Form and Function of the Dinoflagellate Transverse Flagellum

1. Prolozooi.. 32(2), 1985, pp. 290-296 Q 1985 by the Society of Protozoologists

Form and Function of the Dinoflagellate Transverse Flagellum’ GREGORY GAINES*2 and F. J. R. TAYLOR**

*Department of Botany, Southern Illinois University, Carbondale, Illinois 62901, USA and **Departments of Oceanography and Botany, University of British Columbia, Vancouver, British Columbia V6T I W5, Canada

ABSTRACT. A reexamination of the dinoflagellate transverse flagellum in relation to swimming in more than 50 species, using a television recording system, has revealed the following new facts: the flagellar beat always proceeds counterclockwise when seen from the cell apex; the cell always rotates in the direction of the flagellar beat, and fluid is propelled in the opposite direction. These observations can be explained by the actions of flagellar mastigonemes not included in previous models. The shape of the flagellar wave is not isotropic. New explanations are offered for other morphological features of the cell as they relate to swimming.

LTHOUGH there have been numerous studies of the A swimming behavior of dinoflagellates (9, 10, 12, 13), rel- atively few have dealt with flagellar action. The flagella of dinoflagellates have several unusual and enigmatic features. There are usually two morphologically distinct flagella per cell, inserted either apically (desmokonts) or ventrally (dinokonts). One en- circles the cell wholly or partly and is referred to as the transverse (or undulating) flagellum. The other (longitudinal) flagellum beats posteriorly.

Electron microscopy has shown that the transverse flagellum consists of an axoneme laterally connected to a fibrous (“ac- cessory” or “striated”) strand (2 1,22) by a thin, flexible, ribbon- like membrane. The flagellum is probably unattached at its distal end but held close to the cell surface by tension (28). In addition, the flagellum possesses delicate hairs (mastigonemes) on the outer edge of the axoneme (21, 22, 28). Although the hairs are individually simple, they may occur in complex arrangements, e.g. Oxyrrhis marina (6). They are fine and solid, in contrast to the tripartite, “stiff,” tubular mastigonemes of chrysomonads, and resemble those of euglenoids (5,27). The latter are presumed to be too thin to remain rigidly perpendicular to the axoneme (1 6). Rees & Leedale (28) recorded some mastigonemes of Pe- ridinium cinctum (0. F. M.) Ehr. 1838 as being “ca. 20 nm” thick, which is similar in size to those of chrysomonads although they did not comment on this. The number, arrangement, and rigidity of the mastigonemes of most dinoflagellates are not known. With scanning electron microscopy (SEM), the strands appear to connect the flagellum to the cell surface (1, 30, 3 l), but this is probably an artifact of the drying process.

The shape of the transverse flagellum, when active, has been difficult to ascertain. Klebs (1 9) correctly identified the flagellum, which previously had been thought to be a ring of cilia (4, 8); however, he erroneously thought it to be a simple filament. This formed the basis of a spiral model accepted by Peters (26), Metzner (25), and Jahn et al. (17). With electron microscopy, Pitelka & Schooley (27) showed the flagellum to be ribbon-like and they first saw mastigonemes. Leadbeater & Dodge (2 1, 22) found a fibrous strand on the edge of the ribbon opposite to the axoneme and assumed that the axoneme wound helically around the fibrous strand, the ribbon thus forming a helical surface. Taylor (31) showed by SEM that the axoneme does not spiral around the fibrous strand. It stays exterior to it throughout its beat. With SEM, two wave forms have been seen: undulatory

I This work was supported by a Grant-in-Aid of Research from Sigma Xi, the Scientific Honor Society, a research grant from the American Philosophical Society, and funds from the Southern Illinois University at Carbondale Office of Research Development and Administration and Department to G. Gaines; and by National Science and Engineering Council of Canada Grant A-6137 to F. J. R. Taylor. We thank Karen Schmitt of the S.1.U.-C. Research Photography and Illustration Facility and Leslie Christian for the line drawings.

Current address: Department of Oceanography, University of Brit- ish Columbia, Vancouver, B.C. V6T 1 W5, Canada.

(14, 30, 3 1) and helical (1, 28); however, even in the latter, only the axoneme is helical. The whole flagellum (the propulsive surface) forms a traveling series of cups opening postero-sinistrally, as shown in the model of LeBlond & Taylor (23). (For illustrations of directional terminology, see Fig. 1 .) In order to permit this unusual axonemal motion, the ribbon membrane must be capable of considerable expansion and contraction, apparently assisted by infoldings of the plasma membrane (28).

Kofoid (20), Peters (26), and Jahn et al. (1 7) stated that the direction of rotation of the cell appears to reverse during swimming. This implies reversals of beat, contrary to the unidirec- tional, base-to-tip flagellar beat found in the majority of flagellates (29). Metzner (25), Peters (20), and Jahn et al. (1 7) depicted the flagellum as a spiral projecting partly beyond the edges of the cingulum, as is commonly seen in SEM views (Figs. 2, 3). The forward thrust, known to be produced by the transverse flagellum (10, 17), was attributed to the backward motion of the projecting waves in conjunction with the forward motion of the inner regions pushing fluid against the anterior face of the cingulum. With the discovery that the ribbon surface is not helical, a new model was required. LeBlond & Taylor (23) attributed the forward thrust of the transverse flagellum to the inclined, advancing outermost edges (described as “hemi-helical”) of the flagellar ribbon.

Kofoid (20) reported that cell rotation in dinoflagellates is predominantly dexiotropic (from the organism’s left towards its right, or clockwise when seen from the cell apex). Peters (26) and Metzner (25), however, stated that cell rotation is predominantly leiotropic (from the organism’s right towards its left) and that apparent changes in direction were seen. Jahn et al. (17) reported that Ceratium spp. turn dexiotropically and erroneously claimed that Kofoid (20) asserted that dinoflagellates are predominantly leiotropic. Because Jahn et al.’s (17) model and direction of flagellar rotation could only produce dexiotropic cell rotation (the flagellum pushing against its base), they attributed rotational exceptions to cell asymmetry but did not venture a more detailed analysis. LeBlond & Taylor (23) based their model on the generally accepted idea that rotation was dexiotropic, their own observation being limited to electron micrographs.

In view of the contradictions in studies of dinoflagellate transverse flagella, we have undertaken a comprehensive analysis of flagellar motion in dinoflagellates and in this report we offer a new model of flagellar propulsion in this group.

MATERIALS AND METHODS Observations of swimming were recorded using a Sony DXC-

1850 Trinicon video camera system and Panasonic NV-8950 videocassette recorder connected to a Zeiss Photomicroscope and Zeiss Inverted Microscope. This provided a time-resolution of x0 sec. Measurements were made from scanning electron micrographs and from freeze-frame images projected on a video monitor calibrated with a stage micrometer. Fluid flow was

290

GAINES & TAYLOR-DINOFLAGELLATE TRANSVERSE FLAGELLUM 29 1

P

with a mixture of 0.5% (v/v) glutaraldehyde, 1% (w/v) osmium tetroxide, 0.2 M NaC1, 5 mM CaCl,, buffered with 0.05 M sodium cacodylate. Fixation was camed out in the cold and followed bv collection of cells on Nucleouore filters. rinsing with

A

V

P

D

Fig. 1. Examples of directional terminology used in this paper. (a) Ventral view showing the two rotational possibilities. (b) Left side view.

Key to Fig. 1 : A = anterior/apical, D = dorsal; DD = dexiotropic rotation; L = 1eWsinistral; LL = leiotropic rotation; P = posterior/antapical; R = right/dextral.

visualized by suspending 0.5-pm latex spheres in the fluid sur- rounding the cells. In some cases, low (less than 0.4%) concentrations of methyl cellulose (4000 cp) were used to slow the cell movement and to prevent aggregation of latex particles.

Scanning electron microscope preparation involved fixation

- progressively decreasing concentrations of seawater, dehydra- tion with acetone, critical-point drying through carbon dioxide, and sputter-coating with gold. Cells were examined at 15 kV with an IS1 Alpha-9 SEM.

Observations were made on cultures from the North East Pacific Culture Collection in Vancouver, British Columbia and on communities of field material collected at Friday Harbor, Washington during August 1982 and 1983 (Table I). The field material was usually concentrated gently by the method of Dod- son & Thomas (7).

RESULTS Our observations on living material show that the form of

the transverse axonemal wave is invariably helical. Each point on the axoneme rotates clockwise when viewed from the flagellar base towards its tip. Identical SEM preparations can produce both appearances referred to earlier: helical and undulating, depending on the species. For example, Peridinium cinctum and Ensiculifera sp. always show helical transverse flagella (Figs. 2, 3) (1,28) whereas Gymnodinium sanguineum and others always show an undulating flagellar wave (Figs. 4, 5) (1 4). The undulating appearance seems to be a relaxation artifact, judging by our analysis of the species listed in Table I. The helix is not isotropic, the outer edges being more vertical than their inner counterparts (Figs. 2, 3, 6-9), in agreement with Berdach (1). This configuration means that there is a greater velocity of the outer edges compared to that of the inner ones. The usual wave- length of the healthy beat appears to be 3-6 pm, in agreement with the data of Peters (26).

Invariably, the flagellar wave travels from the base to the tip (Fig. 10). No reversals in wave direction were seen. Occasion- ally, when the flagellum stopped beating, there was a slight recoil (e.g. in Protoperidinium excentricum ), but no sustained re-

Figs. 2, 3. SEM of flagellar wave form of Ensiculifea sp. seen in dorsal (2) and apical (3) views. Scales = 10 pm.

292 J. PROTOZOOL.. VOL. 32, NO. 2, MAY 1985

TABLE I. Dinoflagellate species specifically examined in this study.

Amphidinium asymmetricurn Kofoid & Swezy, 192 1

A . carterae Hulburt, 1957 Ceratium azoricum Cleve, 1900 C. fusus (Ehr.) Dujardin, 184 1 C. longipes (Bailey) Gran, 1902 Ceratocorys horrida Stein, I883 Cochlodinium polykrikoides

Margalef, 1961 Crypthecodinium cohnii (Seligo)

Chatton, in Grase, 1952 Dinophysis acuminata Claparkde

& Lachmann, 1859 D. acuta Ehrenberg, 1839 D. fortii Pavillard, 1923 D. infundibulus Schiller, 1928 D. rotundata Claparkde & Lach-

mann, 1859 Ensiculifera sp. Erythropsidinium agile (Hertwig)

Silva, 1960 Gambierdiscus toxicus Adachi &

Fukuyo, 1979 Gonyaulax grindleyi Reinecke,

1967 G. polyedra Stein, 1883 “Gonyaulax” rugosum Wailes,

Gymnodinium sanguineum Hira-

G. simplex (Lohmann) Peters,

Gyrodinium corallinum Kofoid

G. spirale (Bergh) Kofoid & Swe-

Heterocapsa sp. Katodinium fingiforme (Anissi-

Kofoidinium sp. Nematodinium armatum Kofoid

Noctiluca scintillans Macartney,

1928

saka, 1922

1930

& Swezy, 1921

zy, 1921

mova) Loeblich, 1965

& Swezy, 1921

1810

Oblea sp. Ostreopsis sp. Oxyrrhis marina Dujardin, 184 1 Polykrikos beauchampii (Chat-

ton) Loeblich, 1980 P. kofoidii Chatton, 19 14 Prorocentrum concavum Fukuyo,

P. gracile Schiitt, 1895 P. lima (Ehr.) Dodge, 1975 P. micans Ehrenberg, 1833 P. rnexicanum Tafall, 1942 Protogonyaulax catenella (Whe-

don & Kofoid) Taylor, 1979 P. tamarensis (Lebour)

Taylor, 1979 Protoperidinium conicum (Gran)

Balech, 1974 P. crassipes (Kofoid) Balech,

1974 P. denticulatum (Gran & Braa-

rud) Balech, 1974 P. depressum (Bailey) Balech,

1974 P. excentricum (Paulsen) Balech,

1974 P. pellucidum (Bergh) Balech,

1974 P. steinii (Jargensen) Balech,

1974 Scrippsiella subsalsa (Ostenfeld)

Steidinger & Balech, 1977 S. sweeneyae Balech ex Loeblich,

1965 S. trochoidea (Stein) Loeblich,

1976 Symbiodinium microadriaticum

Freudenthal, 1928 Warnowia violescens (Kofoid &

Swezy) Lindemann, 1928 Zygabikodinium lenticulatum

Loeblich & Loeblich, 1970

1981

versals occurred. Also, occasionally the fibrillar strand contract- ed, pulling the flagellum close to the cell surface and stopping the beat. This was most clearly evident in those species pos- sessing wide girdle lists, (e.g. Dinophysis spp., Ceratocorys hor- r ida) , in which the flagellum when beating extends far from the cell surface. In Prorocentrum, which has no cingulum, the flagellum passes first over the least excavated (left, according to Butschli [3]) valve, as shown by Biecheler (2) and Soyer et al. (30). In benthic Prorocentrum species (e.g. P. l ima) , the flagellum beats principally anteriorly and is much shorter than in planktonic species (e.g. P. micans), where the flagellum extends posteriorly. In dinokonts the flagellum invariably beats to the cell’s left, extending to a variable degree around the cell, depending on the species (e.g. the flagellum does not completely encircle the cell in many species of Protoperidinium and Ce- ratium ). In the multiple-flagellated genus Polykrikos, the several transverse flagella beat approximately in synchrony.

All cell rotation in the dinoflagellates seen by us has been leiotropic (i.e. in the direction of the transverse flagellar beat) (Fig. 11). In view of the large number of species and the phy- logenetic diversity of this material, the invariably leftward en-

Figs. 4, 5. Flagellar wave forms of (4) Gymnodinium sanguineum and (5) Gyrodiniurn uncatenum seen in dorsal view by SEM. Scales = 10 pm.

circlement of the cell by the transverse flagellum, and the con- sistent base-to-tip direction ofbeat, we believe this to be universal in dinoflagellates, despite reports of predominantly dexiotropic rotation by Kofoid (20) and Jahn et al. (1 7).

The posterior component of fluid flow caused by the transverse flagellum is as described by Jahn et al. (17). The circum- ferential component, however, travels in the opposite direction from the flagellar wave, contrary to their assumption.

DISCUSSION Our observations require revisions in the LeBlond & Taylor

(23) model of the mechanics of the dinoflagellate transverse flagellum. The fact that they used a hemi-helical, instead of helical, edge-shape does not require significant modification. It should be stressed that although the outer edge undulates in a helical fashion, the inner edge of the ribbon does not beat and is curvilinear. Consequently, the propulsive surface is not helical; it is more like the edge of the fin of a ray or skate. Together with previous authors, we have seen that the fluid adjacent to the cingulum is propelled across the cingulum towards the posterior of the cell; however, the lateral cingular flow is from the tip to the flagellar base and not in the direction of wave prop- agation assumed by Jahn et al. (17) and LeBlond & Taylor (23). The only other flagellate group in which fluid flow is directed opposite to the flagellar wave motion is the Chrysomonadida (1 8). The reversed fluid flow associated with the chrysomonad anterior flagellum has been attributed to rigid mastigonemes (27), whose net propulsive effect is opposite to and greater than that of the axoneme (Fig. 12a). The current interpretation of

GAINES & TAYLOR- DINOFLAGELLATE TRANSVERSE FLAGELLUM 293

Figs. 6-9. Video photographs of beating transverse flagella of various dinoflagellates, showing the predominantly vertical outer loops and their respective wavelengths. (6) Dinophysis sp. (7) Protoperidiniurn depressum (8) Zygabikodinium lenticulatum (9) Warnowiu violescens. Scales = 10 pm.

the locomotive effect of the tubular mastigonemes (e.g. 15, 16) relies upon their stiffness or active manipulation. Their actions are oarlike and analogous to the mechanics of the parapodia of polychaete worms (1 1). Thrust is produced by the rapid movement of the mastigoneme as it snaps around the outer bends of the flagellum. In dinoflagellates the fine mastigonemes cannot be seen with the light microscope and are not preserved in an extended state in SEM preparations. At present, they can only be considered in a theoretical sense, with the assumption that they remain more or less normal to the axoneme. Previous authors have not included them in their models of motion.

The strong antero-posterior flow from the transverse flagellum does seem to be due to the traveling series of postero-sinistrally directed propulsive faces of the ribbon, moving from base-to- tip as the axoneme beats clockwise. The power stroke occurs outside the cingulum, where the distance from the axoneme to the fibrous strand is greatest and part of the wave projects beyond the girdle rims. This effect is enhanced by the asymmetry of the beat, which is faster at this point, thus increasing the drag and consequently the water movement. The inward curves of

the flagellum appear to have very little effect on fluid flow as compared to the outward curves, both because of the smaller propulsive surface of the ribbon and because of the lower velocity of return.

We believe that the reports of reversals during swimming ( 1 7, 20,25,26) are due to changes in the plane of swimming relative to the observer, as we have seen similar illusions when using low magnifications. The transparency of the cell permits views of its lower surface, resulting in the optical reversal of its apparent sense of rotation. Thus, a cell may appear to change rotation direction without stopping. Frame-by-frame television observations clearly show the latter to be a focusing artifact. This does not apply to the direction of travel of the cell, which is altered or even reversed by the longitudinal flagellum (13, 26). Ceratium spp. are capable of sustained backward swimming using the longitudinal flagellum (24, 26, Taylor, unpubl. obs.).

We can only attribute the leiotropic cell rotation to the mod- ifying influence of the mastigonemes on the outer edge of the flagellum, analogous to the chrysomonad anterior flagellum (Fig. 12a); however, the dinoflagellate axoneme describes a helix, not

294 J. PROTOZOOL., VOL. 32, NO. 2, MAY 1985

Fig. 11. Tracings from successive video frames of Protoperidiniurn conicurn swimming, showing the leiotropic rotation of the cell. Time interval between frames is % sec. Sequence is from bottom to top.

Fig. 10. Tracings from successive video frames of Protoperidiniurn conicurn (antapical view of the girdle), showing the leiotropic direction of transverse flagellar beat. One wave is blackened for reference. Time interval between frames is xs sec. Sequence is from bottom to top. Arrow indicates direction of cell rotation.

a planar wave as in chrysomonads, and it has mastigonemes only on one side (Fig. 12b, c). Assuming that the mastigonemes remain approximately in the same plane as the ribbon, the trans- lation of flow should be similar to that on one side of the chrysomonad flagellum. Some feathering of the mastigonemes may also occur, but its effects can only be determined by direct observation or theoretical modeling. Our model of the dinoflagellate transverse flagellum, including beat asymmetry and mastigonemes, is depicted in Fig. 13. The number and arrangement of mastigonemes are speculative (18, 27, 28).

The dexiotropic flow and leiotropic cell rotation necessitates reinterpretation of associated morphological features such as cingular and list configurations. The posterior redirection of flow postulated by LeBlond & Taylor (23) to result from the enlarged left sulcal list cannot arise because the direction of flow in the cingulum is the reverse of that assumed by them. The enlarge- ment of the list appears to protect the sulcus from lateral flow,

and its curvature, when present, follows the streamlines of fluid flow around the rotating cell. The lists also appear to serve as protection for the flagella. Contraction of the fibrous strand causes the transverse flagellum to pull inwards, associated with stoppage of the beat. The longitudinal flagellum of Cerutium also retracts into the sulcus by contraction of a similar fibrous strand (24). The commonness of left-handed cingular displace- ment can no longer be explained as a posterior channeling of cingular flow.

The relative strengths of the posteriorly directed and laterally directed forces generated by the transverse flagellum can be appreciated by considering those forms having strong displace- ments of the ends of the transverse flagellum (e.g. Prorocentrum, Gyrodinium). Swimming in these forms is not noticeably dif- ferent from that in other forms, indicating that the net posterior propulsive effect of the ribbon face is much greater than the net anterior propulsive effect of the mastigonemes. Otherwise, the posterior inclination of the encircling flagellum should result in a reversal of swimming direction. Prorocentrum swims in the same manner as dinokonts, indicating that the cingulum is not necessary for forward swimming, contrary to the model of Jahn et al. (17).

Jahn et al. (17) and LeBlond & Taylor (23) attributed the

GAINES & TAYLOR-DINOFLAGELLATE TRANSVERSE FLAGELLUM 295

H

a

W H

Fig. 12. Comparison of chrysomonad flagellum (a) with dinoflagellate transverse flagellum seen in (cell) apical (b) and lateral (c) views. Direction of axonemal wave travel is left-to-right. Arrows refer to net direction of fluid flow. Scales = 1 fim.

Fig. 13. Lateral view of model of transverse flagellum proposed here, showing beat asymmetry and mastigonemes according to Berdach (1) and Rees & Leedale (28). In this view, the axonemal wave travels from left to right, and the organism rotates in the same direction. The cell apex is beyond the top of the figure. Scale = 1 pm.

dextral torsion of the cingulum seen in many genera (e.g. Go- nyaulax, Cochlodinium) to the force ofthe flagellar base pushing against the basal insertion. In view of our observations, this cannot be invoked. It is also not a case of streamlining, as the cell rotates contrary to the smoothest flow down the cingulum. Instead, torsion may be an adaptation to increase the power of the transverse flagellum by increasing its length. The only way this can be accomplished is by increasing the number of revo- lutions around the cell described by the cingulum and transverse flagellum. In cells having torsion the transverse flagellum usually extends for much of the cingular distance (e.g. Cochlodinium). In Ceratium, in which most of the forward thrust seems to be due to the longitudinal flagellum and turning is minimal, the cingulum is often incompletely developed (3 1) and the flagellum is short.

We have not considered the function of the longitudinal flagellum in this paper, although it clearly has a very important role to play in dinoflagellate locomotion. With the exception of a few genera, such as Ceratium (above), the longitudinal flagellum appears to act primarily as a rudder; however, this includes not only directional change but also the stabilizing of the cell during transverse flagellar beat and some forward propulsion of its own.

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Received 6 XII 83; accepted 22 III 84

J. Protozool.. 32(2), 1985, pp. 296-305 0 1985 by the Society of Protozoologists

Cytokinesis of ThuricoZa foZZicuZa~a (Ciliophora, Peritrichida) SIMONE EPERON

Biology of Protists, University of Geneva, CH-I21 I Geneva 4, Switzerland

ABSTRACT. Thuricola folliculata is a sessiline, loricate peritrich ciliate. Its somatic pellicle consists of annular transverse crests and includes the plasma membrane, the alveolus, the electron-dense epiplasm, and the subepiplasmic layer. Cytokinesis occurs along an oral-aboral median plane where two fissures develop, one at the oral and one at the aboral end of the peritrich. Each fissure results from simultaneous formation of two furrows on opposite sides of the cell. At the end of cytokinesis, both trophozoites remain joined to each other by an intercellular bridge. During cytokinesis, microfilament bundles appear at the level of the subepiplasmic layer in the fission plane; they are distributed in two arcs, one oral and one aboral, and may be responsible for the formation of the four furrows. The cross-sectioned microfilament arc is 1 pm wide and about 0.1-0.2 pm thick at first and later can be more than 1 pm in diameter; it shows many microfilaments, 3-10 nm in diameter and oriented parallel to the fission plane, and also many dense corpuscles 25-55 nm in diameter. Then both arcs join each other to form a microfilament ring. This ring is delimited by discontinuous dense borders and a boundary layer. The microfilament ring seems structurally analogous to the contractile ring of various dividing cells, where it works like a sphincter. The dense corpuscles, the discontinuous dense borders, and the boundary layer of T. folliculata have not been reported in any other ciliates.

YTOKINESIS of animal cells occurs by furrowing (22). folliculata (0. F. Miiller, 1786) has previously been studied (6, C Active constriction has been postulated as the principal 8). In this cell, cytokinesis can be observed in vivo and moni- mechanism involved in furrowing, and strong support for this hypothesis comes from the discovery of a ring of contractile microfilaments below the fission furrow (23,35). This ring works like a sphincter and is observed in invertebrate eggs as well as in vertebrate cells (15, 32, 36); it is composed of a contractile bundle about 0.1 pm thick and 10 pm wide, which is in turn comprised of microfilaments 3-1 0 nm in diameter. The mechanism of cell constriction seems analogous to muscular contraction since in some cells the actin filaments of the contractile ring appear to slide relative to one another (1 2, 32). To date, contractile rings in protozoa have been described in Amoeba proteus (13), Nassula sp. (41), Tetrahymenapyriformis (27,43), T. thermophila (1 6), T. paravorax (24), Paramecium aurelia (17), and Stentor c o e r u l m (4). Instead of a contractile ring, a band of microtubules has been observed in Amphidinium carterae (28).

The binary fission of the sessiline loricate peritrich Thuricola

tored when desired; observations by serial sectioning for electron microscopy can be made on cells in a known fission stage. In the present work, cytokinesis is described by light- and transmission electron microscopy.

MATERIALS AND METHODS The culture method for T. folliculata is described elsewhere

(6, 7, 14). Dividing T. folliculata were isolated and observed in vivo by phase contrast microscopy. To obtain a frontal view of cytokinesis (Fig. I), cells were sometimes treated with 0.5% pronase dissolved in culture medium (Fig. 4).

For transmission electron microscopy, cells were fixed either for 60 min in 2% (w/v) OsO,, buffered at pH 7.2 with Michaelis buffer (Na-veronal and Na-acetate at a final concentration of 7.14 mM), or they were fixed for 60 rnin in 2-2.7% (v/v) glutaraldehyde, buffered at pH 7.2 with Ssrensen buffer (composed either of 14 mM NaH,PO, and 36 mM Na,HPO, or of 19 mM NaH,PO, and 48 mM Na,HPO,), rinsed first in Ssrensen, then in Michaelis buffer, and postfixed for 60 min in 2% (w/v) OsO,, buffered at PH 7.2 with Michaelis buffer. Cells were dehydrated in ethanol followed by ProPYlene oxide and embedded in won. Sections were stained for 20 min in uranyl acetate and 15 min

I I am grateful to Professor G. de Haller for his encouragement in this work. I thank Drs. R. K. Peck and J. Fahmi for the careful reading and constructive criticism of the manuscript, MS. Y. Eperon, H. Bruesmann. E. Martinez. M. Vautravers. and E. Wider for their efficient tech&al assistance. Supported in part by Swiss National Funds of Scientific Research grant 3.6 5 7 .O. 80.

in lead citrate (30, 33). All figures are of the ciliate Thuricola folliculata. In the trans-

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