Studies On Cell Structure And Polymorphic Macronuclear DNA In Territricha Stramenticola (Ciliophora: Hypotrichida) Under Different Physiological Status

Soil protozoan as a large assemblage of protozoa plays an important role in the soil ecosystem. There are similarities and difference between soil protozoa and aquatic protozoa. Especially, soil ciliates, their unique living conditions, cell structures and physiological characters could adapt to the soil life. Encystment and excystment are one part of the soil protozoa life history, which takes an important effect on its survival. At present the scientists in many fields focus on ciliates lived in water, but there are no more studies on soil ciliates. Most studies on the ciliates remained in the ecological functions, community characteristics and species distribution, etc., only a few small ciliates were observed on physical characteristics and its internal structure. By means of studying cortical structure and mode of cytoplasm, organelles and nuclear ultrastructure, etc. we want to know how they adapt to the soil environment and understand the relationgship between soil ciliates and freshwater ciliates.In our experiments, the occurrence of resting cysts was found.This kind of resting cysts had special cyst wall, which could adapt the dry soil environment. This is to further reveal the cell structure of genetic changes of soil ciliates, the establishment of cell model, species classification,and we can have more understandings of their place in the system.Studying soil ciliates and the status of the process of evolution has important biological significance. In this study, we used Territricha stramenticola as the experimental material of soil ciliates, applying fluorescence labeling method(FLUTAX), anti-a tubulin antibody fluorescence labeling, scanning electron microscopy(SEM),transmission electron microscopy(TEM) as well as the RAPD technology, not only in the microscopic level and sub-microscopic level, to observe and compare the structure and ultrastructure of the external morphology of the normal cells and resting cysts, the ciliate cortex, the cytoplasm and its various organelle of Territricha stramenticola, but also, from gene level, the macronuclear DNAs of the normal cell and resting cyst of Territricha stramenticola were compared and studied.The results are as follows:The microtubular organelles in the cortical ciliature of the soil ciliate T. stramenticola were visualized by direct fluorescent labeling of FLUTAX and immuno-fluorescent labeling using anti-a tubulin antibody. The distribution pattern of base-associated microtubules were found to be in accordance with that of adoral zone membranelles (AZM), undulating membranes(UM), frontal-ventral-transverse cirri (FVTC), left-right marginal cirri (L-RMC) and dorsal kineties (DK). The cytoskeleton of AZM and UM was composed of membranelle brackets, membranelle-associated microtubules and the skeleton of UM. The anterior longitudinal microtubules (ALM), posterior longitudinal microtubules (PLM) and transverse microtubules (TM) were located in the basal part of FVTC. ALM, PLM, TM and sprouted microtubules (SM) were situated in the basal parts of LMC and RMC. ALM and PLM were visualized in the basal part of DK. The base of transverse cirri (TC) contained comparatively developed bundles of TM. The occurence of SM and the location of TM in the basal parts of L-RMC might be of species specificity. The microtubules and a layer of microtubule bellow cell's cortex could be used to combine the microtubular cytoskeleton network with cell's various ciliatures used for movement. The presence of these microtubular structures might be one of the cell's adaptive characteristics to soil environment.The surface ultrastructure of the hypotrichida ciliate Territricha stramenticola is observed by using scanning electron microscopy. The results show that the structural mode of the frontal cirri of Territricha stramenticola was similar to that of Oxytrichids. The midventral cirrus line, consisting of 8 zigzag-distributed ventral cirri, was located in the middle of the ventral surface. During the morphogenesis of ciliature, the old cilia experienced the process of gradual degeneration when the new structure began to occure and develope. A study of resting cysts in Territricha stramenticola has been made by using scanning electron microscopy. It is revealed that the cyst wall of Territricha stramenticola is composed of two layers named from outside to inside:ectoeyst(EC) and endoeyst(EN). The EC is composed of many bamboo-like transparent square fibrous structures.They are thin and regular. But the EN is thick and transparent. The results indicate that the old cilia plays an important role not only in the occurrence of the ciliature primordia, but also in the structural development of new cilia. There are a lot of spine-like structures at the terminal part of frontal surface region where no cilium is present. At the remaining non-ciliated surface region of Territricha stramenticola, there distributed a large number of unevenly arranged shallow pits. These morphological characteristics might be the adaptive change to the interaction between the ciliate and the soil environment. The formation of the resting cells is also a result to adapt to the soil environment.The internal ultrastructure of Territricha stramenticola was observed by using transmission electron microscopy.We found that its surface membrane was composed of the plasma membrane and the cell membrane. We also observed that there was dense fiber bundles close to ventral surface membrane in the Territricha stramenticola.Its lips, ventral cirri and mouth cortex surface membrane gathered under the formation of thick fiber bundle or fiber tape.These fiber bundle structures allowed Territricha stramenticola's surface membrane to be reinforced caterpillar to adapt its crawling life in the soil.The cyst of Territricha stramenticola contained large amounts of mitochondria cyst caterpillars and food bubbles, which might be related to food digestion and energy storage. Largest nuclear elongated cylindrical, nuclear membrane was not smooth, showing wavy. Chromatin granules scattered in large nuclei, arranged rather sloppy, and some family reunion into a high electron density of the sizes of the clumps, nucleolus high number of scattered large nucleus; membrane was divided into two layers, inner nuclear membrane and outer nuclear membrane. Inner nuclear membrane was thicker, but outer nuclear membrane was very thin.The nuclear pore-like structure made the nuclear membrane into the dotted line. After the formation of cysts, cyst wall became two layers.This kind of structure had not been found in other ciliates. Membrane consists of two layers of its composition. During hollow area, of which there were many small membrane separation,was their uniform square chamber.They arranged in neat rows like a square with a circle circuit in cells peripheral.Cysts have two autophagic vacuoles:organelle autophagic vacuoles, and cytoplasmic autophagic vacuoles.The macronuclear DNA from normal cells and resting cysts of Territricha stramenticola was comparatively analyzed by using RAPD technology in the paper. The results showed that no fragments produced using 2 random primers,134 fragments were amplified from macronuclear DNAs of the normal cells and the resting cysts using 30 random primers, of which 54 specific fragments belonged to normal cell and 80 fragments were specific to the resting cyst. There were 20 fragments appearring in both the normal cells and resting cysts. The similarity value was 30%. The data indicated that the structures of the macronuclear DNAs in Territricha stramenticola perhaps had major changes in the process of cyst formation, which were closely related to the change in structural features and metabolic activities and to the physiological and biochemical changes in the resting state.