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.     

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.     

3种不同食性淡水鱼类谷胱甘肽S-转移酶Pi、Mu、Theta型cDNA序列的克隆分析及表达

鱼类谷胱甘肽S-转移酶(glutathione S-transferase,GST)是鱼类一种重要的Ⅱ相去毒酶,在催化毒素与还原谷胱甘肽(GSH)加合去毒代谢过程中具有关键作用。采用RT-PCR及RACE法,分离、克隆得到草鱼、尼罗罗非鱼pi、mu、theta型GST(GSTpi、GSTmu、GSTtheta)基因、鲢鱼GSTmu、GSTtheta基因的cDNA部分序列并推测各自对应的氨基酸序列。氨基酸序列同源性比较和系统进化分析均表明,鲢鱼、草鱼、尼罗罗非鱼与鱼类GST同源性较高,与哺乳类、鸟类、两栖类GST同源性较低,可能与鱼类GST基因在水环境毒素去毒代谢中承担的特殊功能有关。而不同种鱼类GSTtheta的同源性明显要较GSTpi、GSTmu的同源性低,可能与不同淡水鱼类食性及对毒素耐受性不同有关。用实时荧光定量PCR(RT-PCR)检测三种鱼肝脏中三型GST基因组成型表达水平,发现三种鱼各型之间皆有一定差异,尼罗罗非鱼肝脏整体GSTs基因表达很低,GSTtheta显著低于草鱼(P<0.05),GSTmu显著低于鲢鱼(P<0.05)。本研究为从分子水平上研究不同型谷胱甘肽S-转移酶基因在不同食性淡水鱼类体内代谢去毒过程中的作用提供了基础。     

Hepatic and branchial glutathione S-transferases of two fish species: Substrate specificity and biotransformation of microcystin-LR

Liver and gills of roach (Rutilus rutilus) and silver carp (Hypophthalmichthys molitrix) were examined for glutathione S-transferases (GSTs) contents and their substrate specificity and capacity to biotransform microcystin-LR (MC-LR). GSTs and other glutathione (GSH) affine proteins were purified using a GSH-agarose matrix and separated by anionic chromatography (AEC). Substrate specificities were determined photometrical for 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), 4-nitrobenzyl chloride (pNBC) and ethacrynic acid (ETHA). Biotransformation rate of MC-LR was determined by high performance liquid chromatography (HPLC). Roach exhibited different hepatic and branchial GST activities for used substrates (DNB, pNBC and DCNB) compared to silver carp but not for ethacrynic acid. It suggests that, both fish species have similar amount of pi and/or alpha class, which were the dominant GST classes in liver and gills. Gills of both fish species contained a higher number of GST isoenzymes, but with lower activities and ability of MC-LR biotransformation than livers. GST isoenzymes from roach had higher activity to biotransform MC-LR (conversion rate ranging up to 268 ng MC-LR min^? 1 mL^? 1 hepatic enzyme) than that isolated from silver carp. Without any prior contact to MC-LR or another GST inducer, roach seems to be better equipped for microcystin biotransformation than silver 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.     

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.     

微囊藻毒素与细菌内毒素对草鱼淋巴细胞凋亡的联合毒效应

采用体外暴露方式,以草鱼淋巴细胞为试验对象,研究了水体中微囊藻毒素(MC-LR)与细菌内毒素(LPS)复合作用对鱼免疫系统的影响.结果表明： MC-LR和LPS在单一与复合暴露下都能够诱导草鱼淋巴细胞发生凋亡,呈现细胞凋亡典型的阶梯状DNA电泳特征.但对比复合暴露与单一暴露的凋亡率可以发现,MC-LR与LPS复合暴露会发生协同作用,并呈显著剂量-效应关系,其中复合暴露Ⅳ组凋亡率为单一暴露Ⅰ(MC-LR)和Ⅱ(LPS)组的2-1和3.3倍.MC-LR可协同LPS抑制谷胱甘肽硫转移酶(GST)活性,引发细胞内活性氧(ROS)和丙二醛(MDA)水平上升,导致DNA损伤,致使淋巴细胞阻滞于G0期,增殖受到明显抑制,加速鱼淋巴细胞的凋亡,提高细胞凋亡率.表明MC-LR能够协同LPS加剧其对鱼体免疫细胞毒性,对水产养殖业产生严重不利影响.     

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.     

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。     

草鱼IRE1-like基因的克隆、组织表达及其对微囊藻毒素-LR的响应

为了研究内质网应激相关基因需肌醇酶1(Inositol-requiring enzyme 1, IRE1-like)的结构和生物学功能及其在草鱼(Ctenopharyngodon idella)响应微囊藻毒素-LR(MC-LR)中的作用,研究根据草鱼转录组测序结果得到该基因家族成员IRE1-like的EST序列,采用RACE技术获得了草鱼IRE1-like基因的cDNA全长序列(登录号:MG797683)。该基因序列全长3595 bp,包括3093 bp开放阅读框,编码1030个氨基酸,分子量为116.24 kD,理论等电点为6.26,具有跨膜结构和信号肽。草鱼IRE1-like基因包含Luminal (39—307 aa)、STKc (569—834 aa)和RNase (837—915 aa)三个结构域。同源性和系统进化树结果表明草鱼IRE1-like基因与斑马鱼IRE1-α基因亲缘关系最近。荧光定量PCR(qRT-PCR)检测结果表明,草鱼IRE1-like基因在肝脏组织中的表达量最高,在肌肉、脾脏、鳃和心脏等其他8种不同组织中也均有表达。不同剂量MC-LR诱导草鱼24h和96h...     

Social facilitation of learning in mixed-species schools of common carp Cyprinus carpio L. and Nile tilapia Oreochromis niloticus (L.)

The process of learning to associate a visual cue with food was studied in groups of common carp Cyprinus carpio and Nile tilapia Oreochromis niloticus by means of a classical conditioning paradigm. In the first phase of the study, fishes were conditioned in either single-species or mixed-species groups to associate a blinking blue light with food. In the second phase, group composition was altered so that only single species were reconditioned, composed of individuals previously paired with the same or the other species. In the first phase, in the single-species groups, Nile tilapia rapidly formed an association between the visual cue and food, whereas common carp failed to do so. In mixed-species groups, both Nile tilapia and common carp associated a visual cue with food. In the second phase of the study, Nile tilapia retained that ability irrespective of whether they had previously been trained in single-species or mixed-species groups, common carp previously trained with Nile tilapia, were not repelled by the blinking blue light and gradually approached it from a distance, whereas common carp previously trained in homogenous groups continued to be repelled by the blinking light. Fish arrivals and departures from the feeding zones as well as fish position in the testing apparatus were analysed and contrasted in single-species and mixed-species groups outside the training sessions, revealing mutual impacts of one species on the other in mixed-species groups. The findings of this study demonstrated interspecific social facilitation of learning to associate a visual cue with food.     

Comparative antioxidant enzyme study in freshwater fishes. I. Distribution of superoxide dismutase, peroxide-decomposing enzymes and lipid peroxidation in herbivorous fishes

The activities of Cu,Zn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase were comparatively studied in organ homogenates from two herbivorous fishes, viz., the grass carp and the silver carp. 2. The protein contents and lipid peroxidation of organ homogenates were also compared. The comparative measurements primarily provide control values for subsequent toxicological examinations. 3. The highest total superoxide dismutase activities were found in the kidney, spleen and liver in the grass carp, and in the kidney and liver in the silver carp. 4. The antioxidant enzyme activities and other parameters of the organ homogenates appear to be independent of the feeding mode, but are rather characteristic of the fish variety.     

Control of bloom in a tropical lake: grazing efficiency of some herbivorous fishes

Using a limnocorral study, Microcystis grazing by silver carp, bighead and tilapia was examined in an eutrophic shallow lake permanently infested with a Microcystis bloom. Twelve fingerlings of each fish species were introduced into the limnocorrals placed in the lake, in triplicate, and their grazing effects were examined over a period until the limnocorral bloom declined substantially and remained unchanged. Introduction of fish, regardless of species, into the limnocorrals resulted in sharp reductions (60–93%) of the initial Microcystis population on day 3 or 7. Maximum gross and net clearance of Microcystis were by silver carp followed by bighead and tilapia. Short-term high retention of Microcystis by tilapia indicated that the clearance effect did not last long due to a high rate of defaecation of undigested Microcystis . Nearly 6–180% nutrient enrichment of the limnocorrals was attributable to the defaecation of test fishes suggesting their ichthyoeutrophication potentials, which were in the following order: tilapia>bighead>silver carp. It is concluded that silver carp is most suitable for clearing Microcystis in the lake because of its minimal ichthyoeutrophication effect.     

The metabolic response in fish to mildly elevated water temperature relates to species-dependent muscular concentrations of imidazole compounds and free amino acids

Fish species show distinct differences in their muscular concentrations of imidazoles and free amino acids (FAA). This study was conducted to investigate whether metabolic response to mildly elevated water temperature (MEWT) relates to species-dependent muscular concentrations of imidazoles and FAA. Thirteen carp and 17 Nile tilapia, housed one per aquarium, were randomly assigned to either acclimation (25 °C) or MEWT (30 °C) for 14 days. Main muscular concentrations were histidine (HIS; P<0.001) in carp versus N-α-acetylhistidine (NAH; P<0.001) and taurine (TAU; P=0.001) in tilapia. Although the sum of imidazole (HIS+NAH) and TAU in muscle remained constant over species and temperatures (P>0.05), (NAH+HIS)/TAU ratio was markedly higher in carp versus tilapia, and decreased with MEWT only in carp (P<0.05). Many of the muscular FAA concentrations were higher in carp than in tilapia (P<0.05). Plasma acylcarnitine profile suggested a higher use of AA and fatty acids in carp metabolism (P<0.05). On the contrary, the concentration of 3-hydroxyisovalerylcarnitine, a sink of leucine catabolism, (P=0.009) pointed to avoidance of leucine use in tilapia metabolism. Despite a further increase of plasma longer-chain acylcarnitines in tilapia at MEWT (P=0.009), their corresponding beta-oxidation products (3-hydroxy-longer-chain acylcarnitines) remained constant. Together with higher plasma non-esterified fatty acids (NEFA) in carp (P=0.001), the latter shows that carp, being a fatter fish, more readily mobilises fat than tilapia at MEWT, which coincides with more intensive muscular mobilization of imidazoles. This study demonstrates that fish species differ in their metabolic response to MEWT, which is associated with species-dependent changes in muscle imidazole to taurine ratio.     

The impacts of invasive Nile tilapia (Oreochromis niloticus) on the fisheries in the main rivers of Guangdong Province,China

Nile tilapia (Oreochromis niloticus) is one of the most widespread invasive fish species, and this species has successfully established populations in the major rivers of Guangdong Province, China. Field surveys and manipulative experiments were conducted to assess the impacts of Nile tilapia on fisheries. We determined that the increase of Nile tilapia in these rivers not only affects the CPUE (catch-per-unit-per-effort) of the fish community and native fish species but also reduces the income of fishermen. In the manipulative experiments, we observed that the growth of native mud carp decreased in the presence of Nile tilapia. Our results suggest that the invasion of Nile tilapia negatively affected the fishery economy and native fish species, and suitable control measurements should be taken.     

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。       

The effect of rice-seeding rate and fish stocking on the floodwater ecology of the trench of a concurrent,direct-seeded rice-fish system

Concurrent rice-fish systems in the Mekong Delta, Vietnam, are characterized by a rice field surrounded by a trench (on average 1000 m², covering 15–20% of the field surface). Rice is direct seeded and fish are reared in polyculture. The most common species for polyculture are silver barb Barbodes gonionotus (Bleeker), common carp Cyprinus carpio L. and Nile tilapia Oreochromis niloticus (L.). In a 1996–1997 dry season experiment, the effects of (1) absence or presence of a fish polyculture, and (2) two different rice-seeding rates (100 and 300 kg pre-germinated rice per ha) on the trench floodwater ecology were studied. Fish were stocked in six out of twelve rice-fish plots at the Co Do experimental rice-fish station (10° 10 N and 105° 20 E). Silver barb, common carp and Nile tilapia were stocked in polyculture at densities of 3150, 310 and 550 fish/ha, respectively. Several hydrological and biological parameters of the trench floodwater were measured. About 65% of the observed variation in the trench floodwater could be attributed to the fishes stocked and changes in rice-seeding rate. The presence of a fish polyculture increased the water turbidity of the trench floodwater, mainly through suspension of mineral and organic material through fish perturbation. At the lower rice-seeding rate, this resulted in a lower primary production. Photosynthetic activity, dissolved oxygen am and pm showed significant decreases, but only at the lower rice-seeding rate in the presence of fish. Fish polyculture presence resulted in lower ortho-phosphate concentrations, higher phytoplankton communities and higher Protozoa numbers, most probably due to a higher availability of nutrients through fish defecation and perturbation. The rice-seeding rate had a significant impact on pH am, pH pm and temperature pm readings. The survival rate for Nile tilapia and the gross production for Nile tilapia and common carp was higher at the lower rice seeding rate, most probably due to a better aquatic environment of the field. In terms of fish production, the trench floodwater environment was better than the field floodwater. The trench is a very important part of the rice-fish system. It serves as a refuge and supplies reasonable amounts of phytoplankton and zooplankton to the fish.     

Predation by Nile perch Lates niloticus (L.) on Oreochromis niloticus (L.), Cyprinus carpio (L.), Mugil sp. and its role in controlling tilapia recruitment in Egypt

Vulnerability of three fish groups (tilapia, common carp, mullet) to predation by Nile perch Lates niloticus was evaluated. The study was conducted in aquaria and in cages placed in concrete ponds. Common carp Cyprinus curpio was the more vulnerable to predation by Nile perch followed in order by mullet ( Mugil cephulus and M. cupito ) and Nile tilapia Oreochromis niloricus . Medium-size Nile perch (30–32 cm t.l. ) were able to consume tilapia up to 14 cm t.l. , while tilapia of 15 cm total length were not eaten. Ratios of 1 : 10 to 1 : 15 between Nile perch and ready-to-spawn females of O. niloticus were found to be adequate to control tilapia reproduction.