The Fine Structure of Ceratium Tripos, a Marine Armored Dinoflagellate I

JOURNAL OF ULTRASTRUCTURE RESEARCH 50, 58-64 (1975)

The Fine Structure of Ceratium tripos, a Marine Armored Dinoflagellate

I. The Cell Covering (Theca)

RICHARD WETHERBEE 1 Department of Botany, University of Michigan, Ann Arbor, Michigan 48104

Received February 14, 1974, and in revised [orm July 12, 1974

In this, the first of three consecutive papers, the complex cell covering of Ceratium tripos is examined at different stages of development. During eytokinesis, the cleavage furrow is surrounded by a system of membranes which, together with the theeal plates, will comprise the future cell covering. The outermost membrane completely surrounds the cell and lies over a single layer of large flattened vesicles. An additional membrane, the theeal membrane, lies within the vesicles just below the region of eventual plate formation. The thecal membrane gradually becomes discontinuous in mature cells. Sutures are observed during the initial differentiation of the cell covering. Near the completion of cytokinesis, the portion of cytoplasm still joining the daughter cells is enclosed by only one membrane of the cell covering, the plasma membrane.

INTRODUCTION therefore suggest that separation at the This is the first in a series of papers on generic level could be determined largely the fine structural features of morphology by variations in the morphology of the and development in Ceratium tripos. Spe- vesicles and the extent to which plate cial attention is given here to the complex material is deposited within them. Several association of membranes and plates which investigators have raised questions about comprise the cell covering. The taxonomy Dodge and Crawford's (3) interpretation of of armored dinoflagellates has been deter- the basic construction of the cell covering. mined largely by thecal morphology: the The most notable difference of opinion number, orientation, and ornamentation of concerns the exact location of the cytoplas- the armored plates. During recent years, mic or plasma membrane (PM) (7, 8, and several workers have used the transmission 12). As early as 1901, Kofoid (9) described and scanning electron microscope in an a membrane beneath the thecal plates as attempt to substantiate or correct previ- the probable location of the plasma mem- ously questionable taxonomic criteria (1, 2, brane. Most recent investigators have 3, 7, and 8). Extensive observations by tended to agree with this interpretation (7, Dodge and Crawford (3) on numerous 8, 10, and 12), although Dodge and Craw- members of the Dinophyceae revealed a ford (3) designate the outer continuous uniformity in the construction of the theca membrane as the PM. The thecal plates throughout the class, and reaffirmed its are therefore produced above or below the usefulness as a taxonomic criterion. All PM depending on one's interpretation. In flagellated members of the Dinophyceae addition, Kalley and Bisalputra (8) have are enclosed by a continuous outer mem- questioned the assumption that the plates brahe which lies over a single layer of are always enclosed within mere- flattened vesicles. Dodge and Crawford (3) branebound vesicles. Their examination of Peridinium trochoideum often revealed 1Present address: Department of Botany, Univer- plates which were not completely sepa- sity of Melbourne, Parkville, Victoria 3052, Australia. rated by the typical suture membranes (8). 58 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. CELL COVERING OF CERATIUM 59

In almost all cases investigators have thecal plates are synthesized shortly there- worked only with mature cells. In the after. The basic structure of this covering is present investigation, the membranes of shown in Fig. 1-6 and illustrated in Dia- the cell covering were studied from their grams 1 and 2. It consists of four unit initial differentiation during cytokinesis membranes which overlie a dense region of and cell shape development through the cytoplasm lined with microtubules. The formation of the thick thecal plates. The outermost unit membrane in this system, results revealed a pattern of membranes the plasma membrane (PM), is continuous similar to those described by Dodge and around the cell and delimits it from the Crawford (3). However, an additional surrounding environment. The layer im- membrane, the thecal membrane, was ob- mediately beneath the PM, the "outer served within each vesicle during early plate membrane" (OPM), is continuous stages of development. along predetermined lines with the "inner By following the development of the cell plate membrane" (IPM), to form a number and of the cell covering in particular, it was of large flattened vesicles. Mature thecae also possible to substantiate the claim of occasionally reveal discontinuities between Dodge and Crawford (3) that the outermost the IPM and OPM which has led to their membrane is the plasma membrane. Dur- designation as separate membranes (ar- ing one stage in development, a portion of row, Fig. 6). The size, shape, and number the cell is enclosed within only one mem- of vesicles is characteristic of a species, and brane of the cell covering, the plasma their differentiation occurs early in devel- membrane. opment. Each vesicle encloses both a thecal plate and an additional membrane, the MATERIALS AND METHODS "thecal membrane" (TM). The point of Ceratium tripos (O.F.M.) Nitzsch was isolated contact between two adjoining vesicles is from samples taken from the north end of the Cape Cod Canal, Bourne, Massachusetts in June, 1970. termed a suture (arrows, Figs. 1, 2, and 5), Cultures were grown in f/2 enriched seawater medium and consists of two unit membranes, one (6) minus silica in a regime of 16 hr light and 8 hr from each vesicle. A vesicle may therefore darkness. Cultures are presently maintained by Dr. be visualized as resulting from the union of Robert R. L. Guillard, Woods Hole Oceanographic the OPM and the IPM at a suture. The Institution, Woods Hole, Massachusetts 02543. Cells begin mitosis 2 hr before the end of the dark period TM, which is discontinuous at the sutures, and complete cytokinesis several hours after the lies just above the IPM and below the beginning of the light period. Development of the region of thecal plate formation. A continu- vegetative cell shape and cell wall synthesis pro- ous layer of microtubles lies beneath the ceeded for an additional 6 hr or more. Cultures were covering system during cytokinesis and fixed at various times during this period and individ- ual cells were selected for sectioning depending on development of the characteristic cell their state of development. Approximately one-third shape (MT, Figs. 1 and 2). This layer of the cells divided at any one time. becomes dispersed into small groups of one The procedure for preparation of C. tripos for to four in maturing thecae (MT, Figs. 4 electron microscopy was the same as described for and 5). Polysiphonia (13). Sections were cut on a diamond knife with a Porter Blum MT-2 ultramicrotome and The most notable alteration in thecal viewed with a Zeiss 9A electron microscope. structure during development concerns the TM which is prominent during cytokinesis OBSERVATIONS and subsequent formation of the cell shape The complex cell covering of Ceratium (TM, Figs. 1-3), but becomes progressively tripos, with the exception of the thecal discontinuous in maturing cells (TM, Figs. plates, is completely differentiated during 4 and 5). cleavage and very early development. The With the exception of the PM, the asso- TM IPM

FIG. 1. The cell covering before the beginning of plate synthesis. The plasma membrane (PM) is the outermost membrane underlain by large vesicles composed of the outer plate membrane (OPM) and the inner plate membrane (IPM). The thecal membrane (TM) lies within the vesicles and is discontinuous at the suture (arrow). A dense layer of cytoplasm lined with microtubles (MT) lies beneath the covering system. × 67 365. FIG. 2. The cell covering following initial plate synthesis. The membranes of the cell covering are clearly exposed. The thecal membrane (TM) is discontinuous at the suture (arrow). × 82 575. 6O CELL COVERING OF CERATIUM 61

Suture (Figs. 10 and 11). When this situation PM'~ // exists, the final location of the combined diffuse regions mentioned above becomes O000©O000000000000OO the area of attachment between the differ- ~ entiating daughter cells (Fig. 9). At this DIAGRAM 1. The orientation of the membranes of point the only membrane continuous be- the cell covering before thecal plate synthesis. The tween the two daughter cells is the PM plasma membrane (PM) is continuous around the entire cell. The outer plate membrane (0PM) fuses (black arrow, Fig. 9). No other membrane with the inner plate membrane (IPM) to form large separates the cytoplasm from the environ- flattened vesicles which contain an additional mem- ment. Vesicles (white arrows, Fig. 9) do not brane, the thecal membrane (TM). A dense layer of form sutures in this region'. cytoplasm containing microtubules (MT) lies under the cell covering. DISCUSSION

Suture/~ The thecal structure observed in C. PM~ opM) A tripos is basically similar to that described by Dodge and Crawford (3) in their survey of the Dinophyceae. The outermost mere- brane is considered the plasma membrane • oooo and lies over a single layer of large vesicles DIAGRAM 2. The structure of the cell covering fol- in which the thecal plates are synthesized. lowing plate formation. Thecal plates are formed The presence of an additional membrane within the vesicles and above the thecal membrane within the vesicles, the TM, has not been (TM) which becomes discontinuous in mature walls. The microtubule layer becomes dispersed into small reported before, although perusal of other packets of 1-4. published micrographs indicates that it is frequently present. The fact that only ma- ciated membranes of the covering are ab- ture thecae are customarily studied may sent from a portion of the cell surface near explain why the TM has not been previ- the completion of cytokinesis when two ously described. Although it is prominent diffuse regions differentiate along the lead- during early development in Ceratium tri- ing edge of the cleavage furrow (Fig. 7). As pos, the TM tends to become less evident seen in Fig. 8 (black arrow), there are no in aged cells. In one of the few reports additional membranes under the PM in available on developing thecae, Dodge and these regions. Though vesicles are present Crawford (4) described a cross section (white arrows, Fig. 8), they are separated through a horn of Ceratium hirundinella in from one another and do not form typical which initial plate formation was evident sutures. Frequently Ceratium daughter and the TM had become discontinuous cells remain attached so that the actual within the vesicles. Though the TM ap- separation following cytokinesis occurs peared similar to other membranes of the concurrently with, or subsequent to, the covering system, it was described as "a completion of cell shape development discontinuous layer of dark-staining mate-

FIG. 3. The ceil covering in the cleavage furrow. Note the unit membrane structures of the TM and other membranes. × 83 025. FIG. 4. The cell covering following cell shape development with the microtubules dispersed into small groups. The TM is discontinuous but still exhibits unit membrane structure, z 86 895. FIG. 5. Mature cell covering showing thick thecal plates and a suture (arrow). The thecal membrane (TM) is discontinuous and only a few microtubules (MT) are found beneath the covering. × 86 085. FIG. 6. Mature wall where the suture (arrow) has become discontinuous and the thick thecal plates are no longer entirely enclosed within vesicle membranes• x 84 735. Fro. 7. Two diffuse regions which originate near the completion of cytokinesis along the leading edge of the cleavage furrow, x 14 602. FIG. 8. Only the plasma membrane (black arrow) is continuous around the diffuse region while the vesicles (white arrows) do not form typical sutures, x 56 295. Fla. 9. Area of attachment between cells which do not separate following cytokinesis. Only the plasma membrane (black arrow) is continuous between daughter cells while the vesicles (white arrows) are separate from each other and do not form typical sutures, x 23 364. 62 CELL COVERING OF CERATIUM 63

FIG. 10. Area of attachment (arrow) between two daughter cells which have recently divided but have remained attached, x 3 474. FIa. 11. Two daughter cells almost completely differentiated which have remained attached (arrow). x 1 372. rial" which Dodge and Crawford (4) sug- layer of the cell covering termed the pel- gest is possible "plate precursor material." licular layer. It is reportedly present in If a recently differentiated covering had typical armored species and consists of one been observed it is likely that this structure continuous unit lying beneath the vesicles. would have been identified as an addi- This structure was not observed in C. tional membrane. In Dodge and Crawford's tripos. (3) survey of thecal structure, at least three There are two different types of cyto- other organisms appeared to have a mem- kinesis in the armored dinoflagellates (I2). brane within the thecal vesicles. This is The first results in each daughter cell most noticeable in Amphidinium retaining approximately one-half of the herdmanii (plate 1D, 3) where the authors parent theca while regenerating the miss- point out that material is present, but ing moiety. In the second method the make no reference to what appears to be a parent cell either divides up within its wall, unit membrane lying at the base of the or sheds the wall (ecdysis) before division. vesicles. Other micrographs which give In this case, the daughter cells must regen- indications of this type of structure include erate an entirely new theca. Ceratium unknown species "Plymouth D" (Plate 2A, tripos is representative of the former in 3), Woloszynskia coronata (Plate 3E, A, method. A subsequent paper in this series in 3), and possibly Glenodinium foliaceum will illustrate how, during cytokinesis, (Plate 3F, in 3) though in this case the complete continuity is maintained between membrane within the vesicles does not the membranes of the new daughter cell appear discontinuous at the sutures. wall and those of the retained parent wall. Though the TM is likely present in these The PM in Ceratium is therefore always and other dinoflagellates, only studies continuous around the cell, even during which follow the cell covering throughout division. Assuming that this membrane development can reveal this with cer- has a similar location in all dinofiagellates, tainty. cells discarding the parent wall at division Loeblich (12) has described another would have to regenerate those membranes 64 RICHARD WETHERBEE located above the region of separation, phenomenon often observed in aged cells presumably before the loss of the parent of C. tripos (Fig. 6). Therefore I designated structure. The continuous membranes ob- the upper and lower membranes of the served below the vesicles of some dinofla- vesicles as two separate units (OPM and gellate cells (3, 10) might therefore be IPM), though the interpretation of the present in anticipation of division. In Gyro- basic thecal structure proposed by Dodge diniurn cohnii (10) the parent cell encysts and Crawford appears correct throughout before dividing into two or more daughter most of development. cells. At the completion of division each Subsequent communications will dem- new cell is surrounded by four unit mem- onstrate additional features of thecal struc- branes. It should be noted that during and ture, including the disposition of covering subsequent to cytokinesis the two outer membranes during plate synthesis. membranes of the cyst wall remain intact. I would like to express my appreciation to Dr. It therefore appears that dinoflagellates Gordon E. McBride and Dr. Robert R. L. Guillard for which divide in such a way will have assistance during the course of this research. developed their new covering system before The procedure for preparation of Ceratium for the parent theca is discarded. It is almost electron microscopy was suggested by Dr. Everett impossible to draw a comparison with Anderson, Department of Anatomy, Harvard Medi- cal School, Boston, Massachusetts. Ceratium, because no definitive work has This work was submitted in partial fulfillment for been done on dinoflagellates which divide the Ph.D. degree in Botany at the University of in this manner. Michigan. The results of this investigation substan- Support during preparation of the manuscript was tiate the claims of Dodge and Crawford (3) provided by NSF GB 4550 to Dr. John West. that the outermost membrane of the theca REFERENCES is the plasma membrane. No other mem- 1. Cox, E. R. AND ARNOTT, H. 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