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.     

Sequential ultrastructural and biochemical changes induced in vivo by the hepatotoxic microcystins in liver of the phytoplanktivorous silver carp Hypophthalmichthys molitrix

Up to now, in vivo studies on the toxic effects of microcystins (MCs) on the ultrastructures of fish liver have been very limited. The phytoplanktivorous silver carp was injected i.p. with extracted hepatotoxic microcystins (mainly MC-RR and -LR) at a dose of 1000 microg MC-LReq. kg(-1) body weight, showing a time-dependent ultrastructural change in liver as well as significant increases in enzyme activity of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). We observed for the first time the occurrence of a large amount of activated secondary lysosomes, which might be an adaptive mechanism to eliminate or lessen cell damage caused by MCs through lysosome activation. Quantitative and qualitative determinations of MCs in the liver were conducted by HPLC and LC-MS2, respectively. MCs concentration in the liver reached the maximum (114.20 microg g(-1) dry weight) after 3 h post-injection, and then rapidly dropped to 7.57 microg g(-1) dry weight at 48 h, indicating a depuration of 99% accumulated MC-LReq. On the other hand, a decrease trend in glutathione (GSH) concentration was observed in the liver of silver carp while the activity of glutathione S-transferase (GST) increased significantly after injection. The high tolerance of silver carp to MCs might be due to the high basic GSH level in their liver, and/or an increased GSH synthesis.     

鲢鱼可溶性谷胱甘肽S-转移酶(sGST)基因cDNA全序列与5′调控区的克隆与分析

淡水鱼类可溶性谷胱甘肽S-转移酶(sGST)在微囊藻毒素去毒代谢过程中具有独特 的关键作用,因而也称为微囊藻毒素去毒酶. 从淡水食毒藻鱼类鲢鱼(Hypophthalmichthys molitrix)肝脏通过简并引物克隆微囊藻毒素去毒酶基因cDNA核心序列,应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列,最后通过序列拼接获得鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全序列. 序列分析结果表明,鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全长920 bp,其中5′-UTR长74 bp,3′-UTR长174 bp,编码区长672 bp,编码223个氨基酸. 应用基因组步行法,在鲢鱼克隆得到淡水鱼类微囊藻毒素去毒酶基因5′侧翼区878 bp序列. 与哺乳动物及海水鱼sGST基因不同,鲢鱼微囊藻毒素去毒酶基因的5′侧翼区,发现存在多个脂多糖反应元件(LPSRE),表明来源于毒藻的脂多糖可能对鲢鱼微囊藻毒素去毒酶基因表达有潜在调控作用.     

Effect of total dissolved gas supersaturation on the survival of common carp (Cyprinus carpio) and silver carp (Hypophthalmichthys molitrix)

Flood discharge results in total dissolved gas (TDG) supersaturation downstream of a dam during the flood period. Fish suffer death from gas bubble disease (GBD) caused by TDG supersaturation. Nonetheless, current studies mainly attach importance to the survival of benthic fish affected by TDG supersaturation in the Yangtze River in China. Few studies have attempted to investigate the survival of pelagic fish influenced by TDG supersaturated water and compare the tolerance characteristics to TDG supersaturation between benthic and pelagic fish. To identify the survival of fish species that inhabit the various water layers affected by TDG supersaturation, silver carp (Hypophthalmichthys molitrix) (pelagic fish) and common carp (Cyprinus carpio) (benthic fish) were chosen to conduct an acute exposure experiment of four different TDG supersaturation levels (125%, 130%, 135% and 140%). The findings illustrated that the two fish species both exhibited evident aberrant behaviours of maladjustment in TDG supersaturated water. Obvious GBD symptoms were also found in the test fish. The survival probability of silver carp and common carp decreased with increasing levels of TDG supersaturation. The median survival time (ST₅₀) values of the silver carp exposed to four levels of TDG supersaturated water (125%, 130%, 135% and 140%) were 26.84, 7.96, 5.56 and 3.62 h, respectively, whereas the ST₅₀ values of common carp were 53.50, 26.00, 16.50 and 11.70 h, respectively. When compared with common carp, silver carp had a weaker tolerance to TDG-supersaturated water and were vulnerable to GBD. It shows that levels above 125% are not safe for common carp survival. In terms of the tolerance threshold value, silver carp merits further investigation because it showed lower tolerance to TDG than did common carp.     

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.     

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.     

Significant genetic differentiation between native and introduced silver carp (<Emphasis Type="Italic">Hypophthalmichthys molitrix</Emphasis>) inferred from mtDNA analysis

Silver carp Hypophthalmichthys molitrix (Cyprinidae) is native to China and has been introduced to over 80 countries. The extent of genetic diversity in introduced silver carp and the genetic divergence between introduced and native populations remain largely unknown. In this study, 241 silver carp sampled from three major native rivers and two non-native rivers (Mississippi River and Danube River) were analyzed using nucleotide sequences of mitochondrial COI gene and D-loop region. A total of 73 haplotypes were observed, with no haplotype found common to all the five populations and eight haplotypes shared by two to four populations. As compared with introduced populations, all native populations possess both higher haplotype diversity and higher nucleotide diversity, presumably a result of the founder effect. Significant genetic differentiation was revealed between native and introduced populations as well as among five sampled populations, suggesting strong selection pressures might have occurred in introduced populations. Collectively, this study not only provides baseline information for sustainable use of silver carp in their native country (i.e., China), but also offers first-hand genetic data for the control of silver carp in countries (e.g., the United States) where they are considered invasive.     

Hypoxia and recovery perturb free radical processes and antioxidant potential in common carp (Cyprinus carpio) tissues

The effects of hypoxia exposure and subsequent normoxic recovery on the levels of lipid peroxides (LOOH), thiobarbituric acid reactive substances (TBARS), carbonylproteins, total glutathione levels, and the activities of six antioxidant enzymes were measured in brain, liver, kidney and skeletal muscle of the common carp Cyprinus carpio. Hypoxia exposure (25% of normal oxygen level) for 5h generally decreased the levels of oxidative damage products, but in liver TBARS content were elevated. Hypoxia stimulated increases in the activities of catalase (by 1.7-fold) and glutathione peroxidase (GPx) (by 1.3-fold) in brain supporting the idea that anticipatory preparation takes place in order to deal with the oxidative stress that will occur during reoxygenation. In liver, only GPx activity was reduced under hypoxia and reoxygenation while other enzymes were unaffected. Kidney showed decreased activity of GPx under aerobic recovery but superoxide dismutase (SOD) and catalase responded with sharp increases in activities. Skeletal muscle showed minor changes with a reduction in GPx activity under hypoxia exposure and an increase in SOD activity under recovery. Responses by antioxidant defenses in carp organs appear to include preparatory increases during hypoxia by some antioxidant enzymes in brain but a more direct response to oxidative insult during recovery appears to trigger enzyme responses in kidney and skeletal muscle.     

Cloning and characterization of the carp prolactin gene

A carp genomic DNA clone containing the carp prolactin (Prl) gene was isolated with carp Prl cDNA as a probe. The organization of the carp Prl gene was determined by restriction nuclease mapping and nucleotide sequencing. The Prl gene comprises approx. 2.8 kilobasepairs (kb) of DNA including the 5'-flanking region, five exons, four introns and the 3'-flanking region. Analysis of the 5'-flanking region reveals (1) the sequence TATATAAT at positions -38 to -31 upstream from the cap site which was found to be a guanine residue, and (2) the palindrome, CTCATTGCATATACAAATGAG at positions -79 to -59. The carp Prl gene matches with the reported cDNA except for one difference in coding region and five in the 3'-flanking region, while the encoded amino acid sequences are identical. The arrangement of exons and introns is very similar to that seen in carp GH as well as mammalian Prl, which, however, have much longer introns.     

Cloning, characterization and expression analysis of interleukin-10 from the common carp, Cyprinus carpio L.

Interleukin (IL)-10 was cloned from the common carp (Cyprinus carpio L.) using IL-10 primers from carp head kidney following stimulation with concanavalin A and lipopolysaccharide. The cDNA consisted of a 1096 bp sequence containing a 55 bp 5' untranslated region and a 498 bp 3' untranslated region. An open reading frame of 543 bp encoded a putative 180 amino acid protein with a putative signal peptide of 22 amino acids. The signature motif of IL-10 is conserved in carp sequence. A 2083 bp genomic sequence of carp IL-10 was found to contain five exons interrupted by four introns. With the exception of much more compact introns, the genomic structure was similar to that of mammalian IL-10. By homology, phylogeny and genomic analyses, the carp gene cloned was designated as IL-10. Carp IL-10 was expressed in head, kidney, liver, spleen and intestine during the resting phase. The gene was also expressed in head kidney and liver following in vitro stimulation with lipopolysaccharide.     

Swimming performance of adult bighead carp Hypophthalmichthys nobilis (Richardson, 1845) and silver carp H. molitrix (Valenciennes, 1844)

Although the movement of invasive bighead carp (Hypophthalmichthys nobilis) and silver carp (H. molitrix) in the Upper Mississippi River system is dependent on their ability to swim through its numerous lock‐and‐dams, the swimming performance of adults of these species is at present unknown. Using a large (2,935‐L) mobile swim tunnel, the swimming performance of adult bighead and adult silver carp was quantified at water velocities that challenged them to exhibit either prolonged and/or burst swimming (76–244 cm/s) with fatigue times of less than 10 min. Simple log‐linear models best described the relative swim speed to fatigue relationships for both species. Under these conditions, the swimming performances of adult bighead and silver carp were similar to several species of adult fishes native to the Mississippi River system, but relatively low (<3 total body lengths per second, TL/s) compared to previously studied juveniles and sub‐adult bigheaded carps (3–15 TL/s). The decline in endurance with water velocity was three times greater in bighead carp (slope = ?2.98) than in silver carp (slope = ?1.01) and the predictive ability of the bighead model was appreciably better than the silver carp model. The differences in adult swimming performance between the two species were coincident with behavioral differences (e.g. breaching in silver carp but not in bighead carp). The swimming performance data of adult bighead and silver carp can now be used to evaluate whether their passage through manmade river structures including the gates of lock‐and‐dams in the Upper Mississippi River might be reduced.     

Gut milieu shapes the bacterial communities of invasive silver carp

Silver carp is an invasive fish present in the Gobindsagar reservoir, India and has a profound impact on aquaculture. Understanding taxonomic diversity and functional attributes of gut microbiota will provide insights into the important role of bacteria in metabolism of silver carp that facilitated invasion of this exotic species. Microbial composition in foregut, midgut, hindgut and water samples was analysed using 16S rRNA gene amplicon sequencing. The bacterial communities of water samples were distinct from gut microbiota, and unique microbial assemblages were present in different regions of gut depicting profound impact of gut environment on microflora. Proteobacteria was the most abundant phyla across all samples. Ecological network analysis showed dominance of competitive interactions within posteriors region of the gut, promoting niche specialization. Predictive functional profiling revealed the microbiota specialized in digestive functions in different regions of the gut, which also reflects the dietary profile of silver carp.     

清河水库鲢、鳙鱼种群动态研究Ⅰ.生产量的估计

本文根据Robson-Chapman公式估算了清河水库鲢、鳙可捕群体的残存率,计算了非捕捞群体的残存率和种群生产量。清河水库非捕捞群体平均年残存率约为0.25,可捕群体的平均年残存率鲢为0.32、鳙为0.38。鲢、鳙鱼种群生产量分别为141和110kg/ha,种群年P/B系数分别为1.15和1.09。鲢、鳙鱼获量仅为其生产量的33%和34%,库存生物量的36%。     

Expression of the polymeric Immunoglobulin Receptor (pIgR) in mucosal tissues of common carp (Cyprinus carpio L.)

The mucosal immune system seems to be an important defence mechanism for fish but the binding of IgM in mucosal organs is poorly described in fish. In this study the gene encoding the polymeric Immunoglobulin Receptor (pIgR) in carp has been isolated and sequenced from a liver cDNA-library and aligned with other species. The pIgR of carp consists of 2 Ig domains, a transmembrane and an intracellular region, together 327 amino acids. In situ hybridisations with sense and anti-sense DIG-labelled pIgR RNA probes were performed on liver, gut and skin of common carp (Cyprinus carpio L.) and in these organs only anti-sense probes were found to hybridise. In liver the majority of hepatocytes was stained around the nucleus. In gut and skin, staining could be detected around the nucleus of the epithelial cells, but in gut also a subpopulation of lymphoid cells was stained in epithelium and lamina propria. The specific in situ hybridisation of the epithelia and hepatocytes coincides with the in situ binding of FITC-labelled carp IgM to the same cells. RT-PCR results indicate the expression of the pIgR gene in all lymphoid organs of carp, but not in muscle. Macrophages/neutrophils enriched by adherence or sorted B cells (MACS) did not show expression of the pIgR gene and are excluded as the pIgR expressing lymphoid cells in the intestine. The relevance of pIgR staining and gene expression in mucosal organs is discussed.     

Development of silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) genetic maps using microsatellite and AFLP markers and a pseudo-testcross strategy

Silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) are two of the four most important pond-cultured fish species inhabiting the major river basins of China. In the present study, genetic maps of silver carp and bighead carp were constructed using microsatellite and AFLP markers and a two-way pseudo-testcross strategy. To create the maps, 60 individuals were obtained from a cross of a single bighead carp (female) and a single silver carp (male). The silver carp map consisted of 271 markers (48 microsatellites and 223 AFLPs) that were assembled into 27 linkage groups, of which 22 contained at least four markers. The total length of the silver carp map was 952.2 cM, covering 82.8% of the estimated genome size. The bighead carp map consisted of 153 markers (27 microsatellites and 126 AFLPs) which were organized into 30 linkage groups, of which 19 contained at least four markers. The total length of the bighead carp map was 852.0 cM, covering 70.5% of the estimated genome size. Eighteen microsatellite markers were common to both maps. These maps will contribute to discovery of genes and genetic regions controlling traits in the two species of carp.     

Unusual structure of the biliary system in the liver of the grass carp and the silver carp

It is known that the bile canaliculus in the liver of almost all vertebrates is made up of membranes of two or more adjacent liver cells. Studying the liver cell ultrastructure of lasting and fed grass carp and silver carp, it was demonstrated that a bile canaliculus is formed by deep invagination of a cell membrane of one hepatocyte. The membrane forms microvilli along the bile canaliculus. The bile canaliculus is seen in the centre of liver cell cytoplasm on the cross section and stretches from the centre of the liver cell cytoplasm to the cell membrane on the longitudinal section. The bile canaliculus is connected with a small duct cell, which is distinct from a liver cell in its small size, little amount of cell organelles and the presence of cytoplasmic filaments. The terminal part of the biliary tract consists of one liver cell and one bile duct cell. The part of the tract adjacent to the terminal one is composed of two or three small bile duct cells devoid of basal membrane. Thus, the liver parenchyma is constituted of a net of numerous bile ducts. In the portal tract, there is a large bile duct, consisting of 12-13 bile duct cells, surrounded by basal membrane and connective tissue cells.     

草鱼免疫相关基因Prkrip1的克隆和表达分析

随着草鱼养殖规模的扩大, 草鱼的病毒性疾病极大地影响着草鱼的产量。开展鱼类病毒免疫反应相关功能基因的研究意义重大。研究首先通过同源克隆的方法从草鱼中克隆到了一段Prkrip1基因的EST序列, 进一步通过RACE、长片段PCR和Genome walking的方法获得了该基因的全长cDNA序列、基因组DNA序列和启动子区序列。氨基酸序列分析显示, Prkrip1含有3个核定位信号和一个双链RNA结合区, 并具有与PKR结合的保守N端区; 荧光报告基因的表达证实我们所克隆到的启动子区是有活性的, 可用于后续该基因的转录调控分析; Real-time PCR分析发现, Prkrip1 基因在草鱼的肝和血中表达量最高, GCRV感染后在大部分免疫组织中均上调表达, 说明该基因确实与病毒感染相关。研究结果为Prkrip1基因在硬骨鱼类的功能研究提供了线索, 也为鱼类天然免疫反应中调控PKR信号通路的系统研究提供了理论依据。       

Comparative study on the gut microbiotas of four economically important Asian carp species

Gut microbiota of four economically important Asian carp species(silver carp, Hypophthalmichthys molitrix; bighead carp,Hypophthalmichthys nobilis; grass carp, Ctenopharyngodon idella; common carp, Cyprinus carpio) were compared using 16 S rRNA gene pyrosequencing. Analysis of more than 590,000 quality-filtered sequences obtained from the foregut, midgut and hindgut of these four carp species revealed high microbial diversity among the samples. The foregut samples of grass carp exhibited more than 1,600 operational taxonomy units(OTUs) and the highest alpha-diversity index, followed by the silver carp foregut and midgut. Proteobacteria, Firmicutes, Bacteroidetes and Fusobacteria were the predominant phyla regardless of fish species or gut type. Pairwise(weighted) UniFrac distance-based permutational multivariate analysis of variance with fish species as a factor produced significant association(P0.01). The gut microbiotas of all four carp species harbored saccharolytic or proteolytic microbes, likely in response to the differences in their feeding habits. In addition, extensive variations were also observed even within the same fish species. Our results indicate that the gut microbiotas of Asian carp depend on the exact species, even when the different species were cohabiting in the same environment. This study provides some new insights into developing commercial fish feeds and improving existing aquaculture strategies.