Studies On Microtubular Cytoskeleton,Extrusome And Resting Cyst In Hypotrich Ciliate Diaxonella Pseudorubra

Ciliates are the most advanced heterotrophic protist. To adapt to the changeable environment, the surface of ciliates not only evolved to have complicated microtubules structure, but also produced complex the under-pellicle fiber system and other various organdies structure. The microtubules are the main components of the cell cortex, including the ciliature microtubules and the non-ciliature microtubules. To date, the related researches mainly focused on the cortical microtubular cytoskeletons of lower ciliates, such as Paramecium, Tetrahymena. However, the data are very limited about microtubular cytoskeletons of the hypotrich ciliates. Study on the assembling mechanism of the microtubular ciliatures will supply basic information about the structure and function of ciliatures, and meanwhile will help understanding the elucidation of heredity and regulation of ciliatures in ciliates.The cortical organelles in ciliates have some differentiation and specialization Mucocysts which are distributed widely in the cortex of Diaxonella pseudorubra. Mucocysts are membrane-limited organelles under the cortex. When cells are stimulated, the substance of mucus will be ejected out of the mucocyst. The mucocysts play an important role in the life activity of ciliates. To date, the related researches are limited within species Urostyla grandis. On the contrary, the data is very rare about the mucocysts in hypotrich ciliates. The study on morphological characteristics, morphogenesis, and the location of mucocysts in hypotrich ciliates can supply the new data for further elucidation of the diversity and heredity of cell organelles, and the relationship between function of organelles and cellular activity. Cyst formation is a common phenomenon in nypotncn cmaies. when me environment changes and has bad impact on the life activities of hypotrich ciliates, the nliates will gradually stop movement and feeding, and shrink its cell, differentiate and secrete substances to form cyst. To date, besides researches on Euplotidae, no (?)ports about the other cyst formation process and the differentiation of the cortex surface structure have been present. In this study structure of ciliature microtubular prganelles, cyst wall and the type of cyst in Diaxonella pseudorubra were observed by using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results of this study will help further revealing the structural differentiation of the Urostylina ciliate in changeable environments and extreme conditions.In the aim to provide basic information for the researches about the formation and regulation of cellular structures in ciliates, the ciliature microtubule organelles, the mucocyst and the resting cysts in Diaxonella pseudorubra were clearly illustrated using FLUTAX direct-fluorescent labeling, SEM and TEM. Results are as follows:1The morphology and morphogenesis of microtubular organellesThe microtubular organelles of hypotrich ciliate, Diaxonella pseudorubra, were visualized by using FLUTAX (fluorescence taxoid) labeling. The results showed that these organelles consist of adoral zone of membranelles (AZM), frontal-ventral-transverse cirri (FVTC) and the base associated microtubules of these ciliatures. The microtubular cytoskeleton of AZM comprises membranelle brackets and its associated microtubules. There are four types of microtubules at the base of FVTC:anterior longitudinal microtubules (ALM), posterior longitudinal microtubules (PLM), transverse microtubules (TM) and radiating microtubules (RM). These different types of microtubules differ from each other in developmental degree. The base associated microtubules of marginal cirri (MC) contain ALM and PLM. All of the old AZM was renewed during the process of morphogenesis. AZM of the proter was formed at the site where the inner of the old AZM. At the end of the differentiation of the cell, some part of old FC, TC and MC remain unchanged for a long time. Meanwhile, our results also indicate that the microtubular organization of Diaxonella pseudorubra is quite different from other ciliates. Moreover, during the period of differentiation and maturation of the new structures, the collapse of old cirri may contribute to the new structure’s orienting and constituting, while the function of old cirri is losing gradually.2The ultrustructure of ciliature microtubular organelles and cytoplasm organelles in Diaxonella pseudorubraThe cortical pellicle, cytoplasm beneath pellicle, cortical microtubular organelles and other organelles in hypotrich ciliates Diaxonella pseudorubra were observed by using TEM. The cortical pellicle has three membranes. The trophic cell of Diaxonella pseudorubra is smoothly rippled in surface, with small depressions in the places where cilia emerge surrounded by protrusions. The cilia is made of ciliary shafts, basal plate, matrix, matrix of base proximal electron dense things, matrix of base nearly connected and matrix of central link structure; cytoplasm beneath pellicle is formed of a tube sample structure or fiber appearance structure and in close to the cytoplasmic side which is separated by membranous structure. Different organelles, such as mitochondria, the endoplasmic reticulum, mucocyst, have different morphology in cytoplasm. In addition, Diaxonella pseudorubra contains more than one bubble macronuclears. During morphogenesis, each of the macronuclears appears a replication band, from one end beginning to copy, after copying, the macronuclears fusion does not take place. However, those feathers of Diaxonella pseudorubra were quite different from Euplotes eurystomus. In addition, there is only one macronuclears and the replication band is fusion at the middle line in Euplotes eurystomus. The reasult reveals that compared with others ciliates, Diaxonella pseudorubra show the evolution of the low level. The aim of the present study is to further elucidate the diversity of the ciliature microtubular organelles, and also provide the new data for classification in hypotrich ciliates.3The ultrustructure of mucocystThe mucocyst of Diaxonella pseudorubra was observed by using TEM. The mucocyst is ellipsoid-like extrusome surrounded with an smooth plasma membrane and containing crystal substance. The extrusome was located in the cytoplasm of vegetable cells. Three parts can be recognized in the mucocyst according to the electron density of the crystal substance within mucocyst:head, body and tail. The observation on mucocysts displaying different developmental phase suggests that: mature mucocyst muld move towards pellicle and fuse its head part plasma membrane with the pellicle; the fusion could result in an opening of mucocyst on the pellicle; the enclusion of mucocyst was extruded out of the organelle; finally the remaining empty mucocyst could become part of the pellicle and no longer be observed. The results indicate that:the endoplasrnic reticulum might bear some relations with the occurring and development of mucocysts; the mucocysts in Diaxonella pseudorubra is different from Urostyla grandis in their location and morphology; the mucocysts could have some contribution to the pellicle renewal and resting cyst formation of the ciliate.4The differentiation of microtubular and cytoplasm organelles during the process of resting cyst formationThe changes of the Diaxonella pseudorubra morphology and the structure of cortex microtubular and Cytoplasmic organelles during encystment was observed by using SEM and TEM. The results indicate that:during encystment, the cells transforming from the long oval shape into ball form, most of the cortex microtubular is disappeared, forming the "Urostyla-type cysts"; The cyst of Diaxonella pseudorubra contained large amounts of mitochondria and autophagic vacuoles, which might be related to food digestion and energy storage. The chromatin within the macronuclei (MAC) is condensed, a large proportion being attached to the nuclear membrane. Inside the cytoplasm, there exist.clearly visible mitochondria and autophagic vacuoles. Containing fully or partially structured endoplasmic reticulum inside, these autophagic vacuoles present certain morphological traits of food vacuoles. Thus, it could be concluded that autophagic vacuoles might be food vacuoles in their special form, providing nuturients and engery that the restin cyst repaire. The results provide base datum for elucidating how Urostylina ciliate could adapt to their environments under the extreme conditions.