Isolation and characterization of six microsatellite markers in the large yellow croaker (Pseudosciaena crocea Richardson)

Six polymorphic microsatellite markers were isolated and characterized using an enriched library technique in the large yellow croaker (Pseudosciaena crocea Richardson, 1864), a commercially important marine fish in China. They showed PIC (polymorphism information content) ranging from 0.064 to 0.885 (average of 0.580) and allele numbers ranging from two to 13 (average of 7.5), which were useful for the studies on population genetics and selective breeding of the large yellow croaker.     

Eleven new highly polymorphic microsatellite loci in the yellow croaker,Pseudosciaena crocea

We developed 11 new microsatellite markers in Pseudosciaena crocea by screening an enriched genomic library using nonradioactive polymerase chain reaction (PCR) techniques. All loci were found to be polymorphic with an average of 14.9 alleles per locus (range four to 30). The mean observed and expected heterozygosities were 0.86 (range 0.57–1.00) and 0.90 (range 0.62–0.98), respectively. Four loci showed significant Hardy–Weinberg disequilibrium. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for population genetic studies of P. crocea.     

大黄鱼对几种饲料蛋白原料消化率的研究

以白鱼粉为主要蛋白源制成参考饲料,以0.1%的Cr2O3为外源性指示剂,按参考饲料和实验原料70%∶30%的比例配制成实验饲料,测定了大黄鱼（Pseudosciaena crocea）对白鱼粉、红鱼粉、肉骨粉、豆粕、花生粕、菜籽粕和棉籽粕的表观消化率。实验大黄鱼（平均初始体重15.0±1.6g）养殖于海水浮式网箱（1.5m×1.5m×2.0m）中,分别以各实验饲料投喂实验鱼5周,然后采用挤压法收集粪便。实验结果表明各原料表观干物质消化率在43.6%至70.0%之间,能量消化率在42.9%到82.6%的范围内。实验原料的蛋白质表观消化率在70.7%到92.4%之间,其中白鱼粉和红鱼粉的蛋白消化率最高（分别为92.4%和89.3%）,而棉籽粕则最低（70.7%）。脂肪的表观消化率在61.3%到90.5%之间,其中棉籽粕最低（61.3%）。磷的表观消化率相对较低,仅白鱼粉和红鱼粉在53.2%以上,其余皆低于37.5%。总之,大黄鱼对这几种原料的表观消化率存在较大差异,但蛋白消化率均较高,因此,可作为大黄鱼的饲料蛋白源在实际饲料配制中使用。     

大黄鱼耐低温性状相关微卫星标记的筛选

鱼类的耐低温性状是一种重要的经济性状。为了初步分析大黄鱼的耐低温性状, 文章采用15对荧光微卫星标记, 以SSR-PCR方法对大黄鱼低温耐受组和正常对照组F₁代共40个个体进行了耐低温性状遗传差异分析。结果显示, 标记LYC0002在两组样品中共扩增出5个等位基因(片段大小分别为112 bp、110 bp、108 bp、106 bp和104 bp), 其中LYC00021_{12 bp}等位基因在低温耐受组的出现频率达60%, 而在正常对照组中的频率为零, 表明该等位基因对大黄鱼的温度敏感特性有较明显的偏好性, 可能与某种耐低温基因存在一定的连锁关系。此外, 对LYC0002_{106 bp、}LYC0002_{108 bp}、LYC0002_{110 bp} 和 LYC0002_{112 bp} 4个等位基因分别进行了回收、克隆及测序。序列比对结果显示, LYC0002_{112 bp}等位基因含有10个(CA)重复单元, 而其他3个等位基因依次缺失1个(CA)重复单元, 说明LYC0002在本研究样本中的突变方式为微卫星逐步突变模型(Stepwise mutation model, SMM)。     

大黄鱼人工诱导雌核发育后代的微卫星标记分析

通过冷休克法诱导2组大黄鱼雌核发育,并采用6对微卫星标记引物 （C7、C13、C27、C44、D19、F27）对雌核发育家系G1、G2中24尾鱼苗及双亲进行PCR扩增,并进行微卫星标记比较分析。结果表明：冷休克诱导雌核发育二倍体获得了35.3%的孵化率和45日龄9.9%的成活率;有4对引物可扩增出清晰的亲本差异条带,其中引物C44、C27扩增结果显示G1子代中均未出现雄鱼基因,全部为雌核发育产物,C27扩增结果显示G2子代中也未出现雄鱼基因,但D19扩增结果显示G2子代中有3尾出现雄鱼基因,属正常受精个体,两家系后代的基因纯合率分别达93.8%和72.9%,平均为83.3%。研究表明雌核发育是促进基因纯合的一个有效途径,微卫星标记技术是是鱼类雌核发育鉴定和遗传分析的有效方法。     

岱衢洋大黄鱼染色体核型分析

为补充岱衢洋海域产大黄鱼(Pseudosciaena crocea)的细胞遗传学数据,采用植物血球凝集素(8~10μg/g)及秋水仙素(1~2μg/g)活体腹腔注射培养,头肾细胞制片、空气干燥法制作染色体标本,显微观察象山港网箱养殖的岱衢洋产大黄鱼的染色体核型,用Micromeasure3.3软件测量染色体相对长度与臂比。结果显示,岱衢洋大黄鱼二倍体染色体数目为48,核型公式为2n=24st+24t,NF=48,染色体相对长度最长为5.53,最短为2.60,未发现异型染色体和随体。本研究的岱衢洋产大黄鱼染色体核型与以往报道的大黄鱼核型存在差异。     

Characterization of OmpK, GAPDH and their fusion OmpK-GAPDH derived from Vibrio harveyi outer membrane proteins: their immunoprotective ability against vibriosis in large yellow croaker (Pseudosciaena crocea)

AIM: To investigate the immunoprotection of three recombinant proteins derived from the Vibrio harveyi outer membrane proteins (OMPs) OmpK, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and their fusion OmpK-GAPDH as vaccine candidates from vibriosis of large yellow croaker (Pseudosciaena crocea). METHODS: The ompK gene, of which the leader sequence was omitted, was fused with the gapdh gene. Three recombinant proteins r-OmpK, r-GAPDH and r-OmpK-GAPDH were expressed and purified. Western blots were carried out to detect the specificity of the antibodies raised against the recombinant proteins; Fish were immunized with recombinant proteins and challenged by native V. harveyi. The immunoresponse to the recombinant proteins were determined by ELISA and phagocytic activity assay. CONCLUSIONS: The fusion protein r-OmpK-GAPDH can afford greater protection against the wild V. harveyi than r-OmpK or r-GAPDH alone or their mixture in humoral and cellular immunity, indicating that OmpK and GAPDH could produce a synergistic immunoprotection against vibriosis of large yellow croaker (Pseudosciaena crocea) when fused into OmpK-GAPDH with a linker. SIGNIFICANCE AND IMPACT OF THE STUDY: It has been realized that a multi-component OMP antigen can induce a higher frequency of immune effectors than a single OMP. The results presented here bring forth a good suggestion for the subunit vaccine design based on the OMPs of gram-negative pathogens.     

大黄鱼病原哈维氏弧菌单克隆抗体的制备及其应用

用甲醛灭活哈维氏弧菌（Vibrio harveyi）GYC1108-1制备免疫原免疫8周龄雌性BALB/c小鼠,利用淋巴细胞杂交瘤技术,用间接酶联免疫吸附试验（ELISA）对杂交瘤进行筛选,阳性克隆经2－3次亚克隆后,共获得5株针对GYC1108-1的单克隆抗体。选取1B7制备小鼠腹水抗体,其细胞上清及腹水效价分别为1∶3200和1∶32000。同样用灭活的哈维氏弧菌免疫新西兰大白兔,5次免疫后颈动脉采血,离心取血清,并用饱和硫酸铵法进行纯化,制备了兔抗哈维氏弧菌多克隆抗体,其ELISA效价为1∶51200。利用1B7单克隆抗体和兔抗哈维氏弧菌多克隆抗体以及山羊抗小鼠HRP酶标二抗,建立了检测哈维氏弧菌的三抗体夹心酶联免疫吸附试验（TAS-ELISA）方法。该方法对哈维氏弧菌的最小检出浓度为1×104个/mL。用该TAS-ELISA方法检测大黄鱼（Pseudosciaena crocea）样品,54尾患病大黄鱼中有45尾检出哈维氏弧菌,而14尾健康大黄鱼都没有检出哈维氏弧菌。由此可见,本试验建立的TAS-ELISA方法,可以用于患病大黄鱼哈维氏弧菌的快速诊断。     

养殖大黄鱼细菌茵群分离鉴定与分析

对新鲜大黄鱼感官、化学、微生物品质和细菌菌群组成进行定性和定量分析。结果表明,细菌总数为5．51±0．25log10cfu／g,挥发性盐基氮为7．84±2．25mg／100g。分离获得279株细菌,84．2％是革兰氏阴性菌,少量革兰氏阳性菌被检出(6．1％)。优势菌群是肠杆菌科(14．7％)、气单胞菌属(12．5％)、不动杆菌属(11．5％)、摩氏杆菌属(11．1％),并出现了一定比例的假单胞菌属、嗜麦芽窄食单胞菌和其他细菌。肠杆菌科出现较多,表明大黄鱼细菌污染主要来自养殖海区,受非原有菌污染较重,应引起重视。     

养殖大黄鱼细菌菌群分离鉴定与分析*

对新鲜大黄鱼感官、化学、微生物品质和细菌菌群组成进行定性和定量分析。结果表明,细菌总数为5．51±0．25log10cfu／g,挥发性盐基氮为7．84±2．25mg／100g。分离获得279株细菌,84．2％是革兰氏阴性菌,少量革兰氏阳性菌被检出(6．1％)。优势菌群是肠杆菌科(14．7％)、气单胞菌属(12．5％)、不动杆菌属(11．5％)、摩氏杆菌属(11．1％),并出现了一定比例的假单胞菌属、嗜麦芽窄食单胞菌和其他细菌。肠杆菌科出现较多,表明大黄鱼细菌污染主要来自养殖海区,受非原有菌污染较重,应引起重视。     

船舶噪声对鲈鱼和大黄鱼血浆皮质醇水平的影响

在实验室条件下研究了模拟船舶噪声对养殖鲈鱼、大黄鱼血液中皮质醇分泌量的影响。结果表明,噪声刺激鲈鱼和大黄鱼后,鱼类血液中皮质醇含量先上升、后下降,后逐渐恢复原有水平或维持在较高的水平。鲈鱼血液中皮质醇水平在155dB的胁迫条件下于刺激1h左右达峰值,为112.23ng/mL,大黄鱼血液皮质醇水平峰值出现在刺激后20min-1h,值为17.18ng/mL。鲈鱼血液中皮质醇水平(CL155,CL138)受重复噪声刺激后随时间(T)回归方程:CL155=33.507e0.2425T(r=0.8939,P0.01);CL138=36.187e0.1862T(r=0.8002,P0.01),大黄鱼血液中皮质醇水平(CH155,CH138)受间歇噪声刺激后随时间(T)回归方程为:CH155=3.1208e0.2535T(r=0.8833,P0.01);CH138=2.8369e0.1706T(r=0.8064,P0.01)。鲈鱼(CL)、大黄鱼(CH)血液中皮质醇水平与噪声源强(S)的回归方程为:CL=33.05154e0.2461S,r=0.9563;CH=3.9706e0.2401S,r=0.9114。     

大黄鱼与小黄鱼细胞色素b基因全序列的比较分析

对大黄鱼、小黄鱼线粒体细胞色素b基因进行了PCR扩增及序列测定,得到1140bp的全序列。大黄鱼和小黄鱼的碱基组成相似,前者T、C、A、G含量分别为28.4%、33.0%、23.2%和15.4%,A+T含量为51.6%;后者T、C、A、G含量分别为26.7%、34.1%、23.8%和15.4%,A+T含量为50.5%。大、小黄鱼cytb基因中三联体密码子中碱基的使用频率很相似,第一位较均一,第二位富含T,第三位富含C。大小黄鱼cytb基因存在明显差异,序列相似性仅为88.95%;两序列间具有126个差异位点;碱基转换/颠换率为3.1,碱基替换多发生在密码子第三位;碱基转换中C\T显著高于A\G,表现出转换偏歧。     

三种消毒剂对大黄鱼致病性弧菌的体外杀灭作用研究

为了解不同消毒剂对大黄鱼中致病性弧菌的抑菌效果。采用纸片扩散法比较了聚维酮碘(PVP-I)、聚六亚甲基双胍(PHMB)和过氧化氢(H2O2)对溶藻弧菌和副溶血性弧菌的抑菌效果,再以肉汤稀释法测定了三种消毒剂的最低抑菌浓度(MIC)、最低杀菌浓度(MBC)及MBC/MIC比值,在此基础上,获得了抑菌效果相对较优的H2O2、PHMB对溶藻弧菌、副溶血性弧菌及其混菌的杀菌动力学曲线。结果表明,H2O2(3%)对两种弧菌具有最大的抑菌圈直径(19~21mm),PHMB(30mg/mL)次之(约为15mm),PVP-I(10%)的抑菌效果最弱(小于10mm)。3种消毒剂的MIC值由小到大依次为PHMB相似文献   

黄姑鱼（ Nibea albiflora） 与大黄鱼（ Pseudosciaena crocea） 精子超微结构的观察与比较

应用扫描电镜（SEM）与透射电镜（TEM）观察了黄姑鱼和大黄鱼精子的超微结构。结果显示,黄姑鱼和大黄鱼精子无论在形态、大小还是超微结构上都十分相似。黄姑鱼和大黄鱼精子均由头部、中段和尾部（鞭毛）3部分组成。精子头部形状近似椭圆形,无顶体,细胞核呈肾形。中心粒复合体位于细胞核背侧,近、远端中心粒相互垂直,远端中心粒分化成基体并形成轴丝。中段的袖套呈筒状,4～5个圆形的线粒体围绕轴丝呈环形排列。精子尾部为单鞭毛,轴丝为典型“9＋2”结构,鞭毛表面质膜形成不规则侧鳍。     

Histological development of the digestive system of the Amazonian pimelodid catfish Pseudoplatystoma punctifer

The organogenesis of the digestive system was described in the Amazonian pimelodid catfish species Pseudoplatystoma punctifer from hatching (3.5 mm total length, TL) to 41 days post-fertilization (dpf) (58.1 mm TL) reared at 28°C. Newly hatched larvae showed a simple digestive tract, which appeared as a straight undifferentiated and unfolded tube lined by a single layer of columnar epithelial cells (future enterocytes). During the endogenous feeding period, comprised between 20 and 96 h post-fertilization (3.5 to 6.1 mm TL), the larval digestive system experienced a fast transformation with the almost complete development and differentiation of most of digestive organs (buccopahrynx, oesophagus, intestine, liver and exocrine pancreas). Yolk reserves were not completely depleted at the onset of exogenous feeding (4 dpf, 6.1 mm TL), and a period of mixed nutrition was observed up to 6 to 7 dpf (6.8 to 7.3 mm TL) when yolk was definitively exhausted. The stomach was the organ that latest achieved its complete differentiation, characterized by the development of abundant gastric glands in the fundic stomach between 10 and 15 dpf (10.9 to 15.8 mm TL) and the formation of the pyloric sphincter at the junction of the pyloric stomach and the anterior intestine at 15 dpf (15.8 mm TL). The above-mentioned morphological and histological features observed suggested the achievement of a digestive system characteristic of P. punctifer juveniles and adults. The ontogeny of the digestive system in P. punctifer followed the same general pattern as in most Siluriform species so far, although some species-specific differences in the timing of differentiation of several digestive structures were noted, which might be related to different reproductive guilds, egg and larval size or even different larval rearing practices. According to present findings on the histological development of the digestive system in P. punctifer, some recommendations regarding the rearing practices of this species are also provided in order to improve the actual larval rearing techniques of this fast-growing Neotropical catfish species.     

Effects of starvation on lipid accumulation and antioxidant response in the right and left lobes of liver in large yellow croaker Pseudosciaena crocea

The present study aimed to determine the effects of starvation on lipid content and antioxidant responses in the right and left lobes of liver in large yellow croaker. Fish were divided into three groups: the control fish fed normally and the fish starved for 4 and 12 days. The set of biomarkers were determined, including crude lipid and MDA contents, and mRNA levels and activities of copper and zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR). Starvation for 12 days decreased lipid content and increased MDA content and mRNA levels and activities of antioxidant enzyme genes tested in both lobes of liver. No significant difference in these biomarkers between both lobes of liver was observed in fish starved for 12 days. However, there were significant differences between both lobes of liver in lipid and MDA contents, activities of CAT and GR, and expression levels of Cu/Zn-SOD and GR in fish starved for 4 days. These observed differences between starved and fed fish and between both lobes of liver could be important biomarkers that contributed in separating starved from fed fish and short-term starved from long-term starved fish, respectively. Our study emphasized the same lobe of the liver should be sampled when evaluating biomarkers during starvation in fish.     

Molecular cloning and functional characterization of a RabGTPase in large yellow croaker (Pseudosciaena crocea)

The molecular mechanisms of the immune system against pathogens in large yellow croaker (Pseudosciaena crocea) are not well known, despite its economic importance as an aquaculture species. In this investigation, a Rab gene (named as LycRab gene) was obtained from this fish, which exhibited high homology with Rab8 of other species. It was expressed in Escherichia coli, and the specific antibody was raised using the purified fusion protein (GST-LycRab). The LycRab protein, containing characteristic signatures of Rab proteins with 5 GTP-binding domains, had GTP-binding activity. The LycRab gene was ubiquitously expressed in all analyzed tissues as revealed by Western blot, although expression levels varied from tissue to tissue. Real-time PCR revealed that the LycRab gene was up-regulated after immunization with poly I:C, formalin-inactive Gram-negative bacterium Vibrio parahaemolyticus or bacterial lipopolysaccharides (LPS), suggesting that LycRab protein might play an important role in large yellow croaker defense against pathogens infection. This discovery might contribute better understanding to the molecular events involved in fish immune responses.     

Ontogeny of the digestive tract during larval development of yellowtail flounder: a light microscopic and mucous histochemical study

The histological development and mucous histochemistry of the alimentary tract in larval yellowtail flounder were studied using light microscopy. Samples were taken when the larvae were first offered food at 3 days post-hatch, then at 7, 10, 29, 36, and 46 days post-hatch, at which time they were metamorphosing. Regional partitioning of the digestive tract into the buccal cavity, pharynx, oesophagus, post-oesophageal swelling (PES), intestine, and rectum was complete by day 10. Goblet cells were present only in the buccal cavity, pharynx and intestine by day 7, but increased in number and distribution as development continued. By day 29, the posterior zone of the oesophagus had a marked increase in goblet cell density and mucosal folding. At the transition from oesophagus to PES/stomach stratified epithelium with goblet cells changed abruptly to a columnar epithelium with no goblet cells. Multicellular glands in the PES of 36-day larvae allowed it to be defined as a stomach. The distinct brush border of columnar epithelium and the presence of goblet cells characterize the intestine and rectum. All goblet cells throughout the digestive tract were strongly positive for acid mucins as was the luminal layer of the stratified epithelia lining the buccal cavity, pharynx and oesophagus. The PES/stomach epithelium stained weakly for neutral mucins. No mucin staining was associated with the gastric glandular epithelium. The brush borders of the intestine and rectum were strongly positive for combinations of neutral and acid mucins.