j. cell sci. printed in great britaindivision chromophyta (chadefaud, i960; christensen, 1962,...

J. Cell Sci. 17, 191-219 (1975) 191Printed in Great Britain

OBSERVATIONS ON THE ULTRASTRUCTURE

OF THE CHOANOFLAGELLATE CODOSIGA

BOTRYTIS (EHR.) SAVILLE-KENT WITH

SPECIAL REFERENCE TO THE

FLAGELLAR APPARATUS

D. J. HIBBERDCulture Centre of Algae and Protozoa, 36 Storey's Way,Cambridge, CB3 oDT, England

SUMMARY

The ultrastructure of the choanoflagellate Codosiga botrytis is described with particularreference to the flagellar appendages, the flagellar rootlet system, the transition zone, the basalbody and accessory centrioles, and the stalk. The controversial early reports of flagellar appen-dages in this species have been confirmed and they have been detected in 2 further species,Salpingoeca frequentissima and Monosiga sp. The appendages consist of a delicate bilateral vane2 fim wide on either side of the axis, composed of extremely fine overlapping or interwovenfibrils.

The flagellar root system consists of a large number of radiating microtubules associated withbands of electron-dense material near the basal body; striated roots are absent. The micro-tubules extend from several separate foci, those in any one group originating near a compositeelectron-dense band, and for a distance of 300 nm from the basal body they are separated byblocks of interstitial material.

The flagellar basal body forms one of a diplosome pair of centrioles. The triplet microtubulesof the accessory centriole are embedded in amorphous electron-dense material and the whole isenveloped in a sheath of similar appearance. The existence of a third centriole close to thediplosome pair is also reported. The relatively complex structure of the flagellar transitionalzone is described.

The stalk is composed of a core of circular lacunae, which may or may not contain finger-likeprotoplasmic extensions of the posterior end of the cell, surrounded by a continuation of thesheath material which encloses the remainder of the protoplast. In the stalk only there is afurther closely sheathing layer about 15 nm thick which is regularly striated, the spacing of thestriations in shadowcast material and sections being about 3 times that measured by negativestaining.

The structure of choanoflagellates differs widely from that of the algal class Chrysophyceae,the group in which they are included in some classifications, and from the remainder of thealgae; they do not appear to have a place in either the algae or the plant kingdom. The structureof Codosiga botrytis is briefly compared with that of sponge choanocytes and collared cells in theMetazoa and some of the possible phylogenetic implications of this are indicated.

INTRODUCTION

The choanoflagellates, or collared flagellates, are a clearly circumscribed group ofunicellular, marine, brackish or freshwater monads, the most distinctive characters ofwhich are a single anteriorly directed flagellum surrounded by a ring of tentacles (thecollar). The protoplast is colourless and is enclosed in a periplast which may be either

192 D.J.Hibberd

a relatively simple, close-fitting sheath, or an elaborate basket-like lorica composed ofribs of silica (Leadbeater, 1972 a). The taxonomic position and phylogenetic relation-ships of choanoflagellates have been the subject of much interest and speculation andthese organisms still have a firm place in classifications of both the plant and animalkingdoms. In the algae they form a Subclass in the Chrysophyceae (golden-brownflagellates) in the system of Bourrelly (1968) and the Class Craspedophyceae in theDivision Chromophyta (Chadefaud, i960; Christensen, 1962, 1966); in the Protozoathey are included in the Order Choanoflagellida in the Class Zoomastigophorea(Honigberg et al. 1964).

The controversy concerning the phylogeny of these organisms stems from thediscovery by James-Clark (1868) of cells in sponges with an apparently identicalstructure, and arguments concerning the relationship of the sponges to choanoflagel-lates continue to the present day. Phylogenetic speculation has recently taken a newturn with the demonstration of collared cells not only in sponges but in a wide rangeof metazoan groups (reviewed by Norrevang & Wingstrand, 1970, and Lyons, 1973)and it has been suggested (Norrevang & Wingstrand, 1970) that the choanocyte-likecell is a fundamental cell type in metazoans, probably derived phylogenetically fromsome flagellate ancestor.

Notwithstanding the general interest of choanoflagellates and the desirability ofdetailed comparison with choanocytes in other groups and with the algae with whichthey are said to be related, detailed information on their generaljinternal structure islacking. Recent work has mostly been concerned with examination of the externalmorphology of species important in the marine nanoplankton (Throndsen, 1970;Leadbeater, 1972a, b; Thomsen, 1973; Leadbeater & Morton, 1974a), though someof these papers include sections which show the general internal structure of thespecies concerned. The only studies using modern methods concerned specificallywith internal morphology are those of Laval (1971) and Leadbeater & Morton (19746),the latter dealing mainly with the process of ingestion.

The present communication provides an account of the internal and external ultra-structure of Codosiga botrytis (Ehr.) Saville-Kent. This is a common and widelyoccurring freshwater representative of the group and there are several early accountsof its morphology as seen in the light microscope (Saville-Kent, 1882; James-Clark,1867, 1868; Fisch, 1885; France^ 1897; Lapage, 1925; de Saedeleer, 1927, 1929)summarized by Hollande (1952). Codosiga botrytis is the type species of the Genus andtherefore is of particular interest since the taxonomy of the whole group, particularlythe forms lacking a complex lorica, is much confused. It was also of great interest toexamine this particular species to clarify the structure of the lateral flagellar appendagesfirst described by Petersen (1929) and Vlk (1938) from light microscopy of materialprepared by Loffler's method for bacterial flagella and later by Petersen & Hansen(1954) from electron microscopy of mordanted and fuchsin-stained material. Owingto the relatively poor resolution of this method, the detailed structure of these putativeappendages could not be determined and there has therefore been much speculationas to their true nature, because of the phylogenetic importance of flagellar appendagesin the algae, and also because of their apparent absence from all other choanoflagellate

Infrastructure of Codostga botrytis 193

species examined subsequently. The increased resolution obtained by shadowcastingwhole mounts with chromium rather than with gold/palladium alloy and the use ofnegative staining in the present study has enabled this long-standing problem to belargely resolved.

MATERIALS AND METHODS

The present lack of information on the internal structure of choanoflagellates can almostcertainly be attributed to lack of sufficient quantities of material for embedding, since theseorganisms, whilst common in nature, rarely occur in quantity, and only 2 species, both marine,have so far been brought into culture (Gold, Pfister & Liguori, 1970; Leadbeater & Morton,19746). Certain species, however, live attached to other free-living animals and plants and it istherefore possible to obtain material indirectly by collecting these host organisms in bulk. Themajority of the material used for the present study was a net plankton sample from Grasmere inthe English Lake District collected on 20 June 1973, which consisted mainly of the diatomAsterionellaformosa var. formota Hassall on which the Codostga grew in relatively large quantity.The material was posted to Cambridge and was processed on the day following collection.Supplementary observations on the flagella of Monosiga sp. and Salpingoeca frequentissima(Zach.) Lemm. were made on samples similarly collected from Blelham Tarn on 20 November1973 and 30 April 1974 respectively.

For light microscopy, material was examined alive using phase-contrast illumination. Materialfor shadowcasting was prepared by fixing a suspension of the plankton, concentrated by centri-fugation, on a coated grid by exposure to the vapour of 2 % osmium tetroxide for 30 s-i min.Excess liquid was removed from the grid after allowing the material to settle, and the prepara-tion was then allowed to dry before shadowcasting with chromium. Material for negativestaining was fixed in osmium tetroxide vapour in the same way, after which a solution of 2 %uranyl acetate in distilled water was applied for 15-30 s, all but a thin film being removed withfilter paper before drying. Material for sectioning was prepared by centrifuging the Asterionellabearing the choanoflagellate into a pellet which was then fixed for 17 min in a mixture con-taining equal parts of 2 % glutaraldehyde and 1 % osmium tetroxide in 0-05 M cacodylatebuffer, pH 7, the fixatives being chilled and mixed immediately before fixation. The pellet wasthen rinsed in 3 changes of buffer over a period of 30 min, gradually increasing the strength ofthe buffer to o-i M, after which it was treated with 2 % osmium tetroxide in o-i M cacodylatebuffer, pH 7, for 2 h, dehydrated in an ethanol series and embedded in Epon. All stages offixation and dehydration to 95 % ethanol were carried out at o°~4 °C. Sections were cut with adiamond knife on a Reichert OmU2 ultramicrotome, stained in uranyl acetate and Reynold'slead citrate and examined on an AEI EM801 electron microscope. Observations are based on atotal of 420 electron micrographs.

Identity of materialCodosiga botrytis and Salpingoeca frequentissima are well denned taxa and the material used

here clearly identifies with previous descriptions of these species. In the absence of any distinc-tive feature and in view of the present confused state of the taxonomy of choanoflagellates ingeneral, it has not been possible to identify positively the species of Monosiga used for observa-tions of negatively stained flagellar appendages.

OBSERVATIONS

General morphology

The structure of C. botrytis as seen in the light microscope is well documented andnew data are included here only to form a basis for the observations from electronmicroscopy and to demonstrate the identity of the material with previously publisheddescriptions.

13 C E L 17

i94 D.J.Hibberd

The majority of cells of C. botrytis in the present collection occurred singly (Figs.2, 3, 5), with only a small proportion in pairs borne on a common stalk (Fig. 4).Clusters of up to 5 cells which are typical for this species were present in later collec-tions from the same locality. The cell body is roughly pyriform with a truncateanterior end (Figs. 3, 5) which sometimes appears slightly depressed. The majority ofcells are 8-10 fim long and 5-7 fim wide. A single flagellum arises from the middle ofthe anterior end of the cells (Figs. 3-6). Its average length is 30 fim (range 23-46 fim)and it bears a distal hair point approximately 3 fim long (Fig. 6). The flagellum eitherbeats rapidly in an approximate sine wave (Fig. 3) or slowly, stiffly and jerkily. Whenat rest it is fully extended and held rigidly (Fig. 5).

The flagellum is surrounded by a funnel-shaped collar approximately 11 fim long(Figs. 3,5) which is made up of 35-40 equally spaced cylindrical tentacles, 150-200 nmin diameter (Figs. 6, 11), which are inserted into the side of the cell slightly below thetruncate anterior end (Figs. 5, 8, 15). The tentacles narrow to a diameter of 100-125 nmat the point of attachment to the cell (Figs. 8, 15). Although their contents are indirect contact with the densely granular cytoplasm of the cell body, they appear tocontain only amorphous or fibrous-reticulate material (Figs. 11, 15, 16) which intransverse sections sometimes appears to form very faint lacunae or circular profiles.The contents of the tentacles only very occasionally faintly appear to be arrangedlongitudinally. In cells flattened by coverslip pressure, the collar contracts and itsdistal opening becomes narrower than its base (Fig. 4).

The posterior end of the cell body is drawn out into a thin stalk by means of whichit is attached to the cells of Asterionella (Fig. 3); the posterior ends of cells occurringin pairs fuse into a single stalk at a distance of approximately 1-5 /tm from the cell body(Fig. 4). The stalk is about 9 fim long in the majority of cells or cell pairs (range 5-10/im)though in a few it appears to be absent, with the Codosiga cells completely sessile onthe diatoms. The diameter of the stalk is approximately 800 nm as measured fromshadowcast material (Figs. 36,38). The detailed structure of the stalk is described below.

Cells occurring in pairs are held together not only by the stalk but by a laterallinkage of the cell bodies towards their posterior ends by a cytoplasmic bridge, whichis just visible in the light microscope (Fig. 4). Favourably oriented shadowcast pre-parations (Fig. 7) show that the bridge contains a median block of dense materialwhich in sections (Figs. 8, 9) appears as a partition midway along the bridge. Thismeasures approximately 280 x 60 nm and contains transverse pores or less-denseareas about 20 nm in diameter (Fig. 9).

The protoplasts are each enclosed in a delicate close-fitting sheath (Figs. 8, 10)which is hardly visible in the light microscope and which appears amorphous inshadowcast preparations (Fig. 7). Sections show that the sheath continues beyond theanterior end of the cell body to surround the tentacles (Fig. 11). Sheath material alsocovers the anterior end of the cell and further material extends anteriorly in a shallowdish-like curve from the point of flagellar insertion (Figs. 8, 15). The sheath materialis composed of 2 layers; the inner, which always appears to be present, is only moder-ately electron-opaque (Figs. 8, 10), whereas the second layer which lies immediatelyagainst it is very electron-opaque (Fig. 8) and appears to be a secondary development

infrastructure of Codosiga botrytis 195

since it is formed more extensively on the outer faces of the cell pairs which from theirshapes appear to have originated from a relatively recent division (Fig. 8). Thestructure of the sheath material covering the stalk is described below.

With the exception of the flagellar apparatus and the stalk, the internal structure ofthe cell is relatively simple. The most conspicuous organelle is the spherical nucleuswhich measures approximately 2 /*m in diameter (Figs. 4, 8, 10). It lies centrally, inthe anterior third of the cell and contains a single, dense, spherical nucleolus up to1 /tm in diameter (Figs. 4, 8,10). A single Golgi body, composed of 5-6 rather inflatedcisternae, lies immediately anterior to the nucleus and posterior to the flagellarapparatus (Figs. 8, 15). Profiles of vesicles 75-100 nm in diameter occur in relativelylarge numbers in the region surrounding the Golgi body (Fig. 15). The mitochondriawhich mostly occur in the cytoplasm surrounding the nucleus (Fig. 13), have flatplate-like cisternae (Fig. 15). Two or three large spherical vesicles almost alwaysoccur in the tapering posterior part of the cell (Figs. 4, 5) though they always appearcollapsed in sections (Fig. 8). Since they have not been seen to discharge externally,they are possibly food vesicles. However, although they do contain granular material,no recognizable contents, such as more or less intact bacteria, have been seen withinthem. Small vesicles containing amorphous material and dense bodies 200 nm indiameter are generally distributed throughout the cell (Figs. 8, 10). The remainder ofthe cell volume is densely packed with ribosomes (Figs. 8, 15).

Flagellar apparatus

Flagellar appendages. Shadowcast preparations of the flagellum of Codosiga botrytisreveal the presence of a delicate bilateral wing-like process or vane (Figs. 12-14),which extends along the flagellum from inside the collar, usually to a point approxi-mately two-thirds along its length where it is abruptly truncated. It is possible, how-ever, that it normally extends further than this and that the distal part has been lost,since it appears extremely delicate, often being completely absent or present only asremnants, and even the most nearly intact specimens show signs of disintegration(Figs. 12, 14). The vane could not be detected in sectional material and therefore theexact point at which it originates at the proximal end of the flagellum is not known.The width of the vane on each side of the flagellar axis is approximately 2 /tm, givingan overall width of axis plus vane of about 4-5 fiva. The vane appears to be composedof 2 sets of interwoven or overlapping fine fibrils each of which lies at an angle of about65°-70° to the axis, and which therefore cross at an angle of about 4Oc-5O° (Fig. 14).The vane is not homogeneous, however, since there appears to be very little or no over-lapping of fibrils in a zone 500 nm wide on either side of the flagellum (Fig. 14) andthe fibrils in this region appear more loosely arranged. In addition, the vane is ridgedlongitudinally, with the ridges on opposite sides of the vane spaced at the samedistance from the flagellar axis (Fig. 14). The arrangement of the ridges in otherspecimens follows the same general pattern as that illustrated in Fig. 14, a narrowdistal zone about 200 nm wide followed by a broad zone about three times this widthbeing particularly evident, but their distances from either the edge of the vane or fromthe flagellar axis do not correspond exactly.

13-2

IQ.6 D. J. Hibberd

Owing to their extreme fineness, the diameter of the vane fibrils cannot be deter-mined accurately from shadowcast preparations and attempts to negatively stain themin Codosiga were unsuccessful. However, a similar vane was also found in a speciesof Monosiga, also epiphytic on Asterionella, and although it is lost or disintegrates in

mt

Fig. i. A 3-dimensional reconstruction of the flagellar rootlet system and transitionzone in Codosiga botrytis. a, A-tubule; ax, axosome; b, B-tubule; c, C-tubule; ca, com-posite arc; cf, central filament; »', interstitial material; mt, microtubule; tp, transverseplate.

this species much more readily than in C. botrytis, negative staining was successful(Fig. 13). This gives a fibril diameter of 3-5 nm but owing to the state of disintegrationof the vane it has not been possible to determine how, if at all, the fibrils interweave.A vane of similar overall dimensions to that in C. botrytis has also been found inSalpingoeca frequentissima but in this case no fibrillar substructure could be detected.

Flagellar axis, basal body and transition zone. Cross-sections of the flagellum show

Ultrastructure of Codosiga botrytis 197

the usual 9 peripheral doublet microtubules and 2 separate central microtubules(Fig. 31). The flagellum is approximately 255 nm in diameter but the flagellarmembrane often appears to be extended laterally into one or two low ridges (Fig. 31).The whiplash point of the flagellum has not been transected but 3 or 4 microtubulescan be seen in negatively stained preparations of this region.

The basal body is constructed in the normal way as a cylinder of 9 tilted tripletmicrotubules (Figs. 20, 24). The point at which the triplet structure ends has not beendetermined from serial tangential sections of the anterior end of the cell but in longi-tudinal sections there is always an abrupt change of density about at the level at whichthe cell membrane begins to form a cone (Figs. 21, 22, 27). This change in appearanceis almost certainly due to a diminution in the total number of microtubules and ittherefore seems likely that the triplet microtubules change to doublets at this point byloss of the C-tubule. At this level delicate strands appear to link the distal end of thebasal body to the cell membrane (arrows in Fig. 22).

The interval between the termination of the C-tubules and the origin of the centraltubules of the axoneme, the transition zone, has a relatively complex structure. At thedistal end of the cone of protoplasm from which the flagellum arises, the cell mem-brane, which continues from this point as the flagellar membrane, is tightly con-stricted around the axoneme so that the overall diameter of the flagellum is only 210 run.Immediately above this level, the flagellar membrane is laterally inflated for a distanceof 100 nm so that the overall maximum width of the flagellum becomes approximately450 nm (Figs. 21, 22, 27, 30). At the level of constriction, a single partition, thetransverse plate, crosses the lumen. This has a central lenticular thickening, theaxosome, which measures 75 x 22-5 nm (Figs. 21, 27). The lumen of the transitionzone proximal to the transverse plate contains flocculent material of relatively lowelectron density (Figs. 21, 27) which appears to extend from the axoneme for a dis-tance of about 15 nm before it spreads laterally to fill the lumen (Fig. 27).

The two central tubules of the axoneme appear to originate about 150 nm above theaxosome and are linked to it by a thin strand of electron-opaque material (Figs. 27-30).The transverse section through this part of the transition zone illustrated in Fig. 30shows that spokes run radially from this central strand to the centres of the doubletfibres, and that fainter strands extend between the spokes and also interconnect thedoublets in a circle. The doublet fibres are also linked by short connexions betweenthe A- and B-tubules. The profile in Fig. 30 probably lies immediately above thetransverse plate, since a part of the flagellar membrane close to the doublets is includedin its thickness; the doublets are connected to this by short arms of dense materialwhich extend outwards from their centres.

In the majority of sections passing through or close to the flagellar basal body, theprofile of a second basal body, or centriole lying approximately at right angles to theflagellar basal body can be seen (Figs. 15, 18, 21, 24, 27). The accessory centriole isapproximately 350 nm long and transverse sections show 9 tilted triplet microtubules(Fig. 28) exactly as in the basal body. In this case, however, the tubules are embeddedin amorphous electron-dense material and the whole is enveloped in a sheath of similarmaterial approximately 40 nm thick separated from it by an electron-transparent

198 D. J. Hibberd

space of similar width (Fig. 28). An exact transverse section through one extremeend of this centriole has been obtained only once and shows that the profiles ofthe C-tubules are very weak at this point and that the sheath material is missing(Fig. 29). From their structure and mutual orientation, the flagellar base and accessorycentriole are interpreted as a diplosome pair, one of which has become a basal body.

Examination of a number of series of sections through this region of the cell hasconsistently revealed the presence of a further centriole close to the diplosome pair butin a slightly more posterior position. This is illustrated in serial longitudinal sectionin Figs. 21-23 and in serial transverse section in Figs. 24-26. This third centriole has asimilar length to the accessory centriole but it does not appear to be enveloped inamorphous electron-dense material.

Microtubular rootlet system. A complex system of radiating microtubules and bandsof electron-dense material occurs around the basal body in C. botrytis and this can beseen even in low-power longitudinal and tangential sections (Figs. 8, 11). Longitudinalsections at higher magnifications through or near the basal body show a stack of about5 microtubules about 160 nm high (Fig. 18) which appears in either longitudinal ortransverse section, and sometimes appears longitudinally and transversely sectionedon opposite sides of the same basal body (Fig. 17). These microtubules appear tooriginate near a band of dense material about 200 nm long and 65 nm wide which liesclose and parallel to the basal body (Figs. 15, 17, 21), though the resolution of themicrographs is not sufficient to determine whether their ends lie against the outerface of the band or a short distance away from it. The proximal end of the band islevel with the proximal end of the basal body and suitably oriented sections show thatit has a compound structure, being composed of 2 outer layers 20 nm thick and 2 verythin layers equally spaced between them (Fig. 17). This band appears in tangentiallongitudinal section in Fig. 23.

From their origin near the composite band to a distance of approximately 300 nmfrom the basal body the microtubules are separated by blocks of amorphous electron-dense material arranged in vertical rows (Figs. 15, 17, 21). Three of these rows arearrowed in Fig. 17 but the exact number is difficult to determine in longitudinalsection. At a greater distance from the basal body than the bands of interstitialmaterial the microtubules lie closer together, the microtubule bundle tapering from athickness of 200 nm near the basal body to approximately 120 nm where it underliesthe plasmalemma.

Tangential sections through these structures are relatively rare but more informa-tive. They reveal that the microtubules are not evenly distributed round the basalbody but extend from 5 or more separate foci (Figs. 19, 20). The groups of micro-tubules are of unequal size and are apparently arranged asymmetrically around thebasal body (Fig. 19). In all of the relatively few tangential sections through thisstructure, one of the groups has been found to be larger than the rest, with the micro-tubules radiating through an angle of almost 180°. The arrangement of the densematerial associated with the microtubules can be seen most clearly in this largestgroup, though that in the others appears to be identical.

In tangential sections the thickness and lamellate structure of the central composite


band appear the same as in longitudinal sections but in this plane the band appearsperfectly semicircular in shape, with a radius of 75 nm (Fig. 19). In addition, thereappears to be an electron-dense spot at the geometrical centre of the band (Fig. 19),though not enough examples have been seen to determine whether this is a generalfeature. The amorphous material separating the microtubules also appears as a seriesof arcs and these have the same centre as the composite arc. There are 3 main arcsspaced at approximately equal intervals of 45 nm from the compound band, the middleone being the densest and thickest, and the outermost the least dense and usuallyincomplete or not very clear. In addition, tangential sections reveal the presence ofless well defined arcs in median positions between the 3 main arcs and between theinnermost of these and the compound arc. It is possible, however, that the latterintermediate arc is not a true structure but merely an appearance resulting from theclose proximity of the ends of the microtubules if they originate in this position andnot immediately against the face of the central compound arc.

After leaving this zone the radiating microtubules run in a straight line under theplasmalemma of the anterior end of the cell to a distance of at least 1-2 fim from thebasal body (Fig. 20). Those from adjacent complexes therefore cross over (Fig. 20) andprobably intermesh shortly after leaving the outermost band of interstitial material,though the exact manner in which they do so has not been determined. About 30-40microtubules can be seen in tangential sections such as that illustrated in Figs. 19 and20, and assuming that these are stacked 4-5 deep there is probably a total of 120-200microtubules in the whole root system. The microtubules curve under the shoulderand down the sides of the cell for some considerable distance, though the point atwhich they terminate is not known. About 130-160 microtubules have been countedin transverse sections of the cell at the level of the tentacles and so the majority if notall of them are probably still present at this level. These microtubules are evenly dis-tributed around the periphery of the cell in ones, twos and threes and are not arrangedregularly in arcs of 4 as has been described for Salpingoecapelagica Laval (Laval, 1971).

Stalk

The stalk of Codosiga botrytis has been found to have an unexpectedly complexstructure. The central part, at least at the proximal end, consists of finger-like proto-plasmic extensions of the tapering posterior end of the cell (Figs. 8, 32). These fingersappear to contain only fibrillar-reticulate material (Fig. 32) and so contrast with themajority of the cell cytoplasm which is densely packed with ribosomes. Transversesections of the stalk at a greater distance from the cell show a variable number of moreor less circular lacunae surrounded by the stalk sheath (Figs. 33, 37). Profiles with 5, 7and 9 lacunae have been seen and it is possible that this number is related to thenumber of cells borne on the stalk. The lacunae do not appear to contain the proto-plasmic fingers when seen in transverse section and it is evident from shadowcastpreparations that the latter may extend only into the most proximal region of thestalk. Occasional preparations have been seen, however (Fig. 36), in which it is equallyclear that they may also extend for varying distances inside the sheath and it is thereforeprobable that the lacunae represent the empty spaces from which the protoplasmic

200 D. J. Hibberd

fingers have been withdrawn. It has not been possible to determine whether thiswithdrawal occurs naturally or is simply a preparation artifact.

The fibrillar sheath material of the cell body continues down the whole length of thestalk and there also the very dense outer layer may or may not be present. In addition,there is a denser and more compact sheathing layer lying closely against the lacunarpart of the stalk (Fig. 34). In longitudinal section this layer appears about 15 runthick and is composed of subunits with an average spacing of 32 nm (Fig. 34). Theseappear as rectangular or L-shaped electron-dense blocks about 24 nm wide separatedby electron-transparent spaces about one-third of this width in vertical longitudinalsection (Fig. 34.) The zones have similar dimensions in vertical tangential section(Figs. 34, 37) where it can also be seen that they are pitched at an angle of about 200

to the horizontal. It can thus be seen that it is this layer which is responsible for thestriated appearance of the stalk in shadowcast preparations (Figs. 35, 36, 38), wherethe average spacing of the striations is also about 32 nm, and where they are alsotilted at a shallow angle to the horizontal.

The stalk also appears striated in negatively stained material (Fig. 39). In this casehowever, the average spacing of the striations is only 10-13-5 nm and the materialappears as alternating light and dark bands, two complete sets of which are equivalentto one complete set of the units visible in vertical longitudinal or vertical tangentialsection. The reason for this difference is not clear but it must be due to a more subtlearrangement of the striated material than has been resolved by means of sections.

DISCUSSION

The basic arrangement of organelles in C. botrytis is similar to that determined inthe electron microscope for this species by Fjerdingstad (1961a) and for a number ofother choanoflagellates (Laval, 1971; Leadbeater, 1972a, b; Leadbeater & Manton,1974; Leadbeater & Morton, 1974a, b). All species so far described have a more orless central, spherical nucleus with a single nucleolus, a single Golgi body lyingbetween the anterior end of the nucleus and the flagellar basal body, a single apicallyinserted flagellum with a relatively long hair point, a circular collar of tentacles sur-rounding the flagellum, mitochondria with flattened cristae, and cytoplasm verydensely packed with ribosomes. The most important new observations made here areon the structure of the flagellar apparatus, particularly the flagellar vane, the micro-tubular rootlet system and the transition zone, and on the stalk.

Flagellar vane

It has been gratifying largely to confirm the early observations of Petersen & Hansen(1954) of lateral structures of a basically fibrillar nature on the flagellum of C. botrytis,since the 2-/im-broad dense zone on both sides of the flagellum described by them hassometimes been regarded as a possible artifact or simply as mucilage. Petersen &Hansen themselves were uncertain whether the zone was composed of hairs whichcrossed on drying, whether it was an envelope with a fibrous reticulate structure, orwhether it was in one plane or projected on all sides of the flagellum. It is now virtually


certain that the zone is a flat vane with an organized structure composed of adheringfine filaments of a very narrow diameter, though it has not yet been possible to confirmthis by means of sections, and it is also not yet certain whether the fibrils are inter-woven or whether they merely overlap. The vane certainly does not resemble any ofthe varied flagellar appendages described for motile cells in the algae (See Manton,1965, for a review; Hibberd, Greenwood & Griffiths (1971); Hibberd & Leedale,1972), and is completely unlike the tripartite tubular flagellar hairs characteristic ofthe Chrysophyceae and other algal classes (Bouck, 1971, 1972). It most closely re-sembles the flagellar appendages described for the choanocytes of certain sponges andthis will be discussed more fully below.

Except for the light-microscope observations of Petersen (1925, 1929) on Salping-oeca sp. and the present observations on Monosiga sp. and Salpingoeca frequentissima,there are no reports of flagellar appendages in other choanoflagellate species. Withrespect to their apparent absence from marine species, including Codosiga gracilis(Kent), de Saedeleer, Leadbeater & Morton (19746) have pointed out that it is possiblethat the vane could be destroyed during preparation owing to some complicationresulting from the saline environment, and it is also apparent from the present studythat the vane is much more delicate in some other freshwater species than it is inC. botrytis. The possibility still remains that the vane is restricted to freshwater formsbut this explanation seems much more unlikely in view of the constant structure andoccurrence of flagellar appendages in other groups.

Microtubular rootlets

Previous information on the structure of flagellar roots in choanoflagellates is scant.Fjerdingstad (1961a) in his study of C. botrytis reported a number of microtubulesconnected with the basal body, though the structures in this region appear mostly notto have been preserved, and Leadbeater & Morton (1974a) describe microtubulesradiating from a ring around the flagellar base in Acanthoeca spectabilis Ellis but nofurther details are given. Laval (1971) described microtubules radiating along curvedpaths from the basal body in Salpingoeca pelagica, these being inserted on a compoundosmiophilic ring. The outermost part of this structure can be seen from Laval'smicrographs to be composed of 3 bands of dense material, though whether this ieallyforms a ring around the flagellar base is not entirely clear and the resolution of themicrographs is not high enough to allow detailed comparison with the present study.More recently Leadbeater & Morton (19746) have described a system of radiatingmicrotubules around the basal body in Codosiga gracilis and in this case have illustratedvery clearly that the microtubules originate near the innermost of 4 concentric ringsof osmiophilic material surrounding the flagellar basal body and not from severalseparate foci as in C. botrytis. The outer 3 rings in C. gracilis are, as in C. botrytis,blocks of dense-staining material inserted between the microtubules. The flagellarrootlet system in Codosiga botrytis therefore resembles that in C. gracilis and Sal-pingoecapelagica in its general form, but is so far unique in that the points of origin ofthe microtubules do not form a complete ring around the basal body. One result ofthis arrangement is that a greater number of microtubules can be incorporated in the

202 D. J. Hibberd

system than would be possible if they were inserted on a circular structure at the samedistance from the flagellar base as the composite arcs. The number of tubules involvedin the structure in C. botrytis must, in any case, be larger than in C. gracilis or Sal-pingotca pelagica since they are stacked 4-5 deep near the basal body, compared withthe 2 or 3 layers in C. gracilis (Leadbeater & Morton, 19746) and the apparent singlelayer in S. pelagica (Laval, 1971). Comparison of the root system in these 3 speciessuggests that the number of microtubules involved may be proportional to cell size.

Microtubules radiating from flagellar basal bodies are common in a wide range oforganisms (Pitelka, 1969, 1974) but this particular arrangement appears to be re-stricted to and characteristic of the choanoflagellates. The compound arc in Codosigaprobably belongs to the range of structures collectively described as dense plaques byWolfe (1972), structures against which both mitotic and non-mitotic microtubulesabut and probably originate in a variety of positions in a variety of cell types (seeWolfe, 1972, for a review). They have only rarely been found near to basal bodies,however, and even in these cases the resemblance between the plaque structures andthe system in Codosiga is not very strong, as comparison with the striated fibre linkingthe basal bodies in the green flagellate Chlamydomonas described by Ringo (1967) andthe laminated cap in the ciliate Nassula described by Tucker (1970) will illustrate.However, in the light of the general resemblance referred to, it would clearly be of thegreatest interest to elucidate the process of cell division in a choanoflagellate.

Striated root fibrils, attached to the proximal end of basal bodies and penetratingdeeply within the cell are a conspicuous component of the flagellar apparatus in a verywide range of flagellated and ciliated cells (Pitelka, 1969, 1974; Wolfe, 1972) and theirabsence in C. botrytis, C. gracilis (Leadbeater & Morton, 19746) and apparently alsoin Salpingoeca pelagica (Laval, 1971) is therefore noteworthy. Since striated rootfibrils are commonly supposed to provide anchorage and support for flagella, any suchfunction in C. botrytis must be provided entirely by the microtubular rootlet system,and this is one possible reason for its relatively elaborate structure.

Transition zone

The structure of the basal body and transition zone in C. botrytis agrees in generalwith that described for a wide range of flagellate cells, and appears to be a modifiedform of Pitelka's (1974) 'Type II ' basal body in which the C-tubules terminate at thelevel of deflexion of the cell membrane (the base of the cone into which the flagellumis inserted in Codosiga), the doublet microtubules continuing upwards for 200 nm ormore (150 nm in Codosiga) before a transverse plate appears and the central axonemaltubules begin. In Pitelka's 'Type I ' basal body the transverse plate occurs near thesite of termination of the C-tubules and is approximately level with the cell membrane.Unusual features of the transition zone in C. botrytis are the conical projection of thecell and the laterally dilated flagellar membrane above it, the relatively long distancebetween the transverse plate and the central pair of tubules and the thin strand linkingthese structures. Since C. botrytis has a normal 9 + 2 axoneme, it is a clear exception tothe general rule established by Barnes (1961) which has since largely been sub-stantiated (Wolfe, 1972), that all cilia or derivations of cilia which possess a diplosomal

Ultrastructwe of Codosiga botrytis 203

basal structure also possess a 9 + 0 fibril pattern. This indicates that C. botrytispossibly derives from a biflagellate progenitor, the second flagellum having becomecompletely reduced.

Stalk

The striated structure of the stalk in C. botrytis was not resolved by Fjerdingstad(1961 a) in his study of this species, but Leadbeater & Morton (19746) have recentlydescribed a diagonal pattern, with a spacing of 25 nm, in shadowcast stalks ofCgradlis. This spacing is of the same order as that measured here but it is not knownwhether there is a similar reduction in this interval when measured from negativelystained mateiial. More detailed studies of the stalk aie clearly required in order todetermine more fully its structure and the function of the various components.

Tentacles

The tentacles of C. botrytis appear to contain only amorphous or fibrous materialwith no granular component, and thus differ from those in Acanthoeca spectabilis forwhich Leadbeater & Morton (1974a) describe central dense-staining granules and finefibrillar or tubular structures, and those in C. gracilis which contain ribosomes andindistinct fibrils or tubules, the latter penetrating deep into the cytoplasm (Leadbeater& Morton, 19746). Since so few species have been sectioned, it is not yet clear whethergenuine differences between species exist or whether the appearance of the contentsvaries with preparation. It does seem certain, however, that the tentacles do notcontain microtubules as normally defined, and those described as running from thebase of the tentacles in C. botrytis by Fjerdingstad (1961 a) can now be seen probablyto represent part of the microtubular rootlet system. The narrowing of the tentaclesat their point of attachment to the cell body in C. botrytis has also been illustrated forAcanthoeca spectabilis (Leadbeater & Morton, 1974a) and it is possible that this will befound to be a general featuie in choanoflagellates.

Sheath

The membranous sheath in C. botrytis is similar to the 2-layered sheath describedby Leadbeater (1972 a) for Monosiga ovata Kent but it is more substantial than theextremely delicate one in C. gracilis (Leadbeater & Morton, 19746). All these sheaths,however, have a similar relatively loose, fibrous-reticulate composition and areclearly different from the firmer envelopes with a more condensed structure inSalpingoecapelagica (Laval, i97i)and S. frequentissima (personal observations). Thisdifference supports the present distinction (Bourrelly, 1968) between the familyMonosigaceae, which includes species with mucilaginous envelopes often invisible inthe light microscope, and the Salpingoecaceae, which comprises species with firmsmooth envelopes.

Cytoplasmic bridge

The bridge described by Fjerdingstad (1961 a) as interconnecting only the envelopesof cell pairs in C. botrytis, is almost certainly homologous with the cytoplasmic link

204 D. jf. Hibberd

described here. There are no other reports of a similar structure and its function andorigin are unknown, though it is almost certainly a remnant of the division process.

Comparison with the algae, sponge choanocytes and metazoan collared cells

A full discussion of the possible taxonomic and phylogenetic implications of the newfacts on the structure of C. botrytis is not within the scope of the present communica-tion, but it may be useful to draw attention to some of the major issues on which theybear and to some of the more obvious gaps in our knowledge of the groups concerned.

The question of the inclusion of choanoflagellates within the algae, either in theChrysophyceae or as a completely separate class, has been reviewed by Leadbeater(1972 a) who concluded that the evidence for this is tenuous and that the choano-flagellates should probably be placed with the zooflagellates within the animal king-dom. More recently, Leadbeater & Manton (1974) have suggested that mitochondrialsubstructure alone is sufficient to rule out any close affinity between the choano-flagellates and all the classes of algae included in the division Chromophyta byChristensen (1962, 1966), and that they should be deleted from the plant kingdom.The present results on the structure of the flagellar apparatus strongly support thesepoints of view and on the basis of the structure of the flagellar appendages alone itnow seems unlikely that choanoflagellates are even remotely related to the Chryso-phyceae or any of the other classes of algae, though the position of a small number ofvery poorly known pigmented forms with an apparently similar organization tochoanoflagellates still remains enigmatic.

With regard to the possible relationship between choanoflagellates and 9ponges, thesimilarity in the ultrastructure of the collar and the general arrangement of organellesin choanoflagellates and sponge choanocytes has recently been pointed out by Laval(1971). The only notable difference in ultrastructure is the apparent absence of anucleolus from the nucleus of sponge choanocytes (Rasmont, 1959; Brill, 1973). Inthe light of the new information on Codosiga interesting new comparisons on thestructure of the flagellar apparatus are revealed. Most noteworthy is the similaritybetween the vane in Codosiga and the flagellar appendages described for spongechoanocytes by Afzelius (1961a, b), Feige (1966, 1969) and Brill (1973), whichresemble it in width, division into zones parallel to the flagellar axis and in the appar-ently meshed structure and narrow diameter of its constituent fibres. This would seemto add considerable weight to the argument that the 2 groups are phylogeneticallyrelated, but it must be pointed out that a full comparison cannot yet be made as it hasbeen impossible to detect the vane of Codosiga in sections and that in sponge choano-cytes has not been studied by means of shadowcast whole mounts or negative staining.Indeed, a significant difference between the vane in the 2 groups appears to be therelative massiveness and durability of the vane in choanocytes compared with thedelicacy and the apparent ease with which the structure is lost in choanoflagellates.The flagellar root systems in the 2 groups may also be organized in a similar way sinceFjerdingstad (1961 b) and Garonne (1969) have described microtubules radiating fromthe flagellar basal body in sponge choanocytes, though these are apparently notassociated with dense interstitial material and Afzelius (1961a), Fjerdingstad (19616)


and Brill (1973) have demonstrated that the flagellar membrane in choanocytes isextended into projecting ridges similar to those described for C. botrytis by Fjerding-stad (1961 a) and in the present paper. Lack of detailed information on sponge choano-cytes limits further comparison but in view of the similarities pointed out above andthe still enigmatic position of the sponges in the animal kingdom, further comparativeinvestigations are greatly desirable.

On the basis of the discovery of collared cells in a variety of Metazoa, Norrevang &Wingstrand (1970) have proposed that not only the sponges but all metazoans may haveevolved from a choanoflagellate ancestor, and Lyons (1973) has suggested that collaredcells may form a distinct cell type in forms as diverse as choanoflagellates and man.However, the collared cells of the coral described by Lyons (1973) and those of thebracheolaria larva of the echinoderm described by Norrevang & Wingstrand (1970)and those described in the papers cited by these authors, in most cases possess aconspicuous cross-banded flagellar root extending from the basal body deep into thecytoplasm, but they do not appear to possess a complex microtubular root system or aflagellar vane. In view of the conservativeness of flagellar structure in general it ispossible that differences of this nature are phylogenetically significant and it is by nomeans clear that they ' hardly concern the choanocyte pattern as such' (Norrevang &Wingstrand, 1970), since it is also possible that a microvillar ring has evolved morethan once in response to the need to ingest external particles. The fact that spongechoanocytes differ from metazoan collared cells in the same ways as choanoflagellatesis also relevant to discussions concerning their phylogeny and affinities with theremainder of the Metazoa.

Thanks are due to Dr J. W. G. Lund of the Windermere Laboratory of the FreshwaterBiological Association for providing the material used in this investigation and to Dr B. S. C.Leadbeater for access to unpublished information and for commenting on the manuscript.

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AFZELIUS, B. A. (19616). Flimmer flagellum of the sponge. Nature, Lond. 191, 1318-1319.BARNES, B. G. (1961). Ciliated secretory cells in the pars distalis of the mouse hypophysis.

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FEIGE, W. (1966). Ober breitfliigelige Anhange an der Choanocyten-geissel von Spongilliden.Naturwissenschaften 53, 617-618.

FEIGE, W. (1969). Die Feinstruktur der Epithelien von Ephydatia fluviatilis. Zool.Jb. (Anat.) 86,177-237.

FISCH, C. (1885). Untersuchungen uber einige Flagellaten und verwandte Organismen.Z. iviss. Zool. 42, 47-125.

FJERDINGSTAD, E. J. (1961a). Ultxastructure of the collar of the choanoflagellate Codonosigabotrytis (Ehrenb.). Z. Zellforsch. mikrosk. Anat. 54, 499-510.

FJERDINGSTAD, E. J. (19616). Ultrastructure of choanocyte collars in Spongilla laaistris (L.).Z. Zellforsch. mikrosk. Anat. 53, 645-657.

FRANCE, R. H. (1897). Der Orgamsmus der Craspedomonaden. Budapest: K6nigliche UngarischeNaturwissenschaftliche Gesellschaft.

GARONNE, R. (1969). Une formation paracristalline d'ARN intranucleaire dans les choanocytesde l'^ponge Haliclona rosea O.S. (Demosponge, Haploscleride). C. r. hebd. Sdanc. Acad. Set.,Paris 269, 2219-2221.

GOLD, K., PFISTER, R. M. & LIGUORI, V. R. (1970). Axenic cultivation and electron microscopyof two species of choanoflagellida. J. Protozool. 17, 210-212.

HIBBERD, D. J. & LEEDALE, G. F. (1972). Observations on the cytology and ultrastructure of thenew algal class, Eustigmatophyceae. Ann. Bot. 36, 49-71.

HIBBERD, D. J., GREENWOOD, A. D. & GRIFFITHS, H. B. (1971). Observations on the ultra-structure of the flagella and periplast in the Cryptophyceae. Br. phycol.J. 6, 61-72.

HOLLANDE, A. (1952). Ordre des Choanoflagelles ou Craspedomonadines. In Traitd de Zoologie,Anatomie, Systematique, Biologie (ed. P. P. Grass6), pp. 579-598. Paris: Masson et Cie.

HONIGBERG, B. M., BALAMUTH, W., BOVEE, E. C, CORLISS, J. O., GOJDICS, M., HALL, R. P.,KUDO, R. R., LEVINE, N. D., LOEBLICH, A. R., WEISER, J. & WENRICH, D. H. (1964). Arevised classification of the Phylum Protozoa. J. Protosool. 11, 7-20.

JAMES-CLARK, H. (1867). On the spongiae ciliatae as infusoria flagellata; or, observations on thestructure, animality and relationship of Leucosolenia botryoides, Bowerbank. Mem. BostonSoc. nat. Hist. 1, 305-340.

JAMES-CLARK, H. (1868). On the spongiae ciliatae as infusoria flagellata; or, observations on thestructure, animality, and relationship of Leucosolenia botryoides, Bowerbank. Ann. Mag. nat.Hist., $th Ser. 1, 133-142; 188—215; 250-264.

LAPAGE, G. (1925). Notes on the choanoflagellate Codosiga botrytis Ehrbg. Q.jfl microsc. Sci.69, 47I-5°8-

LAVAL, M. (1971). Ultrastructure et mode de nutrition du choanoflagell^ Salpingoeca pelagica,sp. nov. Comparison avec les choanocytes des spongiaires. Protistologica 7, 325-336.

LEADBEATER, B. S. C. (1972a). Fine-structural observations on some marine choanoflagellatesfrom the coast of Norway. J . mar. biol. Ass. U.K., 52, 67-79.

LEADBEATER, B. S. C. (19726). Ultrastructural observations on some marine choanoflagellatesfrom the coast of Denmark. Br. phycol.J. 7, 195-211.

LEADBEATER, B. S. C. & MANTON, I. (1974). Preliminary observations on the chemistry andbiology of the lorica of a collared flagellate (Stephanoeca diplocostata Ellis). J. mar. biol. Ass.U.K. 54, 269-276.

LEADBEATER, B. S. C. & MORTON, C. (1974a). A light and electron microscope study of thechoanoflagellate Acanthoeca spectabilis Ellis and A. brevipoda Ellis. Arch. Mikrobiol. 95, 279-292.

LEADBEATER, B. S. C. & MORTON, C. (19746). A microscopical study of a marine species ofCodosiga James-Clark (Choanoflagellata) with special reference to the ingestion of bacteria.Biol. J. Linn. Soc. 6 (in Press).

LYONS, K. M. (1973). Evolutionary implications of collar cell ectoderm in a coral planula.Nature, Land. 245, 50-51.

MANTON, I. (1965). Some phyletic implications of flagellar structure in plants. Adv. bot. Res. 2,i-34-

NORREVANG, A. & WINGSTRAND, K. G. (1970). On the occurrence and structure of choanocyte-like cells in some echinoderms. Acta zool., Stockh. 51, 249-270.

PETERSEN, J. B. (1925). Om svingtraadene hos flagellater. Nuova Notarisia 36, 191-193.PETERSEN, J. B. (1929). Beitr3ge zur Kenntnis der Flagellaten geisseln. Bot. Tidsskr. 40, 373-389.


PETERSEN, J. B. & HANSEN, J. B. (1954). Electron microscope observations on Codonosigabotrytis (Ehr.) James-Clark. Bot. Tidsskr. 51, 281-291.

PITELKA, D. R. (1969). Fibrillar systems in Protozoa. In Research in Protozoology, vol. 3 (ed.Tze-Tuan Chen), pp. 279—388. Oxford: Pergamon.

PITELKA, D. R. (1974). Basal bodies and root structures. In Cilia and Flagella (ed. M. A.Sleigh), pp. 437-469. London and New York: Academic Press.

RASMONT, R. (1959). L'ultrastructure des choanocytes d'^ponges. Amtls Set. nat. (Zool.) 12,253-262.

RINGO, D. L. (1967). Flagellar motion and fine structure of the flagellar apparatus in Chlamy-dommas.J. Cell Biol. 33, 543-571.

SAEDELEER, H. DE (1927). Notes de Protistologie. I. Craspedomonadines: Material syst6matique.Annls Soc. r. zool. Belg. 58, 117-147.

SAEDELEER, H. DE (1929). Notes de Protistologie. II. Craspedomonadines: Morphologie etphysiologic Reel Jnst. zool. Torley-Rousseau 2, 241-287.

SAVILLE-KENT, W. (1882). A Manual of the Infusoria, vol. 1, pp. 1-472. London: D. Bogue.THOMSEN, H. A. (1973). Studies on marine choanoflagellates. I. Silicified choanoflagellates of

the Isefjord (Denmark). Ophelia 12, 1-26.THRONDSEN, J. (1970). Marine planktonic acanthoecaceans (Craspedophyceae) from Arctic

waters. Nytt Mag. Bot. 17, 103-111.TUCKER, J. B. (1970). Morphogenesis of a large microtubular organelle and its association with

basal bodies in the ciliate Nassula. J. Cell Set. 6, 385-429.VLK, W. (1938). t)ber den Bau der Geissel. Arch. Protistenk. 90, 448-488.WOLFE, J. (1972). Basal body fine structure and chemistry. Adv. Cell molec. Biol. 2, 151-192.

(Received 8 July 1974)

208 D. J. Hibberd

Figs. 2-5. Phase-contrast light microscopy of Codosiga botrytis.Fig. 2. A living colony of Asterionella with 2 attached cells (arrows), x 300.Fig. 3. A living cell showing the pyriform shape and truncate anterior end, the beat

of the flagellum, the collar, the stalk and the foot attached to an Asterionella cell,x1000.

Fig. 4. A living cell-pair flattened by coverslip pressure showing the central nucleus(n), the parabasal Golgi body (jg), posterior vacuoles, the cytoplasmic bridge ibr), andthe narrowed and partially retracted collar, x 1500.

Fig. 5. A living cell showing the lateral attachment of the collar, the posteriorvacuoles and the stiffly held resting flagellum. x 1000.Figs. 6, 7. Shadowcast whole cells of Codosiga botrytis.

Fig. 6. The electron-microscope equivalent of Fig. 5, showing the distal hair pointof the flagellum and the tentacles of which the collar is composed, x 2500.

Fig. 7. Part of a cell-pair showing the amorphous lorica and the cytoplasmic bridgewith a dense median thickening, x 7500.


C E h 17

2io D.J.Hibberd

Figs. 8—II. General morphology of Codosiga botrytis.Fig. 8. Longitudinal section through a cell-pair showing the spherical nucleus {n)

containing a single nucleolus, the parabasal Golgi body {g), mitochondrial profiles (»«),posterior vesicles (v), the cytoplasmic bridge (br), the laterally attached tentacles (t)and the close-fitting sheath (s). x ioooo.

Fig. 9. Part of the cell-pair in Fig. 8. at higher magnification to show the structureof the median partition of the cytoplasmic bridge, x 50000.

Fig. 10. Transverse section at the level of the nucleus. Labels as in Fig. 8. x 20000.Fig. 11. Tangential section of the anterior end of a cell showing the ring of equally

spaced tentacles closely invested by the sheath, and the rootlet system (arrow) sur-rounding the flagellar basal body. Labels as in Fig. 8. x 20000.

Ultrastructure of Codosiga botrytis 2 1 1

14-2

212 D.J.Hibberd

Fig. 12. Chromium-shadowcast preparation of the flagellum of Codosiga botrytis toshow the general appearance of the flagellar vane, x ioooo.Fig. 13. Negatively stained preparation of part of the partially disintegrated flagellarvane in Monosiga sp. showing the extremely narrow diameter of the component fibrils,x 200000.

Fig. 14. Chromium-shadowcast preparation of the flagellar vane of Codosiga botrytisshowing the arrangement of the fibrils. The double arrows indicate the directions inwhich the fibrils lie and the small arrows indicate the more obvious longitudinal ridges.The bracket indicates the part of the vane on either side of the flagellum in which thefibrils do not appear to overlap in the same way as in the remainder, x 25000.

2i4 D.J.Hibberd

Figs. 15-20. Detailed structure of the anterior end of Codosiga botrytis.

Fig. 15. Longitudinal section passing through the flagellum (/) the basal body (bb)and one tentacle (i). cz, accessory centriole; g, Golgi body; m, mitochondria; mt,microtubule. x 40000.

Fig. 16. Part of a transverse section between the point of insertion of the tentaclesand the anterior end of the cell showing the peripheral arrangement of the microtubulesand the contents of a tentacle, x 50000.

Fig. 17. Longitudinal section of the flagellar rootlet system showing microtubules inlongitudinal and transverse section, the composite arc (ca) and 3 vertical bands ofinterstitial material (arrowed), x 75000.

Fig. 18. Section passing obliquely through the basal body and accessory centriole{cz) and transversely through a 5-deep stack of flagellar root microtubules. x 60000.

Fig. 19. Tangential section of the flagellar rootlet system illustrated in Fig. 11 athigher magnification showing the 5 sets of radiating microtubules surrounding theflagellar basal body. In the laigest of these the origin of the microtubules at or nearthe composite arc can be seen. The 3 main arcs of interstitial material are arrowed.ca, composite arc. For further explanation see text, x 75 000.

Fig. 20. Section passing tangentially through the basal body and rootlet systemshowing more clearly the path of the microtubules. Two points at which tubulesoriginating from adjacent foci appear to overlap are arrowed, x 50000.


15

mt .

2i 6 D.J.Hibberd

Figs. 21-23. Serial longitudinal sections through the basal body and rootlet systemshowing the accessory centriole of the diplosome pair (c2) and a further centriole (C3)in oblique section. The level at which the C-tubule of the basal body is apparently lostis arrowed in Figs. 21 and 22. A composite arc (ca) appears in tangential section inFig. 23. The small arrows in Fig. 22 indicate the delicate strands linking the basalbody to the cell membrane, x 50000.

Figs. 24-26. Successively deeper transverse sections through the anterior end of a cellshowing the flagellar basal body (bb), the accessory centriole of the diplosome pair inoblique section (c2) and a third centriole (C3) in longitudinal section, x 50000.

Fig. 27. Median longitudinal section through the basal body, transition zone andproximal part of the flagellum. C2, accessory centriole. See text for further explanation,x 60000.

Figs. 28, 29. Longitudinal sections from the same series through the anterior end of thecell passing transversely through the accessory centriole.

Fig. 28. The main part of the centriole with the triplet microtubules embedded inamorphous material and the sheath of similar material surrounding the whole,x 50000.

Fig. 29. An extreme end of the centriole showing the absence of the sheath and thevery weak profiles of the C-tubules. x 50000.

Fig. 30. Transverse section through the flagellar transition zone immediately abovethe transverse plate, x 75000.

Fig. 31. Transverse section through the axoneme showing the 9 + 2 structure and theprojecting ridges of the flagellar membrane, x 75000.

21

Ultrastructure of Codosiga botrytis

22 iT'^tL t 23217

2i 8 D.J.Hibberd

Figs. 32-39. Structure of the stalk in Codosiga botrytis.Fig. 32. Longitudinal section through the extreme posterior end of the cell showing

profiles of 4 protoplasmic fingers, x 40000.Fig. 33. A transverse section at an unknown level showing the core of lacunae sur-

rounded by the 2 layers of the sheath material, x 50000.Fig. 34. Oblique longitudinal section showing the striated layer in vertical section

(upper part) and tangential section (lower part), x 75000.Fig. 35. Shadowcast whole mount of the posterior end of the cell and the proximal

part of the stalk. The cell protoplasm appears to terminate before the striarions begin,x 25000.

Fig. 36. Shadowcast whole mount of a stalk containing protoplasmic fingers ofvarying length, x 25 000.

Fig. 37. Oblique longitudinal section of the distal part of the stalk and the footwhich is still attached to a cell of Asterionella. The striated layer can be seen in tangen-tial section in the upper part of the figure, x 40000.

Fig. 38. Shadowcast whole mount of a stalk showing striarions with a periodicity ofabout 34 nm. x 50000.

Fig. 39. Negatively stained stalk showing striarions with a periodicity of about15 nm. x 75000.

j. cell sci. printed in great britaindivision chromophyta (chadefaud, i960; christensen, 1962,...

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