不同生境下青海湖裸鲤(Gymnocypris przewalskii)两亚种鳃形态差异及其功能适应

该研究比较观察青海湖裸鲤(Gymnocypris przewalskii)两亚种(青海湖裸鲤指名亚种(Gymnocypris przewalskii przewalskii)和青海湖裸鲤甘子河亚种(Gymnocypris przewalskii ganzihonensis))鳃的形态及显微结构。结果显示,指名亚种的第一鳃弓鳃耙数多、密集且较长,而甘子河亚种则耙数少、稀疏且短;两者的鳃丝间距及鳃小片间距无明显差异,指名亚种的鳃丝较长;指名亚种扁平上皮细胞形态以不规则椭圆形为主,鳃丝表面有黏液细胞,其上分布的氯细胞数量多于甘子河亚种,而甘子河亚种扁平上皮细胞的形态以六边形为主,鳃丝表皮未观察到黏液细胞。由此推测,青海湖裸鲤两亚种鳃形态的差异与其食性和栖息环境相适应。     

黄河裸裂尻鱼群体遗传结构和Cyt b序列变异

测定了来自黄河上游和柴达木盆地托索湖的裸裂尻鱼共16个个体的Cytb基因全序列(1141bp),探讨了种群结构和遗传多样性。用MEGA2.1软件分析了碱基组成和序列变异;以青海湖裸鲤、花斑裸鲤和极边扁咽齿鱼为外类群,用PAUP*4.0b10程序构建了单倍型NJ树;用Arlequin Ver.2000程序计算了群体间遗传变异值(Fst)和Nm值以及群体分化概率值。结果显示,来自柴达木水系托索湖的裸裂尻鱼没有形成单系群,Fst=0.204(P0.05),Nm=1.95。初步判断,黄河和柴达木水系托索湖的裸裂尻鱼未显著分化,支持将柴达木裸裂尻鱼(Schizopygopsis kessleri)归并入黄河裸裂尻鱼(Schizopygopsis pylzovi)的形态学结果。两种群核苷酸多样度()分别为0.0012和0.0026,表现为较低水平。根据校正的分子钟推测,黄河和托索湖裸裂尻鱼群体分歧时间为距今7万年左右的更新世末期,结合地理分布的资料和古地质事件,对黄河裸裂尻鱼群体分布水系间的历史联系进行了分析。       

青海湖裸鲤类胰岛素生长因子IGF-Ⅱ的原核表达

青海湖裸鲤(Gymnocypris przewalskii)俗称湟鱼,属鲤科裂腹鱼亚科裸鲤属,是我国最大内陆咸水湖青海湖中唯一的经济鱼类,其生长极其缓慢,平均每生长到500g需要近10年[1]。青海湖裸鲤具有明显的生殖洄游,每年     

黄河裸裂尻鱼肌酸激酶M-CK cDNA的克隆及组织表达分析

肌酸激酶(CK)能够催化磷酸基在二磷酸腺苷(ADP)和磷酸肌酸之间的可逆性转移,在细胞能量代谢过程中发挥重要作用。本研究利用RT-PCR和RACE方法克隆了黄河裸裂尻鱼(Schizopygopsis pylzovi)肌酸激酶基因全长c DNA序列,并进行了生物信息学分析和系统发育关系研究。黄河裸裂尻鱼肌酸激酶c DNA全长1 599 bp,开放阅读框(ORF)1 143 bp,编码380个氨基酸,具有典型的N-端结构域和C-端催化结构域。氨基酸序列同源性分析表明,黄河裸裂尻鱼肌酸激酶与斑马鱼(Danio rerio)和鲤(Cyprinus carpio)M3-CK有较高的同源性,其序列一致度达94%以上。系统发育分析显示,黄河裸裂尻鱼肌酸激酶与斑马鱼和鲤的M3-CK聚在一支,形成一个单系群,初步判定克隆获得的黄河裸裂尻鱼肌酸激酶基因可能与鲤和斑马鱼M3-CK是直系同源基因。利用Real-time PCR方法检测和分析了黄河裸裂尻鱼主要组织肌酸激酶m RNA表达水平,肌肉、肠、眼、心中肌酸激酶转录本表达较高,在肝胰脏和脑中表达微弱。肌酸激酶在肌肉、肠、和心中高表达与其能量代谢功能相适应,而在眼组织中的高表达是否与肌酸激酶的其他功能相关,有待于进一步研究。     

黄河裸裂尻鱼肥胖基因克隆及其对青藏高原季节性冷环境的适应性表达

目的克隆黄河裸裂尻鱼肥胖基因(obese gene,ob),分析编码蛋白leptin的序列特征,检测ob mR-NA的组织特异性表达和不同环境温度下肝胰脏和白肌ob mRNA的相对表达水平,为探究黄河裸裂尻鱼在青藏高原季节性寒冷环境下的生态适应机制提供科学数据。方法利用RT-PCT、5’-RACE和3’-RACE法获得ob基因全长cDNA序列。通过已知cDNA序列设计半定量RT-PCR和real-time PCR引物,进行表达研究。结果黄河裸裂尻鱼ob基因序列全长为1 337 bp,其中开放阅读框(ORF)长513 bp,编码170个氨基酸。黄河裸裂尻鱼ob mRNA在心脏、肾脏、脂肪、肠、精巢、肝胰脏、鳃、脑和肌肉9个被检组织中均有表达,其中在心脏中表达最强,肌肉组织中表达最弱。栖息水域环境温度下降,将显著抑制黄河裸裂尻鱼肝胰脏和白肌ob mRNA的表达。结论黄河裸裂尻鱼ob基因特殊的组织表达特征可能与其特定组织的能量代谢及其特殊的生物学功能有关。Leptin在黄河裸裂尻鱼能量代谢调节和季节性环境适应方面发挥着重要作用。     

利用Cyt b基因序列探讨柴达木裸裂尻鱼的分类学地位

长期以来,柴达木裸裂尻鱼(Schizopygopsis kessleri)的分类学地位存在争议。为此,测定了柴达木裸裂尻鱼格尔木河及托素湖种群24个个体及黄河裸裂尻鱼(S.pylzovi)黄河及大通北川河种群12个个体的Cytb基因全序列。通过遗传距离和系统发育分析,探讨了柴达木裸裂尻鱼的分类学地位。结果显示,柴达木裸裂尻鱼与黄河裸裂尻鱼在系统进化树上并未形成相应的单系群;柴达木裸裂尻鱼与黄河裸裂尻鱼种间平均遗传距离为0.54%,明显低于裂腹鱼亚科其他属鱼类的种间遗传距离及本属其他种间遗传距离,表明来自柴达木盆地格尔木河和托素湖的裸裂尻鱼与黄河裸裂尻鱼之间没有达到种级水平的显著分化。综合地质学资料和柴达木水系与黄河水系现今的隔离格局,建议将柴达木裸裂尻鱼作为黄河裸裂尻鱼的一个亚种,即S.pylzovi kessleri。     

青海湖裸鲤体腔寄生蠕虫群落研究

青海湖裸鲤体腔为三种寄生蠕虫寄生,即裂头绦虫裂头蚴,舌状绦虫裂头蚴和对盲囊缄虫的三期幼虫,这几种蠕虫不同大小的青海湖裸鲤中数量差异很大,将其看作一个组分群落,并根据其发生数量进行模糊聚类和极点排序,结果将其分为二个亚组分群落,即体长小于１５０ｍｍ的青海湖裸鲤体腔蠕虫亚组分群落和体长大于１５０ｍｍ的个体的体腔蠕虫亚组分群落。这与宿主的行为和食性改变有密切关系,舌状绦虫与另两种蠕虫间有显著负协调关系。     

黄河裸裂尻鱼肌肉生长抑制素基因克隆及表达分析

肌肉生长抑制素(myostatin,MSTN)属于转化生长因子β(TGF-β)超家族中的一个成员,是骨骼肌生长发育的负调控因子。该文以黄河裸裂尻鱼肌肉总RNA为模板,采用RT-PCT、5’-RACE和3’-RACE法获得MSTN基因全长cDNA序列为2180bp,包含长为1128bp的开放阅读框,编码375个氨基酸。以肌肉总DNA为模板,通过PCR法进一步获得了MSTN基因的2个内含子序列,分析表明,黄河裸裂尻鱼MSTN基因与其他脊椎动物具有相似的基因结构(包括3个外显子和2个内含子)。黄河裸裂尻鱼MSTN具有脊椎动物MSTN的共同序列特征,含有1个蛋白酶水解位点RXXR和8个位于TGF-β功能区域保守的半胱氨酸残基。氨基酸序列同源性分析表明,黄河裸裂尻鱼MSTN序列与其他鲤科鱼类MSTN具有较高的同源性;而与哺乳动物和禽类的MSTN同源性较低。系统发育分析表明,黄河裸裂尻鱼MSTN与其他鲤科鱼类聚于同一进化支。RT-PCR分析表明,该基因在黄河裸裂尻鱼9个被检组织中均有表达,但在心、肾、肠、精巢中表达量较高。Real-TimePCR分析显示,MSTN基因在胚胎中的相对表达量,随胚胎发育阶段的不同而有所差异,暗示MSTN的功能可能并不局限在对肌肉生长发育的负调控作用,可能还有其他功能。     

黄河裸裂尻鱼细胞色素C氧化酶Ⅰ、Ⅱ和Ⅲ亚基基因的克隆及序列特征分析

采用RT-PCR技术克隆获得了黄河裸裂尻鱼(Schizopygopsis pylzovi)CO Ⅰ、Ⅱ、Ⅲ基因的编码序列,并对此进行了初步分析.结果表明,黄河裸裂尻鱼CO I基因全长为1 551 bp,开放阅读框(ORF)由基因全长组成,编码516个氨基酸;COⅡ基因全长为691 bp,开放阅读框为690 bp,编码2...     

小瓜虫在澳洲龙纹斑鳃器官上的分布及其影响

【目的】小瓜虫病是澳洲龙纹斑苗种阶段危害巨大的寄生虫病。探究小瓜虫在澳洲龙纹斑鳃器官上的分布及其影响可以丰富小瓜虫的致病性及病理学方面的研究内容,也可以为渔业生产中小瓜虫病的防治提供参考。【方法】采用光镜及扫描电镜技术确定病原,并观察小瓜虫在澳洲龙纹斑鳃丝、鳃小片及鳃盖上的分布情况及这些器官的变化情况。【结果】小瓜虫侵染澳洲龙纹斑的鳃器官后,分布在鳃丝、鳃小片及鳃盖的表面,上皮细胞之下及鳃小片之间,或是包裹在黏液细胞里,但侵染后期在鳃丝及鳃小片上的数量明显少于鳃盖。侵染后期,鳃丝、鳃小片及鳃盖出现一定程度的膨胀变形,黏液细胞分泌增多,鳃小片末端膨大变形甚至黏连融合,顶端充血呈球状或棒状。【结论】对于鳃部而言,侵染后期小瓜虫主要分布在澳洲龙纹斑的鳃盖上。小瓜虫主要通过侵染引发澳洲龙纹斑鳃部器官的变形膨大,使其丧失正常的功能,造成血液循环受阻、渗透压调节失衡,最终导致鱼体缺氧死亡。     

Relations between histopathology and parasitaemias in Oncorhynchus mykiss infected with Cryptobia salmositica, a pathogenic haemoflagellate

Ichthyophthirius multifiliis is a ciliated protozoan parasite that infects the skin and gills of freshwater fish. This report describes the unusual finding of I. multifiliis within the peritoneal cavities of experimentally infected channel catfish Ictalurus punctatus. Twenty catfish fingerlings were exposed to I. multifiliis theronts using a standardized protocol. Five infected fish and 2 control fish were killed at various time points after infection and their tissues examined. Formalin-fixed, paraffin embedded sections were processed for light microscopy and immunohistochemical detection of I. multifiliis immobilization antigen. Trophonts were observed in skin and gill sections of all exposed fish. Parasites were associated with epithelial hyperplasia, focal areas of cellular disruption and necrosis. In addition to these usual sites of infection, individual trophonts were unexpectedly found within the peritoneal cavities of 4 fish. Staining for parasite antigen facilitated their detection within abdominal adipose tissue or adjacent to intestines. This discovery is interesting as it suggests I. multifiliis may be found in tissues other than the skin and gills during the course of a normal infection.     

Mitochondrial cytochrome b Sequence Variation and Phylogenetics of the Highly Specialized Schizothoracine Fishes (Teleostei: Cyprinidae) in the Qinghai-Tibet Plateau

The complete 1140 bp mitochondrial cytochrome b sequences were obtained from 39 individuals representing five species of all four genera of highly specialized schizothoracine fishes distributed in the Qinghai-Tibet plateau. Sequence variation of the cytochrome b gene was surveyed among the 39 individuals as well as three primitive schizothoracines and one outgroup. Phylogenetic analysis suggested that the group assignment based on 1140 bp of the cytochrome b sequence is obviously different from previous assignments, and the highly specialized schizothoracine fishes (Schizopygopsis pylzovi, Gymnocypris przewalskii, G. eckloni, Chuanchia labiosa, and Platypharodon extremus) form a monophyletic group that is sister to the clade formed by the primitive schizothoracine fishes (Schizothorax prenanti, S. pseudaksaiensis, and S. argentatus). The haplotypes of Schizopygopsis pylzovi and G. przewalskii were paraphyletic based on cytochrome b data, which most likely reflected incomplete sorting of mitochondrial DNA lineages. The diploid chromosome numbers of Schizothoracinae were considered in phylogenetic analysis and provided a clear pattern of relationships. Molecular dating estimated for highly specialized schizothoracine fishes suggested that the highly specialized schizothoracine fishes diverged in the late Miocene Pliocene to Pleistocene (4.5 × 10⁴–4.05 × 10⁶ years BP). The relationship between the cladogenesis of highly specialized schizothoracine fishes and geographical events of the Qinghai-Tibet plateau is discussed.     

Influence of Anguillicola crassus (Nematoda) and Ichthyophthirius multifiliis (Ciliophora) on swimming activity of European eel Anguilla anguilla

We investigated the swimming activity of 70 European eels Anguilla anguilla in relation to natural infection with 2 parasite species: the eel-specific swimbladder nematode Anguillicola crassus and the non-specific skin and gill protozoan Ichthyophthirius multifiliis. We measured how long individual eels exposed to a water current in a swimming channel with a steady-stream profile could withstand the water current. The parasites affected the swimming behaviour of eels in different ways. The maximum period of time the fish were able to swim against the current was not correlated with infection by A. crassus. In contrast, infection with I. multifiliis reduced the swimming time. The protozoan has a higher pathogenicity than the swimbladder nematode, at least in closed systems, where I. multifiliis is able to spread within a few days. Reduction in swimming capacity after infection with the ciliate averaged 47 % compared to capacity prior to infection. Thus, our results do not support the previously suggested strong negative relation between swimming activity of eels and intensity of A. crassus infection, at least in the short-term. However, there are indications in the literature that the pathological effects of A. crassus on the eel swimmbladder may involve a higher energy demand, possibly manifested in a prolonged spawning migration. As a result, eels heavily infected with this parasite may arrive too late at the spawning site to participate in mating. This could ensure a selection of 'good genes'.     

多子小瓜虫的形态发生及其寄生导致翘嘴鳃组织病理变化

为阐明小瓜虫的形态发生及其对鳃组织的危害过程,本文开展了多子小瓜虫的光镜观察和翘嘴鲌鳃组织病理切片方面的研究。光镜形态研究发现小瓜虫运动时呈球形,直径500-800 m;前端具有耳廓状胞口和内陷的袋状胞咽;环境温度为18℃时,滋养体经1h左右发育成包囊,体表形成一层透明胶膜,并以虫体中央的分裂沟为界进行二分裂;子代虫体从8细胞期进入16细胞期时,包囊内部形成内胞膜将子代虫体等分为两个集团。组织切片结果显示虫体呈卵圆形,挤压了周围的包裹细胞,导致组织变形;鳃小片黏连,鳃丝上皮细胞受到破坏,严重区域鳃丝结构被完全破坏。本研究解析了小瓜虫的形态发生与发育过程以及对宿主危害的组织病理结构与机制,为揭示小瓜虫的侵染、发育和成熟规律奠定了理论基础,并为小瓜虫病害的有效防控提供理论资料。     

Partial cross protection against Ichthyophthirius multifiliis in Gyrodactylus derjavini immunized rainbow trout.

Partial cross protection against a skin-parasitic ciliate has been recorded in rainbow trout previously immunized with an ectoparasitic platyhelminth. The susceptibility to infection by Ichthyophthirius multifiliis differed significantly between naive and Gyrodactylus derjavini immunized rainbow trout. Fish partly immune to the ectoparasitic monogenean G. derjavini became less infected and experienced lower mortality than naive fish when exposed to I. multifiliis infections. In vitro studies on immobilization of theronts using decomplemented (heat-inactivated) serum from G. derjavini immune or non-immune hosts showed no immobilization. However, untreated serum from both immune and non-immune fish containing intact complement immobilized theronts (titre 128-256). In addition, non-specific priming of the host response with interleukin (IL-1), bacterial lipopolysaccharide (LPS), concanavalin A (Con A) or mannan did confer a partial resistance to I. multifiliis infection. This will suggest that non-specific factors including complement could be partly responsible for the host response against infections with this ciliate.     

低氧胁迫和螺原体感染对中华绒螯蟹存活和细胞凋亡的影响

为了探究低氧胁迫对螺原体(Spiroplasma eriocheiris)感染中华绒螯蟹(Eriocheir sinensis)的影响, 以中华绒螯蟹为研究对象, 在低氧胁迫后, 取中华绒螯蟹肝胰腺和鳃组织加入组织固定液, 进行HE染色。在低氧条件下使螺原体感染中华绒螯蟹, 计算螺原体拷贝数, 并对血淋巴细胞进行细胞凋亡、细胞坏死和线粒体膜电位检测。结果显示, 与对照组相比, 处于长时间低氧胁迫状态下的中华绒螯蟹肝胰腺组织疏松, 出现大量小空泡, 鳃轴结构弥散, 组织结构被破坏。此外, 低氧组的中华绒螯蟹感染螺原体后的死亡速度相对于常氧组明显加快, 血细胞内的螺原体数量、线粒体膜电位、血细胞凋亡率和坏死率相较于常氧组均显著升高。以上研究说明低氧胁迫可以加速螺原体的感染, 使河蟹死亡速度变快, 使血淋巴细胞凋亡和坏死更显著, 不利于河蟹的生理生化。     

Cellular and humoral factors involved in the response of rainbow trout gills to Ichthyophthirius multifiliis infections: molecular and immunohistochemical studies

The parasitic ciliate Ichthyophthirius multifiliis infecting skin, fins and gills of fish induces a protective immune response in rainbow trout (Oncorhynchus mykiss) surviving the infection and a similar protection can be conferred by i.p. injection of live theronts. A combined molecular and immunohistochemical approach has been used in this work for pinpointing cellular and humoral immune factors in gill tissue involved in the response and indicating interactions between the systemic and local responses. Fish were immunized by intra-peritoneal injection of live I. multifiliis theronts, control fish were injected with PBS and subgroups were treated with the immuno-suppressant hydrocortisone before fish were challenged with live theronts. Significant up-regulations of genes encoding IgM, IgT, C3, SAA, IL-8, IL-22 and IFN-γ were induced by immunization and challenge. Hydrocortisone treatment had a significant down-regulating effect on genes incoding IgT, IgM, CD4, CD8, IFN-γ, IL-8 and IL-22 in all groups. Immunohistochemistry, using monoclonal antibodies to detect cellular markers, demonstrated active involvement of CD8, MHC II, IgT and IgM positive cells in gill tissue. Putative T-cells (CD8 positive cells) were detected in the intraepithelial lymphoid tissue located at the base of gill filaments and in hyperplastic gill tissue but following infection a clear efflux of these cells was detected. MHC II positive cells were distributed across the gill filaments and accumulated in hyperplastic tissue but hydrocortisone treatment affected their density negatively in both immunized and non-immunized fish. IgT positive cells were present in the epithelial lining of the gill lamellae (suggesting a primary role of this protein in the mucosal defence against the ciliate) whereas IgM positive cells were found only in gill arterioles and the lamellar capillaries. The present work indicates an intensive activity and specialized function of immune cells (B-cells, T-cells and macrophages) and humoral elements such as immunoglobulins IgT and IgM which are orchestrated by cytokines in gill tissue reacting against I. multifiliis.     

青海湖裸鲤不同年龄鉴定材料的年轮特征

通过各种年龄鉴定材料比较研究了青海湖裸鲤的年轮特征。微耳石和星耳石可用于鉴定年龄,而矢耳石易碎,不适合鉴定年龄。4种年龄鉴定材料对青海湖裸鲤年龄的判别能力为:背鳍条>微耳石>臀鳞>脊椎骨。对8龄以下个体,用背鳍条磨片鉴定年龄效果较佳;对8龄以上个体,臀鳞、背鳍条磨片和脊椎骨上的年轮计数明显低于微耳石磨片,微耳石磨片是高龄青海湖裸鲤较为可靠的年龄鉴定材料。       

Treatment of ichthyophthiriasis after malachite green. II. Earth ponds at salmonid farms

We tested formalin, chloramine-T-formalin and Desirox-formalin, for use against white spot disease (ichthyophthiriasis) caused by Ichthyophthirius multifiliis at 3 salmonid farms (Salmo salar and S. trutta smolt reared in earth ponds). I. multifiliis disappeared from most individuals 4 to 5 wk after the first treatment (and after the first I. multifiliis were found) with all chemicals, indicating that combinations of these chemicals, and even formalin alone, can be used to lower the parasite burden in earth ponds to such a level that no mortality occurs. This was the case when the fish were treated frequently at the beginning of the infection. Treatment can be stopped once the fish have achieved immunity to ichthyophthiriasis. The developing immunity was also revealed by the distribution of ciliates in the course of the disease. At the beginning of the infection I. multifiliis individuals were randomly distributed among the fish, but after 2 to 3 wk, when all the fish were infected, ciliates had increased in numbers and were aggregated in such a way that some fish carried quite heavy burdens. However, over 60% of the fish were free of the parasites after 4 to 5 wk, and had few or no ciliates, meaning that the distribution was even more aggregated. Sea trout had fewer parasites than salmon, and they also recovered from infection earlier even though the treatments and ponds were similar, indicating variation in resistance to I. multifiliis between fish species. It was also evident that the chemicals and their concentrations must be planned carefully to suit the conditions at each farm.     

Scuticociliata infection in the weedy sea dragon Phyllopteryx taeniolatus

The weedy sea dragon Phyllopteryx taeniolatus at an aquarium in Kanagawa Prefecture were found infected by protozoan ciliate, 2001. The infected fish in particular, showed sloughing of the epidermis. Fish with intense infections showed sloughing of the dorsal fin, depigmentation of skin, anal distension and accumulation of ascitic fluid in the body cavity. In biopsies, ciliates were detected only in fresh mounts of abdominal dropsy and in the mucus on the body surface. Histopathological studies revealed ciliates mainly infected the dermis, and induced extensive detachment of the epidermis from the skin. Based on the arrangement and shape of the buccal structure and the number of somatic ciliature, ciliates isolated from the fishes belonged to the order Scuticociliatida. We discuss the major factors that bring about the death of weedy sea dragons.