J. Gen. App!. Microbio!., 39, 571-582 (1993)

PRASINOCOCCUS CAPSULATUS GEN. ET SP. NOV., A NEW MARINE COCCOID PRASINOPHYTE

HIDEAKI MIYASHITA,' * HISATO IKEMO T0,' NORIHIDE KURANO,' SIGETOH MIYACHI,2 AND MITSUO CHIHARA"3

'Marine Biotechnology Institute , Kamaishi Laboratories, Kamaishi 026, Japan 2Marine Biotechnology Institute , Bunkyo-ku, Tokyo 113, Japan 3Japanese Red Cross College of Nursing , Shibuya-ku, Tokyo 150, Japan

(Received July 2, 1993)

A new genus, Prasinococcus, and a new species, P, capsulatus, are de- scribed on the basis of specimen that appeared in enriched cultures inoculated from water samples in the western Pacific Ocean. Cells of this unicellular alga are spherical and surrounded with a large amount of gelatinous matrix. The pigment composition, which includes chlorophylls a and b, prasinoxarithin, Mg 2,4-diviriylphaeoporphyrin a5 monomethyl ester (Mg 2,4-D) and 5,6-epoxy-3,3 '-dihydroxy-5,6,7',8'-tetrahydro$-E- caroten-11',19-olide (uriolide), is close to that observed in the Mamiel- lales (). The cell is non-flagellate and has a firm , but no scales on the cell body. The pyrerioid has a characteristic structure in which a mitochondrial lobe and outer membranes protrude into the matrix. The cell wall has a projecting appendage like a circle collar surrounded by distinctive holes penetrating the cell wall. Since the cell contains no 3-deoxy-manno-octulosonic acid (KDO), the wall is considered to have a different origin from the cell coverings which consist of fused scales in 1 etraseimis species. It has a characteristic way of asexual reproduction in which, after cell division, one cell remains within the parent cell wall while the other is extruded. This suggests that this alga be placed tentatively in the Pycnococcaceae (Order Marniellales) on the basis of the pigment composition and morphological characteris- tics, but the taxonomic position of coccoid prasinophytes rrray need to be reconsidered.

Since the cells of prasinophycean algae are generally covered with scales instead of cell walls, they are considered to be the most primitive forms of

* Address reprint requests to: Mr . Hideaki Miyashita, Marine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Kamaishi 026, Japan.

571 572 MIYASHITA et al. VOL. 39 green-colored algae. With few exceptions, a scaly cell with an apical depression having two or four flagella with bluntly rounded ends is the typical feature of the Prasinophyceae. Cells of Tetraselmis species have a theca rather than body scales, but the origin of theca is thought to be scales because of the presence of 2-keto sugar acids (2) and the active production of scales in the Golgi body (3). Recently, two taxa of coccoid prasinophytes, Bathycoccus prasinos (4) and provasolii (8), were described. These two taxa were placed tentatively in the order Mamiellales on the basis of the presence of scales and the pigment composition, respectively. This article focuses on the description of the characteristics of another related taxon of non-flagellated coccoid algae, Miyashita et Chihara gen. et sp. nov., isolated from pelagic waters in the western Pacific Ocean.

MATERIALS AND METHODS

Isolation and cultivation. During a cruise by the research vessel Sohgen-maru of Marine Biotechnology Institute, in November and December, 1990, seawater samples were collected at 64 localities in the western Pacific Ocean, from 31 ° N to 12° S. One liter of seawater was filtered through a nitrocellulose membrane filter (pore size 0.45 ,am, Toyo Roshi, Tokyo, Japan), and the filter was then incubated in several seawater-based and artificial seawater media at 25°C under continuous illumination (150 1aE m-2 sec -') . Samples were brought back to the Kamaishi Laboratories, and serial transfers were carried out to isolate the dominant species in each culture. Microalgal species in these enrichment cultures were recorded by light microscopic photographs and classified on the basis of their morphological features. Isolation was carried out by pipetting an individual cell under microscop- ic observation to establish the unialgal culture. The cells were cultured in a 500 ml flat oblong glass vessel with ESM medium (16) at 25°C under fluorescent illumina- tion (14 h day-', 80,uE m-2 sec-') and used for following experiments. Electron microscopy. For transmission electron microscopy (TEM), two methods were employed for cell fixation. One was the simultaneous glutaraldehyde/ osmic acid fixation described by Melkonian and Preisig (9), in which cells were suspended in 0.1 M phosphate buffer (pH 7.0) containing 2.0% glutaraldehyde, 0.7% 0804 and 1.0% NaCI at 0-4°C for 30-60 min. In the second method, cells were prefixed with 1.0% glutaraldehyde solution containing 0.1 M phosphate buffer (pH 7.0) and 1.5% NaCI. The cells were then suspended in the same buffer containing 1.0% 0504. Before embedding in Spurr's resin (1S), the fixed cells were buried in 1.0% agar and dehydrated with a 50-100% ethanol series. Ultrathin sections were cut with a diamond knife on a Porter-Blum Mt-1 Ultramicrotome, collected on slot grids and double-stained with uranyl acetate and lead citrate (13). Observations were carried out with a Hitachi H-7000 transmission electron micro- scope. Pigment analysis. Pigment composition was analyzed using the reverse-phase 1993 Prasinococcus capsulatus gen. et sp. nov. 573

HPLC method described by Fawley (S). Cells were collected by centrifugation and the pigments were extracted with cold methanol. The extract was centrifuged, and 100 al of supernatant was injected into a TSKge1 ODS-80Ts column (25 cm X 4.6 mm I.D., 5,am particle size, Tohsoh, Tokyo, Japan). The HPLC system (Tohsoh) consisted of a CCMP pump, a Rheodyne injector, a packed column, a UV detector (UV-8010) and a system controller (SC8010). A photodiode array UV-visible detector system (SPD-M6A, Shimadzu, Kyoto, Japan) was also used to measure the spectra of eluted pigments. Solvents used were A) methanol : water (9 :1), and B) 100% methanol. Samples were eluted with 100% solvent A for 3 min, then passed through a linear gradient from 100% solvent A to 100% solvent B in 10 min. The initial flow rate of 1.0 ml min-' was increased to 2.0 ml min -' at 20 min. The eluted pigments were detected by the absorbance at 440 nm. The retention times and the spectra of each peak of eluted pigment were compared with those from two reference strains, Mantoniella squamata and Micromonas pusilla, which were kindly supplied by Dr. Isao Inouye (University of Tsukuba). Detection of 3-deoxy-manno-2-octulosonic acid. Cells were harvested by cen- trifugation and washed once with filtered seawater and twice with methanol. The precipitates were dried and resuspended in 5% HCl methanol solution (Wako Chemicals, Tokyo, Japan). Samples were incubated in sealed tubes at 85°C for 18 h. The insoluble materials were removed by centrifugation and the supernatant was dried in vacuo. Detection of 3-deoxy-manno-2-octulosonic acid (KDO) in the extract was performed as described by Becker et al. (2). The samples were trimethylsilylated with pyridine/chlorotrimethylsilane/hexamethyldisilazane (5: 1: 1) for 30 min at room temperature and analyzed with capillary gas-liquid chroma- tography (GC-14A; Shimadzu, DB-1 column: 30m X 0.25 mm I.D., J & W Scien- tific, California, U.S.A.). The presence of KDO in the samples was identified by comparing the chromatograms with those of trimethylsilylated methyl glycosides prepared from KDO (Sigma, St. Louis, U.S.A.) and the methanolysates from Mantoniella squamata from Dr. I. Inouye and Tetraselmis chui CS-26 obtained from CSIRO Marine Laboratories, Tasmania, Australia.

RESULTS

Distribution A new species of a non-flagellated coccoid alga, Prasinococcus capsulatus, which has a cell wall and a distinct polysaccharide capsule around the cell, was isolated from pelagic waters throughout the course of the cruise (Fig. 1). It was found in seawaters from surface to a depth of 200 m. The alga was observed in microscope preparations in the enrichment culture samples from 34 of 64 survey sites. It did not grow in cultures using artificial seawater media. Isolations were made from nine samples: that obtained from the surface water at Site No. 4 (27°48' N, 142°03' E, water temperature 23.2°C) was selected as the type strain of this new marine alga. All the observations and analyses described in this paper were 574 MIYASHITA et al. VOL. 39 conducted on this type strain. The type strain is maintained at Marine Biotechnol- ogy Institute, the Karnaishi Laboratories, Japan.

Light microscopy The alga is unicellular. Cells are solitary, non-flagellate, spherical, occasionally ovoid or subspherical, 3.5-5.5 urn and rarely reaching 8,urn in diameter, and yellow-green in color. They are surrounded with a thick gelatinous envelope which is easily observed in the cell suspensions mounted in Indian ink (Fig. 2). The capsule is ellipsoidal or drop-shaped, ranging 10-15,am wide and 12-26 ,am long. The cells have a single chloroplast which is parietal and cup-shaped. It is deeply

Fig. 1. Distribution of P. capsulatus in the western Pacific Ocean. The alga was found at 34 points (Q) and isolated from 9 points (.) of the 64 survey points.

Fig. 2. P. capsulatus (phase contrast). A) Cells in culture medium. B) Cells mounted in Indian ink. Scale bar= l0,um. Fig. 3. P. capsulatus (electron micrographs). A) Vertical section of cell showing the arrangement of organelles. Scale bar= LO tem. B) Section through nucleus showing the arrangement of pyrenoid and nucleus. Scale bar =1.0,um. C) Part of cell showing the detailed structure of pyrenoid matrix. Scale bar =0.5 ICm. D) Part of cell showing the three layers of cell covering and the collar-shaped appendage. Scale bar =0.5/em. E-G) Serial section of holes around the collar-shaped appendage viewed from the anterior. Scale bars =0.5,um. ch, chloroplast; co, collar-shaped appendage; ge, gelatinous matrix; go, Golgi body; h, hole; m, mitochondrion; n, nucleus; py, pyrenoid; s, starch; w, wall. 1993 Prasinococcus capsulatus gen. et sp. nov. 575 576 MIYASHITA et al. VOL. 39 1993 Prasinococcus capsulatus gen. et sp. nov. 577 cleft into two lobes, and has one pyrenoid at the base of the chloroplast cup (Fig. 4).

Electron microscopy An individual cell is surrounded with a cell wall and a thin layer of electron- dense materials. A thick gelatinous matrix covers the surface of the outermost wall layer (Fig. 3A, B, D). No distinct scales were observed. The cell wall has a circular projection on the side opposite to the pyrenoid (Fig. 3A). The projection appears as a round collared lid, with 8 to 14 holes along its outerside (Fig. 3D-G). These holes penetrate the cell wall (Fig. 3A). A thin layer of electron-dense materials covers the entire cell wall. The collar appendage and the rims of the holes appears to be formed by the piling of electron-dense materials (Fig. 3A, D). The cup-shaped chloroplast with a pyrenoid at its base occupies more than half of the cell volume. A deep cleavage in the middle forms two large lobes of chloroplast (Fig. 4). Each lobe extends almost to the top end of the cell, and thylakoids occupy a large part of the chloroplast (Fig. 3A). The pyrenoid matrix is transversely elliptical in longitudinal section, ranging from 0.8 to 1.2,um wide, and is surrounded by a starch sheath. The cytoplasm extends as a bifurcate intrusion through an opening in the starch sheath. The chloroplast and mitochondrial membranes are observed within the cytoplasmic channel in the pyrenoid (Figs. 3, 5). A nucleus, a mitochondrion, a large Golgi body and many vesicles occur in the cytoplasm (Figs. 3, 4). The nucleus is boat-shaped and located adjacent to one of the chloroplast sinus. The mitochondrion is bifurcate extending from the pyrenoid and placed inside the chloroplast lobes. The Golgi body is at the center of the cell and occupies a large part of cytoplasm. Vesicles are located at the upper side of Golgi body near the cells collar.

Asexual reproduction The process of the asexual reproduction is shown in light and electron micrographs (Fig. 6), and schematically illustrated (Fig. 7). When the type strain is cultured under 14 h light/1 h dark cycles at 25°C, the chloroplast and pyrenoid start to divide (Fig. 6F; Fig. 7B) at the beginning of the dark regime. The mother cell divides longitudinally into two daughter cells (Fig.

Fig. 4. P. capsulatus, schematic illustration showing the distribution of organelles. ch, chloroplast; co, collar-shaped appendage; ge, gelatinous matrix; go, Golgi body; h, hole; m, mitochondrion; n, nucleus; py, pyrenoid; v, vesicle; w, wall. Fig. 5. P. capsulatus, part of the cell showing the structure of the pyrenoid matrix. ch, chloroplast; cy, cytoplasm; m, mitochondrion; py, pyrenoid; s, starch. Fig. 6. P, capsulatus, photo- (A-E) and electron- (F-K) micrographs showing the process of asexual reproduction. Scale bars=1.0,im. 578 MiYASxiTA et al. VoL. 39

6A, G). At first, the two daughter cells lie side by side, their chloroplast cups facing the same direction. One of the daughter cells then enlarges and the smaller cell rotates about 45 ° so that the bottom of the chloroplast cup moves toward the larger cell (Fig. 6H; Fig. 7C). The smaller cell then protrudes through a hole of the parent cell wall (Fig. 7D, E). The hole is located opposite to the cell wall projection (Figs. 3, 7) indicating that the circle projection does not function like a cyst aperture. The cytoplasm passes through the hole, followed by the chloroplast and pyrenoid (Fig. 7E). Finally, the smaller cell is completely squeezed out from the mother cell wall (Fig. 7F). The larger cell remains in the mother cell wall and expands to fill the empty space. These processes are completed in 4 h from the initiation of the dark regime. The size of cells remaining in the mother wall is 4- 5 , im in diameter, which is 0.3-0.7 ,am larger than the cell squeezed out. At this time, a cell wall could not be seen on the smaller cell at least with the fixation methods we employed. A firm cell wall is seen, however, before it moved to the edge of the parent capsule (Fig. 7H). Cell separation (Fig. 7A-H) is completed in 2 h from the end of the dark regime by removing the smaller cell from parent capsule. The capsule layer of the smaller cell is thin, but grew thicker as the cell aged. Sexual reproduction has not been observed.

Photosynthetic pigments The following photosynthetic pigments were detected in the type strain of P. capsulatus by HPLC (Fig. 8) : chlorophylls a and b, carotenes, an unknown xanthophyll, lutein, zeaxanthin, violaxanthin, prasinoxanthin, neoxanthin, 5,6- epoxy-3,3 '-dihydroxy-5,6,7', 8 '-tetrahydro/3-E-caroten-11',19-olide (uriolide) and Mg 2,4-divinylphaeoporphyrin as monomethyl ester (Mg 2,4-D). This pigment composition resembles that of MMsquamata except for the absence of "unidentified Ml" (14) and "unknown A" (5), and the presence of large amount of the unknown xanthophyll.

3-Deoxy-manno-2-octulosonic acid 3-Deoxy-manno-2-octulosonic acid (KDO) was detected in the methanolysate of the cell extracts from Tetraselmis chui (CS-26) and M. squamata. KDO was not detected in P. capsulatus.

Growth characteristics Nine isolates of P. capsulatus grew well at 25°C, but did not grow at temperatures below 15°C or above 35°C. Cells preferred a slightly alkaline pH for growth, the optimum pH being 8.5. Cells did not grow at a pH either below 6.5 or above 9.5.

Diagnosis Prasinococcus Miyashita et Chihara gen. nov. 1993 Prasinococcus capsulatus gen. et sp. nov. 579

/ Fig. 7. P. capsulatus, schematic illustrations showing the sequential process of asexual reproduction (from A to H).

Fig. 8. HPLC separation chromatograms of pigments. A) P. capsulatus. B) MMsquamata.

Alga unicellularis; cellulae sphaericae, solitariae, parieti, sine flagello, sine squama; chloroplastus unus, parietalis, bilobatus, cupulatus; pyrenoides vagina amyli circumcincta, canale invasa; canalis furcata, cytoplasme, chloroplasto et 580 MIYASHITA et al. VOL. 39 mitochondrio completa; reproductio asexualis per fissionem binariam; pigmenta chloroplasti ex chlorophyllis a et b, magnesio 2,4-divinylphaeoporphyrino, prasino- xanthino, et uriolide pro pigmento rnajore constantes. Species typica: Prasinococcus capsulatus Miyashita et Chihara.

Alga unicellular; cells spherical, solitary, walled, without flagella, without scales; chloroplast single, parietal, bilobed, cup-shaped; pyrenoid covered with a starch sheath, invaded by a cavity; cavity bifurcated, filled with cytoplasm, chloro- plast and mitochondrion; asexual reproduction by binary cell division; photosynthe- tic pigments containing chlorophylls a and b, Mg 2,4-divinylphaeoporphyrin a5 monomethyl ester, prasinoxanthin, and uriolide as major pigments. Type species: Prasinococcus capsulatus Miyashita et Chihara.

Prasinococcus capsulatus Miyashita et Chihara sp. nov. Cellulae flavo-virentes, 3.5-5.5,um diametro, vagina gelatinosa ellipsoidea circumcinctae, 10-15,um latae, 12-26 urn longae. Holotypus: Figurae 3, 4.

Cells yellow-green, 3.5-5.5 um in diameter, surrounded by an ellipsoidal gelatinous envelope, 10-15 urn wide, 12-26 urn long. Type locality: Lat. 27°48' N; Long. 142°03' E; western Pacific Ocean; surface water. Etymology: Prasinococcus; Latin prasinus, leek-green + Latin coccus, coccus. capsulatus; Latin capsularis, capsule-like. Distribution: widely distributed in temperate to tropical pelagic water from surface to considerable depths.

DISCUSSION

The genus Prasinococcus is characterized by 1) a spherical cell with cell wall, 2) the lack of flagella and basal bodies, 3) the lack of scales on cell body, 4) a circular projection on the cell wall surrounded by many holes penetrating the cell wall, 5) a pyrenoid with a cytoplasrnic channel containing a mitochondrial lobe and the chloroplast membranes, 6) the asexual reproduction in which one cell retains the old cell wall and capsule while the other is squeezed out as a naked cell, and 7) the presence of chlorophylls a and b, prasinoxanthin, uriolide and Mg 2,4-D. The cell of P. capsulatus does not contain 3-deoxy-rnanno-2-octulosonic acid (KDO) which is generally detected in scales of prasinophycean cells as well as in the cell coverings of Tetraselmis species (2). No scale-like particles with their distinct shape were observed around the cell in electron microscopic observations. These results indicate that the cell wall of P. capsulatus does not originate from fusion of scales. Since the new naked daughter cell which is squeezed out from the mother cell of P. capsulatus forms a new cell wall and grows continuously, the stage 1993 Prasinococcus capsulatus gen. et sp. nov. 581

with a cell wall is not considered to be the cyst stage, as it is in a few other prasinophycean algae (1,12) . The pigment composition resembles that of Mantoniella sauamata (Fig. 8), Micromonas pusilla (data not shown) and Mamiella gilva (5). The order Mamiel- lales, with MMgilva as the type, was described by Moestrup (10) on the basis of the lack of an inner layer of square or diamond-shaped scales. Recently, Guillard (8) proposed to emend the circumscription of the order Mamiellales to include coccoid prasinophytes containing prasinoxanthin and Mg 2,4-D, since the presence of both pigments was the typical characteristic of the Mamiellales as exemplified in the above-mentioned three genera (6-8). Two species have been described as prasinophycean coccoid algae, Bat hycoccus prasinos Eikrem et Throndsen (4) and Pycnococcus provasolii Guillard (8). Both taxa were placed in the Mamiellales according to the emended definition. The former taxon is distinguished from P, capsulatus by the presence of a layer of spiderweb-like scales on the cell body and the absence of a pyrenoid. The latter is similar to P. capsulatus in the presence of a cell wall, the absence of scales and the structure of the pyrenoid. The pyrenoid matrix of both algae has a channel filled with cytoplasm and membranes of both the chloroplast and the mitochondrion (Fig. 5). This characteristic structure was also found in marina (11). P. provasolii was reported to have a in some stages (8) but in contrast a cell stage having flagella or basal bodies is not observed in P. capsulatus. Moreover, P. capsulatus reproduces in peculiar way as shown in Figs. 6 and 7. It is also noteworthy that the cell wall of P. capsulatus has a collar-like appendage which is surrounded by a circle of holes (Fig. 3). P. provasolii was reported to have an "operculum-like" region (8), but that structure is different from the collared projection of P. capsulatus. The thick gelatinous envelope as well as the presence of uriolide are also characteristic of P. capsulatus. Taking these characteristics into consideration, it seems to be appropriate to establish a new genus for this pelagic alga in the order Mamiellales. The distinctive manner of cell separation and the characteristic cell structure, the cell wall not containing KDO, and a circular projection on the cell wall surrounded by holes, distinguish it from the other genera in the Mamiellales. P. capsulatus is tentatively placed in the family Pycnococcaceae of the Ma- miellales according to the emended definition of Guillard (8). This emendation was based on the similarities of the pigment signature between P. provasolii and certain taxa of the Mamiellales. It was recommended by Fawley (5), however, that the taxonomic position of P. provasolii in the order Mamiellales be reassessed, since the presence of prasinoxanthin and Mg 2,4-D is not restricted to the Mamiellales. The taxonomic position of P. capsulatus should be reconsidered when further detailed information on this and related algae is available.

The survey of the western Pacific Ocean in 1990 by R. V. Sohgen-maru was performed under the management of Research Institute of Innovative Technology for the Earth (RITE) as a part of the 582 MIYASHITA et al. VOL. 39

Research & Development Project on Biological COZ Fixation and Utilization supported by New Energy and Industrial Technology Development Organization (NEDO), Japan. We are grateful to Professor Richard E. Norris, Botanical Research Institute of Texas, for his reading of the manuscript and Dr. Isao Inouye, University of Tsukuba, for his valuable comment and advice. We would also like to thank Ms. Mika Atsumi for her technical assistance in isolating algae, Mr. Yoji Kikuchi for his help in taking electron micrographs, and Ms. Mayumi Fukushi for her assistance in maintaining this algal culture.

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