Observations by Scanning Electron Microscopy of Surface Events During Encystment and Excystment in Gastrostyla Steinii (Cliophora, Hypotrichida)

Observations by Scanning Electron Microscopy of Surface Events during Encystment and Excystment in Gastrostyla steinii (Cliophora, Hypotrichida)

FATIMA KNAIPPE, LAURENCE TETLEY and MARGARET MULLIN

Department of Zoology, University of Glasgow, Glasgow G12 8QQ, SCOTLAND

Received 11 November 1991/ Accepted 21 January 1992

Key words: Ciliate/ excystation, encystation, morphogenesis

ABSTRACT The surface of the protozoan Gastrostyla steinii undergoes profound morphological changes during the encystment and excystment processes. Resorption of the various ciliary structures occurs progressively during differentiation of the free living cell into the resting cyst. In ciliar disassembly, layers of the cyst wall are deposited on the surface of the encysting organism. At excystation, the hatching trophozoite emerges from the protective wall through a cleft after the formation of the ciliary organelles.

INTRODUCTION The cryptobiosis state in nature accounts for the wide distribution of organisms capable of “dedifferentiating” themselves into a cyst form throughout a great variety of environments. Encystment and excystment are essential to the biological cycle of several pathogenic and free living protozoa. Despite the importance of the cyst form in biocenosis, insufficient studies have been undertaken on the cellular and molecular mechanisms which regulate cell differentiation among encysting organisms. Pioneering studies carried out by Weyer (1930) on the free-living ciliate Gastrostyla steinii demonstrated that major morphological changes occur in the cytoplasm and nuclei during the two stages of the ciliate life cycle. More recently, transmission electron microscopy data (Walker et al., 1980) on the events occuring during cyst wall formation as well as on the morphogenesis of the cytoplasmic organelles and nuclei, has amplified the current knowledge on those mechanisms in G. steinii. However, none of these studies has focused on the precise description of surface morphogenesis during the protozoan differentiation. The present work attempts to analyze by scanning electron microscopy the surface events that take place during encystation and excystation in G. steinii.

MATERIALS AND METHODS Gastrostyla steinii was isolated as cysts from the intestine of Rhinocricus padbergii (Diplopoda). The intestine contents were placed in petri dishes containing mineral water. Newly formed trophozoites were

68 MORPHOGENESIS IN GASTROSTYLA STEINII kept, without strict sterility conditions, in mineral water plus lettuce powder as the nutritive medium, and then used when the logarithmic phase of the cell growth was attained. Resting cysts were collected from aging cultures after 5 or 6 days of in vitro trophozoite maintenance. To obtain encysting organisms, a syracuse dish containing a drop of distilled water with active swimming trophozoites was placed on a hot plate and the temperature then gradually increased to 40OC. Samples were taken every 20 minutes and the specimens fixed for one hour at room temperature with 2.5% (v/v) glutaraldehyde in 0.1M phosphate buffer pH 7.4. They were then rinsed twice in the same buffer plus 2% (w/v) sucrose, post-fixed for another one hour with 1% (w/v) osmium tetroxide in 0.1M phosphate buffer and washed with distilled water. The specimens were transferred into a polycarbonate filter apparatus then treated with 0.5% (w/v) aqueous uranyl acetate for 30 minutes, rinsed with distilled water, dehydrated in acetone and critical point dried. To trigger excystation, resting cysts were placed in mineral water containing a trace of peptone and left for 4 hours at room temperature. Fixation in glutaraldehyde and osmium was followed by uranyl acetate treatment as described above. After extensive washing, rapid freezing was accomplished by plunging specimens in distilled water on 10mm diameter glass coverslips into liquid propane at –180OC. Frozen samples were then freezedried at –75OC under vacuum (10-6 torr) for 12 hours and slowly brought to room temperature over 5 hours. Critical point and freeze dried specimens were mounted on stubs with double-sided tape and cool sputter-coated with a 50nm layer of gold/palladium prior to viewing on a modified Philips SEM 500 operating at 3 or 6 kilovolts.

RESULTS AND DISCUSSION Trophozoites of G. steinii are elongated cells (110µm x 40µm) with the ciliary organelles grouped in distinct arrays. When the cells were incubated at 40OC, the first signs of “dedifferentiation” could be observed after only 20 minutes. At the beginning of encystment, the ciliate shape changed from ellipsoid to round by shortening, although some ciliary structures could still be seen (Fig. 1). The surface of encysting organisms became wrinkled (Fig. 2) and globs varying in size appeared (Figs 1, 3, 5). The adoral zone of membranelles and cirri belonging to the fronto-ventral-transverse group as well as part of the marginal ciliature were still visible in different stages of resorption during encystation (Fig. 3). Distinction between dorsal and ventral surfaces became blurred as resorption of each of the ciliary structures proceeds (Figs 4, 5, 6). Next, the encysted ciliate appeared as a round resting cyst, in which the typical ciliary structures of the free form were absent (Figs 7, 8). Encystment in G. steinii has been showed to slow down at low temperatures while 60% (v/v) D20 speeds up the process in the same temperature range (Walker et al., 1980) but not at room temperature; however, these authors did not test the effect of temperature alone on encystment. Using temperatures above 37OC we succeeded in showing an increase in the speed of the cell “dedifferentiation”, which led to the completion of the typical rounded resting cyst after 3 hours at 40OC. In our hands, encysted organisms were approximately one-third the volume of the trophic form, ranging from 40 to 50µm in diameter. This finding is not in agreement with the dimensions found in another G. steinii strain (Walker et al., 1980), in which the average size for the resting cyst was 27 to 29µm. The disassembly of the microtubular system and/or its primordia, the mitochondrial aggregation together with lipid and carbohydrate storage characterize encystment in Oxytricha bifaria (Verni et al., 1984; Weir, 1945). Oxytricha fallax (Hashimoto, 1962), Stylonychia mytilus (Walker et al., 1975),

69 MORPHOGENESIS IN GASTROSTYLA STEINII

Pleurotricha sp. (Matsusaka, 1976) and Gastrosytla steinii (Walker et al., 1980). Synthesis de novo and/or repolymerization of the microtubular primordia should occur in an early stage of the “redifferentiation” process, since the trophozoite ciliature is completely reformed when the organism emerges from the resting cyst. Based upon the capability of resorption of the microtubular system and its primordia, Gastrostyla steinii is placed in the kineto-resorbing group (Walker et al., 1980), in contrast with Diophrys scutum which is known to belong to the non-kinetosome-resorbing group (Walker and Maugel, 1980). In G. steinii, excystation consists mainly in the previous formation of the somatic-oral infraciliature and ciliature while macronuclear fission and cytoplasmic reorganization take place. These stages seem to be coupled with the rehydration of the cyst, which becomes evident by the resumption of contractile vacuolar activity and the swelling of the cytoplasm, which will cause dispersion of the mitochondria and rupture of the cyst wall. Experiments designed for the triggering of excystation revealed sequential stages during the emergence of G. steinii. Firstly, a cleft appeared in the cyst wall (Fig. 9), probably due to inner pressure created by the increasing volume of the excysting organism. No visible structure on the surface of the resting cyst was found to indicate where the cleft would occur. As the cleft continued to enlarge, the emergent trophozoite (Fig. 13) gradually escaped from the outer cyst wall leaving behind the empty shell (Figs 10-14). At this stage all the newly formed ciliary structures could already be seen. Strong movements of the cirri may contribute to the force necessary to liberate the trophozoite from the innermost cyst layer. The escape of the organism ready to emerge from the cyst wall can happen in several interesting ways (Corliss and Esser, 1974). In the case of some ciliates, the emergence takes place through a characteristic pore that possesses a sort of “removable plug”. In other cases, the “cyst membranes” are simply destroyed, presumably due to the water reabsorption. Furthermore, in other examples, enzymatic digestion of the cyst wall is one of the final stages of the excystment process. In many cases, a combination of both processes with other factors are certainly involved. Our work indicates that the newly formed G. steinii cell reaches the external environment through a cleft in the cyst wall, which allows it to escape from the protective layers. Nevertheless, we cannot say whether the appearance of the cleft and the subsequent disruption of the cyst wall are caused solely by the rehydration of the cytoplasmic volume or whether enzymatic digestion plays a part. The size of the excysted ciliate ranges form 110 to 150µm and it already possesses fully formed ciliary organelles. Frequently, globular structures were visualized on the surface of the encysting organisms. Whether those structures are related to the mechanisms of transportation of the cyst wall components to the surface of the cell is still to be verified. Further comparative studies of different protozoa at the ultrastructural level will certainly be necessary to address the questions surrounding the surface events that occur during “dedifferentiation and redifferentiation” of many unicellular organisms.

FIGURES Fig.l: Dorsal view of an encysting organism at the beginning of the “dedifferentiation” process. Dorsal (arrowhead) and ventral (arrow) ciliary structures can still be seen as well as globular formations (*) on the cell surface. 600x Fig.2: Detail of morphogenesis at the anterior region of an encysting cell, when the adoral zone of membranelies (AZM) as well as frontal cirri (arrow) are in an advanced stage of resorption. The irregular surface of the “dedifferentiating” call can also be observed (arrowhead). 1800x Fig.3: The encysting organism changes its shape from an ellipsoid to a rounded form, although the majority of the ventral (arrowhead) and marginal (arrow) ciliary organelles can still be observed. Globular

70 MORPHOGENESIS IN GASTROSTYLA STEINII formations (*) are prominent on the trophozoite dorsal surface at this stage. 600x Fig.4: Image taken from an angle appropriate for visualizing the remnants of the dorsal ciliature (arrowhead) as well as the adoral zone of membranelles (AZM) and part of the oral membrane (arrow), originally belonging to the trophozoite ventral surface. 1000x

71 MORPHOGENESIS IN GASTROSTYLA STEINII

Fig.5: Rounded encysting organism showing remnants of the membranelles (AZM) and of the marginal ciliature (arrow). Globular formations can be easily seen (*) in this image. 800x Fig.6: A group of encysting cells in which the typical ciliary structures of the trophozoite are in different stages of resorption (arrowhead). 600x Fig.7: View of the process of “dedifferentiation” after completion with the cyst wall already formed. The double arrow points to the fibrilar material, constitutive of the ectocyst layer. This micrograph shows the loss of cytoplasmic material (arrow) and other structures (*) while sealing of the cyst wall occurs. 800x

72 MORPHOGENESIS IN GASTROSTYLA STEINII

Fig.8: General aspect of a resting cyst showing its characteristic irregular surface resulting from the wrinkle of the outermost cyst layer (ectocyst). 1000x Fig.9: Detailed image of an early stage of the excystation process when a cleft on the cyst wall occurs. The emergent (*) trophozoite can be identified inside the cyst. 2000x Figs 10-12: Different stages of the trophozoite (*) exit from the protective covering. 1000x, 1200x and 700x respectively. Fig.13: Side view of the same image shown in Figure 12 showing the enlargement on the cyst wall due to the trophozoite (*) emergence. 1000x Fig.14: An empty cyst left behind after the escape of the free swimming cell. 1200x

ACKNOWLEDGEMENTS To Professor Keith Vickerman (Department of Zoology, Glasgow University, Scotland) for providing all the laboratory facilities necessary to achieve this study; Dr Maurilio Jose Soares (Institute Oswaldo Cruz, Rio de Janeiro, Brazil) for his valuable suggestions and encouragement throughout this work.

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