Molecular cloning of CD4 from ginbuna crucian carp <Emphasis Type="Italic">Carassius auratus langsdorfii</Emphasis>

ABSTRACT:   The clonal triploid ginbuna crucian carp Carassius auratus langsdorfii , a naturally occurring gynogenetic fish, is a useful model for studying T-cell-mediated immunity. CD4, a T-cell receptor (TCR) coreceptor, is a membrane-bound glycoprotein found on helper T-cells, and assists in the binding of major histocompatibility complexes. In the present study, full-length cDNAs encoding the CD4 molecule from the S3n strain of ginbuna crucian carp were cloned and characterized. 5'-rapid amplification of cDNA ends (RACE) and 3'-RACE yielded two distinct cDNA clones of CD4 homolog from the ginbuna, and these sequences share 95% identity at the amino acid level. These ginbuna CD4 molecules consisted of a signal peptide, immunoglobulin superfamily (IgSf) like domains, a transmembrane domain, and a cytoplasmic domain similar to other known CD4. A tyrosine protein kinase p56^lck binding motif is conserved in the cytoplasmic tail of ginbuna CD4. Phylogenetic tree analysis indicated that ginbuna CD4 sequences are closely related to CD4L-1 from other fish species. Expression of ginbuna CD4 mRNA was detected in the gill, thymus, head kidney, trunk kidney and peripheral blood leukocytes, indicating that its expression pattern is similar to that of ginbuna TCRβ mRNA. The results suggest that ginbuna CD4 sequences are useful as molecular probes for helper T-cells.     

Availability of culture filtrate protein-10 (CFP-10) secreted from Mycobacterium pseudoshottsii for mycobacteriosis diagnosis in ginbuna crucian carp Carrasius auratus langsdorfii

Mycobacteriosis in cultured fish is a challenge for the aquaculture industry worldwide. Treatment by chemical administration is difficult and no effective vaccine has been developed. Therefore, detection and isolation by early diagnosis are important for prevention of the spread of the disease. In mammals, interferon gamma release assays have been established for detection of tuberculosis; these tests are based on the delayed-type hypersensitivity (DTH) response against culture filtrate protein-10 (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) of Mycobacterium tuberculosis. On the other hand, little is known about the fish immune response against the ESAT-6 and CFP-10 proteins of mycobacteria, although these responses should find application in the diagnosis of mycobacteriosis in fish. In the present study, we identified ESAT-6 and CFP-10 from Mycobacterium pseudoshottsii and cloned the corresponding genes. Intraperitoneal injection of the corresponding DNA plasmid constructs in ginbuna crucian carp yielded increased expression of the fish interferon-γ1-1-encoding gene (IFN-γ1-1). In contrast, IFN-γ1-1 expression accompanied by DTH response was observed only in the CFP-10-DNA plasmid-injected fish. Furthermore, fish that had been prophylactically injected with CFP-10-DNA plasmid exhibited increased survival of M. pseudoshottsii infection. Taken together, these results suggested that CFP-10 may facilitate diagnosis of mycobacteriosis.     

Amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> interleukin-10 negatively suppresses host cell-mediated immunity

Interleukin-10 (IL-10) is an anti-inflammatory cytokine and negatively regulates cell-mediated immunity (CMI) induction by inhibiting cytokine production in type 1 T helper cells. IL-10 genes have been isolated from several fish, and inflammatory cytokine inhibition by IL-10 has been well examined. However, a CMI regulator of IL-10 in fish has not yet been identified. In this study, we cloned the IL-10 gene in amberjack Seriola dumerili and analyzed its function using its recombinant protein (rIL-10). In an in vitro culture experiment, gene expression of inflammatory cytokines was suppressed in leukocytes incubated with rIL-10 compared with cells that only received Nocardia seriolae stimulation. This result suggests amberjack IL-10 has conserved function as an inflammatory cytokine inhibitor. Bactericidal activity of amberjack cells against intracellular pathogen stimulation was decreased in a rIL-10 dose-dependent manner. Furthermore, a significant reduction in the T-bet/GATA-3 ratio was observed in N. seriolae living cell (LC)?+?rIL-10-injected fish. Taken together, these results suggest amberjack rIL-10 suppresses CMI induction both in vitro and in vivo. In addition, the number of IgM⁺ cells among spleen leukocytes in N. seriolae?+?rIL-10-injected fish was higher than in only N. seriolae LC, suggesting that Th2-dominant immunity was induced by adding rIL-10.     

Deficient melanin production contributes to the absence of melanophores in early development of red carp

Red carp and red crucian carp are ornamental fish with a red body color. Unlike in red crucian carp, no melanophores are observed in red carp embryos or larvae. To explore the roles of the mitfa gene in body color formation in red carp, we investigated the structural characteristics and physicochemical properties of the mitfa gene in 16 kinds of fish. The mitfa amino acid sequence similarity between red carp and red crucian carp was 95.6%, and this was 91.5% similar between carp and zebrafish. Compared with red crucian carp, red carp showed lower tyrp1 messenger ribonucleic acid (mRNA) expression but similar mitfa mRNA expression in the body pigment stage of the embryo. Moreover, mitfa⁺ cells as well as melanocytes could be observed in cultured embryo cells derived from red carp and red crucian carp. Our data show that the absence of melanophores in red carp is not the result of mitfa gene deletion or mutation, increasing our understanding of the molecular and genetic mechanisms of coloration in cyprinid fish.     

Isolation and expression analyses of the <Emphasis Type="Italic">Sox9a</Emphasis> gene in triploid crucian carp

To investigate the evolutional significance of Sox9 in fish, we isolated and characterized Sox9a cDNA and genomic clones in triploid crucian carp. The cDNA encoded a protein of 457 amino acids with an HMG box and showed more than 60% amino acid sequence identity with known vertebrate Sox9 proteins. Triploid crucian carp and vertebrate Sox9s showed similar gene structure, and two introns in the coding region were located at conserved positions. On the basis of the amino acid sequences, Sox9a can be categorized into the same subgroup of Sox-E proteins as Sox8, 9, and 10. Interestingly, the expression of triploid crucian carp Sox9a was predominantly observed not in the ovary but in the testis by Northern blot and RT-PCR analysis. The expression analysis of Sox9a suggested that it may seldom contribute to the formation of normal functions of spermatozoa, but it may play an important role in the development of testicular tubules. Besides the testicular expression, Sox9a was also shown to be expressed in many other tissues including the brain, kidney, and heart of triploid crucian carp, indicating that Sox9 may have unique functions in some specific tissues during development.     

异育银鲫内参基因的筛选

内参基因在实时荧光定量PCR(qRT-PCR)中具有校准的作用,然而鲤疱疹病毒Ⅱ型感染异育银鲫内参基因目前仍未见研究报道。采用qRT-PCR技术检测不同处理条件下异育银鲫组织和尾鳍细胞GAPDH、EF-1α、18S rRNA和β-actin 4个候选内参基因在组织和尾鳍细胞的转录水平,利用软件geNorm、Norm Finder、Best Keeper和Delta Ct分析了其表达量的稳定性,以筛选出健康异育银鲫不同组织以及鲤疱疹病毒Ⅱ型感染的肾脏、脾脏和异育银鲫尾鳍细胞在不同时间均较稳定的内参基因。geNorm稳定值以及4个内参的表达量Ct值分析显示,健康异育银鲫脑、脾脏、肾脏、肌肉、鳃、肠、肝脏和心脏组织中β-actin和EF-1α都是比较稳定的内参基因;鲤疱疹病毒Ⅱ型感染肾脏和尾鳍细胞不同时间点,内参基因β-actin稳定性最佳;鲤疱疹病毒Ⅱ型感染脾脏不同时间点时,EF-1α稳定性最佳。分别以4个候选内参基因为内参分析PIN1基因在肾脏组织不同感染时间的相对表达量,结果进一步证实,PIN1基因在肾脏组织感染不同时间点以β-actin为内参时,其表达量呈下降趋势,与cDNA文库测序中的表达量分析结果一致,各时间点的表达量差异极显著(P<0.01)。本试验结果有利于研究异育银鲫在不同处理条件下基因的表达分析,可为获得精准的结果奠定理论基础。     

Effect of Coriolus versicolor polysaccharides on the hematological and biochemical parameters and protection against Aeromonas hydrophila in allogynogenetic crucian carp (Carassius auratus gibelio)

The effect of dietary intake of Coriolus versicolor Polysaccharides (CVP) on the hematological and biochemical indices of Allogynogenetic crucian carp (Carassius auratus gibelio) was investigated. Fish were fed CVP supplemented diets (0, 0.25, 0.5, 1.0, 2.0 or 4.0 g CVP kg^?1) for 56 days. The RBC, WBC counts, hemoglobin content, ESR in blood and TP, ALT, AST, ALP, GLU, CHO, TG, and BUN in serum were measured on day 0, 14, 28, 42, and 56. After feeding of 56 days, fish were infected with Aeromonas hydrophila and mortalities were recorded. The results indicated that feeding crucian carp with suitable dose of CVP enhanced the RBC, WBC counts, hemoglobin and TP content, ALP activity, and decreased the ESR, ALT, AST, GLU, CHO, TG and BUN. There was no effect in fish at low dose (0.25 g kg^?1). Unexpectedly, the higher CVP dose used here (2.0 and 4.0 g kg^?1) has a negative effect in fish. The results of challenge experiment indicated that a moderate level of CVP in the diet (1.0 g kg^?1) was the most effective to enhance the survival of fish after infected with A. hydrophila. In summary, the use of CVP, as dietary supplements, can improve the innate defense of crucian carp providing resistance to pathogens.     

不同倍性鲫鲤MeCP2基因分子克隆及时空表达分析

为了探寻不同倍性鱼生殖育性的表观遗传调控规律,实验基于不同倍性鲫鲤精巢的转录本信息库设计引物克隆了不同倍性鲫鲤的MeCP2基因。序列比对结果发现,在远缘杂交的过程中基因组发生了遗传重组和变异。基于序列公共部分设计引物,通过半定量PCR和实时定量PCR的方法分析了MeCP2基因在不同倍性鱼发育过程中的时空表达特征。结果还发现,MeCP2基因在所有组织中都有表达,但在脑组织中的表达量最高;纵向对比性腺的发育过程,MeCP2基因的表达量会随着性腺发育成熟而下降;横向对比不同倍性鱼的性腺发育,MeCP2基因在三倍体鱼卵巢中的表达量明显高于二倍体和四倍体鱼,但在精巢中的表达量随鱼的倍性增加而增加。研究表明,MeCP2基因的表达与不同倍性鱼的卵巢发育密切相关,这为研究鱼类生殖不育的分子机理及其在水产育种上的应用奠定了坚实的基础。     

鲤鲫杂交回交鲫染色体核型及DNA含量的分析

将性成熟的鲤鲫杂交鱼一代（F1）的雌鱼与雄性红鲫鱼杂交,获得了回交鲫子代,分析了平均体质量94.2g的1龄回交鲫子一代的细胞染色体核型,测定了其DNA含量。肾细胞直接制片法表明：回交鲫子代染色体由147条组成,即3n=147,NF=222,其核型公式为：3n=51m＋24sm＋27st＋45t。流式细胞记数法结果表明：在20尾鱼中有14尾回交鲫子代细胞的DNA含量是对照鱼（红鲫鱼）的1.5倍,占总鱼数的70%;6尾在2～3倍体之间,非常接近三倍体,占总鱼数的30%。本结果与其细胞染色体核型分析结果基本一致,说明该鱼是以三倍体为主的回交种。     

Chlorella additive increased growth performance,improved appetite and immune response of juvenile crucian carp Carassius auratus

This study was designed to investigate effects of dietary Chlorella meal (CM) additive on growth performance, immune responses and appetite regulation of juvenile crucian carp Carassius auratus (initial body weight: 1.27 ± 0.03 g). Four experimental diets were formulated to contain 0% (CM0), 1% (CM1), 2% (CM2) and 4% Chlorella meal (CM4), respectively. Each diet was randomly assigned to triplicate groups with 40 juvenile fish per fibreglass tank for 8 weeks. Weight gain rate, specific growth rate and feed intake increased with increasing dietary CM levels. In contrast, FCR (feed conversion rate) declined with dietary CM levels. No significant differences were observed in moisture, crude protein, crude lipid and ash contents of muscle and liver tissues. Dietary CM addition increased activities of acid and alkaline phosphatase in liver and kidney. Dietary CM up‐regulated the mRNA expression levels of NKEF‐B, MCHII and IgM in kidney, and increased the mRNA levels of NPY and agouti gene‐related protein in the brain, but down‐regulated mRNA levels of MC4R, LEP, LEPR, CART1, CART2 and CCK8 genes. Based on these observations above, this study indicated that dietary CM additive increased growth performance, immune responses and appetite of crucian carp. The results, for the first time, demonstrate a role for the central nervous system in the control of food intake in fish fed dietary Chlorella meal.     

Stimulation of glycerol kinase in grass carp preadipocytes by EPA

This study was conducted to assess the effect of eicosapentaenoic acid (EPA) on grass carp preadipocyte glycerol kinase (GyK) expression, as well as to explore the mechanism. Here, we cloned partial sequence of grass carp GyK gene and analyzed its tissue distribution. The result showed that GyK gene expressed most in the liver, followed by adipose tissue and the kidney. Besides, 400 μM oleic acid (18:1n-9, OA) was used to establish a hypertrophic preadipocyte model. GyK gene expression and enzyme activity were significantly enhanced after model cells were treated with 100 μM eicosapentaenoic acid (20:5n-3, EPA) for 6, 12, and 24 h. Meanwhile, peroxisome proliferative-activated receptor (PPAR)γ, adipose triglyceride lipase (ATGL), and the two isoforms of grass carp HSL gene were first identified by Sun et al (2016), and they defined the two isoforms as HSLa and HSLb. Therefore, maybe HSLa and HSLb are appropriate.. The content of triglyceride was dramatically increased by EPA treatment for 24 h. Further, a competitive ATGL antagonist, HY-15859, attenuated the increase in GyK induced by EPA at 12 h. Surprisingly, the enhanced lipolysis and PPARγ gene expression induced by serum deprivation were paralleled by an increase in GyK gene expression, whereas a stabilization in GyK enzyme activity. Other fatty acids, including docosahexaenoic acid, alpha-linolenic acid, linoleic acid, and OA also promoted GyK gene expression. Moreover, an irreversible PPARγ antagonist, GW9662, was used to investigate the role of PPARγ in GyK induction. Data showed that GW9662 abolished the induction of GyK by EPA at 12 h. Together, these data suggested that EPA elevated grass carp preadipocytes GyK expression. ATGL and PPARγ contributed to the induction of GyK. PPARγ may be a key regulator in response to GyK expression induced by EPA.     

Effect of fish meal replacement by Chlorella meal with dietary cellulase addition on growth performance,digestive enzymatic activities,histology and myogenic genes’ expression for crucian carp Carassius auratus

This study was conducted to investigate the effects of fish meal (FM) replacement by Chlorella meal (CM) with dietary cellulase supplementation on growth performance, digestive enzymatic activities, histology and myogenic genes’ expression in crucian carp Carassius auratus (initial body weight: 2.90 ± 0.02 g, mean ± SEM). Six isonitrogenous diets were formulated at two cellulase levels (0 and 2 g kg^?1). At each cellulase level, CM was added at three levels of 0, 533.1 and 710.8 g kg^?1 to substitute 0, 75 and 100% of dietary FM respectively. Each experimental diet was randomly assigned to triplicate groups with 25 juvenile fish per fibreglass tank for 8 weeks. Dietary CM substitution significantly increased growth, feed utilization, amylase activity and the expression of Myod, Mrf4 and Myf5, but reduced the Myog expression. Dietary cellulase addition increased hepatosomatic and viscerosomatic index, lipase activity and the expression of Mrf4, but reduced trypsin activity and the expression of Myog and Myf5. Dietary CM substitution enlarged the cell size and also caused some karyopyknosis in liver. Our results showed that CM could totally replace FM in diets; dietary cellulase supplementation at the level of 2 g kg^?1 played a subtle role in improving growth and feed utilization for crucian carp.