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].     

Characterizations of four toll‐like receptor 4s in grass carp Ctenopharyngodon idellus and their response to grass carp reovirus infection and lipopolysaccharide stimulation

In this study, the subcellular localization, tissue distribution and response to grass carp reovirus (GCRV) infection and lipopolysaccharide (LPS) stimulation of four grass carp Ctenopharyngodon idellus toll‐like receptor 4 (tlr4) genes were investigated. All four genes were constitutively expressed in all tissues studied, but the subcellular localization and tissue exhibiting the highest expression differed for each protein. Following GCRV infection, all the four tlr4s were upregulated in all tissues examined, and stimulation of C. idellus kidney (CIK) cells with LPS resulted in downregulation of all four tlr4s. These results provide a foundation for further investigation of tlr4 genes in bony fishes.     

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.     

Characterization,expression analysis and localization pattern of toll‐like receptor 1 (tlr1) and toll‐like receptor 2 (tlr2) genes in grass carp Ctenopharyngodon idella

In this study, the toll‐like receptor 1 (tlr1) and toll‐like receptor 2 (tlr2) genes of grass carp Ctenopharyngodon idella were cloned and characterized. tlr1 and tlr2 were found to be highly expressed in immune system organs such as spleen, middle kidney and heart kidney. The expression level of tlr1 and tlr2 was found to be up‐regulated at the later stage of viral challenge process. Moreover, subcellular localization indicated that Tlr1 and Tlr2 shared similar localization pattern and both of them may locate in the plasma membrane of transfected cells.     

草鱼胶原凝集素基因的克隆及其功能分析

从草鱼Ctenopharyngodon idella肝肾cDNA文库中克隆得到胶原凝集素基因。草鱼胶原凝集素全长cDNA为1128bp,其中5′非编码区229bp,3′非翻译区104bp,最大开放阅读框为795bp,编码264个氨基酸。系统进化分析表明草鱼胶原凝集素与斑马鱼的亲缘关系最近。根据草鱼胶原凝集素序列特征,克隆了包含糖基识别域(CRD)的cDNA,并进行原核表达、纯化获得其重组蛋白PCRD。进行PCRD与6种细菌的凝集和糖抑制实验,结果表明半乳糖、葡萄糖、甘露糖和麦芽糖4种糖都会使PCRD与嗜水气单胞菌的凝集明显下降甚至极大地干扰凝集;麦芽糖使金黄色葡萄球菌的凝集明显下降,而肽聚糖和甘露糖会使凝集受到抑制;此外,PCRD的凝集反应不依赖Ca2+。     

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。       

不同生境草鱼肠道微生物组成和群落特征分析

【目的】分析不同生境来源的草鱼前肠、中肠和后肠的微生物组成和群落特征。【方法】利用16S rRNA高通量测序技术比较河流、湖泊、高密度池塘养殖与水库低密度养殖4种不同生境来源的草鱼其前、中、后肠的微生物组成和群落特征。【结果】Venn图、稀释性曲线和Alpha指数分析结果显示,前肠微生物群落多样性以养殖生境草鱼更高,而后肠微生物群落多样性以自然生境草鱼更高。不同生境草鱼前肠微生物组成和群落特征差异较大,自然生境的草鱼前肠微生物优势菌群都是不动杆菌属和贪铜菌属;高密度池塘养殖生境优势菌群是鲸杆菌属、希瓦氏菌属;低密度水库养殖生境的草鱼前肠优势菌群是链球菌属、消化链球菌属等。【结论】不同生境的环境因子不同,养殖方式不同,饵料不同,导致草鱼前肠微生物组成和群落特征的差异较大,该结果可为草鱼肠道微生物的研究提供基础数据。高密度养殖生境的草鱼存在潜在的感染风险,建议优化养殖模式以减少有害菌的产生。     

Cloning and preliminary functional studies of the JAM-A gene in grass carp (Ctenopharyngodon idellus)

Grass carp (Ctenopharyngodon idellus) is a very important aquaculture species in China and other South-East Asian countries; however, disease outbreaks in this species are frequent, resulting in huge economic losses. Grass carp hemorrhage caused by grass carp reovirus (GCRV) is one of the most serious diseases. Junction adhesion molecule A (JAM-A) is the mammalian receptor for reovirus, and has been well studied. However, the JAM-A gene in grass carp has not been studied so far. In this study, we cloned and elucidated the structure of the JAM-A gene in grass carp (GcJAM-A) and then studied its functions during grass carp hemorrhage. GcJAM-A is composed of 10 exons and 9 introns, and its full-length cDNA is 1833 bp long, with an 888 bp open reading frame (ORF) that encodes a 295 amino acid protein. The GcJAM-A protein is predicted to contain a typical transmembrane domain. Maternal expression pattern of GcJAM-A is observed during early embryogenesis, while zygote expression occurs at 8 h after hatching. GcJAM-A is expressed strongly in the gill, liver, intestine and kidney, while it is expressed poorly in the blood, brain, spleen and head kidney. Moreover, lower expression is observed in the gill, liver, intestine, brain, spleen and kidney of 30-month-old individuals, compared with 6-month-old. In a GcJAM-A-knockdown cell line (CIK) infected with GCRV, the expression of genes involved in the interferon and apoptosis pathways was significantly inhibited. These results suggest that GcJAM-A could be a receptor for GCRV. We have therefore managed to characterize the GcJAM-A gene and provide evidence for its role as a receptor for GCRV.     

Development of microsatellite DNA markers of grass carp (Ctenopharyngodon idella) and their cross-species application in black carp (Mylopharyngodon piceus)

Thirty-four microsatellite DNA loci were developed for grass carp (Ctenopharyngodon idella) and used to determine the diversity of 42 individuals of grass carp and 16 individuals of black carp collected from Jingzhou fragment (the middle reach) of Yangtze River. All the 34 loci were polymorphic in grass carp. The number of alleles found in grass carp at these loci ranged from 2 to 24. The observed and expected heterozygosities varied from 0.238 to 1.000 and from 0.162 to 0.945, respectively. Thirty of the 34 loci cross-amplified the DNA of black carp (Mylopharyngodon piceus), and 21 of them revealed polymorphism (more than one allele each locus).     

Dietary lipid requirement on non-specific immune responses in juvenile grass carp (Ctenopharyngodon idella)

This study aimed to evaluate the dietary lipid requirement and its effects on liver oxidative status and non-specific immune responses of juvenile grass carp (Ctenopharyngodon idella). Purified diets with five dietary lipid levels (0%, 2.5%, 5%, 7.5% and 10%, fish oil/corn oil = 1:1) were each fed to triplicate groups of grass carp (mean initial weight: 6.57 ± 0.01 g) in a recirculating rearing system maintained at 27.5 ± 0.5 °C for 10 weeks. Percent weight gain was highest (P < 0.05) with 5% lipid and lowest in fish fed the lipid free control diet. Feed efficiency (FE) and protein efficiency ratio (PER) in fish followed the same pattern of percent weight gain. Fish fed with lipid containing diets had better non-specific immune response indexes (e.g. phagocytic activity, plasma peroxidase and lysozyme activity) and low-level of liver oxidation status than fish fed with the control diet. But excess dietary lipid supplement would bring over metabolic burden to liver. After the feeding trial, fish were challenged by Aeromonas hydrophila. Fish fed control diet obtained significantly (P < 0.05) lower survival rate. The survival rate was highest with 7.5% lipid. The results of this study indicated that proper dietary lipid supplementation enhanced the immune response of grass carp and improved the survival rate in the bacterial challenge, but excess dietary lipid may elevate liver oxidation rates of grass carp. Analysis by second-order regression of percent weight gain indicated that the optimal dietary lipid level in juvenile grass carp (6.6–35.5 g) is about 6.5%.     

Antigenic analysis of grass carp reovirus using single-chain variable fragment antibody against IgM from Ctenopharyngodon idella

Grass carp (Ctenopharyngodon idella) is an important species of freshwater aquaculture fish in China. However, grass carp reovirus (GCRV) can cause fatal hemorrhagic disease in yearling populations. Until now, a strategy to define the antigenic capacity of the virus’s structural proteins for preparing an effective vaccine has not been available. In this study, some single-chain variable fragment antibodies (scFv), which could specifically recognize grass carp IgM, were selected from a constructed mouse naïve antibody phage display cDNA library. The identified scFv C1B3 clone was shown to possess relatively higher specific binding activity to grass carp IgM. Furthermore, ELISA analysis indicated that the IgM level in serum from virus-infected grass carp was more than two times higher than that of the control group at 5–7 days post infection. Moreover, Western blot analysis demonstrated that the outer capsid protein VP7 has a specific immuno-binding-reaction with the serum IgM from virus-infected grass carp. Our results suggest that VP7 can induce a stronger immune response in grass carp than the other GCRV structural proteins, which implies that VP7 protein could be used as a preferred immunogen for vaccine design.     

Purification and characterization of a rhamnose-binding lectin with immunoenhancing activity from grass carp (Ctenopharyngodon idellus) ovaries

A rhamnose-specific lectin was isolated from ovaries of the grass carp (Ctenopharyngodon idellus). The grass carp lectin possesses a molecular mass of 205 kDa. It is composed of six subunits each with a molecular mass of 35 kDa. The N-terminal amino acid sequence of the grass carp shows similarity to those of other fish species with 26-35% amino acid identity. It is mitogenic toward murine splenocytes and peritoneal exudate cells.     

SNP‐based susceptibility–resistance association and mRNA expression regulation analyses of tlr7 to grass carp Ctenopharyngodon idella reovirus

Eleven single nucleotide polymorphisms (SNP) in Ctenopharyngodon idella toll‐like receptor 7 (citlr7) gene, containing two in the 5′‐flanking region, three within the single intron and six distributed in the coding sequence (CDS), were identified. A case–control study of 73 susceptible individuals and 67 resistant individuals was conducted to test the SNPs‐based susceptibility–resistance association and mRNA expression of citlr7 to grass carp reovirus (GCRV), showing that both 820 A/G and 1726 A/G were significantly correlative sites in genotype (P < 0·05). Multifactor dimensionality reduction (MDR) analysis suggested the exertion of antiviral effects of 820 A/G might rely on SNPs interactions of citlr7 and C. idella toll‐like receptor 8 (citlr8). Combining the mortality rate and citlr7 mRNA expression, it was suggested that 1726 GG‐genotyped individuals might be more resistant than 1726 A/G genotyped individuals, indicating the selection on synonymous mutations in 1726 A/G might be susceptibility–resistance‐type specific. In addition, haplotype analysis uncovered no significantly correlative haplotypes in citlr7. These findings may provide an in‐depth insight for the further functional research of citlr7. The potential genetic markers identified may contribute to the molecular and transgenic breeding of C. idella.     

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.     

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.     

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.     

Validation of reference genes of grass carp Ctenopharyngodon idellus for the normalization of quantitative real-time PCR

Expression of four reference genes of grass carp, including β-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA (18S) and elongation factor-1 alpha (EF1α), was studied in tissues of normal individuals and bacteria-infected individuals. EF1α had the most stable expressions followed by 18S rRNA then GAPDH; ACTB had the least stability. After being infected with bacteria, the grass carp showed minimal changes in expression levels of EF1α in the liver and head kidney, while ACTB had the most stable expressions in spleen but the least stable in liver. EF1α is thus the optimal reference gene in quantitative real-time PCR analysis to quantitate the expression levels of target genes in tissues of grass carp.