PROROCENTRUM IIJARIAE-LEBOURIAE COMB. NOV. 315 5. HOFFMAN, L. R. 1967. Obscrvationson thc fine struc? ture of Oedogonium. III. Microtubular elements in the chloroplasts of Oe. cal'diacum. ]. Phycol. 3:212-21. 6. -- 1968. Obscrvations on thc fine structure of Oedogonium. IV. The maturc pyrcnoid of Oe. cal'diacum. Trans. Am. Microsc. Soc. 87:178-85. 7. -- 1968. Observations on the finc structure of Oedogoniurn. V. Evidcncc for thc de novo formation of pyrcnoids in zoosporcs of Oe. cardiacum. ]. Phycol. 4: 212-8. 8. -- &: MANTON, 1. 1962. Observations on the finc structure of the zoosporc of Oedogonium cm'diacu/Il with special reference to the flagella apparatus. ]. Exptl. Bot. 13:443-9. 9. HOLDSWORTH, R. H. 1968. The presence of a crystalline matrix in pyrenoids of the diatom, Aclmanthes brevipes. ]. Cell Bioi. 37:831-7. 10. -- 1971. The isolation and Ipartial chal'acterization ]. Pilycol. 10,315-322 (1974) of the pyrenoid protein of Eremosphaera Vi,?idis. ]. Cell Bioi. 51:499-513. 11. KOWALLIK, K. 1969. TIle crystal lattice of the pyrenoid matrix of Procentrum micans. ]. Cell Sci. 5:251-69. 12. LUFT, J. H. 1961. Improvements in epoxy resin em? bedding methods. ]. Bioph"s. Biochem. Cytol. 9:409-14. 13. PICKETT-HEAPS, J. D. 1968. Microtubule-like structures in the growing plastids of chloroplasts of two algae. Planta 81:193-200. 14. RETALLACK, B.&: BUTLER, R. D. 1970. The development and structure of pyrenoids in Bulbochaete hi/oensis. ]. Cell Sci. 6:229-41. . 15. -- 1972. Reproduction in Bulboclzaete hiloensis (Nordst.) Tiffany. I. Structure of the zoospore. Arch. Mikrobiol. 86:265-80. 16. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. ]. Cell Bioi. 17:208-12. MICROMORPHOLOGY OF A SMALL DINOFLAGELLATE PROROCENTRUM MARIAE-LEBOURIAE (PARKE 8c BALLATINE) COMB. NOV.1?2?8 Maria A. Faust Smithsonian Institution, Chesapeake Bay Center for Environmental Studies Route 4, Box 622, Edgewater, Maryland 21037 SUMMARY The surface structw'es of the bivalvate dinOflagel? late Prorocentrum mariae-lebouriae m'e described in detail. It has an almost spheroidal shape in face view, a compressed saucer-shape in side view, with a distinct striated band at the edge of the cell. Its surface i$ covered with small spines in a Tegular pat? tern,with 450 nm distance between pairs. The spines are 100-120 nm wide and 200-300 mn long. There are 600-700 spines on each valve. At the antel'ior cell end, one of the valves has a V-shaped depression which contains a specialized $iruclttre aCCOl1unO? dating the 2 flagellar pores. The flagellar pores al'e enclosed by 8 small, thick plates held together and to the valves by sutures. The flagellar pore area consists of 2 distinct structures: an apical collal' possessing a curved for/wd plate and a larger stntcture composed of an unbranched plate. There are 2 flagella?' canals located between the flagellar pOl'e plates. Beneath each flagellar canal lies a row of 11 microtubules. A row of microtubules forming a microtubular 1 Received March 7, 1974; revised May 28, 1974. "Supported in pal't by a grant from the program for Re? search Applied to National Needs of the National Science Foundatioh and by the Smithsonian Institution Environlllcntal Science Program. S Published with the approval of the Secretary of the Smithsonian Institution. cylinder is situated adjacent to the oblong flagella?' canal neal- a. simple pusule. The microtubular cylin? der encircles electron dense bodies. The bases of the longitudinal and tmnsverse flagella appear to lie at an angle to each other. The above features al'e illustrated with transmission and scanning electron micrographs. INTRODUCTION The fine structure of the surface of the bivalvate dinoflagellate genera Prorocentrum and Exuviaella appears to have a highly characteristic and critical taxonomic value in algal classification (1,2,7,13-17). Light microscopy has not provided sufficient infor? mation because of the small size of these organisms. Electron microscopic examination of several species indicated distinct differences in architecture, size, ornamentation of the valves, and the organization of the apical flagellar pore area. Small spines were reported on the surface of the valve of E. mariae? lebouriae (2,J,8,1J), smooth cell surface of P. micans Ehrenb. (3). Later, Dodge (5) extended the descrip? tion of the valve surfaces to P. balticum and P. obtusidens; both were covered with evenly spaced small spines. Dodge (7) also described the flagellar pore structure of P. mariae-lebouriae, composed of a number of slfiall thick plates. 316 MARIA A. }'AUST Hulburt (12) recognized the difficNlty in separating small Exuviaella and Prorocentrum species, which were collected from the natural environment; using ~he light microscope, because of their extensive varia? tions in cell shape, size, and the presence and absence of anterior spine. As the ultrastructural informa? tion on Prorocentrum and Exuviaella species in? creased', their identity as distinct and separate genera was not warranted. Abe (1) and Doclge &: Bibby (7) recognized this, and proposed that the .2 genera should be merged into the prior genus Proro? centrum Ehrenberg. They defined the most unique features of the combined genera: the distinct parallel orientation of the flagella (7), the presence of a simple sack pusule (6), and the characteristic apical flagellar pore structure (1,7,13). The above char? acteristicsare regarded as unique features for Proro? centrum species and distinguish them from other algae. The wrrent investigation revealed that parallel orientation of the flagella might not be universal for this genus. The above-described criteria were used to identify the organism responsible for a dense bloom in the Rhode River arm of the Chesapeake Bay. In this study light, transmission, and scanning electron microscopes were used to examine the structure of this small flagellate identified as P. mariae-Zebouriae (Parke &: Ballantine) comb. nov. This investigation revealed that the apical pore area of this organism is more complex than recognized previously and provides additional information on this small bivalvate dinoflagellate. MATERIALS AND METHODS Bay water samples were collected from 1 m below surface and fixed immediately in 4% glutaraldehyde in OJ M phosphate buffer (pH 6.8). Cell dimensions were determined by mea? suring the length and width of 100 cells using an eye-piece micrometer with brightfield optics of a Carl Zeiss light microscope at a magnification of X1000. Light micrographs were taken with the Nomarski Interference 11ilter with the above microscope using Kodak High Contrast Copy film. },ixed cell suspensions were prepared for electron micros? copy. Cells were postfixed with 1% aqueous osmiulU tetroxide and embedded in. epon as described previously (II). Thin sections were stained with lead citrate or uranyl acetate and examined in a Philips 300 electron microscope. , Negative staining was calTied out in distilled water. One drop of cell suspension was mixed with a drop of 2% phos? photungstic acid buffered at pH 7.0 and applied to l'ormvar? carbon coated grids. Trichocyst band spacing was determined with an ocular micrometer using 10 different prints at X 96,000. Replicas WI:I'I: prepared of glutaraldehyde fixed cells as described by Gantt (10). After several washings in distilled water, cells were applied to freshly cleaved mica, shadowed in. a Denton Vacuum Evaporator (Denton Vacuum Inc., Cherry Hill, N.J.). Replicas were floated on distilled water and then transferred to chromic acid for 60 min to remove organic material. This was followed by 2 distilled water rinses. The replicas were pickcd up on Formvar-coated grids and were examined in the electron microscope. Longitudinal sections of 20 randomly selected cells were photographed at a magnification of X 36,500. The width and length of the 2 apical plate structureS wcre Illeasurl:d on the photographs and the average measurements determined. 11ixed celh used for scanning electron microscopy Were also postfixed' with I% osmium tetroxide fOI' 15 min and washed several times in distilled water. A small drop of cell suspension was placed directly on a stub previously lightly coated with acetone-solubilized adhesive from 3M double-stick Scotch tape. Specimens on stubs were plunged directly into liquid nitrogen and quickly frozen. Specimens were dried in Pearse tissue dryer (Edwards High Vacuum Ltd.) to complete dryness as reported previously (18). Specimens have been examined with a Cambridge Stereoscan II Scanning electron microscope (Engis Equipment Company) using 10 kv accelerating voltage and a 200-po apertme. OBSERVATIONS The cell shape of P. rnariae?Zebouriae as seen through light microscope equipped with Nomarski Interference filter and with the electron microscope is illustrated in Fig. 1 2, 3, and 4, respectively. Proro? centrum rnariae-lebouriae is almost spheroidal in face view; it is stI10ngly compressed and saucer-shaped in side view; it has a very distinct striated band at the edge of the cell; and its surface is covered with small pI1ojections in a regular pattern. ~ FIG. 1-3. The almost spherical cell outline of P. mar:iae-Iebourille is evident in light micrographs taken with Nomarski Inter? ference filter. FIG. l. The surface of the organism is covered with small projections and ridges are exhibited at the cell periphery. The 2 flagella .are not visible in this illustration. X 1500. IIIG.2, 3. The separated bivalves have the same outline as of Fig. 1. The striated band at the edge of each valve shows a regular periodicity and appears thicker relative to the whole valve, and Fig. 3 has a V-shaped depression at the anterior cell end, into which tile flagellar pore structures arc fitted. X 1500. }1l(;. 4. Strongly compressed and flattened shape of P.1Il1iriae-Ieboll1'ille is pictured in this scanning electron micrograph. The surface of the cell has an evenly distributed pattern, composed of raised bulges mark the tiny spines. A clear view of the cell margin is seen 011 one of the saucer-shaped organism. Amorphous material afjlhered to their surface of the cell is debris from the natural environment..X 3600. FIG. 5. The anterior end of the cells of P. rIlllriae-Iebourilie has apical plates, 2 flagellar pores, tiny spines, and trichocyst pores (m'l'oUls) on the valve surface. The apical collar (e) frames the flagellar pore in the rear. A second structure, the apical spine (a), additionally protrudes from the apical plates, near the periphery of the second flagellar pore. X II,OOO. 11lG. 6. In thin section the V-shaped flagellar pore area is composed of 8 small thick plates variously sized and shaped, enclosing the circular and oblong flagellar pores. The 8 small plates are held together and to the valves by tightly fitted sutures. The 8 plates have been observed in various sections, but not all of them shown here. x 35,000. FIG. 7. The flagellar pore area in longitudinal section is shown. The 2 -flagellar canals arc located between the apical plates (P). Beneath each flagellar canal (fe) lie a row of 11 microtubules (m'l'ows). X 35,000. PlWROCENTRUM AfARlAE?LElJOURlAE COMB. NOV. 317 318 MARIA A, FAUST ThULE I. Comparison of cell size, sUI'face structure, (Iud tl'ic!locysts of Prorocentrum mariae-lciJouriac and E. mariac-lcbourhlc. Spines on thecal surface Organism Cell size (/lm) True pores Diameter No. nm No. Length mn Width (nm) Spaced apllrt (nm) Trichocyst band spacing Mlljor Minor (nm) (nm) ne,ference E. marille?leboul'iae (14-17) X (11-15) E. marine-lebom'illc -- P. Jlwriae-leboul'iae (18-20) X (1G-17) Not observed 20 200--250 500 30 150-200 600--700 600 200-250 300-500 150 200-300 100--120 2.1)0 700 450 66 60 17 15 Parke & Ballantine (15) Dodgc (3) Present investigator The projections are composed of spines covering the cell surface revealed in negatively stained prep? aration (PTA) and platinum-carbon replicas (Fig. S, 9). These spines are evenly distributed over the cell surface with 450 nm distance between pairs. The spines are about 100-120 nm wide and 200? 300 nm long. There are about 600-700 spines on each valve. Numerous trichocyst pores are also found scattered through out the surface of the valve (Fig. 5, 8, II). Data concerning surface features of the valve of E. mariae-lebouriae has been reported previously (3,15) and comparison ismade in Table 1 witl~ the new information on similar features of P. mariae-lebouriae. The data reported previously differ from ours as follows: cell size of P. mm'iae? lebou1'iae is larger than of E. mariae-lebouriae;cliam? eter of true pores are narrower and more numerous per valve; length and width of spines are shorter and they occur more frequently on the valve surface; trichocyst band spacing found at shorter intervals. The cell of P. mariae-lebouriae possesses 2 saucer? shaped valves with a smooth inner surface (Fig. 2, 3). The above observations were confirmed in scanning electron micrographs at higher magnifications (not shown). The valve in thin sections has uniformly dense granular appearance (Fig. 6,7, 10, 12), its thickness varies from one cell to the other. The border of each valve has a uniform width and con? sists of a pattern of evenly spaced ridges. The border on 1 valve completely overlaps the other valve and the 2 fit together around the cell producing the saucer-shaped appearance of the organism (Fig. 4). The valve of P. mariae-lebouriae is covered by an outer membrane (Fig. II) which is continuous over the entire cell surface. The cell membrane was lost during embedding iIi most of the thin sections, but has been shown to be present in other prepara? tions (7). One valve is thickened at the anterior end of the cell and has a V-shTA) electron micrograph. Due to the absence of the outer mcmbrane the spincs are very po.intcd and 2 of them aTe broken. X 95,000. FIG. 9. The evenly spaced pines on the valve shown in platinum-carbon repUca at lower magnification. Here the outer mem? brane covers the valve surface and the spines have a blunted shape which is their normal appearance. X 35,000. FIG. 10. Longitudinal section showing the apical collar of the flagellar pore area. This is a forked, sUghlly curved, solid plate. Indications of tbe outer membrane are shown (an?Olus). The position of the flagellar bases is seen in the cytoplasm (1). A glancing section through a portion of one of the flagella displays Ule arrangement of :lxonemcs. Transverse section through the second flagellum Illay indicate that the flagellar bases lie at an angle to cach other. Chloroplast (Gil) is also present. X 35,000. FIG. 11. Thc apical coUar (e) slllTounded by tbe outer membrane shown in this canning electron micrograph. Evenly dis? tributcd spines and trichocyst pores (an-olUs) dominate the cell slll'race. X 12,000. 320 MARIA A. FAUST dulating double membrane walls and vesicles posi? tioned close to the cell vacuole. The position of the longitudinal and transverse flagellum inserted into the cytoplasm is shown in Fig. 10. The position of one flagellum suggests that it may emerge from the cell through the circular flagellar pore. Additionally, a glancing section through the length of the flagellum displays the ar? rangement ofaxonemes. A transverse section through the second flllcgellum indicates that the flagellar bases lie at an angle to each other. Other ultrastructural features of E. mariae? lebouriae and P. mariae-lebotwiae previously de? scribed (3-7,9,15) appear to be similar than those observed of P. mariae-lebouriae causing a dense bloom in the Rhode River. The above organisms have 2 large multilobed chloroplasts and large pyrenoids situated midway between the base and apex, one at each side of the cell. The pyrenoids occur in the swollen part of the chloroplast, trans? versed by several pairs of chloroplast lamellae. The pyrenoid matrix consists of rows of particles forming a paracrystalline structure described previously by Dodge & Crawford (8). No starch was found to be associated with the pyrenoids, but starch granules were found within the cytoplasm. The chloroplast thylakoids occur in threes and run parallel across the chloroplast (Fig. 12). The nucleus is situated in the posterior end of the cell. It is a typical meso? caryotic nucleus, spherical in shape, with nUl11erous chromosomes situated within the granular nuclear matrix (3). The golgi bodies, the mitochondria, and trichocysts are all typical in structure and need not be described in detail. DISCUSSION The electron microscope is necessary to define ultrastructural features of the genera of Proro" centr1.lm and Exuviaella (2-5,8,13). Examinations of several species by the above investigators indicate that there is no sharp distinction between the 2 genera. Many features of these organisms are re? garded as typical of the Dinophyceae (1,5,13), but others, such as the size and ornamentation of the valves, the organization of the apical flagellar pore area,and the flagellar structures, are not. In this study, using various microscopical tech? niques, it was possible to show that the bivalvate surface of P. mariae-lebouriae collected from the Rhode River estuary is covered by small spines. The same type of spine, of different dimensions, already has been observed in specimens of E. rnariae? lebouriae and P. TlIa1'iae-lebouriae and in species of P. balticum and P. obtusidens, but not in all Proro? centrum species examined (3,5,7). Scanning electron microscopy revealed additional information on the features of the flagellar pore area of P. mairae-lebouriae, which was found more complex than previously described of other speci? mens of P. mariae-lebouriae (7,13). The use of the term apical collar to describe the flared plate was chosen because of the structural and architectural characteristics revealed with the scanning electron microscope. A similar structure of different elimen? sions has been described as a winged spine for P. micans (3). A second structure, a single spine, also fringes the oblong flagellar pore of P. mariae-lebouriae. Pre? sumably this large and wider structure has been observed with the light microscope, identified as an apical tooth or spine of other Prorocentrum and Exuviaella species by Hulburt (12), Martin (14), and Pavillard (16). The double-spine structure observed with the electron microscope by Dodge 8c Bibby (7) is also designated as an apical spine. In our prepara? tions both structures are present on the same or? ganism. This may be unique for P. rnariae-lebou.riae found in the Rhode River, but it could also be more widespread in other Prorocentrurn species and not observed so far. The existence of the 8 thick plates of the flagellar pore area makes it a more complex feature of P. mariae-lebottriae described in the present investigation than in other specimens of P. rnariae-lebouriae. Only 4 or 6 plates were identified previously in the latter organism (7). Location of the longitudinal and transverse ~ FIG. 12. Longitudinal section through the apical flagellar pore area reveals a large straight structure, a single spine, pro? truding from the apical plates (P). The thickened value has a uniform dellSl' granular appearance. The flagellar canal (fe) is ad, jacent to a row of microtubular cylinder (me), fibrous bodies (b), and mitochondria (m). The chloroplast lamellae (Cit) consist of 3 thylakoids running parallel across the chloroplast. Cell vacuole (V) consisting of electron deuse material lie between the micro? tubular cylinder (me) and the chloroplast. x 35,000. FIG. 13. Intimate arrangement of the apical f1a.gellar pore area between the valves shown in a scanning electron micl'llgraph. Tihe large siugle spine shown in l'ig. 12 and 13 is the same structure positioned around the oblong flagellal' pore (0). The circular flagel? lar pore (C) and apical collar complete this structure. X 22.000. FIG. 14. The single spine, obsel'ved from another angle, surrounded by numerous spines of the valve surface seen in a scanning electron micrograph. X 24,000. l-rG. 15. Transverse section of a microtubular cylinder .(me) consists of 40 mictolubules and encircles electron dense bodies (arrow). It is positioned beneath the valve adjacent to a cell val;Uolc(V) and fibrous bodies (b). X 61,000. FIG. 16. Longitudinal section of the microtubular c.ylinder (lIle) runs parallel to the valve, beginning at one end by the simple pllsule (P), adjacent to the flagellar canal (te). mitochondria (m), and fibrous bodies (b). The simple pusule is constructed of double-membrane walls and vesicles. X 346,000. PROROCENTR AI J\IA./UAE-LEI30 RIAE COMB. NOV. 321 322 MARIA A. FAUST flagella has been observed in thin sections. The bases of the 2 flagella appear to lie at an angle to each other in P. rnariae-lebouriae. This observation is different from the flagellar structure of P. triesti? num, the only other organism examined in detail (7). The flagellar system shows some diversity o? organization in dinoflagellates. examinecl (5). The observations of the flageUar structures of Proro? centrum species are obviously too incomplete for any generalization. Although only a few Prorocentrum species have' been studied, it is believed that the apical flagellar pore area will be unique to this group as are the surface structures of the bivalves and the flagellar structure. It is expected that the above ultrastruc? tural features, while essentially similar, will vary according to the species and may become useful in taxonomic characterization. ACKNOWLEDGMENT I wish to express my gratitude to Dr. J. D. Dodge, Uni? versity of London, for his help in identifying the organism. Thllnks are due to Dr. E. Gantt, Radiation Biology Laboratory. and Dr. J. W. Pierce, Division of Sedimentology at the Smithsonian Institution, for permission to use their electron microscope; to Walter Brown for operating the llCanning electron microscope; to ClaUdia A. LipschUltz and JoAnne Battista for their valuable suggestions related to the manu? llCripts; and' to Dr. E. B. Small. University of Maryland, for use of their facilities. REFERENCES 1. ABE, T. H. 1967. The armored Dinoflagellata. II. Prorocentridae and Dinophysidae (A). Publ. Seto. Mar. Bioi. Lab. 14:369-89. 2. BRAARUD. T., MI\RKAU, ~ .? &: NoROLI, E. 1958. A note on the thecal structure of &xuviaeUa baltica Lohm. NyU Mag. Bot. 6:43~6. 3. DODGE, J. D. 1965. Thecal fine-structure in the dino- flagellate genera Prorocentrum and ExuviaeUa. j. Mar. Bioi. Assoc. U;K. 45:Q07-14. 4. --- 1968. The fine structure of chloroplasts and pyrenoids in some marine dinoflagellates. J. Cell Sci. 3: 41-8. I 5. --- 1971. Fine structure of the Pyrophyta. Bot. Rev. 37:481~508. 6. --- 1972. The ultrastructure of the dinOflagellate pusule: A unique osmo-regulatory organelle. Protoplasma 75:285-302. 7. -- & BIBBY, B. T. 1973. The Prorocentrales (Dino. phyceae). 1. A comparative account of fine structure in the genera Prorocentrum and Exuviaella. Bot. ]. Linn. Soc. 67:175-87. 8. DODGE, J. D. &: CRAWFORD, R. M. 1970. A survey of thecal fine structure in the Dinophyceae. Bot. ]. Linn. Soc. 63: 53-67. 9. --- 1971. A fine structural survey of dinoflagellate pyrenoidsand food reserves. Bot. .1. Linn .. Soc. 64:105-15. 10, GANtT, E. 1971. Micromorphology of the periplast of Ch1'oomonas sp. (Cryptophyc?ae). ]. Phycol. 7:177-84. 11. ---, EDWARDS, M. R.,&: PROVASOLI. L. 1971. Chloro? plast stt'ucture of the Cryptophyceae. Evidence for phyco? biliproteinswithin hnrathylakoidal spaces. ]. Cell Bioi. 48:280-90. 12. HULBURT,E. M. ,1965. Three closely allied dinoflagel? lates. ]. Phycol. 1:95-6. 13. LOllBLICH, nI, A. R. 1969. The amphiesma or dino? flagellate cell covering. P1'OC. N. Am. Paleont. Conv. G: 867-929. 14. MARTIN, G.W. 1929. Dinoflagellates from marine and brackish waters of New Jersey. U11iv. Iowa Stud.. Studies in Natural History. 12:1-32, pIs.F8. 15. PARKE, M.&: B.AJ;LANTlNll,D. 1957. A new marine dino? flagellate: Exuviaella mariae'leboul'iae n. sp. .1. Mm'. Bioi. Assoc. fJ.K.36:643-50. 16. PAVILLARD, J. 1916. Recherches Stll' les peridinien de Golfe du Lion. Trav. Ins!. Bot. Univ. Montpelliel' Se1'ies InixteMem. 4:9-70. 17. SCHILLE.R, J. 1933. In Rabenhorst, L., K1'Yptogllll1tJ1Z Flora von Deutschland, osterreich und del' Schwciz 10 (3) (1) 1-.617, text figs. 1-6$1. 18. SMALL, E. B. &: MARSZALEK, D. S. 1969. 'Scanning electron microscopy of fixed, frozen and dried protozoa. Science 163:1064-5.