THE ADULT WORM

Paired schistosome male and female worms can live for a long time, Schistosoma mansoni and S. japonicum mainly in the portal vein and S. haematobium in veins of the urogenital system. Although the literature is unclear as to the exact length of time worms can survive (half-lives of several years have been estimated and some individual cases of several decades recorded (Gryseels et al. Reference Gryseels, Polman, Clerinx and Kestens2006), the relatively prolonged survival of worms in the bloodstream of the host suggests a very successful adaptation to the environment. Because the control of schistosomiasis by immunological means has been a clear objective for a long time, most research has focused on the interactions of the parasite with the immune system. Consequently, the influence of, and interactions between the parasite and other host factors, such as hormones, blood pressure, blood iron, ion and gas concentrations, and the multifarious factors involved in blood clotting, have received much less attention. However, it is known that the parasite requires cholesterol, iron, essential fatty acids, purines, essential amino acids and vitamins, all of which it acquires from the host bloodstream (Rumjanek, Reference Rumjanek, Rollinson and Simpson1987). That the parasite also responds to changes in the metabolism of the host is indicated by results from the genome project, showing that the parasite has genes for receptors for host hormones (Verjovski-Almeida et al. Reference Verjovski-Almeida, DeMarco, Martins, Guimarães, Ojopi, Paquola, Piazza, Nishiyama, Kitajima, Adamson, Ashton, Bonaldo, Coulson, Dillon, Farias, Gregorio, Ho, Leite, Malaquias, Marques, Miyasato, Nascimento, Ohlweiler, Reis, Ribeiro, Sá, Stukart, Soares, Gargioni, Kawano, Rodrigues, Madeira, Wilson, Menck, Setubal, Leite and Dias-Neto2003). To date, the respective locations of the receptors that facilitate uptake of different host factors by the parasite are unknown, but have been assumed to be in or on the surface membrane (Davies and Pearce, Reference Davies and Pearce1995; LoVerde et al. Reference LoVerde, Niles, Osman, Wu, Secor and Colley2005).

The morphology of the adult parasite is highly complex, and the great morphological differences between male and female worms (i.e. dimorphism) are reflected and accentuated by similarly great differences in gene expression (Grevelding et al. Reference Grevelding, Sommer and Kunz1997; LoVerde et al. Reference LoVerde, Niles, Osman, Wu, Secor and Colley2005; Fitzpatrick and Hoffmann, Reference Fitzpatrick and Hoffmann2006) and metabolism (Cornford, Reference Cornford1985; Rumjanek, Reference Rumjanek, Rollinson and Simpson1987). The organ systems (gut, nervous system and excretory system) of male and female worms have different distributions in the whole organism and may have very different mechanisms regulating metabolic pathways. For example, the excretory system is developed into a highly complex organelle in both sexes, but its regulation and response to drugs is different between males and females (Sato et al. Reference Sato, Kusel and Thornhill2002; Oliveira et al. Reference Oliveira, Kusel, Ribeiro and Coelho2006). However, descriptions of this and other features of female worms are relatively rare, as work has often only been done on the male.

Electron microscopy

Hockley and McLaren (Reference Hockley and McLaren1973) showed that the surface of the adult is a double bilayer, the outer bilayer being prominent in electron micrographs because it bound uranium salts, which was, in turn, possibly due to its high phospholipid content. Using an electron microscope, the juxtapositioning of the two bilayers gave the appearance of a pentalaminate structure. However, very few cytoskeletal structures could be seen beneath the inner bilayer, and the properties of the narrow area between the two lipid bilayers were not defined.

Using freeze fracture methods, Hockley et al. (Reference Hockley, McLaren, Ward and Nermut1975) carried out a painstaking study of the distribution of intramembranous particles in all of the monolayers in the double bilayer of the adult worm, studied either as fresh tissue or after fixation in glutaraldehyde. These authors showed that the innermost and outermost monolayers of adult worms contained the majority of intramembranous particles, and they speculated that the outermost monolayer had the properties of an inner monolayer of a mammalian cell (Hockley et al. Reference Hockley, McLaren, Ward and Nermut1975). However, this proposal is not considered likely, since, although some of the particles in the outermost monolayer may have been derived from the host (i.e. were host antigens), the study by Harnett et al. (Reference Harnett, Kusel and Barrowman1985) showed the presence of parasite-specific antigens in the outer surface of the glutaraldehyde-fixed adult male worm. Subsequently, it has been shown that the outermost monolayer of schistosomes is rich in lectin-binding sites and has a glycocalyx (Schmidt, Reference Schmidt1995), thus being distinct from a cytoplasmic monolayer.

The conclusions from electron microscopy and freeze-fracture studies are thus that the double bilayer consists of 2 apposed bilayers, with a glycocalyx on the outermost monolayer, but a region of very uncertain composition between the bilayers.

Identification of surface components

Early studies (Snary et al. Reference Snary, Smith and Clegg1980; Roberts et al. Reference Roberts, MacGregor, Vojvodic, Wells, Crabtree and Wilson1983) were carried out with the enzyme lactoperoxidase, which, in the presence of hydrogen peroxide and radioactive iodine, catalyses the labelling of surface protein and lipid. The advantage of using radioactive iodine and lactoperoxidase is that only proteins that are exposed in the outer monolayer become labelled, since lactoperoxidase has a molecular weight of 90 kDa and will not penetrate membrane pores. Therefore, the iodine should not label constituents of inner bilayers. Using this method, Roberts et al. (Reference Roberts, MacGregor, Vojvodic, Wells, Crabtree and Wilson1983) showed that the outer bilayer of freshly perfused adult worms carried 13 proteins that could be labelled. After renewing the outer bilayer in vitro, as a result of turnover induced by cationised ferritin, they showed that 3 additional proteins were accessible to the labelling. These proteins were obscured by the presence of molecules, proposed to be derived from the host.

Through the application of genomic and proteomic technologies, Braschi et al. (Reference Braschi, Curwen, Ashton, Verjovski-Almeida and Wilson2006) were able to identify proteins in various membrane fractions. They fractionated purified tegumental membranes by 3 consecutive chemical extractions to yield a final pellet (designated 4) that contained most of the integral membrane proteins. Three other fractions contained (1) cytosolic proteins, (2) cytoskeletal proteins and (3) membrane-associated proteins. The identity of many proteins in each of these fractions was established by proteomic analysis. The presence of a calcium pump (fraction 4) and several calcium-binding proteins (fractions 1 and 3) suggested a role for calcium in the regulation of tegumental function, evidence for which has also come from biochemical data (Cunha et al. Reference Cunha, Meyer-Fernandes and Noel1992). No cytokine receptors, such as the TGFβ receptor (Davies and Pearce, Reference Davies and Pearce1995; LoVerde et al. Reference LoVerde, Niles, Osman, Wu, Secor and Colley2005) were found, and the glucose transporter SGPT1 was found, but not SGPT4. Braschi and Wilson (Reference Braschi and Wilson2006) went on to evaluate the location of some surface-exposed proteins by labelling with biotin reagents which, although membrane impermeant, may diffuse through membrane pores. Two biotin reagents, a short and a long form, were used and labelled proteins were isolated in sequential chemical steps to yield 5 fractions. The proteins were isolated from each extract after binding to a streptavidin column. Possible locations for some of the labelled proteins could be suggested. Thus, 3 enzymes, alkaline phosphatase, phosphodiesterase and ATP-diphosphohydrolase were located in the inner bilayer and it was proposed that phosphorylated substrates gained access to these enzymes, as did the biotin reagents, through aqueous pores. The presence of pores, measuring 13·4 angstroms in diameter, had previously been suggested by Davies and Pearce (Reference Davies and Pearce1995). There is no direct evidence for these pores. Indeed, the work of El Ridi and Tallima (Reference Tallima2006) suggests that alterations in the sphingomyelin metabolism are sufficient to alter the exposure of enzyme proteins, antigens and receptors. Thus, pores may not exist! Box 2 suggests some experiments to determine whether the pores have reality.

Tetraspanins and annexins were located between the bilayers, tethering of the two bilayers being their suggested function (Braschi and Wilson, Reference Braschi and Wilson2006). Sm29, a secreted protein, was also placed between the bilayers. Host proteins, such as IgM, IgG3, IgG1 and complement component C3, were located in the outer bilayer. Altogether, 24 proteins were grouped according to possible functions, such as membrane structural proteins, membrane enzymes, transporters, cytoskeletal proteins and cytosolic proteins (Braschi and Wilson, Reference Braschi and Wilson2006).

It is of interest to compare these very recent results with the earlier evidence obtained using the lactoperoxidase method. Roberts et al. (Reference Roberts, MacGregor, Vojvodic, Wells, Crabtree and Wilson1983) reported that 13 proteins could be labelled with lactoperoxidase on freshly perfused worms, and 3 additional proteins were detected after stimulation of membrane turnover. As lactoperoxidase cannot penetrate into the inner membrane, these proteins are very likely to be located in the outer bilayer, though there have been reports of internal proteins being labelled by this method (for example, in Aeromonas (Dooley and Trust, Reference Doolley and Trust1988)), but usually lactoperoxidase and biotin labelling give similar results (Apps et al. Reference Apps, Percy and Perez-Castineir1989). All of the proteins detected employing the lactoperoxidase method have molecular weights corresponding to those found in the study by Braschi et al. (Reference Braschi and Wilson2006). However, in this study, only 4 proteins (IgM, IgG3, IgG1 and complement C3 (C3c/C3dg fragments) were located in the outer bilayer. This discrepancy could perhaps be resolved by using precise methods (cf. Braschi et al. Reference Braschi and Wilson2006) to identify which proteins are present in the outer bilayer, following their isolation by the methods described in Roberts et al. (Reference Roberts, MacGregor, Vojvodic, Wells, Crabtree and Wilson1983), in which cationised ferritin is allowed to stimulate the turnover of this layer.

Alkaline phosphatase is a glycoprotein that occurs in the outer monolayer of mammalian plasma membranes (Hirano et al. Reference Hirano, Iiizumi, Mori, Toyoshi, Sugiura and Iino1985). Braschi and Wilson (Reference Braschi and Wilson2006) detected this enzyme in the inner bilayer of the schistosome. Thermodynamic considerations, which allow very little mixing of hydrophobic and hydrophilic moieties (Singer and Nicolson, Reference Singer and Nicolson1972), indicate that its carbohydrate chain is unlikely to cross into the outer bilayer. Thus, the bulk of the molecule may lie in the space between the bilayers. This argument holds for any other glycoprotein suggested to be located in the inner bilayer, such as ATP-phosphohydrolase, acetylcholinesterase and the heavy subunit of the amino acid transporter (Skelly, Reference Skelly, Secor and Colley2005).

Some proteins that were expected to be labelled were not detected by Braschi and Wilson (Reference Braschi and Wilson2006), for example, the glucose transporter SGPT4, the amino-acid transporters, and caveolin. This could have been due to the low abundance of these molecules in the membrane preparation, the insensitivity of the technique, or the protected state or burial of the lysines and other amino-groups with the potential to react with the biotin probes.

The inner bilayer of the double bilayered surface of schistosomes appears to have conventional lipid membrane properties. The outer bilayer has been shown to be secreted on to the inner bilayer and has been termed the membranocalyx (Wilson and Barnes, Reference Wilson and Barnes1977; Roberts et al. Reference Roberts, MacGregor, Vojvodic, Wells, Crabtree and Wilson1983). The freeze fracture studies of Hockley et al. (Reference Hockley, McLaren, Ward and Nermut1975) suggest that the majority of intramembranous particles are found in the outermost monolayer of the outer bilayer (membranocalyx) and innermost monolayer of the inner bilayer. Further work on the isolation of the outer bilayer (membranocalyx) is needed to explain why few proteins are found in the outer monolayer using the biotin labelling technique. The function of some molecules in the space between the two bilayers may be to anchor the outer to the inner bilayer; the properties of the tetraspanins and annexins, found there by Braschi and Wilson (Reference Braschi and Wilson2006), would be ideal for this since both tetraspanins and annexins interact with membranes and could serve as molecules connecting the bilayers (Braschi et al. Reference Braschi and Wilson2006). Since many membrane enzymes and receptors are glycoproteins (Skelly and Wilson, Reference Skelly and Wilson2006), the hydrophilic glycanic residues are likely to be located in the aqueous environment within this inter-membrane space.

Lipid composition

Evidence of the presence and quantity of specific lipids is of crucial relevance for understanding the structure and physiology of the membrane (Allan et al. Reference Allan, Payares and Evans1987; Brouwers et al. Reference Brouwers, Skelly, Van Golde and Tielens1999). There is a large amount of sphingomyelin in the isolated membrane (Allan et al. Reference Allan, Payares and Evans1987), and it may have a stabilizing role with cholesterol, as was shown for vesicles and monolayers, and for mammalian membranes (Albi et al. Reference Albi, Peloso and Magni1999). Sphingomyelin can act as a signalling molecule (Testi, Reference Testi1996) as well as a molecule that can stabilize membrane rafts. Liberation of ceramide from sphingomyelin can stimulate vasodilator pathways in mammalian smooth muscle, as well as activate vesicle membrane fusion pathways and pathways for calcium release. Fluorescence quenching studies showed that sphingomyelin was likely to be present in the outermost monolayer of the adult membrane (Redman and Kusel, Reference Redman and Kusel1996), so that any signalling event would have to cross both bilayers. The proportion of the phosphatidylcholine fraction of the surface membrane is not very different from other plasma membranes (Allan et al. Reference Allan, Payares and Evans1987). Brouwers et al. (Reference Brouwers, Skelly, Van Golde and Tielens1999) showed that this fraction is rapidly labelled with radioactive fatty acids, and these fatty acids can be transferred after deacylation to form triglycerides. Little radioactive lipid was found in the growth medium, and it was concluded that no rapid release of surface membrane was occurring by sloughing. This finding is different from that of Kusel et al. (Reference Kusel, Mackenzie and McLaren1975), who postulated that sloughing occurred in the form of small vesicles. Further work is required to resolve this discrepancy.

The lipid 5-octadecenoic fatty acid is rare in nature. Its presence in the worm is almost entirely restricted to the single phosphatidylcholine species, 1-palmitoyl-2-(5-octadecenoyl) phosphatidylcholine, which is abundant in, and confined to the outer tegumental membrane. Although schistosomes do not synthesize fatty acids de novo, the parasite probably manufactures 5-octadecenoic acid in the cells beneath the tegument (Brouwers et al. Reference Brouwers, Versluis, Van Golde and Tielens1998) as it is absent from the host's blood. The octadecenoic acid may have a signalling function in the parasite (Brouwers et al. Reference Brouwers, Versluis, Van Golde and Tielens1998) or have an effect on host red cell or endothelial cell membranes, or even in signalling between parasites. In mammalian cells, phosphatidylcholine has both a structural and a signalling function (Billah, Reference Billah1993). This lipid is likely to be a major component of both bilayers, so any signalling pathway involving phosphatidylcholine could act through a single or a double bilayer.

The uptake and distribution of fatty acids in the tissues of schistosomula and adult worms may involve Sm14, a fatty acid-binding protein that has been a potential vaccine candidate and whose crystal structure has been determined (Angelucci et al. Reference Angelucci, Johnson, Baiocco, Miele, Brunori, Valle, Vigorosi, Troiani, Liberti, Cioli, Klinkert and Bellelli2004). These authors found an interesting increase in affinity of fatty acids for this protein at a low pH, and suggested that this property could allow it to transfer fatty acids from host plasma to worm tissues (Angelucci et al. Reference Angelucci, Johnson, Baiocco, Miele, Brunori, Valle, Vigorosi, Troiani, Liberti, Cioli, Klinkert and Bellelli2004). A low pH is expected in the microenvironment of the inner membrane surface, but it may be influenced by the membrane-bound carbonic anhydrase that is present in the membrane fraction (Braschi and Wilson, Reference Braschi and Wilson2006). Moffat and Kusel (Reference Moffat and Kusel1992) showed an uptake and transfer of fluorescent fatty acids from the membrane to the gut. Lipid bodies, small membrane-bound lipid droplets, are thought to be crucial in intracellular signalling and regulation of metabolism in mammalian cells, but their function in schistosomula has not been established. In leucocytes, these bodies are sites of eicosanoid metabolism (Weller and Dvorak, Reference Weller and Dvorak1994) and thus they may be very relevant factors facilitating early schistosome migration, since eicosanoids have been shown to inhibit the exit of Langerhans cells from the skin (Angeli et al. Reference Angeli, Faveeuw, Roye, Fontaine, Tessier, Capron, Wolowczuk, Capron and Trottein2001).

Cholesterol is also found in the isolated tegument (Allan et al. Reference Allan, Payares and Evans1987). It seems that cholesterol can be taken up from medium containing bovine serum albumin as a carrier (Haseeb et al. Reference Haseeb, Eveland and Fried1985). Cholesterol can stabilize membranes by interacting with sphingomyelin, and is often found in lipid rafts in mammalian plasma membranes associated with sphingomyelin, glycolipid and proteins anchored by the GPI moiety. Cholesterol can be shown to be in the outer monolayer of the adult membrane by staining with fluorescent filipin (El Ridi et al. Reference El Ridi, Tallima, Mohamed and Montash2004). The manner by which cholesterol is inserted in schistosome membranes is different from that in erythrocytes, because streptolysin O, a bacterial toxin specific for cholesterol and that lyses erythocytes, does not damage the schistosomulum or adult surface (Kusel et al. Reference Kusel, Stones and Harnett1981). Amphotericin B is a polyene antibiotic from Streptomyces nodosus and binds to sterols in membranes. It can damage the schistosomulum surface, but not the adult surface (Kusel et al. Reference Kusel, Stones and Harnett1981). Therefore, it can be concluded that the lipid-protein organization of the adult membrane is very different from that of the schistosomulum and both are distinct from the erythrocyte membrane.

The organization of proteins and lipids in the bilayers

Probes have been employed to dissect the organization of membranes. Such probes have included (i) fluorescent molecules whose structures allow access to different regions and different depths of the bilayers, (ii) enzymes with specificities towards various lipids, a variety of toxins which bind to particular lipids, and (iii) some proteins and fluorescent lectins which bind to specific sugars on glycoproteins and glycolipids (Kusel and Gordon, Reference Kusel and Gordon1989).

In investigations of the lipid phase of the double bilayer of living parasites, membrane-active compounds (Dingle and Lucy, Reference Dingle and Lucy1965), and a variety of detergents have been used as disruptive agents to worm surfaces labelled with radioactive chromium and iodine-labelled lectin (e.g. wheat germ agglutinin), to assess the extent and nature of the damage so induced (Kusel et al. Reference Kusel, Stones and Harnett1982). From this work, it was suggested that the bilayers were composed of domains, which might be considered to be equivalent to the lipid rafts and appear to be universally present in membranes (Simons and Ikonen, Reference Simons and Ikonen1997). Rafts are enriched in proteins with glycosyl phophatidylinositol (GPI)-anchors. In the model proposed by Braschi and Wilson (Reference Braschi and Wilson2006), all GPI moieties are located in the inner bilayer and, by extrapolation from other organisms and mammalian cells (Magee and Parmryd, Reference Magee and Parmryd2003), might be expected to be associated with rafts. Racoosin et al. (Reference Racoosin, Davies and Pearce1999) treated surface membranes with detergents and demonstrated the existence of a fraction that is detergent-resistant, a feature of lipid rafts. Furthermore, they identified caveolin, a raft-associated protein in mammalian membranes, as a molecule that concentrates in this fraction. The importance of the heterogeneity of rafts in cells has been emphasized by Pike (Reference Pike2004), who suggested that the observed great variability in raft composition was dependent upon the structure of the proteins present in the membrane at a particular time. Some rafts are cholesterol-free, and the composition of all rafts can undergo alteration depending on changes in environmental conditions. The existence of rafts in schistosome surface membranes is of great interest, since their presence may explain not only the great variability in lipid composition between individual parasites (Tallima et al. Reference Tallima and El Ridi2005), but also heterogeneity on the surface of an individual schistosome. Different rafts are thought to have different functions in the membrane, and a non-genetic variability in the surface of schistosomes may thus occur due to the dynamic nature of the rafts. The process of transmembrane signalling in lipid rafts is described in Box 3. The heterogeneity in surface properties among individual parasites is illustrated clearly by the patterns of binding of antibody by ‘clones’ of schistosomula (Vieira and Kusel, Reference Vieira and Kusel1991), and in the range of susceptibilities of individual lung stages in a population to treatment with methyl-beta-cyclodextrin that removes cholesterol from the surface membrane (El Ridi et al. Reference El Ridi, Tallima, Mohamed and Montash2004). Many other examples of this heterogeneity are described in the article by Jones and Kusel (Reference Jones and Kusel1989).

If we consider the surface of the adult male schistosome, for instance, the membrane occupies a large surface area, covering a large number of various sensory organs, spines and tubercles and is greatly pitted and folded (McLaren, Reference McLaren1980). The same applies to earlier life-cycle stages, such as the schistosomula and lung stage, but not the eggshell. To cover so many structures and present such a pitted surface with many curves and folds, membrane composition is likely to vary throughout the worm surface, leading to differences in membrane permeability in different areas (Kusel and Gordon, Reference Kusel and Gordon1989) Rafts in the membrane are likely to allow for such versatility in membrane structure and these ideas have been reviewed for mammalian cells by Morris et al. (Reference Morris, Cox, Mombelli and Quinn2004). We hypothesize that rafts of various compositions and structures account for the variability in expression of an antigen or a receptor among individual schistosomes. There were clear differences in membrane fluidity parameters between tail, dorsal and head regions of the surface of the individual adult male. This may be due to variations in protein/lipid composition or the distribution under the bilayered membrane of the cytoskeleton. These variations may explain why certain areas are more readily damaged by poly-L-lysine than others (Jones et al. Reference Jones, Helm and Kusel1988). Differences between the dorsal surface and the gynaecophoric canal are exemplified by the morphology and the different antigens expressed (Osman et al. Reference Osman, Niles, Verjovski-Almeida and LoVerde2006). This heterogeneity may also explain why it was not possible to select for parasites highly resistant to protective immunity (Lewis et al. Reference Lewis, Heiny and Sher1985) and to praziquantel (Cioli et al. Reference Cioli, Botros, Wheatcroft-Francklow, Mbaye, Southgate, Tchuente, Troini, El-Din, Sabra, Alton, Engels and Doenhoff2004), since the expression of an antigen or a calcium channel might depend not only on the genetic make-up of the parasite, but also on heterogeneity of its distribution on the surface.

A particular motivation for the study of membrane turnover in schistosomes has been that, since the adult parasite is long-lived, any membrane proteins lipids and carbohydrates released into the bloodstream from the surface membrane may be immunogenic and could be central to inducing protective immunity. A very good account of research on schistosome membrane turnover is given in Skelly and Wilson (Reference Skelly and Wilson2006). Much of the in vitro work may have been studying membrane damage (Kusel and MacKenzie, Reference Kusel, Mackenzie and McLaren1975; Kusel et al. Reference Kusel, Mackenzie and McLaren1975; Wilson and Barnes, Reference Wilson and Barnes1977; Roberts et al. Reference Roberts, MacGregor, Vojvodic, Wells, Crabtree and Wilson1983). The membrane-derived molecules released from the parasite were analysed in a very imaginative way by Cutts and Wilson (Reference Cutts and Wilson1997). In this study, adult S. mansoni pairs or single male and females were surgically transferred into naïve mice, and the identity of antigens released was predicted/inferred based on the specificity of the antibodies stimulated. Dominant antigens released from the worms ranged from 14 to 208 kDa. The origin of most of these antigens was shown by immunofluorescence to be the gut (i.e. 32, 38, 53 and 67 kDa antigens) and/or the tegument (i.e. 14, 18, 19, 22 and 24 kDa antigens). Monoclonal antibodies made against the tegumental antigens showed some cross-reactivity with other tissues, such as the protonephridial (excretory) system (Riengrojpitak et al. Reference Riengrojpitak, Vojvodic, Boot and Wilson1989), indicating excretory antigens may be released from both the surface membrane and the latter system. This possibility must be examined further by molecular techniques, for example, by whole mount in situ hybridization. Other in vivo work measuring the loss of host erythrocyte antigens (Saunders et al. Reference Saunders, Wilson and Coulson1987) revealed a turnover time of 131 h. This result is similar to estimates based on the rates of exchange of host antigens measured by a very sensitive radioimmunoassay in experiments in which mouse-derived worms were transferred to the portal veins of monkeys (Smithers et al. Reference Smithers, Terry and Hockley1969).

Kusel and Mackenzie (Reference Kusel and Mackenzie1975) detected ‘soluble’ membrane-derived antigens in culture medium in vitro. These may represent the ‘ectodomain shedding’ reported for mammalian cells (Blobel, Reference Blobel2002) and may have been mediated by surface proteases. Perez and Terry (Reference Perez and Terry1973) showed massive tegumental changes during damage of the surface with anti-host antigen antibody; the damage caused by antibody and complement induced the production of membraneous vesicles in what is likely to be a repair process.

Membrane synthesis and turnover has also been studied using a detergent fractionation technique to separate the ‘membranocalyx’ fraction from the inner membrane fraction (Zhou and Podesta, Reference Zhou and Podesta1989). Examination of the fractions by electron microscopy and by biochemical analysis demonstrated effects of complement C3 and serotonin on the synthesis of each fraction. Synthesis of the membranocalyx was stimulated by C3, and the synthesis of the inner bilayer by serotonin (Young and Podesta, Reference Young and Podesta1986). Sands and Kusel (Reference Sands and Kusel1992) showed that serotonin, however, had an effect on the fluidity of the outer monolayer (the ‘membranocalyx’), indicating that the response of the membrane can be complex.

The identification of the protease calpain in the membrane (Siddiqui et al. Reference Siddiqui, Zhou, Podesta, Karcz, Tognon, Strejan, Dekaban and Clarke1993) suggested that this molecule is involved in membrane assembly. Braschi and Wilson (Reference Braschi and Wilson2006) also suggested that calpain was located in the surface membrane, but to the inner bilayer and suggested that it was involved in cytoskeletal remodelling. Siddiqui et al. (Reference Siddiqui, Phillips, Charest, Podesta, Quinlin, Pinkson, Lloyd, Pompa, Villalovos and Paz2003b) have developed a DNA immunization procedure using the large subunit of calpain (Sm-p80). This work was based on the imaginative idea that an inhibition of calpain may interfere with membrane synthesis. This immunization protocol gave about 40% protection (Siddiqui et al. Reference Siddiqui, Phillips, Charest, Podesta, Quinlin, Pinkson, Lloyd, Pompa, Villalovos and Paz2003a).

Cytoskeleton

At the proximal region of the surface membrane adjacent to the cytoplasm (innermost monolayer), interactions with the cytoskeleton are to be expected, which has been the subject of a fine review by Jones et al. (Reference Jones, Gobert, Zhang, Sunderland and McManus2004). Although the function of many cytoskeletal proteins is not clear, microtubules, actin, severin and fimbrin have been found in preparations of the worm surface (Braschi et al. Reference Braschi, Curwen, Ashton, Verjovski-Almeida and Wilson2006), such that important membrane-cytoskeleton interactions are to be expected, particularly during organelle fusion or endocytosis. An interesting suggestion by Jones et al. (Reference Jones, Gobert, Zhang, Sunderland and McManus2004) is that adult worms suffer a collapse of the cytoskeleton when removed from the host. This proposal is consistent with the extreme fragility of the worm surface in vitro, compared with its apparent robustness in vivo, alluded to by Skelly and Wilson (Reference Skelly and Wilson2006). Studies of the cytoskeleton of worms fixed in vivo are awaited with interest. In their excellent review of the cytoskeleton, Jones et al. (Reference Jones, Gobert, Zhang, Sunderland and McManus2004) stated: “Schistosomes express many of the classic cytoskeletal and motor proteins found in eukaryotes, but it is most plausible that we do not observe them in the distal tegument simply because we have not looked for them, because we do not know how to look for them, or because we cannot preserve the tegument adequately”. A recent paper by Pica-Mattoccia et al. (Reference Pica-Mattoccia, Valle, Basso, Troiani, Vigorosi, Liberti, Festucci and Cioli2007) has now opened up a new phase in the understanding of cytoskeletal function in schistosomes.

The aim of the work of Pica-Mattoccia et al. (Reference Pica-Mattoccia, Valle, Basso, Troiani, Vigorosi, Liberti, Festucci and Cioli2007) was to provide evidence from the living schistosome that praziquantel acts on calcium channels. This hypothesis was supported by results from a study of the interaction of praziquantel with L-type calcium channels in schistosomes expressed in a heterologous system (Xenopus) (Greenberg, Reference Greenberg2005). Pica-Mattoccia et al. (Reference Pica-Mattoccia, Valle, Basso, Troiani, Vigorosi, Liberti, Festucci and Cioli2007) initially tested this hypothesis by incubating living male worms for 1 h in growth medium containing calcium channel blockers, and subsequently incubating the culture with praziquantel overnight, washing and then observing for a further 7 days. This protocol showed that certain blockers (nifedipine and nicardipine) functioned to partially, but significantly inhibit the effect of praziquantel, suggesting that an ‘L’ type calcium channel was involved in the activity of praziquantel. Interestingly, nifedipine and nicardipine incompletely inhibited the effect of praziquantel, suggesting that, although considerable homology exists between mammalian and schistosome channels, these do not behave in the same way, an important lesson learnt from studying homologous channels in different organisms. Pica-Mattoccia et al. (Reference Pica-Mattoccia, Valle, Basso, Troiani, Vigorosi, Liberti, Festucci and Cioli2007) argued that, since mammalian L-type calcium can be regulated by the actin cytoskeleton, cytochalasin D and other inhibitors of actin function might inhibit the action of praziquantel. These authors found that cytochalasin D completely obviated the effects of praziquantel; thus, the calcium channel appeared to be regulated by the actin cytoskeleton. Although many details of this regulation are unknown, this discovery links the cytoskeleton with a vital function in the schistosome membrane. Therefore, investigations into the mechanism by which actin regulates the calcium channel will reveal a great deal about all aspects of membrane structure and function. Lipids also may be involved here, since lipid rafts have been implicated in calcium channel activity (Davies et al. Reference Davies, Douglas, Hendrich, Wratten, Tran, Foucault, Koch, Pratt, Saibil and Dolphin2006). Lipid–protein interactions will also need to be considered.

Endocytosis

In vitro studies of schistosomula or adult worms have yielded little evidence for the occurrence of endocytosis in the surface membrane. Cushley and Kusel (Reference Cushley and Kusel1987) could not demonstrate any entry of ricin into either stage of the parasite, although binding of fluorescent ricin to the surface of living worms was shown. Ribeiro et al. (Reference Ribeiro, Coelho, Vieira, Powell and Kusel1998) showed the uptake of a fluorescent marker for endocytosis, called FM 1-43, into vesicles, but no uptake of protein could be shown. Tan et al. (Reference Tan, Thornhill, Al-Adhami, Akhkha and Kusel2003) could find endocytosis in the excretory system of the schistosomulum, while damage to the surface leads to uptake of external protein by the excretory epithelium of adult worms (Oliveira et al. Reference Oliveira, Kusel, Ribeiro and Coelho2006). However (Thors et al. Reference Thors, Jokiranta, Meri, Kairemo, Meri and Linder2006), using an assay for the detection of host immunoglobulins in freshly perfused adult worms, demonstrated their presence within the worm close to the tegument. Uptake by endocytosis, or ingestion followed by endocytosis by gut cells, may have occurred.

The lysosome/autophagosome system seems to have similar properties to that of the mammalian cell. However, there may be significant differences between schistosomes and mammals (Al-Adhami et al. Reference Al-Adhami, Noble, Sharaf, Thornhill, Doenhoff and Kusel2005). Soluble N-ethyl maleimide sensitive factor attachment receptor proteins (SNARE), which mediate vesicle fusion in mammalian cells, were not found in the labelling studies conducted by Braschi and Wilson (Reference Braschi and Wilson2006).

In dealing with the host antigen hypothesis, some possible alternative functions for the host molecules (e.g. signalling, or forming a glycocalyx which might be additional to or instead of immune concealment/disguise) have been considered. Host-derived protease inhibitors, such as serpins (Modha and Doenhoff, Reference Modha and Doenhoff1994) and alpha-2 macroglobulin (Damian et al. Reference Damian, Greene and Hubbard1973) may also have distinct roles, such as the inhibition of damage from host proteases or to regulate membrane-bound parasite proteases involved in such processes as membrane turnover. Whether host-derived proteases such as contrapsin (Modha and Doenhoff, Reference Modha and Doenhoff1994) have functions distinct from those expressed by inherent schistosome genes (Blanton et al. Reference Blanton, Licate and Aman1994) remains to be determined. Similarly, complement binding is not necessarily related to immune evasion, as Young and Podesta (Reference Young and Podesta1986) showed that C3 can increase membrane turnover and may be activating signalling cascades.

The excretory system

The excretory system is extraordinarily well developed in adult male and female schistosomes. It has extensive ramifications very close to the tegument, and carries epithelial cells close to the surface membrane (Figs 1 and 2D and E). Its structure will be discussed briefly, followed by its functions, which may be very diverse in relation to the surface, the tegument and the worm as a whole.

Fig. 1.

Top panel shows, by fluorescence microscopy, the labelling of collecting ducts and branches of the excretory system of male adult Schistosoma mansoni with the fluorescent dye resorufin. Side branches issuing from the collecting ducts end very close to the surface (S). Bottom panel shows a male adult Schistosoma mansoni by bright field microscopy, with a clear view of the collecting ducts of the excretory system.

Fig. 2.

(A) Schistosomulum produced by syringe passage (Colley and Wikel, Reference Colley and Wikel1974) stained with LysoTracker to show the presence of large lysosomes (acid-rich vesicles) (LY) (Al-Adhami et al. Reference Al-Adhami, Noble, Sharaf, Thornhill, Doenhoff and Kusel2005). (B) Schistosomulum produced by syringe passage (Colley and Wikel, Reference Colley and Wikel1974) without fluorescence, stained with methylene blue. (C) Schistosomulum produced by syringe passage (Colley and Wikel, Reference Colley and Wikel1974). This has been slightly damaged by poly-L-lysine in the presence of Texas red-labelled bovine serum albumin (BSA) (Tan et al. Reference Tan, Thornhill, Al-Adhami, Akhkha and Kusel2003), and shows uptake of the fluorescent protein through the nephridiopore (N) into the excretory tubules (Tub) and adjacent tissue. (D) Adult worms incubated with poly-L-lysine, in the presence of Texas red-labelled bovine serum albumin (BSA). This shows slight areas of damage indicated by the blue fluoresecence of Hoechst 33258, and uptake of the fluorescent protein into the excretory tubules (Tub) in the damaged areas. (E) Higher magnification of the labelled tubules is also shown. (G) Indicates uptake of the fluorescent protein by the activities of the gut.

The excretory system of adult S. mansoni (both female and male) has been well described morphologically (Wilson and Webster, Reference Wilson and Webster1974; Bogers et al. Reference Bogers, Nibbeling, van Marck and Deelder1994). In brief, the functional unit is the flame cell, which leads into a fine tubule lined with epithelial cells. These tubules connect to larger tubules, similarly lined, until the tubules combine to form a large ciliated collecting duct. The collecting duct empties its contents into the external environment through a nephridiopore distal to a muscular contractile vesicle. The function of the system is assumed to be excretory, and the beating of the flame cells has been shown to vary with the osmotic pressure of the medium surrounding the parasite (Wilson and Webster, Reference Wilson and Webster1974). Some studies (Finken et al. Reference Finken, Sobek, Symmons and Kunz1994; Schechtman et al. Reference Schechtman, Winnen, Tarrab-Hazdai, Ram, Schinder, Grevelding, Kunz and Arnon2001; Skelly and Shoemaker, 2001 b; Wippersteg et al. Reference Wippersteg, Kapp, Kunz, Jackstadt, Zahner and Grevelding2002) have indicated the presence of signalling molecules in the excretory system, which may be of importance both in the response of the parasite to the host, and to the interactions between adult male and female schistosomes. Mecozzi et al. (Reference Mecozzi, Rossi, Lazzaretti, Kady, Kaiser, Valle, Cioli and Klinkert2000) have demonstrated that calcineurin is localized on the flame cells of the schistosomula and adult worms, and Schechtman et al. (Reference Schechtman, Winnen, Tarrab-Hazdai, Ram, Schinder, Grevelding, Kunz and Arnon2001) found protein 14-3-3, a molecule that regulates many signalling pathways, in the protonephridial system of the female schistosome.

P glycoproteins (P-gp) have also been detected in schistosomes (Bosch et al. Reference Bosch, Wang, Tao and Shoemaker1994; Greenberg, personal communication). For instance, Sato et al. (Reference Sato, Kusel and Thornhill2002, Reference Sato, Kusel and Thornhill2004) have shown that the excretory system of S. mansoni functions with the aid of P-gp and multi-resistance proteins (MRP) to pump out particular fluorescent substrates. This group of proteins is often over-expressed in drug-resistant tumour cells, and their function is to reduce the concentration of cytotoxic drugs in the cytoplasm of these cells (Box 6). The role of phosphorylation in the regulation of these activities is unclear, but the finding that 14-3-3 is present in the female excretory system (Schechtman et al. Reference Schechtman, Winnen, Tarrab-Hazdai, Ram, Schinder, Grevelding, Kunz and Arnon2001) suggests an interaction leading to the regulation of the P-gp activity. Furthermore, as P-gps can transport steroids (Borst et al. Reference Borst, Zelcer and van Helvoort2000), they may be involved in male–female interactions. The presence of active P-gps and multi-resistance proteins in the excretory system invites the speculation that their activity might respond to changes in the environment. Thus, these molecules could act as the sensors that monitor environmental change. In this context, Greenberg (personal communication) has shown by Western blotting that praziquantel treatment appears to induce the expression of P-gp molecules.

Excretory system in adult male and female worms

When adult worms in culture medium are labelled with the fluorescent dye resorufin, the excretory system of the male is clearly and rapidly revealed (Fig. 1). Unpaired females also show clear excretory system activity. However, it was observed that, although some dye had penetrated the surface of paired females, they were rarely labelled in their collecting ducts and tubules (Oliveira et al. Reference Oliveira, Kusel, Ribeiro and Coelho2006). These findings suggest that the male is inhibiting the excretory activity of the female worm. When the male was damaged by a drug (e.g. praziquantel) or suffered a metabolic inhibition by cooling, the activity of the female excretory system increased substantially from its previous state, even though it remained paired with the male. Therefore, we hypothesize that, when paired, the male profoundly decreases the diffusion of resorufin into the female excretory system. Cornford (Reference Cornford1985) reported that acidic amino acids were not absorbed by paired adults, although single unpaired males and females showed full transport activity. Some mechanism involving changes in membrane protein conformation may occur when pairing takes place, with signals being transmitted to transport and excretory systems.

The importance of external calcium and phosphate ions

The activity of the adult excretory system is clearly seen in RPMI medium when the parasites are labelled with resorufin. In a medium without calcium ions (e.g. Dulbecco's Modified Eagle's Medium, DMEM), the excretory tubules are inactive. However, with the addition of calcium ions, the tubules regain their activity (Kusel et al. Reference Kusel, Oliviera, Coelho, Mattos, Reis, Lima and Ribeiro2006). Similarly, phosphate ions are also essential (Kusel et al. Reference Kusel, Oliviera, Coelho, Mattos, Reis, Lima and Ribeiro2006). This evidence suggests that the external surface can respond and transport external calcium and phosphate ions (Kusel et al. Reference Kusel, Oliviera, Coelho, Mattos, Reis, Lima and Ribeiro2006). A membrane-bound calcium and phosphate sensor may be present (Garrett et al. Reference Garrett, Capuano, Hammerland, Hung, Bron, Herbert, Nemeth and Fuller1995). There are some candidates that might be involved in the calcium/phosphate effect on the excretory system. Reports of surface enzymes sensitive to calcium have been in the literature for some time (Cunha et al. Reference Cunha, Meyer-Fernandes and Noel1992) and recent proteomic data for the surface membrane (Braschi and Wilson, Reference Braschi and Wilson2006) suggest the presence of a calcium ATPase (Accession no. C604128.1). Alkaline phosphatase has been implicated in the transport of phosphate through the membrane (Peerce et al. Reference Peerce, Fleming and Clarke2003). However, there may be a different explanation for the apparent linkage between the surface membrane and the excretory system. It has been shown that the tegumental membrane potential depends crucially on the presence of glutamine and an optimum phosphate concentration (Lane et al. Reference Lane, Pax and Bennett1987). Thus, the membrane potential of the excretory tubules may be linked to the surface in the same way as are the muscle membranes. Depolarization of the surface membrane may lead to depolarization and lack of function of P-gps.

Effect of removal of cholesterol from the surface

It has been shown that treatment of the adult male worm with methyl-beta-cyclodextrin (MCD), which disrupts lipid rafts in cell membranes (El Ridi et al. Reference El Ridi, Tallima, Mohamed and Montash2004), caused the excretory system to become active in its excretion of resorufin (Oliveira et al. Reference Oliveira, Kusel, Ribeiro and Coelho2006). This finding could be explained by suggesting that the disruption of rafts in the bilayer(s) of the surface alters calcium transport into the tegumental cytoplasm, altering the activity of the excretory system. It has been shown for blood cells (Unruh et al. Reference Unruh, Li, Mutch, Sharia, Grigoriou, Sanval, Brown and Deans2005) that the function of CD20 (which is putatively a store-operated calcium channel) is affected by the integrity of lipid rafts. However, the observation may mean that the P-gps are directly involved in cholesterol metabolism. This is a possibility, since P-gps are known to pump steroids (Debry et al. Reference Debry, Nash, Neklason and Metherall1997). P-gps are present in rafts in multidrug-resistant mammalian cells, and alteration of cholesterol levels can cause a change in activities and in raft localization (Troost et al. Reference Troost, Lindenmaier, Haefeli and Weiss2004).

Damage to the surface by poly-L-lysine

Incubation with damaging concentrations of poly-L-lysine has been shown to rapidly induce activity of the excretory system (Kusel et al. Reference Kusel, Oliviera, Coelho, Mattos, Reis, Lima and Ribeiro2006), which may be due to an influx of calcium and phosphate ions into the tegumental cytoplasm. It could not be replicated by the calcium ionophore ionomycin, although the latter did give a stimulus that caused sustained activity of the excretory system. Repair of the membrane may involve removal of Ca⁺⁺ to low levels by intracellular pumps, as indicated by the repair of mammalian membranes, which needs a low calcium ion concentration (Reddy et al. Reference Reddy, Caler and Andrews2001).

The effects of praziquantel

Praziquantel completely inhibits the function of the excretory system of the adult male schistosome, as demonstrated by resorufin staining (Oliveira et al. Reference Oliveira, Kusel, Ribeiro and Coelho2006). The mechanism is likely to be due to the profound inhibition of the male excretory system by the excess of calcium entering the worm tissues or released from intracellular stores. The male parasite can recover rapidly from this inhibition, perhaps by pumping calcium ions from tissues, either out of the worm or back into intracellular stores. The enzyme systems responsible for the pumping of calcium ions are unknown, but there are numerous candidate membrane-bound enzymes represented in the transcriptome (Verjovski-Almeida et al. Reference Verjovski-Almeida, DeMarco, Martins, Guimarães, Ojopi, Paquola, Piazza, Nishiyama, Kitajima, Adamson, Ashton, Bonaldo, Coulson, Dillon, Farias, Gregorio, Ho, Leite, Malaquias, Marques, Miyasato, Nascimento, Ohlweiler, Reis, Ribeiro, Sá, Stukart, Soares, Gargioni, Kawano, Rodrigues, Madeira, Wilson, Menck, Setubal, Leite and Dias-Neto2003). These enzymes are vital for the function of the excretory system. Further work will elucidate their identity. In human submandibular gland cell membranes, store-operated calcium (SOC) channels are located in rafts associated with caveolin (Lockwich et al. Reference Lockwich, Liu, Singh, Jadlowiec, Weiland and Ambudkar2000). It would be interesting to establish whether caveolin in the schistosome membrane (Racoosin et al. Reference Racoosin, Davies and Pearce1999) is associated with calcium channels of this type. Recent work has suggested that the actin cytoskeleton may control the activity of SOC channels (Cioffi et al. Reference Cioffi, Wu, Alexeyev, Goodman, Zhu and Stevens2005) and the adenyl cyclase associated with caveolin (Head et al. Reference Head, Patel, Roth, Murray, Swaney, Niesman, Farquhar and Insel2006).

Pica-Mattoccia et al. (Reference Pica-Mattoccia, Valle, Basso, Troiani, Vigorosi, Liberti, Festucci and Cioli2007) have shown that the treatment of adult male schistosomes with cytocalasin D (which binds to the end of F-actin chains) prevents the action of praziquantel and, by implication, inactivated calcium channels, which are the putative sites of action of PZQ (Greenberg, Reference Greenberg2005). This exciting finding suggests that the cytoskeleton of the adult schistosome has a function in the control of the activity of calcium channels, so that, if the cytoplasmic concentration increases, the dissociation of the actin cytoskeleton prevents further ingress of calcium in a feedback loop with the calcium channels. This finding might explain why immature stages are less susceptible to praziquantel, if the feedback mechanism has not developed in these forms. In contrast to male worms after treatment with PZQ, adult females, that were paired with males, showed increased activity of their excretory systems (Oliveira et al. Reference Oliveira, Kusel, Ribeiro and Coelho2006). The female has a system that is much less sensitive to the calcium ion concentration. This finding may explain why unpaired female worms are less susceptible to PZQ than unpaired male worms (Gonnert and Andrews, Reference Gonnert and Andrews1977). This seems also to be the case for cercariae and parasites between 7 days and 4 weeks old (Kusel et al. Reference Kusel, Oliviera, Coelho, Mattos, Reis, Lima and Ribeiro2006).

Membrane damage and repair and the activity of the excretory system

The damage and repair of the adult schistosome membrane has been studied by electron microscopy (Perez and Terry, Reference Perez and Terry1973; Wilson and Barnes, Reference Wilson and Barnes1977; Roberts et al. Reference Roberts, MacGregor, Vojvodic, Wells, Crabtree and Wilson1983; Popiel et al. Reference Popiel, Irving and Basch1985) and, recently, by using the uptake of fluorescent proteins as an indication of the production of lesions by poly-L-lysine (Oliveira et al. Reference Oliveira, Kusel, Ribeiro and Coelho2006). When such lesions were induced by poly-L-lysine in schistosomula (Tan et al. Reference Tan, Thornhill, Al-Adhami, Akhkha and Kusel2003), it was concluded that the excretory system absorbed environmental protein into its tubules through the nephridiopore, and this protein was endocytosed and transferred into the body of the parasite. As a consequence of damage to adult worms fluorescent proteins were endocytosed from the tegumental cytoplasm into the epithelial cells of the excretory system and passed into the nephridiopore (Wippersteg et al. Reference Wippersteg, Ribeiro, Liedke, Kusel and Grevelding2003). It is possible that the excretory system is acting as an organ system active in the repair of the lesion. Certainly, the activity in pumping resorufin is increased by surface membrane damage (Oliveira et al. Reference Oliveira, Kusel, Ribeiro and Coelho2006) and thus might be expected to remove ions and metabolic end products, for example, lactic acid. However, it is possible that the excretory epithelium removes denatured proteins, lipids and carbohydrates that are released due to worm damage, and has an anti-apoptotic effect by reducing the concentration of effector molecules, such as cytochrome c. Further work, to establish the fate of such molecules within the parasite, would be of great importance and may reveal new vesicle cycling mechanisms.

The repair of the surface membrane is likely to involve vesicle fusion, lipid synthesis and turnover (Redman et al. Reference Redman, Kennington, Spathopoulou and Kusel1997; Brouwers et al. Reference Brouwers, Skelly, Van Golde and Tielens1999) as well as protein and carbohydrate chain synthesis. Few studies have examined repair as such, but the rapid turnover rates reported for the surface membrane may have been a reflection of inadvertently inflicted damage. Electron microscopy of tissues subsequent to lesions (cuts) made in the tegument (Popiel et al. Reference Popiel, Irving and Basch1985) has shown the involvement of the membranous bodies in the repair process. Evidence from mammalian cells suggests that lysosomes can be involved in membrane repair in calcium-linked exocytosis (Reddy et al. Reference Reddy, Caler and Andrews2001). Many cytoplasmic vesicle structures may be involved (Keefe et al. Reference Keefe, Shi, Feske, Massol, Navarro, Kirchhausen and Lieberman2005). In schistosomes, it is possible that vesicles cycled from the excretory system, lysosomes and other vesicles may be involved in membrane repair. In mammalian cells damaged by perforin many organelles contribute to the repair process (Keefe et al. Reference Keefe, Shi, Feske, Massol, Navarro, Kirchhausen and Lieberman2005), so that the cell undergoes apoptosis rather than necrosis. Clearly, much more work is required to address this topic in schistosomes. Very few biochemical studies have been carried out on membrane repair. The results of an in vitro study of sphingomyelin synthesis (Redman et al. Reference Redman, Kennington, Spathopoulou and Kusel1997) have suggested a rapid rate of membrane repair but another study (Brouwers et al. Reference Brouwers, Skelly, Van Golde and Tielens1999) did not report any such evidence. This is an important field for further investigations.

CERCARIAE AND SCHISTOSOMULA

The recognition and penetration of the mammalian skin (Stirewalt, Reference Stirewalt1974) is the first step in a complex process of transformation from a free–living cercaria to a schistosomulum, which migrates from the skin through a variety of tissues and finally locates in the bloodstream of the host. Cercariae form a population with variable metabolic properties, and they penetrate and migrate at different rates. Cercariae of S. japonicum migrate much more rapidly than those of S. mansoni (see Gui et al. Reference Gui, Kusel, Shi and Ruppel1995).

The penetration of human skin by schistosome cercariae has been studied by Whitfield et al. (Reference Whitfield, Bartlett, Khammo, Brain, Brown, Marriott and Clothier2003), who showed that the cercarial tail often penetrates the epidermis and is not cast off, as is usually found for other mammalian skins and other trematode species. Whitfield et al. (Reference Whitfield, Bartlett, Khammo, Brain, Brown, Marriott and Clothier2003) suggested that this behaviour may have relevance to the phenomenon of concomitant immunity, since the antigens from the tail musculature or its very active excretory system (Stirewalt, Reference Stirewalt1974) may induce a specific immune response in the host. A stimulating discussion on cercarial penetration of human and other skins was published by McKerrow (Reference McKerrow2003) and Curwen and Wilson (Reference Curwen and Wilson2003).

Enzymes are required from the parasite to complete the migration of the schistosomulum, and present evidence shows that those of S. japonicum could be very different from those found in S. mansoni and S. haematobium (see Chlichlia et al. Reference Chlichlia, Schauwienold, Kirsten, Doenhoff, Fishelson and Ruppel2005). Intriguingly, it seems that, as a result of a natural infection, no antibodies are produced in the host against the cercarial elastase (Bahgat et al. Reference Bahgat, Franklow, Doenhoff, Li, Ramzy, Kirsten and Ruppel2001). This enzyme hydrolyses specific anti-parasite immunoprecipitating antibodies, putatively IgG (Darani et al. Reference Darani, Curtis, McNeice, Price, Sayers and Doenhoff1997) and also IgE (Pleass et al. Reference Pleass, Kusel and Woof2000). When antibody responses were raised in mice against elastase, some protection against challenge infection was achieved (Darani et al. Reference Darani, Curtis, McNeice, Price, Sayers and Doenhoff1997).

Signals from the host must be received for normal development. Careful recent studies (Knudsen et al. Reference Knudsen, Medzihradszky, Lim, Hansell and McKerrow2005; Curwen et al. Reference Curwen, Ashton, Sundaralingam and Wilson2006) have built on the much earlier work of Stirewalt (Reference Stirewalt1974) on the process by which the cercariae enter the host skin and on the involvement of the acetabular glands in this process. Curwen et al. (Reference Curwen, Ashton, Sundaralingam and Wilson2006) have identified new peptidases and proteases likely to be involved in skin penetration. Furthermore, venom allergens similar in homology to chemokines, and potassium channel blockers may affect the T cell response to the cercariae (Curwen et al. Reference Curwen, Ashton, Sundaralingam and Wilson2006). In view of the high calcium ion concentration in the secretions, it was interesting that Knudsen et al. (Reference Knudsen, Medzihradszky, Lim, Hansell and McKerrow2005) identified a calcium-binding protein, calcium ATPase, and calreticulin. Paramyosin was also detected, and Deng et al. (Reference Deng, Gold, LoVerde and Fishelson2003) demonstrated an important role for parasite surface-bound paramyosin in inhibiting complement activation. Schistosomula have also a surface bound elastase (Ghendler et al. Reference Ghendler, Parizade, Arnon, McKerrow and Fishelson1996) which may be related to the above-mentioned secreted elastase, but the function of which has not been fully investigated.

During penetration the cercaria releases from membrane, glands or the excretory system phospholipids which were identified as lysophosphatidylserine and which can interact with Toll-like receptor 2 on dendritic cells (Van der Kleij et al. Reference Van der Kleij, Latz, Browers, Kruize, Schmitz, Kurt-Jones, Espevik, de Jong, Kapsenberg, Golenbock, Tielens and Yazdanbakhsh2002). This interaction allows presentation of parasite antigens in a mode that favours the induction of regulatory T cells and the production of IL-10. This very interesting work shows us how important it is to keep an open mind about the function of parasite molecules. Lysophosphatidylserine can act as a detergent and one possible function is that it lyses host cells, and one might therefore have speculated that if it reacts with membranes of effector cells and destroys them, it functions as a mechanism of immune evasion. For example, lysophosphatidylcholine produced by adult worms has been shown to lyse erythrocytes (Golan et al. Reference Golan, Brown, Cianci, Furlong and Caulfield1986; Brouwers et al. Reference Brouwers, Skelly, Van Golde and Tielens1999). However, it seems from the work of Van der Kleij et al. (Reference Van der Kleij, Latz, Browers, Kruize, Schmitz, Kurt-Jones, Espevik, de Jong, Kapsenberg, Golenbock, Tielens and Yazdanbakhsh2002) that lysophosphatidylserine has a signalling function. Thus the earliest stage of the parasite is adapted to modulate the host's immune response in an extremely sophisticated manner. This is further seen as eicosanoids are also released and these can interact with dendritic cells to prevent their migration to lymph nodes and also to favour a Th2 response (Angeli et al. Reference Angeli, Faveeuw, Roye, Fontaine, Tessier, Capron, Wolowczuk, Capron and Trottein2001). These molecules are synthesized from arachidonic acid that may be derived from the skin of the host or from membrane phospholipids in the cercariae, the arachidonic acid being originally derived from the snail or from linoleic acid (Fusco et al. Reference Fusco, Salafsky and Delbrook1986). Thus, the host, especially through skin-derived arachidonic acid, is contributing to the survival of the parasite in its modulation of the immune response! This is a good example of the ‘welcome mat’ concept that was envisaged by Davies et al. (Reference Davies, Hall, Targett and Murray1980). The transfer of macromolecules in vitro to the membrane of the schistosomulum from red blood cells and from radioactively-labelled epithelial cells has been observed by Goldring et al. (Reference Goldring, Kusel and Smithers1977a). The glycocalyx fixes complement very vigorously and because it is shed rapidly, there is little surface membrane damage. Where complement binds to membranes, and this is relevant to the metabolism of the adult as well as the schistosomulum, sublytic membrane attack complexes (MAC) can signal across the membrane and have a growth effect (Bohana-Kashtan et al. Reference Bohana-Kashtan, Ziporen, Donin, Kraus and Fishelson2004).

It is not clear how active the excretory system is during its development in the schistosomulum. Tail loss from the cercaria exposes the nephridiopore and damaged schistosomula take up protein through this orifice (Tan et al. Reference Tan, Thornhill, Al-Adhami, Akhkha and Kusel2003) and this uptake may occur during skin penetration. No excretion of resorufin has been found in schistosomula at this stage (Kusel et al. Reference Kusel, Oliviera, Coelho, Mattos, Reis, Lima and Ribeiro2006). A range of molecules having profound effects on the host are released from the schistosomulum during migration and from the adult when in the portal vein and its vascular ramifications in the gut and liver. What is the origin of these molecules in the structures of the parasite?

The immunomodulatory lysophosphatidylserine is produced by the adult worms and eggs and perhaps schistosomula (Van der Kleij et al. Reference Van der Kleij, Latz, Browers, Kruize, Schmitz, Kurt-Jones, Espevik, de Jong, Kapsenberg, Golenbock, Tielens and Yazdanbakhsh2002), but the mechanism of release of the molecule has not been studied. Similarly, the lung stage produces ‘excretory products’ which can decrease the permeability of lung endothelial cells and have an anti-inflammatory effect, but their origin is not known (Trottein et al. Reference Trottein, Descamps, Nutten, Dehouck, Angeli, Capron, Cecchelli and Capron1999a, Reference Trottein, Nutten, Angeli, Delerive, Tessier, Capron, Staels and Capronb). The authors suggested these molecules from the parasites may be eicosanoids and prostaglandins. Angeli et al. (Reference Angeli, Faveeuw, Roye, Fontaine, Tessier, Capron, Wolowczuk, Capron and Trottein2001) described the formation of prostaglandin D2 (PGD2) by schistosomula and the inhibition that this excretory product induces on the Langerhans cells in the skin. These authors also mentioned that PGD2 synthase is secreted into the skin during penetration, and this could increase PGD2, a molecule derived from the host by the enzyme secreted by the parasite. It transpired that PGD2 synthase was in fact a glutathione S-transferase isoform (Herve et al. Reference Herve, Angeli, Pinzar, Wintjens, Faveeuw, Narumiya, Capron, Urade, Capron, Riveau and Trottein2003).

Neuropeptides, morphine-like compounds and renin-angiotensin peptides are secreted by all stages of the parasite and these compounds may have an immunomodulatory function (Salzet et al. Reference Salzet, Capron and Stefano2000; Duvaux-Miret et al. Reference Duvaux-Miret, Stefano, Smith, Mallozzi and Capron1992). An apoptosis-inducing molecule of about 23 kDa molecular weight is secreted and induces apoptosis in the effector cells which gather around the parasite (Chen et al. Reference Chen, Rao, He and Ramaswamy2002). It is a distinct possibility that the excretory system and its epithelia are actively involved in the release of many of these molecules, and further research is needed to determine whether this is the case. If so, regions of the excretory system may be targets of protective immunity. Currently nothing is known unequivocally about those compounds excreted through the nephridiopore.

Other molecules can be taken up from the skin cells during penetration, and there is a massive release of glycocalyx during the penetration process. Skin lipids, proteins (Smith and Kusel, Reference Smith and Kusel1979) and blood group substances can be incorporated into the membrane. This means that the skin schistosomulum is a very different organism from the mechanically produced form. No proteomic comparisons have been made between skin-transformed and mechanically transformed schistosomula, but DNA arrays of cultured and lung-derived schistosomula have been compared (Chai et al. Reference Chai, McManus, McInnes, Moertel, Tran, Loukas, Jones and Gobert2006). Certain genes (e.g. heat shock protein 70, the anti-inflammatory Sm16 and paramyosin) are expressed at a higher level in organisms taken from the living host when compared with the in vitro-transformed and cultured schistosomula. The acquisition of, or interaction with, molecules from the host may be a cause of transmembrane signalling and thereby cause changes in transcription. Shaker et al. (Reference Shaker, Wu, El-Shobaki, Ashour, Khattab, Draz, Kamel and Wu1998) have shown increases in the rates of cell division in parasites cultured in portal blood compared with those maintained in peripheral blood and they showed that portal blood contains a factor of 38 kDa which stimulates cell division in cultured 20-day-old worms. Rocha and Coelho (Reference Rocha and Coelho1980) inoculated schistosomula directly into the portal system of mice and these showed an enhanced rate of development compared to those schistosomula derived from the normal transcutaneous infection process. During migration through skin schistosomula can stimulate dermal endothelial cells to produce IL-7, which in turn can stimulate the development of the parasite and increase its fecundity (Wolowczuk et al. Reference Wolowczuk, Nutten, Roye, Delacre, Capron, Murray, Trottein and Auriault1999). It is not currently understood how the secreted molecules are involved in the development of the parasite during and after penetration. Some could be vaccine candidates but their suitability in this regard depends on their accessibility to the immune effectors. The cercarial secretions, if injected into the skin, do not protect against an infection (Harn et al. Reference Harn, Cianci and Caulfield1989; Fallon et al. Reference Fallon, Teixeira, Neice, Williams, Hellewell and Doenhoff1996). The penetration of the skin and subsequent migration can be enhanced if cercarial extracts or the medium in which mechanically-transformed cercariae are incubated are injected into the penetration site prior to exposure of the skin to cercariae (Fallon et al. Reference Fallon, Teixeira, Neice, Williams, Hellewell and Doenhoff1996), possibly because constituents in the injected fluid, particularly proteases, induce an inflammation which involves influx of neutrophils and eosinophils (Teixeira et al. Reference Teixeira, Doenhoff, McNeice, Williams and Hellewell1993). Also released into the skin are factors that decrease inflammation such as the anti-inflammatory protein Sm16 (Rao and Ramaswamy, Reference Rao and Ramaswamy2000) and eicosanoids (Angeli et al. Reference Angeli, Faveeuw, Roye, Fontaine, Tessier, Capron, Wolowczuk, Capron and Trottein2001). Migration of the parasite through host tissues appears to be a delicate balance between pro- and anti-inflammatory effects of molecules released by the parasite (Mountford and Trottein, Reference Mountford and Trottein2004). Harrop and Wilson (Reference Harrop and Wilson1993) cultured S³⁵ methionine-labelled schistosomula and showed that in the first 24 h few proteins were synthesized. Between 1 and 7 days numerous proteins (from <20 kDa to >100 kDa) were synthesized and released. These were immunogenic, but many were unidentified and their function unknown.

During early transformation, remarkable changes occur in the structure of the cercarial membrane, as first described by Hockley and McLaren (Reference Hockley and McLaren1973). Some of the molecular events involved in this process have been elegantly described by Skelly and Shoemaker (Reference Skelly and Shoemaker2001a). For example, relocation of the glucose transporter SGTP4 from intracellular vesicles into the surface membrane by membrane fusion. Other changes in the membrane may result in the transient expression of particular proteins that are not usually found in mature membranes (Skelly and Wilson, Reference Skelly and Wilson2006). These molecules could be ‘cryptic’ antigens and possible vaccine targets in view of their brief expression on the surface. Tetraspanin in the membrane may be exposed at this stage and thus achieve some degree of protective immunity (Tran et al. Reference Tran, Pearson, Bethony, Smyth, Jones, Duke, Don, McManus, Correa-Oliveira and Loukas2006). Also, a molecule named Sm Fes, which is an fes/fps tyrosine kinase has been discovered in the tegument of both miracidium and cercaria and is suggested to have a signalling function during host penetration and parasite transformation (Bahia et al. Reference Bahia, Mortara, Kusel, Andrade, Ludolf, Kuser, Avelar, Trolet, Dissous, Pierce and Oliveira2007).

The profound environmental changes experienced by the parasite as it passes from fresh water to the skin and tissues of the mammalian host are accompanied by secretions from the acetabular glands and the re-organization of parasite tissues following these secretory processes. There are also surface membrane and cytoskeletal changes (Skelly and Shoemaker, Reference Skelly and Shoemaker2001a) as well as re-oganization of tissue near the nephridiopore after the loss of the tail of the cercaria, immediately following skin penetration. The tissue remodelling and perhaps the repair processes are likely to be carried out by the lysosomal/autophagosome system. This remodelling was first described for helminths by Threadgold and Arme (Reference Threadgold and Arme1974) and Bogitsh (Reference Bogitsch1975) in gastric epithelial cells. In mammalian cells and yeast, the lysosome and autophagosome pathway is induced by signals acting on PI3 kinase. This pathway is also present in the schistosomulum, as was shown by inhibitors of the PI3 kinase-stimulated vesicular pathway, such as wortmannin, 3-methyl adenine, and N-ethylmaleimide (Al-Adhami et al. Reference Al-Adhami, Noble, Sharaf, Thornhill, Doenhoff and Kusel2005). In this work, the inhibitors had to be present in the transforming fluid for them to be active, and thus may have entered the transforming parasite via the nephridiopore (Tan et al. Reference Tan, Thornhill, Al-Adhami, Akhkha and Kusel2003, Fig. 2C).

Lysosomes and autophagosomes have not been found in cercariae or miracidia, but they form rapidly during transformation of the cercariae into schistosomula (Al-Adhami et al. Reference Al-Adhami, Noble, Sharaf, Thornhill, Doenhoff and Kusel2005, Fig. 2A) or the miracidia into mother sporocysts (Mattos et al. Reference Mattos, Kusel, Pimenta and Coelho2006). The signals triggering the formation of these organelles are unknown, but are likely to be related to the effects of environmental change. The cultivation of schistosomula in Eagle's medium containing 5% foetal calf serum increases the number of these organelles, whereas culturing them in a starvation medium lacking amino acids leads to the formation of very large lysosomes. These observations were made using quantitative fluorescence with lysosome- and autophagosome-specific fluorescent probes, but Western blot analysis of parasite extracts lent support to the conclusion that there was an induction of lysosome specific (Lamp-1 and MAP-LC3) protein synthesis (Al-Adhami et al. Reference Al-Adhami, Noble, Sharaf, Thornhill, Doenhoff and Kusel2005).

The migration of schistosomula through tissues and the bloodstream brings the surface membrane into contact with a variety of host cell membrane-bound and plasma macromolecules. Some of these may act as signals for metabolic pathways or stimulate the parasite nervous system, hence signalling environmental change. This signalling can have a profound effect on gene expression, as was shown when in vitro-grown schistosomula were compared with those derived from the infected host using a transcriptomic approach (Chai et al. Reference Chai, McManus, McInnes, Moertel, Tran, Loukas, Jones and Gobert2006). Among the genes upregulated from in vivo parasites are those responding to stress (e.g. hsp70) and those associated with immune evasion, such as Sm16, suggested to be an anti-inflammatory protein. During development in the skin, the schistosomulum requires the presence of IL-7, in order to develop to maturity (Wolowczuk et al. Reference Wolowczuk, Nutten, Roye, Delacre, Capron, Murray, Trottein and Auriault1999). Later during development in the liver, the parasite also requires the presence of factors from a subset of CD4+ cells (Hernandez et al. Reference Hernandez, Kim, McKerrow and Davies2004). This is another example of a close interplay between the host and the parasite. Thus, it is necessary to be aware of these signals and explore the nature and location of their receptors in the future.

SCHISTOSOME EGGS: POTENT IMMUNOGENS AND THROMBOGENS

Much of what has been discussed indicates that the survival of adult worms is most likely aided by their immunological, and indeed also haemostatic inertness (i.e. the parasite does not trigger the blood clotting mechanisms of the host). The contrast with the potent immuno- and thrombogenicity of schistosome eggs could hardly be more stark. The egg presents to the host a surface through which a variety of molecules are secreted and with which host cells can interact. There is also evidence that secreted molecules can modify host lipid metabolism and promote angiogenesis, which may aid egg excretion and preserve liver and intestinal functions.

VARIABILITY WITHIN PARASITE POPULATIONS

Individual cercariae in a population, even if derived from a monomiracidial infection, differ from each other in a variety of biochemical, metabolic (Jones and Kusel, Reference Jones and Kusel1989; Al-Adhami et al. Reference Al-Adhami, Doenhoff, Thornhill, Akhkha, White and Kusel2001) and immunological (Vieira and Kusel, Reference Vieira and Kusel1991) properties. For example, the individuals of a population stained with an antiserum developed in rabbits against snail haemolymph gave a wide range of values when the bound antibody was quantified on individual cercaria by the binding of a fluorescent anti-rabbit antibody (Vieira and Kusel, Reference Vieira and Kusel1991). Some of this variability is due to the physical modification to the surface during the release from the snail. Some variability may be due to the position in the snail that the daughter sporocyst occupies during growth. A further origin of cercarial diversity may be due to genomic instability. Variability at the DNA level among individual cercaria was first shown by Grevelding (Reference Grevelding1995) and was detected by examining W1 and W2 tandem repeat sequences occurring in individual cercariae using a polymerase chain reaction (PCR)-based approach. The sequence variability among individual cercariae was thought to occur during the differentiation of the cercariae in the daughter sporocyst, and it was suggested that genome instability could be a method of introducing variation in the cercarial population (Grevelding, Reference Grevelding1995). What are the consequences of this genetic variability? It could reflect a substantial variability among individual parasites in their ability to repair the surface membrane after damage and may thus be relevant to the ability to vaccinate against parasitic infections. Variability in the susceptibility of individuals to be damaged by polycations has been described (Jones et al. Reference Jones, Helm and Kusel1988), although the genetic basis for this variability is unknown. It was also found that the rates of development of the individual parasites may differ (Clegg, Reference Clegg1965).

Although some recent results are promising (Tran et al. Reference Tran, Pearson, Bethony, Smyth, Jones, Duke, Don, McManus, Correa-Oliveira and Loukas2006), attempts to develop a vaccine against schistosomes have not yet led to a fully effective product and there is a lack of a convincing explanation for this. In conventionally used mouse models, the levels of protection induced by immunization with candidate vaccine proteins (whether surface antigens, oxidative enzymes, or receptors) have usually not exeeded 50% reduction in the worm burden of mice when compared with unvaccinated control mice. The inevitability of 50% or more worms surviving any and every vaccination protocol may be explained by 2 hypotheses. (1) The different migration routes taken by the challenge larvae, or the different periods of time the larvae spend in the skin, mean that some larvae are exposed to immune effector mechanisms for longer periods than others (Wilson, Reference Wilson, Rollinson and Simpson1987). (2) Variation among larvae in their susceptibility to damage from the immune system, because of differences in some aspect of metabolism linked to their rates of development or of repair of membranes and other lesions. Whether of genetic or non-genetic origin, the variability in metabolism (Al-Adhami et al. Reference Al-Adhami, Doenhoff, Thornhill, Akhkha, White and Kusel2001) of the cercariae and schistosomula will affect the way they can interact with the tissues and immune system of the host, which could in turn influence their survival rates. Attempts to select cercariae resistant to immune damage have been unsuccessful (Lewis et al. Reference Lewis, Heiny and Sher1985; Cioli, personal communication), which suggests that this type of variability is of a non-genetic origin.

These hypotheses could be tested and might lead to the following conclusions. If hypothesis 1 were correct, vaccination that yields complete protection will always be impossible because we cannot control the migration routes or periods spent in the skin. If hypothesis 2 were correct, we may be able to vaccinate if we understand some of the metabolic processes involved in development or repair of membrane damage and target them using vaccine strategies. However, as for the adult worm, very little is known about membrane repair processes in schistosome larvae.

AREAS FOR WHICH THERE IS LITTLE UNDERSTANDING, BUT WHICH ARE OF GREAT FUTURE IMPORTANCE

CONCLUSIONS AND PERSPECTIVES

In this review, we have tried to introduce some suggestions about the life of the immature and adult parasite, as follows. (1) There is a constant communication (‘cross-talk’) between host and parasite. Host molecules binding to the parasite may be involved in signalling. Serum cholesterol can be regulated by the presence of eggs in the bloodstream and in tissues and thus eggs may indirectly control those adult membrane properties, (such as antigen expression) which are dependent on cholesterol. (2) The parasite surface is a membrane, but one composed of a very heterogeneous mixture of lipid rafts through which signalling to the cytoplasm of the tegument occurs. Some signalling may be modified by changing raft structure according to serum composition. (3) This membrane may contain relatively few receptor molecules, but they are stimulated by ligand binding, the stimulation being greatly magnified in the cytoplasm and throughout the worm via the activity of PgP in the excretory system. The extraordinarily extensive excretory system thus acts as a mechanism to increase the effects of outer surface changes in the male worm. The female excretory system seems to be inhibited in the male-female pair, and may not be responsive to environmental change until the female is separated from the male. (4) The community of schistosome pairs in the portal system sense each other by macromolecules secreted via the membrane, gut and excretory system. (5) The male adult worm provides both stimulatory and inhibitory signals to the female worm, perhaps by membrane raft or sensory organ perturbation, or through the glycocalyx. (6) The adult worm surface has a glycocalyx that transduces signals to the parasite during environmental change. The macromolecules in the glycocalyx contain the Lewis X epitope, also found in egg secretions and circulating adult worm antigens. The significance of this distribution remains to be elucidated. Figs 3 and 4 provide a diagrammatic summary of some of these aspects.

The parasite is seen as an organism in such a dynamic equilibrium with the host that it needs help from the host at every stage of its development. In some ways the host can benefit from the presence of the parasite.

Fig. 3.

This diagram shows some features of the tegument and associated excretory system and highlights some structures and processes that warrant further investigation. The index numbers refer to a few informative references on each subject. (1) Schmidt (Reference Schmidt1995). (2) Kovbascnuk and Spring (Reference Kovbasnjuk and Spring2000) and Platts and Duling (2004). (3) Wuhrer et al. (Reference Wuhrer, Balog, Catalina, Jones, Schramm, Haas, Doenhoff, Dunne, Deelder and Hokke2006b). (4) Racoosin et al. (Reference Racoosin, Davies and Pearce1999), Thi et al. (Reference Thi, Tarbell, Weinbaum and Spray2004) and Tallima and El Ridi (Reference Tallima, Salah and El Ridi2005). (5) Braschi and Wilson (Reference Braschi and Wilson2006). (6) Davies and Pearce (Reference Davies and Pearce1995). (7) Lockwich et al. (Reference Lockwich, Liu, Singh, Jadlowiec, Weiland and Ambudkar2000). (8) Jones et al. (Reference Jones, Gobert, Zhang, Sunderland and McManus2004). (9) Hockley (Reference Hockley1973). (10, 11) Mair et al. (Reference Mair, Maule, Day and Halton2000, Reference Mair, Niciu, Stewart, Brennan, Omar, Halton, Mains, Eipper, Maule and Day2004). (12) Schechtman et al. (Reference Schechtman, Winnen, Tarrab-Hazdai, Ram, Schinder, Grevelding, Kunz and Arnon2001). (13) Roberts et al. (Reference Roberts, MacGregor, Vojvodic, Wells, Crabtree and Wilson1983), Popiel et al. (Reference Popiel, Irving and Basch1985) and Oliviera et al. (Reference Oliveira, Kusel, Ribeiro and Coelho2006). (14) Skelly (Reference Skelly, Secor and Colley2005). (15) Greenberg (Reference Greenberg2005) and Pica-Mattoccia et al. (Reference Pica-Mattoccia, Valle, Basso, Troiani, Vigorosi, Liberti, Festucci and Cioli2007). (16) Hockley and McLaren (Reference Hockley and McLaren1973). (17) Braschi and Wilson (Reference Braschi and Wilson2006). (18, 19) Wilson and Webster (Reference Wilson and Webster1974), Bogers et al. (Reference Bogers, Nibbeling, van Marck and Deelder1994) and Mecozzi et al. (Reference Mecozzi, Rossi, Lazzaretti, Kady, Kaiser, Valle, Cioli and Klinkert2000). (20, 21) Sato et al. (Reference Sato, Kusel and Thornhill2002, Reference Sato, Kusel and Thornhill2004). (22, 23, 24) Finken et al. (Reference Finken, Sobek, Symmons and Kunz1994), Bogers et al. (Reference Bogers, Nibbeling, van Marck and Deelder1994) and Oliveira et al. (Reference Oliveira, Kusel, Ribeiro and Coelho2006). (25) Tan et al. (Reference Tan, Thornhill, Al-Adhami, Akhkha and Kusel2003) and Oliveira et al. (Reference Oliveira, Kusel, Ribeiro and Coelho2006).

Fig. 4.

This diagram is a summary of some areas for future investigations in skin penetration by cercariae, and subsequent migration and development of schistosomula in the mammalian host. As in Fig. 3, the index numbers refer to informative references in each subject. (1) Cercariae-variability, Grevelding (Reference Grevelding1995) and Al-Adhami et al. (Reference Al-Adhami, Doenhoff, Thornhill, Akhkha, White and Kusel2001). (2,3) Schistosomula in skin, Angeli et al. (Reference Angeli, Faveeuw, Roye, Fontaine, Tessier, Capron, Wolowczuk, Capron and Trottein2001), Al-Adhami et al. (Reference Al-Adhami, Noble, Sharaf, Thornhill, Doenhoff and Kusel2005), Mountford and Trottein (Reference Mountford and Trottein2004) and Curwen et al. (Reference Curwen, Ashton, Sundaralingam and Wilson2006). (4,5,6) Schistosomula in the lung, Wilson (Reference Wilson, Rollinson and Simpson1987), El Ridi and Tallima (Reference El Ridi and Tallima2006), Trottein et al. (Reference Trottein, Descamps, Nutten, Dehouck, Angeli, Capron, Cecchelli and Capron1999a, Reference Trottein, Nutten, Angeli, Delerive, Tessier, Capron, Staels and Capronb) and Chai et al. (Reference Chai, McManus, McInnes, Moertel, Tran, Loukas, Jones and Gobert2006). (7,8,9) Parasite in the portal vein, Shaker et al. (Reference Siddiqui, Phillips, Charest, Podesta, Quinlin, Pinkson, Lloyd, Paz, Villalovos and Pompa1998), Wuhrer et al. (Reference Wuhrer, Balog, Catalina, Jones, Schramm, Haas, Doenhoff, Dunne, Deelder and Hokke2006), LoVerde et al. (Reference LoVerde, Niles, Osman, Wu, Secor and Colley2005) and Knobloch et al. (Reference Knobloch, Kunz and Grevelding2006). (10,11) Egg secretions, egg extravasation, Schramm et al. (Reference Schramm, Gronow, Knobloch, Wippersteg, Grevelding, Galle, Fuller, Stanley, Chiodini, Haas and Doenhoff2006), Kanse et al. (Reference Kanse, Liang, Schubert, Haas, Preissner, Doenhoff and Dennis2005), Robijn et al. (Reference Robijn, Wuhrer, Kornelis, Deelder, Geyer and Hokke2005), Wood and Bacha (Reference Wood and Bacha1983), Ngaiza et al. (Reference Ngaiza, Doenhoff and Jaffe1993) and Ashton et al. (Reference Ashton, Harrop, Shah and Wilson2001).

The help the parasite receives is not just from its use of the immune system, although that has had most prominence in the literature, but the whole environment of the host contributes, even the blood pressure, haemodynamics and CO₂ concentration. We have emphasized that the function of a parasite molecule may be very different from that expected from application of current hypotheses. Even though a protein has some homology with a mammalian protein, it may have a very different function in the parasite (Popovici et al. Reference Popovici, Isnardon, Birnbaum and Roubin2002). This has also been shown clearly with the properties of the schistosome calcium channels that have important differences from homologous mammalian channels (Pica-Mattoccia et al. Reference Pica-Mattoccia, Valle, Basso, Troiani, Vigorosi, Liberti, Festucci and Cioli2007). Issuing from this review, future work might be suggested as shown in Figs 3 and 4. For the tegument (Fig. 3) (1) demonstration of the size and protein composition of the pores in the outer bilayer of the adult surface membrane; (2) the identity of proteins in the outer bilayer; (3) the demonstration of endocytosis in vivo or in vitro in the surface membrane, and investigate the function of caveolae; (4) investigations into the signalling from the surface membrane through rafts and through the glycocalyx; (5) the role of the excretory system in removing drugs and secreting immunomodulatory molecules; (6) is a P-glycoprotein in the excretory system or in the membrane a key molecule in regulating parasite metabolism?; (7) what are the mechanisms of membrane repair, and what is the role of lysosomes in the process?; (8) use of a variety of methods to demonstrate the presence of lipid rafts in the parasite surface membrane, and the existence and significance of their heterogeneity; (9) the role of sense organs in regulation of metabolism.

For other aspects of parasite biology, future areas are shown in Fig. 4: (1) what changes occur in human skin during penetration and how does it differ from the skin of the mouse model? (2) what are the changes in development that occur during parasite migration? (3) what signalling systems are active in the lung stage; (4) what is special about the portal vein for development? (5) what is the origin and function of the molecules excreted by the egg? (6) how do eggs traverse the capillaries and tissues? (6) how does the egg damage mammalian cells? (7) how do calcium channels function in schistosomula and eggs?