Ultrastructural observations on merogony of Protoentospora ptychoderae Sun, 1910 (Apicomplexa, Coccidia) from the enteropneust Glossobalanus minutus (Kowalewsky, 1866)

European Journal of

Europ.J.Protistol. 25, 338-344 (1990) June 29, 1990

PROTISTOLOGY

Ultrastructural Observations on Merogony of Protoentospora ptychoderae Sun, 1910 (Apicomplexa, Coccidia) from the Enteropneust G/ossobalanus minutus (Kowalewsky, 1866) Jesus Benito and Isabel Fernandez Departamento de Biologfa AnimalI, Facultad de Biologfa, Universidad Complutense, Madrid, Espana

SUMMARY The ultrastructure of the merogony of Protoentospora ptychoderae Sun, 1910 from the coelom of the enteropneust Glossobalanus minutus (Kowalewsky, 1866) is described. Trophic forms dedifferentiated into meronts by losing components of both the apical complex and the pellicle. Prior to merozoite formation, multinucleate meronts showed a discontinuous pellicle inner membrane complex. Merozoites formed by ectomerogony or external budding. In daughter merozoites the subpellicular microtubules were the first apical complex structures that were differentiated; rhoptries, conoid and polar ring differentiated later, and lastly micronemes appeared. The new merozoite had a trilaminate pellicle with a micropore, and showed approximately 60 subpellicular microtubules, a short conoid, at least five rhoptries, one paranuclear Golgi apparatus and one nucleus including one nucleolus. No parasitophorous vacuole was found at any stage of merozoite development. The organellogenesis events are discussed and compared with already known data from other Coccidia.

Introduction During our studies on the trunk gut of Glossobalanus minutus (a marine, benthic, vermiform hemichordate) we observed that the body cavity appears frequently infected by a protozoan parasite identified as Protoentospora ptychoderae. Although such occurrence has been reported since many years ago [5, 11,21], this organism has been poorly studied. Originally considered as a presumptive member of Haplosporidia [5], P. ptychoderae was later identified as a possible member of Sarcodina by Sun [22] who named it and described its life cycle. Both views have been refuted by us in a recent ultrastructural study [3] stating their apicomplexan nature. This study represents the first report dealing with the ultrastructure of the merogony of this organism. It therefore contributes to the knowledge of the cytological features, life cycle and systematic position of this interesting parasite. Material and Methods Adult specimens of Glossobalanus minutus (Kowalewsky, 1866) naturally infectedwere collectedfrom medium-coarsesand 0932-4739/90/0025-0338$3.50/0

in the intertidal zone of Luanco (Asturias,North coast of Spain). Shortlyafter capturing, their trunks (from branchial to the hepatic region)were dissectedin order to obtain smalltissue blocks which were immersed in the fixative. Fixation was made in 4% glutaraldehyde in sea water with 6.5 gil NaCl added at 4°C followed by postfixation in 1% osmium tetroxide in sea water with 6.5 gil NaCI added. Tissue pieces were then dehydrated in acetone series, stained "in block" with uranyl acetate and embedded in Araldite via propylene oxide. One micrometer thick sections stained with an alkaline solution of toluidine blue were used to select the appropriate areas. The ultrathin sections, obtained with an LKB III ultratome, were post-stained with uranyl acetate and lead citrate and examined using a Philips EM 201 electron microscope. Results Merogonic stages of Protoentospora ptychoderae Sun, 1910 developed within the coelomic muscle cells of the enteropneust Glossobalanus minutus (Kowalewsky, 1866). The parasites lay free in the host cell cytoplasm without a membrane lined parasitophorous vacuole (Figs. 1,2). Trophozoites (Fig. 2) were slightly elongate in shape, averaging 6 X 3 urn, and surrounded by a pellicle © 1990 by Gustav Fischer Verlag, Stuttgart

Ultrastructure of Merogony of Protoentospora ptychoderae (Apicomplexa) . 339

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4 Fig. 1. Section through a coelomic muscle cell (MC) containing a trophozoite. P = parasite, x 6300. - Fig. 2. Trophozoite. A = amylopectin granules, HC = host cell cytoplasm, M = mitochondria, N = nucleus, Arrow-head = micropore, Fine arrow = micronem es, Large arro w = rhoptries, x 17800. - Fig. 3. High magnification of the three-layered pellicle of the parasite. 1M, OM = inner- and outer-membranes of the pellicle, Arrow -head = subpellicular microtubules, X 89000. - Fig. 4. Young meront with a large nucleus (N) showing a prominent nucleolus (n). AC = apical complex organ elles, Arrows = cisternae of endoplasmic reticulum, Arrow-heads = mitochondria, x 8000. - Fig. 5. Golgi apparatus (G) of the young meront. ER = endoplasmic reticulum cisternae, N = nucleus, W = thick walled vacuole, x 11 000.

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Ultrastructure of Merogony of Protoentospora ptychoderae (Apicomplexa) . 341 made up of an outer membrane and two closely apposed inner membranes (Fig. 3). The pellicle was interrupted by a micropore at the anterior region of the parasite. Most of the apical complex structures (conoid, subpellicular microtubules, rhoptries and micronemes) of the infective stages were found. The subpellicular microtubules extended to the posterior end of the parasite and were easily recognized on either side of the nucleus. Micronemes were scattered in the peripheral cytoplasm of the parasite. The cytoplasm, densely packed with ribosomes, contained few cisternae of endoplasmic reticulum and amylopectin granules. Th e relatively large nucleus was located at the posterior half of the parasite, and showed a dense nucleoplasm with several clumps of heterochromatin. Swollen mitochondria with light matrices and few vesicular cristae were found anterior to the nucleus. In the dedifferentiation process of the trophozoite stage into meront, significant ultrastructural changes occurred. Earl y meronts (12.5 X 7 urn) (Fig. 4) retained the threelayered pellicle, and remnants of the apical complex pers isted. The nucleus, which was large and with an irregular profile, contained an eccentric nucleolus (1.6 urn in diameter), and the chromatin appea red diffu se. Close to the nucleus, a well developed Golgi apparatus could be observed (Fig. 5 ). Proliferation of th e endoplasmic reticulum cisternae and mitochondria was evident in the less condensed cytoplasm, and lipid inclu sion s were identified. Vacuole s with light contents and bounded by one or more limiting membranes were also found. A later development stage was a rounded meront (averaging 10 urn in diameter) with sections of five nuclei (Fig. 6). The outer membrane unit of the pellicle remained intact, but some interruptions of the inner membrane complex were evident in some places, and the apical complex organelles were lost . In addition, nuclei were located at the periphery of the parasite and their nucleoplasm contained osmiophilic particles distributed throughout. Sometimes the perinuclear envelope was observed to be connected with strands of cytoplasmic rough endoplasmic reticulum and thick-walled vacuole s were often seen closely apposed to the nuclear envelope. Mitochondria with dense matrices remained at the margins of the parasite . In a subsequent pha se of development (Figs. 7, 8), the meront had an irregular shape (measuring 19 x 7 urn) because of the presence of cytopl asmic lobules produced by invaginations or infoldings of the periphery. Budding merozoites (Figs. 9,10) were limited by the plasmalemma and the early inner membrane compl ex. The differentiation process of the ap ical complex stru ct ures took place

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with the formation of the subpellicular microtubules, near which were located one or two centrioles. Two presumptive rhoptry anlagen, multimembraned vacuoles and small vesicles arising from the Golgi apparatus or from the nuclear envelope were also present. A conical protuberance of the nuclear envelope ma y be attributed to remains of the centrocone. At this stage, the meront's cytoplasm contained free ribosomes, smooth and rough endoplasmic reticulum and lipid inclusion s, but no amylopectin granules. Mitochondria were also scattered throughout. During elongation of dau ghter merozoites (Figs. 11, 12), the apical complex constituents (conoid, polar ring com plex and rhoptries) become organized. Three helically arranged tubules appeared in the conoid, which was covered by a canopy-like layer, and the subpellicular microtubules seemed to be attached to an electron-dense structure. At this moment, rhoptries were roughly spherical and appeared partially condensed or showing a striated substructure; the ductules were already formed. A well developed Golgi apparatus was found anterior to the nucleu s which contained a dense nucleoplasm and a small, eccentric nucleolus. 2-8 merozoite sections were found lying free in the ho st cell cytoplasm (Fig. 13). Every merozoite was enclosed by a trilaminate pellicle, where a micropore was found. About 60 subpellicular microtubules, micronemes, amylopectin granules and lipid inclu sion s could also be observed. The merozoite sections showed several mitochondrial profiles with in which electron-dense inclusions were sometimes found (Fig. 15). On the other hand, the presence of a centriolar structur e is indicated in Fig. 14.

Discussion The majority of the parasites found in the body cavity of

Glossobalanus minutus correspond to stages described by us in a recent ultrastructural study [3] as Protoentospora growing trophozoites, but merogony stages have been infrequently observed and located onl y in the gonadal region. The earliest recognizable stage of the merogonic proces s of P. ptychoderae shows the general features of the Apicornplexa infective form s, and th e cytological changes occurring during the dedifferentiation process into rneronts are in general acco rda nce with those reported in other Coccidia. Protoentospora production of merozoites is preceded by a multinucleat e stage in which the trimernbr anal pellicle of the infecti ve form s appears to be interrupted at some bod y places, as in the primary meronts of Dactylosoma [4] and Schellackia [15] .

Fig. 6. Meront with fivenuclei (N). Theinner membrane complex of the pellicle appearsinterruptedin someplaces (arrows), X 9000.Fig. 7. Meront with merozoites buddingbyexogenesis. The nuclei (N) are locatedperipherally and do not have distinctnucleoli. Mu = musculature, x 9400. - Fig. 8. Partial view of the parasite periphery. Note the interruption of the pellicle inner membrane complex (between arrows). HC = host cell cytoplasm, Mi = micropore, Mu = process of a muscular cell, X 38 350. - Fig. 9. Detail of a merozoite budding. Ce = centriole, G = Golgi apparatus, Mu = musculature, N = nucleus, Pe = pellicle, RhP = probable rhoptry precursors, ** = host cellcytoplasm, X 20000 . - Fig. 10. Detail of the pellicle of a buddingmerozoite. HC = host cellcytoplasm, IM, OM = inner- and outer-membranes of the pellicle, N = nucleus, Arrow = subpellicular microrubules, X 45000.

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Ultrastructure of Merogony of Protoentospora ptychoderae (Apicomplexa) . 343 Although nuclear division during merozoite formation has not been observed in this study, centriolar structures usually associated with such processes and with merozoite differentiation [7, 9, 19] have been detected. Such centrioles are also present in fully developed Protoentospora merozoites. The merozoites of the present species develop by exogenesis or ectomerogony as described in Klossia helicina [14], Dactylosoma ranarum [4], Isosporan parasites [13] and in several Eimeria species [19]. However, and unlike other Coccidia [8, 19] the conoid has not been found in the former stages of merozoite development but during the elongation process of daughter merozoites, together with the apical ring complex. In the course of their formation, the rhoptries show a heterogeneous appeareance as it has been already noted in Eimeria [8], Globidium [17] and Aggregata [18]; micronemes are the last characteristic organelles appearing during merogenesis. On the other hand, the association of the Golgi complex with the nucleus during merozoite development is another feature described in the genus Eimeria [8, 12]. Such association persists in the differentiated Protoentospora merozoites. The newly formed merozoites have a system of about 60 subpellicular microtubules as in Aggregata eberthi [18], and the short conoid resemble those of Eucoccidium dinophili [2], Haemogregarina sp. [1], A. eberthi [18] and Klossia helicina [14]. Furthermore, mitochondria with osmiophilic inclusions of unknown nature inside were also reported in K. helicina merozoites

[14]. Although the Coccidia are usually enclosed within a parasitophorous vacuole, all merogony stages of Protoentospora develop in close contact with the host cell cytoplasm. This interesting feature has also been noted by us in growing trophozoites of this organism [3]. As in the latter, the host cell does not appear to be affected by the parasite since no morphological or structural changes of the host cellcytoplasm have been detected. The lacking of a parasitophorous vacuole has been also described in Eucoccidium dinophili sporozoites [2], endogenous stages of Eimeria stigmosa [10] and meronts of several Sarcocystis species [6, 16,20], among others. The cytologicaldata presented in this study confirm that Protoentospora ptychoderae belongs to the order Eucoccidia as we have already suggested in a previous paper [3], but we are somewhat hesitant about assigning the suborder and the family without essential information concerning the gamogonic and sporogonic developments. Unfortunately, the nature and particular characteristics of the host, Glossobalanus minutus, do not allow current para-


sitological procedures, such as inoculation or incubation, to be used. Therefore, we depend on hazardous findings of single, isolated stages of the life cycle, which is yet fully unknown. Acknowledgements We thank Dr. F. Pardos for excellent technical assistance. References 1 Baker J. R. and Laison R. (1967): The fine structure of the

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14,233-238. 2 Bardele C. F. (1966): Elektronenmikroskopische Untersuchungen an dem Sporozoon Eucoccidium dinophili Grell. Z. Zellforsch. Mikrosk. Anat., 74,559-595. 3 Benito J. andFernandez 1. (1989): Theultrastructural identity of Protoentospora ptychoderae Sun, 1910 (Apicomplexa,

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Fig. 11. Developing merozoite sections. N = nuclei, x 8300. - Fig. 12.Partial view of a forming merozoite withthe apical complex structures differentiated. C = conoid, D = rhoptry ductules, G = Golgi apparatus, N = nucleus, Arrow = polar ring complex, Arrowhead = canopy-like layer, Large arrow = micropore, x 37000. - Fig. 13. Merozoite sections lying free in the host cell cytoplasm (HC). Mu = musculature. Arrow indicates thepresence of a centriolar structure, X 6100. - Fig. 14. Detail ofthecentriolar structure (Ce) shown in Fig. 13, taken from a different section of the same parasite, x 36100. - Fig. 15. Cross-section through the anterior region of a new merozoite. Note the dense inclusion within the mitochondria (M). Arrows = rhoptries, Arrow-head = micronemes, X 22 600.

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13 Milde K. (1979): Light and electron microscopic studies on Isosporan parasites (Sporozoa) in sparrows (Passer domesticus L.). Protistologica, 15, 607-627. 14 Moltmann U. G. (1980): Light and electron microscopic studies on the merogony of Klossia helicina (Coccidia; Adeleidea) in snailkidneytissuecultures. Z. Parasitenkd., 62, 165-178. 15 Ostrovska K. and Paperna 1. (1987): Ultrastructural studies on the merogony of Schellackia cf. agamae (Lankesterellidae, Apicomplexa) from the starred lizard Agama stellio. Ann. ParasitoI. Hum. Comp., 62, 380-386. 16 Pacheco N. D. and Fayer R. (1977): Fine structure of Sarcocystis cruzi schizonts. j. Protozool., 24, 282-288. 17 Porchet-Hennere E. (1977): Ultrastructural study of the schizogony in Globidium gilruthi. Protistologica, 13, 31-54.

18 Porchet-Hennere E.et RichardA. (1971): Laschizogonie chez Aggregata eberthi. Etude en microscope electronique, Protistologica, 7, 227- 259. 19 Senaud J. et Cerna Z. (1969): Etude ultrastructurales des merozoites et de la schizogonie des Coccidies (Eimeriina) : Eimeria magna (Perad 1925) de I'intestin des lapins et E. tene/la (Railliet et Lucet, 1981) des coecums des pulets. j. Protozool., 16, 155-165. 20 Senaudj. et CernaZ. (1978): Le cycle de development asexue de Sarcocystis dispersa (Cerna,Kolarovaet Sule, 1977) chez la souris: etude au microscope electronique. Protistologica, 14, 155-176. 21 Spengel]. (1893): DieEnteropneusten desGolfes von Neapel. Monogr., 18, 755-756, Berlin. 22 SunA. (1910): UbereinenParasiten aus der Korperhohlevon Ptychodera minuta. Arch. Protistenkd., 20, 132-134.

Key words: Ultrastructure - Apicomplexa - Merogony - Protoentospora - Enteropneust host Jesus Benito, Departamento de Biologia Animal I (Zoologfa de Invertebrados), Facultad de Biologia, Universidad Complutense, 28040 Madrid, Espana