J. Eukaryot. Microbial.. 48(3), 2001 pp. 348-361@ 2001 by the Society of Protozoologists

Ultrastructural Characteristics of the In Vitro Cell Cycle of the ProtozoanPathogen of Oysters, Perkins us marinus

INKE SUNILA," ROSALEE M. HAMILTONb and CHRISTOPHER F. DUNGANb

'State of Connecticut, Department of Agriculture, Bureau of Aquaculture, P.O. Box 97, Milford, Connecticut 06460, USA, andbMaryland Department of Natural Resources, Cooperative Oxford Laboratory, 904 S. Morris Street, Oxford, Maryland 21654, USA

ABSTRACT. Ultrastructural characteristics of vegetative and zoosporangial stages of cultured Perkinsus marinus, a pathogen of theeastern oyster, Crassostrea virginica, were examined by transmission electron microscopy. An axenic cell cnlture was propagated frominfected Chesapeake Bay oyster hemolymph. Different stages of the in vitro cell cycle, including schizonts and different size trophonts,were examined. Trophonts had spherical nuclei with wide perinuclear spaces, mitochondria with tubular cristae, and vacuoles withvacuoplasts. There were micropores on the. inside of cell walls. A tubular network in the cytoplasm connected lomasomes to vacuoles,and contained vacuoplast precursor material. Vacuoplasts and precursor material diminished when cell cultures were not fed, suggestinga function in metabolite storage. Cells divided by schizogony or binary fission. Daughter cells in a schizont were not alike, and mayspecialize for different functions. Some of the daughter cells in a schizont died. Some hypnospores, directly isolated from infectedoyster hemolymph enlarged in Ray's fluid thioglycollate medium, and were induced to zoosporulate. Zoosporangia contained varicose,hypha-like structures, whose apical tips gave rise to prezoospores. Ultrastructural characteristics of the vegetative and zoosporangialstages did not resemble any apicomplexan parasites other than members of the genus Perkinsus.

Key Words. Apicomplexa, cell culture, dermo disease, parasite, transmission electron microscopy.

P ERKINSUS marinus is an oyster parasite that causes sig-nificant mortalities in populations of the eastern oyster,

Crassostrea virginica, on the east coast of the United Statesfrom the Gulf of Mexico to Long Island Sound (for review seeFord and Tripp 1996). Besides the eastern oyster, P. marinusor related species infect 12 other bivalve mollusc species, in-cluding the clams Mya arenaria and Macoma balthica (An-drews 1954). Previously, P. marinus was named Dermocysti-dium marinum (Mackin et al. 1950) and Labyrinthomyxa ma-rina (Mackin and Ray 1966). Currently, P. marinus is classifiedin the class Perkinsea, phylum Apicomplexa (Levine 1978),based on ultrastructural characteristics of the apical complex inflagellated zoospores described by Perkins (1976).

Zoospores (zoites), which have only been observed in vitro,represent a free-swimming stage in the life cycle of the parasite.When P. marinus cells from infected oyster tissue explants areincubated in Ray's high-salt modification of fluid thioglycollatesterility test medium (RFTM, Ray 1952, 1963), they undergocharacteristic enlargement to form thick-walled hypnospores(prezoosporangia). Numerous biflagellate zoospores differenti-ate within some RFTM-enlarged hypnospores when they aretransferred to seawater (Perkins and Menzel 1966) or to in vitropropagation medium (La Peyre 1996). Mature zoospores havenot been reported to proliferate in vitro, but do so upon infect-ing oyster tissue explants and losing zoospore characteristics(Perkins 1988; Perkins and Menzel 1966). The function of zoo-spores in the natural life cycle of P. marinus remains unknown.Within oyster tissues, trophozoites (trophonts) proliferate veg-etatively: after several internal cell divisions, four to 64 daugh-ter cells are liberated through a tear in the schizont wall, andeach daughter cell enlarges to divide and repeat the cycle (Per-kins 1996). .

The ultrastructure of P. marinus in vivo was described byPerkins (1969, 1988, 1996). He described an immature uninu-cleate trophont with a fibrogranular wall, two centrioles, tubu-lovesicular mitochondria, and lipid droplets. The mature tro-phont has a vacuoplast within an eccentric vacuole, which push-es the nucleus close to the cell wall, giving the characteristicsignet ring configuration. Azevedo (1989) described the in vivoultrastructure of a related pathogen, Perkinsus atlantic us of theclam Ruditapes decussatus from Portugal, and Blackbourn,Bower, and Meyer (1998) the in vivo ultrastructure of Perkin-sus qugwadi, a pathogen of the Japanese scallop Patinopecten

Corresponding Author: I. Sunila-Telephone number: 203-874-0696;FAX number: 203-783-9976; E-mail: dept.agric@snet.net

yessoensis from Canada. Kleinschuster et al. (1994) culturedanother Perkinsus species, isolated from Macoma balthica, andprovided electron micrographs of in vitro zoosporangia and ma-ture zoospores.

Several research groups have recently established P. marinuscell cultures (Gauthier and Vasta 1995; La Peyre, Faisal, andBurreson 1993). The P. marinus in vitro cell cycle includesnutrient-stimulated enlargement and schizogony of mothercells, followed by release of multiple daughter cells, which sub-sequently enlarge and repeat the. division process (Dungan andHamilton 1995; La Peyre 1996). However, no comprehensivestudies describing ultrastructural characteristics of different lifecycle stages of cultured P. marinus cells have been published.Such information is essential when evaluating P. marinus tax-onomy. The classification of P. marinus is still questionable,and based on molecular genetic and morphological character-istics, some authors suggest its closer relationship to dinofla-gellates than-to'apicomplexans (Goggin and Barker 1993; Ree-ce et al. 1997; Siddall et al. 1997). Based on the current clas-sification of P. marinus, terminology used herein is that definedfor apicomplexan life stages (Vivier and Desportes 1990),which was also adopted by Ford and Tripp (1996) in their re-cent review of oyster diseases. It defines the uninucleate stageas a trophont, which transforms into a multinucleate stagecalled a schizont (or a meront). The present article is based ona cell culture (American Type Culture Collection #50439)maintained at the Cooperative Oxford Laboratory (Dungan andHamilton 1995). This report describes the ultrastructure of cul-tured P. marinus vegetative and zoosporangial stages.

MATERIALS AND METHODS

Perkins us marinus cell culture. Perkinsus marinus strainATCC 50439 was isolated from infected Chesapeake Bay C.virginica hemolymph, axenic ally propagated in vitro at 28 °Cin an optimized, 650 mOsmlkg Dulbecco's Modified Eagle(DME)/Ham's F-12 liquid growth medium supplemented to 3%(v/v) fetal bovine serum (FBS) (DME/F-12) without antibiotics,and cryopreserved (Dungan and Hamilton 1995). Cell typesharvested and examined are presented diagrammatically in ref-erenceto the cell cycle (Fig. 1). For this study, cryopreserved,5-fLm diam. trophonts (Fig. IG) were thawed, inoculated intoflasks, and periodically harvested, fed with fresh medium ordiluted, to obtain cell populations dominated by specific celltypes of interest.

Hypnospores and zoosporangia (Fig. IB, C). Hypnosporesand zoospores of P. marinus were produced in vitro by incu-

348

\

1

SUNILA ET AL.-PERKINSUS MARINUS IN VITRO ULTRASTRUCTURE 349

G

H

/ SASW 6MO

~

Fig. 1. A diagrammatic drawing of the cell cycle of cultured Perkinsus marinus and the cell types harvested. A. P. marinus trophozoite frominfected oyster hemolymph. B. Enlarged hypnospore following incubation in Ray's fluid thioglycollate medium (RFTM). C. Zoo sporangium andzoospores from enlarged hypnospore transferred to Dulbecco's Modified Eagle (DME)/F12 medium. D. Large mother cell from vegetative in vitrocell cycle in DME/F12 medium. E. Early schizont (internally subdividing mother cell) containing daughter cells. F. Mature schizont with enlargingdaughter cells rupturing lysed mother cell membranes. G. Cluster of daughter cells enlarging to repeat the division process. H. Stationary phase(inactive) schizonts limited by scarce nutrients. I. Stationary phase small daughter cells after 6 mo in sterile artificial seawater (SASW).

bating 0.5-ml samples of infected oyster hemolymph (Fig. lA)with 2.0 ml of antimicrobial-supplemented Ray's fluid thiogly-collate medium (RFTM) in covered 24-well plates for 8 d at28 °c. Antimicrobial supplements to RFTM included penicillinG 100 U ml-l, streptomycin sulphate 100 /-Lgml-l, gentamicinsulphate 50 /-Lg-l,chloramphenicol 2.5 /-Lgml-1, and 1% (v/v)of saturated aqueous nystatin. Enlarged hypnospores (Fig. IB)were harvested and washed by centrifugation (Dungan andRoberson 1993), and then inoculated into DME/F-12 mediumsupplemented to 3% (v/v) FBS and with penicillin, streptomy-cin, chloramphenicol, and gentamicin to concentrations speci-fied above for RFTM. Over the following 6 d, a low percentage(= 10%) of enlarged hypnospores underwent asynchronouszoo sporulation after germ tube extrusion, progressive subdivi-sion of sporoplasm, and daughter cell elaboration of flagellarappendages (Perkins 1996; Perkins and Menzel 1966). On day

six after hypnospore transfer from RFTM, the DME/F-12 su-pernatant medium containing zoosporangia was harvested andfixed (Fig. Ie).

Vegetative cells (Fig. ID-I). Ten days after thawing andexpanding a frozen aliquot of cells (Fig. IG), a late log-phasepopulation of 20-/-Lmdiam. schizonts (Fig. IF) was sampled andfixed. This schizont population was thinned and fed to encour-age rapid completion of schizogony and release of daughtercells. Resulting rosettes of log-phase, sibling, 5-/-Lmdiam. cellscontaining refractile cytoplasmic granules and vacuoplasts wereharvested and fixed 24 h later (Fig. IG). This small-trophontpopulation was also diluted, fed, and propagated for 72 h. Re-sulting large trophonts (average diam. 16 /-Lm)with large re-fractile vacuoplasts and smaller refractile cytoplasmic granuleswere harvested and fixed (Fig. ID). These large trophonts werealso diluted, fed, and incubated for 24 h, when they initiated

10-

RFTM

)'=""

A

C12

B C

No FE EO

E ",,7 iV".50it .Pi" 'n

350 J. EUKARYOT. MICROBIaL., VOL. 48, NO.3, MAY-JUNE 2001

schizogony and began to subdivide internally. A sample of 16-fLm diam. log-phase early schizonts and mature trophonts fromthis population was harvested and fixed (Fig. IE). A sample ofearly stationary-phase, 1O-fLm diam. schizonts from a separatepopulation that had not been diluted or fed for 5 d (Fig. 1H)was harvested and fixed, as was a late stationary-phase popu-lation of small, 3-fLm diam., refractile trophonts that had beenmaintained at 28 DC in sterile, 20%0 artificial seawater (SASW)for 6 mo (Fig. 11).

Electron microscopy. Two aliquots of each cell pellet wereharvested by centrifugation at 430 g for 5 min, washed with20%0 sterile artificial seawater (SASW) and pelleted. Sampleswere fixed with 2.5% (v/v) glutaraldehyde (pH 7 in sodiumcacodylate-buffered 20%0 SASW) for transmission electron mi-croscopy (TEM). After fixation, cell pellets were stabilized byembedding them in 2% (w/v) low gelling temperature agarosein cacodylate-buffered 20%0 SASW. Samples were post-fixedin 0.5% (w/v) OS04 in sodium cacodylate buffer, dehydratedthrough graded ethanols and propylene oxide, and embedded inepoxy resin. Thick sections were stained with methylene blue-azure lIlbasic fuchsin stain, and thin sections with uranyl ace-tate and lead citrate. Sections were viewed with a lEOL 1200

TX TEM operating at 60 kV.

RESULTS

Hypnospores and zoosporangia (Fig. IB, C). Zoosporangiacontained numerous prezoospores. Zoosporangia were sur-rounded by a thick cell wall, which was slightly undulating,with cytoplasmic papillae extending into the internal wall, andflattened at the plugged opening of the discharge tube (Fig. 1C,2, 3). Many RFTM-enlarged hypnospores (90%) did not dif-ferentiate into zoosporangia. They retained a large vacuole withvacuoplasts inside (Fig. 1B, 3). Prezoospores proliferated bysuccessive binary fissions. Rows of prezoospores could be seenstill attached to each other with continuous plasma membranes,but each individual cell had developed vacuoles, mitochondria,and flagella apparatus (Fig. 4). Prezoospores had spherical nu-clei, often multiple nucleoli, and round mitochondrial profiles,which often protruded into the vacuole. There were two flagellaapparatuses in the cells, usually at some distance from eachother. Longitudinal and cross-sections of developing flagellawere seen inside differentiating zoospores. Between prezoos-pores there were rare sections of external flagella, and numer-ous small vesicles (Fig. 5). Inside the zoosporangium, therewere varicose, hypha-like structures. At the apical tips of thehyphae, round prezoospores were seen budding (Fig. 6). Or-ganelles such as vacuoles, vacuoplasts, mitochondria, and cy-toplasmic tubular network with vacuoplast precursor materialdid not differ from those in stages of the vegetative cell cycledescribed below.

Mature, log-phase schizonts (Fig. IF). The first sample(Fig. IF) was collected 10 d after thawing a frozen aliquot ofcells, and consisted of late-log phase 20-fLm schizonts and rareenlarged trophonts. Schizonts were composed of several (over10) small daughter cells, which had clear vacuoles and smallvacuoplasts visible by light microscopy (Fig. 7). Each daughtercell had a spherical nucleus with a prominent nucleolus andgranular chromatin. Fibrillar and granular areas of the nucleoluscould be distinguished in several cells. A wide perinuclearspace with distinct nuclear pores surrounded the nucleus. Elon-gated mitochondria with tubular cristae had round cross-sectionprofiles. Most of the cytoplasm was filled with free ribosomesand a few small membrane-bound vacuoles (Fig. 8). All daugh-ter cells were surrounded by a common schizont cell wall. Vac-uoplasts were small or absent. Some cells had lipid droplets(Fig. 9). No active Golgi apparatus was present in these cells.

Small, log-phase trophonts (Fig. IG). Twenty-four hoursafter diluting and feeding schizonts, schizogony was completedas daughter cells enlarged, ruptured schizont walls, and released5-fLm trophonts (Fig. 1G). Sequential steps in schizogony anddaughter-cell release, as seen by light microscopy of live cells,are shown in Fig. 10. Some of the sibling cells formed rosettesand were still surrounded by remnants of a common schizontcell wall. Cells had dense nuclei, prominent nucleoli, and mem-brane networks filling the vacuoles in addition to the vacu-oplasts. Some cells had lipid droplets. Cell walls were eitherthick or thin (Fig. 11). Under higher magnification, numerouscross-sections of a tubular network with dense accumulations

on one side (vacuoplast precursor material) were seen scatteredin the cytoplasm. Micropores were detected protuding into thecytoplasm under the cell wall. Vacuoplasts had developed tofill most of the vacuoles. A vesicular lomas orne structure

formed beneath the cell wall (Fig. 12).Large, log-phase trophonts (Fig. ID). Seventy-two hours

later, trophonts had enlarged to 16 fLm in diam. (Fig. 1D). Thesecells had signet-ring morphology, characterized by eccentricnuclei with prominent nucleoli clearly visible in live cells bylight microscopy, as well as granular cytoplasm and large vac-uoles with relatively small vacuoplasts (Fig. 13). Trophonts hada round nucleus and nucleolus, a vacuoplast that filled most ofthe vacuole, and several lipid droplets. Vacuoplasts had elec-tron-lucent inclusions, were irregularly shaped, and continuedinto the cytoplasm through invaginations connected to the tu-bular network containing vacuoplast precursor material (Fig.14). Some trophonts had an apical structure, which gave thema polar appearance (Fig. 15, 16), but which probably repre-sented adherent schizont wall remnants. This structure, whichoverlaid a thin, continuous new cell wall, was composed ofthick, discontinuous cell-wall material with folds in each endand fibrillar material on its surface. Beneath the apical structure,there were sponge-like structures. Dense vacuoplast precursormaterial was concentrated at the apical end, while the nucleuswas situated in the opposite end. There were mitochondria dis-tributed throughout the cell (Fig. 15). Cell walls were relativelythin.

Young, log-phase schizonts (Fig. IE). Young log-phaseschizonts with subdividing cytoplasm were harvested (Fig. IE).Live early schizonts observed by light microscopy showeddaughter cell walls internally subdividing schizont cytoplasm(Fig. 17). Large subdividing schizonts had different types ofcells inside. Pleomorphic daughter cells fitted together in a puz-zle-like manner within a common schizont cell wall (Fig. 17,18, 19). The most common cell type had a spherical nucleus,granular chromatin, and a prominent nucleolus. There was awide perinuclear space with distinct nuclear pores. Irregularlyshaped vacuoles contained vacuoplasts (Fig. 19). Cytoplasmictubular networks containing vacuoplast precursor material werepresent, and a few lipid droplets. Some cells inside a schizontwere swollen with rupturing membranes and irregularlyclumped chromatin indicating necrosis (Fig. 18). Some cellswere condensed with intact membranes and dense, uniformlypacked chromatin. indicating apoptotic cell death. Some of thecells had structures resembling sponge-like aspects of rhoptries.

Stationary-phase schizonts (Fig. IH). A stationary-phasepopulation of 1O-fLm diam. cells, which had not been fed for 5d, was harvested (Fig. IH). These cells had spherical nucleiwith prominent nucleoli and wide perinuclear spaces. Vacuoleswere well-defined and contained remnants of vacuoplasts (Fig.20, 21). A tubular network connected the vacuoplast to the cy-toplasm. Vacuoplast precursor material in the cytoplasmic, ram-ifying tubular network was scarce or absent. Some cells hadextremely elongated mitochrondria (Fig. 20). Prominent loma-

-

SUNILA ET AL.-PERK/NSUS MAR/NUS INVITRO ULTRASTRUCTURE 351

, ~

..61&

J

,...-

~.

Fig. 2. Zoosporangium of Perkins us marinus. Hundreds of prezoospores are surrounded by a thick cell wall (W). Arrowheads point todeveloping prezoospores. P, plug at discharge pore. A montage of four exposures. Bar = 6 [Lm. TEM.

352 J. EUKARYOT. MICROBIOL., VOL. 48, NO.3, MAY-JUNE 2001

~I

38

=-\

---.

..

..."

,.

Fig. 3-6. Zoosporu1ation of Perkinsus marin us. 3. A zoosporangium (Z) with a discharge tube (D) and hundreds of prezoospores inside.Hypnospores (H) have a large vacuole with vacuop1asts. Bar = 20 [Lm.Light microscopy, 1 [Lm-thick section. 4. Prezoospores divide by successivebinary fissions. Arrowheads point to budding. B, basal body; N, nucleus; V, vacuole; W, wall of zoosporangium. Bar = 1 [Lm. TEM. 5.Prezoospores have spherical nuclei (N) and vacuoles (V). Arrowheads indicate cytoplasmic and extracellular flagellar figures; B, basal body; M,mitochondrion; W, wall of zoo sporangium. Bar = 1 [Lm. TEM. 6. Note hypha-like structures inside zoosporangium. Prezoospores were seenbudding at the apical tips of these structures (arrowheads). V, vacuole; VP, vacuoplast. Bar = 2 [Lm. TEM.

b

SUNILA ET AL.-PERK/NSUS MAR/NUS IN VITRO ULTRASTRUCTURE 353

Fig.7-9. VegetativecellcycleofculturedPerkinsus marinus:mature, log-phase schizonts (Fig. IF). 7. Live mature schizont (F) containingmultiple daughter cells (G) which have enlarged to rupture their containing schizont wall. Bar = 10 J1.m.Differential interference contrast. 8.Daughter cell with spherical nucleus (N), wide perinuclear space, and a vacuole (V). M, mitochondrion; VP, vacuoplast. Bar = I J1.m.TEM. 9.Daughter cells within schizonts were surrounded by a common wall (C). Bar = 4 J1.m.TEM.

somes were present, opening between the plasma membraneand the cell wall (Fig. 21).

Stationary-phase trophonts (Fig. 11). A stationary-phasecell population, which had been held at 28 °C in SASW (20-30%0) for 6 mo, consisted of small, 3-fLm diam. trophonts (Fig.

11). These cells had round nuclei with abundant heterochro-matin against the nuclear membrane and a prominent nucleolus.Cell walls were thick, and several cells had condensed, sepa-rating the plasma membrane from the cell wall (Fig. 22). Vac-uoles were present, but vacuoplasts had disappeared, leaving

10

354 J. EUKARYOT. MICROBIOL., VOL. 48, NO.3, MAY-JUNE 2001

c/

11

~.

Fig. 10-12. Vegetative cell cycle of cultured Perkins as marinas: small log-phase daughter trophonts (Fig. IG). 10. Three live cells showingthe sequence of schizogony and daughter trophont release: schizont (E), ruptured mature schizont (F), and cluster of small daughter trophonts(G). Bar = 20 [Lm. Hoffman modulation contrast. 11. Some trophonts had thick (arrowhead), some thin cell walls (C) and vacuoles (V) withvacuop1asts (VP). Remnants of schizont cell walls were present. Bar = 2 [Lm. TEM. 12. Small trophont with a lomasome (L) under cell wall,tubular network with vacuop1ast precursor material, and a large vacuop1ast (VP) inside the vacuole (V). MP, micropore; N, nucleus; NS, nucleolus;P, precursor material; PS, perinuclear space. Bar = 1 [Lm. TEM.

~

SUNILA ET AL.-PERK/NSUS MAR/NUS INVITRO ULTRASTRUCTURE 355

!11

16

,

':)1'

14

.. 15~ ...

Fig. 13-16. Vegetative cell cycle of cultured Perkinsus marinus: large log-phase trophonts (Fig. ID). 13. Live large trophonts with largevacuoles (V), vacuoplasts (arrowhead), nuclei with prominent nucleoli (N), and refractile cytoplasmic bodies. Bar = 10 [Lm.Differential interfer-ence contrast. 14. Trophonts had spherical nuclei (N), large irregular vacuoplast, and lipid droplets (L). NS, nucleolus; P, precursor materiaL Bar= 2 [Lm TEM. 15. Urn-shaped trophont after binary fission with cell wall (C) material still attached to the plasma membrane. N, nucleus; S,sponge-like aspects. Bar = 4 [Lm.TEM. 16. Remnants of cell wall materiaL Higher magnification of Fig. 15. Bar = I [Lm.TEM. L-

356 J. EUKARYOT. MICROBIaL., VOL. 48, NO.3, MAY-JUNE 2001

Fig. 17-19. Vegetative cell cycle of cultured Perkinsus marinus: early subdividing, log-phase schizonts (Fig. IE). 17. Live subdividing earlyschizont showing division furrows between developing daughter cells. Bar = 5 [Lm. Differential interference contrast. 18. Multicellular stageschizonts had different types of cells inside, some of which were necrotic (Ne). Nuclei (N) were spherical and vacuoles (V) had irregularvacuoplasts. Sponge-like aspects (S) were present in some cells. Bar = 2 [Lm.TEM. 19. DeveJoping daughter cell magnified from Fig. 18. Typicaldaughter cells inside schizonts had spherical nuclei (N) with wide perinuclear spaces (PS) and irregular vacuopJasts inside the vacuoJe (V). C,cell wall; M, mitochondrion. Bar = 500 nm. TEM.

,I

~

357SUNILAETAL.-PERKINSUS MARINUS IN VITRO ULTRASTRUCTURE

-~ ~-_u_-~--~_u_- -- ~

"'.

Fig. 20-21. Vegetative cell cycle of cultured Perkinsus marinus: stationary-phase schizonts unfed for five days (Fig. IH). 20. A daughter cellwith tubular network connecting the vacuoplast (VP) with the cytoplasm. N, nucleus; M, mitochondrion; P, precursor material; V, vacuole. Bar= I fLm~TEM~ 21. A daughter cell with remnants of the vacuoplast (VP) and a lomasome (L) under the cell wall. M, mitochondrion; N, nucleus;V, vacuole. Bar = 500 nm. TEM.

fibrous remnants behind (Fig. 23, 24). Tubular cytoplasmic net-works were empty of the vacuoplast precursor material. Virus-like particles were present in the nuclei of this sample (Fig. 23).Nuclear particles had a light halo around them and a densercore with hexagonal symmetry. Their diameters were uniformly55 nm. Few cell division figures were observed in this sample.Figure 24 shows a late telophase figure with two nearly sepa-rated new nuclei, chromatin condensation against the nuclearmembranes, and a microtubular network between the nuclei.Overlapping mitotic spindles formed a mid-body between thecells (Fig. 25).

Cell divisions. During the vegetative cell cycle, cells dividedeither by binary fission or schizogony, the latter being the mostcommon. During binary fission, a daughter cell budded fromthe trophont, producing a mother trophont surrounded by athick cell wall, and the daughter cell by a plasma membrane)(Fig. 26, 27). During schizogony, plasmotomy initially was syn-chronous, forming two-cell, four-cell and eight cell-stages with-in a common schizont wall (Fig. 28, 29).

DISCUSSION

Descriptions of ultrastructural characteristics of different ap-icomplexans are numerous, since this phylum includes parasitescausing important human diseases, such as toxoplasmosis(Toxoplasma gondii), diarrhea (Cryptosporidium spp.) and ma-laria (Plasmodium spp.). Perkinsus marinus is currently clas-sified in the phylum Apicomplexa based on a specific structureof the zoospore, the apical complex, which is characteristic forall species of this phylum (earlier Sporozoa). The apical com-plex is thought to aid infective zoospores in host invasion. Zoo-spores are similar in all members of the Apicomplexa, althoughthe structure of other cell stages is highly variable.

The zoospore apical complex has a polar ring connected withmicrotubules, a cone-shaped conoid, made up of spirally ar-

ranged microtubules, and paired rhoptries. According to Siddallet al. (1997), the conoid of P. marinus and other Perkinsusspecies is not conical, which together with other supportingmorphological and molecular-genetic disparities refutes classi-fication of these organisms as apicomplexans. However, mem-bers of one basal apicomplexan order (Plasmodium spp.) lackthe conoid altogether (Scholtyseck and Mehlhorn 1970), indi-cating conoid presence and ultrastructure to be a variable fea-ture in the apicomplexan.

Regardless of slight morphological similarities between thesegroups, molecular systematics group apicomplexans, dinofla-gellates, and ciliates in a single alveolate clade (Gajadhar et al.1991), Cavalier-Smith (1998) suggested abandoning the nameApicomplexa and moved Perkinsus to the proposed phylum Di-nozoa as a separate subphylum Protalveolata, together with aseparate subphylum Dinoflagellata. Recently, Noren, Moestrup,and Rehnstam-Holm (1999) proposed a new phylum Perkin-sozoa for Perkinsus and other perkinsid protozoans, which aredistinct from both dinoflagellates and apicomplexans, yet shareDNA sequence homology and some morphological character-istics with both of these other alveolate subgroups.

Although the purpose of the investigation reported here isdescriptive, not systematic, ultrastructural characteristics of thein vitro vegetative cell cycle of P. marinus described herein didnot resemble apicomplexan parasites other than other Perkinsusspecies. Organelles of the apical complex or traces of theseorganelles (polar rings, micronemes, rhoptries) were usuallystill present in the immobile stage of other apicomplexans(Friedman et al. 1995; Kim and Paperna 1993a; Scholtyseckand Mehlhorn 1970). A distinct peripheral cytoplasmic layer ofzoites, the pellicle, may be present also in meronts (Kim andPaperna 1993b). A lomasome described in the present paperand by Perkins (1969) has not been reported for other apicom-plexans. It is possible, though, that the sponge-like vesicles

~

358 J. EUKARYOT. MICROBIaL., VOL. 48, NO.3, MAY-JUNE 2001

23

.4

Fig. 22-25. Vegetative cell cycle of cultured Perkinsus marinus: stationary-phase trophonts unfed for six months (Fig. 11).22. Trophonts withempty vacuoles (arrowheads); C, cell walls. Bar = 2 [Lm. TEM. 23. Trophonts had virus-like nuclear particles (arrowheads). N, nucleus; V,vacuole. Bar = 1 [Lm. TEM. 24. Dividing cell. This late telophase trophont had two nuclei (N) still connected to each other and condensedchromatin under nuclear membranes. Arrowheads point to chromatin. Bar = 500 nm. TEM. 25. A higher magnification of Fig. 24. Bar = 200nm. TEM.

(Fig. 16, 18) and the tubular network described in this studycould represent rhoptries and micronemes (Scholtyseck andMehlhorn 1970). Micropores were present and were describedby Perkins (1969), who called them cell wall vesicles. Thestructure of the cells described in this report, among which no

condensed chromosomes were observed, holds no resemblanceto dinoflagellates, whose nuclear organisation is characterizedby persistently condensed chromatin pattern.

The ultrastructure of mature P. marinus zoospores is not de-scribed in this paper because it has been previously described

.

SUNILA ET AL.-PERK/NSUSMAR/NUS INVITRO ULTRASTRUCTURE 359

29Fig. 26-29. Vegetative cell cycle of cultured Perkinsus marinus: cell divisions. 26. Some trophonts divide by binary fission. The mother cell

was surrounded by a thick cell wall (C), and the daughter cell just with a plasma membrane. N, nucleus. Bar = 2 [Lm. TEM. 27. A highermagnification of Fig. 26. C, cell wall. Bar = 500 nm. 28. Some of the cells divide by schizogony. A four-cell schizont. L, lipid droplet; N, nuclei;V, vacuole; VP, vacuoplast. Bar = 1 [Lm.TEM. 29. A multi-cell schizont. Arrowheads point to nuclei, Bar = 2 [Lm.TEM. :..

360 J. EUKARYOT. MICROBIOL., VOL. 48, NO.3, MAY-JUNE 2001

in detail by Perkins (1976) and zoospores are not a part of thein vitro cell cycle. Ultrastructures of P. marinus zoosporangiahave not been previously described in detail. Pioneering worksby Perkins and Menzel (1967) originated from the early daysof electron microscopy, and technical aspects are inadequate.Kleinschuster et al. (1994) provided an electron micrograph ofa zoosporangium of another Perkins us sp. from the clam M.balthica in which the zoosporangium appeared to contain celldebris from degenerating and necrotic cells. Zoosporangia of P.qugwadi parasitizing Japanese scallops, P. yessoensis (Black-bourn, Bower, and Meyer 1998), have only slight resemblanceto zoosporangia of P. marinus of this article. Perkinsus qugwadizoosporangia did not have a discharge tube. They were 12 fLmin diam. whereas P. marinus zoosporangia were 85 fLm(Perkinsand Menzel 1967). Cross-section of a P. qugwadi zoosporan-gium displayed 16 prezoospores while there were 250 prezoos-pores in a cross-section of P. marinus zoo sporangium (Fig. 2).Azevedo (1989) described zoosporulation of P. atlanticus fromthe clam Ruditapes decussatus. His observations of prezoos-pores undergoing successive binary fissions to produce numer-ous zoospores are in accordance with the results of this study,where bipartitioning of prezoospores was observed (Fig. 4).

Our observations of peculiar hypha-like structures, whichgave rise to prezoospores, suggest an additional mechanism forzoospore production besides binary fission (Fig. 6). Such hy-pha-like structures appeared frequently in each of several zoo-sporangia studied, but are not described for other members ofthe phylum Apicomplexa. It is possible that the genu~ Perkinsusestablishes an evolutionary link between the Alveolata (apicom-plexans, ciliates, and dinoflagellates) and the fungi. Ray (1952)felt that infrequent in vitro observations of budding cells andshort, thick hyphae established D. marinum (P. marinus) as afungus. Moreover, recent work shows that P. marinus is toler-ant of several antibacterial agents at high concentrations, but invitro proliferation is inhibited by antimycotics, such as ampho-tericin B and cycloheximide, far below concentrations recom-mended for control of fungal contamination in tissue cultures(Dungan and Hamilton 1995). However, molecular systematicsconsistently failed to support a close relationship between Per-kinsus spp. and fungi (Goggin and Barker 1993; Ragan et al.1996).

Virus-like particles were observed in the nuclei of severalcells. They occurred exclusively in nuclei and did not resemblethe cytoplasmic, dense, membrane-bound virus-like particles(125 nm in diam.) described in P. atlanticus (Azevedo 1990).Virus-like particles reported in this paper had morphologicalsimilarity to virus-like particles described by Perkins (1969) invivo. He described particles with five-fold or six-fold symmetrywith a limiting dense line and a light cortex and medulla sep-arated by a dense line (49 nm). The diameter of the particleshe described is close to the size of particles described in thisreport (55 nm); the difference might reflect different fixationmethods. The origin and function of these particles remain un-known.

Schizont daughter cells (Fig. 18, 29) were not necessarilyalike. Morphological differences between the cells might reflectdifferent functions. Cells with sponge-like aspects might pro-liferate into zoosporangia while the rest remain as hypnospores.Some daughter cells in a schizont were eliminated by cell deathvia different pathways: classical necrosis (Fig. 18) and by ap-optosis. Occurrence of the latter was interesting because itwould require communication between daughter cells. Death ofthese cells resembled formation of degenerating polar bodiesduring ovogenesis and might provide a selection mechanism toeliminate unwanted genetic material. Death of some of the

daughter cells in schizonts explains odd numbers of cells withinthe schizont wall, which puzzled Perkins (1996).

Binary fission by P. marinus has not been observed in vivo(Perkins 1996). Rare cells (1 %) undergoing binary fissions werefound among large, log-phase trophonts (Fig. ID); schizogonywas the more common mode of cell division. It is possible thatbinary fission of the parasite does occur in infected oyster tis-sues, but is not reported because of the rarity of getting a his-tological section through the mother-daughter cell axis. Apical,polar structures of large trophonts (Fig. 15) might result frombinary fission. They looked like remnants of cell wall surround-ing the mother trophont in Fig. 26.

Several stages of endomitosis were observed. Molon-Noblotand Desportes (1980) described nuclear division of another ap-icomplexan (Grebnickiella gracilis) in detail, including dupli-cation of centrocones. These are peculiar structures consistingof an extranuclear cone-shaped microtubular spindle adjacentto the nuclear envelope with centrioles at the top. They de-scribed thickening of chromatin on the nuclear membrane underthe centrocones, condensation of chromosomes, and depositionof fragments of the nuclear membrane on the surface of thechromosomes. They reported nine singlet tubules in the centri-oles. In our material, centrioles had classic 9 X 3 + 2 structure,which was also reported by Perkins (1969) in vivo. We sawthickening of chromatin under the nuclear membrane, but con-densation of chromosomes and formation of metaphase plateswas never observed. This is unusual, because more than 12,000log-phase cells were studied. Perkins (1969), based on his invivo observations, speculated that his persistent failure to ob-serve mitotic figures in P. marinus cells was due to rapid nu-clear division. However, it is possible that P. marinus endo-mitosis represents a type where simultaneous condensation isabsent and endoreduplication and condensation cycles occurnon-synchronously between chromosomes (for description ofnon-synchronous condensation pattern during endomitosis seeTherman, Sarto, and Kuhn 1986). Therman, Sarto, and Stub-blefield (1983) noted that endomitosis shows considerable var-iability among different organisms. In paraffin sections of P.marinus-infected oysters, there is no visible endomitosis, al-though parasite endomitosis is readily observed in sections ofoysters infected with another protozoan parasite, Haplospori-dium nelsoni (Kernstab-stage, Farley 1967).

The vacuoplast, intracytoplasmic tubular network with pre-cursor material, micropores, and lomasomes must be consideredas a single functional complex. The tubular network connectsthe vacuoplast with the cytoplasm (Fig. 20). The size of thevacuoplast was determined by the nutritional state of the cell.Vacuoplasts decreased in cells starved for 5 d and disappearedfrom cells starved for 6 mo. Simultaneously, the amount ofprecursor material decreased in the tubular network. The tubularnetwork leads to lomasomes, which open into the space be-tween the plasma membrane and the cell wall and may absorbnutrients from the culture medium or host. Micropores oftenhad apparent vacuoplast precursor material inside and seemedto be continuous with the tubular network. The function of thevacuoplast for food storage is consistent with a similar func-tional interpretation by Perkins (1996).

ACKNOWLEDGMENTS

Electron microscopy was carried out at the Center of Mi-croscopy and Image Analysis at George Washington Univer-sity, Washington, D.c. We warmly thank Ms. Kim Insley fromthe Maryland Department of Natural Resources for line graph-ics (Fig. 1). This work was supported, in part, by award #NA86RGO037 from the Sea Grant College Oyster Disease Re-search Project. The views expressed herein are those of the

:.

SUNILA ET AL.-PERKINSUSMARINUS IN VITRO ULTRASTRUCTURE 361

authors, and do not necessarily reflect those of NOAA or itssubagencies.

LITERATURE CITED

Andrews, J. D. 1954. Notes on fungus parasites of bivalve mollusks inChesapeake Bay. FroG. Natl. Shellfish. Assoc., 45:157-163.

Azevedo, C. 1989. Fine structure of Perkinsus atlanticus n. sp. (Api-complexa, Perkinsea) parasite of the clam Ruditapes decussatus fromPortugal. J. Parasitol., 75:627-635.

Azevedo, C. 1990. Virus-like particles in Perkinsus atlanticus (Apicom-plexa, Perkinsidae). Dis. Aquat. Org., 9:63-65.

Blackbourn, J., Bower, S. M. & Meyer, G. R. 1998. Perkinsus qugwadisp. novo (incertae sedis), a pathogenic protozoan parasite of Japanesescallops, Patinopecten yessoensis, cultured in British Columbia, Can-ada. Can. J. Zool., 76:942-953.

Cavalier-Smith, T. 1998. A revised six-kingdom system of life. Bioi.Rev., 73:203-266.

Dungan, C. F. & Hamilton, R M. 1995. Use of a tetrazolium-based cellproliferation assay to measure effects of in vitro conditions on Per-kinsus marinus (Apicomplexa) proliferation. J. Eukaryot. Microbiol.,42:379-388.

Dungan, C. F. & Roberson, B. S. 1993. Binding specificities of mono-and polyclonal antibodies to the protozoan oyster pathogen Perkinsusmarinus. Dis. Aquat. Org., 15:9-22.

Farley, C. A. 1967. A proposed life cycle of Minchninia nelsoni (Hap-losporida, Haplosporidiidae) in the American oyster Crassostrea vir-ginica. J. Protozool., 14:616-625.

Ford, S. E. & Tripp, M. R. 1996. Diseases and defence mechanisms.In: Kennedy, V. S., Newell, R I. E. & Eble, A. E, (ed.), The EasternOyster, Crassostrea virginica. Maryland Sea Grant College, CollegePark, MD. 581-660.

Friedman, C. S., Gardner, G. R., Hedrick, R P., Stephenson, M., Caw-thorn, R J. & Upton, S. J. 1995. Pseudoclossia haliotis sp. n. (Api-complexa) from the kidney of California abalone, Haliotis spp. (Mol-lusca). J. Invert. Pathol. 66:33-38.

Gajadhar, A. A., Marquardt, W. C., Hall, R, Gunderson, J., Ariztia-Carmona, E. V. & Sogin, M. L 1991. Ribosomal RNA sequences ofSarcocystis muris, Theileria annulata, and Crypthecodinium cohn;;reveal evolutionary relationships among apicomplexans, dinoflagel-lates, and ciliates. Mol. Biochem. Parasit., 45:147-154.

Gauthier, J. D. & Vasta, G. R. 1995. In vitro culture of the easternoyster parasite Perkinsus marinus: optimization of the methodology.J. Invert. Patho/., 66:156-168.

Goggin, C. L & Barker, S. C. 1993. Phylogenetic position of the genusPerkinsus (Protista, Apicomplexa) based on small subunit ribosomalRNA. Mol. Biochem. Parasit., 60:65-70.

Kim, S. H. & Paperna, I. 1993a. Endogenous development of Goussiatrichogasteri (Apicomplexa: Eimeriidae) in the intestine of gouramiTrichogaster trichoterus. Dis. Aquatic. Org., 17:175-180.

Kim, S. H. & Paperna, I. 1993b. Development and fine structure ofintracytoplasmic and epicytoplasmic meronts, merozoites, and youngmacrogamonts of the cichlid fish cladder coccidium Goussia cichli-darum. Dis. Aquat. Org., 15:51-61.

Kleinschuster, S. J., Perkins, F. 0., Dykstra, M. J. & Swink, S. L 1994.The in vitro life cycle of a Perkinsus species (Apicomplexa, Perkin-sidae) isolated from Macoma balthica (Linneaus, 1758). J. ShellfishRes., 13:461-465.

La Peyre, J. E 1996. Propagation and in vitro studies of Perkinsusmarinus. J. Shellfish Res., 15:89-101.

La Peyre, J. E, Faisal, M. & Burreson, E. M. 1993. In vitro propagation

of the protozoan Perkinsus marinus, a pathogen of the eastern oyster,Crassostrea virginica. J. Eukaryot. Microbio/., 40:304-310.

Levine, N. D. 1978. Perkinsus gen.n. and other new taxa in the proto-zoan phylum Apicomplexa. J. Parasito/., 64:549.

Mackin, J. G. & Ray, S. M. 1966. The taxonomic relationship of Der-mocystidium marinum, Mackin, Owen and Collier. J. Invert. Patho!.,8:544-545.

Mackin, J. G., Owen, H. M. & Collier, A. 1950. Preliminary note onthe occurrence of a new protistan parasite Dermocystidium marinumn. sp. in Crassostrea virginica (Gmelin). Science, 111:328-329.

Molon-Noblot, S. & Desportes, I. 1980. Etude ultrastructurale des mi-toses gamogoniques de la Gregarine Grebnickiella gracilis Gr. Par-asite de la Scolopendre Scolopendra cignulata L Considerations surles mitoses schizogoniques des sporozoaires (Apicomplexa). Protis-tologica, 16:395-411.

Noren, F., Moestrup, 0. & Rehnstam-Holm, A. 1999. Parvilucifera in-fectans Noren et Moestrup gen. et sp. novo (Perkinsozoa phylumnov.): a parasitic flagellate capable of killing toxic microalgae. Europ.J. Protisto/., 35:233-254.

Perkins, E O. 1969. Ultrastructure of vegetative stages in Labyrintho-myxa marina (= Dermocystidium marinum), a commercially signif-icant oyster pathogen. J. Invert. Patho/., 13:199-222.

Perkins, E O. 1976. Zoospores of the oyster pathogen, Dermocystidiummarinum. 1. Fine structure of the conoid and other sporozoan-likeorganelles. J. Parasito/., 62:959-974.

Perkins, F. O. 1988. Structure of protistan parasites found in bivalvemolluscs. Am. Fish. Soc. Spec. Publ., 18:93-111.

Perkins, F. O. 1996. The structure of Perkinsus marinus (Mackin, Owenand Collier, 1950) Levine, 1978 with comments on taxonomy andphylogeny of Perkinsus spp. J. Shellfish Res., 15:67-87.

Perkins, F. O. & Menzel, R. W. 1966. Morphological and cultural stud-ies of a motile stage in the life cycle of Dermocystidium marinum.FroG. Natl. Shellfish. Assoc., 56:23-30.

Perkins, E O. & Menzel, R. W. 1967. Ultrastructure of sporulation inthe oyster pathogen Dermocystidium marinum. J. Invertebr. Pathol.,9:205-229.

Ragan, M. A., Goggin, C. L, Cawthorn, R. J., Cerenius, L, Jamieson,A. V. c., Plourdes, S. M., Rand, T. G., Soderhiill, K. & Gutell, RR 1996. A novel glade of protistan parasites near the animal-fungaldivergence. FroG. Natl. Acad. Sci. USA, 93:11907-11912.

Ray, S. M. 1952. A culture technique for the diagnosis of infectionswith Dermocystidium marinum (Mackin, Owen, and Collier) in oys-ters. Science, 116:360-361.

Ray, S. M. 1963. A review of the culture method for detecting Der-mocystidium marinum, with suggested modifications and precautions.FroG. Nat/. Shellfish. Assoc. 54:55-69.

Reece, K. S., Siddall, M. E., Burreson, E. M. & Graves, J. E. 1997.Phylogenetic analysis of Perkinsus based on actin gene sequences. J.Parasitol., 83:417-423.

Scholtyseck, E. & Mehlhorn, H. 1970. Ultrastructural study of charac-teristic organelles (paired organelles, micronemes, micropores) ofsporozoa and related organisms. J. Parasitenk., 34:97-127.

Siddall, M. E., Reece, K. S., Graves, J. E. & Burreson, E. M. 1997.'Total evidence' refutes the inclusion of Perkinsus species in thephylum Apicomplexa. Parasitology, 115:165-176.

Therman, E, Sarto, G. E. & Kuhn, E. M. 1986. The course of endo-mitosis in human cells. Cancer Genet. Cytogenetics, 19:301-310.

Therman, E., Sarto, G. E. & Stubblefield, P. A. 1983. Endomitosis: areappraisal. Hum. Genet., 63:13-18.

Vivier, E. & Desportes, I. 1990. Phylum Apicomplexa. In: Margulis,L, Corliss, J. 0., Melkonian, M. & Chapman, D. J. (ed.), Handbookof Protoctista. Jones & Barlett, Boston. 549-573.

Received 07/15/99, 09/22/00, 02/12/01; accepted 02/14/01