J. Cell Set. a, 265-272 (1967) 265

Printed in Great Britain

FURTHER OBSERVATIONS ON THE FINE

STRUCTURE OF CHRYSOCHROMULINA CHITON

WITH SPECIAL REFERENCE TO THE

HAPTONEMA, 'PECULIAR' GOLGI STRUCTURE

AND SCALE PRODUCTION

IRENE MANTONBotany Department, University of Leeds

SUMMARY

A more detailed study of' peculiar' Golgi structure in this organism has been attempted thanin any similar species, using modern fixation methods. Where comparisons are possible there isclose resemblance to the related genus Prymnesium, to a degree sufficient to suggest that thisparticular type of Golgi system is likely to be a major phyletic indicator for the class Hapto-phyceae. Certain stages of scale production have been traced and some developmental aspectsof the whole system are discussed, though full interpretation is deferred. Some additionalfeatures of haptonema structure are described for the sake of completeness in characterizing theparticular strain of the species under investigation. These include information on the internalstructure of the haptonema tip.

INTRODUCTION

In a previous communication (Manton, 1966a) attention was primarily directed toa reinvestigation of the somewhat unusual structure of the pyrenoid in this speciesby means of more modern methods than those available when it was first described(Parke, Manton & Clarke, 1958). The present investigation extends the enquiry toother cell components, less specialized in the sense that while characteristic of thespecies they are also characteristic of the group Haptophyceae. A phyletic as well as afine-structural interest may thus be expected.

The methods used are exactly as in Manton (1966a) and the material is also thesame, namely a descendant of the type culture, No. 146 in the Plymouth collection,which has been maintained for many years in Leeds. Since 1962 the culture conditionshave been those found by experiment to yield rapid growth and copious scale produc-tion to meet the requirements of a chemical investigation on the scales (Green, 1966;Green & Jennings, 1967). Some changes in scale morphology and arrangement seemto have been induced by this treatment, to which further reference will be made below.It is enough to emphasize here that the observations to be described, like those ofManton (1966a), refer to the deviant Leeds material, which is otherwise fullyvigorous and normal in behaviour. A detailed comparison with the state of the cultureas originally described and as still maintained under the conditions of slow growth usedat Plymouth will follow later.

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OBSERVATIONS

The haptonema

It will be convenient to refer to the haptonema at the outset, since this is the mainorganelle hitherto recognized as diagnostic of the new class Haptophyceae (Christensen,1962, 1966), at least in the motile stages. The fine structure is known in a general way,from osmium-fixed material embedded in methacrylate, in three genera, namelyChrysockromulina (Parke, Manton & Clarke, 1958, 1959; Manton & Leedale, 1961a,b; Manton & Parke, 1962; Parke, Lund & Manton, 1962), CrystalloRthus (Manton &Leedale, 1963a) and Prymnesiutn (Manton & Leedale 19636). The newer techniqueshave so far been applied only to the latter (Manton, 1964) and since the small sizeof the haptonema in this genus makes it specially favourable for detailed anatomicalstudy Prymnesium parvum is at present the only species for which full information,including numerical and structural details of both tip and base of the organelle, havebeen elucidated.

A haptonema is by definition (Parke, Manton & Clarke, 1955) a filamentous appen-dage arising near the flagella but thinner than these and with different properties andstructure. In the genus ChrysochromuUna it is always long enough to be helicallycoiled when quiescent, though when extended the length varies from species to species.In some, for example C. kappa, C. minor and C. brevifihim (Parke et al. 1955), thehaptonema is shorter than the flagella. In others it is several times longer than theflagella (for example C. alifera, C. ephippium, C. strobilus (Parke, Manton & Clarke,1956, 1959) and C. parva (Parke et al. 1962)). In the remaining described species,notably C. ericina (Parke et al. 1956), C. polylepis (Manton & Parke, 1962), C. pring-sheimii (Parke & Manton, 1962) and C. chiton (Parke et al. 1958) the haptonemais longer than the flagella but less than twice as long. The functional significanceof the organelle is by no means fully understood in any of these cases though inall of them it can serve from time to time as a temporary organ of attachment to asurface.

Many species react to fixation by discarding the haptonema while it is still helicallycoiled but broken off at the base from the subtending cell. An example of a haptonemain this condition will be found in Fig. 1. A median section through such a helix willdisplay two parallel rows of profiles of the haptonema itself, part of one such rowbeing illustrated in Fig. 3. When accurately transected the internal structure canbe seen to consist mainly of a central core containing a ring of 7 tubes or fibres,outside which are 3 concentric unit membranes, with a clear space between themiddle and inner membrane suggesting that these bound a cavity, probably withosmotic properties. The nature of this cavity has not been fully demonstrated,though in Prymnesiutn it was thought to be derived perhaps from a cisterna of endo-plasmic reticulum. The agreement of the present findings both with those forPrymnesium and with previous information for the present species (Parke et al. 1958)is close.

While detailed exploration of the haptonema extremities has not been a majorobjective of the present enquiry one fortunate section giving a slightly oblique

Fine structure of Chrysochromulina 267

longitudinal view of the distal tip is reproduced in Fig. 2 to represent this part of theorganelle for the first time in this genus. As in Prymnesium (Manton, 1964) thehaptonema tip is rounded, with the cavity between the two inner membranes passingwithout interruption over the domed end of the core in which the tubes or fibres endblindly. The structure of the haptonema base is not represented fully here since thisregion is known in a preliminary way in two other species, namely C. parva andC. ericina. Part of a longitudinal view of a haptonema base entering the cell beside aflagellar base is however included incidentally in Fig. 5. Only the fibres and not themembranes enter the cell in this as in other species. They do so immediately besidethe fiagellar bases and end abruptly at the same level as these.

The scales

Sections of detached scales fill the field of Fig. 2. There are two morphologicaltypes of scale, clearly distinguishable as such in a whole mount (Fig. 4), but recogniz-able with equal certainty in most planes of section. There are large scales with shorterect rims directed towards the convex side of the slightly domed scale-plate (Fig. 2,top), though in a whole mount (Fig. 4) such rims have commonly collapsed on to thefield. There are also a greater number of smaller scales, each with a rim stronglyinflexed towards the concave surface of the subtending plate; in a whole mount thevisibility of the rim in this type of scale thus depends on which way up the scale islying, the two surfaces contrasting strongly in appearance (Fig. 4). In both types ofscale the surface of the plate carries a pattern of radiating ridges, slightly moreclosely packed on the large scales than on the small but otherwise very similar in thetwo and visible on both surfaces.

Most cells after embedding are naked, having lost their scales during processing. Itis, however, not difficult to find the occasional cell in which the scaly covering is stillintact and one such was in fact reproduced as Fig. 1 in Manton (1966a). Part ofanother is illustrated here in Fig. 5. When undisturbed the scales exhibit a veryuniform arrangement, confirmed now in many examples. The smaller scales areundermost, forming from one to three layers, with all the component scales orientedwith their rims outwards with respect to the subtending cell surface. The large scales,on the other hand, form a single outermost layer in which all are oriented with theirrims directed inwards, i.e. towards the subtending cell surface. This curiouslyinverted orientation of the two types of scale with respect to each other representsone of the most constant and unexpected differences between the Leeds state of theculture and that originally described. Interpretation of the significance, if any, of thisdifference is not yet possible but it will be sufficient for the present purpose if thefacts pertinent to the Leeds condition have been clearly demonstrated.

The Golgi system

The existence of ' peculiar' Golgi in this species was shown in a preliminary way inManton (1966a), since the essential structure happened to be just visible in one low-power view of a complete section inserted to illustrate the general topography of the

268 /. Manton

cell as a whole (Fig. i, Manton, 1966 a). When C. chiton was first described (Parkeet al. 1958) the characteristic Golgi structure subsequently encountered in all othermarine species of the genus had not yet been recognized. When it was detected inanother species, C. strobilus (Parke et al. 1959), the adjective 'peculiar' was applied toindicate that the structure was not understood. That it was also imperfectly preservedwith the older techniques has become apparent since application of glutaraldehydeto this type of organism (Manton, 1966 a, b).

A preliminary glance at Fig. 7 will show that the Golgi system as a whole is stronglypolarized in one direction and that the central parts of a row of adjacent cisternae arelocally distended by means of some unidentified metabolite which at certain times ofday can stain strongly after the treatment applied. Once seen, such distended regionswill easily be recognized in all the remaining illustrations, whether the dense metaboliteis represented within them or not. The cisternal walls in the distended regions areundoubtedly more delicate than those elsewhere and they do not normally remainintact after osmic fixation. Their collective breakdown then results in production of acentral cavity within the Golgi system in which the remaining membranes appear toend blindly, as previously described (Parke et al. 1959). Such a cavity can now berecognized as a fixation artifact, although the physical basis behind the 'peculiar'condition is real enough.

The number and position of the ' peculiar' dilations varies somewhat from cell tocell, as does the character of their contents. Where there is more than one such region(Fig. 6) some relation to an impending division may be suspected. After prolongeddarkness (Fig. 5, 18 h darkness) the distensions appear virtually empty and areperhaps about to break down, since they are much farther from the forwardly directedmargin than is usual. Under less extreme conditions of light or dark the commonestappearance is that of Fig. 6, namely dense material distributed unevenly on thebounding walls and less-dense material occupying the centres of the dilated regions.The capacity for intense staining encountered in Figs. 7 and 9 is limited to a briefperiod at the end of a long exposure to light, i.e. in the late afternoon or early eveningof a 16-h day. This relation to light has been tested more than once and seems to besignificant. Unfortunately, intense staining is not found in every cell fixed at thesetimes, and though differences between adjacent cells within one and the same blockmay perhaps be explicable in terms of differences of developmental stage, it is difficultto exclude some entirely accidental local effects of the treatment applied. The mainsignificance of this stainability, when encountered, is thus limited at the present stageof the enquiry to the evidence which it presents that ' peculiar' dilations are genuinestructures expressing some local, metabolically significant differentiation within theGolgi system. The nature of the metabolite and its functional significance for the cellas a whole remain unknown.

The direction towards which the cisternae of the central region are polarized seemsto be defined by the position of the entering flagella and haptonema (see especiallyFig. 5). Towards these the forwardly directed edges of the central cisternae arebunched, these edges themselves having a narrow, smooth appearance (see especiallyFig. 7), suggesting that they may be regions of cisternal growth rather than of differen-

Fine structure of Chrysochromulina 269

tiation. The opposite edges (Fig. 7) show the normal type of dilation accompaniedby abstriction of so-called 'hairy' vesicles or diverticula as encountered in manyother organisms including Prymnesium (Manton, 19666) from which C. chitondiffers conspicuously only in the greater number of cisternae carrying the ' peculiar'dilations.

When seen in planes of section permitting examination of the cisternae remote fromthe centre, the whole Golgi system is found to be not only polarized but heterogeneous.On one side (left in Fig. 6) the system is bounded by cisternae of rough endoplasmicreticulum (ER) locally continuous with the nuclear envelope. This subtending ER ismost easily recognized in the appropriate type of longitudinal section (Figs. 6, 7), butwith care it can also be made out in transverse sections (Figs. 9, 10). Between thesubtending ER and the Golgi centre some undisturbed flattened cisternae are present(Figs. 6, 7 and 9). In the other direction, that is, on the opposite side of the distendedcentre, the cisternae responsible for scale production are encountered. Scales are notalways present in them (Figs. 5, 6), in which case the cisternae affected differ fromothers only in their generally swollen appearance and in their contents of coarsedistorted tubes, sometimes replaced by vesicles (Fig. 9). When scales arepresent they may occur singly (Figs. 8, 9), in which case they occupy cisternae remotefrom the Golgi centre, suggesting that such scales represent the last of a batch, most ofwhich have already been discharged. In favourable cases (Figs. 10, 11) normallyencountered, in this material, only when fixed in the late afternoon or early evening, arow of scales, each in a different cisterna, can be found. In such cells scale productionis in full swing and a series of developmental stages, the youngest nearest to the Golgicentre, can frequently be recognized.

Fig. 10 is the best example of this condition reproduced here, selected because noless than 5 almost mature scales can be recognized as such. They have been numberedfor convenience, no. 1 being mature and probably already preparing for liberation, tojudge by the other contents in the cisterna it occupies, while no. 5 is still too young tobe individually identifiable. Though the whole section is cut transversely at a levelwhich does not include the ' peculiar' dilations, there is no difficulty in identifyingthe right side of this specimen as that containing them, since the position of thesubtending ER is clearly seen. If now the morphology of the numbered scales iscompared there is no difficulty in recognizing nos. 1 and 3 as small scales and nos. 2and 4 as large ones. Further, all the scales are oriented with respect to the Golgicentre in a uniform manner. In the scales of both types, the morphologically outersurface is directed away from the Golgi centre and the morphologically under-surface towards it. The inverted orientation of the rim-carrying surface in the twotypes of scale is thus already present at the site of formation.

A second example of the mutual orientation characteristic of the two types of scalewill be found in Fig. 11, although this specimen is illustrated here for other reasons.The act of scale liberation is not an easy one to study directly, yet the left-hand side ofFig. 11 seems to show some aspects of it. The scale-containing vesicles remote fromthe Golgi centre are becoming larger and more complicated internally as the cell sur-face is approached. At the surface itself (Fig. 11, top left), a bulge over a vesicle

270 /. Manton

containing at least one scale among other contents, suggests that scale liberation isabout to occur here. The site of this bulge cannot be far from the flagellar baseswhich, though not contained in this section, are indicated by the direction of polariza-tion of the main cisternae.

Another manifestation of activity suspected to be of developmental significance isvisible on the right of Fig. 11. The position of the subtending ER on the extremeright is not in doubt and between this and the Golgi centre lies an unusual thicknessof undistended cisternae, in this case, however, characterized by a curiously whorledarrangement. It is suggested that such swirls might be part of a process of cisternalreplication which must take place at least once in each division cycle, yet about whichnothing is so far known. A somewhat similar interpretation could be put forward withrespect to the left-hand side of Fig. 8 (i.e. again that lying between the Golgi centreand the subtending ER), although the morphological details and level of cutting aredifferent. A great deal more evidence will nevertheless be required before either ofthese sections can be fully understood. Attention is drawn to them here mainlybecause they represent part of the range of diverse appearances for which develop-mental explanations will ultimately be required.

DISCUSSION

Some obvious difficulties of interpretation have already been mentioned at the endof the previous section and these may serve to show why conclusions based on themicrographs illustrated here have been made somewhat cautiously. A Golgi system ofthis type is complex in more ways than one and any attempt to solve all problemssimultaneously would lead inevitably to mistakes. It is essential first to unravel thevarious functions as well as the various stages before a coherent picture in a functionaland developmental sense will become possible.

With regard to functions these are clearly multiple. The structural aspects of thedistal edges of the central cisternae are so like those in any other Golgi system that onemust suppose that common functions are still being carried out, in spite of andconcurrently with the more spectacular peculiarities of this particular system. Limitingenumeration of the latter, we have on the one hand the highly characteristic centraldilations to which the word 'peculiar' was originally applied, and on the other thearray of scale-producing cisternae. It is important to emphasize that there is no reasonat present to link these two peculiarities together. Scale production within Golgicisternae can be carried on in other groups, notably Prasinophyceae (Manton, 1966 c),without any trace of central dilations. Moreover, within the Haptophyceae there is noevidence of correlation between the size or number of central cisternae affected byswellings and scale characters of any so far detectable kind. On the other hand, themarked difference in the prominence of the central cisternae between the presentspecies and Prymnesium parvum (Manton, 19666) suggests that there may perhapsbe a correlation between haptonema length and the size of the peculiar Golgicentre, though a great deal more comparative evidence would be required to provethis. Moreover, even if it were proved, the scarcity of accurate information

Fine structure of Ckrysochromulina 271

about the functional or biochemical implications of either of these structuresmakes further interpretation on these lines meaningless at present. The possibility isnevertheless a main reason for the introduction of some information regarding thehaptonema (Figs. 1-3) in the particular strain of the present species used for thispaper.

With regard to scale production, the very close agreement with recent observationson Prymnesium parvum (Manton, 19666) is noteworthy. There can be little doubtthat the Golgi system in both genera is constructed on an identical plan, althoughinformation for the present species is now considerably more detailed. Evidence isindeed accumulating to suggest that this highly characteristic Golgi system mayprove to be as important a diagnostic character for the group Haptophyceae as is thepossession of a haptonema. If this is true, however, a greater body of comparativeinformation will be needed, and should shortly become available. Further interpreta-tion can therefore safely be deferred.

REFERENCESCHRISTENSEN, T. (1962, 1966). Alger. In Botanik (Systematisk Botamk), vol. 2 (ed. T. W.

BScher, M. Lange & T. Serensen), 178 pp. Copenhagen: Munksgaard.GREEN, J. C. (1966). An Investigation into the Nature of the Scales of Certain Chrysophycean

Flagellates. Ph.D. thesis, University of Leeds.GREEN, J. C. & JENNINGS, D. H. (1967). A physical and chemical investigation of the scales

produced by the Golgi apparatus within and found on the surface of the cells of Ckrysochro-mulina chiton Parke et Manton. J. exp. Bot. (in the press).

MANTON, I. (1964). Further observations on the fine structure of the haptonema in Prym-nesium parvum. Arch. Mikrobiol. 49, 315-330.

MANTON, I. (1966a). Further observations on the fine structure of Chrysochromulina chiton,with special reference to the pyrenoid. J. Cell Sci. 1, 187-192.

MANTON, I. (19666). Observations on scale production in Prymnesium parvum. J. Cell Sci. 1,375-380.

MANTON, I. (1966c). Observations on scale production in Pyramimonas amylifera Conrad.J. Cell Sci. 1, 429-438.

MANTON, I. & LEEDALE, G. F. (1961a). Further observations on the fine structure of Chryso-chromulina ericina Parke & Manton. J. mar. biol. Ass. U.K. 41, 145-155.

MANTON, I. & LEEDALE, G. F. (19616). Further observations on the fine structure of Chryso-chromulina kappa and Chrysochromulina minor with special reference to the pyrenoids.J. mar. biol. Ass. U.K. 41, 519-526.

MANTON, I. & LEEDALE, G. F. (1963 a). Observations on the microanatomy of Crystallolithushyalinus Gaarder & Markali. Arch. Mikrobiol. 47, 115-136.

MANTON, I. & LEEDALE, G. F. (19636). Observations on the fine structure of Prymnesiumparvum Carter. Arch. Mikrobiol. 45, 285-303.

MANTON, I. & PARKE, M. (1962). Preliminary observations on scales and their mode of originin Chrysochromulina polylepis sp.nov. J. mar. biol. Ass. U.K. 42, 565-578.

PARKE, M., LUND, J. W. G. & MANTON, I. (1962). Observations on the biology and finestructure of the type species of Chrysochromulina (C parva Lackey) in the English LakeDistrict. Arch. Mikrobiol. 43, 333-352.

PARKE, M. & MANTON, I. (1962). Studies on marine flagellates. VI. Chrysochromulina prings-heimii sp.nov. J. mar. biol. Ass. U.K. 43, 391-404.

PARKE, M., MANTON, I. & CLARKE, B. (1955). Studies on marine flagellates. II. Three newspecies of Chrysochromulina. J. mar. biol. Ass. U.K. 34, 579-609.

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PARKE, M., MANTON, I. & CLARKE, B. (1956). Studies on marine flagellates. III. Three furtherspecies of Chrysochromulina. J. mar. biol. Ass. U.K. 35, 387-414.

PARKE, M., MANTON, I. & CLARKE, B. (1958). Studies on marine flagellates. IV. Morphologyand microanatomy of a new species of Chrysochromulina. J. mar. biol. Ass. U.K. 37, 209—228.

PARKE, M., MANTON, I. & CLARKE, B. (1959). Studies on marine flagellates. V. Morphology andmicroanatomy of Clirysockrormdina strobilus sp.nov. J. mar. biol. Ass. U.K. 38, 169-188.

(Received 31 October 1966)

Fig. 1. Detached coiled haptonema and a scale, from a whole mount. MicrographD 5935, x 10000.Fig. 2. Field of detached scales of both types (see especially thick arrow); an obliquelycut flagellum in the centre with the distal tip of a coiled haptonema nearby (thinarrow). Micrograph D 1937, x 50000.Fig. 3. Three profiles of a coiled haptonema showing the characteristic internalstructure of 3 concentric unit membranes surrounding a ring of 7 tubes or fibres; forfurther description see text. Micrograph D 1931, x 100000.

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Fig. 4. Three scales from a whole mount, shadow cast (reversed print) showing a scaleof the larger type flanked by two scales of the smaller type, one (right) showing therimmed surface and the other (left) the opposite surface; for further description seetext. Micrograph D5940, x 30000.Fig. 5. Part of a cell cut longitudinally with a flagellar base if) and haptonema base (/;)entering the cell near the Golgi body; part of the nucleus (w) and profiles of mitochon-dria and plastids also visible; on the cell surface (above) the various scale layers inposition and showing their mutual arrangement; for further description see text.Micrograph D 5802, x 30000.

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Fig. 6. Part of a longitudinal section showing the position of the Golgi system (centre)in relation to the nucleus (n), and subtending endoplasmic reticulum (er); other visibleorganelles include two mitochondria (m) and two chloroplasts, one with a pyrenoid(py) partly included in the section. For further description see text. MicrographD2147, x about 25000.

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Fig. 7. The central region of a Golgi system fixed in the late afternoon, showing the'peculiar' dilations filled with a dense metabolite; subtending endoplasmic reticulum(cr) right; for further description see text. Micrograph D5875, x 50000.

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Fig. 8. Longitudinal section through a Golgi system with the subtending endoplasmicreticulum (er, left) and an obliquely cut flagellar base (/) above; a single scale-containingvesicle (arrow) carrying a well-marked 'hairy' diverticulum; for further descriptionsee text. Micrograph D 1899, x 30000.Fig. 9. Transverse section through a Golgi system otheiwise comparable to that ofFig. 7, showing the subtending endoplasmic reticulum (er, right) and a single scale-containing vesicle (arrow), left. Micrograph D 1948, x 30000.

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Fig. 10. Transverse section through a Golgi system with active scale-production (5scales numbered), cut at a level not including the 'peculiar' dilations but clearlyshowing the subtending endoplasmic reticulum (er, right); for further description seetext. Micrograph C8360, x 30000.Fig. 11. Section showing some of the dilations of the Golgi centre with scale-containingvesicles (left) and stages thought to be replicating vesicles between the Golgi centreand the subtending endoplasmic reticulum (right). Three internal scales marked byarrows, the uppermost occupying part of a vesicle thought to be about to liberate itscontents to the cell exterior. For further description see text. Micrograph 05904,X35000.

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