长江刀鲚寄生的异钩铗虫分子鉴定及形态学研究

为了获知长江刀鲚寄生的异钩铗虫的分类学地位,采用光学、扫描电子显微镜和分子遗传扩增28S rRNA基因5’端部分序列相结合的方法,对长江刀鲚寄生的异钩铗虫进行种属鉴定和形态学研究。结果显示,后吸器与体前区分不明显,含有四对不对称的吸铗,其中一侧的前2个开放几丁质吸铗明显大于其余6个封闭的吸铗,虫体尾部带有二对端钩,卵两端具有较长的极丝。序列分析显示,林氏异钩铗虫与六棘异钩铗虫相似度为100%,邻接法构建分子进化树中处在同一分支。经形态及分子综合分析鉴定,此寄生虫为林氏异钩铗虫。     

常见病害防治方法

病名：固着类纤毛虫病病原：最常见的为聚缩虫、单缩虫、微孢子虫、吸管虫、累枝虫和钟形虫等。症状：虾类（南美白对虾、罗氏沼虾、青虾等）被固着类纤毛虫附着时,虾体外观呈黑色,体表有灰黑色绒毛。当大量虫体寄生在虾体、鳃、附肢时,轻者病虾活动能力下降,不摄食、不蜕壳,生长缓慢,影响鳃的呼吸,重则与细菌性疾病并发,引起虾的死亡;成虾感染寄生虫后体表粗糙,     

鳗鱼车轮虫病防治新方法

鳗鱼患车轮虫病虽没有患拟指环虫病对鳗鱼影响那么严重,治疗也没那么艰难,但因虫体繁殖力强,易侵及多数鳗鱼并造成伤亡,因而也是威胁鳗鱼养殖的重要外寄生虫。从欧洲鳗、日本鳗到美洲鳗,鳗苗、黑仔、幼鳗到成鳗等各阶段,皆易受到本虫之侵袭而导致较重大的病害。该虫传播速度快,一旦鳗池有本虫侵入,即使开始时仅有数尾发病,也会很快蔓延全池的鳗鱼。因此若不能及早正确诊断本病而施以对策,将会给养鳗场带来较重大的损失。车轮虫主要寄生于鳗鱼的鳃、鳍及体表,并不侵入体内。鳗苗及幼鳗多见寄生于体表及鳍,而成鳗多见寄生于鳃部,少量寄生时对…     

网箱养殖翘嘴红鲌小瓜虫病的防治

在集约化水产养殖中,养殖业户常常面临寄生性原虫的困扰,其中小瓜虫是致病性最强的原虫之一。小瓜虫学名为多子小瓜虫,是一种遍生性（周身遍布纤毛）纤毛虫,通常寄生于淡水鱼类的体表、鳍和鳃上,形成小白点,所以在生产上称之为“白点病”。该寄生虫会引起鱼的活动异常、上皮增生、呼吸困难以及机械损伤,继而带来病菌的继发感染。     

斜管虫病的预防与治疗

<正>淡水鱼类的斜管虫病病原体只有一种,即鲤斜管虫。鲤斜管虫属于纤毛虫门,是一种单细胞寄生虫,主要寄生于鳃、鳍、体表缝隙。斜管虫病为低温季节流行病,是鱼苗、鱼种的大敌,会引起苗种严重死亡。养殖中由于环境条件限制和高密度精养,水体恶化,鱼体抗病能力下降,斜管虫病经常暴发。一旦发生如不及时控制,鱼的死亡率超过80%,尤其在小面     

额尔齐斯河银鲫寄生指环虫18S rDNA序列测定及 系统发育研究

为验证依靠形态鉴定的指环虫种类的正确性,进行了分子生物学鉴定,并研究了系统发育。2009-2014年在额尔齐斯河采集的银鲫(Carassius auratus gibelio)鳃部的指环虫,依靠形态鉴定为坏鳃指环虫(Dactylogyrus vastator)和伸展指环虫(Dactylogyrus extensus)。18S rDNA序列与Gen Bank中18S rDNA序列同源性比较结果,坏鳃指环虫同源性为99.36%(457/460),碱基转换率为0.65%(3/460);伸展指环虫同源性为100%(472/472)。用MEGA4.1软件分析和计算3科8属19种单殖吸虫18S r DNA序列的遗传距离。3个科单殖吸虫种类的种间遗传距离0.006~0.238,指环虫科的遗传距离0.007~0.047,指环虫科与锚首虫科的遗传距离0.097~0.182,指环虫科与鳞盘虫科的遗传距离0.164~0.235。系统发育研究结果,坏鳃指环虫和中间指环虫首先聚为一支,然后与伸展指环虫聚为一支,最终指环虫科的所有虫种聚为一支。研究结果同时也为额尔齐斯河人工养殖银鲫的病害防治提供依据。     

欧洲鳗败血症的防治对策

本作者深入生产实践,结合科技理论,探讨对欧洲鳗败血症的治疗取得了成功,现把经验介绍如下：一、欧洲鳗败血症主要特点1病原体主要是温和气单胞菌或嗜水气单胞菌,传染力强,传染速度快。2、与寄生虫（如车轮虫、指环虫、小瓜虫等）破坏并发关系密切。3．发生范围大,往往遍及全池、全场,死亡率高,危害严重。4．治疗难度大,疗程较长,病原体较顽固,传染快,24－48小时可传染池鳗20－50％,疗程10－30天。二、欧洲鳗败血症主要特征1体表状况：主要是红鳃。红鳍。由于寄生虫大量寄生在鳃部破坏和药物刺激,鳃细胞脱落,粘液分泌增多,…     

金鱼单殖吸虫病及其防治

单殖吸虫是体型较小的寄生虫。其种类很多,除少数种类营腔寄生(口腔、鼻腔、膀胱)以外,绝大多数寄生在鱼类的体表、鳍条和鳃上。单殖吸虫的幼虫发育,不需经过变态和无性繁殖,也没有中间宿主,而直接发育为成虫。由于单殖吸虫寄生在体表和鳃上,用它的附着器官(钩子和铗子等)钩住鳃丝,破坏鳃组织,刺激鳃细胞分泌过多     

瓶囊碘泡虫的重描述及其与洪湖碘泡虫分子标记的比较

为了完善瓶囊碘泡虫的分类学特征及厘清其与洪湖碘泡虫的分类关系,实验采用形态学、组织学和分子生物学方法对瓶囊碘泡虫进行了重描述,并与洪湖碘泡虫的分子标记进行了系统比较。结果显示,瓶囊碘泡虫寄生于异育银鲫的鳃,形成乳白色的圆形或椭圆形孢囊,直径为1.2～1.4 mm。成熟孢子壳面观呈梨形,前端较尖,后端钝圆,孢子长17.3～19.6 μm,孢子宽7.4～9.9 μm。两个极囊呈瓶状,大小不等。大极囊长6.4～9.7 μm,大极囊宽2.1～3.3 μm;小极囊长5.3～8.9 μm,小极囊宽2.0～3.3 μm。极丝圈数为8～11圈。组织学分析显示,瓶囊碘泡虫寄生于鳃小片间的上皮组织。BLAST分析显示,本研究获得的瓶囊碘泡虫的SSU rDNA序列与GenBank中瓶囊碘泡虫序列的相似性为99.5%～99.8% （KC425223-KC425225,MH329620,JQ690361,JQ690373,KJ725082,MN227351,DQ339482）。系统发育分析表明,瓶囊碘泡虫与洪湖碘泡虫形成姐妹支。瓶囊碘泡虫与洪湖碘泡虫分子标记序列的比较显示,这两种碘泡虫的序列相似性为98.2%～98.8% ,遗传距离为0.014～0.018,存在27个碱基差异。SSU rRNA二级结构分析显示,瓶囊碘泡虫和洪湖碘泡虫不同群体间的二级结构一致,两个物种间的二级结构存在明显差异,表明SSU rRNA二级结构可以作为鉴别瓶囊碘泡虫和洪湖碘泡虫的分子特征。本研究完善了瓶囊碘泡虫在异育银鲫鳃部的详细寄生部位,提出SSU rRNA二级结构可以作为有效鉴别瓶囊碘泡虫和洪湖碘泡虫的分子标记。     

鳜肤孢虫新种的分离和鉴定

为准确鉴定鳜肤孢虫病的病原,实验基于形态学比较,结合寄生特征分析和分子系统学方法开展研究,并观察超微结构。形态学观察结果显示,纺锤形或哑铃形白色孢囊,长约1.9～6.8 mm,寄生于鳍条、皮肤、口腔、鳃盖内外表面和鳃丝;圆形内生孢子,直径约(9.8±1.8)(7.3～13.8)μm;孢子内环状细胞质和直径约(6.5±1.2)(3.9～8.3)μm的圆形折光体,鉴定其为肤孢虫。该物种测量值区别于其他肤孢虫,与寄生于鳜鳃的未定种(HB)测量值基本一致;与广东省和河南省的鳜源鳗鲡肤孢虫(GD和HN)以及HB寄生于相似部位。分子序列比对和系统发育结果显示,该物种与GD、HN、芬兰肤孢虫、鲑肤孢虫和寄生美洲鳗鲡的鳗鲡肤孢虫序列的一致性大于99%;与GD和HN的亲缘关系最近,与鲑肤孢虫次之,与芬兰肤孢虫和鳗鲡肤孢虫的亲缘关系较远。综上,该物种与已报道的寄生与鳜的肤孢虫为同种,并修订寄生鳜的鳗鲡肤孢虫的GD和HN分离株记录;将本研究肤孢虫和HB、GD和HN分离株一同命名为鳜肤孢虫(Dermocystidium sinipercae sp. n.),新种。鳜肤孢虫种内形态差异可能与采样时间和发育阶段有...     

Non-lethal loop-mediated isothermal amplification assay as a point-of-care diagnostics tool for Neoparamoeba perurans,the causative agent of amoebic gill disease

Neoparamoeba perurans is the causative agent of amoebic gill disease (AGD). Two loop-mediated isothermal amplification (LAMP) assays targeting the parasite 18S rRNA and the Atlantic salmon EF1α, used as internal control, were designed. The N. perurans LAMP assay did not amplify close relatives N. pemaquidensis and N. branchiphila, or the host DNA. This assay detected 10⁶ copies of the parasite 18S rRNA gene under 13 min and 10³ copies under 35 min. Five “fast-and-dirty” DNA extraction methods were compared with a reference method and further validated by TaqMan™ qPCR. Of those, the QuickExtract buffer was selected for field tests. Seventy-one non-lethal gill swabs were analysed from AGD-clinically infected Atlantic salmon. The pathogen was detected under 23 min in fish of gill score >2 and under 39 min for lower gill scores. About 1.6% of the tests were invalid (no amplification of the internal control). 100% of positives were obtained from swabs taken from fish showing gill score ˃3, but only ~50% of positives for lower gill scores. The present LAMP assay could be implemented as a point-of-care test for the on-site identification of N. perurans; however, further work is required to improve its performance for lower scores.     

Control of Salmincola californiensis (Copepoda: Lernaeapodidae) in rainbow trout, Oncorhynchus mykiss (Walbaum): a clinical and histopathological study

A stock of rainbow trout, Oncorhynchus mykiss, held at an experimental facility, was found to be heavily infested with the lernmaeapodid copepod Salmincola californiensis. The efficacy and effects of treatment were compared with ivermectin or manual removal of parasites as a means of control of S. californiensis. One group of fish was orally intubated with 0.2 mg ivermectin active ingredient kg-1 fish. A second treatment was administered after a further 14 days. In a second group of fish, parasites were manually removed from the gills using forceps. These fish were sampled for up to 21 days post-first removal of parasites. In the ivermectin-treated fish adult parasites became inactive and changed colour within 18 h of the initial treatment. Copepods began to disappear by day 3 post-treatment and by day 31 almost all embedded female parasites had disappeared. Gills were clinically normal apart from cavitation deformity resulting from parasite attachment. Post-ivermectin treatment, there was an increase in the number of eosinophilic granular cells surrounding the bulla of attached S. californiensis, but from day 31 post-treatment these were replaced by macrophages and epithelioid cells to form a necrotic focus. In manually picked fish there was extensive haemorrhage in the interlamellar spaces as a result of parasite removal. At sites of parasite removal tissue necrosis was minimal and healing was rapid. At the end of the sampling period the structure of the gill was improved. The use of oral dosage with ivermectin is an effective treatment for S. californiensis and could be particularly beneficial for use with endangered salmon broodstocks infested with the parasite.     

多子小瓜虫滋养体糖蛋白残基的种类和分布研究

应用伴刀豆凝集素A、麦胚凝集素、大豆凝集素和荆豆凝集素Ⅰ,检测寄生于金鱼皮肤、鳃和鳍上的多子小瓜虫滋养体糖蛋白残基的种类和分布。研究结果发现,伴刀豆凝集素A和麦胚凝集素免疫阳性染色在金鱼皮肤、鳃和鳍寄生的滋养体上均有分布,麦胚凝集素免疫阳性染色强于伴刀豆凝集素A,未见有大豆凝集素和荆豆凝集素Ⅰ免疫阳性染色。鳃上寄生的滋养体伴刀豆凝集素A免疫阳性染色最强,皮肤次之,鳍最弱。鳍上寄生的滋养体麦胚凝集素免疫阳性染色最强,皮肤次之,鳃最弱。研究结果表明,滋养体有单糖D-甘露糖和D-葡萄糖,以及氨基衍生物乙酰氨基葡萄糖。寄生于鳃上的滋养体D-甘露糖和D-葡萄糖可能较多,寄生在鳍上的滋养体乙酰氨基葡萄糖可能较多。     

不同DNA条形码基因在帘蛤目贝类分类鉴定中的比较分析

DNA条形码基因已经广泛应用在海洋贝类的分类鉴定、系统发育进化、种群遗传分析等领域的研究。为进一步研究评估不同DNA条形码基因在海洋贝类鉴定中的作用,本研究利用从Gen Bank数据库随机下载的帘蛤目COI、16S r RNA、18S r RNA和28S r RNA基因序列,通过传统距离法和单系聚类法结合分析,比较了上述DNA条形码基因在鉴定物种及系统发育进化中的鉴定效率,并以本实验室已获得的部分贝类DNA序列进行了验证。结果表明,根据"10倍法则"和"2%"阈值标准,本研究中COI能够鉴定57.1%物种,16S r RNA能够鉴定60.9%,18S r RNA鉴定16.7%,而28S r RNA无法有效鉴定;多数种COI和16S r RNA基因序列的种间遗传距离和种内遗传距离存在"条形码间隙",而18S r RNA和28S r RNA序列的种间和种内的遗传距离存在显著重叠,没有明显"条形码间隙";聚类分析结果表明,基于COI基因序列,87.9%的个体与同种聚为单系,以16S r RNA序列,65.6%的个体与同种聚为单系,未聚成单系的个体则形成姐妹系,未出现不同种聚为单系现象,能够呈现与形态分类基本一致的系统发生关系;但18S r RNA和28S r RNA呈现的聚类关系相对混乱。相对而言,在鉴定帘蛤目物种时,COI和16S r RNA都能够作为条形码基因,且COI有效性更高,18S r RNA和28S r RNA基因由于种内变异较大,不适于作为条码基因。研究结果为科学选用DNA条形码基因进行帘蛤目贝类的鉴定提供了参考资料。     

The effects of temperature on the uptake and metabolism of benzo[a]pyrene in isolated gill cells of the gulf toadfish,Opsanus beta

The effects of acclimation temperature and acute temperature change on the uptake and metabolism of the procarcinogen benzo[a]pyrene (BaP) by gill cells of the gulf toadfish, Opsanus beta, were examined. BaP was rapidly accumulated by isolated gill cells and uptake rates were directly proportional to BaP concentration in the medium (1 to 100 μg/ml). Uptake rates were higher in cells isolated from fish acclimated to 18°C when compared to cells from 28°C acclimated fish at all incubation temperatures. When cells were exposed to BaP at the respective acclimation temperatures of the fish, uptake rates were similar (0.14 ± 0.01 at 18°C and 0.12 ± 0.01 μg BaP/s/10 mg cells at 28°C). This finding is discussed in view of results which showed a partial compensation of membrane fluidity in plasma membranes isolated from fish from the two acclimation temperatures. At higher incubation temperatures, cells from fish acclimated to 18°C metabolized BaP at a greater rate than those at 28°C (49.6 ± 1.92 and 43.0 ± 2.24 μg/g/8h, respectively, at 23°C). Low but detectable activities of common biotransformation enzymes (aryl hydrocarbon hydroxylase, glutathione-S-transferase) and cytochrome P-450 content were found, however, no significant differences were evident between cells from fish acclimated to different temperatures. To whom to address correspondence     

Recovery of Bacillus mycoides,B. pseudomycoides and Aeromonas hydrophila from common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) with gill disease

During a 3‐month period from June to the end of August 2016, ~5% mortalities were observed in a farm with rainbow trout (Oncorhynchus mykiss Walbaum) and one farm of common carp (Cyprinus carpio L.) in Bulgaria. The disease was manifested by gill ulcers/rot, asphyxiation and bloody ascites. Aeromonas hydrophila was isolated from the internal organs of all the diseased fish. Bacillus mycoides or B. pseudomycoides were recovered from the gill lesions on diseased carp and rainbow trout, respectively, with identification achieved by conventional phenotyping and by sequencing of the 16S rRNA gene. In vivo experiments confirmed that all three organisms were pathogenic to rainbow trout.     

Real-time PCR detection of Parvicapsula pseudobranchicola (Myxozoa: Myxosporea) in wild salmonids in Norway

The myxozoan genus Parvicapsula contains 14 species infecting fish, some of which are known to cause severe disease in farmed and wild salmonids. Parvicapsula pseudobranchicola infections were first reported from seawater-reared Atlantic salmon, Salmo salar, in Norway in 2002 and have since then been an increasing problem. The present study describes a Taqman real-time PCR assay for specific detection of P. pseudobranchicola. The Taqman assay targets the 18S rRNA gene of P. pseudobranchicola and is able to detect as few as ten copies of the target sequence. Using the described assay, P. pseudobranchicola was detected in both farmed and wild salmonids, indicating that wild Atlantic salmon, sea trout, Salmo trutta, and Arctic char, Salvelinus alpinus, may be natural hosts of the parasite. Parvicapsula pseudobranchicola was found in samples from wild salmonids in the far south and the far north of Norway, displaying a wide geographic range of the parasite. Farmed salmonids showed P. pseudobranchicola infection levels many folds higher than that observed for wild sea trout, indicating that farmed Atlantic salmon are subjected to an elevated infection pressure compared with wild salmonids.     

Generation of Paramoeba perurans clonal cultures using flow cytometry and confirmation of virulence

Amoebic gill disease (AGD) in farmed Atlantic salmon is caused by the amoeba Paramoeba perurans. The recent establishment of in vitro culture techniques for P. perurans has provided a valuable tool for studying the parasite in detail. In this study, flow cytometry was used to generate clonal cultures from single‐sorted amoeba, and these were used to successfully establish AGD in experimental Atlantic salmon. The clonal cultures displayed differences in virulence, based on gill scores. The P. perurans load on gills, determined by qPCR analysis, showed a positive relationship with gill score, and with clonal virulence, indicating that the ability of amoebae to proliferate and/or remain attached on gills may play a role in virulence. Gill scores based on gross signs and histopathological analysis were in agreement. No association between level of gill score and specific gill arch was observed. It was found that for fish with lower gill scores based on histopathological examination, gross examination and qPCR analysis of gills from the same fish were less successful in detecting lesions and amoebae, respectively.