The tegument of Schistosoma mansoni: observations ...

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Oct 22, 1973 - cytoplasmic inclusions in relation to tegument function ... tegumental inclusion, termed a membranous body, contributes material to the.

Parasitology (1974), 68, 239-258 With 7 plates

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The tegument of Schistosoma tnansoni: observations on the formation, structure and composition of cytoplasmic inclusions in relation to tegument function R. A. WILSON and P. E. BARNES Department of Biology, University of York, Heslington, York (Received 22 October 1973) SUMMARY

Two major cytoplasmic inclusions, multilaminate vesicles and discoid granules, are present in the tegument of Schistosoma mansoni. These are produced at separate Golgi apparatuses in the tegument cell bodies and move up to the surface by a combination of diffusion and fluid flow. The discoid granules contain neutral mucopolysaccharide and are believed to break down to form the ground substance of the tegument. The multilaminate vesicles are rich in phospholipid and the contents, at least superficially, resemble unit membranes. The multilaminate vesicles are believed to contribute their contents to the multilaminate surface of the worm which appears to be continually replaced. These observations are related to current ideas on membrane turnover and the ability of the worm to acquire a disguise of host erythrocyte glycolipid.

INTRODUCTION

The immune response of the mammalian host appears to be directed chiefly against the tegument of the mature schistosome worm (Smithers, Terry & Hockley, 1969; Clegg, 1972). Smithers et al. (1969), in a series of transfer experiments, demonstrated that the worm is able to evade this immune response by acquiring a covering of host-like antigenic determinants. These 'host antigens' are intimately associated with the surface of the tegument. They are acquired and lost only slowly over a period of days (Smithers et al. 1969) indicating that they are not simply surface contaminants. Clegg (1972) has suggested that the host antigens are actually host erythrocyte glycolipids attached to the schistosome surface plasmalemma. It seems probable, therefore, that the structure and function of the tegument play an important role in the ability of the schistosome to evade the immune response of its host. A number of accounts of the ultrastructure of the schistosome tegument have recently been published (Morris & Threadgold, 1968; Smith, Reynolds & von Lichtenberg, 1969; Silk, Spence & Gear, 1969). These workers have shown that the organization of the schistosome tegument and associated subtegumental cells conforms to that of other trematodes. Spines, small mitochondria and a variety of other inclusions are present in the cytoplasm of the tegument. One apparent difference between the schistosome and other trematode teguments is the structure of the surface plasmalemma which Hockley & McLaren (1973) showed to be

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multilaminate, using uranyl acetate as a 'fixative' additional to glutaraldehyde and osmium. The cercaria-schistosomulum acquires this surface about 3 h after penetration of the mammal host. Hockley & McLaren suggested that one type of tegumental inclusion, termed a membranous body, contributes material to the multilaminate surface which is 'continually breaking down and being reformed'. The aim of the work reported in this paper was to extend the observations outlined above, to investigate the dynamic processes occurring in the tegument and to relate the findings to the work on the evasion of the immune response. The formation and route of transport of the tegumental inclusions are described. Thencontents are characterized and suggestions are made about their ultimate fate and function. MATERIALS AND METHODS

Biological material The life-cycle of Schistosoma mansoni was maintained in the laboratory using LACA strain white mice and the snail Biomphalaria glabrata as hosts. The snails used to establish the colony and a Puerto Rican strain of S. mansoni were obtained from Dr S. R. Smithers, National Institute for Medical Research, London. Mature worms were recovered from mice by incision of the hepatic portal vein after the mice had received anaesthetic doses of Nembutal (Abbott Laboratories) (Smithers & Terry, 1965) followed by cervical dislocation. The worms were handled as little as possible to minimize cell damage. Light microscope histochemistry The distribution of the major classes of cell constituents was examined by light microscope histochemistry on 10 /*m thick sections of worms cut following paraffin wax impregnation. All methods and fixatives were as recommended by Pearse (1960). Acid mucopolysaccharides were detected by Alcian blue staining; other polysaccharides by the periodic acid-Schiff technique with amylase controls; DNA and RNA by the methyl green-pyronin Y technique with ribonuclease controls, and proteins by staining with bromophenol blue. Lipids were detected by staining frozen sections with Sudan black B or Nile blue sulphate solution. Ultrastructure For conventional ultrastructural studies worms were fixed in 4 % glutaraldehyde in 0-1 M phosphate buffer pH 7-2, for 2 h at 4 °C; 20 min after transfer to the fixative the worms were chopped into 1 mm pieces. After fixation worms were washed three times in phosphate buffer and stored overnight at 4 °C. They were then osmicated in 1 % aqueous osmium tetroxide for 2 h at 4 °C (the use of complicated mixtures of salts or iso-osmotic solutions in combination with osmium did not appreciably improve cytological detail). Worms were then washed in three changes of distilled water, dehydrated in a series of ethanols, cleared in propylene oxide and embedded in an Araldite-Epon mixture. Thin sections were cut on Huxley or LKB ultramicrotomes and examined in an A.E.I. EM6B electron microscope. Specimens were stained in 1 % uranyl acetate in 70 and 90 % ethanol

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during dehydration, except where uranyl acetate was used immediately after osmication, as recommended by Hockley & McLaren (1973). All sections were stained on the grid in lead citrate solution for 15 min (Kay, 1965). Evidence for synthesis and secretion Ultrastructural identification of the components of the subcellular system responsible for synthesis, packaging and secretion of macromolecules was facilitated by the use of special fixatives. These included sodium permanganate (Wetzel, 1961) and an osmium tetroxide-zinc iodide mixture (Niebauer, Krawczyk, Kidd & Wilgram, 1969). Electron microscope histochemistry Carbohydrates were detected at the ultrastructural level by three different techniques. Thin sections of worms, fixed in either 2-5 % glutaraldehyde or freshly prepared 4 % formaldehyde in phosphate buffer pH 7-3, were stained by the silver methenamine technique of Rambourg (1967). Sections of worms in which periodic oxidation was omitted were used as controls (Reissig, 1970). Thin sections of glutaraldehyde-osmium fixed worms, some subsequently bleached in hydrogen peroxide, and sections of worms fixed in glutaraldehyde alone, were stained with phosphotungstic acid solution at pH < 1-0 and 5-5 (Pease, 1970). Lastly, small pieces of worm fixed in glutaraldehyde were stained by the PATCO technique (Seligman, Hanker, Wasserkrug, Dmochowski & Katzoff (1965), as modified by Oaks & Lumsden (1971)) and then embedded and sectioned. Pieces of worm incubated in 0-3 % aniline in 0-5 % acetic acid for 2 h prior to staining, were used as controls (Oaks & Lumsden, 1971). Some pieces of tissue were digested with amylase after fixation and prior to staining in order to remove glycogen. There do not appear to be any satisfactory methods for the detection of proteins at the ultrastructural level. However, an attempt was made to stain pieces of glutaraldehyde-fixed worm using Millon's reagent for protein tyrosine (after Pearse, 1960). After staining, the pieces of tissue were osmicated, embedded and sectioned. The permanganate fixation of Wetzel (1961) and the osmium tetroxide-zinc iodide fixation of Niebauer et at. (1969) described above provide information on the distribution of lipid constituents in the worm tissues. Virtually all electron-microscope observations were made upon the posterior half of the body of the male worm. The structure of this region is relatively simple, due to the absence of reproductive organs.

RESULTS

Observations on the tegument The results of this current investigation confirm the broad outlines previously described by other workers. The thickness of the syncytial tegument varies from 0-9 to 3-0 /an, at least some of this variation being due to changes in shape of the living worm. In regions where the underlying musculature is contracted the tegument is thicker and may be thrown into folds. Where the muscles are relaxed the tegument is thinner. The cell bodies associated with the tegument lie beneath the

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muscle layers and are connected to it by narrow tubules of cytoplasm. The cell bodies are roughly ovoid in shape varying in size from 3 to 6 /an by 6 to 12 /on. Their apparent spacing beneath the tegument obviously depends upon the degree o£ contraction of tb.e musculature and varies from 5 to 20 /im. The cytoplasmic tubules connecting the tegument cell bodies with the surface syncytium vary in diameter from 1 to 1 -5 /im. at their junction with the cell bodies, narrowing down to a diameter of 0-3 /im where they pass between adjacent fibres of the musculature (PI. 1 A). In this region, the tubules are lined by a peripheral ring of microtubules orientated parallel to the longitudinal axis. There are many more longitudinal fibres in the musculature of the dorsal surface of the male than in that of the ventral surface. Consequently the cytoplasmic tubules of the dorsal surface vary in length from 8 to 30 /im whilst those of the ventral surface are only about 3 /im long. The tubules passing to the dorsal surface must also follow a more tortuous course between the muscle fibres. Tangential sections through the worm surface at the level of the musculature (PI. 1A) reveal that the cytoplasmic connexions are spaced from 0-2 to 4/im apart, lying in the gaps between transverse muscle fibres. These gaps are approximately 0* 1-0-5 /im wide and the muscle fibres themselves about 1-0 fim wide. The relationship between the spacing of cytoplasmic connexions and that of the tegument cell bodies mentioned above suggests that there may be as many as 50 cytoplasmic tubules per individual tegument cell body. The tegument contains a number of interesting cytological features noted by other workers. The outer surface is highly invaginated to form a series of pits or channels running up to 1-0 /tm into the cytoplasm. The bounding plasma membrane of the inner surface of the tegument rests upon a fibrous basal lamella and is thrown into numerous narrow invaginations which project upwards into the basal cytoplasm of the tegument. These invaginations may broaden out somewhat at their tips to form small extracellular vacuoles deep in the cytoplasm of the tegument. The surface channels and basal invaginations may be separated from each other by as little as 0-25-0-5 /iva. of cytoplasm. The ground substance of the tegument is coarsely granular and of moderate electron density. A number of cytoplasmic inclusions are present within the ground substance. The most prominent of these are the tegumental spines (PI. 2B). A few small mitochondria, 0-2 /im. in diameter, are scattered through the tegument. Previous workers have described up to five further types of cytoplasmic inclusion. The results of this study indicate only two major types of inclusion and the nomenclature of Smith et al. (1969), will be used in describing them. The most common type of inclusion is the discoid granule (PI. 1C) which has been given various names by other workers (rod body: Morris & Threadgold (1968), Reissig (1970),Silk etal. (1969); elongate body: Hockley& McLaren (1973)). The 'ring-like elements' described by Silk et al. (1969) are probably discoid granules sectioned longitudinally. The second and less common type of inclusion, termed a multilaminate vesicle by Smith et al. (1969) has also received a variety of names (spherical body: Morris & Threadgold (1968); circular inclusion: Silked al. (1969); round inclusion: Reissig (1970); membranous body: Hockley & McLaren (1973)).

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There are from 15 to 45 discoid granules per /an2, distributed uniformly throughout the tegument cytoplasm apart from a narrow (2 nm) dense band of material lying immediately beneath the surface plasmalemma. The multilaminate vesicles are usually confined to the inner two-thirds of the tegument cytoplasm, seldom reaching up beyond the base of the surface channels. They vary in number from one to three per /

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