【作者】 栾菊敏；

【导师】 顾福康；

【作者基本信息】 华东师范大学 ， 动物学， 2011， 硕士

【摘要】 原生动物赭纤虫属(Blepharisma)是在1852年首先由瑞士原生动物学家Perry建立的,至今已经有将近160年的研究历史。经过长期研究现在被普遍认为是一个多源的发生系,Corliss基于口区纤毛图示将异毛目分为六个亚目：异毛亚目Heterotrichina、武装亚目Amophorina、考利亚目Coliophorina;丽克亚目Licophorina;斜边亚目Plagiotomina,并且这六个亚目归于旋唇亚纲Spirotricha, Lynn认为异毛纲只包含异毛目。异毛目纤毛虫通常个体较大、肉眼可见、容易培养、有一定程度上的伸缩性、再生能力很强、具有遍布体表的同型纤毛等特点使它们成为实验纤毛虫的理想研究对象。目前对它们的研究也是集中在射出胞器、黏液泡、刺丝泡等方面,而对其它一些特殊胞器如内共生体、色素颗粒的研究并不常见。这些功能性的器官具有各自不同的结构和功能,不同纲目的相似纤毛器功能与结构也不尽完全相同。本实验室在野外采集原生动物的过程中,采集到了一种异毛目纤毛虫美国赭纤虫,它的形态特征与Suzuki(1953)发现的美国赭纤虫(Blepharisma americanum)一致,将之定为该种。在对赭纤虫的研究中,研究比较多的是赭纤虫的信息素成份及功能、聚合酶、Y微管蛋白基因片段及遗传学研究,而对赭纤虫皮层微管、细胞形态发生及体纤毛器在细胞结构形成过程中分化特征则尚不完全清楚。本文应用Flutax荧光紫杉醇直接荧光标记法对美国赭纤虫的形态、细胞发生过程做了详细的观察。运用微分干涉相差显微术显示了虫体表膜下皮层色素颗粒的形态和细胞受到强烈光照时溶胀破裂的细胞结构。同时运用扫描电镜术与透射电镜术对美国赭纤虫细胞表面以及内部结构做了超微结构观察。1美国赭纤虫皮层微管胞器的形态和形态发生应用FLUTAX荧光紫杉醇直接荧光标记显示,美国赭纤虫细胞呈饱满的梨形并呈现明亮的砖红色,前端稍细,后端钝圆。口围带约占整个细胞长度的1/2,起始于细胞前端并延伸至细胞中部,位于细胞腹面前端左侧。波动膜一片,位于虫体细胞右侧,波动膜长度约为口围带长度的2/3。体表布满同型纤毛,体纤毛均匀分布在整个虫体细胞表面并沿着虫体纵向排列,表膜下动纤丝纵贯整个虫体。细胞形态发生过程中,后仔虫口器微管在皮层口围带后第一列体纤毛位置发生,前仔虫口器微管在老口围带位置形成；后仔虫口器发生早于老口围带更新,老口围带在新口围带发生同时逐步更新。新的体纤毛在各个老体纤毛列中部每两根纤毛基体之间发生,此后新、老体纤毛共同组成为新仔虫的体纤毛列。所得结果表明,老细胞的皮层纤毛器结构对新纤毛器微管的发生不仅具有定位和诱导作用,并对新细胞的形成具有物质与结构贡献。前仔虫的口围带经历生理改组等纤毛器瓦解更新的过程,最终形成前仔虫新纤毛器。2美国赭纤虫皮层色素颗粒的显微观察应用微分干涉相差显微术显示,美国赭纤虫营养期细胞有较强的伸缩性,在暗视野下呈现明亮的赭红色,细胞表膜下可见清晰的伸缩泡以及食物泡,赭纤虫细胞的赭红色是由于皮层下色素颗粒而呈现出来的,它们维持着细胞的颜色。皮层色素颗粒为大小均匀的均质圆球形颗粒,均匀排列在细胞表膜下,并沿着细胞纵轴体纤毛列旁动纤丝成对排列。在暗视场下观察时,虫体细胞游动迅速活跃,皮层下色素颗粒清晰可见。在逐渐加强的亮视野下观察时,皮层细胞色素颗粒因受到高强度光线照射,色素颗粒会发生破裂并同时伴随着细胞溶解、胞质外流、细胞破裂并死亡。据结果推测：美国赭纤虫的色素颗粒与细胞颜色有关,皮层下色素颗粒在受到强光照射时所发生的程序性细胞死亡可能是细胞的一种自我防御方式。3美国赭纤虫细胞超微结构的观察。应用扫描电镜术以及透射电镜术显示,美国赭纤虫细胞表面有棘状突起,相邻棘之间为纤毛沟,体纤毛着生于纤毛沟内。虫体细胞表面布满同型的长而密集的体纤毛,纵向均匀分布于体表。进一步放大倍数观察时发现,正常状态下的虫体表面区域平整光滑,无颗粒状的突起。有时也会在虫体细胞表面观察到小的圆形凹痕。在营养状况不良的条件下,虫体细胞会通过结合生殖的方式来繁殖后代。口围带由彼此之间平行排列的纤毛列构成,每一纤毛列由两列平行的纤毛杆组成。美国赭纤虫细胞内皮层下色素颗粒及共生细菌结构十分丰富,除此以外还含有细胞核、线粒体、内质网等结构,细胞内膜系统并不十分发达,含有很多椭圆形食物泡以及很多未消化的食物颗粒。线粒体数量庞大,十分丰富,线粒体内部有很多颗粒状物质。横切面显示纤毛杆也是典型的“9+2”结构,皮层下色素颗粒大小均一均匀分布于表膜以及嵴突下,按照每两个一组的方式均匀排列。细胞质内有很多类似于共生细菌样的水滴状物质以及尚未完全消化的食物泡,动纤丝沿着虫体细胞纵向排列在表膜下。口围带部分的纤毛排列也有一定规律,每两根纤毛杆组成双动基系,色素颗粒多数按照每两个按对分布于双动基系之间,有时也可见三个色素颗粒排列在口围带纤毛之间。更多还原

【Abstract】 Protozoa Blepharisma was first set up by Switzerland protozoa scientist Perry in 1852, it has been studied by scientists for nearly 160 years. It has been widely recognized as a polyphyletic assemblage after long time research. Corliss divided Heterotrichea into six suborder: Heterotrichina、Amophorina、Coliophorina、Licophorina、Plagiotomina according to oral area cilia pattern, and he attributed these six suborder into Spirotricha. Lynn thought that Heterotrichea only contain Spirotricha.Heterotrichea usually has a large individual volume, could be saw by eyes, easily cultivated, has a reasonable excellent flexibility, strong power of regeneration and has the same cilia pattern which covers all over the cell. They were ideal research object on account of these excellent characteristics. Scientists have been done research mainly on extrusome、muciferous body、mucous trichocyst、while there were few research on inter symbiont and granule pigment. These functional organelle have totally different structure and function while similar cilia organelle have different structure and function. In the process of the collection of protozoa, we found a kind of Heterotrichea Blepharisma. americanum. Its morphology characteristics were basically same as Suzuki found in 1953. There were many research on the composition and function of pheromone, polymerase,γtublin gene segment and genetics while the research on cortical, morphogenesis and the differentiation characteristics of body cilia were seldom reported. The author make carefully observation of morphology and morphogenesis using the method of Flutax direct fluorescent-labeled. At the same time make observation of pigment granule under pencile and the swelling and rupture of cell under strong light using differential interference contrast microscopy. At the same time the author make observation of ultra microstructure using SEM and TEM.1 The cortical morphology and morphogenesis of Blepharisma americanum The result of the method of Flutax direct fluorescent-labeled indicate that:BIepharisma americanum has an ample pyriform and the whole cell were red. The frontier of the cell is slim while the real end is round. The length of the adoral zone of membranelles(AZM) take up about 1/2 of the whole cell. It originated from the frontier of the cell and extends to the middle of the cell. It locates on the left of the frontier of the adroal. The umbrane locates at the left of the cell, the length of the UM is about 2/3 of the adroal zone of membrane(AZM). The surface of the cell were covered with same type of cilia. The body cilia which distributed along the cell and cover all over the surface of cell. Kinetodesma under pencile. During the process of morphogenesis, the mouthparts of the opisthe is originated at the first row of body cilia, the mouthparts of proter were founded at the location of old adoral zone of membrane, the morphogenesis of opisthe earlier than the regeneration of old adoral zone of membrane, the regeneration of old adoral zone of membrane happened when the new adoral zone of membrane were generated. New body cilia originated between every two cilia among the old row of body cilia. And then the old and new body cilia composition of the new body cilia together. The result show that:the cortical constructure of old cell not only play the role of location and induction but also make contribution of material and constructure. The adoral zone of membrane of proter experience the process of regeneration.2 The observatin of pigment granule of Blepharisma americanum Differential interference contrast microscopy showed that the cell present red or faint red colors, the cell has excellent flexibility. Contractile vacuole and food vacuole could be easily observed under pencile, the color of cell is on account of the pigment granules, they maintain the color of cell. Pigment granules distribute regularly under the membrane and have basically the same volume. They locate besides the kinetodesma of the cell. Cell swimming actively under dark light and pigment granule could be seen even without microscopy. While the light were generally activated, the pigment granules will collapse and swelling and cytoplasm would be flow out of cell on account of the impact of strong light. The process of the light induced swelling and death of cell is some kind of protection of cell.3 The microstructure observation of Blepharisma americanum The result of the application of TEM and SEM show that the surface of Blepharisma americanum have small granule shape protuberance, there are many grove on the surface of the cell. It is cilia grove and the body cilia were grow among them. The body cilia is the same type and long which covers all over the body. After the magnification show that:the surface area of the cell of normal nutrition stage cell is smooth and there were not any other material attach on the surface, however, sometimes there could be observed some depression. Cells would be conjugation to generation under some bad condition such as the lack of nutrition. Adoral zone of membrane were made up of parallel cilia row, each cilia row were composed by two row of parallel cilia. The pigment granule and inner cell constructure were abundant. Besides there were many food vacuole and food granule which have not yet digest by the cell.更多还原