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The forebody glands and surface features of the metacercariae and adults of Microphallus similis
International Journal for Parasitology. Vol. 9, pp. 553-564. Pergamon Press Ltd. 1979. Printed in Great Britain. 0 Australian Society for Parasirology.
0020-7519/79/1201-0553
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THE FOREBODY GLANDS AND SURFACE FEATURES OF THE METACERCARIAE AND ADULTS OF MICROPHALLUS SIMILIS CAROLINE DAVIES Department of Zoology and Applied Entomology, Imperial College, Prince Consort Road, London SW7 2BB, England (Received 30 January 1979)
Abstract-Dares
C. 1979. The forebody glands and surface features of the metacercariae andadults of
Microphallus similis. International Journal for Parasitology 9: 553-564. Numerous unicellular glands are present in the forebody of the metacercariae and adults of Microphallus similis. The gland cell
bodies lie beneath the tegumental perinuclear cytoplasm and ducts pass upwards penetrating the tegumental distal cytoplasm. Electron dense granules are secreted onto the forebody surface and into the oesophagus via small pits in the tegument. Histochemical tests revealed that the glands contain diastase-resistant neutral mucosubstances, RNA, protein, esterase and small amounts of acid phosphatase; inhibitor studies indicated that the esterase was acetylcholinesterase (EC 3.1.1.7). Ultracytochemistry showed that the mucosubstances were located in the general cytoplasm of the gland cells and that cholinesterase was present in the granules. The forebody of M. similis is covered by large toothed spines whereas the hindbody only bears short peg-like spines. It is suggested that the forebody spines may have an irritating, abrasive effect on the host’s intestinal mucosa and that the secretion of acetylcholinesterase in the region of these spines may produce a ‘local anaesthetic’ effect on the host’s gut by reducing movement in the immediate vicinity of the Auke, so decreasing the likelihood of expulsion. INDEX KEY WORDS: Microphallus similis; unicellular glands; spines; cytochemistry; chemistry; acetylcholinesterase (EC 3.1 .1.7).
INTRODUCTION THE METACERCARIAE of Microphallus similis are encysted in the digestive gland of Carcinus maenas and the adults are found in the small intestine of gulls of the genus Larus (Stunkard, 1957) although various laboratory rodents can also act as definitive hosts (James, 1971). Considerable growth and development occurs within the metacercarial cyst and the mature encysted metacercaria possesses well-defined genitalia in an advanced stage of development so that maturation in the final host is completed in 2-3 days (Davies & Smyth, 1979). A conspicuous feature of microphallids is the presence of numerous unicellular glands in the forebody between the oral and ventral suckers (Strong & Cable, 1972). Similar gland cells (called subcuticular cells) have also been described in the forebody of various strigeids and histochemical studies have shown that they contain non-specific esterases (Ohman, 1965, 1966; Bogitsh, 1966). It has been suggested that the secretion of esterases by strigeids functions in the extra-corporeal digestion of host tissues (Erasmus, 1972). In the present study ultrastructural, cytochemical and ultracytochemical techniques have been used to investigate the structure and possible function of the forebody glands of Microphallus similis.
ultracyto-
MATERIALS AND METHODS Metacercariae of Microphallus similis were recovered from naturally infected Curcinus maenas (supplied by the University of London Marine Biological Station, Millport) and excysted or fed to mice according to the techniques described by Davies & Smyth (1979). Cercariae of M. similis were shed from naturally infected Littorina saxatilis (provided by Dr. B. L. James, University of Swansea). Transmission electron microscopy (TEM). Cercariae, excysted metacercariae and adults recovered from mice 4 days post-infection were fixed, processed and examined according to the methods described by Davies (1978). Scanning electron microscopy (SEM). Excysted metacercariae and 4 day old adults were fixed in cold (4°C) or hot (60°C) 4% glutaraldehyde in 0.1 M-phosphate buffer (pH 7.2-7.4), dehydrated in ethanol, dried in a Polaron Critical Point Drier, mounted on stubs with double-sided Sellotape, coated with gold and examined on a Cambridge Stereoscan Scanning Electron Microscope at 10 kV. Cytochemistry. Excysted metacercariae and 4 day old adults were fixed in 4% glutaraldehyde in 0.1 M-phosphate buffer (pH 7.2-7.4). For the lipid and enzyme tests, the fixed flukes were washed in buffer, embedded in Tissue-Tek OCT compound (Miles Laboratories), cut on a Slee cryostat at 10 urn and collected onto glass microscope slides. For the other tests, the fixed flukes were washed in buffer, dehydrated in ethanol and embedded in the water-soluble plastic embedding medium
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JB4 (Polysciences Inc.). Semi-thin sections were cut on an ultramicrotome at l-2 urn and transferred to glass slides. Unless otherwise stated, the following histochemical tests were carried out according to Bancroft (1975) except that after staining the sections were not dehydrated but were dried and mounted in glycerolgelatin. Neutral mucosubstances: periodic acid-Schiff (PAS) with or without prior digestion with diastase to remove glycogen (Methods 19 and 21). Acid mucosubstances: alcian blue (Method 22). DNA/RNA: Methyl greempyronin (Method 70). Protein: aqueous mercury bromphenol blue (according to Pearse, 1968). Lipid: Oil Red 0 or Sudan Black B in isopropanol (Methods 54 and 55). Acid nhosvhatase (EC 3.1.3.2): kzo-dye method using naphthol AS-B1 phosphate and Red violet LB salt (according to Pearse, 1968). Alkaline phosphatase (EC 3.1.3.1): Azo-dye method using sodium a-naphthyl phosphate and Fast Red Salt TR (Method 101). Non-specific carboxyl esterase (EC 3.1.1.1): indoxyl method using 5-bromo, 4-chloro indoxyl acetate (Method 112). Cholinesterase: acid salt method using acetylthiocholine iodide or butyrylthiocholine iodide (according to Pearse, 1972-after the method of Gomori). In conjunction with the esterase and cholinesterase tests, some slides were pre-incubated in phosphate buffered solutions of various inhibitors at 37°C for 1 h. The effect of the following inhibitors was examined: non specific esterase-silver nitrate; general cholinesterase-diethylp-nitrophenyl phosphate (E600) (provided by Dr. D. Wright, Imperial College Field Station), eserine sulphate (Sigma); acetylcholinesterase (EC 3.1.1.7)-l :5-bis(4allyldimethyl ammoniumphenyl)-pentan-3-one dibromide (284C51) (Burroughs-Wellcome); pseudocholinesterase (EC 3.1.1.8)-tetraisopropyl pyrophosphoramide (iso OMPA) (Koch-Light), lo-(2-diethylaminopropyl) phenothiazine hydrochloride (Lysivane) (May & Baker). In addition to pre-incubation, the reversible inhibitors (284C51, Lysivane and eserine) were also included at the appropriate concentration in the test incubation medium. Ultracytochemistry. Excysted metacercariae and 4 day adults were fixed in the usual way in 4% glutaraldehyde (Davies, 1978) and transferred to 0.1 M-phosphate buffer (pH 7.2-7.4) where they were chopped into small pieces with a razor blade. Pieces of fluke were stained according to the periodic acid-thiocarbohydrazide-osmium tetroxide (PATCO) technique of Seligman, Hanker, Wasserkrug Dmochowski & Katzoff (1965) as modified by Oaks & Lumsden (1971) for the ultrastructural localisation of neutral mucosubstances. Some pieces of tissue were digested with 1% diastase in 0.1 M-phosphate buffer (pH 7.2-7.4) for 1 h at 37°C after fixation and prior to staining to remove glycogen. Staining for cholinesterase was carried out according to the Kasa & Csillik (1966) modification of the Karnovsky (1964) technique using acetylthiocholine iodide; the substrate was omitted in control treatments. In both the PATCO and cholinesterase techniques, thin sections were viewed without further staining.
RESULTS Ultrastructure of gland cells Opening onto the whole surface of the forebody of M. similis, especially in the region of the oral sucker, are the ducts of numerous unicellular glands.
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The fine structure of these gland cells appears identical in the excysted metacercaria and in the adult. The gland cell bodies lie beneath the tegumental cell bodies (tegumental perinuclear cytoplasm) and are irregular in outline with processes ramifying between adjacent parenchyma cells (Fig. 1). The majority of the gland cell is taken up by large, homogeneous, electron dense granules which are membrane bound. The granules vary in shape although most are oval to spherical measuring, on average, 0.4 urn in length. The cell bodies are nucleated and short lengths of granular endoplasmic reticulum (ger), mitochondria and numerous free ribosomes are also present in the cytoplasm.
Although conventional active-looking Golgi complexes were never seen, indistinct areas containing flattened sacs and vesicles were occasionally observed but they did not contain electron dense material. Ducts from the gland cells pass upwards between the tegumental cell bodies and peripheral musculature (Fig. 2). They are packed with granules which are sometimes less homogeneously dense than those in the cell bodies (Fig. 3) and they are lined with microtubules which are frequently seen in cross section (Fig. 4). The ducts penetrate the tegumental distal cytoplasm to which they are joined by short inconspicuous septate desmosomes and they open to the exterior of the fluke at the bottom of small pits in the tegument (Figs. 2 & 3). The granules appear to be extruded, along with some of the cytoplasm, in ‘packets’ which are not themselves membrane-bound (Fig. 3). The ducts also penetrate the tegument lining the oesophagus into which they empty their sections (Fig. 5). Gland cell bodies or the ducts from them were never observed posterior to the ventral sucker. Although the cercariae of M. similis possess cystogenous and penetration glands, cells corresponding to the forebody gland cells of the mature metacercaria and adult were not present. Cytochemistry
Histochemical tests revealed that the gland cells of both metacercariae and adults contain diastaseresistant neutral mucosubstances, protein, RNA and esterase. Small amounts of acid phosphatase were present in the gland cells of the adult but not in those of the metacercaria. The tests for acid mucosubstances, lipid and alkaline phosphatase all proved negative. The type of esterase that was present was investigated using various substrates and inhibitors (Table 1). Similar results were obtained in both excysted metacercariae and adults. Using acetylthiocholine as the substrate, a strong reaction product was obtained in the gland cells and ducts and in the suckers, pharynx and forebody musculature. This reaction was reduced but not abolished by treatment with IO-%I-eserine and 10-7~-E600
FIG. 1. Excysted metacercaria. Nucleated gland cell bodies lie beneath the tegumental cells and contain electron dense granules, mitochondria, ribosomes and granular endoplasmic reticulum (scale bar represents 1 urn). FIG. 2. Adult from mouse. Gland cell ducts pass between the peripheral musculature and penetrate the tegumental distal cytoplasm. The duct contains closely packed granules and is lined with microtubules (scale bar represents 1 urn).
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Forebody glands of Microphallus similis TABLE
I-Microphallussimilis: H~STOCHEMICALTESTSFORESTERASES CHOLINESTERASESINEXCYSTEDMETACERCARIAEAND ADULTS Substrate
Indoxyl acetate Indoxyl acetate Indoxyl acetate Indoxyl acetate Acetyl thiocholine Butyryl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine
Inhibitor
Gland cells
lo-%f-&NO,
lo- %-eserine lo-%-eserine lo-WAgNO, lo- %-eserine lo-%-eserine lo- 5~-E600 lo-‘M-E600 10-3~-284C51 lo- 4~-284C5 1 10-5~-284C51 10-6w-iso OMPA lo-%-is0 OMPA 10-4r+Lysivane lo-%-Lysivane
Key: + + + intensely stained; stained; - not stained.
strongly suggesting that the esterase is a cholinesterase. The results of further inhibitor studies using selective inhibitors for acetylcholinesterase (284C51) and for pseudocholinesterase (Lysivane and iso OMPA) and the fact that butyryl thiocholine was hydrolyzed less efficiently than acetylthiocholine, indicates the presence of a true cholinesterase (acetylcholinesterase, EC 3.1 .1.7.) in both gland cells and muscles. The cholinesterases in the two sites behaved slightly differently in their response to inhibitors (Table l), that of the gland cells being generally more resistant.
Ultracytochemistry
Using the PATCO technique, neutral mucosubstances were localised in the cytoplasm of the gland cells (Fig. 6); the granules themselves were negative. The reaction product appeared to be diastase-resistant which suggests that the mucosubstance was not glycogen. However, some glycogen rosettes were still present in the parenchyma cells, although they were much reduced in number after diastase digestion, indicating that the diastase
+++ + ++ +++ ++ +I+ ++ +I+ ++ +++ +++ +++ +++
+ + moderately stained;
AND
Muscles
+++ +/+ +++ ++ +i+I+ +++ +++ +++ +++ + slightly
digestion may have been incomplete. The granules were positive for cholinesterase using acetylthiocholine as substrate although not all the granules in each gland cell were stained (Fig. 7). Control treatments, without substrate, were negative (Fig. 8). Surface features No obvious differences were noted between the
surface features of excysted metacercariae and adults. Although the shape of the fixed flukes varied considerably, they were generally pyriform in outline with a slight lateral constriction in the region of the ventral sucker (Fig. 9). The surface of the fluke was divided into two distinct regions with different morphologies-the forebody anterior to the ventral sucker and the hindbody posterior to it. The lateral edges of the forebody tend to curl towards the ventral surface giving it a cuplike appearance. The forebody (Fig. 13) was covered with posteriorly-pointing, scale-like spines which were serrated at their tips having 12-13 teeth. These spines were arranged in a quincuncial pattern and embedded in pits in the tegument. In many cases, especially in those flukes which had been fixed cold,
FIG. 3. Excysted metacercaria. Opening of gland cell duct. Some of the granules in the apical portion of the duct are less dense (arrowed). The granules are secreted into a pit in the tegument in the form of a small packet (scale bar represents 1 pm). FIG. 4. Adult from mouse. Cross section of gland cell duct showing peripheral microtubules (scale bar represents 0.5 urn). RG. 5. Excysted metacercaria. Gland cell ducts open into the oesophagus which is lined with tegument (scale bar represents 1 urn).
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they overlapped completely and obscured the structure of the underlying tegument. However, when hot fixative was used some of the flukes were fixed with the forebody extended and small pores could be seen in the tegument (Fig. 13-inset). The diameter of these pores was approx. 0.5 pm which corresponds to the width of the gland cell openings seen in thin sections. The surface of the oral sucker (Fig. 11) was covered in similar toothed spines although the lip of the sucker and its lining were aspinose and in this region the tegument was corrugated. The ventral sucker (Fig. 10) was also spined although in the central cup-shaped region the spines were smaller and less frequent and the surface tegument was folded. The opening of the genital atrium could be seen as a deep furrow on one side of the ventral sucker (Fig. 10). In hot-fixed flukes, which were generally more relaxed than those fixed cold, the protruding male papilla could occasionally be seen; it had a smooth aspinose appearance. Sensory papillae were present over the whole surface of the fluke but were more frequent on the ventral surface of the forebody especially around the suckers (Figs. 10 & 11). In contrast to the large, toothed spines on the forebody, those on the hindbody posterior to the ventral sucker were small and peg-like (Fig. 14) and, although they were evenly spaced, they did not appear to be arranged in any regular pattern. The surface of the hindbody tegument was thrown up into ridges which ran around the spines. Superimposed on this were deeper circumferential or longitudinal corrugations, the distribution of which seemed to depend on the posture of the fluke at the time of fixation (Fig. 9). The surface around the excretory pore was similar to that of the general hindbody except that spines were completely lacking (Fig. 12). DISCUSSION The unicellular glands of Microphallus similis are similar in ultrastructure to gland cells associated with the general tegument or specialised areas of the tegument in other helminths, e.g. the lappets of strigeids such as Apafemon gracilis and Diplostomumphoxini (Erasmus, 1969a, b); the subcuticular cells of Posthodiplostomum minimum (Bogitsh & Aldridge, 1967); the scolex glands of pseudophyllidean cestodes (ohman-James, 1973; Arme &
Threadgold, 1976); the glands associated with the oral sucker of Haplometra cylindracea and Opisthoglyphae ranae (Halton & Dermott, 1967); the forebody and haptoral glands of Aspidogaster conchicola (Halton & Lyness, 1971; Bailey & Tompkins, 1971); and the subtegumental glands of ~eiogym~ophallas minutes (Davies, 1976, unpublished Ph.D. thesis, University of London). However, similarity in ultrastructure does not necessarily imply similarity in function or in the chemical composition of the secretory products produced by the glands. Various functions have been suggested for such gland cells. The lappet glands of A. gracilis and D. phoxini (ijhman, 1965, 1966) and the subcuticular cells of P. minimum (Bogitsh, 1966) secrete esterases and it has been suggested that they function in the extracorporeal digestion of host tissues. The bothria glands of Diphyl~o~othriam ditremam do not produce an enzymic secretion and ~hman-James (1973) has suggested that they may have an adhesive function and aid in attachment. The oral sucker glands of 0. ranae contain neutral and acid mucopolysaccharides (Halton, 1967) and Halton & Dermott (1967) suggest that, amongst other possible functions, the secretions may help protect the parasite against host digestive enzymes. In ~i&rapha~las similis all the evidence suggests that the gland cell secretions are enzymic in nature and that they contain acetylcholinesterase. Mucosubstances are also present in the gland cells but they are located in the general cytoplasm surrounding the granules. It has been recognised (Silver, 1974) that the cholinesterases associated with the nervous system of parasitic heirninths behave differently in their susceptibility to inhibitors than those of vertebrates; in general, they exhibit much greater resistance and higher concentrations of inhibitors are required. Vertebrate cholinesterases are completely inhibited by lo-Weserine and 5 x 10-5~-284C51 (Pearse, 1972) whereas heIminth cholinesterases often show only partial inhibition at these concentrations and require higher concentrations for complete inhibition (Fripp, 1967; Halton, 1967; Lee & Tatchell, 1964). In the present case the gland cell choline&erase appears to be even more resistant than that associated with the neuromuscular system indicating that it is probably of a slightly different type. Apart from their presence in the neuromuscular system of helminths, cholinesterases have also been found in various other sites, e.g. the tegument of
FIGS. 6-8. Excysted metacercariae-ultracytochemistry FIG.
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(scale bar represents 1 pm).
6. PATCO technique. The electron dense reaction product is located in the cytoplasm around the granules. It is also present in adjacent parenchyma cells.
FIG. 7. Localisation of cholinesterase. The gland cell granules are stained although not all granules show a positive reaction (arrows). FIG 8. Localization of choiinester~e~ontrol (without substrate). Negative.
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cestodes (Hymenolepis sp. (Lee, Rothman &Senturia, 1963; Schardein & Waitz, 1965; Rothman, 1966; Bogitsh, 1967), Hydatigera taeniaeformis (Lee et al., 1963; Schardein & Waitz, 1965), Dipylidium car&urn (~hardein & Waitz, 1965), A~oplocep~aia perfoliata (Lee & Tatchell, 1964)) and digeneans (Sch~stosoma sp. (Fripp, 1967), Fasciola hepatica (Halton, 1967)); the hydatid cyst wall of Echinococcus (Schwabe, Koussa & Acra, 1961); the reproductive ducts of Fasciob hepatica (Halton, 1967) and Anoplocephala perfoliata (Lee & Tatchell, 1964); and the excretory glands of various gut parasitic nematodes (reviewed by Yeates & Ogilvie, 1976). iihman (1966) describes a “non-specific esterase of resistant type, possibly a non-specific cholinesterase” in various sites in A. gracilis including the subcuticular cells. Although Bogitsh (1966) describes the esterases of the subcuticular cells of P. minimum as non-specific, he obtained a positive reaction using acetylthi~hoIine iodide as substrate and this reaction was partially inhibited by IO- %-eserine, suggesting a cholinesterase. It seems possible, therefore, that strigeid subcuticular glands may also contain cholinesterases. Various functions have been suggested for the presence of cholinesterases in helminths. In the te~ment it has been suggested that cholinesterase is involved in transport processes and uptake (Rothman, 1966; Bogitsh, 1967; Halton, 1967). Schwabe et al. (1961) suggest that the cholinesterase in the hydatid cyst wall of Echinococcas is involved in permeability control and osmoregulation. Several theories have been put forward to account for the presence of cholinesterases in the excretory glands of nematodes (Yeates & Ogilvie, 1976). These include the suggestions that they may reduce the host’s immune response, that they may help the parasite to feed by increasing the availability of glucose or that they may act as a kind of local anaesthetic on the host gut to reduce peristalsis and so prevent parasite expulsion. The latter hypothesis seems very attractive in the case of Microphallus similis especially when the distribution of the gland cells is related to the surface features of the Auke and its mode of attachment in the host’s intestine. In the seagull it is the forebody of M. similis which is embedded between the host’s intestinal villi while the hindbody projects into the lumen of the intestine (Dr. B. 1;. James, personal
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communication). SEM reveals that the forebody is covered with large, backwardly-pointing, toothed spines. Such spines have been described in other digeneans associated with the gastro-intestinal tract of various hosts e.g. F. hepatica (Bennett, 1975), Metago~~imas yo~oga~ai (Inatomi, Tongu, Sakumoto, Suguri & Itano, 1968), Diplostomum phoxini (Erasmus, 1970) Prosorhynchus thapari (Bilquees, 1976), Cryptocotyle lingua (Koie, 1977), Neophasis lageniformis (Kaie, 1973) and Meiogymnophallus minutus (Davies, 1976, unpublished Ph.D. thesis, University of London). They probably aid in maintenance of position but must also have a very irritating effect on the host’s intestinal mucosa. The presence of these spines in M. similis coincides with the distribution of the cholinesterase secreting glands. The hindbody, which is not in such intimate contact with the host and which is spineless or bears much reduced spines, is not provided with such gland cells. It seems possible, therefore, that the cholinesterase secreted over the surface of the forebody may serve to compensate for the irritating effect of the toothed spines by neutralizing host acetylcholine and so reducing the movement of the villi in the immediate vicinity of the fluke thereby reducing the likelihood of dislodgement and expulsion. Nizami, Siddiqi & Islam (1977) made a quantitative analysis of the levels of cholinesterase in seven species of digeneans and concluded that it is present in “remarkably high quantities” in species which inhabit the gastro-intestinal tract compared with those that parasitize the liver or the swim bladder. These authors also suggest that the presence of cholinesterase may be related to the reduction of intestinal movements in the vicinity of the flukes. The forebody gland cells of M. similis are already well developed in the mature metacercaria and secretory granules can be seen in the ducts. Since
these gland cells are not present in the cercaria, they presumably develop during the early stages of encystment. By the time the metacercariae are mature the glands have accumulated large numbers of granules and no longer possess the well-developed ger-Golgi system normally associated with the active production of secretory granules. Strong & Cable (1972) have suggested that secretions from the forebody gland cells of the metacercariae of Microphallus opacus form the inner layer of the cyst wall;
FIGS. 9-14. SEM of excysted metacercariae. FIG. 9. Excysted metacercaria showing variations in anterior and posterior surfaces, the oral sucker, ventral sucker and excretory pore (scale bar represents SOurn). FIG. 10. Ventral sucker. The lip of the sucker bears large toothed spines and sensory papillae; the central region of the sucker has fewer spines. To one side of the sucker is a groove which forms the genital atrium (scale bar represents 10 urn). FIG. 11. Oral sucker. The lining of the sucker and the lip are spineless, elsewhere the sucker bears toothed spines. Many sensory papillae of varying sizes are present (scale bar represents 10 urn). FIG. 12. Excretory pore. The surface is aspinose and folded (scale bar represents 10 urn).
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this function seems unlikely in M. similis. The precocious development of the lappet gland cells in the metacercariae of D. phoxini has been noted by Erasmus (19696) who suggests that this may allow the lappets to function immediately the metacercaria reaches the definitive host intestine. A similar requirement may be necessary for the establishment of M. similis between the host’s intestinal villi. Acknowledgements-I would like to thank Dr. B. L James of the University of Swansea for providing infected Littorina suxatilis and Dr. Denis Wright of Imperial College Field Station for supplying some of the cholinesterase inhibitors. The work was supported by a grant from the Science Research Council to whom grateful acknowledgement is made.
REFERENCES ARME C. & THREADGOLDL. T. 1976. A unique tegu-
mentary cell type and unicellular glands associated with the scolex of Eubothrium crassum (Cestoda: Pseudophyllidea). Rice University Studies 62: 21-34. BAILEYH. H. & TOMPKINSS. J. 1971. Ultrastructure of the integument of Aspidogaster conchicola. Journal of Parasitology 57: 848-854. BANCROFT5.. D. 1975. Histochemical Techniques. 2nd Edition. Butterworths. London. BENNETTC. E. 1975. Scanning electron microscopy of Fasciola hepatica L. during growth and maturation in the mouse. Journal of Parasitology 61: 892-898. BILQUEESF. M. 1976. A comment on the relationship of Prosorhynchus thapari Manter, 1953 (Trematoda) from Plectorhynchus cinctus (T.S.) off the Karachi coast, with a note on its surface-ultra-structure. Proceedings of the Pakistan Academy of Science 13: 29-33. BOGITSH B. J. 1966. Histochemical observations on Posthodiplostomum minimum, II. Esterases in subcuticular cells, holfast organ and the nervous system. Experimental Parasitology 19 : 64-70. BOG~TSHB. J. 1967. Histochemical localization of some enzymes in cysticercoids of two species of Hymenolepis. Experimental Parasitology 21: 373-379. B~GITSH B. J. & ALDRIDGEF. P. 1967. Histochemical observations on Posthodiplostomum minimum. IV. Electron microscopy of tegument and associated structures. Experimental Parasitology 21: l-8. DAVIESC. 1978. The ultrastructure of the tegument and the digestive caeca of in vitro cultured metacercariae of Fasciola hepatica. International Journal for Parasitology 8: 197-206.
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of the metacercariae of Microphallus similis in vitro and in the mouse. International Journal for Parasitology 9: 261-267. ERASMUSD. A. 1969~. Studies on the host-parasite interface of strigeoid trematodes. IV. The ultrastructure of the lappets of Apatemon gracilis minor Yamaguti, 1933. Parasitology 59: 193-201. ERASMUSD. A. 1969b. Studies on the host-parasite interface of strigeoid trematodes. VI. Ultrastructural observations on the lappets of Diplostomum phoxini Faust, 1918. Zeitschrift fur Parasitenkunde 32: 48-58. ERASMUSD. A. 1970. The host-parasite interface of strigeoid trematodes. IX. A probe and transmission electron microscope study of the tegument of Diplostomum phoxini Faust 1918. Parasitology 61: 35-41. ERASMUSD. A. 1972. The Biology of Trematodes. Edward Arnold, London. FIUPP P. J. 1967. Histochemical localisation of esterase activity in schistosomes. Experimental Parasitology 21: 380-390. HALTOND. W. 1967. Histochemical studies of carboxylic esterase activity in Fasciola hepatica. Journal of Parasitology 53: 1210-1216. HALTOND. W. & DERMOTTE. 1967. Electron microscopy of certain gland cells in two digenetic trematodes. Journal of Parasitology 53: 11861191. HALTOND. W. & LYNE~SR. A. W. 1971. Ultrastructure of the tegument and associated structures of Aspidogaster conchicola (Trematoda: Aspidogastrea). Journal of Parasitology 57: 1198-1210. INATOMIS., TONGU Y., SAKUMOTOD., SUGURI S. & ITANO K. 1968. The ultrastructure of helminth. (2) The body wall of Metagonimus yokogawai takahashi Suzuki, 1930. Japanese Journal of Parasitology 17: 455-460. JAMESB. L. 1971. Host selection and ecology of marine digenean larvae. Fourth European Marine Biology Symposium. (Edited by D. J. CRISP). pp. 179-196. Cambridge University Press, London. KARNOVSKY M. J. 1964. The localization of cholinesterase activity in rat cardiac muscle by electron miscroscopy. Journal of Cell Biology 23: 217-232. K.&sA P. & CsILLIKB. 1966. Electron microscopic localization of cholinesterase by a copper-lead-thiocholine technique. Journal of Neurochemistry 13: 1345-1349. K~IE M. 1973. The host-parasite interface and associated structures of the cercaria and adult Neophasis lageniformis (Lebour, 1910). Aphelia 12: 205-219. Ken M. 1977. Stereoscan studies on cercariae, metacercariae and adults of Cryptocotyle lingua (Creplin, 1825) Fischoeder 1903 (Trematoda: Heterophyidae). Journal of Parasitology 63: 835-839.
FIG. 13. Forebody surface showing scale-like toothed spines arranged in a quincuncial pattern. Inset: in some specimens small pores can be seen in the underlying tegument (arrowed) (scale bar represents 1 pm). FIG. 14. Hindbody
surface showing the ridged appearance of the tegument and the small peg-like spines (scale bar represents 1 pm).
List of abbreviations: a-anterior surface, epexcretory pore, g-granules, ga-genital aperture, gb-gland cell body, gd-gland cell duct, ger-granular endoplasmic reticulum, mt-microtubules, n-nucleus, oe-oesophagus, os-oral sucker, p-posterior surface, pc-parenchyma cell, pm-peripheral musculature, ps-peg-like spines, r-ribosomes, sp-sensory papilla, t-tegumental distal cytoplasm, tc-tegumental cell body, ts-toothed spines, vs-ventral sucker.
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LEE D. L., ROTHMANA. H. & SENTURIAJ. B. 1963. Esterases in Hymenolepis and in Hydatigera. Experimental Parasitology 14: 285-295. LEE D. L. & TATCHELLR. J. 1964. Studies on the tapeworm Anoplocephalaperfoliata (Goeze, 1782). Parasitology 54: 467-479. NIZAMI W. A., SIDDIQI A. H. & ISLAM M. W. 1977. Quantitative studies on acetyl-cholinesterase in seven species of digenetic trematodes. Zeitschrift ftir Parasitenkunde 52: 275-280. OAKS J. A. & LUMSDENR. D. 1971. Cytological studies on the absorptive surface of cestodes. V. Incorporation of carbohydrate-containing macromolecules into tegument membranes. Journal of Parasitology 57: 12561268. OHMAN C. 1965. The structure and function of the adhesive organ in strigeid trematodes. II. Diplostomum spathaceum Braun, 1893. Parasitology 55: 481-502. OHMAN C. 1966. Thestucture and function of theadhesive organ in strigeid trematodes. III. Apatemon gracilis minor Yamaguri, 1933. Parasitology 56: 209-226. OHMAN-JAMESC. 1973. Cytology and cytochemistry of the scolex gland cells in Diphyllobothrium ditremum (Creplin, 1825). Zeitschrift ftir Parasitenkunde 42: 77-86. PEARCEA. G. E. 1968. Histochemistry Theoretical and Applied. 3rd Ed., Vol. I. Churchill, London. PEARSEA. G. E. 1972. Histochemistry Theoretical and Applied. 3rdEd., Vol. II. Churchill Livingstone, London. ROTHMANA. H. 1966. Ultrastructural studies of enzyme activity in the cestode cuticle. Experimental Parasitology 19: 332-338.
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SCHA~EIN J. & WAITZ V. 1965. Histochemical studies of esterases in the cuticle and nerve cords of four cyclophyllidean cestodes. Journal of Parasitology 51: 356-363. SCHWABEC. W., KOUSSAM. & ACRA A. N. 1961. Hostparasite relationships in Echinococcus. IV. Acetylcholinesterase and permeability regulation in the hydatid cyst wall. Comparative Biochemistry and Physiology 2: 161-172. SELIGMANA. M., HANKER J. S., WASSERKRUGH., DMOCHOWSKIH. & KATZOFFL. 1965. Histochemical demonstration of some oxidised macromolecules with thiocarbohydrazide (TCH) or thiosemicarbazide (TSC) and osmium tetroxide. Journal of Histochemistry and Cytochemistry 13: 629-639. SILVER A. 1974. The Biology of Cholinesterases. North Holland, Amsterdam. STRONGP. L. & CABLER. M. 1972. Fine structure and development of the metacercarial cyst in Microphallus opacus (Ward, 1894). Journal of Parasitology 58: 92-98. STUNKARD H. W. 1957. The morphology and lifehistory of the digenetic rrematode, Microphallus similis (Jggerskiald, 1900) Baer, 1943. Biological Bulletin Marine Biological Laboratory, Woods Hole 112: 254-266. YEATESR. A. & OCILVIEB. M. 1976. Nematode acetylcholinesterases. In Biochemistry of Parasites and Host Parasite Relationships (Edited by H. VAN DEN BOSSCHE) Elsevier/North Holland Biomedical, Amsterdam.
0020-7519/79/1201-0553
$02.00/O
THE FOREBODY GLANDS AND SURFACE FEATURES OF THE METACERCARIAE AND ADULTS OF MICROPHALLUS SIMILIS CAROLINE DAVIES Department of Zoology and Applied Entomology, Imperial College, Prince Consort Road, London SW7 2BB, England (Received 30 January 1979)
Abstract-Dares
C. 1979. The forebody glands and surface features of the metacercariae andadults of
Microphallus similis. International Journal for Parasitology 9: 553-564. Numerous unicellular glands are present in the forebody of the metacercariae and adults of Microphallus similis. The gland cell
bodies lie beneath the tegumental perinuclear cytoplasm and ducts pass upwards penetrating the tegumental distal cytoplasm. Electron dense granules are secreted onto the forebody surface and into the oesophagus via small pits in the tegument. Histochemical tests revealed that the glands contain diastase-resistant neutral mucosubstances, RNA, protein, esterase and small amounts of acid phosphatase; inhibitor studies indicated that the esterase was acetylcholinesterase (EC 3.1.1.7). Ultracytochemistry showed that the mucosubstances were located in the general cytoplasm of the gland cells and that cholinesterase was present in the granules. The forebody of M. similis is covered by large toothed spines whereas the hindbody only bears short peg-like spines. It is suggested that the forebody spines may have an irritating, abrasive effect on the host’s intestinal mucosa and that the secretion of acetylcholinesterase in the region of these spines may produce a ‘local anaesthetic’ effect on the host’s gut by reducing movement in the immediate vicinity of the Auke, so decreasing the likelihood of expulsion. INDEX KEY WORDS: Microphallus similis; unicellular glands; spines; cytochemistry; chemistry; acetylcholinesterase (EC 3.1 .1.7).
INTRODUCTION THE METACERCARIAE of Microphallus similis are encysted in the digestive gland of Carcinus maenas and the adults are found in the small intestine of gulls of the genus Larus (Stunkard, 1957) although various laboratory rodents can also act as definitive hosts (James, 1971). Considerable growth and development occurs within the metacercarial cyst and the mature encysted metacercaria possesses well-defined genitalia in an advanced stage of development so that maturation in the final host is completed in 2-3 days (Davies & Smyth, 1979). A conspicuous feature of microphallids is the presence of numerous unicellular glands in the forebody between the oral and ventral suckers (Strong & Cable, 1972). Similar gland cells (called subcuticular cells) have also been described in the forebody of various strigeids and histochemical studies have shown that they contain non-specific esterases (Ohman, 1965, 1966; Bogitsh, 1966). It has been suggested that the secretion of esterases by strigeids functions in the extra-corporeal digestion of host tissues (Erasmus, 1972). In the present study ultrastructural, cytochemical and ultracytochemical techniques have been used to investigate the structure and possible function of the forebody glands of Microphallus similis.
ultracyto-
MATERIALS AND METHODS Metacercariae of Microphallus similis were recovered from naturally infected Curcinus maenas (supplied by the University of London Marine Biological Station, Millport) and excysted or fed to mice according to the techniques described by Davies & Smyth (1979). Cercariae of M. similis were shed from naturally infected Littorina saxatilis (provided by Dr. B. L. James, University of Swansea). Transmission electron microscopy (TEM). Cercariae, excysted metacercariae and adults recovered from mice 4 days post-infection were fixed, processed and examined according to the methods described by Davies (1978). Scanning electron microscopy (SEM). Excysted metacercariae and 4 day old adults were fixed in cold (4°C) or hot (60°C) 4% glutaraldehyde in 0.1 M-phosphate buffer (pH 7.2-7.4), dehydrated in ethanol, dried in a Polaron Critical Point Drier, mounted on stubs with double-sided Sellotape, coated with gold and examined on a Cambridge Stereoscan Scanning Electron Microscope at 10 kV. Cytochemistry. Excysted metacercariae and 4 day old adults were fixed in 4% glutaraldehyde in 0.1 M-phosphate buffer (pH 7.2-7.4). For the lipid and enzyme tests, the fixed flukes were washed in buffer, embedded in Tissue-Tek OCT compound (Miles Laboratories), cut on a Slee cryostat at 10 urn and collected onto glass microscope slides. For the other tests, the fixed flukes were washed in buffer, dehydrated in ethanol and embedded in the water-soluble plastic embedding medium
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JB4 (Polysciences Inc.). Semi-thin sections were cut on an ultramicrotome at l-2 urn and transferred to glass slides. Unless otherwise stated, the following histochemical tests were carried out according to Bancroft (1975) except that after staining the sections were not dehydrated but were dried and mounted in glycerolgelatin. Neutral mucosubstances: periodic acid-Schiff (PAS) with or without prior digestion with diastase to remove glycogen (Methods 19 and 21). Acid mucosubstances: alcian blue (Method 22). DNA/RNA: Methyl greempyronin (Method 70). Protein: aqueous mercury bromphenol blue (according to Pearse, 1968). Lipid: Oil Red 0 or Sudan Black B in isopropanol (Methods 54 and 55). Acid nhosvhatase (EC 3.1.3.2): kzo-dye method using naphthol AS-B1 phosphate and Red violet LB salt (according to Pearse, 1968). Alkaline phosphatase (EC 3.1.3.1): Azo-dye method using sodium a-naphthyl phosphate and Fast Red Salt TR (Method 101). Non-specific carboxyl esterase (EC 3.1.1.1): indoxyl method using 5-bromo, 4-chloro indoxyl acetate (Method 112). Cholinesterase: acid salt method using acetylthiocholine iodide or butyrylthiocholine iodide (according to Pearse, 1972-after the method of Gomori). In conjunction with the esterase and cholinesterase tests, some slides were pre-incubated in phosphate buffered solutions of various inhibitors at 37°C for 1 h. The effect of the following inhibitors was examined: non specific esterase-silver nitrate; general cholinesterase-diethylp-nitrophenyl phosphate (E600) (provided by Dr. D. Wright, Imperial College Field Station), eserine sulphate (Sigma); acetylcholinesterase (EC 3.1.1.7)-l :5-bis(4allyldimethyl ammoniumphenyl)-pentan-3-one dibromide (284C51) (Burroughs-Wellcome); pseudocholinesterase (EC 3.1.1.8)-tetraisopropyl pyrophosphoramide (iso OMPA) (Koch-Light), lo-(2-diethylaminopropyl) phenothiazine hydrochloride (Lysivane) (May & Baker). In addition to pre-incubation, the reversible inhibitors (284C51, Lysivane and eserine) were also included at the appropriate concentration in the test incubation medium. Ultracytochemistry. Excysted metacercariae and 4 day adults were fixed in the usual way in 4% glutaraldehyde (Davies, 1978) and transferred to 0.1 M-phosphate buffer (pH 7.2-7.4) where they were chopped into small pieces with a razor blade. Pieces of fluke were stained according to the periodic acid-thiocarbohydrazide-osmium tetroxide (PATCO) technique of Seligman, Hanker, Wasserkrug Dmochowski & Katzoff (1965) as modified by Oaks & Lumsden (1971) for the ultrastructural localisation of neutral mucosubstances. Some pieces of tissue were digested with 1% diastase in 0.1 M-phosphate buffer (pH 7.2-7.4) for 1 h at 37°C after fixation and prior to staining to remove glycogen. Staining for cholinesterase was carried out according to the Kasa & Csillik (1966) modification of the Karnovsky (1964) technique using acetylthiocholine iodide; the substrate was omitted in control treatments. In both the PATCO and cholinesterase techniques, thin sections were viewed without further staining.
RESULTS Ultrastructure of gland cells Opening onto the whole surface of the forebody of M. similis, especially in the region of the oral sucker, are the ducts of numerous unicellular glands.
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The fine structure of these gland cells appears identical in the excysted metacercaria and in the adult. The gland cell bodies lie beneath the tegumental cell bodies (tegumental perinuclear cytoplasm) and are irregular in outline with processes ramifying between adjacent parenchyma cells (Fig. 1). The majority of the gland cell is taken up by large, homogeneous, electron dense granules which are membrane bound. The granules vary in shape although most are oval to spherical measuring, on average, 0.4 urn in length. The cell bodies are nucleated and short lengths of granular endoplasmic reticulum (ger), mitochondria and numerous free ribosomes are also present in the cytoplasm.
Although conventional active-looking Golgi complexes were never seen, indistinct areas containing flattened sacs and vesicles were occasionally observed but they did not contain electron dense material. Ducts from the gland cells pass upwards between the tegumental cell bodies and peripheral musculature (Fig. 2). They are packed with granules which are sometimes less homogeneously dense than those in the cell bodies (Fig. 3) and they are lined with microtubules which are frequently seen in cross section (Fig. 4). The ducts penetrate the tegumental distal cytoplasm to which they are joined by short inconspicuous septate desmosomes and they open to the exterior of the fluke at the bottom of small pits in the tegument (Figs. 2 & 3). The granules appear to be extruded, along with some of the cytoplasm, in ‘packets’ which are not themselves membrane-bound (Fig. 3). The ducts also penetrate the tegument lining the oesophagus into which they empty their sections (Fig. 5). Gland cell bodies or the ducts from them were never observed posterior to the ventral sucker. Although the cercariae of M. similis possess cystogenous and penetration glands, cells corresponding to the forebody gland cells of the mature metacercaria and adult were not present. Cytochemistry
Histochemical tests revealed that the gland cells of both metacercariae and adults contain diastaseresistant neutral mucosubstances, protein, RNA and esterase. Small amounts of acid phosphatase were present in the gland cells of the adult but not in those of the metacercaria. The tests for acid mucosubstances, lipid and alkaline phosphatase all proved negative. The type of esterase that was present was investigated using various substrates and inhibitors (Table 1). Similar results were obtained in both excysted metacercariae and adults. Using acetylthiocholine as the substrate, a strong reaction product was obtained in the gland cells and ducts and in the suckers, pharynx and forebody musculature. This reaction was reduced but not abolished by treatment with IO-%I-eserine and 10-7~-E600
FIG. 1. Excysted metacercaria. Nucleated gland cell bodies lie beneath the tegumental cells and contain electron dense granules, mitochondria, ribosomes and granular endoplasmic reticulum (scale bar represents 1 urn). FIG. 2. Adult from mouse. Gland cell ducts pass between the peripheral musculature and penetrate the tegumental distal cytoplasm. The duct contains closely packed granules and is lined with microtubules (scale bar represents 1 urn).
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Forebody glands of Microphallus similis TABLE
I-Microphallussimilis: H~STOCHEMICALTESTSFORESTERASES CHOLINESTERASESINEXCYSTEDMETACERCARIAEAND ADULTS Substrate
Indoxyl acetate Indoxyl acetate Indoxyl acetate Indoxyl acetate Acetyl thiocholine Butyryl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine Acetyl thiocholine
Inhibitor
Gland cells
lo-%f-&NO,
lo- %-eserine lo-%-eserine lo-WAgNO, lo- %-eserine lo-%-eserine lo- 5~-E600 lo-‘M-E600 10-3~-284C51 lo- 4~-284C5 1 10-5~-284C51 10-6w-iso OMPA lo-%-is0 OMPA 10-4r+Lysivane lo-%-Lysivane
Key: + + + intensely stained; stained; - not stained.
strongly suggesting that the esterase is a cholinesterase. The results of further inhibitor studies using selective inhibitors for acetylcholinesterase (284C51) and for pseudocholinesterase (Lysivane and iso OMPA) and the fact that butyryl thiocholine was hydrolyzed less efficiently than acetylthiocholine, indicates the presence of a true cholinesterase (acetylcholinesterase, EC 3.1 .1.7.) in both gland cells and muscles. The cholinesterases in the two sites behaved slightly differently in their response to inhibitors (Table l), that of the gland cells being generally more resistant.
Ultracytochemistry
Using the PATCO technique, neutral mucosubstances were localised in the cytoplasm of the gland cells (Fig. 6); the granules themselves were negative. The reaction product appeared to be diastase-resistant which suggests that the mucosubstance was not glycogen. However, some glycogen rosettes were still present in the parenchyma cells, although they were much reduced in number after diastase digestion, indicating that the diastase
+++ + ++ +++ ++ +I+ ++ +I+ ++ +++ +++ +++ +++
+ + moderately stained;
AND
Muscles
+++ +/+ +++ ++ +i+I+ +++ +++ +++ +++ + slightly
digestion may have been incomplete. The granules were positive for cholinesterase using acetylthiocholine as substrate although not all the granules in each gland cell were stained (Fig. 7). Control treatments, without substrate, were negative (Fig. 8). Surface features No obvious differences were noted between the
surface features of excysted metacercariae and adults. Although the shape of the fixed flukes varied considerably, they were generally pyriform in outline with a slight lateral constriction in the region of the ventral sucker (Fig. 9). The surface of the fluke was divided into two distinct regions with different morphologies-the forebody anterior to the ventral sucker and the hindbody posterior to it. The lateral edges of the forebody tend to curl towards the ventral surface giving it a cuplike appearance. The forebody (Fig. 13) was covered with posteriorly-pointing, scale-like spines which were serrated at their tips having 12-13 teeth. These spines were arranged in a quincuncial pattern and embedded in pits in the tegument. In many cases, especially in those flukes which had been fixed cold,
FIG. 3. Excysted metacercaria. Opening of gland cell duct. Some of the granules in the apical portion of the duct are less dense (arrowed). The granules are secreted into a pit in the tegument in the form of a small packet (scale bar represents 1 pm). FIG. 4. Adult from mouse. Cross section of gland cell duct showing peripheral microtubules (scale bar represents 0.5 urn). RG. 5. Excysted metacercaria. Gland cell ducts open into the oesophagus which is lined with tegument (scale bar represents 1 urn).
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they overlapped completely and obscured the structure of the underlying tegument. However, when hot fixative was used some of the flukes were fixed with the forebody extended and small pores could be seen in the tegument (Fig. 13-inset). The diameter of these pores was approx. 0.5 pm which corresponds to the width of the gland cell openings seen in thin sections. The surface of the oral sucker (Fig. 11) was covered in similar toothed spines although the lip of the sucker and its lining were aspinose and in this region the tegument was corrugated. The ventral sucker (Fig. 10) was also spined although in the central cup-shaped region the spines were smaller and less frequent and the surface tegument was folded. The opening of the genital atrium could be seen as a deep furrow on one side of the ventral sucker (Fig. 10). In hot-fixed flukes, which were generally more relaxed than those fixed cold, the protruding male papilla could occasionally be seen; it had a smooth aspinose appearance. Sensory papillae were present over the whole surface of the fluke but were more frequent on the ventral surface of the forebody especially around the suckers (Figs. 10 & 11). In contrast to the large, toothed spines on the forebody, those on the hindbody posterior to the ventral sucker were small and peg-like (Fig. 14) and, although they were evenly spaced, they did not appear to be arranged in any regular pattern. The surface of the hindbody tegument was thrown up into ridges which ran around the spines. Superimposed on this were deeper circumferential or longitudinal corrugations, the distribution of which seemed to depend on the posture of the fluke at the time of fixation (Fig. 9). The surface around the excretory pore was similar to that of the general hindbody except that spines were completely lacking (Fig. 12). DISCUSSION The unicellular glands of Microphallus similis are similar in ultrastructure to gland cells associated with the general tegument or specialised areas of the tegument in other helminths, e.g. the lappets of strigeids such as Apafemon gracilis and Diplostomumphoxini (Erasmus, 1969a, b); the subcuticular cells of Posthodiplostomum minimum (Bogitsh & Aldridge, 1967); the scolex glands of pseudophyllidean cestodes (ohman-James, 1973; Arme &
Threadgold, 1976); the glands associated with the oral sucker of Haplometra cylindracea and Opisthoglyphae ranae (Halton & Dermott, 1967); the forebody and haptoral glands of Aspidogaster conchicola (Halton & Lyness, 1971; Bailey & Tompkins, 1971); and the subtegumental glands of ~eiogym~ophallas minutes (Davies, 1976, unpublished Ph.D. thesis, University of London). However, similarity in ultrastructure does not necessarily imply similarity in function or in the chemical composition of the secretory products produced by the glands. Various functions have been suggested for such gland cells. The lappet glands of A. gracilis and D. phoxini (ijhman, 1965, 1966) and the subcuticular cells of P. minimum (Bogitsh, 1966) secrete esterases and it has been suggested that they function in the extracorporeal digestion of host tissues. The bothria glands of Diphyl~o~othriam ditremam do not produce an enzymic secretion and ~hman-James (1973) has suggested that they may have an adhesive function and aid in attachment. The oral sucker glands of 0. ranae contain neutral and acid mucopolysaccharides (Halton, 1967) and Halton & Dermott (1967) suggest that, amongst other possible functions, the secretions may help protect the parasite against host digestive enzymes. In ~i&rapha~las similis all the evidence suggests that the gland cell secretions are enzymic in nature and that they contain acetylcholinesterase. Mucosubstances are also present in the gland cells but they are located in the general cytoplasm surrounding the granules. It has been recognised (Silver, 1974) that the cholinesterases associated with the nervous system of parasitic heirninths behave differently in their susceptibility to inhibitors than those of vertebrates; in general, they exhibit much greater resistance and higher concentrations of inhibitors are required. Vertebrate cholinesterases are completely inhibited by lo-Weserine and 5 x 10-5~-284C51 (Pearse, 1972) whereas heIminth cholinesterases often show only partial inhibition at these concentrations and require higher concentrations for complete inhibition (Fripp, 1967; Halton, 1967; Lee & Tatchell, 1964). In the present case the gland cell choline&erase appears to be even more resistant than that associated with the neuromuscular system indicating that it is probably of a slightly different type. Apart from their presence in the neuromuscular system of helminths, cholinesterases have also been found in various other sites, e.g. the tegument of
FIGS. 6-8. Excysted metacercariae-ultracytochemistry FIG.
559
(scale bar represents 1 pm).
6. PATCO technique. The electron dense reaction product is located in the cytoplasm around the granules. It is also present in adjacent parenchyma cells.
FIG. 7. Localisation of cholinesterase. The gland cell granules are stained although not all granules show a positive reaction (arrows). FIG 8. Localization of choiinester~e~ontrol (without substrate). Negative.
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cestodes (Hymenolepis sp. (Lee, Rothman &Senturia, 1963; Schardein & Waitz, 1965; Rothman, 1966; Bogitsh, 1967), Hydatigera taeniaeformis (Lee et al., 1963; Schardein & Waitz, 1965), Dipylidium car&urn (~hardein & Waitz, 1965), A~oplocep~aia perfoliata (Lee & Tatchell, 1964)) and digeneans (Sch~stosoma sp. (Fripp, 1967), Fasciola hepatica (Halton, 1967)); the hydatid cyst wall of Echinococcus (Schwabe, Koussa & Acra, 1961); the reproductive ducts of Fasciob hepatica (Halton, 1967) and Anoplocephala perfoliata (Lee & Tatchell, 1964); and the excretory glands of various gut parasitic nematodes (reviewed by Yeates & Ogilvie, 1976). iihman (1966) describes a “non-specific esterase of resistant type, possibly a non-specific cholinesterase” in various sites in A. gracilis including the subcuticular cells. Although Bogitsh (1966) describes the esterases of the subcuticular cells of P. minimum as non-specific, he obtained a positive reaction using acetylthi~hoIine iodide as substrate and this reaction was partially inhibited by IO- %-eserine, suggesting a cholinesterase. It seems possible, therefore, that strigeid subcuticular glands may also contain cholinesterases. Various functions have been suggested for the presence of cholinesterases in helminths. In the te~ment it has been suggested that cholinesterase is involved in transport processes and uptake (Rothman, 1966; Bogitsh, 1967; Halton, 1967). Schwabe et al. (1961) suggest that the cholinesterase in the hydatid cyst wall of Echinococcas is involved in permeability control and osmoregulation. Several theories have been put forward to account for the presence of cholinesterases in the excretory glands of nematodes (Yeates & Ogilvie, 1976). These include the suggestions that they may reduce the host’s immune response, that they may help the parasite to feed by increasing the availability of glucose or that they may act as a kind of local anaesthetic on the host gut to reduce peristalsis and so prevent parasite expulsion. The latter hypothesis seems very attractive in the case of Microphallus similis especially when the distribution of the gland cells is related to the surface features of the Auke and its mode of attachment in the host’s intestine. In the seagull it is the forebody of M. similis which is embedded between the host’s intestinal villi while the hindbody projects into the lumen of the intestine (Dr. B. 1;. James, personal
561
communication). SEM reveals that the forebody is covered with large, backwardly-pointing, toothed spines. Such spines have been described in other digeneans associated with the gastro-intestinal tract of various hosts e.g. F. hepatica (Bennett, 1975), Metago~~imas yo~oga~ai (Inatomi, Tongu, Sakumoto, Suguri & Itano, 1968), Diplostomum phoxini (Erasmus, 1970) Prosorhynchus thapari (Bilquees, 1976), Cryptocotyle lingua (Koie, 1977), Neophasis lageniformis (Kaie, 1973) and Meiogymnophallus minutus (Davies, 1976, unpublished Ph.D. thesis, University of London). They probably aid in maintenance of position but must also have a very irritating effect on the host’s intestinal mucosa. The presence of these spines in M. similis coincides with the distribution of the cholinesterase secreting glands. The hindbody, which is not in such intimate contact with the host and which is spineless or bears much reduced spines, is not provided with such gland cells. It seems possible, therefore, that the cholinesterase secreted over the surface of the forebody may serve to compensate for the irritating effect of the toothed spines by neutralizing host acetylcholine and so reducing the movement of the villi in the immediate vicinity of the fluke thereby reducing the likelihood of dislodgement and expulsion. Nizami, Siddiqi & Islam (1977) made a quantitative analysis of the levels of cholinesterase in seven species of digeneans and concluded that it is present in “remarkably high quantities” in species which inhabit the gastro-intestinal tract compared with those that parasitize the liver or the swim bladder. These authors also suggest that the presence of cholinesterase may be related to the reduction of intestinal movements in the vicinity of the flukes. The forebody gland cells of M. similis are already well developed in the mature metacercaria and secretory granules can be seen in the ducts. Since
these gland cells are not present in the cercaria, they presumably develop during the early stages of encystment. By the time the metacercariae are mature the glands have accumulated large numbers of granules and no longer possess the well-developed ger-Golgi system normally associated with the active production of secretory granules. Strong & Cable (1972) have suggested that secretions from the forebody gland cells of the metacercariae of Microphallus opacus form the inner layer of the cyst wall;
FIGS. 9-14. SEM of excysted metacercariae. FIG. 9. Excysted metacercaria showing variations in anterior and posterior surfaces, the oral sucker, ventral sucker and excretory pore (scale bar represents SOurn). FIG. 10. Ventral sucker. The lip of the sucker bears large toothed spines and sensory papillae; the central region of the sucker has fewer spines. To one side of the sucker is a groove which forms the genital atrium (scale bar represents 10 urn). FIG. 11. Oral sucker. The lining of the sucker and the lip are spineless, elsewhere the sucker bears toothed spines. Many sensory papillae of varying sizes are present (scale bar represents 10 urn). FIG. 12. Excretory pore. The surface is aspinose and folded (scale bar represents 10 urn).
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this function seems unlikely in M. similis. The precocious development of the lappet gland cells in the metacercariae of D. phoxini has been noted by Erasmus (19696) who suggests that this may allow the lappets to function immediately the metacercaria reaches the definitive host intestine. A similar requirement may be necessary for the establishment of M. similis between the host’s intestinal villi. Acknowledgements-I would like to thank Dr. B. L James of the University of Swansea for providing infected Littorina suxatilis and Dr. Denis Wright of Imperial College Field Station for supplying some of the cholinesterase inhibitors. The work was supported by a grant from the Science Research Council to whom grateful acknowledgement is made.
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FIG. 13. Forebody surface showing scale-like toothed spines arranged in a quincuncial pattern. Inset: in some specimens small pores can be seen in the underlying tegument (arrowed) (scale bar represents 1 pm). FIG. 14. Hindbody
surface showing the ridged appearance of the tegument and the small peg-like spines (scale bar represents 1 pm).
List of abbreviations: a-anterior surface, epexcretory pore, g-granules, ga-genital aperture, gb-gland cell body, gd-gland cell duct, ger-granular endoplasmic reticulum, mt-microtubules, n-nucleus, oe-oesophagus, os-oral sucker, p-posterior surface, pc-parenchyma cell, pm-peripheral musculature, ps-peg-like spines, r-ribosomes, sp-sensory papilla, t-tegumental distal cytoplasm, tc-tegumental cell body, ts-toothed spines, vs-ventral sucker.
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