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

FINE STRUCTURE OF THE LONGITUDINAL

FLAGELLUM IN CERATIUM TRIPOS,

A MARINE DINOFLAGELLATE

TADASHI MARUYAMALaboratory 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 onlyundulations but also retractions, has been examined in both the retracted and relaxed states.Although conventional fixation always triggered retraction, the flagellum was found to remainrelaxed 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 thatappeared to be a single fibre under the light microscope is a bundle of four fibres; the 9 + 2microtubular axoneme, the packing material, the striated fibre, and the paraxial fibre. The firsttwo are common in the longitudinal flagella of dinoflagellates, and the axoneme presumablygenerates the undulation. The last two are new and unique to the longitudinal flagellum ofCeratium. The R-fibre, which probably contracts to fold the flagellum during retraction, con-sists of fine filaments, which pursue a loosely spiral course in the contracted state, but alignlongitudinally in the relaxed state. Periodic striations appear only on the extended R-fibre. TheR-fibre shortens to approximately one third of its extended length and pursues a left-handedhelix. The packing material, which sticks to the microtubular axoneme on one side, is connectedwith the R-fibre on the other side at intervals of approximately 5-8 fim. The retraction seemsto 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. Thewave was reported to be planar and to consist of circular arcs and connecting straightsegments (Brokaw & Wright, 1963), as reported also in invertebrate sperm tails(Brokaw, 1965). The flagellum retracts when the cell body, especially the tip of theapical horn, is stimulated mechanically. It is folded from the tip to the base, and isfinally installed in the sulcus on the ventral side of the cell body (Maruyama, 1981).

Light-microscopic observation of the retracted flagellum showed two fibres, theaxoneme and the R-fibre (Maruyama, 1981). The axoneme was presumed to containthe 9 + 2 microtubular axoneme, which generates the undulation. The R-fibre, whichis unique to the longitudinal flagellum of Ceratium, was thought to contract to foldthe flagellum.

The fine structure of the longitudinal flagella in some dinoflagellates, which werenot 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 packingmaterial. However, the fine structure of the longitudinal flagellum in Ceratium has notbeen described, apart from a brief description in a freshwater species, Ceratiumhirundinella, which showed the 9 + 2 microtubular axoneme (Dodge & Crawford,1970).

The present paper describes the fine structure of the longitudinal flagellum inCeratium tripos, in both retracted and relaxed states. It is a complex flagellum con-taining five fibres. The axoneme, which appears to be a single fibre under the lightmicroscope, has been shown to be composed of four fibres including the 9 + 2 micro-tubular axoneme and three associated fibres. The R-fibre consists of fine filamentswhose arrangement is changed during retraction.

MATERIALS AND METHODS

C. tripos was grown as reported previously (Maruyama, 1981). After a month or more ofincubation, cells were harvested by low-speed centrifugation at 1000 to 2000 rev./min for30-60 3 at room temperature in conical centrifugation tubes with screw caps.

Cells were fixed with a phosphate-buffered glutaraldehyde fixative containing 5 % glutar-aldehyde, 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-Nacacodylate (pH 7-4) for 1 h at room temperature. These procedures always triggered retractionof the flagellum (Maruyama, 1981). After washing with a graded series of glucose solutions inthe same buffer, post-fixation with 1 % 0sO4 in the same buffer without glucose for 45-60 minat 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 thatthe cells were embedded in 1-2 % agar prior to dehydration. Some cells in Spurr resin wereplaced 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 Nomarskioptics. 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 remainsrelaxed if it is washed briefly with CaI+-free medium prior to fixation. Two different Cal+freemedia were used; Cat+-free ASW and Ca1+-free low ionic strength medium. The former con-tained 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). Thelatter was composed of 10 mM-ethylenediaminetetraacetic acid (EDTA), 1 M-glucose and60 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 theR-fibre took a left-handed spiral course beneath the folded axoneme when it waslooked at from the outer helical surface. The R-fibre intersects the axoneme atintervals.

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 itdisintegrated into a thin thread within a few minutes at room temperature. Theflagellum 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 fineglass capillary. The thin thread that remained after disintegration could not bereactivated. These findings indicate that the retractile mechanism remains intact inthe Ca2+-free medium until decomposition takes place.

Fig. 1. A light micrograph of a retracted longitudinal flagellum in the sulcus. The cellwas fixed by conventional fixation and embedded in Spurr resin. Nomarski differentialinterference 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 strengthmedium. Unfixed preparation. Phase-contrast optics. Bar, 50 /*m.Fig. 3. A higher magnification micrograph of a fully relaxed longitudinal flagellumfixed 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 todistinguish in the fully relaxed flagellum, probably because it lay close to the axonemeand perhaps because the extended R-fibre is thinner than the contracted one. Thisindicates that the length of the extended R-fibre is roughly equal to that of the axo-neme, which is presumably constant during motion. The extent of contraction of theR-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 wassquashed between the cell body and a coverslip so as to lie in the same focal plane.

i i2 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 contractionwas calculated to be 1/3-2. If the retracted R-fibre makes a helix, the length of oneturn (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 contractedR-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 theflagellum. The length of R-fibre per turn was estimated to be 36 fim. The length ofthe folded axoneme per period measured 115 + 1-5 fim (n = 11). The ratio of con-tracted to extended length would be i:3-2, which agrees with the value obtainedabove from direct measurement on the flattened flagellum. This indicates that ourassumption is reasonable. The distance along the axoneme between the intersectionsof 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 longitudinalflagellum. The folded axoneme, which appeared to be a single fibre under the lightmicroscope, is in fact a bundle of several fibres including the 9 + 2 microtubularaxoneme. This bundle will be referred to as the axonemal complex. It lies beneaththe flagellar membrane of the outer helical surface, as shown previously by scanningelectron microscopy (Maruyama, 1981). Prominent lamellar foldings on the innerhelical surface are filled with a large amount of the flagellar matrix. Sections of thecontracted R-fibre confirmed the view that it takes a helical course. The diameter ofthe 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 aresimilar 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 microtubularaxoneme 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 ofthe 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 fixedby conventional fixation. The axonemal complex consisting of the axoneme (ax), thepacking material (pm), and the striated fibre (sf), faces the flagellar membrane of theouter helical surface (o/u). The paraxial fibre can not be recognized well at this lowmagnification. The R-fibre (Rf) seems to pursue a helical course. The lamellar foldsprotrude from the inner helical surface (ihs). Bar, i-o/tfn.

Longitudinal flagelhim of Ceratium

pm

O

ohs

Rf

ihs

Longitudinal flagelhtm of Ceratium 115

been reported in other dinoflagellates were found in the central core. The tubules are10 nm in diameter and are packed compactly in the bundle. The number of tubulesin a section through the packing material varies from o to over 20; it is greater in theproximal portion and decreases distally. A longitudinal section of the 10 nm tubulebundle usually shows a regular cross-banding pattern (Fig. 6). The overall periodicitywas approximately 59 nm. Each repeat period consists of two similar dark bands17-18 nm in width, a 5 nm transparent band and a wide translucent band of 16-18 nm. Itis, however, not clear whether these bands are on the tubules or on adjacent structures.The peripheral radiating structures sometimes seemed to connect with those darkbands. Viewed from the outer helical surface of the retracted flagellum, the packingmaterial always attaches to the underside of the axoneme. The shape of the packingmaterial 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 fromthe side opposite to the junction between axoneme and packing material; this will bereferred to as the lateral fin. Tangential sections of the packing material show alattice-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 locatedbeside the axoneme (Fig. 5). The longest side measured 73 ± 14 nm (n = 10), and theother two were 51 + 11 nm, and 58 ± 16 nm. It is a triangular prism with cross stria-tions, which have an overall periodicity of 46 ± 2 nm (n = 9) (Figs. 6, 7). Each periodcontains a dark band, which is composed of two lines with an interval of approxi-mately 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 willbe called the striated fibre. It has a unique property: it kinks at several points in thecurved 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 theaxoneme. Tangential or longitudinal sections of this structure did not show anystriation or substructure. This will be called the paraxial fibre.

Fig. 5. A higher-power electron micrograph of the axonemal complex in a retractedlongitudinal 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 of10 nm tubules (t). The lattice-like appearance of the peripheral radiating structure ofthe 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 arrow-head indicates the packing material-R-fibre junction. Bar, 0-5 fim.

n 6 T. Maruyama

All these accessory fibres oridinate in the proximal portion of the flagellum and donot enter the cell body. They are always located around the axoneme to constitute theaxonemal complex. Their angular positions around the centre of the axoneme weredetermined on the electron micrographs. The angles were 155 + 8° (n = 35) betweenthe paraxial fibre and the striated fibre, 73 ± 90 between the striated fibre and thecentre of the packing material, and 133 + 90 between the centre of the packing materialand the paraxial fibre. Because the axoneme invariably faces the flagellar membraneof the outer helical surface and the packing material always attaches to the axonemeat 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 is5-7 nm (Fig. 8). No limiting membrane surrounds it. The filaments swirl looselyaround the centre of the R-fibre in cross-section. Longitudinal sections show that theypursue a roughly spiral course around the axis of the R-fibre. Since the orientation ofthe different filaments is not uniform, the filaments seem to be entangled to make ameshwork.

Fig. 9 shows the basal region of a relaxed longitudinal flagellum. Regular striationsappear on the extended R-fibre. Two or three stripes are seen in each period, spacedat intervals of 20-40 nm (Fig. 10). The variation in overall periodicity of between 111and 162 nm might be related to the degree of relaxation or contraction. Unlike thosein the contracted fibre, the filaments are arranged in longitudinal arrays in the relaxedfibre. The diameter of each filament in the extended fibre was 4-7 nm and was notmuch different from that in the contracted state. Cross-sections of the extendedR-fibres were mostly round, but in the proximal portion were sometimes ovoid orpear-shaped (Fig. 11).

The diameter of the relaxed R-fibre was estimated from the cross or obliquesections to be 0-56±o-i5/tra (n = 16) in the proximal, 0-29 ±0-07/mi (n = 13) inthe middle, and 0-24 ± 0-07 fim (n = 5) in the distal portion. Comparison of thesevalues with those in the retracted state suggest that the relaxed R-fibre is thinner thanthe contracted one.

A partially relaxed R-fibre was sometimes observed (Fig. 12). The striations werenot clear, but the periodicity was approximately 70-80 nm and was shorter than thatin the fully relaxed state.

Examination of successive sections near the intersection of the R-fibre and theaxonemal complex in the retracted flagellum has shown that the R-fibre is connectedwith the packing material on the side opposite to the junction between axoneme andpacking material (Figs. 4, 8). No specific linkage was found between them.

Previous light-microscopic observation showed that the R-fibre originates near thebase and terminates near the tip of the flagellum (Maruyama, 1981). Fig. 13 showsthat it originates at the flagellar membrane a little above the basal body in a retractedflagellum. A space of about 20 nm was observed between the R-fibre and the mem-brane.

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, flagellarmembrane; pm, packing material. Bar, i-o/tm.

T. Maruyama

Longitudinal flagelhm of Ceratium

ft

* " •

tFig. 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 inC. tripos are not clear, a striated rootlet was sometimes observed to originate at theproximal 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 theaxonemal 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. Noteparallel 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 arevague. Bar, ro/im.Fig. 13. The membrane-R-fibre junction (arrowhead) near the base of a retractedlongitudinal 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 theaxoneme and the flagellar membrane, they may be linked together as reported in someother cilia and flagella (Dentler, 1981). The size and microtubular organization of theaxoneme in the longitudinal flagellum are typical of those in various organisms (Sleigh,1962; Afzelius, 1969; Warner, 1974). This fact and the similarity of the undulatorywave-form to those of sperm tails (Brokaw & Wright, 1963; Brokaw, 1965) togethersupport the idea that the undulation of the longitudinal flagellum is generated by theaxoneme (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 inlongitudinal section is similar to that of the paraxial rod of the undulating membranein Trypanosoma (De Souza & Souto-Padr6n, 1980) and of the flagellum in Euglena(Piccinni, Albergoni & Coppelloti, 1975; Bouck, Rogalski & Valaitis, 1978). Thefunctions 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 allalong 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). Thisproperty may be explained if it is a stiff filament, which, once bent, undergoes areduction in rigidity in the bent portion so that it becomes flexible. Although thefunctions of the accessory filaments are not clear, they possibly increase the rigidityof the axonemal complex and may facilitate the re-extension of the retractedflagellum.

From previous observations the R-fibre was presumed to contract to fold theflagellum, 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-fibrepursues 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 ofcell 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 duringcontraction, though there is no firm evidence. The spiral configuration of the con-tracted R-fibre is advantageous for compact folding of the flagellum, because thedistance between adjacent folds of the axonemal complex is determined by the pitchof the spiral instead of its length.

The contraction of the R-fibre is accompanied by a reorganization of the filamentsfrom highly ordered longitudinal arrays to disorganized loosely spiral arrays. Thespiral arrangement of filaments may bring about the helical coiling of the contractedR-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 explanationof the mechanism of contraction of the R-fibre.

A similar structural change was reported in the myoneme of Stentor; protofilamentsof the myoneme that orient longitudinally in the relaxed state transform to randomlyoriented 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) andretracted state (B). A. A side view of a relaxed longitudinal flagellum. It presumablyundulates in a plane perpendicular to the plane of the figure. The left side of theflagellum 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 outerhelical surface. The R-fibre (i?/) pursues a left handed helical course beneath the foldedpacking material that sticks to the underside of the axoneme. ax, axoneme; pm, packingmaterial ;/m, flagellar membrane; 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 similaroptical properties (Weis-Fogh & Amos, 1972). Although ATP-dependent slidingfilament models are widely accepted in skeletal muscle contraction (Hanson & Huxley,1955) and in flagellar motion (Satir, 1968; Summers & Gibbons, 1971), Weis-Foghand 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 mechan-ism 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 flagella are contractile(Simpson & Dingle, 1971; Salisbury & Floyd, 1978; Sleigh, 1979). The striatedrootlet in Ceratium is not connected with the R-fibre, because the R-fibre does notenter the cell body. The possibility that the R-fibre is an extension of the striatedrootlet, 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). Theretraction of the longitudinal flagellum is probably regulated by Ca2+, because it isprevented in Ca2+-free medium.

In conclusion, the longitudinal flagellum probably has two different motile systemsfor two dissimilar motions; a typical 9 + 2 microtubular axoneme for undulation andthe R-fibre for retraction (Fig. 15 A, B). The packing material, which is one of threeaccessory fibres of the axoneme, probably links the two systems together. The R-fibreis unique to the longitudinal flagellum of Ceratium. It is composed of fine filamentswhose arrangement is changed, from longitudinal arrays with regular striations in theextended state, to loose spiral arrays without striation in the contracted state. Theretracted R-fibre pursues a left-handed helical course and lies beneath the foldedaxonemal complex, when viewed from the outer helical surface.

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

REFERENCES

AFZELIUS, B. A. (1969). Infrastructure of cilia and flagella. In Handbook of Molecular Cytology(ed. A. Lima-de-Faria), pp. 1219-1241. Amsterdam, London: North-Holland.

AMOS, W. B. (1971). Reversible mechanochemical cycle in the contraction of Vorticella.Nature, Lond. aa<), 127-128.

BANNISTER, L. H. & TATCHELL, E. C. (1968). Contractility and the fibre systems of Stentorcoeruleus. J. Cell Set. 3, 295-308.

BOUCK, G. B., ROGALSKI, A. & VALAITIS, A. (1978). Surface organization and composition ofEuglena. II . Flagellar mastigonemes. J. Cell Biol. 77, 805-826.

BROKAW, C. J. (1965). Non-sinusoidal bending waves of sperm flagella. J. exp. Biol. 43, 155-169.

BROKAW, C. J. & WRIGHT, L. (1963). Bending waves of the posterior flagellum of Ceratium.Science, N.Y. 143, 1169-1170.

DENTLER, W. L. (1981). Microtubule-membrane interactions in cilia and flagella. Int. Rev.Cytol. 73, 1-47.

D E SOUZA, W. & SOUTO-PADR6N, T . (1980). The paraxial structure of the flagellum of Trypano-tomatidae. J. Parasit. 66, 229-235.

DODGE, J. D. & CRAWFORD, R. (1970). The morphology and fine structure of Ceratium hirundin-ella (Dinophyceae). J. Phycol. 6, 137-149.

EBASHI, S. & ENDO, M. (1968). Calcium ion and muscle contraction. Prog. Biophys. molec. Biol.18, 123-183.

Longitudinal flagellum of Ceratium 123

HANSON, J. & HUXLEY, H. E. (1955). The structural basis of contraction in striated muscle. InFibrous Proteins and Their Biological Significance (ed. R. Brown & J. F. Canielli), pp. 228-264. Cambridge University Press.

HOFFMANN-BERLING, H. (1958). Der Mechanismus eines neuen, von der Muskelkontraktionverschiedenen Kontraktionszyklus. Biochim. biophys. Acta 27, 247-255.

HUANG, B. & MAZIA, D. (1975). Microtubules and filaments in ciliate contractility. In Moleculesand Cell Movement (ed. S. Inou£ & R. E. Stephens), pp. 389-409. New York: Raven Press.

HUANG, B. & PITELKA, D. R. (1973). The contractile process in the ciliate, Stentor coeruleus.I. The role of microtubules and filaments. J. Cell Biol. 57, 704-728.

KRISTENSEN, B. I., NIELSEN, L. E. & ROSTGAARD, J. (1974). Variations in myoneme bire-fringence in relation to length changes in Stentor coeruleus. Expl Cell Res. 85, 127-135.

KUSHIDA, H. (1963). An improved epoxy resin 'Epok 533' and polyethylene gh/col 200 as adehydration agent. J. electron Microsc. 12, 167-174.

LEADBEATER, B. & DODGE, J. D. (1967). An electron microscope study of dinoflagellate flagella.J. gen. Microbiol. 46, 305-314.

LEE, R. E. (1977). Saprophytic and phagocytic isolates of the colourless heterotrophic dino-flagellate Gyrodimum lebouriae.J. mar. biol. Ass. U.K. 57, 303-315.

MARUYAMA, T. (1981). Motion of the longitudinal flagellum in Ceratium tripos (Dinoflagellida):A retractile flagella motion. J. Protozool. 28, 328-336.

NAITOH, Y. & KANEKO, H. (1972). Reactivated triton-extracted models of Parameciwn:Modification of ciliary movement by calcium ions. Science, N.Y. 176, 523-524.

NAITOH, Y. & KANEKO, H. (1973). Control of ciliary activities by adenosine triphosphate anddivalent cations in Triton-extracted models of Paramecium caudatum.J. exp. Biol. 58,657-676.

PETERS, N. (1929). Uber Orts- und Geisselbewegung bei marinen Dinoflagellaten. Arch.Protistenk. 67, 291-321.

PICCINNI, E., ALBERGONI, V. & COPPELLOTTI, O. (1975). ATPase activity in flagella fromEuglena gracilis. Localization of the enzyme and effects of detergents. J. Protozool. 22, 331-335-

REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain inelectron microscopy. J. Cell Biol. 17, 208-211.

SALISBURY, J. J. & FLOYD, G. L. (1978). Calcium-induced contraction of the rhizoplast of aquadriflagellate green alga. Science, N. Y. 202, 975-977.

SATIH, P. (1968). Studies on cilia. III. Further studies on the ciliary tip and a ' sliding filament'model of ciliary motility. J. Cell Biol. 39, 77-95.

SCHOTT, F. (1895). Die Peridineen der Plankton-Expedition. I. Studien iiber die Zellen derPeridineen. Ergebn. D. Plankton-Exped. (Kiel und Leipzig), 1-170.

SIMPSON, P. A. & DINGLE, A. D. (1971). Variable periodicity in the rhizopoast of Naeglerioflagellates. J'. Cell Biol. 51, 323-328.

SLEIGH, M. A. (1962). The Biology of Cilia and Flagella, pp. 242. Oxford: Pergamon.SLEIGH, M. A. (1979). Contractility of the roots of flagella and cilia. Nature, Land. 377, 263-264.SPURR, A. R. (1969). A low viscosity epoxy resin embedding medium for electron microscopy.

J. Ultrastruct. Res. 26, 31-43.SUGI, H. (1961). Volume change during contraction in the stalk muscle of Carchesium. J. Fac.

Set. Tokyo. Univ. (sec. IV), 9, 155-170.SUMMERS, K. E. & GIBBONS, I. R. (1971). Adenosine triphosphate-induced sliding of tubules

in trypsin-treated flagella of sea-urchin sperm. Proc. natn. Acad. Set. U.S.A. 68, 3092-3096.WARNER, F. D. (1974). The fine structure of the ciliary and flagellar axoneme. In Cilia and

Flagella (ed. M. A. Sleigh), pp. 11-37. London, New York: Academic Press.WEIS-FOGH, T. & AMOS, W. B. (i972)- Evidence for a new mechanism of cell motility. Nature,

Lond. 236, 301-304.

{Received 15 April 1982)

5-2