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.     

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.     

Genetic structure,polymorphism identification of LGP2 gene and their relationship with the resistance/susceptibility to GCRV in grass carp, Ctenopharyngodon idella

Laboratory of genetics and physiology 2 (LGP2) is an actual detector and regulator during RNA viral infection in innate immunity. In this study, 5′-flanking region and all introns of LGP2 in grass carp (Ctenopharyngodon idella) were excavated. The genomic CiLGP2 (C. idella LGP2) was 8062 bp in length, with a 364 bp 5′-flanking region, twelve exons and eleven introns. Besides, the promoter activity of the upstream region before initiator codon was identified. By sequencing, six single nucleotide polymorphisms (SNPs) and one 20-bp insertion/deletion polymorphism were detected in CiLGP2. With a challenge experiment, the genotype and allele distributions of these seven polymorphisms were examined. Analytic result revealed only the − 1392 C/G, 494 A/T and 4403 C/T loci were significantly associated with the resistance/susceptibility to grass carp reovirus (GCRV) (P < 0.05). To further identify these correlations, another independent challenge test was performed. The analytic result based on the cumulative mortality demonstrated that the stock in − 1392 GG genotype was more susceptible to GCRV than that in CC genotype, while the stocks in 494 TT genotype and 4403 TT genotype were more resistant to GCRV than that in AA and CC genotype stocks, respectively (P < 0.05). Those significant SNPs might be potential gene markers for the future molecular selection of C. idella strains that are resistant to GCRV.     

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.     

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.     

草鱼Noxa基因克隆及其应答GCRV入侵的表达响应

研究通过获得草鱼Noxa基因(Cinoxa)全长cDNA, 进行序列分析和进化树构建, 使用定量PCR (qPCR)的方法研究其在GCRV刺激下的表达模式。研究发现, 草鱼Noxa基因的蛋白序列与斑马鱼Noxa基因具有高度相似性。通过分析草鱼和斑马鱼的Noxa基因的蛋白三维结构(3D)模型, 研究发现两者具有高度一致的蛋白三维结构模式, 该模型与高等哺乳类中的Bcl-2家族蛋白中C端结构域同源。对Cinoxa在GCRV刺激下的表达模式的研究表明, Cinoxa在GCRV感染后中肾、脾脏和头肾中表达发生显著性变化, 攻毒后24h和120h出现显著上调表达。研究表明草鱼Noxa为斑马鱼Noxa的同源基因, 并且参与了草鱼对GCRV入侵的应答反应, 为深入研究鱼类Noxa应答病毒入侵的功能和转录调控机制奠定了基础。     

Effects of intraperitoneal injection of cortisol on non‐specific immune functions of Ctenopharyngodon idella

After grass carps Ctenopharyngodon idellus were injected with cortisol, with (CBC) and without (C) a cocoa butter carrier, the effects of both slowly and rapidly acting exogenous cortisol on their non‐specific immune functions were investigated. On the one hand, after injection with CBC, the cortisol concentration and lysozyme activity in fish serum were enhanced and were sustained at high levels for a long period (30 days). The killing activity in the serum declined with time, and phagocytosis of head kidney macrophages diminished significantly (P < 0·05 or P < 0·01). The leukocrit values in the high dose group (31·8 mg cortisol fish^?1) increased over time, however, with the maximum average being 5·6% at day 30. The spleen mass index in the high dose group was 0·93 × 10^?3 after 30 days, notably lower (P < 0·05) than that in the control group. In addition, a decrease in resistance to Aeromonas hydrophila infection in cortisol‐treated fish was shown, with the final cumulative mortalities being 54·5 and 66·7% in the low and high dose groups, respectively. On the other hand, there was a decrease in both serum cortisol concentration and lysozyme activity of the experimental fish within 2 weeks after injection with C, where plasma bactericidal activities in the high dose group (31·8 mg cortisol fish^?1) were remarkably lower (P < 0·01) than those in the control group at each sampling, but were increased slightly over time. The results of which were different from those in the CBC trial. Phagocytic activity of head kidney macrophages and spleen mass index decreased significantly (P < 0·05), while there were increases in leukocrit value and cumulative mortality due to A. hydrophila. The results of which were similar to those in the CBC trial. This study indicated that the injection of cortisol depressed the non‐specific immune functions of the grass carp and increased its susceptibility to disease.     

Association between the lack of teeth and the expression of myosins in masticatory muscles of microphthalmic mouse

The purposes of the present study were to elucidate the influences of the deficiency of teeth on masticatory muscles, such as the masseter, temporalis and digastric muscles and compare the influence among masticatory muscles. We analysed the expressions of myosin heavy chain (MyHC) isoform messenger RNA (mRNA) and protein in these muscles in the microphthalmic (mi/mi) mouse, whose teeth cannot erupt because of a mutation in the mitf gene locus. The expression levels of MyHC mRNA and protein in the masseter, temporalis, digastric, tibialis anterior and gastrocnemius muscles of +/+ and mi/mi mice were analysed with real‐time polymerase chain reaction and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, respectively. The mi/mi masseter muscle at 8 weeks of age expressed 4·1‐fold (p < 0·05) and 3.3‐fold (p < 0·01) more MyHC neonatal mRNA and protein than that in the +/+, respectively; the expression level of MyHC neonatal protein was 19% of the total MyHC protein in the masseter muscle of mi/mi mice. In the digastric muscle, the expression levels of MyHC I mRNA and protein in the mi/mi mice were 4·7‐fold (p < 0·05) and 5‐fold (p < 0·01) higher than those in the +/+ mice. In the temporalis, tibialis anterior and gastrocnemius muscles, there was no significant difference in the expression levels of any MyHC isoform mRNA and protein between +/+ and mi/mi mice. These results indicate associations between the lack of teeth and the expression of MyHC in the masseter and digastric muscles but not such associations in the temporalis muscle, suggesting that the influence of tooth deficiency varies among the masticatory muscles. Copyright © 2011 John Wiley & Sons, Ltd.     

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%.     

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.     

Mitochondrial uncoupling proteins regulate angiotensin‐converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin‐converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole‐body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3‐55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8‐fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role.