Assessment of gut microbiota in different developmental stages of Malaysian Mahseer (Tor tambroides)

Malaysian Mahseer (Tor tambroides) has a good prospect for aquaculture because of its high market demand. However, there is a scarce information on gut microbiota associated with Malaysian Mahseer unlike other fish species. Therefore, we constructed and compared gut microbiota in different developmental stages (larval, juvenile, fingerling, yearling, and adult) using culture dependent and PCR‐DGGE fingerprinting technique for better understanding of gut microbiota composition associated with T. tambroides. Culturable gut microbiota composition in all developmental stages were composed of β‐ and γ‐Proteobacteria, and Bacilli. Biodiversity analysis of culturable gut microbiota showed that larval, juvenile, and adult stages have higher diversity than fingerling and yearling stages. Ward's linkage cluster analysis showed that culturable gut microbiota composition in larval and juvenile stages were close to adult stages, whereas fingerling and yearling stage composed same cluster. PCR‐DGGE fingerprinting technique showed that unculturable gut microbiota were constituted by α‐and γ‐Proteobacteria, and Actinobacteria. Ward's linkage cluster analysis showed that unculturable gut microbiota composition in both larval and juvenile stages were distinct from other developmental stages. Our results revealed that gut microbiota composition were varied in different developmental stages of Malaysian Mahseer and continuous shifts of gut microbiota from larval to adult stages.     

微囊藻和小球藻携带WSSV量的变化及对水体游离WSSV的影响

研究了铜绿微囊藻（Microcystis aeruginosa）和蛋白核小球藻（Chlorellapyrenoidosa）携带白斑综合症病毒（whitespotsyndromevirus,WSSV）数量变化及对水体游离WSSV量的影响。试验设置微藻高、低密度组和不加微藻的对照组,分别加入等量的WSSV粗提液,于第2、第24、第72和第120小时以实时荧光定量PCR检测藻液上清和沉淀藻体的WSSV数量。结果表明,微囊藻和小球藻可携带少量WSSV,且随时间延长而减少;微囊藻携带WSSV量与其细胞数呈显著正相关（P〈0．05）,小球藻携带WSSV量与其细胞数相关性不显著（P〉0．05）;2种微藻均有促进水体WSSV数量消减的效果,且小球藻的消减效果优于微囊藻,对养殖对虾白斑综合症（whitespotsyndrome,WSS）的生态防控更有积极意义。     

大鲵虹彩病毒TaqMan实时荧光定量PCR检测方法的建立

利用PCR技术扩增出大鲵虹彩病毒(giant salamander iridovirus, GSIV)主要衣壳蛋白(MCP)编码区长度为1 392 bp 的片段, 克隆到 pMD19-T载体上, 构建重组质粒 pMD19-T-MCP。经PCR鉴定确认正确后, 以10倍梯度稀释 pMD19-T-MCP重组质粒, 作为标准模板进行 TaqMan实时荧光定量PCR扩增, 制作标准曲线, 建立了大鲵虹彩病毒的 TaqMan实时荧光定量PCR检测方法。制作的标准曲线有极好的线性关系, 且线性范围宽, 相关系数为0.990 19。组内重复试验的CT值标准偏差为0.52%。检测结果显示, 该方法对大鲵虹彩病毒的检测有高度的特异性, 与锦鲤疱疹病毒、弗氏柠檬酸杆菌、嗜水气单胞菌以及鲤上皮瘤细胞基因组DNA之间均无交叉反应, 特异性好, 检测总DNA灵敏度为10个病毒核酸分子拷贝数, 约1.1×10-3 pg/μL病毒核酸, 较之常规PCR的敏感度高出约1 000倍。研究建立的大鲵虹彩病毒TaqMan实时荧光定量PCR方法灵敏度高、特异性强, 对大鲵虹彩病毒病的快速诊断与病毒病原定量检测有重要意义。     

脊尾白虾不同发育期mtDNA拷贝数变化特征分析

为了解脊尾白虾(Exopalaemon carinicauda)在不同发育期线粒体基因组(mtDNA)拷贝数的变化特征,本研究共采集其8个时期(受精卵期、溞状幼体Ⅰ期、溞状幼体Ⅱ期、溞状幼体Ⅲ期、溞状幼体Ⅳ期、溞状幼体Ⅴ期、仔虾期和成虾期)的DNA样品,利用TaqMan实时荧光定量PCR技术,对上述各发育期单个细胞中的mtDNA拷贝数进行了测定。检测结果显示,脊尾白虾受精卵期、溞状幼体Ⅰ期、溞状幼体Ⅱ期、溞状幼体Ⅲ期、溞状幼体Ⅳ期、溞状幼体Ⅴ期、仔虾期和成虾期的mtDNA拷贝数的均值分别为2366、2648、2644、2873、3559、9948、6452和8872。进一步统计分析结果显示,mtDNA拷贝数与发育时间存在正相关性,相关系数为0.83。研究表明,随着脊尾白虾不断生长,其单个细胞中mtDNA拷贝数总体呈上升趋势。     

半滑舌鳎创伤弧菌分离鉴定及其荧光定量PCR方法的建立

为确定患病半滑舌鳎的病原,从其体内分离到3株革兰氏阴性菌,将其分别命名为ST-1、ST-3和ST-6,进行细菌理化特性、药敏试验、16S rDNA测序以及组织病理学观察等方面的研究,并建立了外膜蛋白(OMP)基因的SYBR Green I荧光定量PCR检测方法。结果显示,3株分离菌株的理化特性符合创伤弧菌特征,16S rDNA与创伤弧菌标准菌株ATCC29307序列相似性达99%以上,系统发育树分析显示,3株分离株与创伤弧菌自然聚为一支,最终鉴定为创伤弧菌;药敏试验显示,3株菌对头孢克肟、头孢哌酮、链霉素、亚胺培南和氟苯尼考等药物高度敏感。人工回归感染试验表明,分离株具有致病性,且半滑舌鳎、斑马鱼的临床症状与自然患病鱼症状相似;组织病理学观察显示,鳃丝上皮细胞增生,鳃小片肿胀黏连、颗粒细胞和黏液细胞增多,肝脏中央静脉淤血,肾脏出血、坏死,肠道黏膜固有层萎缩、黏液细胞增多等。进一步建立OMP基因的荧光定量PCR方法,结果显示,标准曲线的相关系数为0.995,检测下限为1.88×10~2 CFU/mL。以上结果可为半滑舌鳎弧菌病的致病机理、分子诊断和防治提供科学参考。     

Description and phylogeny of Ceratomyxa anko sp. n. and Zschokkella lophii sp. n. from the Japanese anglerfish, Lophius litulon (Jordan)

Two new species of myxozoans from the Japanese anglerfish, Lophius litulon, are described using myxospore morphology and small subunit rDNA sequences. Ceratomyxa anko sp. n. is a parasite of the gall bladder and had a prevalence of 57%. Mature spores of C. anko sp. n. are arcuate to crescent shaped with valves tapering to rounded tips. A prominent sutural line runs centrally between the round adjacent polar capsules containing the polar filament coiled two to three times. Spore measurements: length 10.8 (9.7-11.9) microm, width 41.9 (36.9-47.2) microm, polar capsule diameter 4.6 (4.1-5.3) microm. Ceratomyxa anko sp. n. can be distinguished from other Ceratomyxa spp. due to its spore dimensions and shape. Zschokkella lophii sp. n. is a parasite of the urinary bladder and had a prevalence of 70%. Mature spores are ellipsoidal to semicircular with bluntly pointed ends. The sutural line is curved or sinuous and the valves have no discernable surface ornamentation. Two almost spherical polar capsules are located separately in the ends of the spore, opening in almost opposite directions and contain the polar filament with five coils. Spore measurements: length 20.1 (16.8-24.0) microm, width 14.9 (12.7-16.8) microm, polar capsule diameter 5.1 (3.6-5.8) microm. Zschokkella lophii sp. n. can be distinguished from other Zschokkella spp. due to the terminal opening of the polar capsules within the spores and the site of infection within the host fish. In the phylogenetic analyses, C. anko sp. n. grouped with other members of the same genus forming a monophyly. Zschokkella lophii sp. n. forms a discrete clade with another Zschokkella sp. that infects the urinary bladder of marine fish. This grouping forms a sister clade to one containing members of the genus Parvicapsula, all of which are parasites of the urinary system in marine fish.     

检测与诊断猪圆环病毒PCR技术的建立及病毒的分离鉴定

本研究建立了检测与诊断猪圆环病毒的PCR方法,并对该方法进行了标准化研究。该方法快速、敏感、特异性强,可扩增10ng的阳性质粒的DNA,可以从30μL病料组织悬液或全血中扩增检测出PCV,并且该方法可以区分PCV1和PCV-2,对其它病原微生物如PPV、PRRSV、SIV以及许多细菌不能检出。应用此方法对6省市的12个猪场的225份病料进行了检测,对阳性病料进行了病毒分离,对分离的病毒进行了电镜观察与序列测定,结果证实分离的病毒为PCV-2。     

The use of a real-time PCR primer/probe set to observe infectivity of Yersinia ruckeri in Chinook salmon, Oncorhynchus tshawytscha (Walbaum), and steelhead trout, Oncorhynchus mykiss (Walbaum)

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM), a common pathogen affecting aquaculture facilities and implicated in large losses of cultured fish. Fisheries scientists continue to gain a greater understanding of the disease and the pathogen by investigating methods of identification and pre- and post-infection treatment. In this study, a real-time PCR probe set for Y. ruckeri was developed to detect daily changes in the bacterial load during pathogen challenges. Two species of fish, Chinook salmon, Oncorhynchus tshawytscha, and steelhead trout, Oncorhynchus mykiss, were exposed to two strains of Y. ruckeri (Hag and SC) during bath challenges. A subset of fish was killed daily for 14 days, and the kidney tissue was biopsied to enumerate copies of pathogen DNA per gram of tissue. While Chinook exposed to either the Hag or SC strains exhibited similar pathogen loads, those exposed to the Hag strain displayed higher mortality (～66%) than fish exposed to the SC strain (～24% mortality). Steelhead exposed to the Hag strain exhibited a greater pathogen load and higher mortality (～42%) than those exposed to the SC strain (<1% mortality). Steelhead challenged with either strain showed lower pathogen loads than Chinook. The study illustrates the efficacy of the probe set to enumerate Y. ruckeri bacterial growth in the kidneys of fish. Also, strains of Y. ruckeri display species-specific growth patterns that result in differential mortality and pathogen load.     

荧光实时定量PCR技术及其在水产养殖研究中的应用

荧光实时定量PCR技术是近年来快速发展起来的一门新技术,它是核酸探针技术、荧光共振能量传递技术(Fluorescence Resonance Energy Transfer,FRET)和PCR技术的有机结合。与常规PCR相比,它具有特异性更强、有效解决PCR污染问题、自动化程度高、能较准确定量等特点。本文综述了荧光实时定量PCR技术的基本原理及几种广泛应用的荧光化学方法,并概述了荧光实时定量PCR技术在水产养殖研究领域的应用现状,并对荧光实时定量PCR技术的应用前景进行了展望。     

杭州湾南岸滩涂贝类养殖环境中微生物数量分布及其类群

研究了杭州湾南岸慈溪滩涂贝类养殖区的微生物在水平、垂直及季节变化的分布规律。结果表明:3个采样区的异养细菌的数量变动在1.62×103~6.62×105cfu.g-1之间,平均值为4.28×104cfu.g-1,细菌数量在春季和夏季的数量偏低,10月份异养细菌的数量开始上升,并于11月份达到最高,表层高于底层;其中以革兰氏阳性菌比例最高,为主要的优势种,具有陆源性特点。各站位反硝化细菌、氨化细菌和硫酸还原菌的检出率均为100%,都维持在一个较高的水平,整体上数量具有表层高于底层的特点,3种微生物在数量上的季节演替与异养细菌具有类似的结果,均为10-11月达到最大。