W&M ScholarWorks

VIMS Articles Virginia Institute of Marine Science

1976

Zoospores of the Oyster Pathogen, Dermocystidium marinum. I. Fine Structure of the Conoid and Other Sporozoan-Like Organelles

Frank O. Perkins Virginia Institute of Marine Science

Follow this and additional works at: https://scholarworks.wm.edu/vimsarticles

Part of the Marine Biology Commons

Recommended Citation Perkins, Frank O., Zoospores of the Oyster Pathogen, Dermocystidium marinum. I. Fine Structure of the Conoid and Other Sporozoan-Like Organelles (1976). Journal of Parsitology, 62(6), 959-974. https://scholarworks.wm.edu/vimsarticles/1991

This Article is brought to you for free and open access by the Virginia Institute of Marine Science at W&M ScholarWorks. It has been accepted for inclusion in VIMS Articles by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. THE JOURNAL OF PARASITOLOGY Vol. 62, No. 6, December 1976, p . 959-974

ZOOSPORES OF THE OYSTER PATHOGEN, DERMOCYSTIDIUM MARINUM. I. FINE STRUCTURE OF THE CONOID AND OTHER SPOROZOAN-LIKE ORGANELLES*

Frank O. Perkins Virginia Institute of Marine Science, Gloucester Point, Virginia 23062

ABSTRACT: An apical complex comparable to that found in the Sporozoa is described from zoospores of Dermocystidium marlnum Mackin, Owen, and Collier, a pathogen of the American oyster (Crassostrea oirginica Gmelin). The complex consists of a conoid, polar ring, up to 39 subplasmalem­ mal microtubules, rhoptries, and micronemes. Micropores and a subpellicular membrane equivalent were also found. Acid phosphatase activity was found in cistemae of the endoplasmic reticulum, inclusion bodies, and vesicles within the conoid lumen. No polysaccharides were detected in the rhoptries and micronemes using the Thiery method. Observations indicate that D. marinum is a protozoan in the sub­ and is most closely related to the coccidian Sporozoasida Leuckart.

Since the initial description by Gustafson et one functional system, possibly the latter al. (1954), nwnerous authors have noted that giving rise to the former (Vivier and Petitprez, coccidians, gregarincs, sp., 1972) . sp., and Frenkelia sp. all have, in one or more Attached to and radiating from the polar cell stages iJ1 the li£e cycle, a hollow, cone-like ring are generally 22 to 24 microtubules which structure at Lhe antedor end of the cell. This lie beneath the "subpellicular membrane" and organelle, termed a conoid, consists of tubular extend most of the distance toward the cell subunits, spirally arranged to form a trun­ posterior. In most of the organisms with cated hollow cone 0.08 µ.m to 0.4 µ.m in di­ conoids are also found 1 to 9 invaginations of ameter at the anterior end and 0.2 µ.m to 0.53 the plasmalemma, the micropores. These µ.m at the posterior end ( Scholtyseck, 1973). specialized stmctures often have a collar of Anterior to the conoid are 2 (possibly 3: electron-dense mate1ial around the distal part Porchet-Hennere, 1975) preconoidal rings con­ of the invaginatiou and another outer cylin­ sisting of elecb·on-dense granular material drical collar fonncd by infolding of the connected to the conoid by a canopy of elec­ "subpellicular membrane." The latter is dis­ b·on-dense material which has an opening at continuous at the proximal end of the invagi.na­ the extreme anterior end. A ring of electron­ tion. dense material, the polar ring, encircles the Collectively the conoid, polar ring, sub­ conoid. The ring is an anterior elaboration pellicular microtubles, micronemes, and rhop­ of the two closely opposed unit membranes tries have been termed the apical complex (''subpellicular membrane") which lie slightly (Levine, 1973). The consistency with which under the plasmalcmma. Two or more fl ask­ the complex has been observed in Protozoa that shaped, membrane-bound sacs of electron­ appear to be related for other reasons, has led dense material converge on the conoid, the to the consb.uction of the subphylum Apicom­ neck of each "flask" lying within the conoid plexa Levine 1970, which includes the piro­ Ju.men. These sbuctw·es, termed rhoptries or plasms, gregarines, and coccidians. The latter paired organelles, are believed to contain lytic group includes sp ecies of Toxoplasma, Sarco­ enzymes which aid the organism in host cell cystis, Besnoitia, , Frenkelia, Ei­ penetration. Scattered throughout the an­ rneria, and . terior ½ or 11.i of the cell are the micronemes In this paper it is shown that the oyster which are cord-like, membrane-bound sacs of pathogen, Dermocystidium marinum, also has electron-dense material. Some workers have the organelles described above and thus has suggested the rhopbi es and micronemes are affinities with the Apicomplexa. There are numerous suggestions in the literature as to Received for publication 26 January 1976. the affinities of the pathogen, ranging through • Contribution No. 786, Virginia Institute of Maline Science, Gloucester Point, Virginia 23062. such diverse groups as the Rhinosporidiaceae 959 960 THE JOURNAL OF PARASITOLOGY, VOL. 62, NO. 6, DECEMBER 1976 of the Endomycctales (Mackin, 1962), Sy11- pared on Formvar-coated grids by suspending a chytriaceae of the Chytricliales (Mackin and pellet of zoospores in distilled water, picking up the ruptured cells In a nichrome wirtl loop, placing BoswolJ, 1956), Labyrinthulia ( Mackin and the loop on the surface of distilled water, and Ray, 1966), Ascomycctes (Mackin, 1951), touching the 1,11·id to the water's surface witJ1in the lJaplosporida (Sprague, 1954), and Entomoph­ wire loop. Grids wore then stained on drops of 2% tJ1oralcs (F. K. Sparrow, Jr. in Ray, 1954). aqueous uranyl acetate for 1 to 5 min. The fow Mackin and Ray ( 1066) have renamed the cells which sprclld on the surface of the wuter had fewer cytoplasmic fragments attached to the conoid organism Labyrinthomyxa marina thus con­ and cytoskeleton than zoospores prepared by any of sidering it to be allied to the Labyrinthulia. the other methods tried, such as those of Porchet­ Their decision resulted from observations of Hennere (1975), Ryley (1969 ), and Angelopoulos gliding cells with labyrinlhulid "tracks," plas­ ( 1970). Dehyclrution of the ruptured zoosporcs by ethyl alcohol did not result in better visualizoUon of moclia, and vegetative division figures similar Lhe cytoskeleton and conoid nor did rupture and to those of Labyrinthomyxa sauvageatii (Du­ extraction on the surface of 10% ( v/ v) aqueous l,osct1 1921). Perkins ( 1974) did not find any triton X-100 or L2% ( w/ v) aqueous hcxylene gly­ laby1inthulid characteristics in the oyster patho­ col. Modified Gomori's cytochemfoal techniques gen, except an unusual kinetosome granule, and ( Kazama, 1973) wertl ustlr 0.1 M Millonig's phosphate buffer at en. 2'1 C. Four buffer rinses over n 60 min period preceded 4, 7). The microtubules are approximately in postfixation with 1% OsO, in oae of the above parallel array and arc obliquely oriented to buffers with 0.2 M NaCl. Postfixation was for 3 the longitudinal axis of the cone, forming angles hr al ca. 24 C. Before dehydration in a graded of inclination ranging from 50° to 88° (N = ethyl alc.-ohol series, cells were held in 2% aqueous 16) depending upon where the measurement unmyl acetate fur 60 111i11. Embeddings were in D11rc11pan ACM ( F lukn ) or Epon 812. Sections is made; the tilt being more pronounced dis­ were stained for 15 min in 2% uranyl acetate pre­ tally than proximally (Fig. 8). pared in 35% ethyl alcohol followed by 5 min in Three or four of the conoid microtubulcs Reynold's lead citrate. extent 0.26 to 0.66 /km 0.45; N 11) For three dimcns.ional studies, serial sections (.x = = were mounted on a Formvar substrate on Mason l,eyoud Lhe lruncaled cone (Figs. 2, 7, 8, 17). and Morton slot (2 1x 1 mm) discs. Reconstructions Attached to the longer microtubules is a single of zoospore structures were then made by tracing row of 16 to 21 (N 6 ) vesicles or mcmbrane­ cell and organelle profiles on clear acrylic plastic = sheets ( Flex-O-Glaze, Warp Brothers, Chicago, bow1d bulbous terminations (25 to 33 nm di­ Ill. ). Cell profllr,s wr,rn then cut out and glued ameter; .x = 30; N = 14 ) of microncmes, herein together in proper sequence to yield n model of the termed conoid-altached microncmes (CAM) . coll surface topography in which could be seen relative positions of orgnnelles. The CAM arc graded in length, those attached Assessments and presentations of mature zoo­ to tJ1e region of the microtubulcs most distal spore structure were based on observations of zoo­ to the conoid being the longest and those fur­ spores which were swimming free of the zoo­ sporangium at the time of fixation. Although thest into the conoid lumen being simple vesi­ micrographs are presented from intrasporangial cles (Figs. 7, 10). The former follow a wavy zoospores, all figures represent fully mature and course diagonally across the cell width, then differentiated structnrcs. Whole mounts of ruptured zoosporcs were pre- terminate in the posterior fourth of the cell, PERKINS-FINE STRUCTURE OF THE ZOOSPORES OF DERMOCYST/DIUM MARINUM 961

F1cUI1Es 1- 19. Eleclrou micrographs and drawings of Den11ocystldlum marinum zoospores. Figures 8, l 1, 12, and 18 are from negatively-stained fragments of ruptured zoospores. Abbreviations: C, conoid; CAM, conoid attached microuemes; CI, membrane-bound inclusions in conoid lumen; D, distal end of of conoid; EM, extended microtubules of conoid to which are attached micronemes; G, granular layer analogous to subpellicular membrane; K, kinetosome; L, labyrinthine complex; LD, lead depositions indicative of acid phosphatase; Lp, lipoid inclusion bodies; M, membranes of flattened vesicles homol­ ogous to subpellicular membrane; MC, microlubules of conoid; Ml, subplasmalemmal microtubules; P, polar mantle; PR, polar ring; Po, posterior ring; R, rhoptry; RM, rectilinear micronemes; T, termina­ tions of micronemes on microtubules of conoid; V, conoid-associated vesicle of endoplasmic reticulum; Va, vacuoplnst; Ve, large anterior vacuole. 1, 2. Longitudinal, medial sections of zoospores. Fig. 1 inset is higher magnification of conoid in Fig. 1. Fig. 1: X 50,000; Fig. 1 inset: X 90,000; Fig. 2: X 134,000. 3, 4. Cross sections of a single apical complex showing distal ( Fig. 3) and proximal ( Fig. 4) ends of conoid. Micrographs are from ca. 60 nm thick sections, numbers 1 and 8 of a series. Note that micro­ tubules of conoid collectively circumscribe a 295° angle at the distal end and a 180° angle at the proximal end. Both Figs.: X 74,000. 5-7. Diagrams of apical complex in longitudinal (Fig. 5) and anterior, frontal (Flg. 6 ) views. Figure 7 shows conoid subsln1cture with polar ring, attached sub­ plasmalemmal mierotubules, and polar mantle. Limits of the latter are indicated by coarse, widely spnced dots. 962 THE JOURNAL OF PARASITOLOGY, VOL. 62, NO. 6, DECEMBER 1976 PERKINS-FINE STRUCTURE OF THE ZOOSPORES OF DERMOCYSTIDIUM MARINUM 963

Cl PR M

~t G C p

Va EM

R

T

RM CAM CD

\...--~------GMt -...e~~~~-----Vc ~--'~ ----v E9!ii~--PR 11::------"'~--CI ...,_....,__~--MC .---~~~--RM

-..;;,.,,.....-~~-R

© 964 THE JOURNAL OF PARASITOLOGY, VOL. 62, NO. 6, DECEMBER 1976

PR

,...______c

traveling beneath the cell surface on the side fluid filled cun: as evidenced by entry of nega­ of the cell opposite the flagellar attachment tive stain into the lumen. The negatively­ points (Fig. 9). The CAM contain electron­ stained microt11h11les have a typical substruc­ dense material organized into a dense core and ture consisting of strands of spherical subunits a lighter medulla. Excluding the bulbous termi­ in parallel array, arranged around the micro­ nations, the cross sectional diameters of Lhe tubule lumen. CAM vary from a mean of 19 nm (N = 17; The overall dimensions of the conoicl are: range = 16 to 24) near the conoid to a mean ( 1) anterior-end width 0.13 to 0.25 µ.m (x = of 42 nm (N = 23; range = 37 to 45) along 0.20; N = 13); (2) posterior-end width 0.18 most of Lhe length. Microtubules comprising to 0.34 1.1.m (x = 0.2!'> µm; N = 12); (3) the conoid are 17 to 26 nm in diameter [N = length ( not including the tlu-ee microtubular 24; x ± (St) (t0.06) = 22 ± l] and have a extensions) 0.21 to 0.33 µ.m (x = 0.29; N = PERKINS-FINE STRUCTURE OF THE ZOOSPORES OF DERMOCYSTIDIUM MARINUM 965

12); and ( 4) length (including microtubular 1, 2, 9, 10, 15), herein referred to as rectilinear extensions) 0.57 to 0.96 µ.m (x = 0.75; N = micronemcs (RM). They differ from the CAM 12). in that the contents are more electron-dense EncircHng most of the conoid is a mantle of and the profiles are nearly sb·aight, most of granular material with the anterior-most por­ them extending from the conoid to the posterior tion forming a functional polar ring to which 10% of the cell. Cross sectional diameters are are attached subplasmalemmal microtubulcs the same in the two classes of sacs (Fig. 15) , (Figs. 1-3, 5-7, 11, 12, 17). Sectioned cells the RM type having a mean of 42 nm (range: contain no evidence of a ring, showing only 35 to 49; N = 11). Antedorly, a few of the microtubular tcrminatfon at the anterior-most microncmes taper to blunt tips within the end of the granular mantle (Figs. 3, 17) conoid lumen, extending within 50 nm of the whereas negatively-stained preparations show plasmalcmma which overlies tho conoid lumen an obvious ring (Figs. 11, 12) with attachment but not connecting to it ( Fig. 2). Most of the of microtubules to the ring (Fig. 11). Although RM terminate postc1ior to or to one side of tl1e there was no clear delineation in negatively­ conoid. The tip contents are generally more stained cells, the polar ring and conoid micro­ electron-lucent tl,an tl,e more posterior regions tubules appeared to be separate entities. In of the sacs. Posteriorly, the micronemes have secti.oned cells subplasmalemmal microtubulcs bulbous tei·minations. were never clearly seen to attach to the conoid. Three to ten rhoptrics consisting of mem­ Tl,ercfore, the polar ring as seen in Figs. 11 brane-bound, electron-dense material are found and 12 is considered to be part of tho granular in the anterior half of the cell with the necks of mantle since the rh1 g could not be part of any the sacs extending into the conoid lumen or other structure as determined from observations along the outside of the conoid (Figs. 1, 15, of sectioned cells. No pre-conoidal rings were 17). Those withm the conoid have anterior observed. terminations in the poste1ior half of the lumen, Up to 39 microtubules have been observed nol near the plasmalemma which overlies the attached to the polar ring in negatively-stained conoid. Total length of the rhoptries ranges whole mounts of ruptured zoosporos and in from 0.5 to 1.6 µ.m . Membrane-bound, clec­ sectioned zoosporos; however, the range of tron-clcnsc inclusions were infrequently found values (20 to 39) was too broad and the values in Lhc conoid lumen (Figs. 1 inset, 14). They too closely spaced to determine a precise num­ resembled the rhoptrics in density and granu­ ber, if there is in fact, a fixed number of micro­ larity; however, neither bucldmg from the tubules. From the termination of the micro­ rhoptrics nor rhoptry fragmentation was identi­ tubules on the granular ring (Fig. 11), they fied. radiate and follow a pathway beneath an Nearly surrounding the apical complex is a overlying, flattened vesicle (Figs. 2, 17) to its U-shapcd vacuole (Figs. 1, 5, 6, 17) contain­ tennination, then follow a path about 10 nm ing amorphous, electron-dense material identi­ beneath the plasmalemma to the posterior end cal in rinc sbucture to the vacuoplasts of veg­ of the cell. Only about 10 microtubules extend etative stages (Perkins, 1969). The complex to the extreme posterior end where there is a is situated in a ridge of cytoplasm which ex­ granular ring about 0.30 µro outside diameter tends through the vacuole. All of the discon­ (Fig. 13). No microtubules were seen attached tinuities in the subunits of tl,e complex ( the to the ring. conoicl, flattened vesicles, and polar ring) face Below the anterior polar ring and paralleling the surface along which the ridge is attached the granular mantle is a flattened vesicle of to the cell body (Fig. 6). Lhc endoplasmic reticulum (ER) (Figs. 5, 6, Between the subplasmalcmmal microtubulcs 10, 13). The vesicle paralJels the granular and tl,c plasmalemma is a labyrinthine network mantle and conoid bemg discontinuous where seen in most sections as a discontinuous, thin, they are incomplete (Fig. 6). clecb·on-dcnse layer (Figs. 16, 17) which is In addition to the CAM, there is a bundle J .5 to 4.2 nm thick (x ± S.t (t0 _05 ) = 2.5 ± of about 20 membrane-bound sacs filled or 0.19; N = 20). In negatively-stained whole nearly filled with electron-dense material (Figs. mounts the labyrinthine substructure of the 966 THE JOURNAL OF PARASITOLOGY, VOL. 62, NO. 6, DECEMBER 1976 layer can be seen (Fig. 18), a characteristic in Gomori's preparations, but rarely in other which is not visible in sections tangential to sectioned cells. Possibly the inclusions arose the cell surface us seen by Ai.kawa and Sterling from fragmentation of rhoptrics

• F1cuRES 8-19. Dcrmocustidimn marin11m cont'd. 8. Uranyl acetate-stained whole mount of oonoid from ruptured zoospore showing tenninations of conoid-atlached micronomcs. Nole inlrnsion ( un­ labeled arrow) of negative slain into conoid microtnbules. Angle of inclination of conoid microtubules ls greater nt distal lhun proximal u11d . X 100,000. 9. Lougitudinal st:clion of zoo~pon: showing rccli­ lincar and conoid-attacl1ed microncmes extending nearly the full length of Lhe cell. X 36,000. JO. Apical complex in longitudinal section. X 140,000. 11, 12. Uranyl acetate-stained whole mounts of conoids and subplasmalemm al microtubules from ruptured zoospores. Note attachment of microtubules to ring portion of polar mantle in Fig. 11 (unlabeled arrow ). Fig. 11 : X 240,000; Fig. 12: X 150,000. 13. Posterior ring of zoospore. Microtubules (unlabeled arrows) approacl1, but do not attach to ring. X 48,000. 14. Zoospore after incubation in modified Gomori's medium used to detect acid phos­ phatase activity. Conoid inclusions show evidence of acid phosphatase whereas the rhoptry and recti­ linear microneme do not, with the possible exception of one region of Lhe rhoptry ( indicated by "LO" ). Normally rhoptrics contain no evidence of acid phosphatase, but are believed to give rise to the cono.id inclns!ons. X 110,000. 15. Cross section of zoospore nt level of kinetosomes (see Fig. 9) showing rhoptries and two types of micrn1111mPs. Note snbpla~malcmmal microtnbnlrs. X 54,000. ·16, 17. Flattened vesicle membranes homologous to subpellicular membrane of apico111plex11n Protozoa. Granular layer which is analogous to the subpcllicular membrane is attached to flattened vesicle ( unlabeled ar­ rows). Fig. 16: X 137,000; Fig. 17: X 76,000. Inset contains high magnification of polar ring region showing convergence and tennfoations of polar mantle and subplasmalemmal microtubnle. X 123,000. 18. Uranyl acetate-stained, ruph.1red zoospore showing labyrinthine complex beneath plasmalcmma. Complex is believed to be same as gronulal' layer seen in Figs. 16 and 17. X 145,000. 19. Micropore ( unlabeled arTow) in posterior end of zoospore. Note electron-dense granular collar around mid-region of !nvngination. X 117,000. PERKINS-FINE STRUCTURE OF THE ZOOSPORES OF DERMOCYSTIDIUM MARINUM 967

.•

.. \

© l

968 THE JOURNAL OF PARASITOLOGY, VOL. 62, NO. 6, DECEMBER 1976 PERKINS-FINE STRUCTURE OF THE ZOOSPORES OF 0ERMOCYSTIDIUM MARINUM 969

® '

.,

® 970 THE JOURNAL OF PARASITOLOGY, VOL. 62, NO. 6, DECEMBER 1976 PERKINS-FINE STRUCTURE OF THE ZOOSPORES OF DERMOCYSTIDIUM MARINUM 971 plex in D. marinum zoospores, the question evidence that flagellated cells of the oyster arises: arc apical complexes truly unique pathogen ru·c a product of meiosis or that organelle groupings or do they have a wider they pnrlicipalc in copulation-isogametic or phylogenetic range thnn [ormcl'ly thought? I hclcrogamclic. believe the organelle complex can still be con­ D. 11wrin11111 zoospores in the absence of an y sidered unique to the Apicomplexn. A con­ other cell type can initiate infections in oysters. siderable number of Protozoa ( Grell, 1973), AlLhough quadriflagcllatc zoospores have been algae (Dodge, 1973; Pickett-Heaps, 1975), observed, they arc rare and arc suspected to and fungi (MadeHn, 1966; Perkins, 1976; be the result of in complete cytokincsis, not Bracker and Littlefi eld, 1973) hnvc hcc•n ex­ copulation, since they arc not numerous enough amined ultrastructurnlly and only the Sporo­ t·o account fo r tho number of infection sites zoasida Leuck::ut 1879 and Pfroplnsmasida had initialed in oysters (Perkins, unpubl. data). been shown to possess an apical complex prior lu spite of Lhc differences cited above, I to this study. Furthermore there nrc severa.l suggest that the presence of : ( l) nn apical additional reasons for i11cluding D. 11wri11um complex, (2) a subpcllicular layer probably in the Apicomplexa. In addi.tion to the apical analogous to the subpellicul ar membrane (see complex, zoospores and trophozoitcs of the discussion below), and (3) rnicropores a.II indi­ oyster pathogen possess micropores (Perk:ins, cate that D. marinum is one of the Apicom­ 1969), zoosporcs have a subpcllicular-likc, plcxa. The presence of a conoicl and the ab­ labyrinthine layer between the plasmalemma sence of a digestive vacuole associated with and subplasmalemmal microtubules, and Lhe the conoicl further in dicates Lhat affinities arc species is parasitic, all of which nre character­ closest lo the coccidinns, not Ll1 0 hcmosporid­ istics of the Apicomplexa. iuns, piroplasms, or grcgmincs. In the absence There are, however, differences from other of adequate information concerning other spe­ members of the Apicomplcxa. D. marinwn cies of the genus Dermocystiditnn, I propose lacks sporozoan schizogony, cndodyogeny, and retention of the name D. mari11um for the pres­ cndopolyogeny in its life cycle, forming cut and pos tponement of any decision concern­ daughter cells by progressive cleavage of a ing where lo place the oyster pathogen within multinuclcated protoplast or by successive bi­ Lhc Apicomplcxa. For the same reason, crea­ partition of the protoplasl (Perkins, 1974). tion of a new genus and species for Lhc oyster Scholtyseck ( 1973) has suggested that cndo­ pathogen would be uuwisc at this time. The dyogeny is a fundamental type of cell division name Labyrinthomyxa marina (Mack:in and in the Sporozoa (Apicomplcxa of Levine, Ray, 1966) shoul d be rejected in the absence 1970 ). Schizogony and cndopolyogcny are of any labyrinthulid charactelistics (see reviews also characteristic of the Apicomplcxa (Aikawa by Perkins, 1974 and Olive, 1975). and Sterling, 1974). Progressive cleavage Much speculation surrounds the role of the could be considered as n mocl ificalion of schi­ apical complex, most workers agreeing that it zogony in that cleavage is initiated from Lhc acts in host cell penetration by some unknown periphery and progresses cenb·ipctally; how­ mechanism (Scholtyscck, 1973; Aikawa and ever, there is no involvement of a subpcllicular Sterling, 1974 ) . The conoid docs not appear to membrane nor is there a residual body left be necessary since some apicomplcxan Protozoa after cleavage as in most sporozoans (Aikawa such as Plasmodium spp. lack the organelle. and Sterling, 1974). Successive bipartition The rhoplTics and microncmcs, however, ap­ (altern ating knryokincses and cytokincscs) does pear to be of fundamental importance. They not occur in the Apicomplcxa (Grell, 1973; may contain lytic agents which, when re­ Scholtyseck, 1973). D. marinum forms bi­ leased, cause localized breakdown of the plas­ flagellated zoospores wilh an anterior flagellum molcmmn, thus permitting peneb·ation (Rob­ possessing mastigoncmcs. Such accessory struc­ erts ct al., 1971; Jadin and Creamers, 1968; tures have not been observed on flagellated Hammond, 1971) or, more probably, may con­ cells of other species in the Apicornplcxa. More­ tain agents which induce phagocytosis of the over, the flagellated cells of apicornplcxa11s nre parasite by the host cell (Jones et al. , J 972; microgamctes, not zoospores. There is no Trager, 1974) . l 972 THE JOURNAL OF PARASITOLOGY, VOL. 62, NO. 6, DECEMBER 1976

Attempts to characterize the contents of D. marinum differs from T. gondit as ob­ rhoptries and microncmcs using cytochemical served by Vivier and Petitprez ( 1972) in that techniques have yielded conflicting results. no polysaccharides were detected in the rhop­ Schrcvcl ( 1968) found acid phosphatase in trics or micronemes. The oyster pathogen may rhoplly-1.ike sacs associntccl with lhc digestive be similar to T. gondii and S. hola11drel in that vacuole of a grcgarinc, Seleniclium holandrei acid phosphatase may be present in the rhop­ Vivier and Schrcvel. The digestive vacuole tlics, assuming that the spheroidal inclusions arises from a pronounced invagination of the in the conoid of D . marinum arc fragmented plasmalemma into the cell body via the conoid rhopb·ies. If so, a hydrolytic role for the lumen. Such vacuoles have also been observed rhoph;es is possible. Suusequenl studies am by Sheffield ( 1966) in the cocciclian, Besnoitia needed to dcmonslJ·atc whether the organelles fellisoni Frenkel, but no acid phosphatase dc­ contain agents whfoh induce phagocytosis of tcm,inalions were made. Chains of small ves­ the parasite b y the host cell or agents which icles were seen in the conoid lumen of Toxo­ permit peneb·ation of the plasmalcmma. plasma gonclii Nicolle and Manceaux by Vi vier The "subpellicular membrane" of the Api­ and Petitprez ( 1972) who suggested that the complcxa was at first reported to be a granular conoid might participate in both host cell pene­ zone beneath the plasmalemma; however, sub­ tration and intake of host cell materials. The sequent, higher resolution studies have shown same workers detected acid phosphatase in a that it is actually two unit membranes discon­ few rhopt1ics, and polysaccharides were de­ tinuous at the nnlerior polar ring and postelior tected in both rhoptries and microncmes using ring. In D. marf11um. the "subpellicnlar mem­ the techniques of Thiery (1967). They con­ brane" analogue is a thin, granular layer. Since sidered the latter observation to be evidence all types of zoospore membranes were easiJy that rhoptrics and micronemes are simiJar struc­ resolved in my preparations, it is unlikely that tures in different stages of development. Light lack of adequate fixation or resolution yielded microscope studies by Nonby ct al (1968) a thin granular layer rather than unit mem­ also show that there ls acid phosphatase ac­ brane substructure. The layer may Le J erived tivity, often at one end of the parasite or from unit membrane degradation in the living scattered throughout lhc cytoplasm, activity cell ns evidenced by its conlinuity with the two which drops upon pcnclration of host cells, fl attened vesicles at the anterior end of the zoo­ then Jises after establishment in the host cell. spore (Figs. 5, 16, 17). The possibility that the The results of Mchlhorn et al. (1974), however, flattened vesicles are homologous to the "sub­ indicate that no acid phosphatase or polysac­ pelicular membrane" must also be considered. charidc is found in the rhopb·ics and micro­ They are in the appropriate subplasmalemmal ncmcs of , Sarcocystis position above the microtubulcs and terminate trmella, Besnnltia jelTisoni, or Frenkclia sp. with directly above the polar ring. In other Api­ tho possible exception of low levels of acid comploxa tho "subpcllicular membrane" fa phosphatase on tho outer surface of the de­ thickened anteriorly and bends downwnrcl to limiting membrane of rhopt1;es in Frenkelia sp. form the polar ring to which the subpcllicular and S. tenella. Ultrastructural localization of microtubules attach. The latter follow a course alkaline phosphatase and ATP-a sc in cyst stages below the "subpcllicular membrane." of S. tenelln indicated that neither enzyme was The granular layer of D . marinum is arranged present in the microncmcs and rhoptrics (Mehl­ into a labyrinthine complex like the "subpel­ ho111 , 1975). licular membrane" of P1asmodium spp. (Aik­ If polysacchariclcs arc present in rhoptrics awa and Sterling, 1974 ) . The "subpelHcular and micronemcs their significance is not membrane" has not been found organized as known; however, tho possible presence of acid a labyrinth in other Sporozoa ( Scholtyseck, phosphatase has been interpreted as further 1973); however, as in other Sporozoa, the "sub­ evidence that the apicaJ complex is used in host pellicular membrane" of Plasmodium spp. is cell pcncb·alion by delivery of lytic enzymes compdsed of two opposed unit membranes. from the rhoptrics and microncmes lo the host The conoid of D. maritwm is similar to those plasmalomma. of other Apicomplexn (Scholtyscck, 1973; PERKINS-FINE STRUCTURE OF THE ZOOSPORES OF DERMOCYSTI DIUM MARINUM 973

Scholtyseck et al., 1970) except that the struc­ BRACKER, C. E., AND L. J. Lrrn.EFmLo. 1973. tw-e is open along one side rather than being a Structural concepts of host-pathogen inter­ faces. In R. J. W. Byrde and C. V. Cutting closed truncated cone as in the other Apicom­ (eds.), Fungal Pathogenicity and the Plant's plexa. The spixally coiled microtubuJes of the Response. Academic l' ress, New York, p . 159- conoid are 17 to 26 nm in diameter as com­ 317. pared to 26 to 30 nm diameter for other Api­ Ci.lAIG, A. S. 1974. Sodium borohydride as an aldehyde blocking reagent for electron micro­ complexa (Scholtyseck, 1973). There are about scope histochemistry. Histochemistry 42: 18 conoid microtubules as compared to the 6 141-144. to 8 recorded by most other workers ( Scholty­ DoocE, J. D. 1973. The fine structure of algal seck, 1973) and ca. 20 found by Porchet­ cells. Academic Press, London, 261 p. Hcnncre ( 1975) in her excellent study of DuooscQ, 0. 1921. Labyrinthomyxa sauvageaui n.g. n.sp., proteomyxee parasite de Laminaria negatively-stained Sacrocystis lenella Railliet lejolisii Sauvageau. Compt Bend Soc Biol 84: coooids. 27-J3. At the anterior end the conoid width is 0.13 GRELL, K. G. 1973. Protozoology. Springer­ to 0.25 µ.m as compared to 0.15 to 0.4 µ.m for Verlag, New York, 554 p. Gus-rAFSON, P. V., H. D. AcAR, AND D. J. CRA,"1.ER. other species; the posterior width is 0.18 to 1954. An electron microscope study of Toxo­ 0.34 µ.m as compared to 0.2 to 0.53 µ.m in plasma. Am J Trop Med I-lyg 3: 1008-1021. others; and the length (not including the three H.A.,1MOND, D. M. 1971. The development and microtubular extensions) is 0.57 to 0.96 µ.m as ecology of and related intracellular compared to 0.08 to 4 µ.m. Other Apieomplexa parasites. In A. M. Fallis (ed.), Ecology and Physiology of Parasites. University of Toronto do not have an extension of the conoid to which Press, Toronto, p. 3-20. are attached micronemes as in D. marinum. JADIN, J. M., AND J. C1'lEEMEl'IS. 1968. Ulb·astruc­ The angle of inclination of conoid microtubules ture et biologie des toxoplasmes intraerylhro­ in the zoospores (50° to 88° ) overlaps the cytaires chez un marnmifere. Acta Trop 25: 267-270. range recorded for other Apicomplexa (70° to Jo.NES, T. C., S. YEH, AND J. G. HmsCH. 1972. 90°); however, the angle of the latter organ­ The interaction between Toxoplasma gondi·i isms is usually the same within the eonoid of a and mammalian cells. J Ex"P Med 136: 1157- given species ( Scholtyseck, 1973) not being a 1172. function of the region of the conoid as in D. KAZA.MA, F. 1973. Ultrastructure of Thrausto­ chytrlum sp. zoospores. Ill. Cytolysomes and marinum. acid phosphatase distribution. Arch Mikrobiol Observations of the changes which occur in 89: 95-104. the apical complex organelles during host cell LEVINE, N. D. 1970. of the sporozoa. penetration are being made. It is known that J Parasitol 56 ( Sect 2) : 208-209. 1973. Introduction, history, and tax­ upon establishment in the host the apical com­ onomy. fo D. M. Hammond and P. L. Long plex a.nd subplasmalemmal granular layer are ( eels. ) , The Coccidia. University Park Press, lost as trophozoites are formed (Perl-ins, 1969). Baltimore, Maryland, p. 1-22. Microporcs are retained and increase in num­ M ACKIN, J. G. 1951. l listopathology of infection of Cras11ostrea virginica ( Gmelin) by Dermo­ ber. cystidium marinum Macki11, Owen, and Col­ lier. Bull Marine Sci Gulf and Caribbean 1: ACKNOWLEDGMENTS 72r-87. The author gratefully acknowledges the ex­ ---. 1962. Oyster disease caused by Dermo­ cystidium marinum and other microorganisms cellent technical support of Mrs. Deborah De­ in Louisiana. Pub] Inst Marine Sci Univ Texas Biasi, Miss Susan Fox, and Miss Peggy Peoples. 7: 132-229. Dr. Jay Andrews is thanked for providing un­ ---, AND J. L. BOSWELL. 1956. The life cycle and relationships of Dennocystidium marinum. infected oysters for ex-perimental infections. Proc Natl Shellfish Assoc 46: 112-115. ---, AND S. M. RAY. 1966. The taxonomic LITERATUR~ CITED relationships of Dermocystidium marinum ArKA.wA, M., AND C. R. STERLING. 1974. Intra­ Mackin, Owen, and Collier. J Invertebr Pathol cellular Parasitic Protozoa. Academic Press, 8 : 544-545. New York, 71 p. MADELIN, M. F. 1966. The fungus ~-pore. Pro­ A.NCELOP0ULOS, E. 1970. Pellicular microtubules ceedings of the 18th Symposium of the Colston in the family Trypanosomatidae. J Prolozol Research Society. Butterworths, London, 338 17: 39-51. p. 974 THE JOURNAL OF PARASITOLOGY, VOL. 62, NO. 6, DECEMBER 1976

M..:10,HORN, II. 1975. Elektronenmikroskopisclrnr L'endozoi:te ( apres colorntion negative). J Nachweis von alkalishcer Phosphatase und Protozool 22: 214-220. ATP-nse in Cystensladien von Sarcocystis RAv, S. M. 1954. Biological studies of Dermo­ tcnella ( Sporozoa, Coccidin) aus der Schlund­ cystidlum marinum, a fungus parasite of oys­ muskulntur von Schafer. Z Parasitenk 4,6: ters. Rice Institute Pamphlet, Special Issue, 95-109. Nov. 1954, p. 1-114. ---, J. SENAUD, AND K ScB0LTYSECK. 1974. ROllERTS, w. L ., C. A. SPEEll, AND D. M. HAM­ li:tude ullrastructurale des coccidies format des MOND. 1971. Penetration of Efmeria lari­ kystes: Toxoplasma go11 clii, Sarcocystis teriella, m.erensis sporozoites into cultured cells as ob­ Besnoitia fellisoni et Frenkelia sp.: distribu­ served with the light and electron microscopes. tion de la phosphatase acide et des poly­ J Parasitol 57: 615-625. saccharides au nivea11 des ultrastruclures d1cz Rvt.i.Y, J. F. 1969. UJLrastructural studies on the le p arasite chez l'hotc. Protistologica 10: sporozoite of Eimer/a /e11ella. Purasitology 21-42. 59: 67-72. Nonnuv, R., L. LmonOLM, AND E. LvoKE. 1008. ScHOLTYSECK, E. 1973. Ultrastrnclure. ln D. Lysosomes of 7'oxoplasma go11dii and their M. Hammond and P. L. Long (eds. ), The possible relation to lhe host cell penetration Coccidia. Eimerla, Isosvora, 'foxovfosma, and of toxoplasma parasites. J Bacteriol 96: 916- related geoern. University Park Press, Balli­ 919. more, Maryland, p. 81-144. OL1vE, L. S. 1975. The Mycetozoans. Academic Press, New York, 252 p. ---, II. ME.lu.HOnN, A.ND K. FRJEDHOFF. 1970. PERKINS, F. 0. 1969. Ultrastructure of vegeta­ The fine slnicture of tl1e conoicl of Sporozoa tive stages in Labyri-nthomyxa marina ( and related orgnoisms. Z Parasilenk 34: = 68-94. Dermocystlditim mariuum), a commercially significanl oyster palhogen. J Invertebr Pathol Scm1EVEL, J. 1968. L'Ullrastructurc de la recion 13: 199-222. a11le1·ieure du ltl gregn1i11e Selenidium el sou 1974. Phylogenetic considerations of int6r~t pour l'ctude de la nutrition chez les the problematic thraustocbytrlnceous-labyrin­ sporozoaires. J Microscopie 7: 391-410. thulid-Dermocystidium complex based on ob­ SREFFJELD, H. G. 1966. Electron microscope servations of fine stmcture. Veroff Inst study of the proliferative form of Besnoitia Meeresforsch Bremerb Suppl 5: 45-63. iellisoni. J Parasitol 52: 583-594. 197fl. Fine structure of lower marine SPRAGUE, V. 1954. Protozoa. fo P. S. Gnltsoff and estuarine fungi. In E. B. G. Jones (ed.), (eel.), Gulf of Mexico. Its origin, waters, and Recent Advances in Aquatic Mycology. Paul marine life. U. S. Fish Wildlife Serv. Fishery Elek Limited, London, p. 279-312. Bull. 89: 243-256. ---, AND R. W. MENZEL. 1967. Ultrastructure TllllilW, J.-P. 1967. Mise en evi