传染性脾肾坏死病毒、鳜鱼蛙病毒和鳜弹状病毒三重PCR检测方法的建立

【目的】建立可同时检测传染性脾肾坏死病毒（infectious spleen and kidney necrosis virus,ISKNV）、鳜鱼蛙病毒（Siniperca chuatsi ranairidovirus,SCRIV）和鳜弹状病毒（Siniperca chuatsi rhabdovirus,SCRV）的三重PCR检测方法,为鳜鱼等养殖品种流行病学调查提供方法支撑。【方法】根据ISKNV MCP基因、SCRIV MCP基因和SCRV N基因设计3对特异性引物,对PCR扩增反应中的退火温度和引物用量进行优化,建立可同时检测 ISKNV、SCRIV和SCRV的三重PCR方法。为验证该方法的敏感性和特异性(备测病毒为IPNV、GCRV、KHV、SGIV、NNV、TiLV和SVCV),对22份疑似感染ISKNV、SCRIV和SCRV的样品分别进行单一和三重PCR检测。【结果】成功建立了三重PCR检测方法,利用该方法可同时检测ISKNV、SCRIV和SCRV,特异性较好,对IPNV、GCRV、KHV、SGIV、NNV、TiLV、SVCV等无扩增;该方法敏感性好,对3种病毒核酸的检测下限均为0.01 ng/μL;利用该方法和3种病毒单一PCR方法同时对22份临床样品进行检测,结果显示2种方法吻合率为100%,其中ISKNV阳性率为27%、SCRIV 阳性率为41%、SCRV阳性率为9%、ISKNV和SCRIV混合感染阳性率均为9%,无ISKNV、SCRIV和SCRV混合感染。【结论】建立的三重PCR检测方法具有特异性强、灵敏度高等特点,可对这3种病毒进行快速鉴别诊断。     

6个不同群体鳜基于SSR的遗传多样性初步研究

旨在了解不同群体鳜遗传多样性及种质资源现状,采用微卫星标记技术,对采自6个不同群体鳜96尾个体的遗传多样性进行了初步分析。结果表明,经筛选的10对引物在6个鳜群体中的平均等位基因数(Na)为2.2000~2.5000,期望杂合度(He)为0.6489~0.7289,多态信息含量(PIC)为0.5614~0.6409 (PIC大鱼0.5);群体遗传分化系数(Fst)为0.0606(0.05小鱼Fst小鱼0.15),平均基因流(Nm)为4.0151;检验表明,除高邮和靖江群体外,其他4个群 体均不同程 度地偏 离Hardy-Weinberg平衡,主要表现为杂合子缺失。构建的UPGMA聚类图显示,铜陵、安庆、太湖、高邮和靖江群体聚为一类,当涂群体聚为另一类。本研究表明6个鳜群体的遗传多样性较高,群体间遗传分化水平中等,群体间存在着一定的基因交流。     

贵州3个斑鳜野生种群形态学差异

为探究贵州境内沅江、柳江和乌江水系野生斑鳜外形差异,基于形态学和框架结构测量数据,运用多元分析法比较3条水系斑鳜种群的形态特征,经数据标准化和参数选择后12项比例性状和12项可数性状进行统计分析。研究结果显示,单因素分析中9项可数性状之间差异显著(P<0.05);主成分中提取了前6个主成分,其累计贡献率为72.668%,前3项主成分对种群间外形差异贡献较大,主要受其尾柄长、眼间距和体宽等性状影响。应用逐步判别方法建立了这3个种群的特征判别函数,其交互验证判别准确率为81.5%。形态学聚类图结果显示,沅江和柳江种群形态关系较近先聚为一支,之后再与乌江种群相聚。差异系数分析表明,乌江种群与沅江和柳江种群在尾柄长/体长性状上的差异达到亚种水平(差异性系数>1.28)。综上可知,贵州3个斑鳜种群在形态学上存在一定差异,其中柳江与沅江形态差异较小,乌江与其差异较大,建立的判别方程可对3个种群进行区分。本研究结果可用于3个水系斑鳜种群的识别,也可为种群保护和育种利用提供理论依据。     

鳜仔鱼视网膜及口腔齿的发育对摄食的适应

采用石蜡切片及HE染色的方法对鳜受精卵及1～30 d仔鱼的视网膜结构和视觉特性进行研究,结果表明:(1)受精后54 h晶状体形成,视网膜还未分化;80 h视网膜分化为外核层、内核层、内网层和神经节细胞层4层结构;98 h形成色素上皮层、视杆视锥层和神经纤维层;124 h外网层形成,视网膜10层结构发育完全。孵化后4 d开始出现少量颌齿和咽齿;5 d和14 d分别观察到腭齿和犁齿;15d时鳃耙形成但形状较小。口腔齿的数量随着仔鱼个体的生长而增加至近于成体。(2)鳜内核层分化成一层水平细胞、数层双极细胞和无长突细胞,属光感受系统不发达的类型。(3)鳜仔鱼在消化系统尚未发育完全时已经开始形成口腔齿,为提高捕食的成功率提供了基础条件,充分表现了个体发育在进化过程中与生存环境相适应的特点。(4)鳜神经节细胞层细胞随个体的生长发育而减少,孵化后29 d,体长18.36 mm时,只有一层神经节细胞,说明此时的仔鱼已适应黑暗环境生活。鳜视觉结构的变化与其从浮游到底层的生态迁移及捕食方式的变化相适应。研究亮点:从组织学研究角度对鳜仔鱼视网膜发育进行了探讨,详细研究了视网膜各组成结构的发育特征,并结合鳜仔鱼齿的发育特征,分析了其对摄食行为的适应,首次解释了伴随着视网膜发育而形成的由浮游生活到底层生活的适应,研究结果以期为鳜在种苗生产过程中正确选择开口饵料和早期动物性饵料,以降低种苗的死亡率等提供理论依据。     

鳜弹状病毒与传染性脾肾坏死病毒双重PCR检测方法的建立

【目的】建立鳜弹状病毒(SCRV)和传染性脾肾坏死病毒(ISKNV)的双重PCR检测方法。【方法】分别针对SCRV N基因和ISKNV MCP基因的保守区域设计特异性引物,通过优化退火温度和引物用量(μL)比,建立SCRV和ISKNV的双重PCR检测方法,并对该方法的敏感性和特异性进行检验。应用建立的双重PCR方法对20份临床样品进行单一和双重PCR检测,比较单一和双重PCR的检测结果。【结果】成功建立了SCRV和ISKNV双重PCR方法,该法特异性较强,对草鱼呼肠孤病毒(GCRV)、锦鲤疱疹病毒(KHV)、神经坏死病毒(NNV)、鲤春病毒血症病毒(SVCV)、新加坡石斑鱼虹彩病毒(SGIV)、罗非鱼湖病毒(TiLV)等鱼类常见病毒无扩增;敏感性较好,对2种病毒DNA的检测下限均为0.01 ng/μL。20份临床样品的双重PCR样品检测结果显示,3份为SCRV阳性,11份为ISKNV阳性,2份为SCRV和ISKNV混合感染,结果与单一PCR检测结果一致。【结论】建立的SCRV和ISKNV双重PCR检测方法特异性较强,敏感性较好,可用于鳜鱼SCRV与ISKNV的快速鉴别诊断和分子流行病学调查。     

Effect of feeding stimulants on growth performance,feed intake and appetite regulation of mandarin fish,Siniperca chuatsi

Mandarin fish is a typical carnivorous fish, it can accept artificial diet after domestication, which is significantly essential to optimize its artificial diet. Nevertheless, only few studies were conducted on artificial diet of mandarin fish. Therefore, an 8‐week feeding trial was conducted to evaluate the influence of feeding stimulants on growth performance, feed intake and appetite regulation of mandarin fish. In this trial, six diets were formulated, a basal diet contained 80% of Fish Meal without feeding stimulants, named as Control, 0.4% inosinic acid meal, 0.4% L‐Alanine meal, 3% Yeast meal, 3% a commercial squid extract meal and 0.4% betaine meal were added into the basal diet to make five experimental diets, which were named as IM, AM, YM, SVO and BM respectively. At the end of feeding trial, SVO group showed higher feed intake, up‐regulated mRNA expression of neuropeptide Y (NPY) and down‐regulated mRNA expression of pro‐opiomelanocorticoid (POMC) significantly as compared to control group, which suggested that squid extract improved feed acceptability and promoted appetite in mandarin fish. Similarly, compared to control group, SVO group showed low food conversion ratio, high weight gain and SGR, indicated that squid extract enhanced the growth performance. Our results suggested that the appropriate level of squid extract addition could contribute to optimize artificial diet in mandarin fish.     

鸭绿江斑鳜胚胎及胚后发育观察

于2005、2006年,连续观察了斑鳜Siniperca scherzeri受精卵的胚胎及胚后发育全过程。结果表明:斑鳜受精卵在22.2~25.2℃水温条件下,孵化所需时间约为144 h,胚胎发育到111 h,口裂已经形成,张合自如;破膜后54 h即可捕食活体鱼苗。斑鳜胚胎期即形成口裂、心脏、胸鳍、鳃盖等器官,在破膜前具备了较其它鱼类先天功能更完善、更复杂的生理结构,这一生理特点为其胚后体质强壮,开口即可捕食,消化活体鱼苗奠定了基础。     

Impact of environmental and innate factors on the food habit of Chinese perch Siniperca chuatsi (Basilewsky) (Percichthyidae)

Laboratory and field investigations were conducted to study the food habit of Chinese perch Siniperca chuatsi (Basilewsky) from first feeding through adult stage. Only fish larvae were consumed by Chinese perch larvae (2–21 days from hatching), and the presence of zooplankton did not have any significant effect on their survival rate. The ability of Chinese perch to feed on zooplankton is clearly limited by some innate factor. Instead of gill rakers, Chinese perch larvae have well‐developed sharp teeth at the first feeding stage, and are well adapted to the piscivorous feeding habit unique to the larvae of Chinese perch, e.g. they bite and ingest the tails of other fish larvae. At the first feeding stage (2 days from hatching), daily rations were both very low, either in light or complete darkness. Although early‐staged Chinese perch larvae (7–17 days from hatching) could feed in complete darkness, their daily rations were always significantly higher in light than in complete darkness. Late‐staged Chinese perch larvae (21 days from hatching) were able to feed in complete darkness as well as in light, similar to the case of Chinese perch yearlings. Chinese perch yearlings (total length, 14–16 cm) consumed prey fish only and refused shrimp when visual cues were available (in light), but they consumed both prey when visual cues were not available (in complete darkness), suggesting that prey consumption by Chinese perch yearlings is affected by their sensory modality in predation. Both prey were found in the stomachs of similar‐sized Chinese perch (total length, 14–32 cm) from their natural habitat, suggesting that shrimp are consumed by Chinese perch at night. Prey selection of Chinese perch with a length >38 cm, which consumed only fish in the field, appears to be based upon prey size instead of prey type. These results suggest that although environmental factors (e.g. light intensity) affect prey detection by Chinese perch, this fish is anatomically and behaviourally predisposed to prey on live fish from first feeding. This makes it a difficult fish to cultivate using conventional feeds.     

斑鳜卵巢发育与血清蛋白磷含量变化关系的初步分析

斑鳜(Siniperca scherzeri)是近年来新开发的名贵淡水养殖鱼类之一,但规模化繁育技术尚未完全解决。为进一步了解斑鳜卵巢成熟发育的规律,利用组织切片技术观察了3月中旬-5月中旬长江斑鳜卵巢的成熟发育过程,并用生化法测定了血清蛋白磷(SPP)的含量,结果表明:这一时期,斑鳜卵母细胞正经历由Ⅲ时相(3月中旬-4月上旬)向Ⅳ时相(4月中旬-4月下旬)、Ⅴ时相(5月中旬)的渐近生长,并发现卵母细胞生长不同步的现象;与此同时,血清蛋白磷含量由0.12 mg/mL逐步上升至0.62 mg/mL。斑鳜血清蛋白磷含量的变化趋势与卵巢的成熟发育过程基本一致,并有着密切的生理相关,血清蛋白磷含量可以作为卵巢发育阶段和成熟程度判定的重要生化指标。