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.     

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.     

Sox genes in grass carp (Ctenopharyngodon idella) with their implications for genome duplication and evolution

The Sox gene family is found in a broad range of animal taxa and encodes important gene regulatory proteins involved in a variety of developmental processes. We have obtained clones representing the HMG boxes of twelve Sox genes from grass carp (Ctenopharyngodon idella), one of the four major domestic carps in China. The cloned Sox genes belong to group B1, B2 and C. Our analyses show that whereas the human genome contains a single copy of Sox4, Sox11 and Sox14, each of these genes has two co-orthologs in grass carp, and the duplication of Sox4 and Sox11 occurred before the divergence of grass carp and zebrafish, which support the "fish-specific whole-genome duplication" theory. An estimation for the origin of grass carp based on the molecular clock using Sox1, Sox3 and Sox11 genes as markers indicates that grass carp (subfamily Leuciscinae) and zebrafish (subfamily Danioninae) diverged approximately 60 million years ago. The potential uses of Sox genes as markers in revealing the evolutionary history of grass carp are discussed.     

Morphology of androgenetic and gynogenetic grass carp, Ctenopharyngodon idella (Valenciennes)

Crosses between female carp x male grass carp resulted in androgenetic grass carp and hybrids. Fertilizing grass carp eggs with carp milt that had been irradiated with ultraviolet light for 15 min at 1.0 mW/cm² produced gynogenetic grass carp. We compared the morphology of experimental progeny with that of the parental species to determine if inheritance was strictly matroclinous in gynogenesis and strictly patroclinous in andro-genesis. Dorsal and anal fin rays, lateral line scales, gill rakers, pharyngeal teeth, and the relative length of the dorsal fin all suggested that gynogenetic grass carp were identical with the maternal species and androgenetic grass carp with the paternal species. Hybrids were intermediate between the parents except that the number of anal rays was identical with that in carp. The number of dorsal rays, the body depth, and the pharyngeal teeth in hybrids were more like those in carp than in grass carp, whereas the number of gill rakers was more similar to that in the paternal species.     

Characterization of oligopeptide transporter (PepT1) in grass carp (Ctenopharyngodon idella)

The oligopeptide transporter (PepT1) is located on the brush-border membrane of the intestinal epithelium, and plays an important role in dipeptide and tripeptide absorptions from protein digestion. In this study, we cloned the PepT1 cDNA from grass carp and characterized its expression profile in response to dietary protein and feed additives (sodium butyrate) treatments. The PepT1 gene encodes a protein of 714 amino acids with high sequence similarity with other vertebrate homologues. Expression analysis revealed highest levels of PepT1 mRNA expression in the foregut of grass carp. In addition, PepT1 mRNA expression exhibited diurnal variation in all three bowel segments of intestine with lower levels of expression in daytime than nighttime. During embryonic development, PepT1 showed a dynamic pattern of expression reaching maximal levels of expression in the gastrula stage and minimal levels in the organ stage. The PepT1 expression showed constant levels from 14 to 34 day post-hatch. To determine whether fish diet of different protein contents may have any effect on PepT1 expression, we extended our research to dietary regulation of PepT1 expression. We found that dietary protein levels had a significant effect on PepT1 gene expression. In addition, PepT1 mRNA levels were higher after feeding with fish meal than with soybean meal. Moreover, in vitro and in vivo sodium butyrate treatments increased PepT1 expression in the intestine of grass carp. The results demonstrate for the first time that PepT1 mRNA expression is regulated in a temporal and spatial pattern during development, and dietary protein and feed additives had a significant effects on PepT1 gene expression in grass carp.     

The tolerance of grass carp Ctenopharyngodon idella (Val.) to seawatert†

Laboratory tests were conducted to determine the tolerance of 2+ grass carp to an oceanic water. It was found that at a temperature of 20.1°C±0.4°C the maximum survival times of the fish in concentrations of seawater equivalent to 17.5 and 10.5 g/1 sodium chloride were 5 h and approximately 24 days respectively. The importance of these results lies in the consideration of the possibility of grass carp migrating through brackish or seawater from one river system to another. Such a migration would seem to be impossible through seawater but may be possible through brackish water.     

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).     

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.     

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.     

The tolerance of grass carp, Ctenopharyngodon idella Val., to aquatic herbicides

Because grass carp may feed selectively on water plants, additional conventional methods may be necessary for effective control of aquatic vegetation. For economic reasons aquatic herbicides are most likely to be used in conjunction with grass carp within an integrated aquatic weed control programme. The acutely lethal toxicities of ten herbicides to grass carp were measured. A preliminary evaluation of the risk to these fish showed that, when used at the recommended rate, the maximum herbicide concentration likely to be found in water should cause little harm to the fish, although attention should be given to the possible inhibition of feeding.     

Digestion of duckweed (Lemna spp.) by the grass carp (Ctenopharyngodon idella)

A feeding trial was conducted in 55-1 aquaria indoors with the grass carp ( Ctenopharyngodon idella Val.) using fresh duckweed ( Lemna spp.) as food. Faeces dry weight was closely correlated with dry weight of duckweed consumed based upon a linear regression. From this regression, the estimated true digestibility of dry matter was 65%. Samples of duckweed and fish faeces were analysed for crude protein and gross energy. Van Soest's Analysis was conducted on composite samples of duckweed and faeces. Apparent digestibilities were 80% for crude protein, 61% for gross energy, 72% for organic cell contents, and 30% for organic cell wall constituents.     

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+。     

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%.