鲟鱼分子鉴定方法的整合应用

鲟形目物种是国家重点保护水生野生动物和CITES附录物种。其人工养殖种群数量众多, 种类丰富, 产品贸易量大, 但种类鉴定困难。本文在厘清当前鲟鱼商业类群的基础上, 通过分析现有种类鉴定方法, 整合了线粒体DNA遗传分析、SNP分析和微卫星DNA分析的鉴定方法, 探讨其鉴定国际贸易所涉鲟鱼的可行性。结果表明: 上述3种方法整合应用可在11种纯种鲟鱼及其正反杂交产生的杂交鲟范围内进行盲检。当前共有贸易鲟鱼36种, 其中杂交鲟14种, 杂交鲟的亲本共涉及9种鲟鱼。整合方法可准确鉴定小体鲟(Acipenser ruthenus)、达氏鳇(Huso dauricus)、施氏鲟(Acipenser schrenckii)、欧洲鳇(Huso huso)、闪光鲟(Acipenser stellatus)、高首鲟(A. transmontanus)两两杂交所产生的杂交鲟, 小体鲟为母本与纳氏鲟(Acipenser naccarii)或富氏鲟(A. fulvescens)或中华鲟(A. sinensis)产生的杂交鲟, 纯种的达氏鳇、高首鲟、富氏鲟和中华鲟, 但无法准确鉴定纯种的西伯利亚鲟(Acipenser baerii)和俄罗斯鲟(A. gueldenstaedti)以及父母本涉及此两种鲟鱼的杂交鲟。由于已开发的分子标记仍有限, 上述结果是对当前CITES贸易所涉鲟鱼鉴定的最大范围, 可以满足一些鲟鱼野生种群保护、贸易产品检测、种质资源管理等情景下的鉴定需求。     

三种鲟科鱼类和两种鲑科鱼类卵黄免疫球蛋白性质的比较

从施氏鲟(Acipenser schrencki)、小体鲟(Acipenser ruthenus)、西伯利亚鲟(Acipenser baeri)、哲罗鲑(Hucho taimen)和金鳟(Oncorhynchus mykiss)五种鱼类卵黄中分离、纯化Ig,并对其分子结构进行了初步研究。结果表明:五种鱼类卵中Ig的分子量分别为施氏鲟524kD、小体鲟468kD、西伯利亚鲟475kD、哲罗鲑为490kD,金鳟498kD,其Ig重链的相对分子量相同,约为97kD。轻链的相对分子量各不相同,施氏鲟为27kD,小体鲟28.5kD,西伯利亚鲟30kD,哲罗鲑28.5kD和金鳟16kD。其中西伯利亚鲟、施氏鲟和小体鲟Ig等电点约为5.85,哲罗鲑和金鳟的Ig等电点约为6.55,其蛋白质结构上具有一定的相似性,且均为糖蛋白     

基于线粒体基因组全序列的鲟形目鱼类(Pisces: Acipenseriformes)的分子系统发育重建

为厘清鲟形目鱼类的系统发育, 研究新测定了中华鲟(Acipenser sinensis)、长江鲟(A. dabryanus)、短吻鲟(A. brevirostrum)、纳氏鲟(A. naccarii)、鳇(Huso dauricus)和匙吻鲟(Polyodon spathula)共6种鲟类的线粒体全基因组序列。联合已测的17种鲟类的线粒体基因组数据, 利用最大似然法和贝叶斯法重建了鲟形目鱼类的分子系统发育关系, 并采用似然值检验对不同的树拓扑结构进行了评价。结果表明, 6种新测鲟类的线粒体基因组大小为16521—16766 bp, 编码13个蛋白质编码基因、22个转运RNA基因和2个核糖体基因, 与大多数已测的鲟类的线粒体基因组结构高度相似。基于23种鲟形目鱼类线粒体基因组数据, 系统发育分析的结果表明: (1)鲟形目的两个科, 匙吻鲟科(Polyodontidae)和鲟科(Acipenseridae)均为单系; (2)鲟科的内部亲缘关系复杂, 鲟属和鳇属的物种均不构成单系群。鲟科鱼类按分子系统发育重建结果可以分为3个类群: 尖吻鲟类(A. sturio - A. oxyrinchus clade)、大西洋鲟类(Atlantic clade)和太平洋鲟类(Pacific clade)。树拓扑结构的检验结果表明, 鲟科的系统发育关系为(尖吻鲟类(太平洋鲟类, 大西洋鲟类))。铲鲟属(Scaphirhynchus)是大西洋鲟类的基部类群。研究也说明线粒体基因组数据在鲟形目鱼类系统与进化研究方面具有重要应用价值。     

鲟分枝杆菌病及其病原研究

20092010年间,我国人工养殖的中华鲟（Acipenser sinensis）、史氏鲟（Acipenser schrencki）和杂交鲟（hybrid sturgeon:A. baeri-A. gueldenstaedtii）暴发了细菌性疾病。患病鲟通过组织切片观察,病原菌的分离、鉴定以及组织样品中病原菌的检测,结果显示从19条患病鲟中分离到49株分枝杆菌。病原菌经过多个保守基因的测序分析和部分生理生化特征的鉴定,共发现有7种分枝杆菌,分别为龟分枝杆菌（Mycobacterium chelonae）、海分枝杆菌（Mycobacterium marinum）、戈登氏分枝杆菌（Mycobacterium gordonae）、偶发分枝杆菌（Mycobacterium fortuitum）、苏尔加分枝杆菌（Mycobacterium szulgai）、猪分枝杆菌 （Mycobacterium porcinum）和Mycobacterium arpuense。在诊断过程中发现两种或三种分枝杆菌同时存在于同一样品中,分子生物学的诊断结果表明分枝杆菌复合感染十分常见,而海分枝杆菌是分枝杆菌复合感染中最为常见的分枝杆菌。分离的病原菌对斑马鱼的攻毒试验结果表明在以上7种分枝杆菌中海分枝杆菌的毒力最强。以上结果表明海分枝杆菌是鲟分枝杆菌病的主要致病菌,分枝杆菌复合感染是鲟分枝杆菌病的主要感染形式。研究中史氏鲟和中华鲟的分枝杆菌病,以及在病鱼体内分离的猪分枝杆菌和M. arupense在国内外均尚未见报道。       

5种鲟鱼免疫球蛋白重链恒定区序列研究

为了探讨几种鲟鱼免疫球蛋白(IgM)所包含的信息与其亲缘和进化之间的关系, 分别对俄罗斯鲟(Acipenser. gueldenstaedtii)、小体鲟(A. ruthenus)、施氏鲟(A. schrenckii)、中华鲟(A. sinensis)和欧鳇(Huso huso)的IgM重链(IgH)恒定区进行了研究。采用RT-PCR的方法对IgH核酸序列进行了克隆, 通过软件获得了相应的IgH氨基酸序列。在分别对这5种鲟鱼免疫球蛋白重链恒定区4个区(CH1～CH4)进行研究后发现, 其CH4区氨基酸序列相似性最高。通过对CH4区序列氨基酸变异期望值(Kaa), 物种分化时间(T)及物种间系统进化树(Phylogenetic Tree)等参数的分析, 将克隆的5种鲟鱼IgH恒定区序列与已发表的西伯利亚鲟同源序列比对(源于NCBI序列)后发现: 西伯利亚鲟与俄罗斯鲟、施氏鲟与欧鳇、小体鲟各构成一个分支, 并与中华鲟相对。实验结果从体液免疫系统的演化这个角度, 反映了被研究的鲟鱼物种间的分类地位、地理分布及进化关系之间的联系。     

盐度对施氏鲟和西伯利亚鲟稚鱼的急性毒性

比较了盐度对施氏鲟(Acipenser schrenckii)和西伯利亚鲟(A. baerii)稚鱼的急性毒性效应.结果表明:施氏鲟稚鱼的96h盐度半致死浓度(LC50)为14.72,西伯利亚鲟为13.08;96h盐度LC50及盐度反应曲线显示,施氏鲟对盐度的耐受力大于西伯利亚鲟;在高盐度(≥14.70)下,2种鲟鱼依次表现出缓慢环游、狂躁环游、活动减弱、身体失衡和活动停止(死亡)等5种行为反应;西伯利亚鲟各行为反应出现的时间均早于施氏鲟,说明西伯利亚鲟对盐度的反应较施氏鲟敏感.     

鲟鱼心外膜脓肿的病理学初步研究

作为新的养殖对象,我国各地先后开展了中华鲟(Acipenser sinensis Gray)、施氏鲟(A.schrenckii Brandt),以及引进的俄罗斯鲟(A.gueldenstaedti Brandt)、杂交鲟等鲟鱼人工养殖,由于养殖刚刚起步且养殖集约化程度较高等诸多因素,出现了多种新的非寄生性疾病,并造成大量死亡,经济损失极其严重,成为当前鲟鱼试养阶段的制约因素。1999年北京市一家鲟鱼养殖试验场鲟鱼患病,经检验和诊断,确诊为“鲟鱼心外膜脓肿”.本文报道了该病的病理解剖和组织病理学研究结果,为疾病的临床检验与诊断及有效控制提供科学依据。       

水下噪音对杂交鲟行为及肠道微生物的影响

为探究水下噪音对杂交鲟行为变化(游泳速率、摄食速率、空间分布)及其肠道微生物的影响,在(145±5) dB, 400 Hz的噪音条件下对杂交鲟进行了0、24h、48h、7d和14d的噪音刺激,随后置于无胁迫噪音的自然环境中恢复48h。结果显示,噪音刺激后杂交鲟游泳速度和摄食速度显著减慢,空间分布发生改变,噪音刺激开始时杂交鲟聚集于远离噪音源的一侧, 3min后却逐渐接近噪音源。噪音对杂交鲟的肠道微生物的丰富度无显著性影响;不同时间段的各组肠道微生物的优势种群存在差异,且随时间变化优势种群反复改变;以上微生物分析结果通过COG预测显示,噪音胁迫48h和7d时“细胞信号传递过程”,“碳水化合物的转运”及“氨基酸的运输和代谢功能”显著低于其他组。结果表明,噪音对杂交鲟摄食速率、游泳速率及空间分布影响显著,改变了其肠道微生物组成和占比,影响了其氨基酸代谢等多条生命活动相关通路。实验模拟了水下各类噪音源的混合噪音对杂交鲟行为和肠道微生物的影响,为探讨杂交鲟的健康生态养殖和逆境生理响应机制提供基础资料。     

黑龙江捕获少见的鲟、鳇鱼大型个体

鲟(Acipenser schrencki)、鳇(Huso dauricus)是黑龙江流域的特产经济鱼类,年产量约20万斤。鲟鱼体重在30—65斤,占48.8％;鳇鱼体重100—400斤,占71.2％。文献记载曾捕获体重达2,000斤鳇鱼,但访问有50年捕鱼史的老渔民,都说从未见过,并说鲟鱼超过百斤、鳇鱼超过千斤的个体极为少见。 1979年汛期生产时,我们在萝北县召兴公社水产     

海水养殖杂交鲟的行为习性

对海水养殖条件下杂交鲟的行为习性进行观察.结果表明,杂交鲟对干露的耐受力较强,干露1 h存活率100%,干露3 h存活率74.1%;食性可以驯化,能以全植物性蛋白源为饲料,耐饥饿能力强;摄食节律明显,在水泥池内养殖形成早晨、傍晚两个摄食高峰,深水网箱养殖在接近早晨、傍晚的平潮或停潮时间内形成两个摄食高峰,在这两个摄食高峰的摄食量超过日摄食总量的50%;水泥池养殖的杂交鲟具有昼伏夜出习性,深水网箱养殖的杂交鲟流急时在网箱底部活动、流缓时到水层中游动;杂交鲟抗流速、抗波浪能力较美国红鱼差、较大黄鱼强.杂交鲟能较快适应海水养殖环境,放入深水网箱养殖的杂交鲟规格宜在400 g左右.     

Generative and somatic production of female sturgeon at a research farm in Primorsky Krai as a basis for manufacturing gastronomic caviar

The generative and somatic characteristics of female sturgeon of five species and three hybrid forms, which were reared at the TINRO Center’s warm-water cage farm at the north of Primorsky Krai and used in caviar production, are considered. Data on the somatic growth, roe output, functional fecundity, mean egg weight, and gonadosomatic index of the female Amur Sturgeon, Kaluga, Siberian Sturgeon of the Lena and the Baikal populations, Sterlet of the Volga population, hybrids of Russian Sturgeon and Siberian Sturgeon, hybrids of Siberian Sturgeon and Amur Sturgeon, and hybrids of Kaluga and Amur Sturgeon are analyzed. The sexual maturity age and interspawning intervals are defined in the studied females. Domesticated females of all the species are shown to mature a few years earlier than those in natural conditions and to have a greater body weight. The interspawning intervals shorten by a few years in domesticated females. Most female Sterlet and some females from both populations of Siberian Sturgeon spawn annually, while the remainder spawn once in 2 years. The interspawning intervals in two species of sturgeon that inhabit the Amur River and in hybrid forms usually last for 2 years. With age, values of characteristics such as roe amount, fecundity, mean egg weight, and roe output relative to body weight grow in females spawning for the second time in the warm-water farm. The maximum roe output is found in female Kalugas at the second spawning. Then the species follow in the order of decreasing of roe output: Amur Sturgeon, hybrids of Amur Sturgeon with Kaluga, hybrids of Siberian Sturgeon with Amur Sturgeon, hybrids of Russian Sturgeon with Siberian Sturgeon, Siberian Sturgeon of the Baikal population, Siberian Sturgeon of the Lena population, and Sterlet. The hybrid between the Russian Sturgeon and Siberian Sturgeon shows the best processability and survivability characteristics; the Amur Sturgeon and Sterlet follow.     

Sturgeon production trials in the waste-heat effluents of a pulp and paper plant in Archangelsk, Russia

Sturgeons were cultured at the Solombalsky cellulose and paper plant (CPP) in Arkhangelsk over a 3 year period. The warm-water effluents of the biological treatment unit were utilized to rear the Siberian sturgeon (Acipenser baerii) and its hybrids with the sterlet (Acipenser ruthenus). Another culture trial included the Russian sturgeon (Acipenser gueldenstädtii) and its hybrids with the beluga (Huso huso). The most profitable culture (in terms of growth rates) was with the Siberian sturgeon and its hybrids with the sterlet. After the second year of culture, Siberian sturgeon and its hybrid averaged about 2.3 kg in wet weight while Russian sturgeons gained less (1.5 kg). One- and two-year old sturgeons grown at the Solombalsky CPP met practically all requirements needed for a successful culture.     

Fecundity of Atlantic sturgeon (Acipenser oxyrinchus Mitchill, 1815) captured by the commercial fishery in the Saint John River,New Brunswick,Canada

Knowledge of fecundity in fishes is important for understanding life history, modeling population dynamics, developing fisheries management and determining conservation status. Sturgeon species are known for their high fecundity but to date only two populations of Atlantic sturgeon (Acipenser oxyrinchus Mitchill, 1815) have been examined for this aspect of their biology. The species is protected in most regions of eastern North America but the Saint John River, New Brunswick supports one of the two remaining commercial fisheries in Canada. Sixty females selected by this fishery were sampled for total length, weight, age, gonad weight and egg number. Absolute fecundity ranged from 153,630 to 1,306,626 eggs, with a mean fecundity of 582,832 ± 261,806 eggs. Absolute fecundity exhibited a significant positive relationship to increasing body weight but not to total length or age. Relative fecundity ranged from 129 to 216 eggs/g gonad weight and although it declined with both increasing body size and age the relationships were not significant. The Gonadosomatic Index (GSI) ranged from 1.3% to 27.0% with a mean of 7.8% ± 4.94 SD. Relative fecundity found in this study was higher and mean GSI lower than reported for other Atlantic sturgeon populations and other sturgeon species. Differences were probably due to location of the fishery downstream from spawning sites and selection of pre-spawning females by the commercial fishery.     

4种鲟鱼养殖亲鱼群体遗传多样性分析

近年鲟鱼人工繁殖技术促进了鲟鱼养殖业的发展,但在选育初期未对后备亲鱼进行遗传背景分析,为了抑制种质资源退化和防止近交衰退,繁育场需要对现有后备亲鱼进行遗传背景分析.本文利用线粒体控制区(D-loop)部分序列对4种养殖鲟鱼后备亲鱼群体(共120个样本)的遗传多样性进行了分析.研究发现不同种鲟鱼D-loop部分序列长度不...     

Immunoglobulin heavy chain constant region of five Acipenseridae: cDNA sequence and evolutionary relationship

The immunoglobulin M (IgM) heavy chain constant region genes of Russian sturgeon (Acipenser gueldenstaedtii), Sterlte sturgeon (Acipenser ruthenus), Amur sturgeon (Acipenser schrenckii), Chinese sturgeon (Acipenser sinensis) and Great sturgeon (Huso huso) were cloned and analyzed with molecular biology and bioinformatics methods. We cloned IGH nucleic acid sequences by RT-PCR using the specific primer, then determined the characteristics and functions of the amino acid sequences. The gene contains four constant region domain-encoding exons (CH1-4), of which CH4 sub-regions were the most conserved in IgM heavy chain constant region domain and had the highest identity within all the experimental species. According to the analysis of the phylogenetic tree, the variation expectation value (K(aa)), and species differentiation time (T) in the CH4 sub-region, we found that Chinese sturgeon and the other five sturgeon form one whole bifurcation of the tree, while, among the five left, Amur sturgeon and Huso sturgeon, Russian sturgeon and Siberian sturgeon (data from GenBank), Sterlte itself forms three other bifurcations. This result can clearly explain the relations of taxonomic status, geographical distribution and evolution among the species studied.     

四个罗非鱼选育品种抗链球菌病能力差异研究

为筛选出抗病力优良的罗非鱼品种, 以奥利亚罗非鱼“夏奥1号”、尼罗罗非鱼“99”埃及品系、吉富罗非鱼“中威1号”和奥尼罗非鱼为研究对象, 33℃水温暂养7d后分别进行无乳链球菌人工感染实验, 连续7d统计累计死亡率, 并于人工感染后0、24h、48h和72h采集血液和组织样本, 研究这4个罗非鱼选育品种抗链球菌病能力的差异。结果显示: 感染7d后奥尼罗非鱼的累计死亡率最低; 奥尼罗非鱼的谷草转氨酶(AST)感染前后始终都低于其余3个品种罗非鱼(P<0.05); 埃及尼罗和奥尼在感染72h后球蛋白(GLO)分别显著升高1.13倍和1.41倍; 奥尼罗非鱼白蛋白/球蛋白(A/G)在感染前后没有显著性变化(P>0.05), 而其余3个品种罗非鱼A/G比值在感染后都显著性降低(P<0.05); 埃及尼罗的碱性磷酸酶(AKP)在感染72h后显著降低(P<0.05), 奥利亚和吉富的AKP表现为先上升后下降, 奥尼的AKP感染前后没有显著性变化(P>0.05); 各品种罗非鱼血清中的乳酸脱氢酶(LDH)感染后都显著升高(P<0.05); 奥利亚、吉富和奥尼罗非鱼的超氧化歧化酶(SOD)感染48h时都显著升高(P<0.05); 奥尼罗非鱼在感染前后溶菌酶(LZM)活性都显著高于其余3个品种罗非鱼(P<0.05)。组织病理学结果显示:吉富和奥尼肝细胞水肿变性, 而奥利亚和埃及尼罗出现大面积肝细胞脂肪变性; 每个品种罗非鱼均呈现严重的脾炎, 奥利亚、埃及尼罗和吉富的脾脏中大量铁血黄素沉积; 每种罗非鱼呈现不同程度的肾小球萎缩, 肾小管上皮细胞变性、坏死。研究表明奥尼罗非鱼抗链球菌病能力最强, 感染后血清中AST水平与肝受损程度呈一定的正相关, LZM水平和罗非鱼抗链球菌病能力呈一定的正相关。     

A note on the fecundity of pallid sturgeon

Fecundity was estimated for two pallid sturgeon, Scaphirhynchus albus, from the Lower Mississippi River (LMR). Sturgeon measured 827 and 886 mm fork length, weighed <3.2 kilograms and had 16 237 and 17 810 ova per kilogram of body mass (based on mean ovum density) with total fecundity estimates ranging from 43 357 to 58 913. Ovum size in the two sturgeon was 2.5–3.0 mm diameter. Pectoral fin ray sections showed distinct annuli and spawning bands. The smaller sturgeon was aged at 10 and the larger fish at 12 years. Spawning bands were present and suggest the smaller sturgeon first spawned at age 8 and the larger fish at age 9. When compared to published results, the data representing specimens from more northern populations suggest that pallid sturgeon from the LMR mature at comparable ages but at smaller sizes and have greater gonadal mass. These features and greater fecundity per mass unit (number of eggs per kilogram body weight) suggest that LMR pallid sturgeon populations exhibit greater potential for population growth and recovery than other populations with slower growth and lower fecundity.     

Discrete phenotypical variability of electrophoretical specters of one of system of myogens in the Acipenseridae family

Analysis of electrophoregrams of water-soluble proteins of white skeletal muscle of 10 species belonging to three genera of the Acipenseridae family has allowed revealing an earlier not described system that can serve as a biochemical marker. This protein system represents a set of five fractions (A, B, C, D, E). A part of the studied species is monomorphous for one of these fractions (sterlet, giant sturgeon, Caspian sturgeon, and Amu-Darya shovelnose had phenotype A, barbel sturgeon—phenotype C, Amur sturgeon—phenotype D). In all individuals of the Amur sturgeon the identical three-component specter (phenotype ACE) was revealed. In the Sakhalin, Siberian, and Russian sturgeons a clearly expressed polymorphism was observed. Among the studied species the Russian sturgeon was characterized by the highest degree of heterogeneity and polymorphism. In the studied sample of this species all five protein variants are found, whereas in the rest of them—only a part of this set.