Fine Structure of the Longitudinal Flagellum in Ceratium Tripos, a Marine Dinoflagellate

J. Cell Set. 58, 109-123 (1982) 109 Printed in Great Britain © Company of Biologists Limited 1982

FINE STRUCTURE OF THE LONGITUDINAL FLAGELLUM IN CERATIUM TRIPOS, A MARINE DINOFLAGELLATE

TADASHI MARUYAMA Laboratory of Microbial Ecology, Department of Biology, Tokyo Metropolitan University, Fukasawa, Setagaya-ku, Tokyo, 158 Japan

SUMMARY The fine structure of the longitudinal flagellum in Cerathtm tripos, which performs not only undulations but also retractions, has been examined in both the retracted and relaxed states. Although conventional fixation always triggered retraction, the flagellum was found to remain relaxed when it was washed briefly with CaI+-free medium prior to fixation. Previous light- microscopic investigation showed that it contained two fibres, the axoneme and the R-fibre. The present study by transmission electron microscopy has revealed that the axoneme that appeared to be a single fibre under the light microscope is a bundle of four fibres; the 9 + 2 microtubular axoneme, the packing material, the striated fibre, and the paraxial fibre. The first two are common in the longitudinal flagella of dinoflagellates, and the axoneme presumably generates the undulation. The last two are new and unique to the longitudinal flagellumo f Ceratium. The R-fibre, which probably contracts to fold the flagellum during retraction, consists of fine filaments, which pursue a loosely spiral course in the contracted state, but align longitudinally in the relaxed state. Periodic striations appear only on the extended R-fibre. The R-fibre shortens to approximately one third of its extended length and pursues a left-handed helix. The packing material, which sticks to the microtubular axoneme on one side, is connected with the R-fibre on the other side at intervals of approximately 5-8 fim. The retraction seems to be regulated by the Cat+ concentration in the flagellum.

INTRODUCTION The longitudinal flagellum of Ceratium moves in two dissimilar ways (Schiitt, 1895; Peters, 1929; Afzelius, 1969; Maruyama, 1981). It undulates during forward or back- ward swimming, and the undulatory waves propagate from the base to the tip. The wave was reported to be planar and to consist of circular arcs and connecting straight segments (Brokaw & Wright, 1963), as reported also in invertebrate sperm tails (Brokaw, 1965). The flagellum retracts when the cell body, especially the tip of the apical horn, is stimulated mechanically. It is folded from the tip to the base, and is finally installed in the sulcus on the ventral side of the cell body (Maruyama, 1981). Light-microscopic observation of the retracted flagellum showed two fibres, the axoneme and the R-fibre (Maruyama, 1981). The axoneme was presumed to contain the 9 + 2 microtubular axoneme, which generates the undulation. The R-fibre, which is unique to the longitudinal flagellum of Ceratium, was thought to contract to fold the flagellum. The fine structure of the longitudinal flagella in some dinoflagellates, which were not reported to be retractile, has been described (Leadbeater & Dodge, 1967; Lee, no T.Maruyama 1977). They consist of the 9 + 2 microtubular axoneme and associated packing material. However, the fine structure of the longitudinal flagellum in Ceratium has not been described, apart from a brief description in a freshwater species, Ceratium hirundinella, which showed the 9 + 2 microtubular axoneme (Dodge & Crawford, 1970). The present paper describes the fine structure of the longitudinal flagellum in Ceratium tripos, in both retracted and relaxed states. It is a complex flagellum containing five fibres. The axoneme, which appears to be a single fibre under the light microscope, has been shown to be composed of four fibres including the 9 + 2 microtubular axoneme and three associated fibres. The R-fibre consists of fine filaments whose arrangement is changed during retraction.

MATERIALS AND METHODS C. tripos was grown as reported previously (Maruyama, 1981). After a month or more of incubation, cells were harvested by low-speed centrifugation at 1000 to 2000 rev./min for 30-60 3 at room temperature in conical centrifugation tubes with screw caps. Cells were fixed with a phosphate-buffered glutaraldehyde fixative containing 5 % glutaraldehyde, o-8-i-o M-glucose, and o-i M-Na phosphate (pH 7-2-7-4) or with a cacodylate- buffered glutaraldehyde fixative containing 5% glutaraldehyde, 1-3 M-glucose, and o-i M-Na cacodylate (pH 7-4) for 1 h at room temperature. These procedures always triggered retraction of the flagellum (Maruyama, 1981). After washing with a graded series of glucose solutions in the same buffer, post-fixation with 1 % 0sO4 in the same buffer without glucose for 45-60 min at room temperature, and dehydration through a graded series of ethanol and n-butylgh/ci- dylether (QY-i) (Kushida, 1963), they were embedded in Spurr low-viscosity resin (Spurr, 1969) or in Quetol 812 (Nisshin EM Co. Tokyo). The viscosity of Quetol 812 was so high that the cells were embedded in 1-2 % agar prior to dehydration. Some cells in Spurr resin were placed on a glass slide, covered with a coverslip. They were hardened by heat at 60 CC for 24 h, and then observed under a light microscope equipped with phase-contrast or Nomarski optics. Lengths of structures were measured on enlarged micrographs. Sections were doubly stained with 1 % uranyl acetate in 50 % ethanol and with lead citrate (Reynolds, 1963), and were observed in a Hitachi H-300 electron microscope operated at 75 kV. Although the longitudinal flagellum is always retracted by conventional fixation, it remains relaxed if it is washed briefly with CaI+-free medium prior to fixation. Two different Cal+free media were used; Cat+-free ASW and Ca1+-free low ionic strength medium. The former contained 477 mM-NaCl, 9-7 mM-KCl, 20-9 mM-MgCl,, 27-6 mM-MgSO|, 5 mM-ethylenglycol- bis-OJ-aminoethyletheO-iV.iV'tetraacetic acid (EGTA), and 30 mM-Tris-HCl (pH 80). The latter was composed of 10 mM-ethylenediaminetetraacetic acid (EDTA), 1 M-glucose and 60 mM-Tris-HCl (pH 80). The fixation procedure was as described above.

RESULTS Light microscopy of the retracted and relaxed longitudinal flagellum The longitudinal flagellum is invariably retracted into the sulcus by fixation (Fig. 1). The axoneme is folded primarily in a plane, but is twisted secondarily in a right- handed helix. Close observation of the tightly retracted flagellum revealed that the R-fibre took a left-handed spiral course beneath the folded axoneme when it was looked at from the outer helical surface. The R-fibre intersects the axoneme at intervals. Although the relaxed flagellum could be fixed by washing it with Ca^-free media Longitudinal flagcllum of Ceratium 111 before fixation, it was unstable in them, expecially in the Ca*+-free ASW, and it disintegrated into a thin thread within a few minutes at room temperature. The flagellum seemed to be relatively stable in the Ca2+-free low ionic strength medium (Fig. 2), and could be reactivated, folding on application of Ca2"1" locally with a fine glass capillary. The thin thread that remained after disintegration could not be reactivated. These findings indicate that the retractile mechanism remains intact in the Ca2+-free medium until decomposition takes place.

Fig. 1. A light micrograph of a retracted longitudinal flagellum in the sulcus. The cell was fixedb y conventional fixationan d embedded in Spurr resin. Nomarski differential interference contrast optics. Rf, R-fibre; ax, axoneme; ihs, inner helical surface; ohs, outer helical surface; s, sulcus. Bar, io/(m. Fig. 2. A fully relaxed longitudinal flagellum in the Ca1+-free low ionic strength medium. Unfixed preparation. Phase-contrast optics. Bar, 50 /*m. Fig. 3. A higher magnification micrograph of a fully relaxed longitudinal flagellum fixed and embedded in Spurr resin. Nomarski optics. Bar,

The R-fibre elongates in the relaxed flagellum and lies beside the axoneme (Fig. 3). Although it was easily observed in the retracted flagellum, the R-fibre was difficult to distinguish in the fully relaxed flagellum, probably because it lay close to the axoneme and perhaps because the extended R-fibre is thinner than the contracted one. This indicates that the length of the extended R-fibre is roughly equal to that of the axoneme, which is presumably constant during motion. The extent of contraction of the R-fibre can be calculated from the lengths of the contracted R-fibre and the corres- ponding axoneme. These lengths were measured in a retracted flagellum, which was squashed between the cell body and a coverslip so as to lie in the same focal plane. ii2 T. Maruyama Seven turns of the contracted R-fibre measured 24-4 fim (35 /im/turn) and the cor- responding axoneme was 78-1 fim long (n-2/im/period). The extent of contraction was calculated to be 1/3-2. If the retracted R-fibre makes a helix, the length of one turn (L) can be computed by the equation: L = {A*n*+B*)i, where A is the diameter of the helix and B is the pitch. Assuming that the contracted R-fibre was a helix, the diameter and the pitch were measured to be io±o-3/im (mean + s.D., n = 11) and i-8±o-7/«n, respectively, in the middle portion of the flagellum. The length of R-fibre per turn was estimated to be 36 fim. The length of the folded axoneme per period measured 115 + 1-5 fim (n = 11). The ratio of contracted to extended length would be i:3-2, which agrees with the value obtained above from direct measurement on the flattened flagellum. This indicates that our assumption is reasonable. The distance along the axoneme between the intersections of the R-fibre and the axoneme is calculated to be approximately 5-8 fim.

Internal structure of the retracted longitudinal flagellum Fig. 4 shows a low-magnification electron micrograph of a retracted longitudinal flagellum. The folded axoneme, which appeared to be a single fibre under the light microscope, is in fact a bundle of several fibres including the 9 + 2 microtubular axoneme. This bundle will be referred to as the axonemal complex. It lies beneath the flagellar membrane of the outer helical surface, as shown previously by scanning electron microscopy (Maruyama, 1981). Prominent lamellar foldings on the inner helical surface are filled with a large amount of the flagellar matrix. Sections of the contracted R-fibre confirmed the view that it takes a helical course. The diameter of the helix appeared to be O-6-I-2 fim and the pitch O-8-I-3 fim, in the middle portion; the length of R-fibre per turn was calculated to be 2-3-4'O fim. These values are similar to those obtained by light microscopy.

Fine structure of the axonemal complex The axonemal complex is composed of four fibres (Fig. 5). The 9 + 2 microtubular axoneme has a typical 9 + 2 array of microtubules and its diameter measured 0-23 + 0-02 fim (n = 30). Some electron-dense material was frequently observed in some of the peripheral doublets, except near the tip. The packing material consists of two parts, a central core and many radiating fin- like structures at the periphery (Fig. 5). A few bundles of fine tubules that have not

Fig. 4. A transmission electron micrograph of a retracted longitudinal flagellum fixed by conventional fixation. The axonemal complex consisting of the axoneme (ax), the packing material (pm), and the striated fibre (sf), faces the flagellar membrane of the outer helical surface (o/u). The paraxial fibre can not be recognized well at this low magnification. The R-fibre (Rf) seems to pursue a helical course. The lamellar folds protrude from the inner helical surface (ihs). Bar, i-o/tfn. Longitudinal flagelhim of Ceratium

ohs

ihs

Longitudinal flagelhtm of Ceratium 115 been reported in other dinoflagellates were found in the central core. The tubules are 10 nm in diameter and are packed compactly in the bundle. The number of tubules in a section through the packing material varies from o to over 20; it is greater in the proximal portion and decreases distally. A longitudinal section of the 10 nm tubule bundle usually shows a regular cross-banding pattern (Fig. 6). The overall periodicity was approximately 59 nm. Each repeat period consists of two similar dark bands 17-18 nm in width, a 5 nm transparent band and a wide translucent band of 16-18 nm. It is, however, not clear whether these bands are on the tubules or on adjacent structures. The peripheral radiating structures sometimes seemed to connect with those dark bands. Viewed from the outer helical surface of the retracted flagellum, the packing material always attaches to the underside of the axoneme. The shape of the packing material is a little different in the proximal region (Fig. 11), where a prominent fin- like structure, which is inconspicuous in the middle or distal region, protrudes from the side opposite to the junction between axoneme and packing material; this will be referred to as the lateral fin. Tangential sections of the packing material show a lattice-like appearance (Figs. 4, 6), which is similar to that seen in other dinoflagellates (Leadbeater & Dodge, 1967; Lee, 1977). In a cross-section of the axonemal complex, an electron-dense triangle is located beside the axoneme (Fig. 5). The longest side measured 73 ± 14 nm (n = 10), and the other two were 51 + 11 nm, and 58 ± 16 nm. It is a triangular prism with cross striations, which have an overall periodicity of 46 ± 2 nm (n = 9) (Figs. 6, 7). Each period contains a dark band, which is composed of two lines with an interval of approximately 6 nm, and two thinner lines, which lie between two adjacent dark bands (Fig. 7). This prism is also unique to the longitudinal flagellum of Ceratium and will be called the striated fibre. It has a unique property: it kinks at several points in the curved portions of the axonemal complex but remains straight in the linear portions (Fig- ?)• In transverse section the fourth fibre is seen as an arch approximately 73 nm long and 13 nm wide (Fig. 5). It usually is attached to one of the peripheral doublets of the axoneme. Tangential or longitudinal sections of this structure did not show any striation or substructure. This will be called the paraxial fibre.

Fig. 5. A higher-power electron micrograph of the axonemal complex in a retracted longitudinal flagellum. The axoneme (ax) is associated with the packing material (pm), the striated fibre (if), and the paraxial fibre (pqf). Two bundles of 10 nm tubules (t) are seen in the central core of the packing material. Bar, 0-2 fim. Fig. 6. An electron micrograph of a longitudinal section of the packing material (pm) and the striated fibre (sf). A regular cross-banding pattern is seen on the bundle of 10 nm tubules (t). The lattice-like appearance of the peripheral radiating structure of the packing material is seen at the top. Bar, 0-2 fim. Fig. 7. A striated fibre (sf) kinked at the arrowhead. Bar, 0-2 fim. Fig. 8. A higher-power electron micrograph of the retracted R-fibre (Rf). The arrowhead indicates the packing material-R-fibre junction. Bar, 0-5 fim. n6 T. Maruyama All these accessory fibres oridinate in the proximal portion of the flagellum and do not enter the cell body. They are always located around the axoneme to constitute the axonemal complex. Their angular positions around the centre of the axoneme were determined on the electron micrographs. The angles were 155 + 8° (n = 35) between the paraxial fibre and the striated fibre, 73 ± 90 between the striated fibre and the centre of the packing material, and 133 + 90 between the centre of the packing material and the paraxial fibre. Because the axoneme invariably faces the flagellar membrane of the outer helical surface and the packing material always attaches to the axoneme at the opposite side, the accessory fibres are straight and parallel to the axoneme.

Fine structure of the R-fibre The contracted R-fibre consists of homogeneous fine filaments whose diameter is 5-7 nm (Fig. 8). No limiting membrane surrounds it. The filaments swirl loosely around the centre of the R-fibre in cross-section. Longitudinal sections show that they pursue a roughly spiral course around the axis of the R-fibre. Since the orientation of the different filaments is not uniform, the filaments seem to be entangled to make a meshwork. Fig. 9 shows the basal region of a relaxed longitudinal flagellum. Regular striations appear on the extended R-fibre. Two or three stripes are seen in each period, spaced at intervals of 20-40 nm (Fig. 10). The variation in overall periodicity of between 111 and 162 nm might be related to the degree of relaxation or contraction. Unlike those in the contracted fibre, the filaments are arranged in longitudinal arrays in the relaxed fibre. The diameter of each filament in the extended fibre was 4-7 nm and was not much different from that in the contracted state. Cross-sections of the extended R-fibres were mostly round, but in the proximal portion were sometimes ovoid or pear-shaped (Fig. 11). The diameter of the relaxed R-fibre was estimated from the cross or oblique sections to be 0-56±o-i5/tra (n = 16) in the proximal, 0-29 ±0-07/mi (n = 13) in the middle, and 0-24 ± 0-07 fim (n = 5) in the distal portion. Comparison of these values with those in the retracted state suggest that the relaxed R-fibre is thinner than the contracted one. A partially relaxed R-fibre was sometimes observed (Fig. 12). The striations were not clear, but the periodicity was approximately 70-80 nm and was shorter than that in the fully relaxed state. Examination of successive sections near the intersection of the R-fibre and the axonemal complex in the retracted flagellum has shown that the R-fibre is connected with the packing material on the side opposite to the junction between axoneme and packing material (Figs. 4, 8). No specific linkage was found between them. Previous light-microscopic observation showed that the R-fibre originates near the base and terminates near the tip of the flagellum (Maruyama, 1981). Fig. 13 shows that it originates at the flagellar membrane a little above the basal body in a retracted flagellum. A space of about 20 nm was observed between the R-fibre and the membrane. Longitudinal flagellum of Ceratium

Fig. 9. A longitudinal section of the basal region of a relaxed longitudinal flagellum. Regular striations are seen on the extended R-fibre (Rf). bb, basal body;/m, flagellar membrane; pm, packing material. Bar, i-o/tm. T. Maruyama Longitudinal flagelhm of Ceratium

*"• t Fig. 14. An electron micrograph of the striated rootlet (r) originating from the proximal portion of the basal body (66). Bar, 0-5 ftm.

The striated rootlet A striated rootlet was reported in Ceratium hirundinella (Dodge & Crawford, 1970). Although the details of the three-dimensional structure around the basal body in C. tripos are not clear, a striated rootlet was sometimes observed to originate at the proximal portion of the basal body and extend toward the cell membrane (Fig. 14). The periodicity of the striations was approximately 33 nm.

DISCUSSION The longitudinal flagellum of C. tripos is a complex flagellum consisting of the axonemal complex and the R-fibre. The 9 + 2 microtubular axoneme in the axonemal

Fig. 10. A higher-magnification electron micrograph of a relaxed R-fibre. Note parallel arrays of filaments and the regular striations. Bar, 0-5 fim. Fig. 11. A cross-section of a relaxed longitudinal flagellum in the proximal portion. The lateral fin (If) protrudes from the packing material (pm). ax, axoneme; Rf, R-fibre. Bar, i-o/tm. Fig. 12. A tangential section of a partially relaxed R-fibre (Rf). The striations are vague. Bar, ro/im. Fig. 13. The membrane-R-fibre junction (arrowhead) near the base of a retracted longitudinal flagellum, where the R-fibre (Rf) originates. Bar, i-o/tm. 120 T. Maruyama complex always faces the flagellar membrane in both retracted and relaxed flagella (Figs. 4, n). Although the present study does not show any connections between the axoneme and the flagellar membrane, they may be linked together as reported in some other cilia and flagella (Dentler, 1981). The size and microtubular organization of the axoneme in the longitudinal flagellumar e typical of those in various organisms (Sleigh, 1962; Afzelius, 1969; Warner, 1974). This fact and the similarity of the undulatory wave-form to those of sperm tails (Brokaw & Wright, 1963; Brokaw, 1965) together support the idea that the undulation of the longitudinal flagellum is generated by the axoneme (Maruyama, 1981). The packing material is unlikely to participate in the retractile flagellar motion, because it is common in non-retractile longitudinal flagella of other dinoflagellates, though they lack the 10 nm tubules and lateral fin. Its lattice-like appearance in longitudinal section is similar to that of the paraxial rod of the undulating membrane in Trypanosoma (De Souza & Souto-Padr6n, 1980) and of the flagellum in Euglena (Piccinni, Albergoni & Coppelloti, 1975; Bouck, Rogalski & Valaitis, 1978). The functions of these are not known, though ATPase activity was shown in the latter (Piccinni et al. 1975). The packing material seems to stick fast to the axoneme all along its length and to the R-fibre at intervals of approximately 58 fim. Therefore, it links two different motile systems together. The striated fibre kinks in curved regions of the axonemal complex (Fig. 7). This property may be explained if it is a stiff filament, which, once bent, undergoes a reduction in rigidity in the bent portion so that it becomes flexible. Although the functions of the accessory filaments are not clear, they possibly increase the rigidity of the axonemal complex and may facilitate the re-extension of the retracted flagellum. From previous observations the R-fibre was presumed to contract to fold the flagellum, though the extent of the contraction was not determined (Maruyama, 1981). Present data show that it shortens to approximately one third of its extended length, though this calculation is partly based on the assumption that the contracted R-fibre pursues a spiral course. This value is similar to that of the stalk contraction in Car- chesium, which shortens to 22-33% °f lXs resting length (Sugi, 1961), and to that of cell body contraction in Stentor, which shortens to 20-25 % of the extended length (Huang & Mazia, 1975). The R-fibre is likely to produce tension actively during contraction, though there is no firm evidence. The spiral configuration of the contracted R-fibre is advantageous for compact folding of the flagellum, because the distance between adjacent folds of the axonemal complex is determined by the pitch of the spiral instead of its length. The contraction of the R-fibre is accompanied by a reorganization of the filaments from highly ordered longitudinal arrays to disorganized loosely spiral arrays. The spiral arrangement of filaments may bring about the helical coiling of the contracted R-fibre. It is not clear whether the conformation of each filament is changed or not. The periodicity of the striations, which are observed only on the relaxed R-fibre, seems to become shorter and finally disappears during the contraction of the R-fibre. The striation may be a result of overlapping of filaments. Although some sliding may Longitudinal flageUum of Ceratium 121 occur among the filaments, this does not seem likely to provide a good explanation of the mechanism of contraction of the R-fibre. A similar structural change was reported in the myoneme of Stentor; protofilaments of the myoneme that orient longitudinally in the relaxed state transform to randomly oriented 10 nm tubules in the contracted state (Bannister & Tatchell, 1968; Huang & Pitelka, 1973; Kristensen, Neilsen & Rostgaard, 1974; Huang & Mazia, 1975). How- ever, there are some differences: no striation was observed in the extended myoneme,

sf-

bb- bb

Fig. 15. A schematic representation of the longitudinal flagellum in relaxed (A) and retracted state (B). A. A side view of a relaxed longitudinal flagellum. It presumably undulates in a plane perpendicular to the plane of the figure. The left side of the flagellum corresponds to the outer helical surface when it retracts, and the other side, the inner helical surface. B. A retracted longitudinal flagellum viewed from the outer helical surface. The R-fibre (i?/) pursues a left handed helical course beneath the folded packing material that sticks to the underside of the axoneme. ax, axoneme; pm, packing material ;/m, flagellarmembrane ; bb, basal body; r, striated rootlet; cm, cell membrane; sf, striated fibre. and no 10 nm tubule is observed in the contracted R-fibre. Kristensen et al. (1974) showed that the myoneme was isotropic in the contracted state but became bire- fringent during the relaxation. The spasmoneme of Vorticellidae also showed similar optical properties (Weis-Fogh & Amos, 1972). Although ATP-dependent sliding filament models are widely accepted in skeletal muscle contraction (Hanson & Huxley, 1955) and in flagellar motion (Satir, 1968; Summers & Gibbons, 1971), Weis-Fogh and Amos (1972) proposed an ATP-independent rubber-like shortening model for J CEL 58 122 T. Maruyama the contraction of the spasmoneme. This latter model could also explain the mechanism of the R-fibre contraction. However, more detailed work including extraction- model experiments is necessary. There is evidence that the striated rootlets of some cilia and flagellaar e contractile (Simpson & Dingle, 1971; Salisbury & Floyd, 1978; Sleigh, 1979). The striated rootlet in Ceratium is not connected with the R-fibre, because the R-fibre does not enter the cell body. The possibility that the R-fibre is an extension of the striated rootlet, is thus ruled out (Fig. 15 A, B). Many motile systems were reported to be regulated by free Ca£+ concentration; e.g. striated muscle contraction (Ebashi & Endo, 1968), ciliary reversal in Paramecium (Naitoh & Kaneko, 1972, 1973), stalk contraction in Vorticellidae (Hoffmann-Berling, 1958; Amos, 1971), and cell body contraction in Stentor (Huang & Mazia, 1975). The retraction of the longitudinal flagellum is probably regulated by Ca2+, because it is prevented in Ca2+-free medium. In conclusion, the longitudinal flagellum probably has two different motile systems for two dissimilar motions; a typical 9 + 2 microtubular axoneme for undulation and the R-fibre for retraction (Fig. 15 A, B). The packing material, which is one of three accessory fibres of the axoneme, probably links the two systems together. The R-fibre is unique to the longitudinal flagellum of Ceratium. It is composed of fine filaments whose arrangement is changed, from longitudinal arrays with regular striations in the extended state, to loose spiral arrays without striation in the contracted state. The retracted R-fibre pursues a left-handed helical course and lies beneath the folded axonemal complex, when viewed from the outer helical surface.

The author wishes to thank Drs S. Takii and Y. Watanabe for their useful suggestions. He is also grateful to Professor M. A. Sleigh of the University of Southampton for reading the manuscript critically and making valuable suggestions and corrections to the English.

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