Developmentaland ComparativeImmunology,Vol. 16, pp. 263-274, 1992 Printed in the USA. All rights reserved.
0145-305)(/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
ADAPTABILITY OF THE COCCIDIAN TO PARASITISM Eliane P o r c h e t - H e n n e r 6
Coelotropha
and T h i e r r y D u g i m o n t
Laboratoire de Biologie Animale, CNRS-URA 148, Universite de Lille Flandres, Artois 59655, Villeneuve d'Ascq, Cedex, France
(Submitted November 1990;Accepted August 1991) 7qAbstractmThe coccidian, Coelotropha durchoni, manages to develop in its host, the polychcate annelid, Nereis diversicolor, because of its ability to circumvent the host's internal defence system. First, it avoids phagocytes by penetrating other cells, principally eleocytes and muscular cells, where it undergoes a phase of intracellular development. After becoming extracellular, a thick coat protects it from being attacked by granulocytes. This coat then breaks and is shed from its surface to permit fertilization. Parasites that have lost their coats and remain unfertilized are surrounded with granulocytes and destroyed by encapsulation. A strict hormonal correlation exists between the biological cycles of the parasite and its host. Thus, the mature spores of the coccidian parasite are disseminated when the worm lays its gametes by rupture of teguments. C. durchoni and N. diversicolor have established a biological equilibrium that permits mutual survival of both partners and constitutes a simple model for the study of the host-parasite relationship.
[]Keywords--Coelotropha durchoni; Nereis diversicolor; Marine invertebrate; Host-parasite relationships; Hormonal correlation; Avoidance of immune processes; Monoclonal antibodies. Introduction
A parasite must overcome numerous obstacles to succeed. First, it is essential to encounter a host that provides a proper physiological environment and for which there exists an adequate site for entry into the body cavity. Second, after entry, Address correspondence to Eliane PorchetHenner6.
it must neutralize the immune surveillance of the host without exerting a pathological effect that could kill the host. Finally, the parasite must adjust its own biological cycle in order to exit from the host at a stage of development suitable for transmission to another host. To face this difficult challenge, " . . . the Coccidia are highly successful parasites maintaining, u n d e r normal circumstances, a well-balanced relationship with their host" (I). One of these coccidians, Coelotropha durchoni, a parasite in the polycheate annelid Nereis diversicolor, a worm living in muddy estuaries, is of special interest because, of several parasites observed in this host, this coccidian is the only one that completes extracellular development in the very hostile environment of the coelomic cavity (2). Many species of Coccidia cause serious and widespread diseases in vertebrates and humans. The ways in which coccidian parasites succeed in living in a phylogenetically ancient invertebrate, the strategies employed to escape host immune surveillance, and the relationship established between them may help to understand similar mechanisms in vertebrate hosts. Indeed, experiments are easier with small nereids, whose anatomy is simpler, than that of mammals: the parasites can be observed from the outside, removed, and implanted. Every stage can be observed in vivo; any substance may be directly injected. In addition, the only endocrine organ, the brain, can easily be removed or grafted, which
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facilitates an analysis of the interactions b e t w e e n the n e u r o e n d o c r i n e and immune systems and the parasites. The confrontation between a coccidian parasite and the internal defence system of invertebrates such as the nereid polychaete enables us to understand the basic immune interrelationships in host animals devoid of antibodies and lymphoid cells responsible for their production. The lifecycle of C. durchoni, a parasite initially observed by Dehorne (3) and Thomas (4), has been subsequently described (5,6). It is the simplest of the coccidian life cycles (Fig. 1). After vegetative growth, g a m o g o n y occurs: Hundreds of biflagellated cup-shaped male gametes are produced in the male gamont; one male gamete fertilizes the large uninucleated female gamete; the zygote becomes an oocyst that divides into about 40 sporocysts, each of them producing 3 0 - 5 0 sporozoites. All phases of the biological cycle of C. durchoni have been extensively studied (5-11). 3a
,@
Figure 1. Biological cycle of C. durchoni. (1), intracellular stage; (2), vegetative stage; (3), female gamont; (3a), (3b), male gamont; (3c), male gamete; (4), oocyst; (5), sporokyst; (6), mature kyst; (7), escape of sporozoites (after 10).
We have now reinvestigated the lifecycle of this parasite, paying special attention to the immune reactions of the host in order to assess this coccidian's adaptability to parasitic life.
Ecology o f Parasites in N. diversicolor T h e t e g u m e n t o f N. diversicolor, though pigmented, is transparent enough to enable us not only to detect the presence of parasites but also to determine the stage of infection. Thus, from the outside we can determine whether the worm is infected by trematodes (near the head), yeast, coccidians in the body cavity, or gregarines in the digestive tract. The immune reactions of the host can also be o b s e r v e d b e c a u s e p a r a s i t e s , when encapsulated by i m m u n o c o m p o nent cells, are invaded by brown pigment (2). Apicomplexa are the most c o m m o n parasites in N. diversicolor. It seems that all worms are infected with gregarines throughout their lifecycles (12). No immune reactions from the host are known to affect gregarines, which live in the digestive tract, where no cells of the internal defence system are thought to occur. They adhere to epithelial cells by specialized anterior structures. Their presence apparently does not affect the life of their host, with the exception of the mechanical pressure caused when they are too numerous. Even starvation caused by numerous parasites does not seem to be a serious problem because worms can fast for several months. In polychaetes, gregarines generally confine themselves to the intestine, where they breed in its lumen. T h e y discharge their o o c y s t s containing sporozoites with the feces into the worm tube from where they can be ingested again. Other gregarines, such as Diplauxis hatti in Perinereis cultrifera (13), go so far as to colonize the coelomic
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cavity in the same way as the coccidian C. durchoni. This phenomenon must be
ancient because polychaetes are sometimes thought to be the first known coelomates (14). Consequently, new and crucial questions arise such as how can the parasite survive in the potentially hostile environment of the coelom, and how can they be transmitted to other hosts when confined to the body cavity? Coccidians are successful parasites and affect all animals including man, whereas gregarines have not developed further than echinodermata. Infections of N. diversicolor by C. durchoni are almost always severe; hundreds of parasites are present in the coelomic fluid of individual animals, causing no apparent pathogenicity. The coelom in nereids is of prime importance since, phylogenetically, the coelom first appears in annelids. Also, nereids are ancient invertebrates, possessing a simple body plan. They are entirely metamerized, meaning that the large coelomic cavity extends all along the body; there is no gonad in N. diversicolor and no endocrine gland, except the brain. Several different types of cells exist in the coelom including gametes (male or female), eleocytes, and granulocytes (15), which are suspended in the coelomic fluid. When infection by C. durchoni occurs, the parasites float among several subpopulations of granulocytes (Fig. 2), which are usually very active in phagocytosing and encapsulating foreign bodies (2,16,17). It is, therefore, quite surprising that these parasites are not destroyed, especially considering the fact that soon after penetration of the coelomic cavity other parasites are encapsulated by numerous sheets of flat granulocytes (2,16). During the encapsulation processes, three different types of granulocytes cooperate (17). Direct oral experimental infection by C. durchoni has been recently achieved. Mature oocysts are isolated from in-
Figure 2. A small coccidia (C) among immunocompetent cells (G) of N. diversicolor, in a drop of coelomic fluid. Scanning electron microscopy. ×1950.
fected worms and fed to young worms in small glass dishes. The absorption of oocysts can be observed microscopically in vivo by compressing the worms between two glass slides. Digestive enzymes facilitate excystment of sporocysts in the gut, the sporozoites pass through the intestinal epithelium (Fig. 3), and enter the body cavity. During this journey, they penetrate and damage cells of the intestine. This behaviour is quite different from that of the sporozoites of
Figure 3. After a meal of oocysts of C. durchoni, the sporocysts open up in the digestive tract of N. diversicolor within a few hours. This sporozoite is penetrating a digestive cell. x9600.
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other coelomic Apicomplexa of marine invertebrates that penetrate between cells (18,19).
Avoidance of the Host Response Intracellular Life Once the parasite enters the coelomic cavity, the sporozoite of C. durchoni has only two possibilities: either to be phagocytosed by granulocytes (which occurs on occasion) or to penetrate a cell (eleocytes, muscle cells, epithelial cells). The first stages of development of C. durchoni are found free in the cytoplasm of eleocytes (Fig. 4a) and muscular cells (9) and not encased in parasitophorous vacuoles. More rarely, they can be observed in epithelial cells, or exceptionally in the ventral nerve cord (unpublished data). Eleocytes are coelomocytes that are very different from granulocytes. They synthesize vitellogenin for the oocytes (20). They are large cells with a high lipid content and may be comparable to fat body cells in arthropods (15). Furthermore, they practice a very specialized phagocytic function in that they engulf sarcolytes, which are free cells detached from the muscle wall (15). It can be hypothesized, however, that the sporozoites are not phagocytosed but instead most probably actively penetrate eleoa
b
cytes, a common trait for infective stages of other coccidian parasites. lntracellular localization is an advantageous mechanism for parasites to escape from their host's immune surveillance system (21,22). Some of them, like Toxoplasma gondii, Leishmania donovani, and Trypanosoma cruzi, live and reproduce in human phagocytes and must avoid the lysosomal hydrolases of their host cell (23). Amastigotes of T. cruzi escape from parasitophorous vacuoles into the cytoplasm of the host cell, where the lysosomal enzymes are at a low level (21). Another means of protection is found by the coccidian T. gondii in macrophages, which prevents fusion of its parasitophorous vacuole by lysosomes (24). This may be due to the special nature of this vacuole, which is not really considered a phagosome (25).
Recognition as S e l f Extracellularly Young C. durchoni can remain dormant for a long time in their host cell before developing freely in the coelom (26). When the host reaches sexual maturity, the intracellular parasite enters into the vegetative stage (Fig. 4a-c). It grows until the host cell bursts, releasing the parasite into the coelomic fluid. The extracellular vegetative stage lacks the organelles that enabled the young invasive sporozoite to move about (10,27) c
Figure 4. Young coccidia in eleocytes. The nucleus of a small parasite looks like that of a sporozoite (a). When growth begins, a nucleolus appears in the nucleus (b,c). (Oo), oocyte; (BV), blood vessel. x970.
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and, therefore only floats among the host's granulocytes (Fig. 2). At this stage in its lifecycle, it has become too large to be phagocytosed but is now susceptible to being encapsulated by sheets of granulocytes (2). U n d e r such extracellular circumstances, parasites may adopt either aggressive strategies or a disguise, or both, to avoid their host's immunoreactions. In the case of aggressive strategies, they can actively suppress the immune response to the host by injecting or secreting immunosuppressants, as nematodes do in their arthropod hosts (28), or parasitoids in insects (29). Molecular disguising strategies are more subtle. The
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parasites may not be recognized as nonself by their host (22). They can be recognized as self by masking their own identity under adoptive molecules from their host or by a true molecular mimicry when they synthesize host-like molecules and present them on their surface (30,31). Development o f a disguising coat. C. durchoni develops a protective coat that is at first fuzzy, then fibrous and granular (Fig. 5a), and finally becomes a thick (up to 2 i~m) two-layer coat around the old vegetative stage (Fig. 5b,c) (11). This coat is not a cystic envelop despite its apparent rigidity (Fig. 6a). Since it exists
i
Figure 5. Electron micrographs of the coat around the vegetative stages of C. durchoni. In early stages, the coat (Co) is made up of one granular sheet (a). Later it is made of two sheets different in their structure (b). Note the different density inside the crypts (Cr). Many tubules arise from the external membrane in the crypts (c). (a) ×12,000; (b) x28,000; (c) x30,O00.
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b
c
Figure 6. The coat of C. durchoni, in vwo micrographs. The surrounding wall of this female gamont, after reaching maturity, retracts from the plasmalemma (a). The female gamont escapes from its coat. Note that a male gamete (arrow) is already attached to the bare surface (b). At sexual maturity, numerous empty coats such as this one, are left in the coelomic fluid (c). After fertilization, the oocysts is surrounded by a thick cystic wall (d). The coat of this spherical coccidia remains bound to the fertilized female gamont. (a)-(c), (e) x800; (d) x700.
throughout the entire vegetative life, i! must be supple enough to permit the growth of the parasite and also permeable to nutrients. It is possible that certain a r e a s o f the p a r a s i t e ' s s u r f a c e
(crypts) (Fig. 5c) are dedicated to nutritive functions (l 1). When the parasite becomes a gamont, the coat is cast off to permit fertilization (8). With the help of the secretion of a
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fluid (10), the vegetative body of the coccidian shrinks (Fig. 6a), the coat splits, and, probably propelled by the pressure of the exudated fluid, the parasite (male or female) rapidly emerges from its coat (Fig. 6b,c). At the time of sexual maturity of C. durchoni, one can find many empty coats in the coelomic fluid (Fig. 6c). Sexual maturity of the parasites in the same host generally is well synchronized and the naked female gamete is soon fertilized by one of the hundreds of male gametes that swim fervently in the coelomic fluid. They often begin to stick to the female even before the coat has been completely cast off (Fig. 6b). They never stick to the coat. After fertilization, the zygote becomes surrounded by a thick cystic envelope (Fig. 6d). At this point of development, even if it is encapsulated by granulocytes, maturation of sporozoites inside the cyst can occur. The coat is not recognized as nonself. The structure of the coat has been described previously (10,11), but its relationship to protection of the parasite from immune processes has not been considered until now. It appears that the coat enables the parasite to evade cellular immune reactions by preventing contact between the plasmalemma of the parasite and that of the granulocytes. Granulocyte attack has never been observed against the coat and, furthermore, loss of the coat can be followed by encapsulation of the naked gamete stage (Fig. 7a). Thus, these observations strongly suggest that the coat is considered as self by the host. A preliminary cytochemical study (unpublished data) has established that the coat is composed of mucopolysaccharides. It has been proved that a thick mucoprotein fibrous layer surrounding the eggs of the parasitoid, Cardiochiles nigriceps, provides them with short-term protection by delaying their encapsulation by hemocytes of their host, He-
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liothis virescens (29). Perhaps this layer carries surface carbohydrates similar to these associated with host structures (such as basement membranes) that then appear as self to host hemocytes. This hypothesis presupposes, as emphasized by the authors, that a recognition system involving carbohydrates operates. Carbohydrate recognition carried out in humoral or cell-bound lectins has been demonstrated to be involved in recognition processes in invertebrates (32). The coat may be of host origin. If the C. durchoni coat is considered as self by granulocytes, it is perhaps because it could well have been produced by the host itself. Even if a direct demonstration that the host participates in building the protective coat is still lacking, this hypothesis should not be discarded. After the discovery of the existence and rejection of a coat by C. durchoni (8), the same phenomenon was found in two other coelomic coccidians of polychaetes, Myriosporides amphiglenae (33) and Eucoccidium ophryotrochae (34). Bardele succeeded in experimentally contaminating a nonnatural host (Dinophilus gyrociliatus, archiannelida) with E. ophryotrochae (34) and under these conditions observed that the coat did not develop around the parasite. According to the conclusions, the coat is actually composed of host material. What kind of signals may trigger the building of the coat around parasites? Perhaps, as with parasitic worms (30,31), the coccidians surround themselves with host tissue during their intracellular life. These adsorbed molecules, once in the coelomic fluid, might initiate the coating. In insects, the contribution of hemocytes to the formation of basal lamina in developing wings has been demonstrated (35). The possible participation of coelomocytes in building a so-called basal lamina around their coccidian parasites must be investigated.
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b
c
d
Figure 7. C. durchoni, cytological aspects. (b-d), immunoperoxidase. Encapsulation of a female gamont by concentric layers of immunocompetent cells (a). After incubation in CC 10 antibody, the surface of the two coccidia is not immunoreactive, unlike the membrane of the granulocytes (b). After incubation in GT 52 antibody, the coelomocytes are not labeled, but the surface of the coccidia (c) and of the spermatocytes (d) are strongly immunoreactive. (a) x600; (b) x400; (c) x300; (d) x200.
Apparent collaboration between host and parasite also has been documented. For instance, the host cell can swell enormously, which may allow the growth of the parasite (36), or even change completely into a true nutritive and protective unicellular organ (37). In these cases, and perhaps also in establishment of a coat around C. durchoni, these relations are of mutual benefit to the two partners. Male gametes-like molecules are found on the surface of C. durchoni. We have studied the surface of C. durchoni for the presence of N. diversicolor-like molecules, using anticoelomocyte monoclonal antibodies in indirect immunoperox-
idase and immunofluorescence assays. But, up to now, we have not found any antigenic similarities using these monoclonal antibodies between the C. durchoni surface and the membranes of granulocytes or those of all N. diversicolor cells (Fig. 7b). However, a striking immunoreactivity was found on the coat of C. durchoni (Fig. 7c) using the GT 52 monoclonal antibody directed against the male gametes of the host (Fig. 7d). Tests with this antibody on empty rejected coats demonstrated a network arrangement of the immunoreactive sites (Fig. 8). This labelling appears similar to the network observed on the surface of C. durchoni gamonts after silver impregnation (7).
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Figure 8. Immunofluorescence after incubation in GT 52 antibody. The remaining coat exhibits labeling in a network. ×650.
Whether this relationship between the surface of C. durchoni and the male gametes of the host (as demonstrated by an immunocrossreactivity) is related to evasion of immune processes cannot yet be established.
Possible Involvement of the Neuroendocrine System A different relationship between C. durchoni and the host male gametes has previously been demonstrated; gametes and parasite development are controlled by brain hormones (26). Under natural conditions, infection by the coelomic coccidian stage occurs in mature worms. Moreover, we established experimentally that C. durchoni can develop in the coelomic cavity of worms of all ages if the brains are removed (26). When the brain is removed, the intracellular stages of C. durchoni undergo considerable growth [in 7 days, there is a sevenfold increase in length (6)] during which the host cell eventually bursts. The vegetative stage of Apicomplexa is characterized by the storage of large spherical or ovoid inclusions (up to 2 ~m in diameter) (Fig. 5b) containing amy-
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lopectin, a polysaccharide related to glycogen and whose structure is well known (38). Amylopectin is particularly abundant in C. durchoni whose female gamonts are literally filled with these inclusions (10,11). Injection of 3H glucose in N.diversicolor is followed by a rapid incorporation in the amylopectin of C. durchoni (39) and in the glycogen of worm cells (15). Durchon demonstrated that the brain of nereids secretes a mitosis and spermatogenesis inhibiting factor (SIF) (40). The secretion of SIF gradually decreases during the worm's life and reduces to zero at sexual maturity (41). Moreover, at sexual maturity the synthesis of glycogen in muscles increases (15) and the level of glucose dramatically falls in the coelomic fluid (41). Similarly, in worm oocytes the activity of the system of conversion of GDP-mannose into GDPfucose increased under the hormonal conditions of maturity. This leads to an enrichment in fucosyl residues, which are the major components of the cortical mucopolysaccharidic granules that characterize the mature oocytes (20). The possibility that the brain of nereids controls mitosis in coelomic parasites at the same time it controls mitotic activity of gametes has been suggested (26,42). Presumably synthesis of the amylopectin, which begins during the intracellular phase of parasite development, requires the same hormonal circumstances as the synthesis of host's glycogen. The construction of the C. durchoni mucopolysaccharide coat may be induced by the same hormonal conditioning as the storage of mucopolysaccharides in the oocytes. As no morphological connection exists in nereids between the brain and the gametes, and since gametes, granulocytes, and coccidians float in the same coelomic fluid, it is probable that all these cells are under the influence of the same messengers in particular secretions from the brain. The question is to deter-
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mine whether the brain secretions act directly on parasites, as e c d y s o n e induces the g a m e t o g e n e s i s of parasitic flagellates in cockroaches (43), or via the immune system. R e c e n t work demonstrates the effective interaction between the i m m u n e , n e r v o u s , and e n d o c r i n e systems not only in mammals but also in invertebrates (44).
Spreading of the Coccidiosis Under natural conditions, the coccidian cycle is completed when the gametes of the worm are mature. Breeding of N. diversicolor then occurs, simultaneously liberating (through rupture of the body walls) oocytes or spermatozoids and mature C. durchoni cysts. This process is also observed in other coelomic parasites of polychaetes. The sporocysts of D. hatti stick to the eggs of P. cultrifera (18), and those of M. amphiglenae cling to the m u c u s - l i n e d tube o f its host, where the newborn larvae can find them as their first food (45). As no predator or vector are involved in the transmission of these parasites, this strict correlation leads to great efficiency in the contamination of new generations of hosts.
encapsulation destroy parasites. C. durchoni, as is the case with coccidian parasites of vertebrates, evades the immune response by invading host cells, and as it d e v e l o p s to its e x t r a c e l l u l a r stage it adopts a disguise. This disguise is not in the form of a discrete molecular mimicry but a structurally distinct strong surface coat. This coat may be produced by the host. The hundreds of coccidia stages that invade the coelomic cavity at sexual maturity certainly exceed the capacities of g r a n u l o c y t e s to e n c a p s u l a t e them. The production of isolating coats certainly save a great amount of the host's energy. The cause of the p h e n o m e n o n of coat rejection, which occurs at maturity, is unknown. It has been shown that the substances secreted around the female gamont attracts the male gametes and causes their adhesion to neutral substrates such as a glass slide (10). This event is a critical stage for the parasite. No description of a similar p h e n o m e n o n is known to us except in two other species of parasitic coccidians in Polychaeta (33,34). The influence of brain messengers (probably neurohormones) on the parasite itself has been d e m o n s t r a t e d (26). It would also be interesting to investigate this possibility for coccidia of vertebrates.
Conclusion In invertebrates, simple cellular immune processes like phagocytosis and
Acknowledgements--The authors thank Y. Himpens and M. Masson for preparing the manuscript.
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cytic vacuoles containing living parasites. J. Exp. Med. 136:1173-1194; 1972. Porchet-Hennerr, E.; Torpier, G. Relations entre Toxoplasma et sa cellule hrte. Protistologica XIX:357-370; 1983. Porchet-Hennerr, E. Corrrlations entre le cycle biologique d'une Coccidie: Coelotropha durchoni; et celui de son hrte: Nereis diversicolor (Ann61ide Polych~te). Z. Parasitenk. 31:299-314; 1969. Porchet-Hennerr, E.; Vivier, E. Ultrastructure comparre des germes infectieux (sporozoites, mrrozoites, schizozoi'tes, endozoites etc.) chez les Sporozoaires. Ann. Biol. 10:77-113; 1971. Nappi, A. J.; Christensen, B. M. Insect immunity and mechanisms of resistance by nematodes. In: Veech, J. A.; Dickson, D. W., eds. Vistas on nematology. Hyattsville, MD: Society of Nematologists, Inc.; 1987:285-291. Davies, D. H.; Vinson, S. B. Passive evasion by eggs of braconid parasitoid Cardiochiles nigriceps of encapsulation in vitro by haemocytes of host Heliothis virescens. Possible role for fibrous layer in immunity. J. Insect. Physiol. 32:1003-1010; 1986. Van der Knaap, W. P. W.; Meuleman, E. A. Interaction between the immune system of lymnaeid snails and trematode parasites. In: Lackie, A. M., ed. Immune mechanisms in invertebrate vectors, Symposium, vol. 56. London: Zoological Society of London; 1986:179198. Yoshino, T. P.; Boswell, C. A. Antigen sharing between larval trematodes and their snail hosts: How real a phenomenon in immune evasion? In: Lackie, A. M., ed. Immune mechanisms in invertebrate vectors, Symposium, vol. 56. London: Zoological Society of London; 1986:221-238. Olafsen, J. A. Invertebrate lectins. Biochemical heterogeneity as a possible key to their biological function. In: Brehelin, M . e d . Immunity in invertebrates. 1985:94-111. Hennerr, E. Stades 6volutifs de deux Coccidies parasites d'Annrlides Polych~tes: Myriosporides amphiglenae gen. n., sp. n., parasite de Arnphiglena mediterranea Clapar~de (Sabellidae), Defretinella eulaliae g e n n . , sp. n., parasite de Eulalia viridis Muller (Phyllodocidae). C.R. Acad. Sci. Paris. 262:890893; 1966. Bardele, C. Notes of the lifecycle in the genus Eucoccidiurn. J. Parasitol. 56:4-20; 1970. Nardi, J. B.; Miklasz, S. D. Hemocytes contribute to both the formation and break down of the basal lamina in developing wings of Manduca sexta. Tiss. Cell. 21:559-567; 1984. Porchet-Hennerr, E.; Ormi~res, R. Etude ultrastructurale des stades hrpatiques de Grasseella microcosmi, coccidie parasite du Tunicier Pyura rnicrocosrnus. P r o t i s t o l o g i c a IX: 187-211 ; 1973. Porchet-Hennerr, E. Etude ultrastructurale de la schizogonie chez la coccidie Globidiurn gilruthi. Protistologica XIII:31-52; 1977.
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38. Schrevel, J. Les polysaccharides de rOserve chez les sporozoaires. Ann. Biol. X:32-44; 1971. 39. Porchet-HennerO, E.; Dhainaut, A. ROsultats pr61iminaires sur l'incorporation de quelques substances radioactives par les trophozoi'tes de la Coccidie Coelotropha durchoni. J. Protozool. 19:71; 1972. 40. Durchon, M. R01e du cerveau dans la maturation gOnitale et le dOclenchement de l'Opitoquie chez les NOrOidiens. Ann. Sci. Natur. Zool. Biol. Animale 18:269-273; 1956. 41. Porchet, M. DonnOes actuelles sur le contrOle endocrine de la maturation gOnitale des NOrOidiens (AnnOlides Polych~tes). Ann. Biol. XV:329-377; 1976. 42. Durchon, M.; Vivier, E. Influence des sOcrOtions endocrines sur le cycle des Gr~garines
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chez les NOrOidiens (AnnOlides Polych~tes). Ann. Endocr. Paris. 25:43-48; 1964. 43. Cleveland, R. L.; Burke, A. W.; Karlson, E Ecdysone induced modification in the sexual cycles of the protozoa of Cryptocercus. J. Protozool. 3:229-239; 1950. 44. Stephano, G. B.; Leung, M. K.; Zhao, X.; Scharrer, B. Evidence for the involvement of opioid neuropeptides in the adherence and migration of i m m u n o c o m p e t e n t invertebrate h e m o c y t e s . Proc. Natl. Acad. Sci. USA 86:626-630; 1989. 45. Porchet-HennerO, E. Correlations entre le cycle de dOveloppement de la Coccidie Myriosporides amphiglenae et celui de son hOte Amphiglena mediterranea (AnnOlide Polych~te), mode de contamination. Protistologica 3:556569; 1967.
0145-305)(/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
ADAPTABILITY OF THE COCCIDIAN TO PARASITISM Eliane P o r c h e t - H e n n e r 6
Coelotropha
and T h i e r r y D u g i m o n t
Laboratoire de Biologie Animale, CNRS-URA 148, Universite de Lille Flandres, Artois 59655, Villeneuve d'Ascq, Cedex, France
(Submitted November 1990;Accepted August 1991) 7qAbstractmThe coccidian, Coelotropha durchoni, manages to develop in its host, the polychcate annelid, Nereis diversicolor, because of its ability to circumvent the host's internal defence system. First, it avoids phagocytes by penetrating other cells, principally eleocytes and muscular cells, where it undergoes a phase of intracellular development. After becoming extracellular, a thick coat protects it from being attacked by granulocytes. This coat then breaks and is shed from its surface to permit fertilization. Parasites that have lost their coats and remain unfertilized are surrounded with granulocytes and destroyed by encapsulation. A strict hormonal correlation exists between the biological cycles of the parasite and its host. Thus, the mature spores of the coccidian parasite are disseminated when the worm lays its gametes by rupture of teguments. C. durchoni and N. diversicolor have established a biological equilibrium that permits mutual survival of both partners and constitutes a simple model for the study of the host-parasite relationship.
[]Keywords--Coelotropha durchoni; Nereis diversicolor; Marine invertebrate; Host-parasite relationships; Hormonal correlation; Avoidance of immune processes; Monoclonal antibodies. Introduction
A parasite must overcome numerous obstacles to succeed. First, it is essential to encounter a host that provides a proper physiological environment and for which there exists an adequate site for entry into the body cavity. Second, after entry, Address correspondence to Eliane PorchetHenner6.
it must neutralize the immune surveillance of the host without exerting a pathological effect that could kill the host. Finally, the parasite must adjust its own biological cycle in order to exit from the host at a stage of development suitable for transmission to another host. To face this difficult challenge, " . . . the Coccidia are highly successful parasites maintaining, u n d e r normal circumstances, a well-balanced relationship with their host" (I). One of these coccidians, Coelotropha durchoni, a parasite in the polycheate annelid Nereis diversicolor, a worm living in muddy estuaries, is of special interest because, of several parasites observed in this host, this coccidian is the only one that completes extracellular development in the very hostile environment of the coelomic cavity (2). Many species of Coccidia cause serious and widespread diseases in vertebrates and humans. The ways in which coccidian parasites succeed in living in a phylogenetically ancient invertebrate, the strategies employed to escape host immune surveillance, and the relationship established between them may help to understand similar mechanisms in vertebrate hosts. Indeed, experiments are easier with small nereids, whose anatomy is simpler, than that of mammals: the parasites can be observed from the outside, removed, and implanted. Every stage can be observed in vivo; any substance may be directly injected. In addition, the only endocrine organ, the brain, can easily be removed or grafted, which
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facilitates an analysis of the interactions b e t w e e n the n e u r o e n d o c r i n e and immune systems and the parasites. The confrontation between a coccidian parasite and the internal defence system of invertebrates such as the nereid polychaete enables us to understand the basic immune interrelationships in host animals devoid of antibodies and lymphoid cells responsible for their production. The lifecycle of C. durchoni, a parasite initially observed by Dehorne (3) and Thomas (4), has been subsequently described (5,6). It is the simplest of the coccidian life cycles (Fig. 1). After vegetative growth, g a m o g o n y occurs: Hundreds of biflagellated cup-shaped male gametes are produced in the male gamont; one male gamete fertilizes the large uninucleated female gamete; the zygote becomes an oocyst that divides into about 40 sporocysts, each of them producing 3 0 - 5 0 sporozoites. All phases of the biological cycle of C. durchoni have been extensively studied (5-11). 3a
,@
Figure 1. Biological cycle of C. durchoni. (1), intracellular stage; (2), vegetative stage; (3), female gamont; (3a), (3b), male gamont; (3c), male gamete; (4), oocyst; (5), sporokyst; (6), mature kyst; (7), escape of sporozoites (after 10).
We have now reinvestigated the lifecycle of this parasite, paying special attention to the immune reactions of the host in order to assess this coccidian's adaptability to parasitic life.
Ecology o f Parasites in N. diversicolor T h e t e g u m e n t o f N. diversicolor, though pigmented, is transparent enough to enable us not only to detect the presence of parasites but also to determine the stage of infection. Thus, from the outside we can determine whether the worm is infected by trematodes (near the head), yeast, coccidians in the body cavity, or gregarines in the digestive tract. The immune reactions of the host can also be o b s e r v e d b e c a u s e p a r a s i t e s , when encapsulated by i m m u n o c o m p o nent cells, are invaded by brown pigment (2). Apicomplexa are the most c o m m o n parasites in N. diversicolor. It seems that all worms are infected with gregarines throughout their lifecycles (12). No immune reactions from the host are known to affect gregarines, which live in the digestive tract, where no cells of the internal defence system are thought to occur. They adhere to epithelial cells by specialized anterior structures. Their presence apparently does not affect the life of their host, with the exception of the mechanical pressure caused when they are too numerous. Even starvation caused by numerous parasites does not seem to be a serious problem because worms can fast for several months. In polychaetes, gregarines generally confine themselves to the intestine, where they breed in its lumen. T h e y discharge their o o c y s t s containing sporozoites with the feces into the worm tube from where they can be ingested again. Other gregarines, such as Diplauxis hatti in Perinereis cultrifera (13), go so far as to colonize the coelomic
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cavity in the same way as the coccidian C. durchoni. This phenomenon must be
ancient because polychaetes are sometimes thought to be the first known coelomates (14). Consequently, new and crucial questions arise such as how can the parasite survive in the potentially hostile environment of the coelom, and how can they be transmitted to other hosts when confined to the body cavity? Coccidians are successful parasites and affect all animals including man, whereas gregarines have not developed further than echinodermata. Infections of N. diversicolor by C. durchoni are almost always severe; hundreds of parasites are present in the coelomic fluid of individual animals, causing no apparent pathogenicity. The coelom in nereids is of prime importance since, phylogenetically, the coelom first appears in annelids. Also, nereids are ancient invertebrates, possessing a simple body plan. They are entirely metamerized, meaning that the large coelomic cavity extends all along the body; there is no gonad in N. diversicolor and no endocrine gland, except the brain. Several different types of cells exist in the coelom including gametes (male or female), eleocytes, and granulocytes (15), which are suspended in the coelomic fluid. When infection by C. durchoni occurs, the parasites float among several subpopulations of granulocytes (Fig. 2), which are usually very active in phagocytosing and encapsulating foreign bodies (2,16,17). It is, therefore, quite surprising that these parasites are not destroyed, especially considering the fact that soon after penetration of the coelomic cavity other parasites are encapsulated by numerous sheets of flat granulocytes (2,16). During the encapsulation processes, three different types of granulocytes cooperate (17). Direct oral experimental infection by C. durchoni has been recently achieved. Mature oocysts are isolated from in-
Figure 2. A small coccidia (C) among immunocompetent cells (G) of N. diversicolor, in a drop of coelomic fluid. Scanning electron microscopy. ×1950.
fected worms and fed to young worms in small glass dishes. The absorption of oocysts can be observed microscopically in vivo by compressing the worms between two glass slides. Digestive enzymes facilitate excystment of sporocysts in the gut, the sporozoites pass through the intestinal epithelium (Fig. 3), and enter the body cavity. During this journey, they penetrate and damage cells of the intestine. This behaviour is quite different from that of the sporozoites of
Figure 3. After a meal of oocysts of C. durchoni, the sporocysts open up in the digestive tract of N. diversicolor within a few hours. This sporozoite is penetrating a digestive cell. x9600.
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other coelomic Apicomplexa of marine invertebrates that penetrate between cells (18,19).
Avoidance of the Host Response Intracellular Life Once the parasite enters the coelomic cavity, the sporozoite of C. durchoni has only two possibilities: either to be phagocytosed by granulocytes (which occurs on occasion) or to penetrate a cell (eleocytes, muscle cells, epithelial cells). The first stages of development of C. durchoni are found free in the cytoplasm of eleocytes (Fig. 4a) and muscular cells (9) and not encased in parasitophorous vacuoles. More rarely, they can be observed in epithelial cells, or exceptionally in the ventral nerve cord (unpublished data). Eleocytes are coelomocytes that are very different from granulocytes. They synthesize vitellogenin for the oocytes (20). They are large cells with a high lipid content and may be comparable to fat body cells in arthropods (15). Furthermore, they practice a very specialized phagocytic function in that they engulf sarcolytes, which are free cells detached from the muscle wall (15). It can be hypothesized, however, that the sporozoites are not phagocytosed but instead most probably actively penetrate eleoa
b
cytes, a common trait for infective stages of other coccidian parasites. lntracellular localization is an advantageous mechanism for parasites to escape from their host's immune surveillance system (21,22). Some of them, like Toxoplasma gondii, Leishmania donovani, and Trypanosoma cruzi, live and reproduce in human phagocytes and must avoid the lysosomal hydrolases of their host cell (23). Amastigotes of T. cruzi escape from parasitophorous vacuoles into the cytoplasm of the host cell, where the lysosomal enzymes are at a low level (21). Another means of protection is found by the coccidian T. gondii in macrophages, which prevents fusion of its parasitophorous vacuole by lysosomes (24). This may be due to the special nature of this vacuole, which is not really considered a phagosome (25).
Recognition as S e l f Extracellularly Young C. durchoni can remain dormant for a long time in their host cell before developing freely in the coelom (26). When the host reaches sexual maturity, the intracellular parasite enters into the vegetative stage (Fig. 4a-c). It grows until the host cell bursts, releasing the parasite into the coelomic fluid. The extracellular vegetative stage lacks the organelles that enabled the young invasive sporozoite to move about (10,27) c
Figure 4. Young coccidia in eleocytes. The nucleus of a small parasite looks like that of a sporozoite (a). When growth begins, a nucleolus appears in the nucleus (b,c). (Oo), oocyte; (BV), blood vessel. x970.
Adaptation to parasitism
and, therefore only floats among the host's granulocytes (Fig. 2). At this stage in its lifecycle, it has become too large to be phagocytosed but is now susceptible to being encapsulated by sheets of granulocytes (2). U n d e r such extracellular circumstances, parasites may adopt either aggressive strategies or a disguise, or both, to avoid their host's immunoreactions. In the case of aggressive strategies, they can actively suppress the immune response to the host by injecting or secreting immunosuppressants, as nematodes do in their arthropod hosts (28), or parasitoids in insects (29). Molecular disguising strategies are more subtle. The
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parasites may not be recognized as nonself by their host (22). They can be recognized as self by masking their own identity under adoptive molecules from their host or by a true molecular mimicry when they synthesize host-like molecules and present them on their surface (30,31). Development o f a disguising coat. C. durchoni develops a protective coat that is at first fuzzy, then fibrous and granular (Fig. 5a), and finally becomes a thick (up to 2 i~m) two-layer coat around the old vegetative stage (Fig. 5b,c) (11). This coat is not a cystic envelop despite its apparent rigidity (Fig. 6a). Since it exists
i
Figure 5. Electron micrographs of the coat around the vegetative stages of C. durchoni. In early stages, the coat (Co) is made up of one granular sheet (a). Later it is made of two sheets different in their structure (b). Note the different density inside the crypts (Cr). Many tubules arise from the external membrane in the crypts (c). (a) ×12,000; (b) x28,000; (c) x30,O00.
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b
c
Figure 6. The coat of C. durchoni, in vwo micrographs. The surrounding wall of this female gamont, after reaching maturity, retracts from the plasmalemma (a). The female gamont escapes from its coat. Note that a male gamete (arrow) is already attached to the bare surface (b). At sexual maturity, numerous empty coats such as this one, are left in the coelomic fluid (c). After fertilization, the oocysts is surrounded by a thick cystic wall (d). The coat of this spherical coccidia remains bound to the fertilized female gamont. (a)-(c), (e) x800; (d) x700.
throughout the entire vegetative life, i! must be supple enough to permit the growth of the parasite and also permeable to nutrients. It is possible that certain a r e a s o f the p a r a s i t e ' s s u r f a c e
(crypts) (Fig. 5c) are dedicated to nutritive functions (l 1). When the parasite becomes a gamont, the coat is cast off to permit fertilization (8). With the help of the secretion of a
Adaptation to parasitism
fluid (10), the vegetative body of the coccidian shrinks (Fig. 6a), the coat splits, and, probably propelled by the pressure of the exudated fluid, the parasite (male or female) rapidly emerges from its coat (Fig. 6b,c). At the time of sexual maturity of C. durchoni, one can find many empty coats in the coelomic fluid (Fig. 6c). Sexual maturity of the parasites in the same host generally is well synchronized and the naked female gamete is soon fertilized by one of the hundreds of male gametes that swim fervently in the coelomic fluid. They often begin to stick to the female even before the coat has been completely cast off (Fig. 6b). They never stick to the coat. After fertilization, the zygote becomes surrounded by a thick cystic envelope (Fig. 6d). At this point of development, even if it is encapsulated by granulocytes, maturation of sporozoites inside the cyst can occur. The coat is not recognized as nonself. The structure of the coat has been described previously (10,11), but its relationship to protection of the parasite from immune processes has not been considered until now. It appears that the coat enables the parasite to evade cellular immune reactions by preventing contact between the plasmalemma of the parasite and that of the granulocytes. Granulocyte attack has never been observed against the coat and, furthermore, loss of the coat can be followed by encapsulation of the naked gamete stage (Fig. 7a). Thus, these observations strongly suggest that the coat is considered as self by the host. A preliminary cytochemical study (unpublished data) has established that the coat is composed of mucopolysaccharides. It has been proved that a thick mucoprotein fibrous layer surrounding the eggs of the parasitoid, Cardiochiles nigriceps, provides them with short-term protection by delaying their encapsulation by hemocytes of their host, He-
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liothis virescens (29). Perhaps this layer carries surface carbohydrates similar to these associated with host structures (such as basement membranes) that then appear as self to host hemocytes. This hypothesis presupposes, as emphasized by the authors, that a recognition system involving carbohydrates operates. Carbohydrate recognition carried out in humoral or cell-bound lectins has been demonstrated to be involved in recognition processes in invertebrates (32). The coat may be of host origin. If the C. durchoni coat is considered as self by granulocytes, it is perhaps because it could well have been produced by the host itself. Even if a direct demonstration that the host participates in building the protective coat is still lacking, this hypothesis should not be discarded. After the discovery of the existence and rejection of a coat by C. durchoni (8), the same phenomenon was found in two other coelomic coccidians of polychaetes, Myriosporides amphiglenae (33) and Eucoccidium ophryotrochae (34). Bardele succeeded in experimentally contaminating a nonnatural host (Dinophilus gyrociliatus, archiannelida) with E. ophryotrochae (34) and under these conditions observed that the coat did not develop around the parasite. According to the conclusions, the coat is actually composed of host material. What kind of signals may trigger the building of the coat around parasites? Perhaps, as with parasitic worms (30,31), the coccidians surround themselves with host tissue during their intracellular life. These adsorbed molecules, once in the coelomic fluid, might initiate the coating. In insects, the contribution of hemocytes to the formation of basal lamina in developing wings has been demonstrated (35). The possible participation of coelomocytes in building a so-called basal lamina around their coccidian parasites must be investigated.
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b
c
d
Figure 7. C. durchoni, cytological aspects. (b-d), immunoperoxidase. Encapsulation of a female gamont by concentric layers of immunocompetent cells (a). After incubation in CC 10 antibody, the surface of the two coccidia is not immunoreactive, unlike the membrane of the granulocytes (b). After incubation in GT 52 antibody, the coelomocytes are not labeled, but the surface of the coccidia (c) and of the spermatocytes (d) are strongly immunoreactive. (a) x600; (b) x400; (c) x300; (d) x200.
Apparent collaboration between host and parasite also has been documented. For instance, the host cell can swell enormously, which may allow the growth of the parasite (36), or even change completely into a true nutritive and protective unicellular organ (37). In these cases, and perhaps also in establishment of a coat around C. durchoni, these relations are of mutual benefit to the two partners. Male gametes-like molecules are found on the surface of C. durchoni. We have studied the surface of C. durchoni for the presence of N. diversicolor-like molecules, using anticoelomocyte monoclonal antibodies in indirect immunoperox-
idase and immunofluorescence assays. But, up to now, we have not found any antigenic similarities using these monoclonal antibodies between the C. durchoni surface and the membranes of granulocytes or those of all N. diversicolor cells (Fig. 7b). However, a striking immunoreactivity was found on the coat of C. durchoni (Fig. 7c) using the GT 52 monoclonal antibody directed against the male gametes of the host (Fig. 7d). Tests with this antibody on empty rejected coats demonstrated a network arrangement of the immunoreactive sites (Fig. 8). This labelling appears similar to the network observed on the surface of C. durchoni gamonts after silver impregnation (7).
Adaptation to parasitism
Figure 8. Immunofluorescence after incubation in GT 52 antibody. The remaining coat exhibits labeling in a network. ×650.
Whether this relationship between the surface of C. durchoni and the male gametes of the host (as demonstrated by an immunocrossreactivity) is related to evasion of immune processes cannot yet be established.
Possible Involvement of the Neuroendocrine System A different relationship between C. durchoni and the host male gametes has previously been demonstrated; gametes and parasite development are controlled by brain hormones (26). Under natural conditions, infection by the coelomic coccidian stage occurs in mature worms. Moreover, we established experimentally that C. durchoni can develop in the coelomic cavity of worms of all ages if the brains are removed (26). When the brain is removed, the intracellular stages of C. durchoni undergo considerable growth [in 7 days, there is a sevenfold increase in length (6)] during which the host cell eventually bursts. The vegetative stage of Apicomplexa is characterized by the storage of large spherical or ovoid inclusions (up to 2 ~m in diameter) (Fig. 5b) containing amy-
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lopectin, a polysaccharide related to glycogen and whose structure is well known (38). Amylopectin is particularly abundant in C. durchoni whose female gamonts are literally filled with these inclusions (10,11). Injection of 3H glucose in N.diversicolor is followed by a rapid incorporation in the amylopectin of C. durchoni (39) and in the glycogen of worm cells (15). Durchon demonstrated that the brain of nereids secretes a mitosis and spermatogenesis inhibiting factor (SIF) (40). The secretion of SIF gradually decreases during the worm's life and reduces to zero at sexual maturity (41). Moreover, at sexual maturity the synthesis of glycogen in muscles increases (15) and the level of glucose dramatically falls in the coelomic fluid (41). Similarly, in worm oocytes the activity of the system of conversion of GDP-mannose into GDPfucose increased under the hormonal conditions of maturity. This leads to an enrichment in fucosyl residues, which are the major components of the cortical mucopolysaccharidic granules that characterize the mature oocytes (20). The possibility that the brain of nereids controls mitosis in coelomic parasites at the same time it controls mitotic activity of gametes has been suggested (26,42). Presumably synthesis of the amylopectin, which begins during the intracellular phase of parasite development, requires the same hormonal circumstances as the synthesis of host's glycogen. The construction of the C. durchoni mucopolysaccharide coat may be induced by the same hormonal conditioning as the storage of mucopolysaccharides in the oocytes. As no morphological connection exists in nereids between the brain and the gametes, and since gametes, granulocytes, and coccidians float in the same coelomic fluid, it is probable that all these cells are under the influence of the same messengers in particular secretions from the brain. The question is to deter-
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mine whether the brain secretions act directly on parasites, as e c d y s o n e induces the g a m e t o g e n e s i s of parasitic flagellates in cockroaches (43), or via the immune system. R e c e n t work demonstrates the effective interaction between the i m m u n e , n e r v o u s , and e n d o c r i n e systems not only in mammals but also in invertebrates (44).
Spreading of the Coccidiosis Under natural conditions, the coccidian cycle is completed when the gametes of the worm are mature. Breeding of N. diversicolor then occurs, simultaneously liberating (through rupture of the body walls) oocytes or spermatozoids and mature C. durchoni cysts. This process is also observed in other coelomic parasites of polychaetes. The sporocysts of D. hatti stick to the eggs of P. cultrifera (18), and those of M. amphiglenae cling to the m u c u s - l i n e d tube o f its host, where the newborn larvae can find them as their first food (45). As no predator or vector are involved in the transmission of these parasites, this strict correlation leads to great efficiency in the contamination of new generations of hosts.
encapsulation destroy parasites. C. durchoni, as is the case with coccidian parasites of vertebrates, evades the immune response by invading host cells, and as it d e v e l o p s to its e x t r a c e l l u l a r stage it adopts a disguise. This disguise is not in the form of a discrete molecular mimicry but a structurally distinct strong surface coat. This coat may be produced by the host. The hundreds of coccidia stages that invade the coelomic cavity at sexual maturity certainly exceed the capacities of g r a n u l o c y t e s to e n c a p s u l a t e them. The production of isolating coats certainly save a great amount of the host's energy. The cause of the p h e n o m e n o n of coat rejection, which occurs at maturity, is unknown. It has been shown that the substances secreted around the female gamont attracts the male gametes and causes their adhesion to neutral substrates such as a glass slide (10). This event is a critical stage for the parasite. No description of a similar p h e n o m e n o n is known to us except in two other species of parasitic coccidians in Polychaeta (33,34). The influence of brain messengers (probably neurohormones) on the parasite itself has been d e m o n s t r a t e d (26). It would also be interesting to investigate this possibility for coccidia of vertebrates.
Conclusion In invertebrates, simple cellular immune processes like phagocytosis and
Acknowledgements--The authors thank Y. Himpens and M. Masson for preparing the manuscript.
References 1. Rose, E. Host immune responses. In: Long P., ed. The coccidia. Baltimore, MD: University Park Press; 1982:329-372. 2. Porchet-Henner& E.; M'Berri, M. Cellular reactions of the Polychaete Annelid Nereis diversicolor against coelomic parasites. J. lnvertebr. Pathol. 50:58-66; 1987. 3. Dehorne, A. Sur l'Aggregata de Nereis diversicolor et sur l'infestation normale de l'6piderme ann61idien par ]es sporozoites. C. R. Soc. Biol. 103:665-668; 1930. 4. Thomas, J. A. Sur le Protozoaire (Coccidie) parasite de Nereis diversicolor. C. R. Soc. Biol. 104:138-141; 1930.
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Adaptation to parasitism
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