Ultrastructural Observations on the Resting Cyst of the Soil Ciliate, Territricha Stramenticola and the Freshwater Ciliate, Urostyla Grandis

Full Length Research Paper

Ultrastructural observations on the resting cyst of the soil ciliate, Territricha stramenticola and the freshwater ciliate, Urostyla grandis

Mixia Yun1, Qili Li1, Xiaocui Zhang1, Chun Sheng2 and Fukang Gu1*

1School of Life Science, East China Normal University, Shanghai 200062, China. 2College of Life and Environment Science, Shanghai Normal University, Shanghai 200234, China.

Accepted 21 February, 2013

The ultrastructure of non-dividing cells and resting cysts of soil ciliate Territricha stramenticola and freshwater ciliate Urostyla grandis were observed using scanning and transmission electron microscopy. The results show that the resting cell of T. stramenticola shared many similarities with that of U. grandis. Inside the cytoplasm of both resting cysts, there existed organelles such as mitochondria and autophagic vacuoles. The resting cyst of the freshwater ciliate U. grandis is spherical or ellipsoid in shape, but its cyst wall is composed of three layers: the ectocyst, the endocyst and the granular layer. However, the cyst wall of T. stramenticola was quite different from U. grandis. The cyst wall of T. stramenticola was composed of the ectocyst and the endocyst; the former was made up of small regular rectangular units tightly packed together like that of beehive, while the latter was an irregular layer of varied thickness between the ectocyst and the pellicle. Such cyst wall, quite unlike those of other freshwater ciliates, might help the cyst keep the moisture inside, thus playing an important role in tiding the resting cells over the unfavorably dryer conditions and in maintaining its existence under extreme circumstances. Those suggested that T. stramenticola would take form of another kind of kinetosome-resorbing cyst. The results of this study would help reveal how soil ciliate T. stramenticola adapt to their environment and structural differences under certain conditions.

Key words: Territricha stramenticola, Urostyla grandis, cyst wall, resting cyst, ultrastructure.

INTRODUCTION

Ciliates are probably the most popular group of protists, of freshwater ciliate such as Euplotidae, Urostylidae, often preferred as lab research objects (Lara and Acosta- Oxytrichae and Oligotrichous have been studied by Mercado, 2011). One of the common features of many researchers (Foissner et al., 1987; Foissner et al., 2005a; ciliates is their tendency to encyst under harsh Foissner et al., 2005b; Foissner et al., 2006; Foissner environmental changes that affect their biological activity, and Pichle, 2006; Foissner et al., 2007; Rios et al., 1985; such as variations in temperature or pH or when food Verni and Rosari, 2011). By contrast, there have been far deficiency arises (Gu and Zhang, 1992; Foissner et al., fewer studies of encystment of soil ciliate. Territricha 2005a; Foissner et al., 2006). Previous studies on the stramenticola belonging to the order Hypotrichida, is a cysts of ciliates have shed light on the biological soil ciliate. After the report on its morphology (Foissner, structure, function and internal regulatory mechanism of 1988) and its taxonomic study (Berger, 1999; Shi, 1999), cell (Foissner et al., 2005b; Foissner and Pichle, 2006; some researchers then undertook detailed research on Foissner, 2009; Foissner et al., 2009). The resting cysts its cortical ciliature, ciliature microtubular and the base- associated microtubules (Shi et al., 2009; Feng et al., 2009), thus providing some valuable reference for further study. *Corresponding author. E-mail: [email protected]. Tel: +86 In the present study, we investigate the ultrastructure of (0) 21 6223 2715. Fax: +86 (0) 216223 3754. the resting cyst of Territricha strammenticola and that of 854 Afr. J. Microbiol. Res.

the freshwater ciliate, Urostyla grandis, using scanning ellipsoid, ranging 150 to 170 μm in length and 60 to 70 electron microscopy (SEM) and transmission electron μm in width, with a flattened ventral side and a slightly microscopy (TEM). It is shown that the resting cyst of T. domed dorsal side. The principal microtubular organelles stramenticola is structurally different from its freshwater of its cortical ciliature include the adoral zone of counterpart, which might reflect the adaptation and membranelles (AZM), undulating membranes (UM), differentiation of the soil ciliate under certain fronto-ventral-transverse cirri (FVTC), left-right marginal environmental conditions. The results provide base cirri (L-RMC), and dorsal kineties (DK), etc. It is clearly datum for elucidating how soil ciliate T. stramenticola shown that the FVTC are arranged in the 8-9-7 could adapt to their environments. corticotype (Figure 1a). The resting cyst is spherical or ellipsoid in shape, varying from 70 to 80 μm in diameter. It is clear that the MATERIALS AND METHODS cortical ciliature has undergone dedifferentiation during the process of cyst formation. At the same time, the AZM Collection, cultivation and encystment and ventral-cortical cytoplasm contract and sink inward,

T. stramenticola was collected from the soil of a pond of Qinglong accompanied with the disappearance of most of the cilia Lake in Wenzhou, Zhejiang Province in June 2007. This soil is from around the area of the AZM and elsewhere. The ciliate is the bottom of the pond and permanently saturated with water. U. now spheric with a wrinkled surface (Figure 1b). Later, grandis was collected from a pond at Zhang’s Mountain Park in the ciliate turns ellipsoid in shape, looking like a dried Bengbu, Anhui Province in August 2010. The water sample was persimmon (Figure 1c). The surface of the newly formed taken directly from the site. In each case, a single ciliate cell was isolated and cultured at 20°C in filtered pond water with a wheat cyst looks like that of the skin of a snake (Figures 1d and grain added to promote the growth of bacterial food organisms. e). The cyst wall is composed of two uniquely structured After the cultured cells reached a relatively high density on the layers, viz. the endocyst and the ectocyst. The endocyst bottom of the cultivation dish (with a diameter of 6 cm), the appears to be an irregular layer of varied thickness exchange of culture solution (filtered pond water) then was stopped between ectocyst and pellicle. On the surface of the to make it “old”; at the same time, the feed was also cut short to pellicle, there are numerous small protrusions (Figure 1f). induce the cells to form cysts on the bottom of the dish. A cross-section of the cyst wall shows that the ectocyst is made up of two thin layers with minute rectangular units SEM and TEM aligned between them (Figure 1g).

Samples for SEM observation in this study were prepared according to Gu and Ni (1993) with some modification. Ciliates at different phases of growth were collected around 8:00 AM and TEM observations washed with filtered pond water and then fixed using a solution of 1% OsO4 (one part) and saturated solution of HgCl3 (5.5 parts) for 5 Under the transmission electron microscope, the AZM of to 7 min. The fixed ciliates were washed three times with 0.1 mol/L T. stramenticola is shown to comprise many phosphate buffer solution (PBS), 5 min each, and dehydrated in a membranelles each composed of three tufts of cilia graded ethanol series. Then the samples were transferred into isopentyl acetate, dried by the critical-point technique, transferred to (Figure 2a). The kinetosomes are connected with each a stub that had been coated with conductive glue, and coated with other and encircled by fibrillar basket (Figure 2b). The gold-palladium. The gold-coated samples were observed and mitochondria are ellipsoid in shape, with their outer photographed with a JMS-5610LV SEM. membrane being smooth and their inner membrane Samples for TEM observation were treated according to the convoluted to form cristae filled with substance of lower procedure reported by Gu et al. (2002) with minor modification. electron density (Figure 2c). Inside the cortical Ciliates were fixed at 4°C in a mixed solution containing 2% OsO4 and 2.5% glutaraldehyde in proportion of 1:1 for 5 min. After being cytoplasmare, there are numerous food vacuoles (Figure washed by 0.1 mol/L PBS, they were then post-fixed in a solution 2d). The macronucleus is cylindrical in shape and has a containing 1% OsO4 for 45 min. Cells were then washed in 0.1 number of nucleoli, evenly distributed chromatin and mol/L PBS, dehydrated, embedded with Epon 812, ultra-thin several conglomerates of higher electron density (Figure sectioned, stained with uranyl acetate and lead citrate. They were 2e). observed and photographed with a JEM100CX TEM. The ectocyst of the resting cyst is made up of closely spaced rectangular units, whereas the endocyst is an irregular layer of varied thickness covering the RESULTS cytoplasmic pellicle (Figure 2f). Some mitochondria, with their tubular cristae gone, becomes structurally loose Structural differentiation of T. stramenticola during (Figure 2g). After encystment, many of the ciliary encystment organelles of the non-dividing cells degenerated and the original microtubular structures disappear. Meanwhile, SEM observations the cytoplasm shrinks with its inner structure becoming denser. The spherical cytoplasmic autophagic vacuoles Non-dividing cells of T. stramenticola are elongate- are retained nearly filling the resting cyst. The endoplasmic Yun et al. 855

Figure 1. Scanning electron micrographs of non-dividing cells and resting cysts of Territricha stramenticola. a. Ventral view of a non-dividing ciliate (AZM, adoral zone of membranelles; UM, undulating membranes; FC, frontal cirri; VC, ventral cirri; TC, transverse cirri; LMC, left marginal cirri RMC, right marginal cirri) (Bar=10 μm). b. An encysting cell. The ciliate is a sphere with wrinkled surface. Arrows indicate the ventral of the resting cyst (Bar=10 μm). c. The resting cyst of the ciliate becomes ellipsoid in shape (Bar=10 μm). d to e. The surface of the newly formed cyst looks like snakeskin. Note the accessory secretions (arrows) (Bar=10 μm). f. Fractured cyst wall show the endocyst and ectocyst. Note the endocyst (arrow) between the ectocyst and the pellicle and the small protrusions on the pellicle (arrowheads) (Bar=5 μm); g. A cross-section of the cyst wall shows that the ectocyst is made up of two thin layers, with minute rectangular units aligned between them (arrows) (Bar=10 μm).

reticulum is dispersed within the autophagic vacuoles and and its width from 40 to 60 mm. The adoral zone of shows signs of being digested (Figure 2h). The membranelles (AZM) is situated at its left front side, the macronucleus is spheroid in shape, and the membrane frontal cirri (FC) are arranged in lines in the anterior between the nucleolus and chromatin disappears. The region of the cell. In addition, there are two rows of chromatin forms granules that are evenly distributed ventral cirri (VC), seven transverse cirri (TC) and four within the macronucleus. Generally, there is only one marginal cirral rows (MC) on both the left and right side nucleolus that occupies nearly half of the internal space (Figure 3a). of the macronucleus (Figure 2i). The size of the resting cyst, around 50 to 60 μm in length, is much smaller than its non-dividing cell. The cyst is spherical at first, having shed most of its ciliary The structural differentiation of Urostyla grandis structures (Figure 3b), then becoming smooth and during encystment ellipsoid in shape (Figure 3c).

SEM observations TEM observations Non-dividing cell of U. grandis is shaped like a spindle,with its body length varying from 150 to 180 mm Non-dividing cell of U. grandis is smoothly rippled in 856 Afr. J. Microbiol. Res.

Figure 2. Transmission electron micrographs of non-dividing cells and resting cysts of Territricha stramenticola. a. The AZM is shown to be made up of many membranelles arranged in order, with each membranelle composed of three tufts of cilia (arrows) (Bar=1 μm). b. The connection between kinetosomes (arrowheads); the kinetsomes taking its roots deep into the cortex beneath the pellicle, is encircled by the surrounding fibrillar baskets (arrows) (Bar=1 μm). c. The ellipsoid mitochondria (MITs). Note the smooth outer membrane and the inner membrane convoluted to form cristae filled with a substance of lower electron density (Bar=1μm). d. Food vacuoles (arrows) at different digestive stages (Bar=1 μm). e. The macronucleus with nucleoli (arrowheads) and evenly distributed chromatin and conglomerates of higher electron density (Bar=1 μm). f. The ectocyst (EC) is made up of closely aligned rectangular units with some space in between (arrows) and the endocyst (EN) covering the cytoplasmic pellicle (Bar=1 μm). g. Some mitochondria, with their tubular cristae gone, becomes loose in structure (Bar=1μm). h. The spherical autophagic vacuoles (AVs), either grouped together or separate, almost completely fill the resting cyst. The endoplasmic reticulum, dispersed within the autophagic vacuole show signs of being digested (Bar=1 μm). i. The chromatin (CH) granules evenly distributed within the macronucleus. A single nucleolus (NU) occupies nearly half the internal space of the macronucleus (Bar=1 μm).

surface, with small depressions in the places where cilia present are several macronulcei, columniform in shape, emerge surrounded by protrusions (Figure 3d). Within the the chromatin within which are loosely distributed. Two or cytoplasm just below the pellicle, in addition to the nuclei, three spherical nucleoli are also present (Figure 3f). mitochondria, endoplasmic reticulum, cytoplasmic bodies The cyst wall of U. grandis is composed of three and vesicles, there are also numerous mucocysts having distinct layers: (1) the ectocyst, which is smooth and higher electron density. Dispersed around the relatively thinner with low electron density; (2) the granule cytoplasmic, some mitochondria are ellipsoid in shape, layer, which is comparatively thicker but has low electron others tubular, with their inner membrane protruding density; (3) the endocyst is an irregular layer between the inward to form the mitochondrial crista (Figure 3e). Also ectocyst and pellicle with varied thickness that shows no Yun et al. 857

Figure 3. Scanning and transmission electron micrographs of non-dividing cells and resting cysts of U. grandis . a: Ventral view of a non-dividing cell (AZM- adoral zone of membranelles; UM- undulating membranes; FC- frontal cirri; VC- ventral cirri; TC- transverse cirri; LMC- left marginal cirri; RMC- right marginal cirri) (bar = 10 μm). b: An encysting ciliate; the cyst is spherical at first, some remnant cilia can be seen in the cyst wall (arrowheads) (bar = 10 μm). c: The surface of the cyst becomes smooth (bar = 10 μm). d: The surface of the non-dividing cell is smoothly rippled with small depressions surrounded by protrusions where the cilia emerge (bar = 1 μm). e: Some mitochondria (MITs) are ellipsoid in shape. Peripheral region of cell shows the mitochondria and mucocysts (arrows) (bar = 1 μm). f: Macronuclei containing loosely distributed chromatin (CH), two or three spherical nucleoli (NU) are present (bar = 1 μm). g: The cyst wall is composed of three distinct layers: the ectocyst (EC), endocyst (EN) and granular layer (GL). The globular or irregular-shaped autophagic vacuoles (AVs) contain partially digested organelles (bar = 1 μm). h: Mitochondria (MITs). Note the cristae look fuzzy in form (bar = 1 μm). i: The chromatin within the macronuclei (MAC) is condensed, a large proportion is attached to the nuclear membrane (bar = 1 μm).

substance of high-density. The cortex of the resting cyst electron density as the chromatin (Figure 3i). has resorbed all the cilia and their associated microtubules and most of the kinetosomes. The AVs, containing partially digested organelles, are globular or DISCUSSION irregular in shape with uneven electron density (Figure 3g). Compared with the non-dividing cells, the The resting cyst of T. stramenticola and its cellular cytoplasmic matrix of the resting cysts has decreased in characteristics of life activity amount considerably and most of the mitochondria congregate in form (Figure 3h). The chromatin within the T. stramenticola was first isolated by Foissner (1988) in macronucleus is condensed, with much of it attached on Austrian soil environment. The ciliate used in our study, the nuclear membrane. Although the number of nucleoli collected from the soil enclosed pond, demonstrates in each macronucleus is variable, they share the same similar characters to the T. stramenticola reported by 858 Afr. J. Microbiol. Res.

Foissner in a general characteristic, thus belonging to the stramenticola, which shows the basic characteristics of same species. The life activities of the soil ciliate are kinetosome-resorbing cysts. Although, unlike the highly subject to the change of soil environment. For freshwater kinetosome-resorbing cysts whose cyst wall example, depending on the moisture of the soil, Colpoda has four layers, the cyst wall of T. stramenticola is cucullus, one of the soil ciliates, would change between composed of two thick layer, suggesting that its resting encystment and excystment in a to-and-fro pattern cyst is yet another type of “kinestome-resorbing cyst”. (Nisbet, 1984). T. stramenticola could live in soil, and its cellular The fact that T. stramenticola can be induced into organelles are quite different from those of its freshwater encystment by the same method applied to freshwater counterparts. In the nature, it is a protective mechanism ciliates such as food shortage or aging of cultures shows for the ciliates to encyst under unfavorable that they share the same cysting mechanism and cellular circumstances. The structure of its ectocyst, made up of characteristics of life activities. In addition, both the rectangular units, not only increases its thickness but also cytoplasm of T. stramenticola and that of U. grandis show makes the cyst wall more resilient. some similar characters. Inside their cytoplasm, there Furthermore, the endocyst might prevent the ciliate exist clearly visible mitochondria and autophagic from water loss. Thus, the unique cyst wall of T. vacuoles. Containing fully or partially structured stramenticola not only increases resting cyst's thickness, endoplasmic reticulum inside, these autophagic vacuoles but also makes it possible for the ciliate to store more present certain morphological traits of food vacuoles. water in order to survive in much dryer conditions. This Thus, it could be concluded that autophagic vacuoles study shows that the resting cyst of T. stramenticola might be food vacuoles in their special form, providing share several common characteristics with U. grandis. nutrients and energy that the cells require (Gu et al., However, the cyst wall of T. stramenticola is quite 2002). different from its freshwater counterparts. The uniqe structure of T. stramenticola might play certain role in its adaptation to its environment. The cyst wall of T. stramenticola and its structural The results of this study will further help reveal the characteristics adaptation mechanism of soil ciliate T. stramenticola to changing environment and its structural differentiation One of the salient features of ciliate’s encystment is the under extreme conditions. dedifferentiation of its cortical ciliature and their associated microtubular structures. However, ciliates of different species do not exhibit the same characteristics ACKNOWLEDGEMENTS of dedifferentiation. Previous studies of the resting cysts of different species This research was undertaken under the support of the of ciliates have proposed that hypotrichs might form two National Natural Science Foundation of China types of cysts, namely “the kinetosome-resorbing cysts” (No.31172042). We would like to give heartfelt thanks to and “non-kinetosome-resorbing cysts”. 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