Analysis of IL-2-1ike factor in lymphocyte culture supernatant of olive flounder, Paralichthys oliveaceus

To study immune mechanism of fish lymphocyte we performed a proliferation assay and ELISA using monoclonal antibody against human IL-2. The result showed that an interleukin-2 (IL-2)-like factor was detected in the supernatant of plant haemoglutinin (PHA)-stimulated lymphocyte culture from peripheral blood, spleen and head kidney of olive flounder,Paralichthys olivaceus. The quantities of IL-2-like factor in the supernatant from different lymphoid tissues were quite different. The IL-2 like factor in the supernatant from cultured head kidney lymphocytes was much higher than those of peripheral blood lymphocytes and spleen lymphocytes (P<0.01). The IL-2 activity was found in either mouse thymocyte proliferation assay or flounder head kidney lymphocyte proliferation assay and shown to have obvious enhancing effect on proliferation of the above two types of cell. The recombinant human IL-2, (rhIL-2) was able to stimulate flounder thymocyte proliferation and used to detect the IL-2 receptor (IL-2R) on the surface of flounder lymphocyte. The cross-reaction between the lymphocytes of flounder peripheral blood and CD25(IL-2R) was detected with flow cytometry and shown that the percentage of CD25-positive cell in peripheral blood was 7.74±0.67%. This work was supported by National “973” Project G1999012003, G19999012006.     

Induction of EROD activity in Paralichthys olivaceus by polychlorinated biphenyl CB-28

Abstract-The test organisms (Paralichthys olivaceus) were exposed to CB-28 with differentspiked concentrations. After 72 h, EROD activities in livers of the test organisms were measured. It wasfound that EROD activities significantly mounted up with the increase of spiked quantity. Therefore,there existed a dose-response relationship between EROD activities and specific pollutant concentrations.In the meantime, the optimal conditions for the analysis of EROD activity were discussed. The limita-tion of EROD activities as a monitoring parameter was also analyzed.     

Characterization,tissue distribution,and expression of neuropeptide Y in olive flounder Paralichthys olivaceus

Neuropeptide Y(NPY) is a 36-amino acid peptide of the neuropeptide Y family that plays key roles in the regulation of food intake. In this study,we focused on NPY m RNA expression changes around feeding time and during food deprivation in olive flounder. The olive flounder NPY m RNA levels were analyzed in different tissues and a high level of expression was detected in the brain. We also demonstrated a correlation between NPY expression levels in the brain and feeding schedule. NPY expression levels in olive flounder maintained on a daily scheduled feeding regimen increased shortly before feeding and decreased after the scheduled feeding time. Compared with the-1 h group before feeding,NPY expression in the 3 h group after feeding decreased significantly( P <0.05). Food deprivation led to an 81.7% decrease in NPY m RNA levels in the 24 h fasted group(P <0.05) and a 91.7% decrease in the 48 h fasted group(P <0.05). Therefore,our study demonstrates that NPY expression is associated with food intake in olive flounder. This result reveals the function of NPY in regulating food intake and its potential importance in olive flounder aquaculture.     

甲基汞在褐牙鲆幼鱼体内蓄积的组织特异性及其对免疫功能和生长的毒性作用

甲基汞(MeHg)对鱼类多种生命过程存在极强的毒性作用,但有关其对海水鱼类免疫功能的毒性作用研究较少。本文利用实验毒理学方法研究了MeHg(暴露浓度0.0, 0.1, 1.0, 10.0, 20.0μg/L;暴露时间30天)在褐牙鲆(Paralichthys olivaceus)幼鱼组织(鳃、肝脏和脾脏)中的蓄积特征及其对幼鱼的四种免疫功能生物指示物(溶菌酶LZM、酸性磷酸酶ACP、碱性磷酸酶AKP和免疫球蛋白MIgM)和生长的毒性作用。结果表明,幼鱼体内MeHg蓄积量具有显著的浓度依赖性和组织特异性,各组织对MeHg的蓄积能力总体上呈现肝脏鳃脾脏的趋势;四种免疫功能生物指示物(活性或含量)对不同MeHg浓度暴露的响应方式各异,且具有显著的组织特异性; MeHg达到一定暴露浓度后显著抑制幼鱼的生长。总体而言,MeHg在褐牙鲆幼鱼体内蓄积,对其产生免疫毒性,并抑制幼鱼的生长。此外,ACP、AKP和LZM等免疫性指标能较好的反映对MeHg暴露的浓度依赖关系和组织特异性,可作为海水环境中MeHg污染风险评估和生物毒性分析的潜在生物指示物。     

用显微注射方法分析牙鲆肌肉发育调节基因MyoD, Myf5的启动子序列

克隆了牙鲆(Paralichthys olivaceus)肌肉发育调节基因MyoD和Myf5的启动子序列,分别为608 bp和1 073 bp。对序列进行分析发现MyoD和Myf5的启动子含有参与肌肉发育调控的基因的结合位点。将MyoD和Myf5的启动子连接到GFP载体上,构建了重组质粒MyoDP-GFP和Myf5P-GFP,并注射到一细胞或两细胞的斑马鱼胚胎中,对这两个基因的启动子进行了瞬时表达研究。结果表明,牙鲆MyoD和Myf5的启动子可以驱动绿色荧光蛋白特异地在斑马鱼肌肉纤维中表达,因此牙鲆608 bpMyoD和1073 bpMyf5的启动子包含着可以驱动MyoD和Myf5正常表达的必需元件,它们是肌肉特异的,并且可以跨物种行使其功能。     

一种新的牙鲆病原菌——兔莫拉氏菌

目前国内外已报道的牙鲆(Paralichthys olivaceus)细菌病的病原有鳗弧菌(Vibrio anguillarum)以及弧菌(Vibrio spp.)属的其他一些种类、爱德华氏菌(Edwardsiella tarda)、屈挠杆菌(Flexibacter maritimus)、肠球菌(Enterococcus seriolicida)、链球菌(Streptococcus spp.)、卡帕奇诺卡氏菌(Nocardia kampachi)、变异微球菌(Micrococcus varians)[1].     

First isolation and characterization of a new species of Vibrio from diseased flounder (Paralichthys olivaceus Temminck et Schlegel)

Appropriate pathogenic bacteria were isolated from diseased (or dead) flounder (Paralichthys olivaceus L) occurring in a mariculture farms of Qinhuangdao, which all diseased fishes expressed bacterial septicaemia. The phenotypic information of the 12 pure cultures was studied extensively, including morphological characteristics, colony characteristics in different media, physiological and biochemical characteristics, and the mole fraction G C ratio of the DNA for representative strain. The results show that the isolates belong to a new species of Vibrio, and are designated as Vibrio qinhuangdaora sp.nov, by the area from which the diseased flounders are collected (Qinhuangdao) based on its biological properties following Rules of International Code of Nomenclature of Bacteria. At the same time,studies on the serum homology ofisolates, and pathogenicity of isolates were conducted by experimental infection. The results show that all the isolates are of serologic similarity, and the isolates have strong pathogenicity to flounder. The studies can extensively reflect the main biological properties of this new species of Vibrio.     

雌牙鲆CYP19A基因启动子“CpG”岛区的单核苷酸多态性及与繁殖性能的关联分析

芳香化酶基因(CYP19)编码的芳香化酶是雄激素转化为雌激素的关键酶,在鱼类性腺发育及繁殖内分泌过程中起着重要的作用。本研究采用聚合酶链式反应-单链构象多态(PCR-SSCP)和放射性免疫方法(RAI)对61尾雌牙鲆进行了CYP19A基因启动子CpG岛区多态性检测,并将其与繁殖性能进行了关联分析。CpG岛区存在3种类型的基因型(pattern1、pattern2、pattern3),经测序发现CpG岛区有以下突变:T-1927插入或缺失、C-1899插入或缺失、T-1772C、C-1766T、TGTCAAC-1752~-1744插入或缺失(11个SNPs连锁突变)。方差分析结果表明:此位点对雌二醇(E2)水平的效应达到显著(P<0.05)水平,经多重比较分析,具有pattern1基因型的个体的雌激素(E2)水平显著低于pattern2和pattern3基因型个体的雌激素水平(P<0.05),同时pattern1基因型的个体的mRNA表达量显著低于pattern2和pattern3基因型个体的mRNA表达量(P<0.05)。以上结果表明,CYP19A基因调控区甲基化位点突变可能导致芳香化酶的表达量,进而影响牙鲆繁殖内分泌过程;同时也阐明了这些与牙鲆繁殖性状相关的SNPs可以用于生产实践中进行标记辅助选择具有优良繁殖性能的牙鲆。