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 (Prasinophyceae). The cell is non-flagellate and has a firm cell wall, but no scales on the cell body. The pyrerioid has a characteristic structure in which a mitochondrial lobe and chloroplast outer membranes protrude into the pyrenoid 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 Pycnococcus 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, Prasinococcus capsulatus 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.