J. Cell Sci. io, 95-122 (1972) qr

Printed in Great Britain

STRUCTURE AND COILING OF THE STALK IN

THE PERITRICH CILIATES VORTICELLA

AND CARCHESIUM

W. B. AMOSTlte Department of Zoology, Downing Street, Cambridge, England

SUMMARY

The stalk of Carcliesium and Vorticella coils by the action of a contractile organelle. Theorganelle lies within a thread of cytoplasm which is encased in a complex extracellular tube.Study with the light microscope and the electron microscope suggests that the structure ofthe tube and the course of the organelle determine the form of the coiling. The contractileorganelle contains a system of interconnected membranous tubules and the cytoplasm aroundit also contains membranous saccules. Both tubules and saccules extend along the length of thestalk.

INTRODUCTION

Vorticella and Carchesium have stalks which are capable of coiling helically. Thecoiling is brought about by the action of a contractile organelle which differs markedlyfrom muscle in its physiological properties. This paper is concerned with the structureof the stalks and seeks to explain some aspects of the coiling in structural terms.Experiments on the activation of the stalks after glycerination are described elsewhere(Amos, 1971, and in preparation).

Faure-Fremiet (1905) examined the stalks of both genera with the light microscopeand found them to be similar. They consisted of a cylindrical sheath containing thecontractile thread, or spasmoneme, which pursued a helical course throughout thelength of the sheath. Longitudinal fibres lay on the inner surface of the sheath on theopposite side to the spasmoneme. Faure-Fremiet regarded these fibres, the bdtonnets,as stiffeners which, because of their position, ensured that the sheath would bend intoa helix, rather than merely collapse, when the spasmoneme contracted. Randall &Hopkins (1962) confirmed his description with the electron microscope in the case ofVorticella, but presented a different account of Carchesium. In Carchesium, theyreported an axial spasmoneme separated equally on all sides from the sheath by anannulus containing many tubular fibres. This structure, which seems ill suited tohelical coiling, is visible in electron micrographs of a related peritrich, Zoothamnium,obtained by Faure-Fremiet, Favard & Carasso (1963). Since the Carchesium studiedhere agrees with Faur^-Fremiet's description of the genus, it seems likely that theorganisms examined with the electron microscope by Randall and Hopkins were notCarchesium but Zoothamnium. The present account of the Carchesium stalk is thereforebelieved to be the first extensive one, though the spasmoneme was described byCarasso & Favard (1966).

96 W. B. Amos

Many studies have been made of the coiling of Vorticella and it has recently beenanalysed with a high speed camera by Jones, Jahn & Fonseca (1970), so observationshere are chiefly confined to the type of deformation involved, and its relation to thestructure of the stalk.

MATERIAL AND METHODS

Carchesium was collected from a pond near Kenn in north Somerset. The species (see Fig. 3)resembled Carchesium polypinum L., as shown by Stein (1854), but the published descriptionsof this species are inadequate for identification. All the organisms used came from the sameclone. They were cultured in a soil extract prepared by autoclaving 80 g of loam in 1 1. ofglass-distilled water, filtering and autoclaving once more. One small drop of fresh milk wasadded to 25 ml of soil extract in a Petri dish before inoculation. Subcultures were made weekly.Vorticella convallaria L. was obtained from the Culture Collection of Algae and Protozoa inCambridge. It was cultured in a salt solution nutrified with the Glaxo preparation ' Complan'.The salt solution contained 17 mM NaCl, 1-2 mM NaHCO3, 0054 mM KC1 and 0-054 mMCaCl2. It was sterilized and poured over a sterile solid layer of 3 % agar, made up in the samesolution, in a Petri dish. One small drop of an autoclaved 2 % suspension of Complan in waterwas added and the culture was inoculated by inserting a glass slip from a previous culture, towhich the organisms had attached themselves. Subcultures were made monthly.

Light microscopy was carried out with a Zeiss Standard WL microscope equipped for phasecontrast and Nomarski's differential interference contrast. Photographs were taken withelectronic flash.

The methods used for electron microscopy were largely conventional. The organisms werefixed between o and 30 C in a solution containing 25 % glutaraldehyde and 27 mM sodiumcacodylate buffer adjusted to pH 7-4 at 2 °C by means of o-i N HC1. After exposure to 2 changesof this medium for a total period of 2 h at 0-3 °C the specimens were washed for 2 h in 4changes of a similar medium, but with 6 % sucrose instead of glutaraldehyde. They were thenpostfixed in 1 % osmium tetroxide with 6 % sucrose and 50 mM veronal acetate buffer. Thebuffer was made up to give a pH of 7 4 at 2 °C. The fixed material was washed for 1 h in thesame medium but without osmium tetroxide and with only 4 % sucrose. In 2 subsequentwashes, each 15 min long, the sucrose concentration was reduced to 2 % and then to zero. Noexperiment was made to test the value of the graded reduction in sucrose concentration, but itis believed that it might have lessened the osmotic stress on the fixed material. Dehydration inethanol, treatment with propylene oxide and embedding in Araldite were carried out accordingto Luft's method (1961). Sections were cut with glass and diamond knives, and grey sectionswere picked up on grids with carbon-coated Celloidin films. They were stained with a saturatedsolution of uranyl acetate in 50 % ethanol for 30-50 min and also with the lead citrate solutionof Reynolds (1963) for 3—5 min. The electron microscope was a Philips EM 200 operated at anaccelerating voltage of 60 kV.

OBSERVATIONS

Principal features

Faure-Fremiet (1905) has described the formation of the stalk in Vorticella, whichbegins with the attachment of the body to a submerged surface. A tube of extra-cellular material is secreted which apparently elongates by the addition of new materialat one end. At the same time, an outpushing of the cytoplasm of the body enters thetube. In Carchesium, but not in Vorticella, the body subsequently divides many timesand the stalk becomes bifurcated after each division, so that a colony of many indivi-duals or zooids is formed. The spasmoneme of each zooid is restricted to its own branch,and there is no cytoplasmic continuity between zooids.

Structure and coiling of peritrich stalk 97

The size and structure of the stalk vary with its position in the Carchesium colony.In the species examined here, the stalk of a newly attached zooid is 7-10/tm in dia-meter, but as it grows in length it increases in diameter to 10-18 /im, SO that the finalform is a slender cone tapered towards the base. The main trunk of the colony loses

Plasma membrane

Spasmoneme

Sheath

Fibrillar matrix

Cytoplasm

Mitochondrion

Batonnet

Fig. 1. Diagram of the main structures in the stalk of Carchesium. The numberof batonnets is reduced for the sake of clarity.

its ability to coil after thirty or so zooids have been produced. It swells somewhat andbecomes filled with a refractive granular material. The chief features of a contractileregion are shown in Fig. 1. Inside the sheath runs the cytoplasmic strand in a left-handed helix, with a pitch of about 140 /tm in the main trunk and 60-100 /im in thebranches. The spasmoneme, which is embedded in the strand, has the form of aribbon running helically, with the outer surface flattened against the inside of the

98 W. B. Amos

sheath. The other surface of the spasmoneme, facing inwards, is covered with a layerof cytoplasm containing many spherical mitochondria. The cytoplasmic strand hasa circular cross-section in which the spasmoneme lies eccentrically, the cytoplasmiclayer being 4 /tm deep on the inner surface of the spasmoneme but so thin on the outersurface that it is barely detectable with the light microscope. The transverse sectionof the spasmoneme is elliptical when the stalk is extended, with the minor axis lyingradially. In the spasmoneme of a branch of a colony the major axis is about 4 /MH andthe minor 1-5 /an. The minor axis increases when the stalk coils, so that the cross-section of the spasmoneme becomes almost circular when the organelle is fully con-tracted (Fig. 5). The batonnets lie longitudinally on the inner surface of the sheath.They are straight and parallel to one another but staggered in such a way that a helicalpalisade is formed (see Figs. 1, 6) which runs around the stalk on the opposite side tothe spasmoneme.

The scopula, the junction between stalk and zooid, is a highly organized region,in which the cytoplasmic strand becomes continuous with the cytoplasm of the bodyof the zooid (Fig. 7). The spasmoneme is continuous with longitudinal myonemes inthe body. Protruding from the base of the body of the zooid are rod-shaped structures,the scopula organelles, which occur quite generally in this position in peritrichs. Theirfine structure is somewhat similar to that of the basal bodies of cilia (Randall &Hopkins, 1962; Faure-Fremiet, Favard & Carasso, 1962).

The base of the main stalk, which is normally attached to the substrate, is sealedby a transverse disk to which the cytoplasmic strand is attached by about 15 elongatedprocesses which splay out from a swelling at its tip. They will be referred to as rootlets,since they seem from their position to anchor the spasmoneme. Rootlets are alsopresent at the end of the cytoplasmic strand in each side-branch of the colony (Fig. 8).The scopula, the holdfast, and the special modifications of aged stalks have not beenexamined further.

The structure of the regions of the stalk which are capable of coiling will now bedescribed. It must be noted that all the observations with the electron microscope weremade on coiled stalks, since contraction of the spasmoneme invariably occurred beforefixation. The following account refers to Carchesium. The small differences in struc-ture in Vorticella are indicated later.

Sheath

The sheath is an extremely thin membrane which is thrown into folds duringcoiling. In the electron microscope, sections perpendicular to its surface show 2 darklines with a clear space between them, the total thickness of the 3 layers being 12 nm.The inner surface is covered with a less-dense layer about 20 nm thick, which appearsto be highly ordered. Oblique sections of this layer show regular parallel lines 9 nmapart, which probably represent longitudinal fibrils (Fig. 17). Transverse sections offibrils are seen rarely and never distinctly, but this may be due to the close proximityof the fibrils to one another and their small diameter.

Structure and coiling of peritrich stalk 99

Fibrillar matrix

The space between sheath and cytoplasm is optically structureless, but, as Randall &Hopkins (1962) discovered in Vorticella, it contains a dense mesh work of fibrils of acharacteristic type. In Carchesium this fibrillar matrix occupies two-thirds of thevolume of the stalk. Each fibril is so thin that the diameter cannot be measured pre-cisely in sections: it is less than 3 nm. Nevertheless, the fibrils in a transverse sectionof the stalk run straight, some as far as 2/tm (Fig. 11), which implies an enormousrigidity, especially since the matrix as a whole is deformed by coiling before fixation.The predominant orientation of the fibrils is longitudinal, but they often intersect atright angles, forming T-shaped or rectangular patterns. It seems from simple observa-tion, without statistical analysis, that the frequency of intersections at right angles istoo high to be accounted for by chance, so that a large proportion of the matrix fibrilsmust be linked to form a rectangular lattice. As Randall and Hopkins discovered, thefibrils have electron-dense beads spaced regularly along their length. In Carchesiumthe beads are about 8 nm in diameter. The mean of 10 measurements of the distancebetween the centres of adjacent beads, involving a total of 54 spacings, was 31-9 nm(S.D. 3-0).

Bdtonnets

These fibrils are not easily seen in life but after the disintegration of the cytoplasmicstrand they become plain under phase contrast (Fig. 6), presumably because thecontrast between them and their surroundings is enhanced by loss of refractivematerial from the matrix. Each one is about 15 fim long and 0-2 /im in diameter.Because of their helical arrangement, a transverse section of the stalk contains acrescentic array of transverse sections of batonnets (Fig. 11). The sections grow smallerat one side of the crescent but not at the other, which indicates that the batonnetstaper to a point at one end, as shown in Fig. 1.

The electron microscope resolves each batonnet into a loose bundle of about thirtysubfibres. The number is not clear because the subfibres are matted together andappear to be partly fused. They have various diameters from 15 to less than 3 nm, andthere is some indication that the subfibres are in turn compounds of even finer fibres.Glancing longitudinal sections of the stalk (Fig. 10) show that most of the subfibresrun longitudinally but some run at various angles across the 2-/im gap betweenbitonnets, forming an irregular interlaced network. The apparent discontinuity of thesubfibres may simply be due to the fact that they run out of the plane of section. Thebundles have transverse, regularly spaced bands of higher density about 4 nm thick.It has not been possible to decide whether the banding is due to dense regions withinthe subfibres or to bridges between them, but since the bands sometimes cross thewhole width of the batonnet the dense regions of adjacent subfibres may perhaps beheld in register by some form of transverse linkage. The mean of 10 measurements ofthe spacing between bands, including a total of 52 spaces, was 27-1 + 2-0 nm. Themeasurement suggests that the subfibres are compounds of the beaded fibres whichoccur in the matrix, the dark bands corresponding to rows of beads.

7-2

ioo W. B. Amos

Membranes

There are no traces of endoplasmic reticulum or Golgi cisternae in the cytoplasmof the stalk. The plasma membrane presents the usual trilaminar appearance with atotal thickness of about 7-5 nm. In coiling it is thrown into extensive folds, as is thesheath. There is a large space between the fibrillar matrix and plasma membrane inmaterial prepared for the electron microscope. This space is an artifact arising fromshrinkage of the cytoplasm by about 30% during preparation. In life the region wherethe space would be can be seen to contain cytoplasm with mitochondria (compareFig. 5 with Fig. 9).

The plasma membrane has the form shown in Fig. 9 in transverse sections of thestalk. Two distinct regions of the membrane, on either side of the strand, are denserthan elsewhere. This appearance is due to a pair of flattened saccules lying on theinner surface of the plasma membrane. One of these saccules is shown at highermagnification in Fig. 18. Their constant position in the transverse section suggeststhat the saccules have the form of a pair of ribbons running throughout the length ofthe cytoplasmic strand and twisting around it in parallel with the spasmoneme. Sometransverse sections appear to show more than two saccules, but this may perhaps beaccounted for by a branching of the ribbons. The saccules are approximately I-I /tmwide. No structure has been seen inside them and their contents are not stained byuranyl acetate or lead citrate. In some sections the separation of the saccular membranesis fairly constant, varying only from 8 to 12 nm for considerable distances along thecross-section and increasing in some regions to about 100 nm. In other sections themembranes are 0-5 /tm apart. In view of the extensive shrinkage of the cytoplasm,mentioned above, it is not at present possible to decide which appearance correspondsto the state in life, if either of them does. But since the mitochondria are often equallywell preserved in both types of section the variation in width may perhaps be genuineand not simply due to variation in preservation. The saccular membrane has the sameappearance and thickness as the plasma membrane, from which it is separated by agap of about 5 nm which does not vary appreciably. This gap is presumably a zone ofadhesion between the saccular and plasma membranes, since they do not come apartfrom one another even when the saccules are highly dilated.

Cytoplasmic microtubules

Isolated sinuous microtubules with an external diameter of about 15 /im have beenobserved, distributed apparently at random in the cytoplasm. Also, structures super-ficially like the basal bodies of cilia are common. They consist of parallel tubulesarranged in a circle to form a cylinder 430 nm high and 124 nm in diameter. From thefrequency with which they are sectioned it can be deduced that there are about 200 ina segment of stalk 50 /tm long. The cylinders are often, though not always, orientedperpendicularly to the plasma membrane and the tubules. There are 9 single tubulesin the bodies, with a tenth placed eccentrically within the cylinder, each tubule havingan external diameter of approximately 22 nm (Fig. 19).

Structure and coiling of peritrich stalk 101

Rootlets

Only one section has been obtained of the rootlets which emerge from the end of thecytoplasmic strand in each branch of the colony. It appears from this section (Fig. 22)that there is a cylinder of 9 single tubules which are only 13 nm in diameter at the baseof each rootlet and that there are densely staining bodies inside the cylinder of tubules.The rootlets themselves are cylindrical structures o-16 ju,m in diameter which protrudefrom the cytoplasmic strand into the fibrillar matrix. They contain 9 tubules whichhave a similar diameter to the basal ones and which end at different levels. Both thebasal and the distal tubules, which are probably continuous with each other, areembedded in a densely staining matrix which has the appearance of a continuation ofthe spasmoneme. A membrane 10 nm thick with a dense outer layer surrounds thetubules and this membrane is in turn clad in a diffuse amorphous coat, spaced 20 nmfrom the surface of the membrane but apparently connected to it by many finefilaments.

Mitochondria

The total mitochondrial area in 5 transverse sections of the stalk was 5-2% of thecytoplasmic area. This may be taken to mean that about 5% of the cytoplasmicvolume is mitochondrial, provided that differential shrinkage of mitochondriaand cytoplasm does not occur during preparation. The mitochondria are spherical.In the terminal branches of actively growing colonies their diameter is about 1-5/tm,but in the main stalk they are smaller and more numerous. As Faure-Fremiet et al.(1962) have observed in the mitochondria of the peritrich Epistylis, the outermembrane is stained less with uranyl acetate and lead citrate than the inner, and thestroma stains much more intensely than the contents of the sinuous villi.

Spasmoneme

The spasmoneme contains a mass of filaments with tubules running through it (seeFigs. 9, 12-15 and 23). It is not separated from the rest of the cytoplasm by a mem-brane. No substructure has been seen in the filaments, which are only about 2 nm indiameter. They are roughly parallel to one another, about 3 nm apart and their cross-sections are locally arranged in straight lines in some transverse sections of thespasmoneme (Fig. 12). The orientation of the filaments in relation to the spasmonemeis roughly longitudinal.

The tubules within the spasmoneme have been described by Favard & Carasso(1965). There are about 150 tubules in a transverse section of the spasmoneme froma branch and 400 in that of a main stalk. The diameter varies from one tubule toanother in the range 38-70 nm, and their distance apart is about 250 nm. Branchingof the tubules has not often been observed but it may be common, since the transversesections often occur in groups of two or three and sometimes have two or three lobes.It seems likely that all the tubules are joined together to form a continuous network.Each tubule is limited by a membrane 1-7-5-2 nm thick in which no structure has yetbeen seen with ordinary fixed material, but a trilaminar appearance was obtained with

102 W.B.Amos

glycerinated preparations. Wisps of an amorphous substance are often visible insidethe tubules.

Vorticella

Although the stalk of Vorticella is much smaller, its structure is similar to that ofCarchesium. It is between ioo and 300 fim long in the species examined, varyinggreatly from one specimen to another. The total diameter is 2-9 /tm and that of thespasmoneme 1-2 /tm. Some features of the fine structure are shown in the electronmicrograph of Fig. 16. The sheath, matrix and batonnets can be identified, as withCarchesium. Within the spasmoneme there is a similar arrangement of tubules andfilaments. The tubules have roughly the same diameter (60 nm) as in Carchesium andthe same separation, and since the spasmoneme is smaller there are only about 60 inthe transverse section.

The course of the tubules is interesting in view of a possible analogy with themembranous systems of striated muscle. Favard and Carasso have concluded fromtheir studies of Carchesium and Vorticella that all the tubules at a given level runparallel to one another at a small angle to the longitudinal axis of the stalk, each endingblindly at both ends near opposite surfaces of the spasmoneme. The evidence for thisview is that longitudinal sections of the spasmoneme, such as that shown in Fig. 15,contain tubules running obliquely. Observations on Vorticella suggest that this maynot be the correct interpretation. When a series of sections at different depths is taken,the angle between the tubules and the longitudinal axis appears to reverse in sign asone proceeds from the near to the far surface of the spasmoneme, suggesting instead ahelical organization (see Figs. 13-15). Measurements on sections of this type revealthat tubules near to the surface of the spasmoneme are at about 300 to the longitudinalaxis of the organelle. If they are continuous along the length of the 9pasmoneme, thetubules must pass around it in a helix. Since Favard and Carasso observed continuityfor as much as 8 jam, it is extremely probable that they do. There is no evidence thatthe tubules ever rise to the surface or communicate with the plasma membrane.

Sections of the junction between the spasmoneme and the myonemes of the bodyshow that the myonemes are structurally similar to the spasmoneme and continuouswith it. Also, the tubules appear to pass from the spasmoneme into the myonemes ofthe body through the junction (see Fig. 23). The fate of the tubules within the bodyhas not yet been discovered.

The coiling of the stalks

The work of Randall and Hopkins on Vorticella showed that after the coiling whichprecedes fixation the palisade of batonnets lies on the outermost part of the helicalstalk, that is, at the highest radius. The same was found to be true when the spon-taneous coiling of living specimens of Carchesium was photographed, as in Fig. 5. Thelength of the palisade in this position, Lc, was calculated from the formula

Lc = A

where N is the number of turns, r the external radius and h the pitch of the helical

Structure and coiling of peritrich stalk 103

stalk. The same stalks were photographed in extension and the extended length, L, ofthe palisade was calculated by a similar method, but this time from the pitch andradius of the palisade in the extended stalk. The mean of 9 determinations of LJLwas 1-02 and the standard deviation 0-05. It may be concluded that the length of thepalisade does not change significantly during coiling. Since this length is equal to theextended length of the spasmoneme, the degree of contraction of the organelle can becalculated from measurements on single photographs of the coiled stalk. Formulaesimilar to the one given above were used to calculate the length of the spasmonemeand of the palisade within the same turn of the coiled stalk. By comparing the 2 lengthsthe extent of contraction was found. The mean of 5 measurements of the contractedlength, expressed as a percentage of the extended, was 36%, and the estimated error8%.

From observations made with phase contrast Sugi (1961) calculated a reduction involume of the spasmoneme of Carchesium of between 24 and 38 % during contraction,but even with the clearer image formed by the Nomarski interference contrast micro-scope the error in the measurement of volume is larger than 40 %. No consistent changein volume was observed in the present work.

The overall effect of the coiling is that the distance from the body to the base of thestalk is reduced by approximately 90%. Also, the body is rotated. To measure therotation small colonies of Carchesium were viewed from above while contracting inresponse to vibrations. In colonies with two or three individuals it was observed thatthe zooids revolved around one another during the uncoiling of their common stalk.The rotation was anticlockwise to an observer looking down the stalk from the zooidstowards the point of attachment. When measurements, each judged to the nearest900, were made on 20 colonies with an average number of about 7 turns, the averagerotation per turn of the coiled stalk was found to be 51°, but the amount varied betweencolonies from 22 to 900.

An abnormal type of coiling was often observed in Carchesium stalks from whichthe zooid had been detached by compression of the living organisms between slideand coverslip. The break invariably occurred at the scopula, and the stalk immediatelybegan to coil near this region. A zone of helical bending about 150/tm long passeddown the stalk, while at the rear of the zone the spasmoneme broke into fragmentsand the stalk became straight. The spasmoneme produces local bending under theseconditions.

DISCUSSION

Coiling

The stalk in these peritrichs is clearly adapted for pulling the body towards thepoint of attachment. The significance of the movement is unknown. It may serve asa defence against predation or mechanical injury. Ideally the coiling should not involverotation, particularly in Carchesium, where the branches of the colony are in danger oftwisting around one another, but a small amount of rotation does occur.

It is instructive to consider what pattern of strain must be produced on the surface

104 ^ - -

of the stalk if there is to be no rotation, and to compare this pattern with the structureof the stalk. The pattern was investigated by means of a model. A square lattice wasdrawn on the surface of a cylinder of plastic foam, with one axis parallel to the axis ofthe cylinder and the other circumferential. A helix of brass wire, left-handed like thecoiled stalk, was threaded through an axial hole in the cylinder in order to deform itinto a helix. The cylinder was provided with disks at each end by which it could beclamped to the wire. It was possible to rotate the disks on the wire and clamp them

•»«*lllllf*«

»*VtfMlllil**»,~*#JJli|H

la• •• •

-*~*«iiii- . . M t f f l l l l

.MMtmiMMM

[••••III• • • » • • « • • * • •

b'Fig. 2. Diagram obtained from the model shown in Fig. 21. The diagram is a projectionof the cylindrical surface of the model. The pattern of strain which occurred in thesurface is revealed by the shape of the quadrilaterals, which were initially square.Compression occurs along the lines a b and a' b'.

so that any desired degree of torsion was imparted to the cylinder (see Fig. 21). Thestrain in the surface was made visible by the distortion of the lattice of squares. Torepresent the pattern of strain in 2 dimensions the distorted squares were measuredand drawn on a diagram (Fig. 2) in which the centres of the bases were drawn equi-distant from one another on a series of equally spaced parallel lines. The diagramrepresents the surface of one complete turn: if extended vertically it would repeat.

An obvious feature of the pattern is a line of maximum compression which runsaround the cylinder once for each turn, along the helix of smallest radius. The greatestextension occurs on another helix, passing through the outermost points of the surface.The diagram suggests a reason for the helical organization of the extended stalk,namely that the line of maximum compression must correspond to a helix on the

Structure and coiling of peritrich stalk 105

extended surface. This is presumably guaranteed by the attachment of the palisadeand perhaps also of the spasmoneme to the rest of the stalk along a helix. To coilwithout rotation of the body the stalk must bend in a definite proportion to theshortening of the spasmoneme. When the spasmoneme is fully contracted, the stalkmust form neither more nor less than one turn for each turn of the extended palisade.The relation between bending and contraction is determined by many factors, in-cluding the resistance to bending of the batonnets. If they were slightly less stiff, thestalk would presumably bend more for a given degree of shortening of the spasmo-neme, and would consequently form more turns than there are in the extendedpalisade. The slight torsion which occurs in Carchesium may perhaps arise in this way.The coiling results from the interaction of mechanically dissimilar parts. It is probablyfortuitous that similar amounts of torsion were observed during the stretching of solidmetal springs (see Jones et al. 1970).

Sugi (i960) observed that coiling in living Carchesium in response to local electricalstimulation was sometimes restricted to the parts of the stalk nearest to the electrode.Also, local coiling in stalks severed from the zooid has been described here. Bothobservations suggest that the spasmoneme is attached to the stalk all along its length,so local contraction can produce local bending. An alternative which seems less likelyis that the spasmoneme can bend actively in a restricted region. If the spasmoneme isindeed attached along its length, the matrix surrounding the cytoplasmic strand, or atleast its peripheral zone, must be solid enough for attachment to be made. This maybe so in spite of the low refractive index of the matrix. Possibly the rectangulararrangement of the matrix fibres produces a high ratio of strength to density.

Unfortunately little is known of the chemical nature of the extracellular materialin the stalks of peritrichs. Sulphur-containing proteins appear to be present, PAS-positive substances and nucleic acids absent (Randall & Hopkins, 1962; Levine, i960;Faure-Fremiet, 1941). On structural grounds, several proteins must be present inCarchesium and Vorticella.

Contraction of the spasmoneme

During coiling the spasmoneme of Carchesium contracts to approximately one thirdof its extended length. The degree of contraction in Vorticella is more difficult tomeasure because of the smaller size of the stalk, but Jones et al. (1970) reported thatthe spasmoneme shortened by only 10% of its extended length in this genus.

The extended spasmoneme in both genera is positively birefringent with respect toits length, but when it contracts it becomes optically isotropic (Schmidt, 1940; Amos,1971). This suggests that contraction involves a disordering or folding of structureswhich were arranged longitudinally in the extended organelle. The structures inquestion can be neither the filaments nor the tubules observed with the electronmicroscope: their orientation is chiefly longitudinal even in maximum contraction.The birefringence must arise either from a substance which, like resilin (see Elliott,Huxley & Weis-Fogh, 1965) has no structure visible in the electron microscope, orfrom a structural change within the filaments or tubules.

Membranous structures have been observed near to bundles of filaments in the

106 W. B. Amos

myonemes of other ciliates. In Spirostomum (Ettienne, 1970) and Stentor (Bannister &Tatchell, 1968) the membrane frequently appears to consist of closed vesicles. Favard& Carasso (1965) report that calcium is stored inside the tubules in the spasmonemeof Carchesium and Vorticella. Their chief evidence is the precipitation of crystalsinside the tubules after treatment with oxalate. Similar evidence has been adducedfor the presence of calcium in the sarcoplasmic reticulum of striated muscle. Thespasmoneme of Vorticella is activated by calcium (Amos, 1971). If, after excitation, thetubules released calcium ions, the whole organelle could be activated rapidly bydiffusion, since no part of the spasmoneme is more than 100 nm from a tubule. Thetubules appear to form a closed system, more similar to the sarcoplasmic reticulumthan to the transverse tubules of a striated muscle fibre, with which Favard and Carassocompared them. Clearly they do not provide a route for expelling calcium from the cell.The observation of Jones et al. (1970) that the spasmoneme can be activated inde-pendently of the myonemes of the body is interesting in view of the continuity betweenthese structures. It suggests that the tubules are not responsible for the longitudinalspread of activation along the stalk, which has been demonstrated by Sugi (i960).Possibly the longitudinal saccules have a conducting function.

Microtuhular structures

It is possible that the rootlets have a common origin in development with scopulaorganelles. The region where they occur may be regarded as a part of the scopulawhich is separated from the rest during the growth of the cytoplasmic strand. Thecylinders in the cytoplasmic strand may perhaps also arise from scopula organelles.Some of the organelles are situated almost inside growing strands (Fig. 7). From thisposition they may perhaps be carried down into the cytoplasmic strand as it issuesfrom the scopula, and may subsequently be transformed into the cylinders. Such atheory implies a rapid multiplication of scopula organelles during growth, since thecylinders in the strand are so numerous.

Like their origin, the function of the microtubular structures is a matter of specula-tion. The rootlets presumably anchor the cytoplasmic strand in the matrix, and thescopula organelles may have a similar mechanical function, though the scopulaorganelles of Epistylis, studied by Faure'-Fremiet et al. (1962), appear to have somecontrol over the deposition of the extracellular material of the stalk.

I thank Professor T. Weis-Fogh for his encouragement and the provision of facilities forthis work, and Dr A. V. Grimstone for his guidance and critical reading of the manuscript.The work was supported by King's College, Cambridge and by a grant from the Departmentof Zoology, Cambridge.

REFERENCES

AMOS, W. B. (1971). A reversible mechanochemical cycle in the contraction of Vorticella.Nature, Lond. 399, 127-128.

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

CARASSO, N. & FAVARD, P. (1966). Mise en evidence du calcium dans les myonemes pedonculairesde cilies peritriches. J. Microscopie 5, 759—770.

Structure and coiling of peritrich stalk 107

ELLIOTT, G. F., HUXLEY, A. F. & WEIS-FOGH, T. (1965). On the structure of resilin. J. molec.Biol. 13, 791-795-

ETTIENNE, E. M. (1970). Control of contractility in Spirostomum by dissociated calcium ions.J. gen. Physiol. 56, 168-179.

FAURE-FREMIET, E. (1905). La structure de l'appareil fixateur chez les Vorticellidae. Arch.Protistenk. 6, 207-226.

FAURE-FREMIET, E. (1941). La nature chimique du p^doncule des Vorticellides. Bull. Soc.zool. Fr. 66, 277-287.

FAURE-FREMIET, E., FAVARD, P. & CARASSO, N. (1962). Etude au microscope electronique desultrastructures d'Epistylis anastatica. J. Microscopie 1, 287-312.

FAURE-FREMIET, E., FAVARD, P. & CARASSO, N. (1963). Images electroniques d'une micro-bioc^nose marine. Cah. Biol. mar. 4, 61—64.

FAVARD, P. & CARASSO, N. (1965). Mise en evidence d'un r^ticulum endoplasmique dans lespasmoneme de cilies peritriches. J. Microscopie 4, 567-572.

JONES, A. R., JAHN, T. L. & FONSECA, J. R. (1970). Contraction of protoplasm. IV. Cine-matographic analysis of the contraction of some peritrichs. J. cell. Physiol. 75, 9-20.

LEVINE, L. (i960). Visualisation of sulfhydryl groups in Vorticella convallaria. Anat. Rec. 138,364-365-

LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. biophys. biochem.Cytol. 9, 409-414.

RANDALL, J. T. & HOPKINS, J. M. (1962). On the stalks of certain peritrichs. Phil. Trans. P.Soc. Ser. B 245, 59-79.

REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain inelectron microscopy. J. Cell Biol. 17, 208-212.

SCHMIDT, W. J. (1940). Die Doppelbrechung des Stieles von Carchesium, insbesondere dieoptische-negative Schwankung seines Myonems bei der Kontraction. Protoplasma 35, 1-14.

STEIN, F. (1854). Die Infusionsthiere auf ihre Entzvickelungsgeschichte. Leipzig: Engelmann.SUGI, H. (i960). Propagation of contraction in the stalk muscle of Carcliesium. J. Fac. Sci.

Tokyo Univ. Sec. 4. Zool. 8, 603-615.SUGI, H. (1961). Volume change during contraction in the stalk muscle of Carchesium. J. Fac.

Sci. Tokyo Univ. Sec. 4. Zool. 9, 155-170.{Received 26 May 1971)

ABBREVIATIONS ON PLATES

bbbccyl

ffi>Ifmmambmt

batonnetsbasal bodiescytoplasmcylinder composed of microtubulesfibrillar matrixfood vacuolefibrils lining the sheathmitochondrionmacronucleu8membranous component of the sheathmicrotubule

my

Ppvirsacshso

sptz

myoneme within the bodypellicleplasma membranerootletssacculesheathscopula organellesspasmonemetubules within the spasmonemezooid

io8 W. B. Amos

Figs. 3 and 4. Light micrographs of a portion of a Carchesium colony. Dark-groundillumination, x 220.

Fig. 3. Extended. The spasmoneme (sp) is visible within the sheath. One zooid (z)has not yet developed a cytoplasmic strand.

Fig. 4. The same zooids in spontaneous contraction.

Structure and coiling of peritrich stalk 109

no W.B.Amos

Figs. 5-8. Light micrographs of the stalk of Carcliesium.Fig. 5. Photographed during spontaneous coiling. The optical section of the helical

stalk shows the spasmoneme (sp), the surrounding cytoplasm (c) containing mito-chondria (m), the sheath (sli) and batonnets (b). The junction between stalk andzoord is at bottom left, x 1940. Interference contrast, shearing direction horizontal.Objects with a higher refractive index than their surroundings have a bright right-hand boundary.

Fig. 6. A decaying stalk. The spasmoneme (sp) is disintegrating and the batonnets(b) have become visible. Phase contrast, x 2200.

Fig. 7. Optical section of the junction between the stalk and a dividing zooid. Thedaughter cell on the left will retain the parental cytoplasmic strand (c), in which thespasmoneme (sp) and mitochondria (m) are visible. The other daughter cell has a newscopula which is uniformly covered with scopula organelles (so). Interference contrast,x 2200.

Fig. 8. Base of a branch of a colony, showing rootlets (r). Other labelling asin Fig. 5. Interference contrast, x 2200.

Structure of coiling of peritrich stalk

ii2 W.B.Amos

Figs. 9-23, except for 21, are electron micrographs of sections.Fig. 9. Transverse section of a Carchesiwn stalk, showing sheath (sli), fibrillar matrix

(/), cytoplasm (c) and batonnets (b). The plasma membrane (pm) appears denser atsx and s2 because of the presence of saccules. Within the cytoplasm are mitochondria(m), a microtubular cylinder (cyl) and the spasmoneme (sp). x 11 700.

Structure and coiling of peritrich stalk

sh

ii4 W.B.Amos

Fig. 10. Glancing section of the coiled stalk of Carcliesium, nearly tangential tothe sheath. The batonnets (b) are sectioned longitudinally, x 43 000.

Fig. 11. Transverse section of the extracellular material of the Carcliesium stalk.Rectangular arrays of matrix fibrils are indicated at rl and r.. x 43 000.

Structure and coiling of peritrich stalk

sh

,'b

m

b

0-5

11; . * , - • • ' '

n6 W.B.Amos

Fig. 12. Transverse section of the spasmoneme of Carchesiwn, showing tubules (t)and filaments between them. The sections of filaments appear to be arranged instraight lines in the region arrowed, x 82000.

Figs. 13-15. Sections of the same gyre of a coiled Vorticella stalk, taken at succes-sively greater depths, x 21000.

Structure and coiling of peritrich stalk 117

M3

14

15

400 nm

v f

n8 W.B.Amos

Fig. 16. Section of a coiled Vorticella stalk. The cytoplasm is poorly preservedand no mitochondria are visible, x 8000.

Fig. 17. A folded region of the sheath of Carchesitim, showing the membrane (111b)and longitudinal fibrils (//). x 68500.

Fig. 18. A saccule (sac), lying beneath the plasma membrane in a Carchesium stalk,x 65000.

Structure and coiling of peritrich stalk 119

pm

120 W.B.Amos

Fig. 19. Transverse section of a cylinder composed of microtubules in the cytoplasmof a Carchesium stalk. A solitary microtubule (mt) is visible nearby, x 67500.

Fig. 20. Longitudinal section of a cylinder similar to that shown in Fig. 19. x 67 500.Fig. 21. Model (see text for explanation) set so that the cylinder is not twisted.

The longitudinal lattice lines do not pass around the wire.Fig. 22. Transverse section of the stalk of Carchesium, showing rootlets surrounded

by the fibrillar matrix. X 53000.

Structure and coiling of peritrich stalk

pm —

Jmfj

400 nm*20

21 <L 22

r *

V

f

400 nm

• -J. " - • • ' •*••

1 2 2 W. B. Amos

mm*

23

Fig. 23. Section of the junction between stalk and zooid in Vorticella, showing con-tinuity between the spasmoneme (sp) and a myoneme (my) of the body. Within thezooid the basal bodies of the ciliary girdle (66), the scopula organelles (so), a foodvacuole (Jv) and the macronucleus (ma) are sectioned, surrounded by the pellicle (/>).x 12600.