Научная статья на тему 'Morphological events during resting cyst formation in the ciliate Colpoda cucullus'

Morphological events during resting cyst formation in the ciliate Colpoda cucullus Текст научной статьи по специальности «Биологические науки»

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Protistology
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COLPODA CUCULLUS / CHROMATIN EXTRUSION / CRYPTOBIOSIS / ECTOCYST / ENCYSTMENT / ENDOCYST / RESTING CYST

Аннотация научной статьи по биологическим наукам, автор научной работы — Funatani Ryoji, Kida Akemi, Watoh Tomoko, Matsuoka Tatsuomi

Morphological events during encystment (resting cyst formation) of Colpoda cucullus were studied. Some of the mitochondria were fragmented within 1 hr after onset of encystment induction. In 1~2 hr, a number of net-like globules (a kind of mucus) were synthesized in the vacuoles (mucocyst) in the cytoplasm and were expelled into extracellular space, and thereby the cell was covered with a thin mucus layer. Thereafter (2~3 hr), the cells were rounded up, and then began to be surrounded by the outermost layer (ectocyst) of the cyst wall, which is possibly derived from pellicle membranes. In this stage, many chromatin granules extruded from the macronucleus and fragmented mitochondria were surrounded by membranes to form autophagosomes, which are probably fused with lysosomes. Ectocyst formation was followed by the formation of a second layer called the endocyst by the excretion of toluidine blue-stained substance between the ectocyst and the plasma membrane. The endocyst layers were formed periodically for several days, and finally the cell was surrounded by several layers of endocysts. A large mass of chromatin was often extruded from the macronucleus more than 7 hr after the onset of encystment induction, and was gradually digested over a few days. The membrane potential of mitochondria disappeared in 7~15 hr after the onset of encystment induction. In the final step (7 days), the organelles were surrounded by amorphous electron-lucent materials, and all ciliary and kinetosomal structures disappeared.

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Текст научной работы на тему «Morphological events during resting cyst formation in the ciliate Colpoda cucullus»

Protistology 6 (3), 204-217 (2010)

Protistology

Morphological events during resting cyst formation in the ciliate Colpoda cucullus

Ryoji Funatani, Akemi Kida, Tomoko Watoh and Tatsuomi Matsuoka

Institute of Biological Science, Faculty ofScience, Kochi University, Kochi, Japan

Summary

Morphological events during encystment (resting cyst formation) of Colpoda cucullus were studied. Some of the mitochondria were fragmented within 1 hr after onset of encystment induction. In 1~2 hr, a number of net-like globules (a kind of mucus) were synthesized in the vacuoles (mucocyst) in the cytoplasm and were expelled into extracellular space, and thereby the cell was covered with a thin mucus layer. Thereafter (2~3 hr), the cells were rounded up, and then began to be surrounded by the outermost layer (ectocyst) of the cyst wall, which is possibly derived from pellicle membranes. In this stage, many chromatin granules extruded from the macronucleus and fragmented mitochondria were surrounded by membranes to form autophagosomes, which are probably fused with lysosomes. Ectocyst formation was followed by the formation of a second layer called the endocyst by the excretion of toluidine blue-stained substance between the ectocyst and the plasma membrane. The endocyst layers were formed periodically for several days, and finally the cell was surrounded by several layers of endocysts. A large mass of chromatin was often extruded from the macronucleus more than 7 hr after the onset of encystment induction, and was gradually digested over a few days. The membrane potential of mitochondria disappeared in 7~15 hr after the onset of encystment induction. In the final step (7 days), the organelles were surrounded by amorphous electron-lucent materials, and all ciliary and kinetosomal structures disappeared.

Key words: Colpoda cucullus, chromatin extrusion, cryptobiosis, ectocyst, encystment, endocyst, resting cyst

Abbreviations: DAPI — 4’,6-diamidino-2-phenyl-indole; AO — acridine orange; ER — endoplasmic reticulum; TB — toluidine blue; TBS — toluidine blue-stained substance; PBS — phosphate-buffered saline; SSS — standard saline solution

Introduction protozoans, when detecting approaching hostile

conditions, promptly transform into resting cysts T o survive on the soil surface, where puddles can resistant to drying, freezing and higher temperatures appear suddenly and dry out rapidly, the terrestrial (Taylor and Strickland, 1936; Gutierrez 2001;

© 2010 by Russia, Protistology

Maeda et al., 2005). They excyst and proliferate when favorable conditions return. Resting cysts of protozoa are a cryptobiotic form (Gutierrez et al., 1990, 2001), whose formation involves a gene-regulated cyto-differentiation (Matsusaka, 1979; Matsusaka et al., 1989; Martin-Gonzalez et al., 1991; Gutierrez et al., 2000) concomitant with the cessation of metabolic activity and, presumably, with the cytoplasmic dehydration.

Species of the genus Colpoda have long been studied with regard to resting cyst formation (encystment). The encystment is induced by an increase in the mainly external Ca2+ concentration in Colpoda cucullus (Yamaoka et al., 2004) or by the overpopulation of vegetative cells in C. duodenaria (Strickland, 1940) and C. cucullus (Maeda et al.,

2005). Ca2+-induced encystment of C. cucullus is suppressed by components released by bacteria (Yamasaki et al., 2004) and components contained in plant leaves, such as certain peptides (Yamasaki et al., 2004; Akematsu and Matsuoka, 2007) and various porphyrins (Tsutsumi et al., 2004; Maeda et al., 2005).

Morphogenesis during encystment of colpodid ciliates has been extensively studied over the last 50 years, with special attention to the cortical structures (Kawakami and Yagiu, ^3a, ^3^ Tibbs, 19б8; Janisch, 1980; Ruthmann and Kuck, 1985; Martin-Gonzalez et al., 1991, 1994; Frenkel, 1994; Delmonte Corrado et al., 199б; Chessa et al., 2002; Diaz et al., 2003; Kida and Matsuoka, 200б). The wall of the resting cyst, which is composed of several layers, contains glycoproteins (Izquierdo et al., 2000; Chessa et al., 2002). The cyst wall has been reported either to originate from pellicular membranes (Kawakami and Yagiu, ^3a; Ruthmann and Kuck, 1985), or to be formed by exocytosis of precursor materials from the mucocyst-like vacuoles (Martin-Gonzalez et al., 1994; Frenkel, 1994). Importantly, the layers of the cyst wall may not be formed in the same manner. In C. cucullus, many large ellipsoidal vacuoles, containing electron-lucent materials or no material at all, are arranged near the cortical region; some of them open into the extracellular space, just after the net-like globules (masses of mucus) are expelled to the outside; the cells are then surrounded by a thin amorphous mucus-like envelope (Kida and Matsuoka, 200б). On the basis of these electron microscopic observations, it was suggested that the outermost thick layer (ectocyst) of the cyst wall is formed by exocytosis of precursor materials contained in the ellipsoidal vacuoles (Kida and Matsuoka, 200б). However, such vacuoles arranged near the cortical region are possibly vacant mucocysts whose contents (net-

like globules) have been expelled. In dry cysts of C. cucullus, the metabolism is completely or almost stopped, as evidenced by the fact that 3-4 year-old cysts excyst (unpublished data). This means that the process of encystment is accompanied by the cessation of mitochondrial activity.

In the present paper, therefore, the process of ectocyst formation is reconsidered. The study also aimed to reveal the timing of the stopping of mitochondrial activity using a dye for the detection of mitochondrial membrane potential. Moreover, a reconstruction process of cell structure during encystment including digestion of organelle was traced by electron microscopy and by the detection of acidic organelles such as lysosomes by AO staining.

Material and methods

Cell culture and encystment induction

Colpoda cucullus was cultured in a 0.1% (w/v) infusion of dried cereal leaves inoculated with bacteria (Enterobacter aerogenes) as food at 23 oC in the dark. For encystment induction, vegetative cells cultured for 1-2 days were collected by centrifugation (1,000~ 1,500 g, 2 min) and subsequently suspended in a standard saline solution (SSS) containing 1 mM CaCl2, 1 mM KCl and 5 mM Tris-HCl (pH 7.2) or the cells were suspended in a solution containing 0.1 mM CaCl2 and 1 mM Tris-HCl (pH 7.2) (Figs 2-4).

Electron microscopy

For prefixation of the vegetative and precystic cells without a cyst wall (0~1 hr after encystment induction), the suspension of cells was mixed with a glutaraldehyde (GA) fixative (б% GA, 1% OsO4, 100 mM cacodylate buffer [pH 7.2], 4 mM sucrose) at a ratio of 1:б (v/v). After a 10-min fixation, the prefixed samples were rinsed 5 times in 100 mM cacodylate buffer (pH 7.2) and postfixed for 2 hr in a postfixative containing 1% OsO4, 100 mM cacodylate buffer (pH 7.2) and 2 mM sucrose. The precystic cells and mature cysts surrounded by the cyst wall (1 hr~2 weeks after encystment induction) were prefixed with GA fixative without OsO4 for б hr, rinsed in 100 mM cacodylate buffer (pH 7.2), and postfixed in a postfixative for a week. The postfixed samples were rinsed several times in distilled water, dehydrated through a graded ethanol series (30, 40, 50, б0, 70, 80, 90 and 100% ethanol) for 15 min each and finally suspended in acetone.

The dehydrated samples were embedded in Spurr’s resin. Ultrathin sections were obtained by using ultramicrotome (Ultracut UCT, LEICA), and stained with 3% uranyl acetate and then with lead citrate (10 min each). The sections were observed under a transmission electron microscope (JEOL, 1010T).

Staining for photomicroscopy

F or vital staining of cells with toluidine blue, 0.1 % toluidine blue (TB) dissolved in SSS was added to an equal volume of cell suspension, and kept for 5~10 min.

For DAPI (4’,6-diamidino-2-phenyl-indole) staining, the cells were fixed in phosphate-buffered saline (PBS) containing 3.7% paraformaldehyde, 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 (pH 7.4) for 30 min, washed twice in PBS by centrifugation (2,000 g, 1~2 min), and then incubated for 30 min in PBS containing 1 % Nonidet P-40. The cells were washed in PBS, and 5 ^l of 0.02% DAPI (dissolved in pure water) was added to 1 ml of cell suspension (final concentration of DAPI 0.0001%).

In order to stain the vegetative cells and precystic cells with acridine orange (AO), the cells were suspended in a saline solution (SSS) containing 0.0001% AO or in the Ca2+-free SSS containing

0.0001% AO. The encysting cells suspended in SSS containing AO were observed at 5, 7, and 21 hr and 3 days under the fluorescence microscope.

The MitoPT Kit (B-Brige International, Inc.) was used to detect mitochondrial membrane potential. MitoPT dye was dissolved in DMSO for a stock solution, and the stock solution was diluted 100 times with 1 mM Tris-HCl (pH 7.2) for staining for 15 min. For staining of all mitochondria irrespective of the membrane potential, Mito Tracker Green FM (Invitrogen) was used. Mito Tracker Green was dissolved in DMSO for a stock solution, and the stock solution was diluted 500 times (1 ^M Mito Tracker Green and 0.2% DMSO in final conc.) with

1 mM Tris-HCl (pH 7.2) for staining for 5 min. After staining, the cells were centrifuged (1,500 g, 2 min) and then suspended in a 1 mM Tris-HCl buffer (pH 7.2) containing 0.1 mM CaCl2 for encystment induction or in the same buffer without CaCl2 for non-induction of encystment.

In order to detect phospholipids contained in the ectocyst layer, HCS LipidTOX phospholipidosis detection reagents (Invitrogen) were employed. The vegetative cells were incubated in the encystment-inducing solution (1 mM Tris-HCl [pH 7.2], 0.1 mM CaCl2) containing dye for phospholipids stain

(500 times diluted), and the ectocyst layer peeled off was observed at 5~6 hr after the onset of encystment induction.

The cells were observed with a fluorescence microscope (BX-50, Olympus) equipped with the filter set WBV for MitoPT staining, WU for DAPI and AO stainings, and WIG for phospholipids staining.

Results

Transmission electron microscopy

The first prominent morphological events after the encystment induction was the fragmentation of some of the mitochondria (Fig. 1d, white arrowhead). It occurred 30~ 60 min after the onset of encystment induction. Prior to fragmentation, the mitochondria became more electron-dense (Fig. 1c, white arrow-head) as compared to normal mitochondria (Fig. 1c). Such fragmentation was not observed in the vegetative cells without encystment induction (Fig. 1a, b). Thereafter, the fragmented mitochondria were surrounded by autophagosomelike structures (Fig. 4a). At 1~2 hr after encystment induction, net-like globules (Kawakami and Yagiu, 1963a; Kida and Matsuoka, 2006) were observed to be enclosed in mucocyst-like vacuoles (Fig. 1e). Numerous endoplasmic reticulum (Fig. 1e) associated with ribosomes that appeared at this stage are probably involved in the synthesis and transport of the components of net-like globules. The net-like globules (masses of mucus) that corresponded to “papilla-like structures” (Chessa et al., 2002) were subsequently expelled into the extracellular space, and the cell was surrounded by a thin mucus envelope (Fig. 2a, b, c) derived from the net-like globules (Fig. 2c). In this stage, many vacuoles (Fig. 2a, b) that seemed to be vacant were located near the peripheral region of the cell and some of them opened to the extracellular space (Fig. 2a, b, arrows). These were probably the vacuoles whose net-like globules had just been expelled.

At the beginning of ectocyst formation (2 hr after the onset of encystment induction), the mucus envelope was lined with a membranous structure, and thereby the outline of the mucus envelope became obvious (Fig. 3a). In this stage, tiny vesicles that are possibly responsible for the lining of the mucus envelope were observed to be released from the cell surface (Fig. 3a). The image showing the ectocyst splitting into two layers (Fig. 3b, arrow) suggests that the ectocyst is composed of layered structures. In addition, new alveolar sacs were

Fig. 1. Transmission electron micrographs of C. cucullus after onset of encystment induction. a, b — vegetative cells; c, d, e — 0.5 hr, 1 hr and 2 hr after onset of encystment induction showing fragmented mitochondria (white arrowheads) and synthesis of mucocyst containing net-like globules, respectively. Abbreviations: al — alveolus, er — endoplasmic reticulum, m — mitochondria, nt — net-like globule.

observed to be formed just beneath the alveoli (Fig. 3c, arrowhead). The vacant vacuoles (Fig. 3b, c), which had contained the net-like globules, seemed to be used for the formation of the pellicle membrane systems including alveoli and ER. The completed ectocyst is about 50 nm thick (Fig. 3d). After the ectocyst has been completed, the first layer of the endocyst was formed between the ectocyst and the plasma membrane (Fig. 3d). At 2~4 hr after the onset of encystment induction, chromatin granules (Fig. 2b) were observed to be extruded from the macronucleus and enclosed in the autophagosomes by being surrounded with ER-like membranes (Fig. 2b, Fig. 4b).

The endocyst layers are formed by the repeated exocytosis of short filamentous precursor materials. A two-day-old immature cyst (Fig. 4c) has the third

layer of the endocyst just being formed by exocytosis (arrow) through duct-like structures. At this cyst age, many autophagosomes still occupied the cytoplasm (Fig. 4d). A week after the onset of encystment induction, the cyst had almost matured (Fig. 5). In this stage, several layers of endocyst were observed, and the cytoplasm was filled with amorphous electron-lucent material and an electron-lucent body (Fig. 5). The mitochondria were surrounded by such amorphous material (Fig. 5).

Staining of cyst wall with toluidine blue or

PHOSPHOLIPIDS STAINING

Toluidine blue (TB) dye is known to form complexes with anionic polysaccharides such as

Fig. 2. Transmission electron micrographs of C. cucullus after onset of encystment induction. 2 hr after onset of encystment induction showing the expelling of net-like globules (a) and the formation of mucus envelope (b, c). Arrows in a and b indicates vacuoles just opening to the outside. Abbreviations: al — alveolus, ap — autophagosome, ch — chromatin granule, ev — mucus envelope, er — endoplasmic reticulum, m — mitochondria, nt — net-like globule, va — vacuole.

Fig. 3. Transmission electron micrographs of C. cucullus after onset of encystment induction. a-d — 2~4 hr after onset of encystment induction showing the formation of ectocyst (a-c) and endocyst (d). Arrow (white) in b indicates the split of an ectocyst; arrowhead in c indicates newly synthesizing alveoli. Abbreviations: al — alveolus, ci — cilia, ev — mucus envelope, ec — ectocyst, en — endocyst, va — vacuole, .

glycosaminoglycans (Shepard and Mitchell, 1976; Terry et al., 2000). Fig. 6a-1 shows TB-stained cells at the early stage of cyst formation (1.5~2 hr after the onset of encystment induction). It has been reported that a huge vacuole opens into the space between the ectocyst and plasma membrane, and subsequently a TB-stained substance (TBS), which is probably an endocyst precursor, diffuses over the entire cell surface within a few minutes after the ectocyst has been completed (Kida and

Matsuoka, 2006). However, as shown in Fig. 6a-

1, TBS-containing vacuole was found to open to several portions in some cysts (arrowheads). The endocyst precursor was not stained when it was enclosed in the vacuoles. It is likely that TB hardly permeates the vacuole membrane. Fig. 6a-2 (inset) shows the ectocyst, peeled off by a mechanical press, which was hardly stained with TBS. Fig. 6b shows the peeled off ectocyst (b-1) and the detection of phospholipids contained in the ectocyst

Fig. 4. Transmission electron micrographs of C. cucullus after onset of encystment induction. a, b — 4 hr after onset of encystment induction showing the formation of autophagosomes; c, d — 2 days after onset of encystment showing endocyst formation and autophagosomes. Arrow in c indicates a duct or vacuole excreting endocyst precursor. Abbreviations: ap — autophagosome, ch — chromatin granule, ec — ectocyst, en-1 — first layer of endocyst, en-2 — second layer of endocyst, m — mitochondria.

Fig. 5. Transmission electron micrographs of C. cucullus after onset of encystment induction. A week after onset of encystment induction showing the features of a nearly matured cyst. Abbreviations: al — alveolus, am — amorphous materials, ec — ectocyst, en-1 — first layer of endocyst, en-2 — second layer of endocyst, en-3 — third layer of endocyst, elb — ellipsoidal electron-lucent bodies, m — mitochondria. .

by phospholipid stain (b-2), with red fluorescence indicating the phospholipids.

MitoPT or mito tracker staining of

MITOCHONDRIA

The stopping of mitochondrial activity was accompanied by a loss of mitochondrial membrane potential that could be visualized by staining with MitoPT dye; red fluorescence indicated the formation of membrane potential, while green fluorescence indicated the collapse of membrane potential. Mitochondrial membrane potential began to be lost at 7 hr after the onset of encystment induction (Fig. 7). At 15 hr, most of the cells emitted green fluorescence, which indicated that mitochondrial membrane potential was lost completely (Fig. 7d). In this case, the image of each mitochondrion could not be observed. Some relatively large particles or aggregations of small particles with bright green fluorescence were observed in vegetative cells and precystic cells in the early stage (Fig. 7b).

As already mentioned, some of the mitochondria were fragmented within 1 hr after the onset of encystment induction (Fig. 1c, d). In order to confirm the reduction of the number of mitochondria, MitoTracker Green was employed (Fig. 8), because the fragmented mitochondria may not be stained with MitoPT dye. At 1 hr after the onset of encystment induction, the number of irregular and somewhat small-sized mitochondria seemed to increase (Fig. 8b).

Acridine orange (ao) staining of autophagosomes

AND DAPI STAINING OF CHROMATIN

Electron-microscopic observation showed that chromatin granules extruded from the macronucleus or mitochondria were surrounded by membranes to form autophagosomes (Figs 2b, 4a, 4b, 4d). The autophagosomes are expected to be fused by lysosomes for digestion. In order to observe lysosome-fused autophagosomes, the cells were stained with AO (Fig. 9). At 5 hr after the onset of encystment induction, various sizes

Fig. 6. Staining of cyst wall with toluidine blue (a) or phospholipid-staining reagent (b). a-1 — TB-stained cells at 1.5~2 hr after onset of encystment induction, showing the formation the first layer of endocyst, arrowheads indicates portions where TB-stained substance (precursor of endocyst) is being excreted; a-2 (inset), ectocyst peeled off; b — a photomicrograph of ectocyst peeled off the б-hr aged precystic cell (b-1) which has been stained with phospholipid-staining reagent and its fluorescence micrograph (b-2). Abbreviations: ven — a large vacuole probably containing endocyst precursor.

of autophagosomes (red-fluorescent vacuoles) were located around a macronucleus (Fig. 9b, ma) and other regions of the cytoplasm. Smallsized autophagosomes almost disappeared at 7 hr, and a few large ones remained or were newly formed (Fig. 9c). As shown on DAPI-stained precystic cells (Fig. 10), a large chromatin mass (Fig. 10, black arrowheads) began to be extruded from a macronucleus in this stage. In some cells, small chromatin granules just extruded from a macronucleus were observed (Fig. 10, white arrowheads). The large autophagosomes (Fig. 9c, d) probably contained extruded chromatin masses.

Some of the immature cysts still contained many small-sized autophagosomes at 3 days after the onset of encystment induction (Fig. 9e).

Discussion

The morphogenetic configuration of the encystment process in C. cucullus is summarized in Fig.

11 on the basis of the present report and a previous study (Kida and Matsuoka, 2006). The first event in the encystment-induced cells is the fragmentation of some mitochondria. It occurs within 1 hr after the onset of encystment induction and is followed by the formation of a kind of mucocyst containing net-like globules (masses of mucus) in the cytoplasm (Fig. 11a). The net-like globules are expelled into the extracellular space by 2 hr after the onset of encystment induction and thereby the cells are surrounded by a mucus envelope derived from the net-like globules (Fig. 11b). Many vacuoles, which have probably expelled net-like globules, are located near the peripheral region (Figs 2a, b; 11b). In a previous report (Kida and Matsuoka,

2006), such electron-microscopic images were interpreted as the beginning of ectocyst formation. In other words, it was believed that these vacuoles contained extremely electron-lucent precursors for the ectocyst layer. However, this idea should be rejected, because the mucus-like envelope is evidently derived from the net-like globules (Fig. 2c), and no image shows that the ectocyst precursor is released from the vacuoles during thickening of the ectocyst (Fig. 3b).

The mucus envelope is soon lined with membranous material, which gradually thickens to form the ectocyst layer. At the beginning of the process, very small vesicles are released from the cell surface (Figs 3 a, 11b). These vesicles are possibly involved in the beginning of the formation of the ectocyst. The microscopic images showing the ectocyst growing (Fig. 3a, b) strongly suggest that the ectocyst layer originates from pellicle membranes as schematically drawn in Fig. 11, where alveoli may be fused to one another, their outer membranes may glue onto the plasma membrane, and the inside layer of the alveoli might become the new plasma membrane. The new alveoli are formed just beneath the new plasma membrane. The ectocyst may become thicker and thicker by the repetition of this process. The fact that the ectocyst layer is hardly stained with TB (Fig. 6), which stains anionic polysaccharides such as glycosaminoglycans deep blue (Shepard and Mitchell, 1976; Terry et al., 2000), and is stained

a С

0 hr 7 hr

10 цт 10 цт

b d

1 hr 15 hr

10 цт 10 [im

Fig. 7. MitoPT staining of the vegetative cells without encystment induction (a) and precystic cells at 1 hr (b), 7 hr (c) and 15 hr (d) after onset of encystment induction.

instead by phospholipids-staining dye supports the hypothesis that the ectocyst is composed of membranes.

In 2~5 hr after the onset of encystment induction (Fig. 11c), the first layer of endocyst is formed by the excretion of the precursor contained in a huge vacuole deeply stained with TB. Endocyst layers are formed repeatedly (probably once per day), until finally the cyst wall is composed of an ectocyst layer including a mucus envelope and

several endocyst layers. The vacuoles containing endocyst precursor open into the space between the plasma membrane and the already-formed layer at several points (see Fig. 6) probably through a number of duct-like structures (Fig. 4c). Chromatin granules and a chromatin mass extruding from the macronucleus and some mitochondria are surrounded by membranes to form autophagosomes that may be subsequently fused with lysosomes for digestion (Figs 4a, b, d, 9, 11b, c, d). As previously

a b

0 hr 1 hr

10 Lim

10 ЦІ71

Fig. 8. Mito Tracker Green staining of the vegetative cells without encystment induction (a) and precystic cells at 1 hr after onset of encystment induction (b).

suggested (Akematsu and Matsuoka, 2008), the biological meanings of chromatin extrusion might be a resetting of the chromatin contents of the macronucleus when encystment induction is forced to occur in the cells in which DNA is being synthesized. Autophagosomes remain in some immature cysts for a few days (Fig. 9). The small red-fluorescent particles seen in the rightmost immature cyst (Fig. 9e) might correspond to autophagosomes observed in the electron micrograph (Fig. 4d). Many particles present on the cell surface (Fig. 9d), which probably correspond to masses of mucus (net-like globules), and mucus-like layers gluing immature cysts to one another (Fig. 9e) are stained green. Previous electron microscopic studies showed that the glue materials for cell-to-cell adhesion are actually fused and crushed net-like globules (Kida and Matsuoka, 2006). The glue material stained green (Fig. 9e) is, therefore, probably made up of crushed and fused net-like globules.

The result that most of the cells emitted green fluorescence at 15 hr after the onset of encystment induction (Fig. 7) indicates that the mitochondrial activity is stopped at this stage. In Fig. 7d, however, no individual mitochondrion can be distinguished. This may be the reason why the MitoPT dye that has accumulated in multimeric forms in the intermembrane space of mitochondria with membrane potential (emitting red fluorescence) might dissociate into monomeric forms (green fluorescence) to disperse to the outside of mitochondria, as the mitochondrial membrane potential disappears. In vegetative cells or in the early stage of encystment, green-fluorescent particles were observed (Fig. 7). In the present electron microscopy, aggregated mitochondria could not be found in the early stage of encystment. In consequence, it cannot be concluded whether or not such particles are aggregated mitochondria.

A previous electron microscopy showed that cilia were resorbed in mature cysts of C. cucullus, but it was not found out whether the kinetosomal structures completely disappeared in the mature cysts (Kida and Matsuoka, 2006). An electron-microscopic image of a nearly matured cyst (Fig. 5) shows that kinetosomes disappear at this stage. It was suggested in the previous report that electron-lucent ellipsoidal bodies (Fig. 5) that accumulate

Fig. 9. Acridine orange staining of the vegetative cells without encystment induction (a) and precystic cells at 5 hr (b), 7 hr (c), 21 hr (d) and 3 days (e) after encystment induction. Abbreviations: ma — macronucleus.

in the central region of a mature cyst are reserve grains (Kida and Matsuoka, 200б). Electron-lucent amorphous materials (Fig. 5) appear in the cytoplasmic space and enclose mitochondria (Fig.

5, 11e). Such amorphous materials seem to be a continuation of ellipsoidal electron-lucent bodies.

One of the functions of such materials may be protection of organelles from drying or freezing, as it is the case with a sugar called trehalose.

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(a) (b) (c) (d) (e)

mb mb ec

Fig. 11. Schematic diagrams showing the encystment process (a-e) and a possible model for ectocyst formation (at the bottom). Abbreviations: mi — micronucleus, ma — macronucleus, m — mitochondria, al — alveolus, nt — net-like globule, ev — mucus envelope, ch — chromatin granule (b) or chromatin mass (d), va — vacuole, ve — vesicle, ap — autophagosome, ec — ectocyst; ven — vacuole containing endocyst precursor, mb — plasma membrane, en-1 — first-synthesized endocyst layer, en-2 — second-synthesized endocyst layer, en-3 — third-synthesized endocyst layer, elb — ellipsoidal electron-lucent body, ax — ciliary axoneme.

Щ1 mm іЯ

Fig. 10. DAPI-stained precystic cells at 10 hr after onset of encystment induction, showing the extrusion of a chromatin mass. Black arrowheads indicates a chromatin mass extruding from the macronucleus, white arrowheads indicates a number of small chromatin granules around the macronucleus. Abbreviations: ma — macronucleus, mi — micronucleus.

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Address for correspondence: Tatsuomi Matsuoka. Institute of Biological Science, Faculty of Science, Kochi University, Kochi 780-8520, Japan, e-mail: tmatsuok@cc.kochi-u.ac.jp

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