Morphology, ultrastructure, and development of the parasitic larva and ...

Cell TissueRes. 206, 487-500 (1980)

Cell and Tissue Research 9 by Springer-Verlag 1980

Morphology, Ultrastructure, and Development of the Parasitic Larva and Its Surrounding Trophamnion of PolypodiumhydriformeUssov (Coelenterata) E.V. Raikova Laboratoryof Cell Morphology,Instituteof Cytologyof the Academyof Sciences,Leningrad,USSR

Summary. The larval stage of Polypodium hydriforme is planuliform and parasitic inside the growing oocytes of acipenserid fishes. The larva has inverted germ layers and a special envelope, the trophamnion, surrounding it within the host oocyte. The trophamnion is a giant unicellular provisory structure derived from the second polar body and performing both protective and digestive functions, clearly a result of adaptation to parasitism. The trophamnion displays microvilli on its inner surface, and irregular protrusions anchoring it to the yolk on its outer surface. Its cytoplasm contains long nuclear fragments, ribosomes, mitochondria, microtubules, microfilaments, prominent Golgi bodies, primary lysosomes, and secondary lysosomes with partially digested inclusions. The cells of the larva proper are poorly differentiated. No muscular, glandular, neural, interstitial, or nematocyst-forming cells have been found. The entodermal (outer layer) cells bear flagella and contain rough endoplasmic reticulum; the ectodermal (inner layer) cells lack cilia and contain an apical layer of acid mucopolysaccharid granules. The cells of both layers contain mitochondria, microtubules, and Golgi bodies; their nuclei display large nucleoli with nucleolonema-like structure, decondensed chromatin, and some perichromatin granules. At their apical rims, the ectodermal cells form septate junctions; laterally, the cells of both layers form simple contacts and occasional interdigitations. The lateral surfaces of entodermal cells are strengthened by microtubules. Key words: Trophamnion - Coelenterate, Polypodium hydriforme - Ultrastructure - Planula - Parasitism.

Polypodium hydriforme Ussov is the only coelenterate endoparasitic in vertebrates

and, at the same time, one of the rare cases of parasitism of a metazoan inside a single cell, a large oocyte. It parasitizes the oocytes of fishes belonging to the Send offprint requeststo: Dr. E.V.Raikova,InstituteofCytology,32 MaklinAvenue,190121Leningrad,

USSR

0302-766X/80/0206/0487/$02.80

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families Acipenseridae (sturgeons and their relatives, Dogiel, 1940) and Polyodontidae (American paddlefishes, Suppes and Meyer, 1975). The parasite undergoes the vegetative phase of its life cycle (from a single binucleate cell to a stolon forming m a n y buds) inside the growing fish oocytes (Raikova, 1958a, 1973). Two stages of this phase, the larva and the stolon, have an inverted position of the germ layers (entoderm outside, ectoderm inside). The larval stage of Polypodium, a parasitic planula, was discovered by Ussov (1887) in sterlet (Acipenser ruthenus L.) oocytes, and later investigated by Lipin (1911). Both authors assumed the planula to have the germ layers in normal position. However, we were able to demonstrate that already in the planula the germ layers are inverted and that, in addition, the planula is isolated from the yolk of the oocyte by a cellular envelope or"capsule" performing protective and nutritive functions (Raikova, 1958b, 1973). Due to parasitism, in Polypodium the stage of planuliform larva lasts up to 2.5 ms, i.e., much longer than that in other coelenterates; during this time, the larva feeds and grows. Preliminary light microscopic data on the feeding mechanism of the planuliform larva of Polypodium within the oocyte have been published earlier in Russian (Raikova, 1958b). The aim of the present work is an ultrastructural investigation of the planuliform larva of Polypodium, with particular attention to structure, function, and origin of its specialized envelope or "capsule" which is so far unique among coelenterates, and which, for reasons pointed out in "Discussion", will be called "trophamnion".

Materials and Methods The material has been collectedin the Volgadelta (nearAstrakhan). In this region the planuliformlarvae of Polypodiumoccur inside early vitellogenic oocytes of the sterlet (AcipenserruthenusL.) in June and July. The infected oocytes are usually larger and darker than healthy ones, which permits their identification before fixation. Infected oocytes of the sterlet were fixed, together with small pieces of the ovary, with 2.5 % glutaraldehyde in 0.1 M phosphate or cacodylate buffer (pH 7.4) during 2 h on ice, and subsequently post-fixed2 h on ice with 1% OsO4in acetate-veronalbuffercontaining saccharose(after Caulfield). The pieceswererinsed with buffer, dehydrated via acetone, and embeddedin Araldite. The ultrathin sections were conventionally stained with uranyl acetate and lead citrate and examined in JEM-7A and JEM100C electron microscopes. For light microscopyof the infectedvitellogenicand previtellogenicoocytesof the stedet, fixations with Zenker's,Champy's,Bouin's, Carnoy's fluids, as wellas with sublimate-aceticacid (95: 5) wereused. The sections were stained with Feulgen, iron hematoxylin, or Mayer's hemalum. Polysaccharideswere revealed by the PAS reaction according to MacManus; acid mucopolysaccharideswere stained with alcian blue or revealed by their metachromasy after toluidine blue staining. At the light microscopic level, the activity of acid phosphatase was revealedin frozen sections of planula-containing oocytes by applying the hexazonium pararosanilin reaction of Barka and Anderson (1962). At the fine structural level, Gomori's method was used for this purpose.

Results Sections through infected early vitellogenic oocytes of the sterlet reveal a bilaminar larva (Fig. 1) lying in a cavity near the oocyte's nucleus. However, the cavity is not in

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direct contact with the yolk but separated from it by the trophamnion or "capsule" (Fig. 2). The cells of the outer layer of the larva bear flagella. Later, this layer becomes the outer layer of the stolon and then, after evagination, the inner layer of the free-living polyp. Thus, it must be considered entodermal. The cells of the inner layer of the larva (facing its body cavity) contain a stratum of acid mucopolysaccharide granules near their apical ends. These granules later mark the ectoderm of flee-living polyps. Consequently, the germ layers of the larva are judged to be inverted. Infected oocytes appear modified in respect to neighbouring healthy ones: they are generally larger and accumulate more yolk, pigment, and glycogen (Raikova, 1963). Most glycogen of the infected oocytes appears to be concentrated in the parasite's body (Fig. 3). However, the cells of the inverted larva never contain ingested yolk granules or lipid droplets. These exist only in the trophamnion surrounding the larva (Raikova, 1958b), thus arguing for the trophic role of this envelope.

1. Fine Structure of the Trophamnion The trophamnion (earlier called "capsule") around the planuliform larva was studied in more detail by electron microscopy. It proved to be a compact layer of cytoplasm, about 6 to 10 ~tm thick, which is closely applied to the yolk-containing cytoplasm of the oocyte (Fig. 4). The boundary between the parasite's trophamnion and the surrounding ooplasm is a single membrane, the plasma membrane of the "capsule" cell, which forms many protrusions, some quite long, into the host's cytoplasm (Figs. 4, 5). Isolation of the parasite from the oocyte yields, at any stage of its development, only the parasite proper, in our case the two-layered larva. The trophamnion always remains with the oocyte; it thus appears to be firmly anchored to the ooplasm with its protrusions. The inner surface of the trophamnion (facing the parasite-carrying cavity) always bears numerous microvilli, 1.0-2.01am in length (Figs. 4, 6-8). The trophamnion is not subdivided into separate cells; no intercellular boundaries have ever been observed in its cytoplasm. In light microscopic sections, the nucleus of the larval trophamnion usually is separated into fragments (Fig. 2). In tangential sections it often has the appearance of ramifying cords (Raikova, 1958b). In ultrathin sections, the nuclear fragments are frequently oblong or ribbonlike (Figs. 4, 6). They are located closer to the inner surface of the trophamnion than to its outer surface. The chromatin is usually decondensed and spread throughout the nucleus in form of fluffy strands interspersed with numerous granules 40-60 nm in size. The granules seem to be perichromatin. The cytoplasm of the trophamnion is lighter than that of the oocyte and contains many free ribosomes and polyribosomes (Fig. 7). The plasma membrane of the outer surface of the trophamnion is underlain with microfilaments, about 3 nm thick, which are especially abundant inside the protrusions anchoring the trophamnion to the ooplasm (Fig. 5). The cytoplasm of the larval trophamnion contains many microtubules (Fig. 7), especially near the nucleus and near the outer surface. Mitochondria are numerous, contain tubular cristae and a dense matrix (Figs. 6, 7). The cristae are often

Figs. 1-3. Sections through infected early vitellogenic sterlet oocytes, showing planula-like larva (p) and its trophamnion (t); o n oocyte nucleus, p c parasite-carrying cavity, e c t ectoderm, e n t entoderm, y yolk, g glycogen. Photomicrographs; 1, 2 iron hematoxylin-eosin staining, 3 PAS reaction. 1, x 30; 2 x 1300; 3 x 400 Figs. 4--6. Electron micrographs of trophamnion (t): 4 general view; 5 protrusion of trophamnion (tp) containing microfilaments into oocyte cytoplasm (oc); 6 part of trophamnial free surface and nucleus (tn); p c parasite-carrying cavity, y yolk platelets, m y microvilli, fi food inclusions, tc trophamnial cytoplasm, l lipid droplets, m mitochondria. 4 x 5500; 5 x 25,000, 6 x 10,500

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longitudinally oriented, unlike those of both oocyte and larval cell mitochondria. The endoplasmic reticulum is poorly developed and is represented by isolated profiles of rough surfaced reticulum, usually associated with the forming surfaces of Golgi bodies (Fig. 7). The cytoplasm of the trophamnion contains many ingested yolk platelets (Figs. 4, 6-8). Typical of the fine structural organization of the trophamnion is the presence, in its cytoplasm, of many prominent Golgi bodies and of vesicles of various sizes with contents of variable density (Figs. 7, 8). Larger vacuoles often have multimembranous walls (Fig. 8) and seem to be secondary lysosomes. Smaller vacuoles with dense contents are frequently adjacent to Golgi bodies and/or to ingested yolk platelets; they are likely to be primary lysosomes (Fig. 7). The lysosomal nature of these vesicles is consistent with the presence of acid phosphatase activity in the cells of the larva and especially in the trophamnion, as revealed at the light microscopic level by a strongly positive hexazonium pararosaniline reaction (Fig. 9). Electron microscopy shows the products of a positive Gomori reaction for acid phosphatase to be localized in the Golgi cisternae and in both the small and the large vesicles, thus confirming them to be primary and secondary lysosomes, respectively (Figs. 10,11). In consequence, the nutrition of the Polypodium larva appears to take place as follows. The trophamnion phagocytizes (apparently with the aid of cytoplasmic protrusions) yolk platelets, lipid droplets, glycogen, and other material from the surrounding cytoplasm of the oocyte. The ingested yolk platetets lose their matrix in the cytoplasm of the trophamnion, leaving behind only the crystalloid element (Fig. 7). Such crystalloids get into contact with primary lysosomes (Fig. 7), the component hydrolases of the latter bringing about gradual digestion of the phospholipid material of the crystalloids. As a result, morphologically identifiable remnants of the yolk platelets in the trophamnion are usually much smaller than intact yolk platelets in the ooplasm (Fig. 4). The same seems true for the lipid inclusions, although no evidence of their digestion with participation of lysosomes was encountered. Digestion of the yolk platelets is completed in secondary lysosomes (Figs. 8, 10), vacuoles containing material of variable density and showing a positive reaction for acid phosphatase. The products of digestion of both the yolk and other nutrients seem to be transported from the trophamnion into the parasite carrying cavity by means of exocytosis, and then absorbed by the entodermal cells of the larva. 2. Fine Structure of the Parasitic Larva The Entoderm. The cells of the outer layer of the larva carry flagella protruding into the cavity containing the parasite (Fig. 13). This is a feature typical for coelenterate entoderm. The entodermal cells, 8 to 10 ~tm high, contain large vacuoles well visible even under the light microscope (Fig. 2); they usually occupy the basal parts of the cells. The nucleus is subapical (Fig. 12) and usually contains a single large nucleolus consisting of fibro-granular nucleolonema. The nucleolus frequently displays a nucleolus-organizing center filled with thin fibrils (Fig. 12). The chromatin rarely forms chromocentres; it usually appears as a fine fibrous network interspersed with small granules. There are also some isolated larger granules (about 60 nm) which

Fig. 7. Cytoplasm of trophamnion, showing Golgi bodies (Gb), fragments of rough endoplasmic reticulum (er), mitochondria (m), free ribosomes and polysomes, microtubules (mr), yolk inclusions (Y0 with adjacent primary lysosome (ly), and microvilli (my). x 37,000 Fig. 8. Cytoplasm of trophamnion containing prominent food inclusions 0q) and digestive vacuoles (v). x 21,000 Fig. 9. Section through infected sterlet oocyte; Hexazonium pararosanilin reaction for acid phosphatase. Reaction localized in planula-like larva (p) and especially in its trophamnion (arrows); y yolk, pl pigment layer, oe oocyte envelope, x 40 Figs. 10 and 11. Electron micrographs of Gomori's acid phosphatase reaction in trophamnion: 10, in secondary lysosomes (sO, 11, in Golgi cisternae (arrow); y yolk ingested, tn trophamnion nucleus. 10, x 23,000, 11, • 51,000

Fig. 12. Entodermal cell nucleus (n) with prominent nucleolus (no containing its organizer (arrowhead) and invagination of inner nuclear membrane (arrow);pc parasitophorous cavity, v intracellular vacuole, rn mitochondria, cb cell boundary strengthened by microtubules, x 12,000 Fig. 13. Flagellum (17) of entodermal cell protruding into parasite-carrying cavity (pc); the kinetosome shows terminal plate (at arrow) and rhizoplast (rp); c second, non flagellated centriole, x 25,500 Fig. 14. Part of entodermal cell nucleus (n) showing invagination of inner nuclear membrane (arrow) and centriole (c) near nucleus. • 18,000 Figs. 15 and 16. Organelles of entodermal cells: 15, microtubules at lateral cell surface; x 40,000; 16 rough endoplasmic reticulum (er) in basal part of cell (0 lateral cell junction); • 27,000

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seem to be perichromatinic. In addition, entodermal nuclei (as well as ectodermal ones) sometimes contain fragments of double membranes of unknown nature (Figs. 12, 14). Each entodermal cell bears a single flagellum with a conventional 9 + 2 structure. The basal body (kinetosome) of the flagellum is also typical. It lacks central microtubules and displays a terminal plate at the level of the cell surface (Fig. 13). A rhizoplast (flagellar rootlet) begins at the basal end of the kinetosome and reaches into the cytoplasm; this is a bundle of microfilaments showing a clear pattern of transverse condensation bands, with a cross striation periodicity of about 75 nm (Fig. 13). The rhizoplast is joined, at various angles, by single microtubules. A second, non-flagellated intracytoplasmic kinetosome is often observed near the base of the flagellated kinetosome (Fig. 13). In addition, a centriole similar in structure to the non-flagellated kinetosome has been seen on several occasions rather far distant from the apical end of the cell, namely, near the side of the nucleus turned towards the basal end of the cell (Fig. 14). The mitochondria are more abundant apically; their profiles are more or less spherical and contain a clear matrix and prominent tubular cristae (Fig. 12). Some channels of the rough endoplasmic reticulum appear widened; some of its cisterns form concentric systems (Fig. 16). The Golgi apparatus is often in form of paired dictyosomes which seem to give rise to many vacuoles. Some of these contain electron dense material and resemble primary lysosomes. The acid phosphatase reaction yields a dense reaction product in such vacuoles. The cytoplasm of the entodermal cells is rich in ribosomes and polyribosomes (Figs. 12-16, 18). The entodermal cells contain many microtubules which seem to have cytoskeletal function; they are connected with the flagellar base and also pass along the lateral surfaces of the cells beneath the plasma membrane (Figs. 12, 15, 16). The apical surface of the cells is rather uneven, forming cytoplasmic protrusions that resemble microvilli. Numerous pinocytotic vesicles with both smooth and coated walls are seen beneath the apical plasma membrane (Fig. 17). Various types of intercellular contacts exist between neighbouring entodermal cells. At their apical rims, junctions with some apposed cytoplasmic material are seen (Fig. 18); these may be poorly developed septate junctions. At lateral cell surfaces the plasma membranes show no special differentiations (simple junctions, Fig. 16); however, interdigitation of adjacent plasma membranes occurs at some points (Fig. 19). The Ectoderm. The cells of the inner layer of the inverted planuliform larva, unlike the ectodermal cells of noninverted planulae of other coelenterates, bear no cilia (Fig. 20). The ectodermal cells, like the entodermal ones, are vacuolated, contain large nuclei with well developed nucleoli (Fig. 20) and often show a nucleolusorganizing zone. The apical layer of acid mucopolysaccharide granules, rather typical of the ectodermal cells (Raikova, 1958b), is absent in some larvae (e.g., Fig. 20). Apparently, it forms during the stage of the planuliform larva. Each granule is surrounded by a membrane (Fig. 21). At the level of the mucopolysaccharide granules, adjacent ectodermal cells form septate junctions. The remaining

Fig. 17. Cytoplasmic protrusions of entodermal cells into parasite-carrying cavity. • 24,000 Figs. 18 and 19. Contacts between entodermal cells: 18, apparently septate junction; x44,000; 19 interdigitation; x 26,000 Fig. 20. Ectodermal cells showing vacuoles (v), nuclei (n) with nucleoli (nO; m g mesoglea, ic inner cavity of larva, x 9600 Fig. 21. Acid mucopolysaccharide granules (pg) at apical ends of ectodermal cells; ic inner cavity of larva, x 22,000

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organelles of the ectodermal cells resemble those of the entodermal ones: rounded mitochondria (Fig. 20), rough endoplasmic reticulum, and Golgi bodies. The apical acid mucopolysaccharide granules appear to be Golgi derivatives. The microtubules, underlying the plasma membranes, are less numerous in the ectoderm than in the entoderm. The cytoplasm contains many vacuoles of various sizes and both smooth and coated vesicles. The ectodermal cells contact the contents of the inner cavity of the larva, filled with a liquid where fixation produces a fluffy precipitate (rig. 20).

The Mesoglea. The layer of mesoglea, varying in thickness from 0.5 to 1.5 lam, separates the ento- from the ectoderm. It contains many 10 nm thick fibrils passing more or less parallel to the cellular layers (Fig. 20). 3. Origin of the Trophamnion The unique trophamnion (earlier called "capsule") has been shown to differentiate already at the unicellular stage of development of Polypodium which parasitizes previtellogenic oocytes (Raikova, 1964 a). The study of these early stages is difficult since infected previtellogenic oocytes do not differ from healthy ones and cannot be electively fixed, e.g., for electron microscopy. All available data on unicellular stages of the life cycle are so far light microscopic and have been obtained by chance, during scanning of a large number of serial sections of the sterlet's ovaries. The earliest known parasitic stage of development of Polypodium is a binucleate cell with differently-sized nuclei (Raikova, 1964a, 1973; Fig. 22, d). These binucleate cells strikingly resemble the binucleate cells that form inside the gonads of free-living polyps (Fig. 22, a-c; Raikova, 1961, 1973). Later, a small portion of cytoplasm becomes separated around the small nucleus; thus, a small cell is formed within the cytoplasm of a large cell (Fig. 22, e). At the same time, the nucleus of the large cell forms a cuplike invagination, and the small cell comes to lie in it (Fig. 22, e, f). Later, the borders of the invagination join (Fig. 22, g), and the small cell, now enclosed in a cavity inside the nucleus of the large cell, starts to divide (Fig. 22, h). Cytophotometrical studies demonstrated the small nucleus and all its known derivatives (from the binucleate cell inside the polyp's gonad to the blastomeres) to be haploid (Raikova, 1965). When and how diploidy is reconstituted in the life cycle of Polypodium, remains unknown. As to the large nucleus, it begins polyploidization while still in the polyp's gonad (2 to 6 n). In parasitic binucleate cells, the large nucleus appears polyploid between 12 and 52 n, depending on the stage. When the large nucleus becomes hollow and encircles several blastomeres, it is already highly polyploid (about 400 n). The derivatives of the small cell form a morula (Fig. 22, i) and later a planula (Fig. 22, j); the mode of gastrulation is, however, unknown (Raikova, 1964b). Thus, the parasitic larva proper develops from a part of the cytoplasm and of the small nucleus of the binucleate cell, which is likely to be a parthenogenetically developing aberrant gamete. As to the trophamnion ("capsule"), it develops from the large nucleus of the binucleate gamete and most of its cytoplasm. The large nucleus undergoes polyploidization, branches, and apparently becomes fragmented, while the cytoplasm of the trophamnion grows intensely (Fig. 22).

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o

b

c

l Fig. 22a-j. Diagram of formation of trophamn'ibn, a Free-living animal with gonads (G). b Gonad containing mature binucleate cells, e Several binucleate cells, d Parasitic binucleate cell within cytoplasm of oocyte, e Two-celled parasitic stage, f Position of small cell in intranuclear cavity of large cell. g Nucleus of large cell completely surrounding small cell. h Division of small cell. i Morula (M) within intranuclear cavity of large cell (LC). j Planuliform larva (P) inside hollow large cell, or trophamnion (73. Approximate magnifications: a ~ x 10; b ~ x 90; c ~ x 780; d-g ~ x 520; h ~ x 390; i ~ x 195; j x 65 (certain proportions exaggerated for the sake of clarity)

T h e binucleate cells, which are the likely p r e c u r s o r s o f the early p a r a s i t i c stages o f Polypodium, d e v e l o p in free-living specimens inside special e n t o d e r m a l g o n a d s (Fig. 22, a - c ; R a i k o v a , 1961). These cells are p r o d u c e d b y two meiotic nuclear divisions o f which o n l y the first is followed b y cytokinesis. T h e cytokinesis c o r r e s p o n d i n g to the 2nd meiotic division is s t r o n g l y d e l a y e d a n d m o d i f i e d a n d c o r r e s p o n d s to the s e p a r a t i o n o f a c y t o p l a s m i c m a s s a r o u n d the small nucleus o f the binucleate cell, a l r e a d y within the fish oocyte (Fig. 22, e). This small cell, with a m i n i m u m o f c y t o p l a s m , c o u l d thus c o r r e s p o n d to the second p o l a r b o d y , b u t it is

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not a polar body since it gives rise to the future embryo. By contrast, the larger product of the delayed division of the 2nd meiocyte, the enveloping cell with the large hollow nucleus, forms no embryo but develops into the trophamnion. It is this cell that may be homologized with the 2nd polar body which undergoes polyploidization and functions as a provisory trophic envelope around the developing embryo.

Discussion

1. Functions and Nature of the Trophamnion Our electron microscopic data, including those on the gradual digestion of the yolk platelets, tend to support our previous supposition (Raikova, 1958 b) regarding the nutritive function of the trophamnion. This opinion is further strengthened by the observation that most of the acid phosphatase activity is localized in the trophamnion and not in the cells of the larva. The presence of microvilli at the inner surface of the trophamnion strongly increases its contact area with the contents of the parasite-carrying cavity and makes likely the transport of soluble nutrients from the trophamnion to the larva. Thus, digestion in larval Polypodium seems to be intracellular and performed by the trophamnion. The other likely function of the trophamnion is protective. It prevents the developing larva from direct contact with the yolk and produces a cavity in the oocyte where the larva can grow and later undergo budding. The microtubules and microfilaments of the trophamnion seem to have a cytoskeletal function; they provide elasticity and rigidity to this envelope and prevent the parasite-carrying cavity from collapsing. The absence of cellular boundaries in the cytoplasm of the trophamnion, demonstrated by electron microscopy, permits the concept that, at least at the larval stage of development, the trophamnion is a single giant hollow cell with a branching and/or fragmented polyploid nucleus, rather than a symplast (i.e. product of fusion of many cells), as supposed earlier (Raikova, 1960). A fine-structural investigation of the trophamnion at later stages of development (budding larva and stolon) has to answer the question whether or not the single trophamnion cell later divides into many smaller cells. Consequently, the trophamnion of Polypodium is a provisory unicellular organ, unique among the coelenterates and very rare elsewhere, which can be considered an adaptation to feeding at parasitic stages of development. Aside from coelenterates, only parasitic polyembryonic Hymenoptera (families Encyrtidae and Platygastridae) show a similar phenomenon of conservation of the polar bodies after meiosis and of their growth into a special envelope around the embryo, the trophamnion, which also serves for the nutrition of the parasite (Iwanowa-Kazas, 1961). Their trophamnion has recently been studied electron microscopically (Kogcielski et al., 1978). The similarity between it and the envelope around t h e Polypodium embryo is strong enough to justify calling the latter by the same term, trophamnion. This similarity involves the ontogenetic origin (from polar bodies), the nutritive function, and the provisory character of the two envelopes. It is beyond

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doubt that phylogenetically the two organs have evolved quite independently from each other, as a result of convergent evolution due to adaptation to a similar mode of life (intracellular parasitism in oocytes).

2. Peculiarities of the Planuliform Larva Besides the formation of the trophamnion, the parasitic mode of life of the planuliform larva of Polypodium produces pronounced effects on its own organization. First of all, the planula of Polypodium is inverted so that the entoderm is outside and nearer to the feeding organ, the trophamnion. Further, the ectoderm of the planula has no cilia which are typical of the planulae of other coelenterates; this is apparently a consequence of the inversion of the germ layers; the ectoderm has lost its external position and a possible role in locomotion. On the other hand, the entodermal cells retain their flagella. The planula of Polypodium also has no specialized cells which would be necessary if the planula were free-living. It also lacks nematocysts, muscular, and glandular cells, described in planulae of other coelenterates (Van de Vyver, 1964; Lyons, 1973). Moreover, the planula of Polypodium also has no neural or interstitial cells, a feature in common with the planulae of Hydromedusae belonging to the Capitata group, superfamily Corynidae (Bodo et Bouillon, 1968), and with the planula of the anthomedusan Eleutheria (Van de Vyver et Bouillon, 1969). Since the trophamnion takes over the functions of phagocytosis and digestion, the entodermal cells of the larva contain very few food inclusions, i.e., small food vacuoles (secondary lysosomes) displaying acid phosphatase activity. They probably reach the entodermal cells from the trophamnion via exocytosis, the parasite-containing cavity, and endocytosis. Being free from their usual digestive function, the entodermal cells of the larva are poorly differentiated. Only some of the cells display a significant development of rough endoplasmic reticulum; these are likely to be the future glandular cells. A similarly low degree of differentiation is characteristic of the ectodermal cells whose only apparent function is, at the larval stage, production and storage of acid mucopolysaccharide granules; in fact, these granules will function only during eversion of the stolon in mature eggs, just before spawning (Raikova, 1960). In the larva, these granules are still not numerous and all belong to one type. In free-living planulae, e.g., in Balanophyllia, similar granules are important for the attachment of the planula to the substratum; they are diverse (4 types) and numerous (Lyons, 1973). The cells of both larval layers have nuclei with decondensed chromatin, large nucleolonema-type nucleoli containing nucleolus-organizing areas, and some perichromatin granules. They also have similar mitochondria, polyribosomes, microtubules, and paired Golgi dictyosomes. Besides these similarities, the ectodermal and entodermal cells also show some fine structural differences, among them the irregular free surface of entodermal cells, probably favoring uptake of soluble nutrients produced by the trophamnion. The entodermal cells of the larva have more microtubules supporting the lateral walls of the cells. This too is a possible consequence of inversion of the germ layers. The cytoskeletal role of the microtubules is also well known both in Hydra and in other invertebrates, e.g., in embryonic cells of the sea urchin (Gibbins et al., 1969; Tilney and Gibbins, 1969).

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References Barka T, Anderson P (1962) Histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler. J Histochem Cytochem 10:741-753 Bodo F, Bouillon J (1968) Etude histologique du d~veloppement embryonnaire de quelques hydromeduses de Roscoff: Phialidium hemisphaerium(L.), Obeliasp. P6ron et Lesueur, Sarsia eximia (Allman), Podocoryne carnea (Sars), Gonionemus vertens Agassiz. Cahiers Biol Marine 9:69-104 Dogie1 VA (1940) New finding places and new hosts of Polypodium hydriforme. (In Russian). Zool Zhurnal (Moscow) 19:321-323 Gibbins JR, Tilney LG, Porter KR (1969) Microtubules in the formation and development of the primary mesenchyme in A rbaciapunctulata. I. The distribution of microtubules. J Cell Bio141:201226 Iwanowa-Kazas OM (1961) Essays on comparative embryology of the Hymenoptera. (In Russian). Leningrad Univ Press, Leningrad Ko~cielski B, Ko~cielska M K, Szroeder J (1978) Ultrastructure of the polygerm of Ageniaspisfuscicollis. Zoomorphologie 89:279-288 Lipin AN (1911) Ober ein neues Entwicklungsstadium von Polypodium hydriforme Uss. Zool Anz 37: 97-99 Lyons KM (1973) Collar cells in planula and adult tentacle ectoderm of the solitary coral Balanophyllia regia (Anthozoa, Eupsammiidae). Z Zellforsch 145:57--74 Raikova EV (1958 a) The life cycle ofPolypodium hydriforme Ussov (Coelenterata). (In Russian, English summary). Zool Zhurnal (Moscow) 37:345-358 Raikova EV (1958b) A histochemical study of the parasitic larva of Polypodium hydriforme Ussov (Coelenterata). (In Russian). Doklady Akad Nauk SSSR 121:549-552 Raikova EV (1960) Morphological and cytochemical investigation of parasitic stages of the life cycle of Polypodium hydriforme Ussov (Coelenterata). (In Russian). Tsitologiya (Leningrad) 2:235-251 Raikova EV (1961) Development of the male gonads and spermatogenesis in Polypodium hydriforme. (In Russian). Tsitologiya (Leningrad) 3:528-544 Raikova EV (1963) Morphological and cytochemical changes in the oocytes of the sterletand the sturgeon under the effect of parasitizing of Polypodium hydriforme Ussov (Coelenterata). (In Russian). Doklady Akad Nauk SSSR 152:985-988 Raikova EV (1964a) Unicellular parasitic stages of the life cycle of Polypodium hydriforme Ussov (Coelenterata). (In Russian, English summary). Zool Zhurnal (Moscow) 43:409~112 Raikova EV (1964b) Early parasitic stages of the life cycle of Polypodium hydriforme Ussov (Coelenterata). (In Russian). Doklady Akad Nauk SSSR 154:742-743 Raikova EV (1965) A cytophotometric study of the DNA content in the cell nuclei of Polypodium hydriforme Ussov (Coelenterata) at various stages of its life cycle. (In Russian, English summary). Zhurnal Obsch Biol (Moscow) 26:546-552 Raikova EV (1973) Life cycle and systematic position ofPolypodium hydriforme Ussov (Coelenterata), a cnidarian parasite of the eggs of Acipenseridae. Publ Seto Marine Biol Lab 20 (Proc. 2rid Internat. Syrup. on Cnidaria): 165-173 Suppes VC, Meyer FP (1975) Polypodium sp. (Coelenterata) infection of paddlefish (Polyodonspathula) eggs. J Parasitol 61 : 772-774 Tilney LG, Gibbins JR (1969) Microtubules in the formation and development of the primary mesenchyme in Arbaeia punetulata. II. An experimental analysis of their role in development and maintenance of cell shape. J Cell Biol 41:227-250 Ussow M (1887) Eine neue Form von Siisswasser-Coelenteraten. Morphol Jahrb 12:137-153 Van de Vyver G (1964) l~tude histologique du drveloppement d'Hydraetinia eehinata (Flem.). Cahiers Biol Marine 5:295-310 Van de Vyver G, Bouillon J (1969) t~tude du drveloppement embryonnaire et de l'histogen+se de Eleutheria diehotoma (de Quatrefages) (Anthomeduse Eleutheriidae). Ann Embryol Morphogen 2:317-327 Accepted December 4, 1979