Ultrastructural studies on the daughter sporocysts of Meiogymnophallus minutus (Digenea: Gymnophallidae)

hternarional

Journalfor

Parasimlogy

Vol. 19, No. 5. pp. 499-508, 1989

Printed in Greaf Britain 0

ULTRASTRUCTURAL MEIOGYMNOPHALLUS

0020-7519/89 163.00 + 0.00 Maxwell Pergamon Mecmilh p/c 1989 Ausfrolian Societyfor Pwmirology

STUDIES ON THE DAUGHTER SPOROCYSTS OF MINUTUS (DIGENEA: GYMNOPHALLIDAE) H. A. AL-SALMAN

and B. L. JAMES*

Marine, Environmental and Evolutionary Research Group, School of Biological Sciences, University College, Singleton Park, Swansea SA2 8PP, U.K. (Received 25 October 1988; accepted 2 February 1989) Abstract-AL-SALMAN

H. A. and JAMESB. L. 1989. Ultrastructural studies on the daughter sporocysts of

Meiogymnophallus minutus (Digenea: Gymnophallidae). International Journalfor Parasitology 19: 499-508. The daughter sporocyst body wall is a syncytial microvillous tegument resting on a basement lamina,

followed by circular and longitudinal muscle fibres and five kinds of subtegumentary cells, namely flame, myoblast, germinal, secretory and digestive cells. The last two have cytoplasmic bridges connecting them to the tegument and their principal function is probably nutrition. Secretory cells probably synthesize hydrolytic enzymes which pass via the tegument into the host’s haemolymph for exogenous digestion of nutrients. Simple nutrients are probably absorbed over the entire surface of the microvilli whereas undigested or partly digested nutrients are absorbed by micropinocytosis at their base. Some nutrients may be digested in the tegument but most pass into the digestive cells which undergo a cycle including synthetic, absorptive, digestive, storage, autophagic and resting phases. Nutrients are supplied to developing embryos by extensions of digestive cells which line the brood chamber. Residual bodies are shed into the host’s haemolymph, or into the brood chamber. In ageing sporocysts, microvilli and later the tegument completely degenerate and subtegumental cells undergo extensive autolysis, finally, the body wall ruptures, releasing the remaining fully formed cercariae. INDEX KEY WORDS: Digenea; Gymnophallidae; Meiogymnophallus minutus; ultrastructure; daughter sporocyst; feeding; digestion.

INTRODUCTION THE daughter sporocyst, unlike other stages in the digenean life cycle, does not possess a gut and so all nutrients must pass through the body wall. This is facilitated by the presence of microvilli that increase the absorptive surface (Kinoti, Bird & Barker, 1971) and by picnotic invaginations of the apical plasma membrane (Kerie, 1971b; Reader, 1975). Previous workers (Bils & Martin, 1966; James, Bowers & Richards, 1966; Kinoti et al., 1971; Koie, 1971b; Bibby & Rees, 1971; Meuleman, 1972; Smith & Chernin, 1974; Reader, 1975; Meuleman, Holzmann & Peet, 1980) have shown that in daughter sporocysts with a birth pore, the body wall is a metabolically active syncytial tegument connected to subtegumentary cells by cytoplasmic bridges and is, thus, basically similar to that of adult Digenea (Erasmus, 1972; James et al., 1966; James&Bowers, 1967a). Imohiosen (unpublished Ph.D. thesis, University College, Swansea, 1969), James (unpublished Ph.D. thesis, University College, Swansea, 1973), Popiel (unpublished Ph.D. thesis, University College, Swansea 1976) and Sannia (unpublished Ph.D. thesis, University College, Swansea, 1977) have shown that the subtegumentary cells *To whom all correspondence should be addressed.

synthesize substances which may include mucopolysaccharides and hydrolytic digestive enzymes which are secreted into the host haemocoel, the former to protect the parasite from the host’s defence responses and the latter for exogenous digestion of nutrients usually derived from the host’s gonad or digestive gland (James, 1965). Reader (1975) demonstrated that excessive secretion of mucopolysaccharide occurs to protect areas of degenerating tegument in the ageing daughter sporocyst. The ultrastructure of the daughter sporocyst and cercaria of Meiogymnophallus minutus from Scrobicularia plana has been briefly described by Sannia (1977, thesis cited above). This, and the brief description of Parvatrema homoeotecnum by Imohiosen (1969, thesis cited above) and Gymnophallus choledochus (= Cercnria dichotoma) by James & Bowers (1967b) and James (1973, thesis cited above) are the only ultrastructural studies on gymnophallid daughter sporocysts. The present investigation describes the daughter sporocyst of Meiogymnophallus minutus in more detail. The extremely rapid development of this parasite, where many thousands of daughter sporocysts produce up to 100,000 cercariae daily (Al-Salman, unpublished Ph.D. thesis, University College, Swansea, 1984; James, Sannia & Bowers, 1977) and where the duration of the 499

H. A. AL-SALMANand B. L. JAMES

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infection in Scrobiculariaplana is probably less than 6 months (Sannia, 1977, thesis cited above; Al-Salman, 1984, thesis cited above), suggests an avid and insatiable appetite for nutrients. Thus, particular attention is paid to feeding and digestion in the daughter sporocyst and to the transfer of nutrients to the developing cercariae (Al-Salman, 1984, thesis cited above). MATERIALS AND METHODS methods for obtaining sporocysts of Meiogymnophallus minutus and electron microscopical techniques have been described previously (Al-Salman & Laboratory

James,

1988). RESULTS

Light microscopy The daughter sporocyst of Meiogymnophallus minutus is an elongate sac with an anterior tapering extremity containing the birth pore and a posterior rounded extremity. The youngest infestation found consisted of hundreds of daughter sporocysts containing germinal balls and therefore must have included mostly second and subsequent generations but, perhaps, some of the first generation. Older infestations consist of daughter sporocysts containing germinal balls and cercariae at all stages in development. Finally, infestations of ageing daughter sporocysts were found containing only fully formed cercariae. Electron microscopy The body wall (Figs. 1, 2, 19) consists of a syncytial tegument resting on a basement lamina, circular and longitudinal muscle fibres and nucleated subtegumentary cells. Some of these cells pass cytoplasmic bridges through the muscle fibres and basement lamina to join the syncytial tegument. The syncytial outer tegument is approximately 0.7 pm thick (Fig. 19). The outer plasma membrane is normally thrown into numerous folds (Figs. 2, 8, 13, 14) bearing blunt irregularly branched microvilli measuring 24 pm long and about 0.05 pm wide. One or more bulges may occur along their length or at their tips (Figs. 2,8). In places, the outer plasma membrane at the base of the microvilli is invaginated (Figs. 2,8,9, 13). There is no nucleus, endoplasmic reticulum or Golgi apparatus in the tegument. The mitochondria are infrequent but more numerous near the inner plasma membrane than elsewhere in the tegument.

They are oval to round in shape with an electron-dense matrix and few cristae (Figs. 2,4, 12, 13). Numerous spherical membrane bound microvesicles occur throughout the tegument. Some (Figs. 47,9) appear to be micropinocytotic in origin and are the most abundant, measuring 0.2 pm in diameter approximately. They are either empty or contain electron light and flocculent material, with electron density at the periphery or at the centre, suggesting different stages of digestion (Figs. 4, 5, 7, 9). Some appear to coalesce and form large digestive vesicles (Fig. 13). The second type are clearly secretory bodies and are usually less frequent and smaller, measuring about 0.14 pm in diameter approximately. Their contents are homogeneously electron dense (Fig. 1). Sometimes (Fig. 1), however, they almost fill the tegument. The inner plasma membrane undulates and rests on a basement lamina (Figs. 1, 2, 12, 13). Occasionally distinct small electron dense hemidesmosomes attach the inner plasma membrane to the basement lamina (Fig. 12). Short tubular invaginations of the inner plasma membrane (Figs. 5, 12) intrude into the tegument, some having ruptured tips (Fig. 10). The basement lamina forms a continuous layer interrupted only by the cytoplasmic bridges which connect the subtegumentary cells to the syncytial tegument (Fig. 9). The body wall musculature is not extensive but consists of circular and longitudinal muscle fibres (Figs. 5, 12) with essentially the same fine structure. They are attached to the basement lamina by hemidesmosomes (Fig. 12). The muscle fibres are connected to subtegumental myoblasts (Figs. 2, 5). The cellular subtegument (Fig. 19) is approximately 6 pm in depth and contains five kinds of cells. These include (1) flame terminal cells with an elongate nucleus and oval to round mitochondria, ribosomes, Golgi apparatus and glycogen granules in the cytoplasm. Cilia and leptotriches of the luminal plasma membrane extend into the excretory tubule (Fig. 7). (2) Myoblasts have a slightly lobed nucleus and cytoplasm packed with glycogen granules together with some mitochondria, free ribosomes and granular endoplasmic reticulum (Figs. 2, 5). (3) Germinal cells (Fig. 14) have a large nucleus surrounded by little cytoplasm. Occasionally these appear free in the brood chamber of the sporocyst. (4) Secretory cells (Figs. 1, 3) which are infrequent, synthesize membrane-bound electron-dense secretory bodies which are delivered to the syncytial tegument

FIGS. 1 and 2. Transmission electron micrographs of body wall of daughter sporocyst of Meiogymnophallus minurus. FIG. 1. Microvillous tegument packed with electron-dense secretory bodies (sb), which appear as electron lucid-bulges in the microvilli (arrows), before being secreted into the host’s haemolymph, and some residual bodies (rb) which also appear to pass out via the microvilli (Fig. 19). Subtegument with two secretory cells (SC) and digestive cells at digestive phase (Dp) in digestive cycle packed with digestive vacuoles (dv), some residual bodies (rb), Golgi (go), microvesicles (mv) and dense bodies (db) in perinuclear cytoplasm. bl, basement lamina. FIG. 2. Microvillous tegument (T) and subtegument containing digestive cells, at absorptive (ABP), storage (Sp) and transition between storage and autophagic (AUP) phases in digestive cycle. bl, basement lamina; Ce, extension of digestive cells lining brood chamber (BC); es, extracellular space in subtegument; ace, origin of cytoplasmic extension of digestive cell; g, glycogen; rb, residual bodies; M, mitochondria; MB, myoblast; N, nucleus. Note small electron-lucid bulges (arrows) in microvilli and picnotic invaginations at their bases (pi).

Nutrition in M. minute daughter sporocysts

501

--

FIGS.1 and 2.

.*

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I-I. A. AL-SALMAN and B. L. JAMES

FIGS. 3-8.

Nutrition in M. minutus daughter sporocysts

FIGS.9-14.

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H. A. AL-SALMAN and B. L. JAMES

(Fig. 1) via cytoplasmic bridges. (5) Large digestive cells (Figs. 1, 2, 6-14, 19) which form the bulk of the subtegument and extend in between the other cells. They contain a large round to oval nucleus with a prominent nucleolus (Figs. 2,11,14). The cytoplasm is extensive and may contain ribosomes, Golgi reticulum, apparatus, granular endoplasmic mitochondria, glycogen granules, secretory bodies and a variety of membrane-bound vesicles. As described below, the contents of the cytoplasm appear to undergo cyclical changes related to nutrition, digestion and storage. They are connected to the tegument by cytoplasmic bridges (Fig. 9) and thin elongate extensions (Figs. 2,7,19) of these cells line the sporocyst lumen or brood chamber. These extensions are rich in glycogen (Figs. 2, 7) which is probably released into the brood chamber and absorbed by the developing cercariae (Fig. 18). Wide extracellular spaces occur between the subtegumentary cells (Figs. 2, 10, 14, 19). Postulated mechanism offeeding

and digestion

In addition to protection from the host’s defence responses, the principle function of the daughter sporocyst body wall must be nutrient uptake and digestion in order to feed the developing germinal balls. Thus most of the ultrastructure suggests adaptation to feeding and digestion. The electrondense secretory bodies synthesized in some subtegumentary secretory cells (Figs. 1,19) sometimes

pass into the tegument in considerable numbers (Fig. 1) and appear to be secreted into the host’s haemolymph (Fig. 1). During secretion, the dense bodies become lucid as they move up the microvilli (Fig. 1, 2) before being pinched off at the tips (Fig. 8). These may be hydrolytic enzymes for exogenous digestion of host nutrients. Glucose, amino acids and fatty acids may diffuse or may be actively absorbed over the entire surface of the microvillus but undigested or partly digested nutrients may be taken up at the base by micropinocytosis (Figs. 2, 8, 9, 13). The ingested micropinocytotic vesicles fuse to form larger microvesicles (Figs. 4, 5, 6, 7, 9). Some of these appear to form digestive vesicles (Fig. 13) and, later residual bodies (Fig. 12) in the tegument. Most microvesicles, however, are transported via cytoplasmic bridges into the subtegumentary digestive cells where they may accumulate and almost fill the cytoplasm. This represents the absorptive phase of the digestive cells (Figs. 2, 6, 8). Prior to absorption, the synthetic phase (Fig. 4) is marked by the presence of some endoplasmic reticulum with few ribosomes, Golgi apparatus, mitochondria and microvesicles presumably containing lysosomal enzymes which condense to form larger electron-dense bodies which later fuse with the incoming microvesicles to form digestive vesicles (Fig. 1). The digestive phase (Fig. 1) is marked by the presence of digestive vesicles of varying sizes which almost fill the cytoplasm. As digestion proceeds the digestive vesicles are

FIGS. 3-8. Transmission electron micrographs of body wall of daughter sporocyst of Meiogymnophallus minutus. FIG. 3. Secretory cell at early stage in development showing granular endoplasmic reticulum (ger), Golgi (go), and microvesicles (mv) involved in synthesis of electron-dense secretory bodies. FIG. 4. Detail of digestive cell at synthetic phase showing Golgi (go) and mitochondria (m) involved in synthesis of microvesicles (mv). er, endoplasmic reticulum; ml, mitochondria and mvl, microvesicles in tegument (T). FIG. 5. Digestive cells at absorptive phase in digestive cycle, showing cytoplasm packed with microvesicles (mv). cm, circular muscles; g, glycogen; lm, longitudinal muscles; m, mitochondria; MB, myoblast; mv, microvesicles in tegument (T); rb, residual bodies; arrows, tubular invagination of inner plasma membrane of tegument. FIG. 6. Digestive cell at absorptive (ABP) and autophagic (AUP) phases in digestive cycle. Note microvesicles (mv) in tegument (T), absorptive cells (ABP) and autophagic vesicles (av) in autophagic cell (AUP) which may contain lytic enzymes. FIG. 7. Digestive cell at absorptive phase of digestive cycle packed with phagosomes, flame terminal cell with cilia (ci) and leptotriches (f) ofplasma membrane in excretory tubule. mv, microvesicles in tegument (T). ce, extension of digestive cells lining brood chamber. FIG. 8. Digestive cell showing transition between storage and absorptive phases in digestive cycle with abundant glycogen (g) and microvesicles (mv) in cytoplasm. Note micropinocytotic vesicles (mp) in tegument and electron-lucid bulges (arrows) in branching microvilli. m, mitochondria. FIGS. 9-14. Transmission electron micrographs of body wall of daughter sporocyst of MeiogymnophaNus minutus. FIG. 9. Cytoplasmic bridge (arrow) between tegument and subtegumentary digestive cell at digestive phases in digestive cycle. Note micropinocytotic vesicles (mp) and microvesicles (mv) in tegument and digestive vacuoles (dv) in subtegument. FIG. 10. Digestive cell at storage phase in digestive cycle showing extracellular spaces (es) in subtegument and invagination of inner plasma membrane of tegument with ruptured extremity (arrow). ce, cytoplasmic extension; g, glycogen; rb, residual body. FIG. 1 I. Digestive cell at storage phase in digestive cycle packed with glycogen (g). N, nucleus; rb, residual body. FIG. 12. Inner tegument and digestive cell in subtegument at early autophagic phase in digestive cycle with much glycogen (g) still in cytoplasm or enclosed in autophagic vacuoles (av). bl, basement lamina; Im, longitudinal muscle; cm, circular muscle; hemidesmosomes (short arrows); ml, mitochondrion in tegument (T); m2, mitochondria in digestive cell; long FIG. 13. Tegument showing microvilli with arrows, tubular invagination of inner plasma membrane of tegument. micropinocytotic vesicles at their bases (arrow) and mitochondrion (m) and digestive cell in subtegument at late autophagic phase in digestive cycle showing autophagic vesicles (av) and cytoplasm without glycogen. Note digestive vesicles (dv) and FIG. 14. Digestive cell at resting phase in digestive cycle, showing residual bodies (rb) in tegument. bl, basement lamina. cytoplasm with little glycogen and little or no evidence ofactivity. Note large extracellular spaces (es) and germinal cell (Cc) in subtegument.

Nutrition

in M. minutus daughter

sporocysts

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FIGS. 15-18. Transmission electron micrographs of various stages in degeneration of body wall in ageing daughter sporocysts of Meiogymnophallus minufus containing healthy fully formed cercariae (C). FIG. 15. Showing vacuolated tegument (T) and subtegument (ST). FIG. 16. Showing increased vacuolation of tegument (T) and subtegument (ST) FIG. 17. Showing tegument has been lost and vacuolated subtegument (ST). and most microvilli lost from tegument. FIG. 18. Investing syncytium (in) of developing cercaria (C) packed with micropinocytotic vesicles (mp). m, mitochondria.

transformed into residual bodies and the digested nutrients accumulate mostly as Pglycogen in the cytoplasm. This represents the storage phase (Figs. 2, 10, 11). The residual bodies may pass presumably through the cytoplasmic bridges into the tegument and then via large bulges in the microvilli (Figs. 1, 19) to be pinched off into the host’s haemolymph or via the extensions of the digestive cells into the sporocyst brood chamber. The glycogen also passes into the cytoplasmic extensions of the digestive cells (Fig. 2) where it is available for the developing embryos (Fig. 18). Glycogen is also digested in autophagic vacuoles in the parenchyma cells during the autophagic phase in the digestive cycle (Figs. 6, 12). Presumably, the resulting glucose diffuses into other subtegument cells for metabolism. A considerable amount is, however, stored as glycogen in the myoblasts (Figs. 2, 5) prior to utilization. After digestion of endogenous glycogen the digestive

cells may pass through a brief resting phase (Fig. 14) before returning to the synthetic and absorptive phases. Although the synthetic, absorptive, digestive, storage, autophagic and resting phases have been described separately, the process is continuous. Thus, many cells exhibit the transition from one phase to another (Fig. 2). Sometimes cells appear to miss out phases, for example, cells showing transition between storage and absorption are present (Fig. 8). All phases in the digestive cycle may occur in the same daughter sporocyst but only in specimens containing developing embryos. The body wall, in ageing daughter sporocysts which contain only fully formed cercariae, appears to stop feeding. The tegument and subtegument become highly vacuolated (Fig. 15). Later the microvilli disappear (Fig. 16) and, finally, the tegument is lost (Fig. 17). Continuing autolysis (Figs. 15-17) in the subtegument will result in the rupture of the body wall and the release of the remaining cercariae.

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H. A. AL-SALMAHand B. L. JAMES

PIG. 19. Diagrammatic repre~ntatio~ of uItrastru~ture of body wall of daughter spotocyst of ~ejog~mno~hal~~ minutus showing microvillous tegument (T) and five kinds of subtegumentary cells, namely flame terminal cells (FC), germinal cells (CC), myoblast (MB), digestive cells(PC), and secretory cells(SC). Digestive ceils undergo a digestive cycle involving synthetic (SP), absorptive (ABP), digestive (DP), storage(S), autolytic (AUP), and resting (RP) phases. Features of synthetic to storage phases are diagrammatically included in the same cell (PC). av, autophagic vesicles; bl, basement lamina; cb, cytoplasmic bridge; ce, cytoplasmic extension lining brood chamber; ci, cilia; cm, circular muscle fibres; dv, digestive vesicles; es, extracellular space; f, leptotriches; g, glycogen; ger, granular endoplasmic reticulum; go, Golgi; h, hemidesmosomes; lb, lucid bulges; lm, longitudinal muscle fibres; m, mitochondrion; N, nucleus; P, phagosomes; rb, residual bodies; sb, secretory bodies.

DISCUSSION The body wall of young daughter sporocysts of ~eiugymnophallus minutes is similar to that of rediae

(Rees, 1966, 1971; Krupa, Bal & Cousineau, 1967; Imohiosen, 1969, thesis cited above; Koie, 1971a) and other daughter sporocysts with birth pores (Bibby & Rees, 1971; Kinoti et al., 1971; Koie, 1971b; Reader, 1975; Sannia, 1977, thesis cited above) in that it consists of a syncytial microvilfous tegument resting on a basement lamina, muscle fibres and subtegumentary cells, including digestive, myoblast, germinal, flame and secretory cells which are connected by cytoplasmic bridges. The body wall musculature of the daughter sporocysts of Meiogymnophallus minutus appears to be more powerfully developed than previously described by Sannia (1977, thesis cited above). Sannia (1977, thesis cited above), however, may have based his description

on older specimens. The cytoplasmic extensions of the parenchyma cells which line the brood chamber have also been recorded in other species (James & Bowers, 1967b; Ksie, 197 1a, b; Reader, 1975; Meuleman & Holzmann, 1975; Sannia, James & Bowers, 1978). They appear to be least developed in Labratrema minimus (= Cercaria bucephaiopsis haimeanus) and Gymnophallus choledochus (= Cercaria dichotoma) where they surround only germinal balls and masses at an early stage in development (James & Bowers, 1967b) and most developed in Cercaria cerastoderma I where they form a central brood chamber within the sporocyst lumen and also surround individual metacercariae (Sannia et al., 1978). Their function, as testified by the presence of glycogen, always appears to be nutrition of the developing embryos. Meuleman & Holzmann (1975) suggest that, in Schistosoma mansoni, the

Nutrition in M. minurus daughter sporocysts investing syncytium surrounding the developing cercaria is formed from cytoplasmic extensions of the subtegument. Kraie (1971a,b) found, in Neophusiu Iageniformis and Cercaria buccini, that they disintegrate as their contents are released into the brood chamber. This does not occur in the present species where glycogen is probably digested before being released as glucose into the brood chamber. At an early stage in development, the daughter sporocyst is mostly full of germinal balls but a few cercariae develop precociously and are released through the birth pore. When all the contained cercariae are fully formed, however, release occurs when the microvilli disappear and the daughter sporocyst ceases to feed, degenerates and ruptures. This also occurs in daughter sporocysts of Meiogymnophalius minutus full of second generation daughter sporocysts (Sannia, 1977, thesis cited above), in the daughter sporocyst of Podocotyle staflordi containing cercariae as described by Gibson (1974) and in all daughter sporocysts without birth pores (Popiel & James, 1978). The rupture and death of the fully formed daughter sporocysts and the release and escape of the cercariae suggest that some long lived primary molluscan hosts may recover from the infestation by Digenea. Since the incidence of infection does not increase with age and infected specimens survive as long as healthy specimens in the laboratory, recovery after infection by Meiogymnophallus minutus seems possible in Scrobicularia

plana.

The role of the microvilli is enigmatic. As stated previously, micropinocytotic vesicles occur at their bases and the release of hydrolytic enzymes and residual bodies may occur at their tips. Although some simple nutrients may be absorbed over the entire surface, there is no direct evidence for this here or in the literature. Some electron lucid bulges (Figs. 1,2,8), which appear to move up from the base to the tip and are extruded, may be the mechanism of mucopolysaccharide secretion. However, synthetic cells containing membranebound flocculent mucopolysaccharide bodies were not found in the subtegument of the daughter sporocysts of M. minutus. Nutrient uptake by micropinocytosis and digestion in the tegument has been observed previously in daughter sporocysts and rediae (Rees, 1966; Southgate, 1970; Bibby & Rees, 1971; KBie, 1971a; Reader, 1975), but as far as we are aware the digestive rhythm in subtegumental digestive cells has not been described in any other species. It has been known for some time that the digestive gland cells in intertidal molluscan hosts undergo a rhythm of digestive activity correlated with the tides (Morton, 1970; Mahmood, unpublished Ph.D. thesis, University College, Swansea, 1980). It is unlikely, however, that the digestive rhythm in the daughter sporocysts of M. minutus is related to rhythm in the host or to the tides, as all phases, from the synthetic to resting, are found in the same daughter sporocyst. If

507

the digestive rhythm in the daughter sporocyst was correlated with the tides, all the digestive cells in all sporocysts taken from a single host would be more or less at the same phase of the cycle. The observation of cells exhibiting transitions from one phase into another excludes the remote possibility that separate specialized cells occur with a single function, namely absorption, digestion or storage. Acknowledgements-We are grateful to Professors E. W. Knight-Jones, J. S. Ryland and J. A. Beardmore for the

provision of research facilities. REFERENCES AL-SALMAN H. A. &JAMES B. L. 1988. Ultrastructure

of the tegument in daughter sporocyst and cercarial embryos of Meiogymnophallus minutus (Digenea: Gymnophallidae).

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Ophelia 9: 165-206. KRUPA P. L., BAL A. K. & COUSINEAU G. H. 1967. Ultrastructure of the redia of Cryptocofyle lingua. Journal of Parasitology 53: 725-734. MEULEMAN E. A. 1972. Host-parasite inter-relationships between the freshwater pulmonate Biomphalaria pfeifferi and the trematode Schistosoma mansoni. Netherlands Journal of Zoology 22: 355427. MEKJLEMANE. A. & HOLZMANNP. J. 1975. The development of the primitive epithelium and true tegument in the mansoni. Zeitschrtft fiir cercaria of Schistosoma Parasitenkunde 45: 307-3 18. MEULEMANE. A., HOLZMANNP. J. & PEET R. C. 1980. The development of daughter sporocysts inside the mother sporocyst of Schistosoma mansoni with special reference to the ultrastructure of the body wall. Zeitschrif fiir Parasitenkunde 61: 201-212. MORTON B. 1970. The tidal rhythm and rhythm of feeding and digestion in Cerastoderma edule. Journal of the Marine Biological Association of the United Kingdom 50: 499-512. POPIEL I. SC JAMES B. L. 1978. The ultrastructure of the daughter sporocyst of Microphallus similis (Jig., 1900) (Digenea: Microphallidae). Parasifology 76: 359-367.

READER T. A. J. 1975. Ultrastructural, histochemical and cytochemical observations on the body wall of the daughter sporocyst of Cercaria helverica XII (Dubois, 1927). Zeitschr(ft fiir Parasitenkunde 45: 243-261. REES G. 1966. Light and electron microscope studies of the redia of Parorchis acanthus Nicoll. Parasitology 56: 589602. REES G. 1971. The ultrastructure of the epidermis of the redia and cercaria of Parorchis acanrhus Nicoll. A study by scanning transmission electron microscopy. and Parasitology 62: 479-488. SANNIA A., JAMES B. L. & BOWERS E. A. 1978. The morphology of Cercaria cerasroderma I nom. nov. (Monorchiidae) (= Cercaria lepidapedon rachion (Cobbold, 1858) sense Lebour, 1908) a rare digenean parasite of the cockle in Britain. Journal of Natural History 12: 487-500. SMITH J. H. & CHERNIN E. 1974. Ultrastructure of young mother and daughter sporocysts of Schisrosoma mansoni. Journal of Parasitology 60: 85-89. SOUTHGATEV. R. 1970. Observations on the epidermis of the miracidium and on the formation of the tegument of the sporocyst of Fasciola hepatica. Parasitology 61: 177-190.