Colpodella vorax: ultrastructure, predation, life-cycle, mitosis, and phylogenetic relationships

Europ. J. Protistol. 38, 113–125 (2002) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ejp

Colpodella vorax : ultrastructure, predation, life-cycle, mitosis, and phylogenetic relationships Guy Brugerolle Biologie des Protistes, UMR 6023, CNRS and Université Blaise Pascal de Clermont-Ferrand, 63177 AUBIERE Cedex, France; E-mail: [email protected] Received: 28 January 2002; 25 March 2002. Accepted: 30 March 2002

The flagellar apparatus of Colpodella vorax comprises two widely separated basal bodies/flagella linked by a pluri-lamellar connector, very short basal bodies and a slightly longer transitional zone. The anterior flagellum arises from a flagellar pit: its basal body is connected to a root of three apically oriented microtubules. Two roots are connected to the basal body of the posterior flagellum: the oblique root composed of a ribbon of 6–7 microtubules is directed towards the anterior flagellar pit and the rostrum, the posterior root of two microtubules is short and directed towards the rear of the cell. The apical complex is composed of a C-ring of interlinked microtubules forming a pseudo-conoid which arises at the apex, as do the associated rhoptries and micronemes. The pellicle is composed of the endoplasmic reticulum beneath the plasma-membrane and of widely separated microtubules arising subapically and present only in the anterior part of the cell. For predation on Bodo and Spumella flagellates, C. vorax attaches itself to the prey by its anterior portion; the pseudo-conoid transforms into a ring of microtubules encircling the attachment zone. The cytoplasm of the prey is aspirated and drawn into a large posterior food vacuole. After feeding the cell encysts and divides, producing four flagellate cells; the mitosis is of the semi-open type similar to that of the apicomplexan Diplauxis hatti. These observations on C. vorax contribute to the ultrastructural definition of the genus Colpodella, which also includes C. gonderi and C. edax (= C. angusta). The species Spiromonas perforans differs from Colpodella in its flagellar apparatus, apical complex, the presence of dinoflagellate-type trichocysts and predatory behaviour. Colpodella is also distinct from the genera Perkinsus, Parvilucifera and Cryptophagus which are bona fide alveolate protists. Key words: Colpodella vorax ; Ultrastructure; Life-cycle; Mitosis; Predation; Apicomplexan protists.

Introduction In aquatic ecosystems, predation plays a major role in the regulation of protist and algal communities (Amblard et al. 1998; Laybourn-Parry and Parry 2000). While the interactions between prey and predator are now better understood (Arndt et al. 2000; Sleigh 2000), the impact of parasitism is incompletely explored in microbial aquatic populations. Several free-living protists have been recognized as predators/parasites of other protists or algae, and comparative ultrastructural studies have

contributed to the understanding of their organisation, life cycle and systematic position (see Brugerolle 2001). Several flagellate genera have the same predatory behavior and broadly the same feeding apparatus, composed of an apical complex reminiscent of parasitic protozoa of the Apicomplexa lineage (Brugerolle and Mignot 1979; Levine 1987; Wolthers 1991; Perkins 1996; Norén et al. 1999; Siddall et al. 2001). Some of these genera, such as 0932-4739/02/38/02-113 $ 15.00/0

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Perkinsus (Perkins 1996), Parvilucifera (Norén et al. 1999) and Cryptophagus (Brugerolle 2001), have been grouped in the Perkinsozoa lineage (Norén et al. 1999), by ultrastructural and sequence comparisons. Several other genera, predators of protists and algae with an apical complex and a complex life-cycle that have been identified under the name Colpodella (Cienkowski 1865) and Spiromonas Aléxéieff (1929), have been recently grouped by Simpson and Patterson (1996, 2002), in one family: the Colpodellidae, and in the genus Colpodella which now comprises seven species. Considering the cytological differences between the species now assembled in the genus Colpodella, a more complete electron microscopic (EM) study seemed necessary in order to characterize this genus more precisely. Moreover, the molecular phylogenetic study of a species of the genus Colpodella by Siddall et al. (2001) showed that these flagellates grouped with the apicomplexans or the ciliates, depending on the type of analysis performed. This was particularly interesting, because the limits and origin of the Apicomplexa lineage are not precisely determined (Levine 1978, 1987; Vivier and Desportes 1990; Perkins et al. 2002), and the presence of a plastid remnant suggests relationships with free-living flagellates containing a vestigial plastid (McFadden and Waller 1997; Hopkins et al. 1999). This was also the first time that a precise molecular phylogenetic study showed, in such a flagellate, a possible origin of the ciliates, a large and homogeneous group of alveolate protists (Lynn and Corliss 1991; Schlegel and Eisler 1996; Lynn and Small 2002). Studies of flagellates with a pellicle and an apical system are of vital importance, since they may represent steps in the evolution and diversification of the three groups of alveolate Protozoa: Dinoflagellates, Apicomplexa and Ciliates (Gajadhar et al. 1991; Schlegel and Eisler 1996; Patterson 1999; CavalierSmith 2000). Precise electron microscopic and molecular studies were thus necessary to characterize and distinguish the species grouped under the name Colpodella Cienkowski redefined by Simpson and Patterson (1996). We here present further observations on the species C. vorax, as well as a comparison with C. gonderi (Foissner and Foissner 1984), C. angusta (Myl’nikov 1991), C. pugnax and C. turpis (Simpson and Patterson 1996, 2002). This comparison is extended to the flagellate previously studied under the name Spiromonas perforans

(Brugerolle and Mignot 1979) and to the Perkinsozoa genera: Perkinsus (Perkins 1996), Parvilucifera (Norén et al. 1999) and Cryptophagus (Brugerolle 2001), the Apicomplexa (Levine 1987; Vivier and Desportes 1990; Perkins 1991; Mehlhorn 1998; Perkins et al. 2002) and the dinoflagellates (Gaines and Elbrächter 1987; Taylor 1987; Roberts and Roberts 1991; Dodge and Lee 2002).

Material and methods Colpodella vorax was collected from pond water containing dead leaves and grass, in the region of Clermont-Ferrand. After three days in the laboratory Colpodella flagellates multiplied in the water, as did Bodo caudatus, Spumella sp. and other flagellates. For several days C. vorax was cultivated in Petri dishes with Bodo caudatus or Spumella sp. as prey. To better determine the predatory behaviour and life-cycle of this Colpodella species, it was cultivated with different prey, such as Synura petersenii, Chilomonas paramaecium, Euglena gracilis and Colpoda cucullus. Small chambers, composed of a ring of vaseline on a microscope slide covered by a coverslip, contained C. vorax mixed with a prey species. Using this method, the organisms could be observed and photographed during several hours and days by phase contrast microscopy. For electron microscopy, a sample of Colpodella in the predatory phase or the encystment phase was collected with a fine pipette using a stereomicroscope. The cells were concentrated by centrifugation and fixed by the addition of 1 vol of 1% glutaraldehyde in 0.1 M cacodylate buffer pH 7 and 1 vol of 1% osmium tetroxide for 1 h. After washing in distilled water, the cells were pre-embedded in 1% agar, contrasted “en bloc” by saturated uranyl acetate in 50% ethanol, then dehydrated in an alcohol series. The final embedding was performed in Epon 812 resin. Ultrathin sections obtained with a Reichert Ultracut S microtome were contrasted by lead citrate for 15 min, carbon coated and observed in a JEOL 1200 EX electron microscope at 80 kV.

Results Light microscopy, predation and life cycle The trophozoite of Colpodella vorax is a dropshaped flagellate 12 µm long by 5 µm wide, with a short rostrum and two flagella, longer than the body and directed towards the rear of the cell (Figs 1, 2). The flagella are inserted at the base of the rostrum on one side of the cell (Fig. 2a). There is no spiral fold or ventral gutter, which distinguishes this species from C. edax and C. angusta. The nu-

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Fig. 1. Phase contrast light microscopic images of the trophozoite and cyst of C. vorax. a, b, c. Trophozoites feeding on Bodo caudatus. d. Single-celled cyst. e. Cysts with two and four cells. f. Fusion of two cells after excystment. Bar = 10 µm.

Fig. 2. Schematic views of several stages of the life cycle of C. vorax from light microscopic observations. a. Trophozoite with two flagella inserted subapically and containing a large posterior vacuole. b. During swimming, the flagellate appears to swing to right and left in an oscillatory motion as it follows a helical path. c. Three trophozoites feeding on a Bodo caudatus cell. d. Feeding ends, most of the prey cytoplasm has been aspirated and accumulates into the posterior vacuole. e. Recently encysted cell with internalized flagella and excentric food vacuole. f. Cyst containing two cells and a central food vacuole. g. Four-celled cyst. h. The cyst envelope opens, releasing four flagellate cells.

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cleus is situated in the anterior half of the cell, and a large, rounded digestive vacuole occupies the posterior portion. The flagellates swim with their pointed anterior in front, the body following a helical path as the flagellate moves forward (Fig. 2b). One to four trophozoites were often seen to attack and to feed on prey such as Bodo caudatus or Spumella sp. (Figs 1a, b, c, 2c, d). They attach to any point of the prey’s surface by their anterior pointed tip, which enlarges. In a few minutes, they aspirate the cytoplasm of their prey, which accumulates in the large posterior digestive vacuole. When satiated, the predators detach from the remnant of the prey, become immobile, withdraw their flagella, round up and encyst. One hour after feeding, the nucleus divides, then a second division occurs, giving four cells by about three hours after encystation (Figs 1d, e, f, 2e, f, g, h). The flagellates begin to move within the cystic envelope before it opens, releasing the young trophozoites (Fig. 2h). The addition of various prey such as Synura, Euglena, Chilomonas and Colpoda showed that C. vorax is not a predator of these species. The conjugation of two cells by their anterior part was also observed several times, but the complete fusion and the development of these doublets could not be followed to completion (Fig. 1f).

Electron microscopy Organisation of the flagellar apparatus The two flagella are inserted subapically: one flagellum, situated more anteriorly than the other, arises from a relatively deep pit (Fig. 3a, b, c) and has a paraxonemal structure in its proximal portion (Fig. 3e, g). The posterior flagellum shows no peculiarities and is directed toward the rear of the cell. The two flagella have very short basal bodies of about 0.15 µm long (Fig. 3e, f, g). Distally, the basal body termi-

nates in a well-marked basal plate, and there is a relatively long transitional zone proximal to the axosome at the beginning of the standard 9 + 2 axonemal structure. There are no flagellar hairs and no spiral ring in the transitional zone, such as are seen in stramenopile flagellates. The two basal bodies are separated by a distance of about 0.8 µm and are connected by a plurilamellar structure (Figs 3h, i, j, 7). This connector is composed of six stacked electrondense lamellae, interlinked by material (Fig. 3i). It is attached to the proximal extremity of each basal body via microfibrillar threads. One flagellar root composed of three microtubules is attached to the anterior basal body and associated with the plasmamembrane of the flagellar pit (Fig. 3k, l); this root is directed toward the tip of the cell but is distinct from the microtubules of the pseudo-conoid (see below). There is also circular microfibrillar material beneath the plasma-membrane around the anterior flagellar pit (Fig. 3k, l). Two roots originate from the posterior basal body; the posterior root is composed of 2–3 rather short microtubules directed towards the posterior of the cell (Fig. 3b). The other, called the oblique root (oR), is composed of a ribbon of 6–7 associated microtubules. It runs forwards just beneath the pellicular surface (Fig. 3b, c, d, k, l), in the direction of the pit of the anterior flagellum, and continues its course in the rostrum, but does not reach the cell apex (Figs 3d, 4 e). The apical complex and pellicle organisation The major component of the apical complex is the pseudo-conoid, which consists of an incomplete cone of about 20–25 interlinked longitudinal microtubules, forming a C-ring in transverse sections (Figs 4a, b, c, d, 7). Each microtubule has two arms, interconnected with its neighbours on the external side of the ring (Fig. 4d). These microtubules arise at the anteriormost level, close to the plasmamembrane, and terminates at the level of the anteri-

Fig. 3. Electron micrographs of C. vorax. a. Longitudinal section presenting the overall organisation of the cell, the apical conoid (C), the subapical insertion of the anterior flagellum (aF), the central nucleus (N), mitochondrial profiles (M) and lipid droplets (L). b, c, d. Anterior longitudinal sections showing the insertion of the anterior flagellum (aF) in a pit, the posterior flagellum (pF), the microtubular C-ring conoid (C) and microtubular flagellar roots: the posterior root (pR) and the oblique root (oR). e, f, g. Longitudinal (Fig. e) and transverse (Figs f, g) sections of the anterior basal body/flagellum showing the short basal body (bB), the transverse plate, the transitional zone (arrowheads) also seen in transverse section (Fig. f), the axosome (a) and the paraxonemal structure (arrow) also shown in transverse section (Fig. g). h, i, j. Sections showing the basal bodies of the two flagella (aF) and (pF) which are linked by a pluri-lamellar connector (arrow) seen in transverse section (Fig. i). k, l. Transverse section showing the anterior flagellum (aF) in its pit, the anterior root (aR), the oblique root (oR), the pellicle (Pe) and the base of the conoid (C). Bar = 1 µm Figs a, b, c, d; 0.5 µm Figs h, i, j, k, l.

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Fig. 4. Structure of the pseudo-conoid and associated organelles. a, b, c. Longitudinal sections of the conoid (C) which is composed of an incomplete ring of microtubules, sometimes protruding out of the pellicle (Fig. c); long micronemes (mi), bulbous rhoptries (bR) and lentil-shaped rhoptries (lR), each attached to the apex by a canaliculus; the pellicular microtubules arise subapically (arrowhead, Fig. c). d, e. Transverse sections at the anteriormost portion of the cell; the pseudo-conoid (C) is composed of a C- ring of interlinked microtubules close to the plasmamembrane (arrow, Fig. d), posteriorly pellicular microtubules are seen beneath the pellicle (arrowheads, Fig. e). f. Transverse section of the micronemes (mi) containing a central filament and the head of a bulbous rhoptry (inset). g. Section of the mitochondrial network (M) with tubulo-vesicular cristae, and the pellicle with endoplasmic reticulum beneath the plasma-membrane (Pe). h. Planar and transverse sections of micropores situated in the anterior flagellum pit. Bar = 1 µm Figs a, b , c; 0.5 µm Figs d, e, f, g, h.

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or basal body. Inside the pseudo-conoid there are three distinct organelles: 30–50 micronemes, 5–10 bulbous rhoptries and 5–10 lentil-shaped rhoptries (Fig. 4a, b, c). The micronemes, with a pinhead and a canaliculus about 1–5 µm long and 30 nm in diameter, are limited by a membrane and contain a cen-

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tral filament (Fig. 4 f). The bulbous rhoptries, with a globular head containing microfibrillar material (Fig. 4 f inset), are also connected to the cell apex by a canaliculus. The lentil-shaped rhoptries have a lentil-shaped head with a dense central plate and are also linked to the apex by a canaliculus (Fig. 4b).

Fig. 5. Predation by C. vorax. a. The trophozoite is attached by its anterior end to a Bodo prey cell (P), where the kinetoplast with flat cristae is recognizable (Bck); a large posterior vacuole contains the ingested cytoplasm; the central nucleus (N), anterior (aF) and posterior flagellum (pF) are also seen. b, c. Anterior section showing the attachment of C. vorax to the prey (P), a ring of interlinked microtubules (arrows) is present around the attachment zone, and pellicular microtubules are seen just posterior to this (Pe, Fig. c). d. A series of micronemes (mi) are attached to the interface between predator and prey. e. Cytoplasmic organelles of the prey (P) are aspirated and drawn toward the posterior vacuole through a channel-like structure (arrowheads). Bar = 1 µm.

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The conoid-like structure very characteristically protrudes more or less at the apex which is not covered by the pellicle (Fig. 4c, d). The cell is delimited by a pellicle comprising the plasma-membrane, a subjacent cisterna of endoplasmic reticulum and pellicular microtubules which are present only in the anterior third of the cell (Figs 3k, l, 4e, g). These pellicular microtubules do not arise at the apex, but at about 0.5 µm from it, and in close contact with the pellicular endoplasmic reticulum (Figs 3k, 4b, c, 7). Several micropores bordered by an electron-dense collar are present in the pit of the anterior flagellum and also in other places in the cell surface (Fig. 4h). The mitochondrial network has tubulo-vesicular cristae (Figs 4g, 5d). The Golgi body (not shown) is situated close to the nucleus, which lies in the anterior half of the cell (Fig. 3 a). Feeding structures and behaviour During feeding, the predator attaches to the prey by its apical portion (Fig. 5a). The zone of attachment is encircled by a ring of 3–6 interlinked microtubules corresponding to the pseudo-conoid microtubules (Fig. 5b, c). The pellicular microtubules, which are widely spaced, are present in the zone posterior to the apical ring (Fig. 5b, c). The separation between the cytoplasms of parasite and prey is due only to the plasma-membrane of the parasite; the plasma-membrane of the prey has disappeared at this point. The rhoptries have disappeared but, in some cases, a set of micronemes were observed attached to this connective membrane (Fig. 5d). Several images showed that the cytoplasm and organelles of the prey are aspirated and engulfed within the parasite, then channelled toward the posterior food vacuole (Fig. 5e). In such a vacuole, the mitochondria of the Bodo with their discoid-shaped cristae remain recognizable (Fig. 5a). Encystment and mitosis After C. vorax has detached from the prey, it ceases swimming, rounds up, and withdraws its flagella. A very thin cystic envelope (Fig. 6a, c) is apparent around the dividing cells, not yet separated from the central food vacuole. Within the cyst nuclear division is followed by partial division into two cells (Fig. 2f) and then, by the stage of Fig. 6a, into four cells each with flagella and a pseudoconoid with 12–14 microtubules. In a more advanced four-cell cyst, each cell has separated from

the others, becomes motile and detaches from the remnant of the central vacuole (Figs 6b, 2g). When they are ripe, the flagellated cells are released from the cyst envelope and are ready to attack another prey (Fig. 2h). C. vorax only divides inside these multiplicative cysts. The mitotic spindle is composed of a cone of microtubules arising from a MTOC structure situated at the base of one basal body (Fig. 6d, e). The microtubules of this cone are directed towards the nuclear envelope, slightly depressed at this point. This stage was often seen in sections, suggesting that it is a lengthy process. Several observations indicate that the spindle microtubules penetrate the nucleus through pores or fenestrae of the nuclear envelope (Fig. 6f), and a complete pole to pole figure was finally obtained (Fig. 6g). This type of mitosis, in which the nuclear envelope is conserved and the spindle is partly outside and partly within the nucleus is termed a semi-open mitosis.

Discussion The goal of this study was to better define the cytological features of the genus Colpodella, and to clarify its relationships to genera with a similar ultrastructural organisation, predatory behaviour and cell cycle. The ultrastructural study of C. vorax demonstrates that the flagellar apparatus of Colpodella comprises two widely separated basal bodies/flagella linked by a plurilamellar desmose. The basal bodies are very short, and there is a relatively longer transitional zone between the terminal plate of the basal body and the axosome which marks

Fig. 6. Views of the cysts (Figs a, b, c) and mitosis (Figs d, e, f, g). a. Encysted cell with four nuclei (N), flagella (F) and a central food vacuole (fV). b. Cyst with four individual, motile flagellates and a central remnant of undigested material; the ensemble surrounded by the cyst envelope (arrow). c. Structure of the very thin cystic envelope (arrow). d, e. Mitotic spindle (arrow) originating from a MTOC attached to a basal body (bB) and terminating on the depressed nuclear envelope (N). f, g. Microtubules of the mitotic spindle penetrating inside the nucleus (arrowhead, Fig. f) and pole to pole view of the spindle microtubules originating from two poles exterior to the nucleus (arrows), traversing the nuclear envelope (nE) and the nucleoplasm (arrowheads). Bar = 1 µm Figs a, b; 0.5 µm Figs c, d, e, f, g.

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the base of the flagellum. The anterior flagellum is inserted in a pit, and its basal body is connected to the anterior microtubular root which is directed towards the apex. A thin posterior root and an oblique root are attached to the posterior flagellum. This oblique root is shown for the first time in a Colpodella species. Very similar features were observed in C. gonderi (= Spiromonas gonderi)by Foissner and Foissner (1984) and in C. angusta (= Spiromonas angusta) by Krylov and Myl’nikov (1986) and Myl’nikov (1991), a species later assigned to C. edax by Simpson and Patterson (1996). These structural and organisational features of the flagellar apparatus are distinctive of the genus Colpodella, they do not occur in dinoflagellates, in the Perkinsozoa: Perkinsus, Parvilucifera and Cryptophagus or in Spiromonas perforans (see below).

Several other features of the apical system are also worthy of consideration and comparison with apicomplexans or apicomplexan-related flagellates. The apical system of C. vorax comprises a pseudo-conoid composed of an incomplete ring of microtubules which arises at the apex, and of secretory organelles, the rhoptries and micronemes. In C. gonderi only the predatory stage was observed (Foissner and Foissner 1984), and the attachment zone with the ring of microtubules was very similar to that of C. vorax. Very similar rostral and feeding structures have been observed in C. angusta by Krylov and Myl’nikov (1986) and Myl’nikov (1991). The incomplete ring of microtubules forming the pseudo-conoid of Colpodella is very similar to that of Perkinsus (Perkins 1996) and is distinct from the complete ring conoid of the Apicomplexa sensu stricto (Vivier and De-

Fig. 7. Schematic reconstructions summarizing the flagellar and apical complex organisation in C. vorax in a longitudinal view (A) and in three successive transverse sections (B). Pseudo-conoid microtubules (C), bulbous rhoptries (bR), lentil-shaped rhoptries (lR), micronemes (mi), pellicle (Pe), pellicular microtubules (Pmt), anterior (aF) and posterior (pF) flagellum, basal body connector (bbC), anterior root (aR), oblique root (oR) and posterior root (pR).

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sportes 1990; Perkins 1991; Mehlhorn 1998). The apical organelles were termed rhoptries and micronemes because of their morphological similarity to the secretory organelles of the apicomplexan genera; they probably represent a plesiomorphic character found in all Apicomplexa, Perkinsozoa, Spiromonas perforans (Brugerolle and Mignot 1979) and possibly in some heterotrophic dinoflagellates (Schnepf and Elbrächter 1992). In the absence of specific studies on the contents and function of these apical organelles in Colpodella, we must suppose that they play a role in the adhesion, penetration and digestion of the prey, functions which they have in Apicomplexa (Blackman and Bannister 2001). The three Colpodella species: C. vorax, C. gonderi, and C. edax have a pellicle comprising the plasma-membrane, a subjacent endoplasmic reticulum and widely separated microtubules present only in the anterior part of the cell, and this pellicle is pierced by micropores. The pellicle is a general feature present in Apicomplexa and apicomplexarelated flagellates (Levine 1987) and in all alveolate protozoa (Gajadhar et al. 1991; Cavalier-Smith 1993, 2000; Patterson 1999). Colpodella species do not have polysaccharidic reserves, such as the starch grains present in many apicomplexan genera (Levine 1987; Vivier and Desportes 1990, 2000; Mehlhorn 1998; Perkins et al. 2002). Colpodella vorax encysts after feeding and divides twice, with a semi-open mitosis, producing four flagellate cells. The conjugation of two cells previously reported by Aléxéieff (1929) and Simpson and Patterson (1996) was observed once again by us, and the fusion of the cytoplasm is confirmed by electron microscopy (not shown). Such observations suggest that there is a sexual phase in the life cycle which remains incompletely known. Among the other representatives of the genus Colpodella, C. pugnax, the type species, and C. turpis which feed on green algae, have the same predatory behaviour and yield four flagellate cells after encystment and division (Simpson and Patterson 1996). It is likely that these two species have the ultrastructural features of C. vorax, C. gonderi and C. edax, unfortunately with the superficial electron microscopic study this is impossible to ascertain. We have also obtained EM photographs of a bacteriophagous species previously described under the name Alphamonas coprocola by Aléxéieff (1916) which shares the ultrastructural features observed in C. vorax. This species was found to be

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synonymous with C. angusta by Simpson and Patterson (1996); we must note that it has not been cultivated and its life cycle is not precisely known. For several reasons, I do not consider the species Spiromonas perforans (Brugerolle and Mignot 1979) = Bodo perforans (Hollande 1938), which feeds on Chilomonas paramaecium, to belong to the genus Colpodella, as proposed by Simpson and Patterson (1996). First, the flagellar apparatus of S. perforans is different, the two basal bodies have a rather normal length (0.36 µm long), they nearly touch at their proximal end and are linked by short striated threads. The flagellar roots are also different, although their ultrastructure and organisation are incompletely understood. Moreover, the apical complex or pseudo-conoid and the secretory organelles, rhoptries and micronemes, show ultrastructural differences from C. vorax. S. perforans possesses three to four Golgi bodies arranged in a semi-circle around a contractile vacuole near the basal bodies, dinoflagellate-type trichocysts and polysaccharidic starch grains at the rear of the cell. However, its predatory behaviour is broadly similar to that of Colpodella although recently an intracellular stage has been observed in the host Chilomonas (unpublished). S. perforans encysts after feeding and the ingested food accumulates in the posterior vacuole as in Colpodella, but it divides several times during encystation, yielding more than four cells. Unfortunately the type of mitosis in S. perforans remains unknown. Colpodella also differs from Perkinsozoa genera: Perkinsus (Perkins 1976, 1996; Azevedo 1989, 1990, Coss et al. 2001), Parvilucifera (Norén et al. 1999) and Cryptophagus (Brugerolle 2001) by the flagellar apparatus, apical complex, type of mitosis (known only for Cryptophagus) and its predatory behaviour, observed in Cryptophagus (Brugerolle 2001), but not clearly established for Perkinsus and Parvilucifera. Molecular phylogeny using SSU rDNA has shown that Perkinsus and Parvilucifera group together and are a sister group of apicomplexans (Norén et al. 1999). A second molecular study using the SSU rDNA sequences of a Colpodella species, which is probably C. vorax because it feeds on Bodo caudatus, showed that Colpodella is phylogenetically close to the apicomplexans, while the couple Perkinsus and Parvilucifera appeared more closely related to the dinoflagellates (Siddall et al. 2001). The same molecular study, including both SSU rDNA and actin sequences, found that

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Colpodella was a sister group of the ciliates and Perkinsus/Parvilucifera a sister group of dinoflagellates (Siddall et al. 2001). From these two studies, we remark that the three genera studied (Colpodella, Perkinsus and Parvilucifera) are bona fide alveolates, as previously shown from ultrastructural studies. They all have a pellicle composed of the plasma-membrane, the subjacent endoplasmic reticulum and microtubules (Gajadhar et al. 1991; Cavalier-Smith 1993, 2000; Patterson 1999). As suggested by Siddall et al. (2001) the morphology present in Colpodella, Perkinsus and Parvilucifera could be “entirely plesiomorphic” within the Alveolata. According to the observations presented in this paper, Colpodella has a semi-open type of mitosis, which is found in several apicomplexans such as Diplauxis hatti (reported by Hollande 1972, and by Raikov 1982, 1994). This type of mitosis is different from the closed mitosis, with an intra-nuclear spindle, of the ciliates and different from the closed multichannelled dinomitosis of dinoflagellates (Raikov 1994), with the notable exceptions of the mitosis in syndinids (Hollande 1974) and Oxyrrhis (Triemer 1982). Since the morphology or ultrastructure could be regarded as providing stronger evidence than molecular phylogeny (Taylor 1999), I am inclined to consider the genus Colpodella as closer to the apicomplexans, because they have a similar type of mitosis. This feature distinguishes Colpodella from the dinoflagellates and the ciliates. Similarly, the genus Spiromonas, with its dinoflagellate-type trichocysts, seems more closely related to the dinoflagellates. From the differing conclusions drawn from molecular phylogeny studies of the genera Perkinsus and Parvilucifera, which are found to be either sister groups of apicomplexans by Norén et al. (1999) or sister groups of dinoflagellates by Siddall et al. (2001), and the lack of information on the type of mitosis of these two genera, it is difficult to decide which are their closest phylogenetic relatives among the alveolates.

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