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。     

青海湖裸鲤和黄河裸裂尻鱼感染多子小瓜虫的病理学比较研究

为探讨不同裂腹鱼类感染多子小瓜虫后的病理学差异, 利用多子小瓜虫对青海湖裸鲤指名亚种(Gymnocypris przewalskii przewalskii)和黄河裸裂尻鱼(Schizopygopsis pylzovi Kessler)实施感染实验, 并对2种鱼进行了深入的病理学研究。研究结果显示: (1) 2种鱼的死亡量均呈现先激增后明显回落的趋势, 青海湖裸鲤死亡高峰在感染后第3至第4天, 黄河裸裂尻鱼死亡高峰在第3至第5天, 青海湖裸鲤的死亡比黄河裸裂尻鱼急剧。(2)感染后2种鱼体表均出现大量肉眼可见的白点。青海湖裸鲤皮肤黏液分泌量明显增加, 体表形成胶状黏液层, 黏液层中见不同细胞期小瓜虫包囊。黄河裸裂尻鱼鳍出现蛀鳍现象, 皮肤出现细菌感染样溃烂。(3)解剖发现, 感染组青海湖裸鲤和黄河裸裂尻鱼肝脏发生病理改变呈淡黄色, 胆有不同程度肿大。(4)组织切片和电镜观察显示, 小瓜虫在鳃部位的寄生导致青海湖裸鲤和黄河裸裂尻鱼鳃丝黏连, 鳃小片和鳃丝上皮细胞萎缩、脱落, 鳃丝结构被严重破坏。小瓜虫在2种鱼皮肤的寄生使寄生部位组织突起, 周围组织塌陷。青海湖裸鲤表皮下层出现空隙, 表皮结构被严重破坏。黄河裸裂尻鱼皮肤表皮细胞出现空泡化病理改变, 失去原有紧密结构, 表皮层和固有层间界限变模糊。综上所述, 小瓜虫的感染对青海湖裸鲤和黄河裸裂尻鱼的鳃和皮肤组织造成严重损伤, 但2种鱼表现的症状和造成的组织损伤类型有明显差异, 这与2种鱼长期适应不同水体环境密切相关。     

水温对多子小瓜虫孵化及幼虫活力的影响

多子小瓜虫引起的白点病是严重危害淡水鱼类的重要疾病之一,该寄生虫的发育繁殖受温度影响极大,因此,温度是影响小瓜虫培养及其疾病发生的重要因素.为了阐明温度在多子小瓜虫生活史中的作用,在实验条件下,研究了水温对该寄生虫包囊形成、孵化和幼虫活力的影响.结果表明:水温在4℃左右或28～32℃,多子小瓜虫都不能孵化,但有少部分虫体以包囊形式存活下来;在9～27℃,随水温升高,包囊彤成和孵化时间缩短,孵化率升高至100%,孵出的感染性幼虫活力增强,但活力维持时间缩短至4 h.因此,建议在多子小瓜虫培养及人工感染试验中,维持23℃左右的水温较合适,这时感染性幼虫使用的有效时限是孵出后4 h内.     

华鲮鳃表面形态结构扫描电镜观察

应用扫描电镜对华鲮Sinilabeor rendahli鳃耙、鳃弓、鳃丝和鳃小片的形态结构进行了观察.鳃耙和鳃弓表面凹凸不平,分布着大量丘突.鳃丝和鳃耙表面有大量粘液细胞分布,鳃丝上皮细胞表面密布微嵴,氯细胞附着在鳃丝表面和鳃小片侧表面.鳃小片薄、表面凹凸不平,垂直排列在鳃丝上.鳃丝和鳃小片的表面形态结构特征有助于提高鱼鳃的气体交换效率.     

休眠期和营养期包囊游仆虫的纤毛器骨架及其微管蛋白

应用光镜和透射电镜术 ,显示了包囊游仆虫休眠细胞中纤毛器骨架的形态 ,并对该纤毛虫休眠细胞和营养细胞的纤毛器及其α、β -微管蛋白进行了免疫荧光定位的比较研究。由免疫荧光显微术显示 ,包囊游仆虫形成休眠包囊后 ,背部毛基体完整地按原有模式保存下来 ;纤毛杆解聚后微管蛋白多集中在细胞皮层 ,小部分均匀散布在细胞质中。据所得结果认为 ,包囊游仆虫形成包囊后 ,微管蛋白主要有 3个去向 ,即 :①处于自噬泡内被逐步消化 ;②以“微管蛋白库”的形式分布于细胞皮层及细胞质中 ;③保留在残留的基体中。此外 ,以往研究中发现的棘毛基部纤维网络未被标记上 ,提示这些纤维体系可能不属于微管系统     

体眠期和营养期包裹游仆虫的纤毛器骨架及其微管蛋白

应用光镜和透射电镜术,显示了包囊游仆虫休眠细胞中纤毛器骨架的形态,并对该纤毛虫休眠细胞和营养细胞的纤毛器及其α,β－微管蛋白进行了免疫荧光定位的比较研究。由免疫荧光显微术显示,包囊游仆虫形成休眠包囊后,背部毛基体完整地按原有模式保存下来;纤毛杆解聚后微管蛋白多集中在细胞皮层,小部分均匀散布在细胞质中,据所得结果认为,包囊游仆虫形成包囊后,微管蛋白主要有3个去向,即：91）处于自噬泡内被逐步消失;（2）以“微管蛋白库”的形式分布于细胞皮层及细胞质中;（3）保留在残留的基体中,此外,以往研究中发现的棘毛基部网络未被标记上,提示这些纤维体系可能不属于微管系统。     

鲻和鲮鳃丝的扫描电镜比较观察

对鲻（Mugil cephalus）和鲮（Cirrhina molitorella）的鳃丝表面结构进行了扫描电镜比较观察,结果表明,鲻鳃丝杆状部比鲮粗．鲻鳃小片高度比鲮低;两者鳃丝表面分泌孔洞口径和密度不同;鲻和鲮细胞外被不同,鲻细胞外被稀疏,鲮的则致密复杂;鳃小片细胞和鳃丝表皮细胞的表面形态存在差异,文章还描述了鳃丝表皮形态特异的细胞。     

斑马鱼鳃的光镜和透射电镜观察

应用光学显微镜和透射电镜对斑马鱼(Danio rerio)鳃的组织结构及鳃丝、鳃小片超微结构进行了观察。结果表明,斑马鱼有4对全鳃,鳃耙呈长锥状,鳃丝呈梳状排列在鳃弓上,鳃小片均匀排列在鳃丝两侧。鳃小片由上皮细胞、柱细胞、内皮细胞和毛细血管网组成,鳃小片基部和血管周围分布有泌氯细胞,胞内有丰富的线粒体和排泄小泡,根据线粒体形态特征和细胞质电子密度可将其分为两个亚型。黏液细胞通常与泌氯细胞对生存在,并且有通外的开口。斑马鱼鳃组织结构与其他硬骨鱼鳃结构相似,其结构和功能有密切的关系。     

多毛类微欧鳃蚕形态的扫描电镜研究

本文系用扫描电镜研究多毛类缨鳃蚕种Sabellidae微欧鳃蚕Oriopsis minuta(Berkeley and Berkeley)的形态。作者观察了整个虫体的外部形态,特别是鳃冠、鳃丝和刚毛的形状,描述了前人在光学显微镜下未观察到的细微结构。微欧鳃蚕标本采自北太平洋东岸、美国华盛顿州、圣胡安岛潮间带。     

珠江口池养梭鱼鳃的光镜、扫描和透射电镜观察

应用光学显微镜、扫描电镜和透射电镜对珠江口池塘养殖梭鱼Liza haematocheila鳃的组织结构、表面形态特征及鳃小片超微结构进行了观察。结果表明梭鱼具有4对鳃,每个鳃由鳃弓、鳃丝、鳃小片和鳃耙组成。梭鱼鳃丝和鳃小片的表面结构和超微结构与其他硬骨鱼类的基本相似,鳃丝表面分布有众多规则或不规则的环形微嵴、沟、坑、孔等结构。鳃丝分为呼吸区和非呼吸区,呼吸区较为平滑,上皮细胞表面无微嵴,呈皱褶状;非呼吸区分布有沟、坑、孔等结构,上皮细胞有较规则的指纹状微嵴。鳃小片是最主要的呼吸场所,由基膜、上皮细胞、内皮细胞、柱细胞和毛细血管网组成。泌氯细胞主要分布在鳃小片基部,并有开口通往外界。本文还探讨了梭鱼鳃的结构与其功能的密切关系。     

Ichthyophthirius multifiliis (Ciliophora) exit from gill epithelium

The first change in the sequence of morphological events occurring as fully developed Ichthyophthirius multifiliis trophonts spontaneously left gill epithelium or as younger trophonts departed, following experimentally induced death of the fish, was the separation of parasites from overlying host cells. Discharge of contractile vacuoles may have played a role in this process. Spaces then appeared between host cells, and individual epithelial cells became vacuolated. Finally, the epithelium ruptured and the parasites swam free. In induced exit after three days residence in the host, departure of the trophont was evident only after autolysis of epithelium had occurred. Induced departure of trophonts after four days residence was more rapid, suggesting an active role for the parasite in exit. Changes in parasite and epithelium observed in induced exit were similar to those in spontaneous departure after five days residence.     

Differential expression of cysteine proteases in developmental stages of the parasitic ciliate Ichthyophthirius multifiliis

An expressed sequence tag database of the freshwater fish parasite, Ichthyophthirius multifiliis (Ciliophora) was analyzed to seek for proteases potentially involved in the invasion and degradation of host tissues during infection. The translation of the database revealed two cathepsin L cysteine proteases (Icp1 and Icp2) of the C1A peptidase subfamily. The analysis of Icp1 and Icp2 sequences suggested that both proteases would be synthesized as preproproteins, with a mature domain of 27.9 and 22.8 kDa, respectively. Their expression level was determined in the trophont parasitic stage, in the tomont reproductive stage, and in the theront infective stage by real-time RT-PCR. ICP1 and ICP2 were significantly upregulated in trophont and theront stages in comparison with the tomont stage. Mature peptides of Icp1 and Icp2 were identified in crude extracts of I. multifiliis trophonts by LC-MS/MS. Zymograms showed three to seven activity bands at the optimum pH of cathepsin L cysteine proteases. Two bands displaying cysteine protease activity were identified by inhibition with E-64. They represented the major proteolytic activity of the trophont stage at pH 5-7, suggesting that cysteine proteases play an important role in the infection process.     

Ichthyophthirius multifiliis (Ciliophora) Invasion of Gill Epithelium1

The sequence of morphologic events associated with Ichthyophthirius rnultifliis invasion of gill epithelium began in the theront with differentiation of secretory mucocysts and the perforatorium. After escaping from the cyst the theront, which stained intensely with Mallory' stain, sought a host. As it approached the host epithelium, the contents of the mucocysts were discharged, enveloping the ciliate in sticky material, which made initial contact with the host epithelium. Rapid penetration by the theront caused disruption of one or more host cells and resulted in a focal necrosis associated with the anterior margin of the ciliate. Within five minutes postexposure, the parasite completed its invasion of the epithelial layer and stained less intensely. The remnants of host cells disrupted during its entry surrounded the trophont until they were ingested by the parasite. Within 40 min postexposure, synthetic activity of the parasite appeared to increase as evidenced by an abundance of organelles, particularly ribosomes and crystalline mucocysts. At this point, the overlying host epithelium appeared normal.     

Ichthyophthirius multifiliis (Ciliophora) Exit from Gill Epithelium1

The first change in the sequence of morphological events occurring as fully developed Ichthyophthirius multifiliis trophonts spontaneously left gill epithelium or as younger trophonts departed, following experimentally induced death of the fish, was the separation of parasites from overlying host cells. Discharge of contractile vacuoles may have played a role in this process. Spaces then appeared between host cells, and individual epithelial cells became vacuolated. Finally, the epithelium ruptured and the parasites swam free. In induced exit after three days residence in the host, departure of the trophont was evident only after autolysis of epithelium had occurred. Induced departure of trophonts after four days residence was more rapid, suggesting an active role for the parasite in exit. Changes in parasite and epithelium observed in induced exit were similar to those in spontaneous departure after five days residence.     

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.     

Prolonged in vitro cultivation of Ichthyophthirius multifiliis using an EPC cell line as substrate

The ciliate Ichthyophthirius multifiliis, which normally requires a fish host to develop from the theront stage to the trophont stage, was cultivated in vitro for part of its life cycle. Experiments were conducted using a laboratory strain of the parasite originally isolated from rainbow trout Oncorhynchus mykiss in a Danish trout farm. Theronts escaping from tomontocysts were kept in water, cell culture media (E-MEM or L-15), or cultures of EPC (Epithelioma Papulosum Cyprini) cells in plastic tissue culture dishes (Nunc multidish plates). In addition, a 2-compartment system, with water separated from tissue culture media by a monolayer of EPC cells on an Anopore Tissue Culture Insert (mimicking the fish epidermis) was tested as an experimental habitat for the parasite. Theronts transformed into trophonts in all treatments except in water alone. However, development was accelerated in wells containing EPC cells, and survival and growth of trophonts were significantly increased compared to water or tissue culture media alone. Further, the 2-compartment system allowed superior performance of the parasites (attachment of parasites to cells and growth from 36 to 46 microm). In all experiments it was found that the presence of host factors (mucus and serum) stimulated parasite development.     

抗菌肽HBβ-C在全雄和杂交黄颡鱼对多子小瓜虫抗性差异中的作用

为研究全雄黄颡鱼(Pelteobagrus fulvidraco)、瓦式黄颡鱼(Pelteobagrus vachelli)和杂交黄颡鱼(黄颡鱼P. fulvidraco♀×瓦氏黄颡鱼P. vachelli♂)对多子小瓜虫(Ichthyophthirius multifiliis)的抗性差异, 通过生物信息学分析黄颡鱼皮肤黏液蛋白质组, 发现其血红蛋白源抗菌肽(HBβ-C)位于血红蛋白β链HBβ的碳端, 共33个氨基酸。利用化学合成的不同浓度的HBβ-C肽段进行体外抗虫实验, 研究发现其能有效杀死滋养体、包囊体和掠食体阶段的多子小瓜虫, 其中15 μg/mL的HBβ-C能在3min内杀死所有滋养体。基因表达量分析显示, 在杂交黄颡鱼的鳃和皮肤组织中, HBβ的mRNA表达量高于全雄黄颡鱼; 但在应对小瓜虫感染的过程中, 全雄黄颡鱼的HBβ mRNA转录水平快速提升, 其表达水平和上升倍率显著高于杂交黄颡鱼。蛋白表达量分析显示, HBβ在全雄黄颡鱼鳃组织中的蛋白表达量明显高于杂交黄颡鱼。免疫荧光定位结果显示, 抗菌肽HBβ-C特异地在红细胞中表达, 可以分泌并附着在滋养体上。综上所述, 相对于杂交黄颡鱼, 全雄黄颡鱼中HBβ具有更高的翻译效率, 可以更高效地应对多子小瓜虫的感染。     

Localized cellular responses to Ichthyophthirius multifiliis: protection or pathogenesis?

The ciliate Ichthyophthirius multifiliis is an economically important parasite in aquaculture as well as in the ornamental fish trade. The parasite has been well studied and details of life cycle, host response and pathogenesis have been characterized. Parasite development within host epithelial tissues initiates localized leukocytic infiltrations, although the relationship between these responses and host resistance is uncertain, and whether or not leukocyte responses play a role in protective immunity is unclear. Here Frank Cross outlines the character of localized cellular responses during I. multifiliis infection and discusses the contribution of such responses to protective immunity and pathogenesis.     

DEVELOPMENT OF THE ENDOPARASITIC DINOFLAGELLATE AMOEBOPHRYA CERATII WITHIN HOST DINOFLAGELLATE SPECIES1

The endoparasitic dinoflagellate Amoebophrya ceratii Koeppen occurs in coastal waters of Nova Scotia within cells of two dinoflagellate hosts, a Scrippsiella species (probably S. trochoidea (Stein) Loeb. III) and Dino-physis norvegica Claparede & Lachman. We describe the development of the endoparasitic stage (the trophont) of A. ceratii within host cells using light and electron microscopy. After entry into the host, the trophont grows and expands until most of the host cell is occupied by the parasite. Growth is marked by a proliferation of trophont nuclei and flagella and by the formation of numerous lobes, each of which possesses a characteristic dinoflagellate amphiesma. The mature endoparasitic trophont is recognized at the light microscopic level as a beehive-shaped structure that consists of numerous lobes of the developing motile sporont cells and a mastigocoel cavity containing the sporont flagella.