海水鱼类寄生纤毛虫刺激隐核虫细胞形态学研究

发布时间：2018-03-21 18:58

本文选题：寄生虫　切入点：纤毛虫　出处：《华东师范大学》2017年博士论文　论文类型：学位论文

【摘要】：刺激隐核虫(CryptocaryonirritansBrown,1951)是一种寄生于几乎所有海水硬骨鱼类原生动物纤毛虫,可引发宿主鱼类刺激隐核虫病,给水产养殖业造成巨大的危害。近年来,重新认识刺激隐核虫的基础生物学特征和病原特性,又受到了人们的广泛关注。刺激隐核虫具有相对复杂的生活史,可分为:寄生于宿主组织中、摄食营养的滋养体时期;脱离宿主后在水中短暂自由运动的包囊前体时期;在包囊内进行细胞分裂增殖的包囊时期;细胞分裂产生的幼虫脱出包囊、再次感染入侵宿主的幼虫时期。目前,关于刺激隐核虫的生活史中各种细胞器的形态和结构,及其在不同生活史时期间的演化规律,仍缺乏较为全面深入的认识;对一些关键的生活史阶段或生命活动过程中所涉及的细胞器及其生物学功能,以及相关的细胞学机制尚不十分清楚。另外,虽然前人的研究己基本确定刺激隐核虫属于前口类,但其在前口类中具体的系统分类地位尚存争议,缺乏必要的形态学证据。本研究选用海水养殖大黄鱼作为感染宿主,建立刺激隐核虫传代培养,对刺激隐核虫各生活史时期内不同生命活动阶段的细胞,采用活体观察、银染技术、荧光标记技术、扫描电镜技术、透射电镜技术以及酶细胞化学技术,从显微、亚显微水平进行了形态学观察,并通过测序建树对刺激隐核虫进行了分子系统学分析。对多种胞器或结构进行了全面系统的描述,发现了较多未报道过的结构或细胞生命活动现象特征,明确了刺激隐核虫的分类地位并给出了形态学定义,为进一步完善并深入阐明刺激隐核虫及鱼类寄生纤毛虫的细胞形态学和基础生物学特征提供了新的资料。主要结果如下:1刺激隐核虫各生活史时期细胞口区相关结构或细胞器除刺激隐核虫滋养体、包囊前体细胞外,幼虫细胞胞口周缘也具有由口周双动基纤毛和口乳头粘联而成的三联体结构;三联体结构均与胞咽内壁肋嵴一一对应,但不与口缘外侧体纤毛列一一对应。毒丝泡在滋养体和包囊前体细胞中不存在,在幼虫细胞中定位于口乳头及口区胞质中,可能参与了感染入侵宿主鱼的生命活动过程,毒丝泡前体分化形成于包囊内细胞分裂后期仔细胞中。滋养体、包囊前体细胞胞口一侧和幼虫细胞反口面具有膜性褶皱结构,其间分布单动基系体纤毛列,该结构可能由该区域体纤毛列相互靠近、纤毛列间表膜特化隆起形成,膜性褶皱前端不与胞口直接连通,可能与刺激隐核虫的寄生特性有关。滋养体、包囊前体和幼虫细胞均具有的口区纤毛刷为3段双动基片段,生有短棒状纤毛,包囊内细胞分裂后期的仔细胞表面可见口区纤毛刷原基;刺激隐核虫口区纤毛刷,与前口类其他类群该结构特征均不相同。2刺激隐核虫各生活史时期细胞表膜及表膜下相关结构或细胞器及细胞核刺激隐核虫的表膜泡不同于自由生纤毛虫的表膜泡,结构较为复杂,不同的生活史阶段具有不同的形态特征:在滋养体摄食营养阶段,表膜泡内容物丰富,其中一类双层膜小泡可通过膜融合的形式通过小孔与细胞外相通,表膜泡可能具有从细胞外摄取液态或小颗粒营养物质的功能,而摄取的营养物质暂时积累于表膜泡中;正在脱离宿主组织的滋养体细胞,表膜泡中产生大量的丝状物排出至细胞外;细胞形成包囊壁前后,表膜泡及其内容物会分阶段、团块状地内化进入细胞质。包囊内细胞分裂过程中表膜泡内容物较少,呈扁平状态。幼虫细胞表膜泡再次变得肿胀,内容物可能来源于胞质中高尔基体和转运小泡的分泌活动。滋养体、包囊前体细胞中不具有黏液囊,成熟的黏液囊定位于幼虫细胞表膜下,可能参与了幼虫细胞入侵感染宿主鱼以及入侵后建立寄生等生命活动,黏液囊前体分化形成于包囊内细胞分裂后期仔细胞中。刺激隐核虫具有多个伸缩泡,不同的生活史时期超微结构有所不同,可能具有不同的生物学功能,可能在滋养体时期发挥较为重要的功能。刺激隐核虫大核在不同的生活史时期形态特征不同,在形成包囊时念珠状的大核融合变形为球形,染色质变得分散。3刺激隐核虫的包囊现象刺激隐核虫呈多层形态的包囊壁,各片层间形态结构较为相似,可能具有类似的生化本质。包囊壁形成过程中未见特异性小泡或特定射出胞器的分泌活动。细胞形成包囊过程中,体纤毛不发生退化或脱落,胞口及口周纤毛也不退化消失。包囊内进行细胞分裂时,会形成暂时性地长细胞链,细胞链长有正在生长分化的体纤毛,无胞口及原基。新的口原基出现于包囊内细胞分裂后期断裂开来的仔细胞表面。繁殖包囊胞质胞器的生命活动非常活跃,可见高尔基体及粘液囊、毒丝泡前体等大量的物质和结构的合成和分化。4刺激隐核虫的系统分类刺激隐核虫口区纤毛刷、膜性褶皱、咽微纤丝等结构形态特征不同于前口类其他科属纤毛虫,具有复杂的生活史和寄生特性,结合分子系统学建树分析,表明刺激隐核虫具有充足的证据支持其建立独立的刺激隐核虫科(Cryptocaryonidae),甚至具有多项目前分类所属前管目(Prorodontida)不具有的细胞形态学特征。本研究系统学分析支持刺激隐核虫存在种属分化的观点。综上所述,本研究深入系统地揭示了刺激隐核虫胞口和胞咽、毒丝泡、膜性褶皱结构、口区纤毛刷、表膜泡、粘液囊、伸缩泡等胞器或结构在各生活史时期的形态特征及其可能的生物学功能,初步阐明了一些重要的尤其是与纤毛虫寄生特性相关的生命活动现象;首次对刺激隐核虫的包囊现象进行了多方面的描述讨论;明确了刺激隐核虫在前口类中的系统分类地位,并给出了刺激隐核虫科形态学定义。为刺激隐核虫细胞形态学和基础生物学等的研究提供了新的基础资料。
[Abstract]:Cryptocaryonirritans (CryptocaryonirritansBrown, 1951) is a parasite in almost all marine teleost ciliates, can cause the host fish cryptocaryonirritans disease, causing great harm to the aquaculture industry. In recent years, the basic biological characteristics and pathogenic characteristics of re understanding of cryptocaryonirritans, and has received extensive attention. Cryptocaryonirritans with life history is relatively complex, can be divided into: parasitic in the tissues of the host, the trophozoite feeding nutrition period; from the host in the water after a short free movement of the protomonts period; for cell proliferation in cysts within the cyst; cyst cell division produced from larvae, re infection the invasion of the host larvae period. At present, the morphology and structure of organelles on the life history of cryptocaryonirritans in the evolution and during different life history, is still missing The lack of more comprehensive and in-depth understanding of its biological functions; the organelles involved some life stages or life activities of key process, and the cytological mechanism is not very clear. In addition, although previous research has identified basic cryptocaryonirritans belong to the export category, but in the mouth before class specific the taxonomy is controversial, the lack of morphological evidence necessary. This study used seawater cultured large yellow croaker as host, the establishment of cryptocaryonirritans subcultured on different life stages of cryptocaryonirritans the life history within the cells, the in vivo observation, silver staining, fluorescence labeling technique, scanning electron microscopy transmission electron microscopy, and enzyme cytochemistry, from the microscopic, ultrastructural level were observed, and the cryptocaryonirritans of system analysis through sequencing of molecular contribution. A variety of organelles or structure of a comprehensive description of the system, found life structure or cell features more not reported, the taxonomic status of cryptocaryonirritans and gives the morphological definition, cell morphology and biological characteristics of the foundation to further improve and clarify cryptocaryonirritans and fish parasitic ciliates provides new data. The main results are as follows: 1 cryptocaryonirritans period the life history of cells or organelles related in structure in addition to c.irritans trophozoites, cyst precursor cells, larvae cell opening circumference also has three conjoined structure by mouth double action based adhesive and cilia and mouth nipple the three CIS structure and cell; pharyngeal wall rib ridge correspondence, but not with the edge of the outer body cilia column correspondence. Toxicyst does not exist in the trophozoites and cysts of precursor cells, in larval cells located in the mouth The nipple and mouth area in the cytoplasm of infected fish may be involved in the invasion of host life process, toxicyst precursor differentiation cysts formed in anaphase offspring cells. Trophozoites of the protomonts cells on one side of the opening and mouth mask anti larval cells membrane fold structure, the distribution of single action base body cilia, the structure may be provided by the regional body cilia listed near each other, cilia column membrane specialization uplift formation, membranous fold front end and cell port directly connected, may be related to the parasitic characteristics of c.irritans. Nourish the body, mouth area and brush the protomonts ciliated cells have larvae the 3 section double moving base fragment, having short rod like cilia, ciliated cells visible on the surface area of mouth Zi in anaphase brush primordium cyst; cryptocaryonirritans mouth area fiber brush, and in other groups before the structure characteristics are not the same.2 cryptocaryonirritans life Table table film film history during the period of the cell membrane and membrane associated structures or organelles and the nucleus of cryptocaryonirritans bubble is different from free ciliates in global, more complex structure, with the characteristics of different life history: in the different stages of the trophozoite feeding nutrition stage, form vesicles content rich, including a kind of double membrane vesicles by membrane fusion and cell form through hole is communicated with the outside surface, bubble may be from outside the cell uptake of liquid or small particles of nutrients, and nutrient uptake temporarily accumulated in membrane vesicles; trophozoite cells are separated from the host tissue, membrane vesicles produced in the table a large number of filaments is discharged to the outside of the cell; cell wall before and after the formation of cysts, surface membrane vesicles and its contents in stages, mass internalized into the cytoplasm. In the process of cell division in the cyst membrane bubble content is less, the flat state. Larvae of the cell membrane bubble again become swollen, the contents may originate from the Golgi secretory activity in cytoplasm and transport vesicles. Trophozoite, cyst precursor cells do not have the bursa, located in larvae under the cell membrane of mature bursa, may establish parasitic life activities involved in the cell invasion of infected larvae the host fish and after the invasion, bursa precursor differentiation within the cyst formed in anaphase cells. Zi c.irritans has multiple life history of global expansion, the ultrastructure of different, may have different biological functions, may play a more important function in the trophozoites of cryptocaryonirritans period. Nuclear in different life history of different characteristics, in the formation of cysts when torulose nuclear fusion deformation is spherical, chromatin became encystment dispersed.3 cryptocaryonirritans of c.irritans was The cyst wall multilayer form, each layer structure is similar, may have similar biochemical nature. In the process of no specific vesicles or specific extrusome secretion of cyst wall formation. Cell formation of cysts of ciliary body is not degraded or off, cytosome and perioral cilia did not disappear. Cell division cysts, will form a temporary cell long chain, cell chain length is the growth and differentiation of cells and ciliary body, no mouth primordium. New oral primordium appeared in the surface within the tomont anaphase apart young cells. The propagation of encapsulation of cytoplasmic organelles life is active visible, Golgi apparatus and bursa, classification system toxicyst synthesis and differentiation of.4 precursor material and the structure of a number of cryptocaryonirritans of cryptocaryonirritans mouth area fiber brush, membranous folds, pharyngeal microfibril structure morphology characteristic is not Is the same as the first class in other ciliates, with parasitic life history and complex characteristics, combined with the molecular phylogenetic analysis showed that the contribution that c.irritans has sufficient evidence to support the establishment of an independent cryptocaryonirritans Department (Cryptocaryonidae), even with multi project before the class before the tube (Prorodontida) cells. Morphological features do not have. This study phylogenetic analysis support cryptocaryonirritans species differentiation point of view. In summary, this study systematically reveals c.irritans and cytopharynx, toxicyst, membranous fold structure, mouth area fiber brush, membrane vesicles, bursa, expansion cell organelles or structure in the period of the life history of the morphological characteristics and biological functions of possible, clarify some important especially life phenomena associated with the characteristics of parasitic ciliates; first encystment of c.irritans. Various descriptions and discussions were made, and the taxonomic status of the stimulation of Cryptococcus in the anterior class was clarified. The morphological definition of the stimulation of Cryptococcus was provided. It provided a new basic information for stimulating the study of cell morphology and basic biology of Cryptococcus.

【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S941.5

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