Studies On The Microscopic And Submicroscopic Structure Of Extrusomes In Three Kinds Of Hypotrichous Ciliate

Extrusomes are membrane-bound peculiar organelles in ciliates of protists and generally found in their cortical cytoplasm or just beneath the pellicle. They are considered to play an important role in the life activity of different kinds of ciliates, such as defensive, offensive and predation by discharging their internal content to the outside of the cell in response to stimulus. The previous researches mainly focused on some extrusomes of lower forms of ciliates, particularly trichocyst extrusomes in Paramecium and mucocyst extrusomes in Tetrahymena. To date, the research data are very limited in the hypotrichous ciliates showing differentiation of the dorsoventral cortex and functional ciliature. To study the origin and morphogenesis, morphology and localization, extrusion mechanism and functions of extrusomes is of great academic significance for the further understanding the diversity and complexity, the genetics and regulation mechanism of the organelles in protozoa cells, and the interaction relationship between the cells and the external environment.By using differential interference contrast microscopy, protargol staining, scanning and transmission electron microscopy and enzymo-cytochemistry, the present paper demonstrates the different types of extrusomes at microscopic and submicroscopic level:trichocyst-like extrusomes in Anteholosticha monilata of Urostylidae, mucocyst extrusomes in Oxytricha granulifera of Oxytrichidae and excretion extrusomes in Architricha indica of Oxytrichidae. The results are as follows: 1Trichocyst-like extrusomes in Anteholosticha monilata(1) Morphology and localization Extrusomes are long rod-shaped and consist of three parts: a body of stratiform structures of uneven electron density, an elongated shaft located at the center of the body and a cap including multilayer membrane-structure. A great number of extrusomes are found to arrange in rows just beneath the pellicle of vegetative cells but less in cyst. The organelles break through the pellicle and discharge their internal substances when suffered from external stimulus. The extruded structures are needle mushroom shaped including a spherical head and a long rod.(2) Origin and function During the process of maturation, small vesicles undergo the collection of the filamentous materials, the differentiation of inner and outer layers, the appearance of dense shaft, the formation of cap structure and finally positioned in their functional areas. So these organelles may originate from membrane-limited vesicle related to Golgi apparatus. The present study reveals that the extrusomes in Anteholosticha monilata discharge inevitably during the experimental process, which suggests the organelles are involved in defense against chemical stimulus; however, no extrusomes are found near the oral region, indicating that the organelles do not play a role in the process of prey capture. The extrusomes are digested inside autophagic vacuole of cyst, suggesting the organelles may provide energy as material in the special physiological conditions.(3) Compared with trichocysts extrusomes Although the extrusomes in Anteholosticha monilata have similarities in morphogenesis, morphology and localization with trichocysts in Paramecium, the extrusomes are sensitive to external stimulus and discharge inevitably lots of mushroom-shaped structure while trichocysts are insensitive and eject needle-shaped inclusion. In addition, these characteristics are consistent with the trichocysts-like extrusomes of Urostylidae ciliates. The results show that the extrusomes in Anteholosticha monilata are identified as trichocysts-like extrusomes and their structure may have similar differentiation characteristics in the close relationship of ciliates.2Mucocyst extrusomes in Oxytricha granulifera(1) Morphology and localization The mature extrusomes are suborbicular vesicles and arrange in rows beneath the pellicle. These organelles are surrounded with a plasma membrane with spike or filament structure and containing high electron dense materials. When suffered from the external stimulus, the inclusions of mucocyst are extruded and stay outside of the cell surface. The structure of the ejection seems to be not smooth and some filamentous structures are often found.(2) Origin and function Due to lots of similar thickness but different length of endoplasmic reticulum structure distribute around the membrane of nuclear, we speculate that the formation of these organelles is connection with endoplasmic reticulum. Oxytricha granulifera is a kind of soil ciliate, which will becomes cyst easily under adverse conditions. The extruded structure are often found on the vegetative cell surface, but not on the cyst surface, which suggests the extruded structure is beneficial to the formation of the cyst wall and contributes to the pellicle retrieval or renewal.(3) Compared with reported mucocyst extrusomes Mucocyst of Oxytricha granulifera, which are membrane-bound vesicles and filled with high election dense materials and can discharge their content when suffered from stimulus, is similar to mucocyst of lower formers of ciliates Tetrahymena and hypotrichous ciliates Urostyla grandis. However, the mucocyst in Oxytricha granulifera are suborbicular with spike or filament structure, which is different from the oval shaped mucocyst in Tetrahymena and from mucocyst Urostyla grandis which has three recognizable parts: head body and tail. In addition, the extrusomes in Oxytricha granulifera have the phenomenon of microfilament occurrence during ejection while the phenomenon is not to be observed in reported mucocyst during the process. 3Excretion extrusomes in Architricha indica(1) Morphology and localization The mature extrusomes are ellipsoidal vesicles and arrange in rows beneath the pellicle. Two parts can be recognized according to its internal structure:a spherical cavity possibly containing structureless substance, occupies about1/4of the extrusomes volume; while the rest part is full of different electron dense materials. When suffered from the external stimulus, the extrusomes are extruded and present a cyathiform-shaped structure including a head with a cavity, a slightly swollen body and a pointed tail.(2) Origin and morphogenesis The observation showed small vesicles are closely bounded up with the endoplasmic reticulum and developing extrusomes migrate to the cell pellicle. Moreover, the results reveal that glucose-6-phosphatase reactive granules are found not only in the endoplasmic reticulum and its nearby vesicles, but in those developing extrusomes in the cytoplasm. We can speculate that the extrusomes of Architricha indica might originate from endoplasmic reticulum.(3) Extrusion mechanism and function Before discharge, the extrusomes expand gradually to separate its membrane from internal content forming a lager space, then the membrane fuse with the pellicle and the internal content break through the pellicle and eject out. During ejection, the entire internal content of extrusomes has not obviously morphological change and the extruded structures of extrusomes are cyathiform and remain outside of the cell surface intactly. The process of ejection of extrusomes in Architricha indica may perform stress response or defensive function, which associates with the activity of mitochondria and contributes to the structure of membrane retrieval or renewal.(4) Compared with reported extrusomes The extrusomes of Architricha indica clearly differ from that of previously reported extrusomes in other ciliates. In their morphogenetic processes, the filamentous substances in vesicles cluster gradually from outer to center area. Before discharge, the extrusomes expand gradually to separate its membrane from internal content and then the membrane fuse with the pellicle. After extrusion, the extruded structure remain outside of the cell surface intactly appear not to "eject" but "secrete" gradually. Therefore, the extrusomes in Architricha indica are excretion extrusomes and are a new type of extrusomes without report in ciliates.4ConclusionBy using the microscopic and submicroscopic methods, the present paper observed and explored the morphology and localization, origin and morphogenesis, extrusion mechanism and functions of different extrusomes in three kinds of hypotrichous ciliate. Based on the comparison of difference and similarities of extrusomes between Anteholosticha monilata and Paramecium and Pseudourostyla ciliates, the extrusomes in Anteholosticha monilata are identified as trichocysts-like extrusomes. The mucocyst extrusomes in Oxytricha granulifera have some obvious characteristics distinguishing from that of Tetrahymena and Urostyla grandis. The extrusomes in Architricha indica are excretion extrusomes by ejected process and extruded structure observation. The present paper supplies the morphological data in detail for the investigation on extrusomes in hypotrichous ciliates. Meanwhile, the results also provide basic information for the further understanding the structure, formation, evolution and regulation of protozoa cells.