The Differentiation Of Cell Structure Of A Soil Ciliate Cyrtohymena Australis Under Different Physiological State

Hypotrichous ciliates are undoubtedly the most advanced group among protozoans, and they share a complicated structure of cortical cytoskeleton and cytoplasm in their cells. The study on differentiation of cell structure in this group of ciliates could be regard as a basis for classification, and in addition, is an important part to research cell morphogenesis, formation of pattern and regulation mechanism of eukaryotic cell. Previous work accumulated a lot of results in this field on aquatic hypotrichous, however, the relevant evidence in soil ciliates is still limited. A soil ciliate, Cyrlohymena australis, was collected and inoculated in our lab, and its life cycle show a close relationship to the soil environment, besides the morphogenesis in asexual reproduction under normal condition, Cyrtohymena australis also process other morphogenesis progress like physiological reorganization, formation of cysts and excystment in accordance with the environmental change. On this basis, this paper takes the ciliates for materials, application of differential interference by microscopy, direct fluorescence labeling, immunofluorescence, scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the differentiation process of cell structure in different physiology conditions had been systematically observed and recorded, in order to further reveal the diversity of cell life activities in different environment and the mechanism of formation and cell organelles regulation provide new information. Results are shown as below.1 Cell structure in vegetative stageThe cortical ciliature microtubular organelles of Cyrtohymena australis is composed of ventral cortical ciliatures and dorsal cortical ciliatures, as well as the ciliature base-associated microtubules. Among of these, a collar and a lapel form the adoral zone membranelles (AZM). Paroral membrane (PM) and endoral membrane (EM) constitute the undulating membrane (UM). The AZM and UM base-associated microtubules formed a adoral microtubules skeleton and a microtubule cytoskeletal network. Abdomen cortex is composed of front-ventral-transverse cirri (FVTC), LMC and RMC; dorsal cortex contains dorsal kineties (DK) and caudal cirri (CC), the pattern of FVTC and DK is different from most aquatic species. Developed posterior longitudinal microtubules (PLM) and anterior longitudinal microtubules (ALM) were separately located at the base part of front and transverse cirri, and LMC and RMC were showed to share a diverse develop level and asymmetric transverse microtubules (TM). The photos of immunofluorescence showed that ?-tubulin was mainly located at the region of cortex ciliature, forming a dimer with ?-tubulin and was the major component of ciliature microtubules and other cortical microtubules. Centrin was mainly situated at the basal body. In addition, cortical granules and vesicles with low density of electrons particle, together with the abundant mitochondrion, food vacuoles, could also be observed in cytoplasm. And furthermore, the microtubule bundles or microtubule band produced periostracum layers of different thickness on the surface of the cell.These results revealed that Cyrtohymena australis possess a unique cortical ciliature localization pattern that different from most aquatic ciliate. Locating at the basal body of the cortex cilia, centrin may be has the most important function of the microtubular organized center (MTOC). The developed microtubule bundles or microtubule might has a function of strengthen and supporting. The presence of this distribution could be one of the cell's adaptive characteristics to soil environment.2 The differentiation of ciliatures organelles in different phases of morphogenesis and physiological reorganizationDuring morphogenesis of Cyrtohymena australis, the ciliature organelles in the cortex experience a whole renew process of assembly and disassembly, The AZM of proter is formed after the disassembly of the lapel of old AZM and in corporation with the old collar membrane. Old UM experience a renew process to form a new UM of proter, whereas the primordia of FVTC and LMC and RMC of proter and opisthe would generate from the same site where the old ciliatures located. Three DK primordia rows formed on the left side of cortex, each consisting of anterior and posterior. The third primordium developed into multi-columns, which generates the dorsal anlagens of proter and opisthe by movement and the ciliogenesis.During the stage of physiological reorganization, the cortical ciliature microtubular organelles of Cyrtohymena australis underwent the process of differentiation and redifferentiation respectively. The old lapel membrane of AZM was disintegrated and then renewed at the same area, The AZM primordium were derived between FC and TC, then AZM primordium moved towards the new lapel of AZM. The new lapel of AZM and UM, together with the undifferentiated collar of AZM, to constitute the new adoral cortical cytoskeleton. New UM primordium differentiated from the old one, the similar situation is also happened during the renew process of FVTC and LMC and RMC.To be concluded, during morphogenesis of Cyrtohymena australis, ventral cortical ciliatures underwent a similar program that were happened in most aquatic species, however, our results also showed that the renew process of AZM in Cyrtohymena australis share a basis characteristic for its speciality, and it can be regarded as a new evidence in studying the final destiny of old AZM structure during morphogenesis. Besides that, we also found that the morphogenesis and differentiation of DK only happened on the dorsal cortex that differ from most aquatic species, it is also worth detecting that whether the distinctive DK structure of Cyrtohymena australis is relate to its tactile movement in soil environment. It showed that two sets of ciliatures were formed in the monogenesis of Cyrtohymena australi in comparison to only one set come into being during physiological reorganization, which share the same regulatory mechanism in the differentiation of ciliary structure.3 The differentiation of cell structure in resting cyst formationAt the beginning of resting cyst formation, anterior and posterior of cell twisted and cell body shrunk, the ciliatures in ventral and dorsal cortex were disassemble gradually, base microtubules and base-associated microtubules were engulfed by the around membrane and then dived into the cytoplasm and formed the autophagic food vacuole. The cyst wall, which contained ectocyst, mesocyst, endocyst and granular layer, enclosed the cell as the form of kinetosome-resorbing cyst and had the amorphous protuberance and rhombic-shaped granules and filamentary structures in the space of ectocyst and mesocyst. In the period of cyst formation, cortical granule, glycogen granules, mitochondrion and plenty of autophagic vacuole in different digestive stages were observed. Two macronucleus (Mac) were found to be closed with each other, together with a few point-shaped micronuclei.This is the first report to show the process of ciliary structure absorbed and the detailed autophagy during resting cyst formation of the soil ciliate Cyrtohymena australis. Presumably, the products of autophagy might provide the resource of energy and materials for the formation of cyst that can regard as a main characteristic in the life cycle of soil ciliates. Morover, the non-fusion macronuclei in resting cyst formation in Cyrtohymena australis also display a species-specific characteristic.