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1988

Structure of Protistan Parasites Found in Bivalve Molluscs

Frank O. Perkins

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Part of the Marine Biology Commons, and the Parasitology Commons American Fisheries Society Special Publication 18:93- 111 , 1988 CC> Copyrighl by !he American Fisheries Sociely 1988

PARASITE MORPHOLOGY, STRATEGY, AND EVOLUTION

Structure of Protistan Parasites Found in Bivalve Molluscs 1

FRANK 0. PERKINS Virginia In stitute of Marine Science. School of Marine Science, College of William and Mary Gloucester Point, Virginia 23062, USA

Abstral'I.-The literature on the structure of protists parasitizing bivalve molluscs is reviewed, and previously unpubli shed observations of species of class Perkinsea, Haplosporidia, and class Paramyxea are presented. Descriptions are given of the flagellar apparatus of Perkin.His marinus zoospores, the ultrastructure of sp. from the Baltic macoma Maconw balthica, and the development of haplosporosome-like bodies in Haplosporidium nelsoni. The possible origin of stem cells of Marreilia sydneyi from the inner two sporoplasms is discussed. New research efforts are suggested which could help elucidate the phylogenetic interrelationships and taxonomic positions of the various taxa and help in efforts to better understand life cycles of selected species.

Studies of the structure of protistan parasites terization of the parasite species, to elucidation of found in bivalve moll uscs have been fruitful to the many parasite life cycles, and to knowledge of morphologist interested in comparative morphol- parasite metabolism. The latter, especially, is ogy, evolu tion, and . A diversity of important for determining the effects of prophy- species and higher taxa contain a great range of laxis and therapy. structures and developmental stages of consider- In most cases, the parasitic state, its pathogenic- able complexity. The most intensive studies have ity, or both cannot be directly transmitted experi- centered on pathogens of commercially important mentally. In cases of successful direct transmission, hosts, and the results have contributed to the for example, of or (perhaps) management of these valuable hosts, primarily , host specificity of the parasite is , by identifying the agent causing mortal- often not known because morphologically distin- ity. Thus, the investigator can identify the caus- guishing structures are lacking and transmission ative agent and conduct epizootiological studies experiments with a single population of parasites in to determine (I) geographical range, (2) frequency several host species have not been attempted. Be- and periodicity of disease expression, and (3) cause infections of bivalves with most parasites controlling physical factors such as salinity and cannot be transmitted, investigations should be con- temperature. Then managers and users of the ducted on host defense mechanisms, identification hosts can be advised how to avoid the disease and of intermediate hosts, and whether the organisms how to minimize the impact of the disease. Unless are truly parasitic or merely opportunistic. protistan pathogens can be di stinguished from one The availability of transmission electron micro- another, the ecological relationships of parasite to scopes beginning in the early 1960s was a major host may be misi nterpreted. advance for protistologists. Most cell stages are Morphologists have not always been successful 2-10 µ.m in size, and distinguishing structures in their attempts at species characterization, be- were often impossible to find by light microscopy. cause the protists within genera have relatively Electron microscopes have provided the ability to few di stinguishing structures. The problem is distinguish major taxa, for example, coccoid uni- compounded by the inability to establish pure cells of the thraustochytriacean protists from the cultures of all but a very few parasitic protista or species of class Perkinsea. of host cell s that could serve as the culture In this presentation, I shall provide an overview medium. The lack of cultures is the greatest of the major groups of protistan parasites with barrier to more complete morphological charac- emphasis on their structure, phylogeny, and geo- graphical distribution. Literature citations wi ll 'Contribution 14 14 VIMS/SMS of the College of direct readers to published descriptions and mi- Wil liam and Mary. crographs of morphological structures. 93 94 PERKINS

Class Perkinsea anterior end and tapers to a pointed posterior end (Plate I, Figure I) . The anterior emerges Perkinsus marinus from a groove located about one-third of the way Levine ( 1978) establi shed the class Perkinsea for from the anterior end toward the posterior end. The the phylum to accommodate the ob- posterior flagellum emerges from a cul-de-sac in the servations of Perkins (1976c), who found that the cell surt'ace at right angles to the anterior flagellum. fl agell ated cells of Dermocystidium marinum, the The kinetosome and base of the flagellum are name then given to a pathogen of the eastern complex. There are no cartwheels in the kineto- virginica, have organelles very similar some lumen; however, a large electron-dense to those of other species in the Apicomplexa. The inclusion body is found in the lumen with cross phylum essentially includes protistan species for- bridging to the A and B microtubules of the triplet merly classified in the Sporozoa. Perkins (1974) blades (Plate I, Figure 2 [d]) . Each triplet blade is noted that flagellated cells are not formed in Der- linked to the adjacent blade by a si ngle bridge. mocystidium sp. from salmon, nor have other The proximal end of the kinetosome is blocked by cyst-forming species of Dermocystidium in fish an electron-dense plate; however, there is no and amphibia been observed to form flagellated basal plate in the interfacial zone between the cell s. Therefore, Levine (1978) created a new ge- kinetosome and flagellum (Plate I, Figure 2). An nus for the pathogen of the and electron-dense, hollow cylinder is present at the renamed the species Perkinsus marinus. base of the flagellum; it is closed at the distal end but This systematic revision has stimulated debate on the validity of the Apicomple xa and Perkinsea may be partially open or closed at the proximal end. by several workers, notably Vivier ( 1982) and A secondary, thin-walled cylinder is found outside myself as related by Canning (1986). Until more is the primary one (Plate I, Figures 2, 2 [b]). known about Perkinsea and other Protista with The central doublet of flagellar microtubules ter- apicomplexan characteristics, the validity of the minates on a plate which is free in the flagell ar class will remain unsettled. However, it appears lumen and located close to the cylinders mentioned that the eastern oyster pathogen is not properly above. At right angl es to the plate and extending placed in the Dermocystidium. distally is a structure that is either a stack of rings or The structures found in the flagellated cells a helically wound filament (Plate I, Figures 2, 2 [aj). (zoospores) of Perkins 11 .1· spp., which are similar Two or more structures, perhaps microtubules, to those found in the Apicomplexa, particularly coated with electron-dense material, extend at the Coccidi a , are the apical complex, micropores, right angles to the long axis of the cell body (Plate rhoptries, micronemes, and subpellicular complex I, Figure 2). They originate from a wedge-shaped, described in detail in Perkinsus marinus by Per- electron-dense structure located at the base of the kins (1976c). They have also been observed in two kinetosomes and between them. If this fila- Perkin.I'll.I' sp. found in Macoma balthica (my mentous extension were shown to be homologous unpublished data) and in Tridacna sp. (R. J. G. to the presumptive vestigal flagella of the micro- Lester, University of Queensland , unpublished gametes of Toxop/asma gondii and magna data). Unlike those of other known Protista, the (Pelster and Piekarski 1971; Speer and Danforth anterior flagellum of Perkin.rns spp. has a row of 1976) , the evidence for including P . marinus in the long, filamentous mastigonemes and a row of Apicomplexa would be strengthened and questions short, spurlike structures along the full length of would be raised as to whether or not the zoospores the flagellum. The repeating units are one spur of P. marinus are isogametes. and five clustered mastigonemes grouped together The complement of the kinetosomal-flagellar (Perkins 1987) . The posterior flagellum lacks structures in Perkinsus spp. has also been ob- mastigonemes. served in the Perkinsus sp. found in Macoma The cell body (2-3 x 4- 6 µm) is rounded at the balthica (Perkins 1968; unpublished data). Fur-

PLATE 1.- Flage ll ar apparatus of Perkin.1·11 .1· marinus zoospores. FIGURE 1.-Phase-contrast micrograph of .... Perkin.1·11 .1· 111ari1111.1· zoospore. A = anterior flagellum; P = posterior flagellum. Bar = 3 µm. FIGURE 2.- Kinetosome and base of anterior flagellum of P. marinus. Bar = 0.5 µm. Lines a-d indicate the sites of cross sections of flagella illustrated in in serts a-d; in sert bar = 0.1 µm. E = electron-dense core in lumen of kinetosome; C = hollow cylinder in flagellar base; P = plate at base of central pair of flagell ar axonemes; Fi = heli cal filament(?); F = bundle of filaments or microtubules. STRUCTURE OF PROTISTAN PARASITES OF BIVALVES 95

® 96 PE RKINS thermore, the electron-dense cylinder in the prox- rations of eastern oyster tissue observed in bright imal end of the kinetosome lumen has been ob- fie ld microscopy because it has characteristic served in the thraustochytriaceous-labyrinthulid Brownian movement and is refringent. It has protists (Perkins 1974). The phylogenetic affinity cytochemical characteristics similar to volutin as between Perkin.rns spp. and the latter group and found in yeast cells. The nucleus has a centrally the importance of the other kinetosomal-flagellar located endosome and is located near the cell structures remain to be determined. wall, giving the characteristic signet configuration Mastigonemes that are fil amentous rather than which led Mackin et al. (1950) to name the patho- tubular are found scattered throughout the protistan gen Dermocystidium marinum. phyla (Moestrup 1982) ; therefore, they appear to The life cycle withi n the eastern oyster consists have no phylogenetic importance, at least at the of successive bipartitioning of the protoplast of phylum level. If they were shown to be important, the mature trophozoite (i.e. , repeating cycles of the alliance of the Perkinsea species with the Api- karyokinesis followed by cytokinesis) to yield 8- complexa would be placed in doubt. The only to 32-cell (rarely 64-cell) sporangia within a flagellated cell s in the Apicomplexa are micro- mother cell wall which then ruptures to yield gametes wh ich appear to lack mastigonemes; how- coccoid or cuneiform immature trophozoites ever, no high-resolution micrographs of negatively (Plate 2, Figures 4, 6-9; Plate 3, Figures 10, 11). stained or shadowed flagella have been published. The vacuoplast and large vacuole presumably Zoospores probably establish infections in the subdivide during the formation of multicellular gill s or mantle by penetrating the gill , labial palp, or sporangia, or sporangium formation occurs from mantle epithelium after losing the flagella and en- stem cell s lacking a large vacuole. Progressive cysting with in or between the cells. The details cleavage (i.e. , repeated karyokinesis followed by have not been determined. I have induced infec- synchronous cytokinesis) may also be involved. tions in uninfected eastern oysters using zoospores When the cell s are placed in fluid thioglycollate (my unpuqlished data) and have observed the medium prepared in seawater with antibiotics to earliest foci of infections in those ti ssues; however, retard bacterial growth, or on rarely observed the cellular detail s were not observed. Infections occasions in moribund eastern oyster tissue, any were detected using the Ray culture method in- stage may enlarge markedly to form 15- to 100- volving fluid thi oglycollate medium (Ray 1952). µm-diameter cells (Plate 3, Figures 12 , 13). Such The earli est cell type observed in eastern oysters cell s have an extremely large vacuole which com- (Perkins 1969 , 1976a) consists of a 2- to 4-µm presses the cytoplasm into a thin layer against the uninucleate coccoid trophozoite, which is pre- cell wall. No vacuoplast remains after the enlarge- sumed to be immature (Plate 2, Figures 3, 5). It is ment is completed. The nucleus assumes a sau- often found within the phagosome of a hemocyte sage-shaped configuration in the thin layer of (Pl ate 2, Figure 5). The trophozoite is delineated by cytoplasm. Numerous small lipoid droplets are a fibrogranular wall in and around which are found scattered throughout the cytoplasm. what appear to be phagosome membranes of the These large cells, called hypnospores after the host. The cytoplasm contains two centrioles with phycologists' term for resting cell s (Mackin 1962) , electron-dense cylinders as in the kinetosomes, are actuall y prezoosporangia and are labile in fluid lomasome, tubulovesicular mitochondria, smooth thioglycoll ate medium, where they survive only endoplasmi c reticulu m, and a few li poid droplets. about 10 days. Upon release into seawater, the The immature trophozoite enlarges to about prezoosporangia initiate zoosporulation. The lipoid 10-20 µm and, in the process, acquires an eccen- droplets dissolve, and a discharge pore (blocked by trically situated vacuole (Plate 2, Figure 6) , often a plug of secondary wall material) and a discharge with a prominent, refringent vacuoplast which tube are formed (Plate 4, Figure 14). The protoplast floats freely in the vacuole fluid. This inclusion is condenses within the cell wall , and the large vac- useful in locating the cell s in fresh squash prepa- uole subdivides and loses its identity. Presumably,

PLA T E 2.-Schi zogony in Perkin.1·1.1 ,1· marinus. F 1GURE 3.- Immature trophozoite. N = nucleus; M = 1111> mitochondrion. Bar = I µm . F1c;uRE 4.-Eight-cell divi sion stage. V = forming vacuoplast; Ce = centriole. Bar = I µm . F1GURE 5.- lmmature trophozoite (arrow) in oyster hemocyte. N = nucleus of hemocyte . Bar = 5 µm. F 1GURE 6.- Mature trophozoite. Arrow indicates large eccentric vacuole without a vacuoplast. N = nu cleus. Bar = 5 µm. F 1GURES 7, 8, 9.- Two-, fo ur-, and eight-cell stages, respectively, resulting from successiv e bipartitioning of th e protoplast. Arrows in dicate cleavage furrows. Bar = 5 µm. STRUCTURE OF PROTISTAN PARASITES OF BIVALVES 97

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0 98 PERKINS

much of the vacuolar fluid is forced into the space distributed without encapsul ation throughout al- between the cell wall and protoplast. Successive most all organs and in the hemolymph. bipartitioning fo ll ows and yields a mass of zoo- The cells of Perkinsus sp. may be coccoid, pyri- spores, which swim around within the mother cell form, or egg-shaped and tend to have larger lipoid wall. Then they escape through the discharge pore droplets in the cytoplasm than those of P . marinus and tube upon dissolution of the plug (Perkins and (Plate 4, Figures 16, 17). In addition, the mature Menzel 1966, 1967) (Plate 4, Figures 14, 15) . trophozoites arc larger (9-48 µm) than those of P. Zoospores have been shown to infect eastern marinus. Otherwise, their ultrastructure is the same oysters and eastern oyster organ explants (Per- as described for P. marinus. They resemble tropho- kins and Menzel 1966) ; however, their importance zoites of P. marinus seen in the first 24 h of in the li fe cycle is not clear. They have only been incubation in fluid thioglycoUate medium. observed experimentally, never in nature. The When Perkinsus sp. is isolated from the host presumed prezoosporangia are so rarely observed and placed in seawater, zoosporulation occurs in that they have not been isolated and induced to mature trophozoites as in P. marinus (Valiulis and zoosporulate, in contrast to Perkinsus sp. in Ma- Mackin 1969) and yields zoospores with the same coma halthic:a . The trophozoites are probably the surface st ructure (Perkins 1968). An identical re- normal agents of disease transmission , as sug- sponse occurs if the cells are induced to enlarge in gested by the ease with which infections can be fluid thioglycollate medium and then isolated in transmitted with infected eastern oyster tissue in seawater to induce zoosporulation. which there are no zoospores (Mackin 1962). I Cell s of P. marinus lose their mitochondria after have attempted to determine whether the zoo- 24 h in thioglycoll atc medium and re-form them spores are isogametes; however, there is no reason during zoosporulation. Mature trophozoites and to believe they are anything but vegetative stages prezoosporangia of Perkinsus sp. retain their mito- except for the presence of a possible third (vesti- chondria (Plate 3, Figure 12; Plate 4, 16) if they are gal) flagellum (see above and Plate I , Figure 2). not treated with the medium. It is not known if the The few quadriflagellated cell s observed appeared mitochondria disappear when Perkinsus sp. cells to result from incomplete cleavage. It is un li ke ly are placed in thioglycoll ate medium. that isogametic copulation had to occur before the flagell ated cell s infected eastern oysters under ex- Perkinsus sp. in Other Bivalve Molluscs perimental conditions. However, one wonders There are only two described species in the why so few zoospores successfully establish foci of genus Perkinsus, P. marinus and P. olseni (Lester infections. When I exposed eastern oysters or and Davis 1981); the latter is found in the blacklip eastern oyster ti ssue explants to millions of zoo- Haliotis ruher in South Australi a. How- spores (my unpublished data), only very light in- ever, after treatment in fluid thioglycollate medium fections resulted. At present, zoospores cannot be (FTM), cells presumed to be prezoosporangia of assumed to be a primary infective cell type in the other Perkin.ms species have been observed in 34 life cycle of the pathogen. species of bivalve molluscs by myself(unpublished data) and other workers (Andrews 1954; Ray 1954; Perkin.1·u.1· sp. in Macoma halthica Da Ros and Canzonier 1985). The hosts occur in The species of Perkinsus found in M. halthica temperate, subtropical, and tropical waters of the can cause mortality in the Baltic macoma, but it is Atl antic and Pacific oceans and Mediterranean not usually as virulent as P. marinus. The host Sea. It is unlikely that protists other than Perkinsus often pseudoencapsul atcs the parasite with con- spp. were observed because ( I) the cell structure of nective tissue fibers and cellular elements, result- prezoosporangia is easy to recognize and is unlike ing in small isolated groups of parasitic cell s in the that of any other known protist cells, (2) the cells connective ti ssue (Plate 4, Figure 18). This is in stain blue or blue-black in Lugol's iodine solution contrast to P. marinus, the cell s of which arc without acid hydrolysis, and (3) cells from selected

PLATE 3.-Un icellular development in Perkin.ms spp. FIGURES 10, 11.-Enlarging trophozoites of P. marinus (Figure IO ; bar = I µm) after rupture of sporangium (Figure 11; bar = 5 µm). V = forming vacuoplast material. Fl(,URES 12, 13.-Prezoosporangia, each cell with an enlarged vacuol e, in host tissue not treated with fluid thioglycoll ate medium . Figure 12 = Perkin.1·11 .1· sp. in Mac:oma ba/thica ; Figure 13 = P. marinus. N = nucleus; M = mitochondrion; L = lipoid droplet. Bar of Figure 12 = I µm; bar of Figure 13 = 5 µm. - STRUCTURE OF PROTISTAN PARASITES OF BIVALVES 99 100 PERKINS

PLATE 4.-Perkinsus sp. from Macoma balthica. FIGURE 14.-Zoosporangium with mature zoospores about to be released. T = discharge tube. Bar = 10 µm. FIGURE 15.-Scanning electron micrograph of zoosporangium discharge pore. Di scharge tube has been removed during specimen preparation. Z = zoospore. Bar = 0.2 µm. FIGURE 16.-Mature trophozoite. N = nucleus; L = lipoid droplet; Ve = vacuole. Bar = I µm. F1GURE 17.-Phase-contrast micrograph of mature trophozoite. Ve = large vacuole. Bar = 10 µm. F1GuRE 18.-Phase-contrast micrograph of emerging mature trophozoites. C = capsule wall. Bar = 10 µm.

FfM cultures that have been isolated and incu- believe that only one species is involved because bated in seawater yielded zoospores typical of P. of the considerable geographical and host isola- marinus (i.e. , bitlagellated with filamentous mastig- tion that characterizes the group. In addition, onemes on the anterior flagellum) as a result of numerous attempts to transmit infections from progressive cleavage. I have observed such zoo- host to host have failed, even when the hosts lived spore formation in Perkinsus spp. from Arca umbo- in the same estuary (Ray 1954). Perkinsus species nata, Pseudochama radians, and Tridacna maxima probably will have to be described and distin- (my unpublished data) in addition to M. balthica guished on the basis of fine structural differences . and C. virginica. Obviously , prezoosporangia from After examining prezoosporangia of Perkinsus each host species should be treated as in step (3) to spp. isolated from 14 different hosts, I believe that confirm the identity of the parasite. there are at least two basic groups. One group Significant structural differences have not been resembles P. marinus, and the other group resem- recognized among the Perkinsu.1· spp. parasitizing bles Perkinsus sp. as seen in M. ba/thica. The the 34 species of bivalve molluscs, probably due mature trophozoites of P. marinus are small er and to the lack of intensive research. It is difficult to have a smaller vacuole:cytoplasm ratio than those STRUCTURE OF PROTISTAN PARASITES OF BIVALVES 101 of Perkinsus sp.; they are coccoid rather than not clear whether V. Sprague (Chesapeake Biology pyriform or egg-shaped. Perkinsus marinus cell s Laboratory, unpublished observations; 1978) meant are scattered throughout the host tissues and are to include in Minchinia only those species with one not confined to pseudocapsules as in Perkinsus anteriorly and one posteriorly directed extension sp. These are weak di stinctions, and further study or those species with any extensions visible under is required; however, there appears to be a basis the light microscope. I suggest that the latter for dividing the species along those lines. It wi ll be characteristic be accepted. This point may be interesting to see whether the larger cells of the relevant in future generic designations of haplo- Perkinsus sp. group behave as prezoosporangia sporidian parasites of bivalve molluscs. Currently when released into seawater (known to occur in it is of importance only with respect to the Perkinsus sp. of Macoma balthica) and if cells parasite Haplosporidium /usitanicum which should from the P. marinus group do not, unless treated be placed in the genus Minchinia (Azevedo 1984). with fluid thioglycollate medium. The only other species of Minchinia parasitic in bivalve molluscs is M. armoricana, which is al- Phylum Haplosporidia ready properly named. Since their di scovery in the late 1800s, the Hap- The spores of Haplosporidium spp. and Min- losporidia have been a troublesome group for tax- chinia spp. are basically alike in structure. The onomists and phylogenists. Numerous taxonomic cell wall is cup-shaped and has a flange at the schemes have been proposed with little agreement anterior end on which rests a lid of wall material as to their taxonomic affinities. The Haplosporidia, continuous with the flange along a limited extent Paramyxea, and Myxozoa may be more closely of the flange , thus forming a hinge. The sporo- related to each other than to other Protista if one plasm inside the spore wall is uninucleate and has accepts the hypothesis that haplosporosomes (de- an unusual organelle, the spherule of unknown scribed below) are unique organelles found only in function at the anterior end. Membrane-bound, these taxa. Perkins (in press) reviewed the perti- electron-dense organelles, the haplosporosomes, nent literature and proposed the following classifi- with bipartite substructure are also found scat- cation scheme based on that of Corliss (1984). tered in the cytoplasm. Spore structure has been described in detail Phylum Haplosporidia (Perkins 1979 ; in press; Perkins and van Banning Class Haplosporea 1981 ). The chief structural feature for distinguish- Order Haplosporida ing species within a genus has been the size of the Family Haplosporidiidae spores. Because the size ranges of species overlap included 33 species and three genera (Hap /o- considerably, host, geographical range, and epi- sporidium, Minchinia and Urosporidium) within zootiological differences are additional characters the family. The first two genera are relevant to used to distinguish species. The structural differ- this presentation. Larsson (1987) described a ence in fibers (ornaments) found attached to and fourth genus (Claustrosporidiu m) from an amphi- around the spore wall is another distinguishing pod after I reviewed the literature. It is unique characteristic (Perkins, in press). When specimen because the sporoplasm is completely enclosed by preparation and resolution are adequate, period- a wall without an orifice. icities are often found which I believe will be of value in distinguishing species (Perkins and van Haplosporidium spp. and Minchinia spp. Banning 1981). All species not yet adequately Spores.-Six species of Haplosporidium and examined need to be observed in thin sections of Minchinia have been described from bivalve mol- well-fixed, mature spores as well as negatively lu scs (Azevedo 1984; La Haye et al. 1984). Nine stained whole mounts. Inadequate attention has other reports deal with parasites that may or may been given the ornamentation, and , generally , not be already described (Katkansky and Warner only micrographs of thin sections are available. 1970; Armstrong and Armstrong 1974; Mix and Prep/asmodial Stages .-The earliest stage of Sprague 1974; Kem 1976; Pichot et al. 1979; Vi- newly establi shed infections consists of a uni- or vares et al. 1982; Bachere and Grizel 1983; Bonami binucleated naked cell about 5 µm in diameter et al. 1985 ; Bachere et al. 1987). The two genera usually found in the gills or labial palps beneath should be distinguished on the basis of the pres- the epithelium or in the connective tissue near the ence and visibility of prominent extensions (tails) basal membrane of the upper gut or midgut epi- of the spore wall under the light microscope. It is thelium. Nuclear structure is the same as in plas- 102 PERKINS

modia. None of the life cycles of the Haplo- the cisterna encloses the forming core by fusing to sporidia have been elucidated; therefore, the pre- create a sphere (Plate 5, Figures 20-25). The cursor cell stage is unknown. Possibly, the early membranes of these bodies are difficult to resolve stages arise from excystment of spores entangled and thus appear different from those of the haplo- in the gi ll s or labial palps or from ingested spores. sporosomes, in which the membrane's tripartite However, direct transmission of infections by substructure is easily resolved. They do not appear means of spores has not been documented. to differentiate into haplosporosomes. Plasmodia .-Nuclear divisions of the preplas- Sporulation.-Spore formation occurs gener- modial stages, coupled with increase in cellular ally after the parasite has spread throughout the mass, result in formation of plasmodia (i .e., naked connective tissue of the host. The plasmodia cells with more than two nuclei). Nuclear division enlarge, acquire numerous nuclei (> 20), and form (Plate 5, Figure 19) involves a mitotic spindle with a thin, electron-dense wall about the thickness of spindle pole bodies free of the nuclear envelope the plasmalemma. The haplosporosomes disap- located in the nucleoplasm and a nuclear mem- pear, and the protoplast completely subdivides brane which is persistent throughout division into uninucleate sporoblasts within the wall . Mei- (Perkin s 1975b; Bonami et al. 1985). Cytokinesis osis may occur during this process because nu- apparently occurs by multiple fission to yield clear pairing, large nuclei , and presumptive syn- daughter cells with variable numbers of nuclei. aptonemal complexes and polycomplexes have However, this occurrence has been deduced after been observed (Perkins 1975a). However, the observations of cell clusters; no cytoplasmic divi- events are not well understood, and more detail in sions have been observed in progress. more species is required before generalizations The cytoplasm contains numerous electron- can be made. dense organelles with a bipartite substructure, How spores are then formed from the uninucle- termed haplosporosomes. They are generally ate sporoblasts is uncertain. I have suggested that spheroidal but may have other forms (Perkins invagination of the sporoblast plasmalemma or 1979). The substructure is unique enough to lead fusion of cytoplasmic vesicles occurs to delimit me to believe thai: they are useful phylogenetic the sporoplasm primordium within a cup-shaped indicators of interrelationships. Haplosporosomes unit of anucleate epispore cytoplasm (Perkins have been found in the Haplosporidia, Paramyxea 1975a). Desportes and Nashed (1983) suggested (see below), and, possibly, the Myxozoa (Current that the sporoblast elongates and the posterior, and Janovy 1977; Kent and Hedrick 1985). anucleate half envelops the anterior, nucleated Another inclusion of unknown function resem- half and becomes separate from it after envelop- bles haplosporosomes because it has a delimiting ment. The suggestion of Desportes and Nashed is membrane with a separate internal membrane. It most convincing; however, I have not yet seen the differs from haplosporosomes because it has a pertinent stages in the species of haplosporidians granular, electron-dense core free of the internal I have studied. membrane and separated from it by an electron- After the spore primordium is differentiated li ght zone (Plate 5, Figure 25). These haplo- into the two halves, first the spore wall and lid , sporosome-like bodies are formed from flattened then the spore wall ornaments, are synthesized in cisternae of the Golgi bodies which, in turn, are the anucleated epispore cytoplasm. Haplo- derived from the nuclear envelope (Plate 5, Figures sporosomes and the spherule are formed in the 20-24). During the synthesis, a cisterna arches sporoplasm early in the process of spore wall (Plate 5, Figure 20) and form s electron-dense ma- formation. Progressive thickening of the cell wall terial on the surface of one membrane. The future and degeneration of the epispore cytoplasm yield core of the body is synthesized free in the cyto- a spore which is capable of survival for at least a plasm. The arch becomes more pronounced until few weeks in seawater. I have observed that

PLATE 5.-0rganelles of Hap/osporidiu111 ne/soni in Crassostrea virginica. FIGURE 19.-Anaphase nucleus. .... Sp = spindle pole bodies; Mi = microtubules of spindle; G = golgi body ; H = haplosporosome ; HB = haplosporosome-like body. Bar = 0.5 µ.m. FIGURE 20.-Haplosporosome-li ke bodies (arrows) forming from cisternae of Golgi body. Bar= 0.1 µ.m. FIGURES 21-24.-Serial sections through two developing haplo- sporosomc-like bodies (arrows). In Figure 24 cisternae ari sing from nuclear envelope are visible near the top of th e mi crograph. The cisternae form the Golgi bodies and haplosporosome-like bodies. Bar = 0.2 µ.m . FIGURE 25.-Mature haplosporosome-like body. Co = granular core. Arrows indicate two membranes. Bar = 0.5 µ.m. STRUCTURE OF PROTJSTAN PARASITES OF BIVALVES 103 104 PERKINS

mature spores of H. costale, isolated from host myxea, , and Paramarteilia , in the class ti ssue, have maintained their structural integrity and six species. The only species thus far de- in seawater with antibiotics (0.1 mg/mL each of scribed from bivalve molluscs are all members of streptomycin sulphate and penicillin G) at 4°C for the genus Marteilia: M. refringens (Grizel et al. up to 1 month. Because the spores were not used 1974a) in edu/is, M. sydneyi (Perkins and to induce infections in organisms or tissue, I do Wolf 1976) in Saccostrea (= Crassostrea) com- not know whether the spores were viable. Death mercialis and possibly Crassostrea echinata can be detected in unfixed preparations because (Wolf 1979), M. maurini (Comps et al. 1982) in the sporoplasm pulls away from the wall and Mytilus ga/loprovincialis, and M. lengehi (Comps condenses. Observations of the fine structure 1977) in Crassostrea cucu/lata . An unidentified confirm that the protoplasm is dead, based on the species of Martei/ia has been found in the loss of structural integrity. Cardium edule (Comps et al. 1975). In attempts to elucidate the life cycles of hap- The life cycles of Marteilia spp. are not known; losporidians, it is important to know if spores can however, the development of spores within the exist for extended periods of time in sediments or hosts thus far identified has been described in detail in seawater. Dye-exclusion tests facilitate detec- for M. reji-ingens (Grizel et al. 1974a, 1974b; Perkins tion of spore death before the spore loses struc- 1976b) and M. sydneyi (Perkins and Wolf 1976). Cell tural integrity (Perkins et al. 1975) . Haplosporid- multiplication other than in spore formation has not ium nelsoni continues to infect imported oysters been noted in any of the five species. at a more or less steady level even where resident Desportes ( 1984) believed that the simplest cell populations of oysters decline to low levels. Thus, is a stem cell which is uni nucleate and amoeboid. there may be a reservoir of infective elements It then supposedly undergoes one nuclear division independent of the oysters. There are no data to fo llo wed by internal cleavage to yield a secondary indicate whether a similar state may exist with cell. It has been suggested that a is other species of haplosporidians. the earliest stage (Perkins 1976b; Perkins and Given the structure of spores, it is unlikely that Wolf 1976). I now question whether the simplest long residence outside the hosts is possible. The or stem cell is as represented by Desportes (1984) sporoplasm is not encased in a continuous wall and Perkins and Wolf(1976). Grizel et al. (1974a, (except perhaps in Claustrosporidium gammari). 1974b) may have been correct when they stated The cap rests on the spore cup flange and is that the earli est stage is a uninucleate cell contain- attached to it by ligaments or fu sed by secondary ing a uninucleate, secondary cell in a vacuole wall material , an arrangement which appears to within its cytoplasm (i.e., primary and secondary lack great strength because sli ght mechanical cell s). The evidence for a stem cell or plasmodium pressure induces the cap to swing open and pro- preceding the primary-secondary cell combina- vide an opening for the sporoplasm to exit from tion is not convincing. Due to its size, thin sec- the spore casing. Haplosporidia appear to be tions of the primary cell could easily not include obligate parasites without a saprozoic phase in the the secondary cell. The plasmodium presented by life cycle, and it is not likely that a resistant, Perkins and Wolf(l976, their Figure I) could have host-free state is present as is found in fungi. been a later stage in sporulation in which the internal cleavage membranes were not visible. Class Paramyxea Further study is required to identify the earliest The taxonomy of the class Paramyxea is in a stage in infections. state of flux. Des po rt es ( 1984) has considered its The primary cell with its secondary cell forms phylogenetic affinities and whether it should be a spores by a series of secondary cell divisions class or phylum. There are three genera, Para- followed by internal cleavages (Plate 6, Figures

PLATE 6.-Sporulation in Marleilia sydneyi. F1GuRt; 26.-Primary cell engaged in fo rmation of secondary

cell s (S). N 1 = primary ce ll nucleus; N2 = secondary cell nuclei; Hv = vermiform haplosporosomes. Bar = I µm. FrGURE 27.-Vermiform haplosporosomes of primary cell. Bar = 0. 1 µm. F IGUR E 28.-Mature spore. N"' = nucleus of middle sporoplasm; N 1 = nucleus of inner sporoplasm; H = haplosporosomes in cytoplasm of outer sporoplasm; Hv = vermiform haplosporosome of middle sporoplasm. Bar = I µm. FIGURE 29.-Vermiform haplosporosomes of Figure 28. Note two delimiting membranes and in completely synthe- sized electron dense matrix. Bar = 0.1 µm. FIGURE 30.-Haplosporosome of outer sporoplasm. Arrows indicate membranes. Bar = 0. I µm.

hr STRUCTURE OF PROTISTAN PARASITES OF BIVALVES 105

'.-r ."' . 0 ( r ./ ••

' >, 106 PERKINS

26, 28). The process has been described by Grizel similar to that of the Myxozoa, the differences et al. (1974a) , Perkins (1976b), and Perkins and being that the former lacks polar capsules and Wolf (1976). The essence of the process is the filaments as well as a spore membrane with valves. multiplication of the secondary cell to form 8-16 Previously, I believed that the presence of haplo- secondary, uni nucleate cells which then form sporosomes in Marteilia spp. warranted inclusion three to four spores within each secondary cell. of the species in the Haplosporidia; however, I Each spore consists of three uninucleate sporo- now recognize that the multicellular condition is a plasms (cells), one within the other and contained more important characteristic which warrants in- within a cell wall (Plate 6, Figure 28). Prior to clusion of the species in the Paramyxea. In addi- rupture and release of the tripartite spores, the full tion , it is now clear that haplosporosomes are cellular complement includes the uninucleate, pri- formed in the Paramyxea and Haplosporidia and mary cell with its internal complement of uninu- probably in the sporoplasms of Myxozoa (Current cleate secondary cells each enclosed by a wall and and Janovy 1977 ; Desser and Paterson 1978 ; Lorn each containing a complement of spores. Presum- et al. 1983), although I have not seen micrographs ably, upon release, the spores di sperse into the of high enough resolution to make a judgment. seawater and infect another host. The host tissue PKX cells, the causative agents of proliferative is in such a degraded state, and mortality among kidney disease (PKD), appear to be stages in the the hosts is so high at the time of sporulation, that life cycle of a myxozoan and, as expected, struc- one must assume that dispersion occurs when tures which appear to be haplosporosomes are crabs or other predators or scavengers eat the found in the primary cell and in the sporoplasm gaping bivalves. The next host is not known. (Seagrave et al. 1980; Kent and Hedrick 1985). Attempts have been made by myself(unpublished Because the function of haplosporosomes is data) and Grizel (1985) to infect edible oysters unknown, the organelles cannot be precisely de- Ostrea edulis by injecting and feeding spore sus- fined; however, I believe that their unique sub- pensions of M. re}i"ingens contained in minced, structure can serve as a phylogenetic marker. infective edible oyster ti ssue. As with the haplo- They are not found in very dissimilar groups of sporidians, transmissions of infections did not protists. An important step toward their definition occur. As suggested by Grizel (1985), an alternate will be to isolate them and characterize them host may be necessary in the life cycle or a period biochemically. It would also be helpful to find the of maturation may be required in the seawater or hosts that are infected by the spores and , by sediments before the spores are infective. means of the electron microscope, examine their If edible oysters are infected by the spores, it is involvement in the establishment of infections possible that the excystment from the spore wall when the spores penetrate the host epithelium or could result in formation of the earliest primary- are carried into the host by hemocytes. If host cell secondary cell stage. The outermost sporoplasm cultures could be used to culture the stem or could degenerate upon entry into the host tissues, primary-secondary cells, the role of haplo- leaving the middle and innermost sporoplasms, sporosomes could be established. I suspect that which then become the primary and secondary they could be involved in lysis of host cells in the cell complement. This suggestion has some cred- case of H. nelsoni, because I have observed them ibility when one notes that (I) the middle sporo- in oyster cells adjacent to plasmodia. plasms of M. refringens and M. sydneyi have Ginsburger-Vogel and Desportes (1979) sug- vermiform, haplosporosome-like organelles as gested that haplosporosomes of Paramarteilia or- does the primary cell (Plate 6, Figures 26-29), (2) chestiae from the amphipod Orchestia gammerel- the innermost sporoplasm has no haplosporo- lus participate in formation of a wall or loosely somes as is the case with the secondary cell , and consolidated layer of material around the spore by (3) the outermost sporoplasm is often degenerate emptying the contents onto the cell surface. The in mature spores (Plate 6, Figure 28). Possibly, the contents appear to include mucopolysaccharides spheroidal haplosporosomes (Plate 6, Figures 28 , as determined by cytochemistry. Azevedo and 30) of the outermost sporoplasm are involved in Corral ( 1985) determined that haplosporosomes of host invasion by inducing either phagocytosis by H. lusitanicum contain glycoproteins. Whether the host cell s or lysis of host cell s as a result of electron-dense inclusions that Ginsburger-Vogel chemical release from the organelles. and Desportes observed are haplosporosomes is Desportes ( 1981) observed that the multicellular questionable, since the typical substructure of hap- (cells within cell s) condition of the Paramyxea is losporosomes was not clearly demonstrated. Much - STRUCTURE OF PROTJSTAN PARASITES OF BIVALVES 107

) I ,(

·4. ' .. ' ..."~ .;:., .. ··". ·. :.r. t:.~----1 .... -.. ~.. ~---·· PLATE 7.-F1GURE 31.-Bonamia sp. from New Zealand oyster. H = haplosporosome; N = parasite nucleus;

N0 = oyster hemocyte nucleus; HB = haplosporosome-like body. Bar = I µm. smaller organelles that I would characterize as The taxonomic affinities of B. ostreae are not haplosporosomes (Ginsburger-Vogel and Des- known, but some workers relate it to the Haplo- portes 1979, their Figure 24) were present in the sporidia due to the presence of haplosporosomes cytoplasm. I have not found evidence of haplo- (Pichot et al. 1980) and multinucleate plasmodia sporosome participation in wall formation in Mar- (Brehelin et al. 1982) . I believe that the species is teilia spp. or in haplosporidians, nor have other probably one of the Haplosporidia. Because cell s workers noted such an involvement. within cell s are not observed, affinitie s with the Paramyxea or Myxozoa appear unlikely. B onamia ostreae The dominant cell type is a uninucleate, naked Bonamia ostreae causes serious mortalities of cell with haplosporosomes, lipoid droplets, tubulo- Ostrea edulis in France, England, The Nether- vesicular mitochondria, and a variable amount of lands, Spain, and Denmark. It appears to have smooth endoplasmic reti(: ulum (Figu re 31) . It is been transported in oysters from (USA) usually found in granular hemocytes where it mul- to , where it was first observed in 1979 in tiplies by binary fi ssion. The nuclear membrane oysters from the port of L'Ile-Tudy in Brittany remains intact during mitosis, whi ch involves an (Comps et al. 1980; El ston et al. 1986). In addi- intranuclear mitotic apparatus consisting of two tion , a species of Bonamia (Plate 7, Figure 3 I) has spindle pole bodies and mi crotubules .. Published been reported to be associated with heavy mor- micrographs of nuclear division (Pichot et al. 1980; tality of the New Zealand oyster Tiostrea lutaria Balouet et al. 1983) resemble those of H. nelsoni from Foveaux Strait along the south coast of New (Perkins 1975b). Cytokinesis may also occur by Zealand (M. Hine, Fisheries Research Center, multiple fis sion since multinucleate plasmodia personal communication). have been observed; however, there is a possibility I08 PERKINS

that the plasmodia observed represented the earli- range of 3-10 µm (except they are larger when est stage leading to sporulation. No spore forma- cultured). I have found that at least one of the tion has been observed, but spores are rarely many species is always associated with the sur- observed in H. nelsoni (Andrews 1982). The hap- face of the mantle and gill tissues of the oyster, losporosome-like inclusions (see phylum Haplo- Crassostrea virginica. Therefore, they have been sporidia, above) are also formed in the New confused with Perkinsus marinus (Mackin and Zealand Bonamia sp. (Figure 31 , HB) . Whether Ray 1966) after oyster tissue infected with P. they are formed by Golgi bodies or by the smooth marinus was placed in nutrient medium and pro- endoplasmic reticulum has not been determined. liferation of labyrinthomorphid protists occurred. In contrast to species of the Haplosporidia and Based on these observations, the authors sug- Paramyxea, the life cycle of B. ostreae can be gested that the name P. marinus be changed to completed experimentally. Direct transmission of Labyrinthomyxa marina. This study was followed infection can be induced by placing infective by others involving descriptions of labyrintho- oysters in aquaria with uninfected ones or by morphids derived from bivalve molluscs and sus- inj ecting infective oyster cell s into healthy oysters pected of being parasites (see Lauckner 1983 for (Pod er et al. 1982 ; Grizel 1985). Whether spores literature review). Because the protists are easily or another host are part of the life cycle in nature cultured in a diversity of nutrient media and remains to be determined. substrates (Perkins 1973) and are difficult to elim- inate from the ti ssue even when the vis- Other Parasites ceral mass tissues (as opposed to gill and mantle Sparks ( 1985) reviewed the literature concern- explants) are used, I believe that their designation ing parasites of noninsect invertebrates, and as parasites must be regarded with doubt, and the Lauckner ( 1983) reviewed the diseases of bivalve relationships should be reevaluated. However, molluscs. The two reviews cover the structure of species of presumptive Thraustochytrium and diverse bivalve mollusc protists including , Labyrinthu/oides have been observed to parasit- sarcodinids, coccidians, zooflagellates, gregarines, ize an , , nudibranch, and abalone microsporidians, and unidentified protists. How- (Polglase 1980; Jones and O'Dor 1983; Bower ever, because the literature lacks detailed struc- 1987; McLean and Porter 1987) . All hosts except tural analyses and contains little ultrastructural the nudibranch were being held in aquaculture information , it is not being considered here. The conditions. Thus, it appears reasonable to con- reader is referred to the two reviews for literature sider the protists as facultative parasites. Never- citations and summaries of what is known of theless, more studies clearly demonstrating species morphology. In addition, Papayanni and whether parasitism occurs in the bivalve molluscs Vivan!s (1987) described the fine structure of the are needed. The isolation and cultivation of laby- parasitic zooflagellate Hexamita nelsoni, found in rinthomorphids from sick individuals cannot be the Mediterranean oysters Ostrea edulis and regarded as prima facie evidence that they are Crassostrea gigas. They established the parasite parasites or agents of disease. Oyster tissue ex- in axenic culture. plants serve as excellent substrates for growth of One group of protists was considered by Lauck- the organisms (Perkins 1973); therefore, a sick ner ( 1983) but not Sparks (1985): the Labyrintho- oyster with lysis of tissues would be expected to morpha, a group of mostly saprophytic protists support proliferation of the ubiquitous protists. which are ubiquitous in marine and estuarine waters all over the world . Olive (1975) reviewed their Acknowledgments structure in detail. The cells form ectoplasmic net- The author thanks P. M. Hine for providing works from distinctive cell cortex organelles termed useful information and the micrograph concerning sagenogenetosomes. The networks are used in glid- Bonamia sp. and acknowledges Susan Arnold and ing motility and are believed to serve in the diges- Patrice Mason for their able technical assistance. tion of food substrates and assimilation of dissolved nutrients. The cell covering consists of circular References plates synthesized in the Golgi apparatus. Motile heterokont zoospores or isogametes are formed. Andrews, J. D. 1954. Notes on fungus parasites of bivalve molluscs in Chesapeake Bay. Proceedings The cell s of most of the species of Thraus- National Shellfisheries Association 45: 157-163. tochytrium, Labyrinthu/oides, Schizochytrium, Andrews, J. D. 1982. Epizootiology of late summer and and other genera are coccoid, mostly in the size fall infections of oysters by Hap/osporidium net- STRUCTURE OF PROTISTAN PARASITES OF BIVALVES 109

soni, and comparison to annual life cycle of Hap- Ostrea edulis L. Comptes Rendus Hebdomadaire losporidium costalis, a typical haplosporidan. Jour- des Seances de l'Academie des Sciences, Serie D, nal of Research 2: 15-23. Sciences Naturelles 290:383-384. Armstrong, D. A., and J. L. Armstrong. 1974. A hap- Corliss, J. 0. 1984. The Protista and its 45 losporidian infection in gaper , capax phyla. Biosystems 17:87-1 26. (Gould), from Yaguina Bay, Oregon. Proceed- Current, W. L., and J. Janovy. 1977. Sporogenesis in ings National Shellfi sheries Association 64:68-72. Henneguya exilis infecting the channel catfish: an Azevedo, C. 1984. Ultrastructure of the spore of Hap- ultrastructural study. Protistologica 13: 157-167. /osporidium lusitanicurn sp. n. (Haplosporida, Hap- Da Ros, L., and W. J. Canzonier. 1985. Perkinsus, a losporidiidae), parasite ofa marine mollusc. Journal protistan threat to bivalve culture in the Mediterra- of Parasitology 70:358-371. nean basin. Bulletin of the European Association of Azevedo, C., and L. Corral. 1985. Cytochemical analy- Fish Pathologists 5:23-27. sis of the haplosporosomes and ve~icle-like droplets Desportes, I. 1981. Etude ultrastructural de la sporula- of Haplosporidium /11 sitanicum (Haplosporida, tion de Paramyxa paradoxa chatton (Paramyxida) Haplosporidiidae), parasite of Helicon pellucidus parasite de l'annelide polychete Poeciloclwetus (Prosobranchi a). Journal of Invertebrate Pathology serpens. Protistologica 17: 365-386. 46:281-288. Desportes, I. 1984. The Paramyxea Levine 1979: an Bachere, E., D. Chagot, G. Tige, and H. Grizel. 1987 . original example of evolution towards multicellular- Study of a haplosporidian (Ascetospora) parasiti z- ity. Origi ns of Life 13:343-352. ing the Australian flat oyster . Aqua- Desportes, I., and N. N. Nashed. 1983. Ultrastructure culture 67 :266-268. of sporulation in Minchinia de111ali (Arvy), an hap- Bachere, E., and H. Grizel. 1983 . Mise en evidence losporean parasite of Denllllium e111ale (Sca- d' Haplosporidium sp. (Haplosporida-Haplospori- phopoda, ); taxonomic implications. Pro- diidae) parasite de l'huitre plate Ostrea edulis L. ti stologica 19:435-460. Revue des Travaux de l'Institut Peches Maritimes Desser, S. S., and W. B. Paterson. 1978. Ultrastruc- 46:226- 232. tural and cytochemical observations on sporogene- Balouet G., M . Poder, and A. Cahour. 1983. Haemo- sis of Myxobolus sp. (Myxosporida: Myxobolidae) cytic parasitosis: morphology and pathology of le- from the common shiner Notropis cornu1 11 s. Jour- sions in the French flat oyster, Ostrea edulis L. nal of Protozoology 25:314-325. Aquaculture 34: 1-14. Elston, R. A., C. A. Farl ey, and M. L. Kent. 1986. Bonami, J .-R. , C. P. Vivares, and M. Brehelin. 1985. Occurrence and significance of bonamiasis in Euro- Etude d'une nouvelle haplosporidie parasite de pean flat oysters Ostrea ed11/is in . l'huitre plate Ostrea edulis L.: morphologie et cytol- Diseases of Aquatic Organisms 2:49-54. ogie de differents stades. Protistologica 21: 161-173. Gin sburger-Vogel, T. , and I. Desportes. 1979. Etude Bower, S. M. 1987. The life cycle and ultrastructure of ultrastructurale de la sporulation de Paramarteilia a new species of thraustochytrid (Protozoa: Laby- orchestiae gen. n., sp. n., parasite de l'amphipode rinthomorpha) pathogenic to small abalone. Aqua- Orchestia gammarc/111 .1· (Pallas). Journal of Proto- culture 67:269-272. zoology 26:390-403. Brehelin, M., J.-R. Bonami, F. Cousserans, and C. P. Grizel, H. 1985. Etude des recentes epizooties de Vivares. 1982 . Existence de formes plasmodiales l'huitre plate Ostrea edulis Linne et de leur impact vraies chez Bonamia ostreae parasite de l'huitre sur l'ostreiculture Bretonne. These. Academic de plate Ostrea edulis. Comptes Rendus de l'Aca- Montpellier, Universite des Sciences et Techniques demie des Sciences, Seri e III , Sciences de la Vie du Languedoc, France. 295:45-48. Grizel , H., M. Comps, J. R. Bonami, F. Cousserans, Canning, E. U. 1986 . Terminology, taxonomy, and life J. L. Duthoit, and M.A. Le Pennec. 1974a. Re- cycles of Apicomplexa. Insect Science and its Ap- cherche sur !'agent de la malad ie de la glande plication 7:319-325. digestive de Ostrea edulis Linne. Science et Peche Comps, M. 1977. Marleilia lengehi n. sp., parasite de 240:7-30. l'huitre Crassostrea rnrnllata Born. Revue des Gri zel , H. , M. Comps, F. Cousserans, J. R. Bonami, Travaux de l'Institut Peches Maritimes 40:347-349. and C. Yago. 1974b. Etude d'un parasite de la Comps, M., H. Grizel, G. Tige, and J.-L. Duthoit. 1975. glande digestive observe au cours de l'epizootie Parasites nouveaux de la glande digestive des mol- actuelle de l'huitre plate. Comptes Rendus Hebdo- lusques marins Mytilus edulis L. et Cardium ed11/e madaires des Seances de I' Academie des Sciences, L. Comptes Rendus Hebdomadaires des Seances Serie D, Sciences Naturelles 279:783- 784. de l'Academie des Sciences, Serie D, Sciences Jones, G. M., and R. K. O'Dor. 1983. Ultrastructural Naturelles 281:179-181. observations on a thraustochytrid fungu s parasite in Comps, M., Y. Pichot, and P. Papagianni. 1982. Recher- the gi ll s of squid (I/lex illecebro.1·11s Lesueur). Jour- che sur Martei/ia maurini n. sp. parasite de la nal of Parasitology 69:903-911. moule My1i/11s gal/oprovincia/is Lmk. Revue des Katkansky , S. C .. and R. W. Warner. 1970. Sporulation Travaux de l' In stitut Peches Maritimes 45:211-214. of a haplosporidian in a (Cras,wsrrea Comps, M. , G. Tige, and H. Gri zel. 1980. Etude ultra- gigas) in Humboldt Bay, Californi a. Journal of the structurale d' un protiste parasite de l'huttre plate Fisheries Research Board of Canada 27: 1320- 1321 . 110 PERKINS

Kent, M. C., and R. P. Hedrick. 1985. PKX, the caus- Papayanni, P., and C. P. Vivares. 1987. Etude cy- ati ve agent of proliferative kidney disease (PKO) in tologique de Hexamita nelsoni Schlicht et Mackin, Pacific salmonid fishes and its affinities with the 1968 (Flagell ata, Diplomonadida) parasite des Myxozoa. Journal of Protozoology 32:254-260. huitres mediterraneennes. Aquaculture 67: 171-177. Kern, F. G . 1976. Sporulation of Min chinia sp. (Hap- Pelster, B., and G. Piekarski. 1971. Elektronenmik- losporida, Haplosporidiid ae) in the Pacific oyster roskopische Analyse der Mikrogametenentwick- Cras.rnstrea gigas (Thunberg) from the republic of lung von . Zeitschrift fiir Para- Korea. Journal of Protozoology 23 :498-500. si tenkunde 37:267-277. La Haye, C. A., N. D. Holland, and N. McLean. 1984. Perkins, F . 0. 1968. Fine structure of zoospores from Electron microscope study of Haplosporidium co- Labyrinthomyxa sp. parasitizing the Macoma matulae n. sp. (phylum Ascetospora: class Stel- balthica. Chesapeake Science 9: 198-208. latosporea), a haplosporidian endoparasite of an Perkins, F. 0. 1969. Ultrastructure of vegetative stages Australian crinoid, 0/igometra serripinna (phylum in labyrinthomyxa marina (= Dermocystidium Echinodermata). Protistologica 20: 507-515. marinum), a commercially significant oyster patho- Larsson, J. I. R. 1987. On Haplosporidium gammari, a gen. Journal of Invertebrate Pathology 13: 199-222. parasite of the amphipod Rivulogammarus pu/ex, Perkins, F. 0. 1973. Observations of thraustochytria- and its relationships with the phylum Acetospora. ceous (Phycomycetes) and labyrinthulid (Rhizo- Journal of Invertebrate Pat hology 49:159-169. podea) ectoplasmic nets on natural and artificial Lauckner, G . 1983. Diseases of Mollusca: Bi valvia. substrates-an electron microscope study. Cana- Pages 520-615 in 0 . Kinne, editor. Diseases of dian Journal of Botany 5 1:485 -491. marine , volume 2. 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