在盛夏,水温在28℃-30℃的情况下也无例外.虫卵发育程度不同,卵的孵化期也有差异.在同等温度28℃-29℃时,草鱼头槽绦虫虫卵的孵化期为1.69±0.17 d;鲤鱼的头槽绦虫虫卵的孵化期为3.98±0.3 d.此外,种群结构也有明显区别,草鱼的头槽绦虫在繁殖季节,体长＜1 cm的幼虫占种群的主体,年终时绦虫全部从宿主体内消失,生活周期从初染至消敛约1年;鲤鱼的头槽绦虫主体全年皆为孕节成虫,在Ⅱ龄商品鱼中继续繁殖.本研究表明,鲤鱼及草鱼的寄生鱊头槽绦虫显示的多样性是因长期适应不同生活环境从而演化为两个不同的亚种[动物学报 53(3):470-480,2007].     

A 15 nucleotide deletion mutation in coding region of the RIG-I lowers grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus

RIG-I (Retinoic acid-inducible gene I) is a pivotal receptor that detects numerous RNA and DNA viruses and plays crucial roles in the induction of type I interferons. In the present study, a deletion mutation in CiRIG-I (Ctenopharyngodon idella RIG-I) coding region was detected, its association with resistance/susceptibility to grass carp reovirus (GCRV) was examined, and possible mechanism was analyzed. A 15-bp deletion mutation was found, and the mutation results in a deletion of five amino acids. To investigate the genotypes and alleles, the relevant PCR products were electrophoresed on 2.5% agarose gel. Three genotypes and two alleles were discovered. The general allele was named as A and the deletion mutation allele was named as B. The deletion mutation cancels a predicted phosphorylation site and changes the secondary structure and the probability of peroxisomal targeting signal 1 in CiRIG-I. To explore the correlation between these genotypes and the resistance of grass carp to GCRV, a challenge experiment was carried out. The cumulative mortality in genotype AA (40.70%) and AB (52.73%) was significantly lower than that in genotype BB (71.43%) (P = 0.032). The result demonstrated that genotype AA and AB were resistant to GCRV, while genotype BB was susceptible. The 15-bp deletion mutation lowers the resistance of grass carp to GCRV. This result might provide a potential genetic marker for further investigation of selective breeding of resistant grass carp to GCRV.     

Zooplankton abundance and diversity in Central Florida grass carp ponds

The effect of the Asian grass carp (Ctenopharyngodon idella Val.) upon the zooplankton in three adjacent experimental ponds (0.139 ha each) was studied for one year. The ponds contained nine species of aquatic macrophytes. Grass carp were stocked into Pond 1 (65 per ha) and Pond 2 (611 per ha) three months after the study was started. At the time of stocking, physichochemical and biological parameters were similar among the ponds.Grass carp did not affect water quality and had little direct or indirect effect upon the zooplankton in the ponds. The abundance and species diversity of zooplankton (number of species, Shannon Index, and Simpson Index) were not significantly different (P < 0.05) between ponds prior to grass carp stocking. After stocking with grass carp, the number of species and species diversity were found to be significantly different (P < 0.05) between Pond 1 (low grass carp stocking rate) and Pond 2 (high grass carp stocking rate). Additionally, significant differences (P < 0.05) in populations between ponds were found for Lecane luna, Monostyla bulla, Lepadella ehrenbergii, Polyarthra sp., and Synchaeta sp. Temporal variation rather than grass carp was probably responsible for those differences. The number of zooplankton per group (Rotifera, Cladocera, and Copepoda) and species diversity were not significantly different (P<0.05) between ponds containing different stocking rates of grass carp.During the study, zooplankton were collected with a shallow-water sampler. No significant differences (P < 0.05) between collections were found for the sampler and a No. 20 mesh nylon zooplankton net.