基于16S rRNA序列探讨我国海鳝科鱼类分子系统进化关系

为从分子水平分析海鳝科鱼类分子系统分类关系,澄清物种分类争议,实验通过PCR扩增获得我国海鳝科9个属26种鱼类16S r RNA序列片段,结合GenBank下载的其他海鳝科鱼类的序列进行分析。结果显示,进化树上,海鳝科主要形成裸海鳝亚科与海鳝亚科两个分支,裸海鳝亚科包括尾鳝属与鞭尾鳝属,海鳝亚科包括裸胸鳝属、裸海鳝属、勾吻鳝属、蛇鳝属等8个属,与形态分类结果一致。裸海鳝亚科分支中,尾鳝属与鞭尾鳝属形成平行的姐妹分支;海鳝亚科分支中,斑马裸海鳝单独形成一支,位于该分支基部,其他种类聚为另一支。分支I中裸胸鳝属、勾吻鳝属、蛇鳝属、海鳝属等属均无法形成单系,揭示这些种属可能是多系起源,与近期的分子水平研究观点相似。研究结果支持将斑马裸海鳝归为裸海鳝属,为单型属物种;虎斑鞭尾鳝归为鞭尾鳝属,与尾鳝属平行进化;拟蛇鳝从长海鳝属分离出来,归为拟蛇鳝属等分类观点。豹纹勾吻鳝与海鳝属关系十分接近,但其形态上还具有多个勾吻鳝属的基本特征,至于是否归为海鳝属,还需后续更多的分子与形态学研究加以验证。     

凡纳滨对虾不同养殖密度高位池水体细菌群落动态

通过对养殖水体环境基因组DNA中细菌16S rRNA基因V4–V5区的高通量测序和生物信息学分析,研究了两种养殖密度的凡纳滨对虾(Litopenaeus vannamei)高位池水体中细菌群落在养殖过程中的动态。结果显示,养殖过程中各菌群相对丰度变化明显,细菌多样性随时间逐渐提高,优势菌群为变形菌门(Proteobacteria)、蓝藻门(Cyanobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)和浮霉菌门(Planctomycetes)。随着养殖时间增长,蓝藻丰度所占比例逐渐减少,而变形菌、拟杆菌和浮霉菌丰度逐渐增大,同时养殖前期高密度池浮霉菌丰度显著高于低密度池(P0.01),而其他菌群无显著差异。结果表明,养殖期前50 d不同养殖密度水体细菌群落差异较大,而后30 d内细菌群落的时间异质性大于空间异质性,这意味着高位池水体菌相被划分为两类,到养殖后期菌相快速转变,养殖密度所带来的影响被减弱。     

基于线粒体基因组COI和12S rRNA序列的魁蚶中国群体分类学地位研究

东北亚地区不同地理种群魁蚶(Scapharca brouhtonii)的分类学地位仍存在较大争议。为探讨魁蚶中国群体的分类地位,测定了魁蚶中国群体线粒体基因组COI和12S rRNA序列,并从GenBank中下载了蚶科26种贝类的同源序列进行分析。以贻贝科的贻贝(Mytilus edulis)为外群,用邻近法和最大简约法构建了蚶科贝类的分子系统进化树,分析其分子系统进化关系和分类等级。结果显示,魁蚶中国群体的COI和12S rRNA基因的碱基组成、密码子使用率及变异位点等与魁蚶日本群体和粗饰蚶属的Anadara sativa相似,且遗传距离很小,进化树中聚为一支,亲缘关系很近,初步确定魁蚶中国群体的分类地位和魁蚶日本群体一致。本研究结果初步阐明了魁蚶中国群体的分子系统进化地位,有利于掌握魁蚶的遗传背景和资源状况,以应用于自然资源的保护和推动养殖业的发展。     

First report of Shewanella sp. and Listonella sp. infection in freshwater cultured loach,Misgurnus anguillicaudatus

A novel disease outbreak occurred among cultured loach, Misgurnus anguillicaudatus in China. The diseased fish displayed lethargy, loss of appetite, black discolouration, anasarca and ventral skin hyperaemia. Clinical signs were observed in most of the fish examined, which consisted of discoloured liver, swollen spleen and petechial haemorrhage in the intestine and peritoneal membranes. The causative agent was found to be two species of bacteria by experimental infection, identified as Shewanella sp. and Listonella sp. The result showed that these were likely to be Shewanella putrefaciens and Listonella anguillarum respectively. Bacterial identification consisted of physiological and biochemical tests as well as 16S rRNA sequence analysis. In addition, antibiotic susceptibility testing was performed, revealing that Shewanella putrefaciens and Listonella anguillarum were susceptible to enrofloxacin, ciprofloxacin, levofloxacin, pipemidicacid and norfloxacin. This is the first report of S. putrefaciens and L. anguillarum from cultured loach.     

基于12SrRNA和ND3基因序列分析巨[鱼丕]遗传多样性及系统进化

为从分子水平研究巨[鱼丕](Bagarius yarrelli Sykes)的遗传多样性和系统进化关系,本实验对采集于怒江、澜沧江、元江3河流5种群中的60个个体进行12S rRNA和ND3基因序列的PCR扩增,同时与红河河口群体12个个体的12S rRNA和ND3基因序列进行比对分析,采用Mega5.02软件对碱基组成、Kimura-2 parameter遗传距离、可变位点等进行分析。应用DnaSP软件进行遗传多样性相关参数的计算。结果显示:12S rRNA和ND3基因序列长度分别为954 bp和346 bp(349 bp),分别定义了51个单倍型和42个单倍型,12S rRNA和ND3序列中的碱基平均含量分别为:T为20.8%、C为25.7%、A为32.1%、G为21.4%和T为29.5%、C为28.2%、A为28.1%、G为14.1%,表现出明显的A+T偏倚性;6个群体的群体内和群体间的遗传距离分别为0.003~0.284(12S rRNA),0.009~0.059(ND3)和0.004~1.062(12S rRNA),0.008~0.143(ND3);12S rRNA基因的51个单倍型和ND3基因的42个单倍型序列总的单倍型多样性(Hd)、核苷酸多样性(Pi)及核苷酸平均差异数(K)分别为0.972和0.937、0.035和0.079、31.414和27.176,均显示出丰富的遗传多样性。结合从GenBank中下载的12S rRNA和ND3基因同源序列的[鱼丕]科9属10种鱼类进行系统发育树的构建,结果表明巨[鱼丕]独自汇聚成一大单系群,怒江潞江坝群体、怒江三江口群体及澜沧江临沧群体间关系较近,澜沧江景洪群体可单独构成一个单系种群,红河河口群体、红河曼耗群体与其它巨[鱼丕]构成一单系种群后再同澜沧江景洪群体汇聚成一支。     

Species identification method for <Emphasis Type="Italic">Scombrops boops</Emphasis> and <Emphasis Type="Italic">Scombrops gilberti</Emphasis> based on polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial DNA

ABSTRACT:   Gnomefish Scombrops boops and Scombrops gilberti are commercially important fishes in Japan, but these species are often confused in the markets because of their morphological similarity. To identify these two species, we performed nucleotide sequencing and restriction fragment length polymorphism (RFLP) analysis on 16S ribosomal RNA (rRNA) gene and the control region in mitochondrial DNA. Five and 12 nucleotide substitutions were observed between species in the 777-bp 16S rRNA gene and 471-bp control region, respectively. Diagnostic restriction sites for discriminating between S. boops and S. gilberti were found in the 16S rRNA gene, but not in the control region. Polymerase chain reaction (PCR)–RFLP analysis using two enzymes, Eco NI and Mva I, clearly discriminated between S. boops and S. gilberti identified by meristic characters. The PCR–RFLP analysis identified most of the 168 Scombrops young caught in the coastal waters of the Izu and Miura peninsulas as S. boops , suggesting that S. gilberti juveniles are rare in this area.     

1株军曹鱼病原弧菌的鉴定及其系统发育树分析

从养殖的患病军曹鱼身上分离出1株病原菌JT2，革兰氏阴性，短杆状，有极生鞭毛，能运动，菌落半透明。经回归感染实验，证明该菌为军曹鱼病原菌。进行了常规生理生化实验，并用API-ID32E系统鉴定该菌。再经Biolog-GN细菌鉴定系统鉴定，结果JT2与鲨鱼弧菌（Vibrio carchariae）相似度最高。为了进一步确定该菌的分类学地位，测定了其16SrDNA序列，分析了相关细菌的同源性，构建其系统发育树。结果表明，该菌株与V．carchariae的亲缘关系最近。综合上述几种方法的鉴定结果，最后鉴定该株菌为鲨鱼弧菌V．carchariae。     

Analysis of bacterial community and bacterial nutritional enzyme activity associated with the digestive tract of wild Chilean octopus (Octopus mimus Gould, 1852)

Information on the bacterial community associated with octopus is very scarce, unlike fish and other molluscs. This study revealed the bacterial community associated with digestive tract of wild Chilean octopus Octopus mimus using a culture‐dependent method and 16S rDNA clone library. Moreover, we analysed the bacterial nutritional enzyme activity of culturable bacteria. A culture‐dependent method showed that the composition of the culturable bacterial community was substantially different between female and male octopus. The predominant species in female octopus were Vibrionaceae and Streptococcaceae, whereas only Vibrionaceae was dominated in male octopus. Bacterial nutritional enzyme activities of culturable bacteria from male octopus were much higher than female octopus. The 16S rDNA clone library analysis showed that the bacterial community of male octopus exhibited a higher diversity than that of female octopus. The genus Mycoplasma was the predominant bacteria in the digestive tract of all octopus samples. The results obtained in this study raise the possibility that each octopus has different food consumption due to different bacterial community and nutritional enzyme activity, although Mycoplasma sp. is one of the predominant bacteria in the digestive tract. Moreover, our results are useful for the future of microbiological investigation associated with the octopus and for probiotics in the octopus aquaculture.     

基于16SrDNA分析的浒苔外生细菌多样性研究

为研究浙江近海浒苔Ulva spp.(Enteromorpha spp.)外生细菌多样性,采用传统的形态学和16S rDNA测序分析的方法,从舟山朱家尖、宁海国华电厂和奉化南沙3个地区分离到可培养的浒苔外生细菌及其周围海水细菌65株。根据细菌菌落特征和革兰氏染色结果等将分离到的细菌分为26种表型。16S rDNA序列测序比对发现:菌株与不动杆菌属(Acinetobacter)、交替单胞菌属(Alteromonas)、弧菌属(Vibrio)、假交替单胞菌属(Pseudoalteromonas)、微小杆菌属(Exiguobacterium)、芽孢杆菌属(Bacillus)、赤细菌属(Erythrobacter)、微球菌属(Micrococcus)、假单胞菌属(Pseudomonas)、深海杆菌属(Idiomarina)、Phaeobacter、Roseivirgaj和Silicibacter等23个属相应菌株具有较高同源性。对不同地区浒苔外生细菌进行了Shannon-Wiener多样性指数分析。研究表明:(1)宁海国华电厂的浒苔外生细菌多样性最丰富,多样性指数为93.98%;(2)浒苔外生细菌与其生活地区密切相关,其群落组成具有地域差异,其优势类群也不尽相同,但均归属于变形菌门。     

加州鲈迟缓爱德华氏菌病的检测和防治

以江苏省吴江市八坼镇区域养殖场加州鲈鱼病鱼的细菌性病原体为研究对象。通过分离纯化,形态学观察,结合16S rRNA基因序列同源性检索,对该病原菌进行初步判断。同时进行药敏实验,了解该病原菌对各种抗生素的耐药性和敏感程度。研究结果:分离纯化得到2株纯菌株L2F和L2P2,革兰染色均呈红色,阴性杆状菌。16S rDNA序列分析,L2F和L2P2菌株与迟缓爱德华氏菌(Edwardsiella tarda)的同源性达99%。初步判断该菌为迟缓爱德华氏菌。药敏实验结果,该菌对磺胺异恶唑、链霉素和利福平等药物不敏感或耐药,对左氧氟沙星、诺氟沙星、恩诺沙星、强力霉素、氟苯尼考、头孢哌酮相对敏感。