Microscopy and the helminth parasite

Micron 35 (2004) 361–390 www.elsevier.com/locate/micron

Review

Microscopy and the helminth parasite David W. Halton* Parasitology Research Group, School of Biology and Biochemistry, Medical Biology Centre, Queen’s University Belfast, Belfast BT9 7BL, UK

Abstract Microscopy has a long and distinguished history in the study of helminth parasites and has made a singularly outstanding contribution to understanding how these complex animals organise their lives and relate to their hosts. Increasingly, the microscope has been used as a powerful investigative tool in multidisciplinary approaches to parasitological problems, placing emphasis on functional correlates rather than anatomical detail. In doing so, microscopy has also uncovered a number of attributes of parasites that are of wider significance in the field of biology. Parasite surfaces have understandably demanded most of the attention of microscopists, largely as a result of the pioneering studies using transmission electron microscopy. Their findings focused the attention of physiologists and immunologists on the tegument and cuticle of helminths and in doing so helped unravel the complex molecular exchanges that are fundamental to understanding host – parasite interactions. Scanning electron microscopy succeeded in augmenting these data by revealing novel microtopographical features of the host– parasite relationship, as well as proving invaluable in helminth taxonomy and in assessing the efficacy of test substances in drug screens. Control of helminth parasites has never been more critical: problems of drug resistance demand urgent action to identify exploitable targets for new generation anthelmintics. In this regard, the neuropeptide signalling system of helminths is envisioned as central to nerve– muscle function, and thereby a crucial regulatory influence on their motility, alimentation and reproduction. The use of immunocytochemistry interfaced with confocal scanning laser microscopy has not only been instrumental in discovering the peptidergic system of helminths and its potential for chemotherapeutic exploitation, but through increasingly sophisticated bio-imaging technologies has continued to help dissect and analyse the molecular dynamics of this and other cellular systems within these important parasites. q 2004 Elsevier Ltd. All rights reserved. Keywords: Ultrastructure; Immunocytochemistry; Parasites; Helminths; Monogenean; Trematode; Cestode; Nematode

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Transmission electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Body surface of parasitic flatworms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Role of the tegument in food acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Role of the tegument in immune evasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Uniqueness of the schistosome tegument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Nematode body surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Scanning electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Taxonomy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Organs of attachment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. In vitro screening of drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Confocal scanning laser microscopy/immunocytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Neuropeptide F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. FaRPergic system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Neuronal pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Neuronal identification and function in nematodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Myoneural junctions in nematodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. Neuroactive substances in reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

* Tel.: þ44-2890-335-792; fax: þ44-2890-335-877. E-mail address: [email protected] (D.W. Halton). 0968-4328/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2003.12.001

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5. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388

1. Introduction Helminths comprise a fairly miscellaneous group of worms, both free living and parasitic, of which the phylum Platyhelminthes (flatworms) and phylum Nematoda (roundworms) form the major phyla. However, the two are evolutionarily quite distinct and differ markedly in their morphology. Flatworms are bilaterally symmetrical and acoelomate, usually dorso-ventrally flattened and hermaphroditic, and they generally lack an anus; in the phylogenetic scheme they form the root stock of the Bilateria. Roundworms, on the other hand, are cylindrical in shape and pseudocoelomate, show a combination of radial and bilateral symmetry and are usually dioecious and possess a through gut; the phylum has no obvious relations in the animal kingdom, although it has been grouped within the ecdysozoan clade of moulting animals. The unifying feature of helminths is that many of their parasitic members, i.e. trematodes or flukes, cestodes or tapeworms and parasitic nematodes, cause disease on a worldwide scale and as such are a common target for chemotherapy in humans, animals and plants. As parasites, helminths have long imposed a heavy burden on the health and economy of the world. Despite decades of intensive research on developing vaccines and chemotherapeutics, we still inhabit a ‘wormy world’ in which helminths are responsible for the widespread suffering and death of a majority of humans and domestic livestock, compounded by their global destruction of food crops (Crompton, 1999; Sasser and Freckman, 1987). While helminth parasites have been the faithful companions of humanity since time immemorial and were first recorded as ‘flat’ (tapeworms), ‘large’ (probably Ascaris) and ‘thin’ (probably Enterobius) by the ancient Greeks and Romans, their identity up to the early 17th century was shrouded in myth and superstition. Physicians considered parasites of the day to have formed from human excretions, akin to warts and boils or, if found internally, to have arisen spontaneously from accidentally swallowed free-living organisms. Thus, Moufet (1634) writes: ‘Some putrefied, superfluous and faecal matter in us is evidently collected, the hand of benevolent nature turns it into worms and in this way purifies the body.’ Natural historians at that time described flukey liver from sheep as being filled with little flounders, believing them to be land-locked fish or leeches. The turning point came in the second half of the 17th century when Francesco Redi, the grandfather of parasitology, and the Dutch lens grinder, Antoni Van Leeuwenhoek pioneered the application of microscopy to parasitology enabling identification of these denizens of the bowels as animals in their own right and, as such, established

the scientific field of helminthology. Thereafter the development of the compound microscope in conjunction with selective staining procedures heralded a wave of scientific achievement, to be followed in the middle of the 20th century by even greater opportunities for discovery of novel structural information at subcellular level through the biological application and use of electron optics. Microscopy has for long dramatically expanded our horizons in the field of helminthology by providing fundamentally important information on the structure and functional correlates of a number of key organ systems of helminths. Most notable among these is their body surface as the primary site of molecular interaction with the host, and their neuromuscular system which increasingly is seen as an exploitable target in novel drug development. Microscopical data have also provided a springboard for biochemical, physiological and molecular studies of helminths and as such have contributed to a number of landmark discoveries, including elucidation of the mechanisms of immune evasion by parasites, notably the human blood fluke, Schistosoma, and the finding of the first known member of the invertebrate neuropeptide F family of peptides in the cestode, Moniezia. In this collective, multidisciplinary manner, microscopy has succeeded in melding the interests of morphologists, biochemists, immunologists, physiologists and, more recently, molecular biologists, into a more integrated and functionally dynamic study of helminth parasites and of parasitism in general. This review is not intended to be comprehensive in any sense but rather it focuses on what are arguably some of the more important discoveries in helminth functional morphology to have accrued these past 40 years by way of transmission- and scanning-electron microscopy, and more recently from use of the confocal scanning laser microscope as an imaging tool in cytochemical procedures.

2. Transmission electron microscopy 2.1. Body surface of parasitic flatworms Prior to the advent of the electron microscope, the outer covering of parasitic flatworms was referred to as a cuticle in that most workers in the field believed it to be an inert but elastic layer of scleroprotein secreted by subsurface (mesenchymal) cells (see Hyman, 1951) This interpretation underpinned the concept of the so-called ‘armour-plated parasite’ which was largely accepted by parasitologists to explain the obvious ability of enteric flatworms to survive hostile environments, such as the host gut. Thus, the cuticle was seen to be largely protective against digestive attack

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from the host. However, in tapeworms, which of course are bereft of gut, it was conceded from physiological evidence emerging in the late 1950s, notably by Read and his coworkers (e.g. Read and Simmons, 1963), that the cuticle must also be of nutritional significance and be capable of absorption, facilitated perhaps by ‘pore canals’ connecting the outside environment to the inner parenchyma. The enigma of the flatworm parasite cuticle was resolved by one of the earliest applications to parasitology of the new and exciting research tool of the day, the transmission electron microscope. The revelation which convincingly challenged the notion that an inert cuticle invested the body of parasitic flatworms was provided by Rothman (1959) who demonstrated that the cuticle of the tapeworm, Hymenolepis diminuta was capable of reducing tetrazolium and that its matrix contained populations of mitochondria. Rothman provided sufficient evidence to justify his renaming the cuticle of H. diminuta as the tegument, a term which has stood the test of time for the outer covering of all other parasitic flatworms examined thereafter. This is largely because the term tegument obviates any connotation of

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non-living and at the same time distinguishes the structure from the less complex epidermis of free-living flatworms. The discovery by Rothman was developed and expounded upon by Threadgold in two seminal papers on the ultrastructure of the tegument of a tapeworm, Dipylidium caninum and the liver fluke, Fasciola hepatica (Threadgold, 1963). His work revealed the tegument to have a unique syncytial organisation such that there is an anucleate surface layer which, at intervals, is connected via cytoplasmic tubes or trabeculae to subsurface nucleated regions or tegumental cell bodies (Fig. 1). A syncytial tegument is universal among trematodes and cestodes but considerable differences in ultrastructure occur between taxonomic groups and even from species to species. In all cases, the tegument is bounded at its outer and inner surfaces by plasma membranes and covers the entire body surface, including that of the foregut in trematodes. Its syncytial arrangement confers a number of advantages of paramount importance to flatworms engaged in parasitism. Thus, the absence of cell boundaries means a syncytium is less susceptible to attack and breakdown by host agents,

Fig. 1. Schematic showing the basic structural organisation of the platyhelminth tegument in which insunken nucleated cell bodies are in cytoplasmic continuity with an anucleate surface layer delimited by a plasma membrane and associated glycocalyx. As the interface with the host environment, this syncytial epithelial layer engages in a multiplicity of functions not least in protecting the parasite against host attack. (Original).

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such as digestive enzymes, detergent bile acids or components of the immune system, than is a layer of epithelial cells with vulnerable intercellular junctions (Fig. 1). Secondly, it means that transport is possible laterally without restriction, enabling metabolic and electrical cooperation. Thirdly, since the nucleated regions of the tegument are insunken they are relatively far distant from any adverse influence of the host. This means that the genetic control of differentiation and proper functioning of the tegument is less likely to be impaired by the host and its hostile products. Finally, the separation of the nucleated regions from one another, although attached to the same cytoplasmic mass, allows regional differentiation and specialisation within the tegument. It also means that new tegumental cells with novel genetic information can move up and fuse into the system and those old cell bodies that are spent can be detached to move out. Complicated as the structure of the tegument may seem, the most significant revelation to emerge from these early electron microscopic studies is that the ultimate boundary between the flatworm parasite and its host is the tegumental surface plasma membrane. This fact revolutionised our understanding of symbiotic relationships in platyhelminths and raised to a new conceptual level the significance of the host – parasite interface. It meant that if the parasite surface is alive, it can respond dynamically to changes in environmental conditions; that protection from potentially

damaging influences is more likely to be subtle and biochemical than as a result of mere physical exclusion. Finally, it opened up the possibility not only of transport of materials into the parasite from the host but of a twoway flux of molecules and information between host and parasite. In changing the name from cuticle to tegument, Rothman and fellow electron microscopists had set the challenge to unravel the very essence of parasitism, namely, the molecular interaction of two heterospecific organisms. As a site where there is likely to be considerable interplay with the host environment, it is not surprising that the tegument is capable of a multiplicity of functions (Fig. 2). Current concepts of tegument ultrastructure are consistent with the view that it serves four important roles: (i) absorption of exogenous material, including nutrients, through membrane digestion and transport, or by endocytosis; (ii) synthesis and secretion of endogenous materials, including components of its surface plasma membrane and glycocalyx, the release and turnover of which have a role in immune evasion; (iii) osmoregulation and excretion through the action of ionic pumps and Naþ/Kþ-ATPase which reside in its surface and basal plasma membranes and therein establish a standing gradient for water and ions; and (iv) provision of a sensory input, insofar as it supports numerous putative sense organs that connect with elements of the worm’s nervous system.

Fig. 2. Schematic summarising the major functions of the platyhelminth tegument, most notable of which are surface digestion/absorption of food materials, osmoregulation and excretion, and the synthesis and secretion of materials particularly those to replenish the surface plasma membrane and associated glycocalyx. (Original).

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2.2. Role of the tegument in food acquisition The absorptive features of the platyhelminth tegument are understandably most apparent in tapeworms since they are without any vestige of gut and rely solely on their body surface for procurement of food (Fig. 3a –c). With few exceptions, adult tapeworms are parasites of the intestine of vertebrates and as such inhabit an environment rich in all dietary requirements. However, they share it with a highly efficient competitor, namely the host mucosal epithelium. The tapeworm tegument, therefore, competes with a highly sophisticated vertebrate transport system. And it does so successfully because, in a sense, the two are comparable in ultrastructure and function. In a very simplistic analogy, the body plan of a tapeworm can be envisaged as a gut turned inside out, with the tegument serving the absorptive functions of the intestinal mucosa (Fig. 4). Its free surface bears a brush-border of finger-like processes or microtriches analogous to the microvilli that adorn the host mucosal cells (Fig. 3d and e). Internally, each is braced by a core of longitudinal microfilaments which connect in the microvillous with a terminal web of similar filaments in the cytoplasm below. Microfilaments in the microvilli contain actin and seem capable of bringing about movement of the microvilli. Were this proved to be the case in cestodes, then microtriches may serve to agitate the microenvironment adjacent to the animal, with obvious physiological significance vis-a`-vis nutrient flux across the tegument surface. Microtriches vary considerably in structure, dimensions and density, both in the developmental stages and between different regions of the scolex (head) and strobila (body) of the adult tapeworm. While details of their origin, development and turnover remain obscure, what is for sure is that microvilli-like microtriches provide significant amplification of the free surface area (some 2 –12-fold, depending on the species and stage); moreover their polymorphism (Section 3) would seem indicative of a diversity of functions, e.g. absorption, protection, attachment, locomotion. As will be discussed later, tapeworms are endowed with a well-developed neuromuscular system and species like the rat tapeworm, H. diminuta are known to move up and down the gut of their host quite considerably, the microtriches providing the worm with an effective degree of purchase against the host mucosa. Thus, in the infected rat, which normally feeds between 10.00 pm and 8.00 am, the worms display diurunal migrations, moving forward to the anterior ileum early morning, so optimising their opportunities for absorbing products of host digestion and later returning to the more posterior portion to assimilate them. In common with the mucosal cell, the tegumental surface is invested with a fuzzy coat of carbohydrate-rich macromolecules, collectively termed the glycocalyx. This is an integral component of the tegument plasma membrane and is derived from glycoproteins and glycolipids that exist as intrinsic molecules within the lipid bilayer (see later).

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Their carbohydrate (oligosaccharides, gangliosides) chains which project from the membrane surface are rich in acidic groups (sialic acid residues) that impart a net negative charge to the parasite’s tegument as a polyanionic coat. Enzyme cytochemistry has demonstrated that the surface plasma membrane of tapeworms is well endowed with intrinsic hydrolytic enzymes, such as glucose-6-phosphatase whose activity in cleaving phosphate esters means products like glucose are more likely to be transported directly into the tegument via coupled digestive –absorptive processes, than float off into the lumen. This gives the glucose molecules kinetic advantage in absorption over identical substrates free in the lumen. The repertoire of digestive enzymes at the tapeworm surface also includes adsorbed host enzymes, such as pancreatic amylase. This is bound as a polycation to the polyanionic glycocalyx of the tapeworm and its binding enhances the host enzyme to digest starch on behalf of the tapeworm. With unlimited food resources, tapeworms produce large quantities of partially oxidised organic acids, such as lactic and propionic, which lower the pH of the host intestine from approximately 6.5 – 6. This has the effect of cutting down the rate of glucose absorption by the host intestine whilst enhancing uptake by the parasite whose tegumental glucose carriers work best at a slightly lower pH. Thus, the parasite regulates to its own advantage the efficiency of its competitor’s transport system. In an evolutionary context, it would seem that millions of years of competition for the same nutrients (e.g. glucose) in the same physiological niche (vertebrate intestine) have resulted in a remarkable convergence of biochemical and ultrastructural organisation between the mammalian gut brush-border and the tegument of the tapeworm, each providing a sophisticated digestive – absorptive surface for the procurement of nutriment. It is evident from ultrastructural studies that the tegument of the other major group of parasitic flatworms, the trematodes, also has an absorptive potential and that in addition to ingesting food materials, those species bathed by media containing soluble nutrients may also extract materials by absorption through the body surface. Indeed, transtegumental absorption of low molecular weight substances has been demonstrated in vitro in a number of instances, notably in the bile-inhabiting liver fluke, Fasciola and the intravascular schistosomes, where most of the sugars examined enter by a saturable mediated transport process showing chemical specificity (Fig. 5c). While the relative importance of the tegument vis-a`-vis the gut to the nutritional economy of the trematode remains unclear, there is some information to indicate the tegument of Schistosoma may be the major route for absorption of host-blood glucose and amino acids, whereas the gut functions primarily in macromolecular digestion and absorption of red blood cells (Fig. 5d). Spine-bearing tubercles distinguish the male dorsal tegument of S. mansoni and serve to give purchase to the worm in the host blood vessel, the male securing

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Fig. 3. (a) SEM of the scolex of the cyclophyllidean tapeworm, Taenia crassiceps showing four well-developed suckers/acetabula (arrow) and an apical rostellum (ro) armed with a double coronet of recurved hooks. (b) SEM of a portion of tapeworm strobila showing constrictions that mark the so-called segments as individual mature proglottids (p). Note posterior portion (arrow) of each proglottis overlaps the anterior part of its neighbour. (c) En face image of the scolex of the pseudophyllidean tapeworm, Eubothrium crassum as seen by SEM. (d) SEM of the tegument surface of a tapeworm showing posteriorly directed, conically shaped microtriches (arrows) and smaller associated microvilli (p). (e) TEM of sagittal sections of microtriches of tapeworm tegument (tg), showing electron-dense cap (cp) separated from shaft (sh), containing a core of microfilaments, by a multilaminar baseplate (unlabelled arrow).

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Fig. 4. Schematic drawing analogy between the ultrastructure of the cestode tegument and the vertebrate mucosal epithelium in that each serves to provide a highly sophisticated digestive–absorptive surface for the acquisition of foodstuffs. (Original).

the female within his embrace in the gynaecophoric canal (Fig. 5f). Ultrastructural cytochemistry has revealed a thin tegumental covering to the tubercles, whereas those areas between the tubercles are elaborated into numerous deep channels and surface folds whose surface plasma membrane is reactive for intrinsic hydrolytic enzymes, including phosphomonoesterases (Fig. 5g). It is these intertubercular regions of schistosome surface that in situ are bathed with host blood and presumably present the male worm with digestive – absorptive sites for food acquisition via the tegument. 2.3. Role of the tegument in immune evasion Compelling evidence that the tegument plays a pivotal role in immune evasion comes from the fact that when its delimiting plasma membrane is breached the trematode or cestode parasite is subjected to extensive damage from the host immune response. Insight into the mechanisms by which flatworm parasites evade the host immune system has come from TEM observations on Fasciola which have revealed the platyhelminth tegument to be a highly active secretory unit (Fig. 6a). One of its secretory products is glycoprotein and pulse-chase TEM autoradiography, using tritiated glucose and galactose, has shown that it is produced through the GER-Golgi apparatus of the tegumental cell for packaging and export to the surface in membrane-bound secretory bodies (so-called T1 bodies) (Hanna, 1980c).

Through membrane fusion (exocytosis) the delimiting membrane of the T1 bodies and their attached secretion become part of the surface plasma membrane of the tegument and its associated glycocalyx (Fig. 2). In Fasciola, there is a continual and fairly rapid production of surface glycoprotein and this enables a turnover of glycocalyx within a matter of a few hours. It is now known that the earliest and most vigorous humoral immune response in sheep, cattle and laboratory rats to Fasciola infection is directed against antigenic determinants in the glycocalyx of the juvenile parasite. This is not surprising since the tegumental glycocalyx totally envelops the parasite during its migration through the liver to the bile duct and represents the only source of antigens available for host recognition. High levels of glycocalyx-specific antibodies build up in the host circulation and attach to the parasite’s surface forming immune complexes that are potentially damaging by virtue of their activity in opsonization and complement fixation. Clearly, the mechanism of glycocalyx turnover in the migrating juvenile is central in protecting the parasite against the host’s immune response. In a series of elegant experiments performed by Hanna (1980a,b,c) the changes in surface antigenicity which accompany development in Fasciola were, for the first time, correlated to its ultrastructure. When the newly excysted juvenile fluke (Fig. 6b) penetrates the host gut wall en route to the liver there is a natural antibody response

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Fig. 5. SEM images of Schistosoma japonicum cercaria (a), S. mansoni cercaria (b), S. mansoni adult pair (c). mb, main body; ta, tail; fu, furcae; mw, male worm; fw, female worm; gc, gynaecophoric canal. (d) SEM of dorsal surface of male S. mansoni showing spiny tubercles (tu). (e) TEM section of the tegument of S. mansoni showing the double plasma membrane (dpm) responsible for the heptalaminate appearance of its surface. (f) TS of male (mw) and female (fm) S. mansoni in a mesenteric blood vessel of an experimentally-infected mouse. Note dorsal tubercles (dtu) of male, and host blood (unlabelled arrow) over the intervening areas of tegument; also relative amounts of blood pigment in the gut caeca (ic) of the two sexes. (g) TEM of the dorsal tegument of male S. mansoni following staining for phosphomonesterase activity (black deposits of lead sulphide). Note deposits are found mainly in the highly folded intertubercular areas of tegument (arrow) and in association with the plasma membranes there. sp, spine; tu, tubercle.

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Fig. 6. (a) SEM of adult liver fluke, Fasciola hepatica. Note oral (os) and ventral suckers (vs), gonopore (unlabelled arrow) and irregular lateral margins to body. (b) SEM of a juvenile liver fluke fixed during excystation via the ventral plug (arrow) of the metacercarial cyst (ct). (c) TEM of juvenile F. hepatica following exposure to antifluke immune sheep serum, showing flocculant complex of adherant immunoglobulin and glycocalyx at the tegument (tg) surface. Note spines (sp). (d) TEM of the outer tegument of F. hepatica following immunogold labelling of monoclonal antibodies (MAbs) developed against specific glycocalyx epitopes. The gold probes (seen as black spheres) tag the MAbs attached to glycoproteins of the T1 secretory bodies (sb) in the tegumental cytoplasm and their presence over the glycocalyx (gl) after 3-h incubation confirms these secretions are released to replace surface glycocalyx.

to its surface antigens. This can best be demonstrated by indirect fluorescent antibody labelling following incubation of worms in immune sheep serum. The adherent immunoglobulin, conjugated with fluoroscein, is distributed over the tegument surface in a reticular pattern of bright

fluorescence which excludes those areas overlying the spines. At ultrastructural level the immune complex produced in this way is seen as flocculant material attached to the surface of the tegument (Fig. 6c). When worms are transferred to a non-immune serum they actively slough this

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layer of immune complex and replace the glycocalyx with a new antigentically similar coat. Confirmation of this came from time-course studies in which monoclonal antibodies developed against specific glycocalyx epitopes were found to attach to the T1 type secretory bodies. Immunogold labelling of the second antibody molecules (goat anti-mouse IgG) enabled the events of synthesis and secretion to be monitored by electron microscopy. Initially, the gold probe was localised over the secretory bodies and Golgi apparatus of the tegumental cell bodies but 3 h later was found concentrated within the surface of the tegument and confined to the secretory bodies and associated glycocalyx (Fig. 6d). This was the first direct ultrastructural evidence that surface antigenicity in a flatworm parasite is due to the glycoproteins that its tegument produces to form the glycocalyx, and confirmed the tegumental cell bodies and surface covering to be functionally related.

The rate of synthesis of glycoprotein and hence the turnover of glycocalyx in Fasciola varies during development of the worm (Fig. 7). It is most rapid during migration of the young fluke through the liver parenchyma of laboratory rats at 3 weeks post-infection (pi), and this is the time when the host immune response is at its height. Rapid turnover of glycocalyx at this time ensures that attached antibody does not remain at the surface long enough to cause damage through complement fixation and/ or attachment of immunocompetent host cells. Once the young fluke is established in the immunologically secluded environment of the bile duct by 12 weeks pi, there is a fall off in production of T1 secretory bodies and a gradual reduction in the rate of glycocalyx turnover (Fig. 7). Clearly, glycocalyx turnover protects pre-bile duct flukes against immunological attack. A second type of secretory body, the T2 body appears in the tegument after the first week of excystment and eventually

Fig. 7. Schematic showing that glycocalyx turnover in the liver fluke, Fasciola hepatica is most rapid during migration of the juvenile worm through the host liver and as such protects pre-bile duct fluke stages from immunological attack (After Hanna, 1980a,b,c).

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becomes the predominant inclusion body of the adult tegument. It does not, however, appear to be involved in immunoprotection but rather may serve to maintain the structural integrity and nutritive function of the surface plasma membrane of the tegument. It is likely that once the fluke is established in the relatively safe environment of the bile duct by 12 weeks pi it can direct more of its energy and resources from protective mechanisms to aspects of growth and reproduction. 2.4. Uniqueness of the schistosome tegument A helminth parasite which has been under the microscope perhaps more than most is Schistosoma, the human blood fluke (Fig. 5c). The associated disease, schistosomiasis, afflicts more than 200 million people in the developing world and, compared to some other tropical diseases, like filariasis, progress in its control has been slow. This is not surprising since in Schistosoma we face the ultimate challenge in a flatworm parasite. By choosing to live in human blood, schistosomes have selected one of the most hostile environments imaginable (Fig. 5f). The fact that they can exist for 20 years or more in an environment which is screened by a highly sophisticated immune surveillance system indicates a biological phenomenon of great subtlety. How is this achieved? A blood fluke would appear to be much more foreign than say a kidney carefully transplanted from one person to another by a surgeon. Yet the kidney survives and functions only through concurrent use of iummunosuppressive procedures. It will not be surprising to learn that this ‘phenomenon of great subtlety’ resides in the tegument of the schistosome (Fig. 5d) and that transmission electron microscopy (TEM) laid much of the important groundwork that enabled workers to unravel some of its complexity. Apart from living in the blood in permanent copula, the single most unique feature of the schistosome and other blood flukes is that their tegument is delimited not by a single plasma membrane and associated glycocalyx, as in the majority of flatworm parasites, but by a double outer membrane (Hockley, 1973). One of the biggest breakthroughs in understanding the nature of the host –parasite interface came with the use of uranyl acetate as a tertiary fixative after routine fixation using glutaraldehyde and osmium tetroxide. This treatment reveals in section the outer membrane of the schistosome tegument as heptalaminate, measuring some 17 nm in thickness and consisting of two closely apposed lipid bilayers (Fig. 5e). Without this tertiary fixation the outer membrane shows a typical trilaminate configuration, indicating that the two lipid bilayers differ in their composition and that the outer one is perhaps less stable than the inner one. In addition to schistosomes, two other families of trematodes live in the blood vascular system of vertebrates and, in both cases, their members have a double outer membrane to the tegument (McLaren and Hockley, 1977). It would seem therefore that

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a double outer membrane is the key to surviving the hazards of living in the blood of the host. To determine the origin of the double outer membrane of blood flukes, it is necessary to look at an earlier stage in the development of the schistosome, namely the fork-tailed cercaria (Fig. 5a and b). This is the free-swimming invasive stage of the parasite which, when released from infected snails, penetrates human skin within minutes and eventually enters the bloodstream. In contrast to the adult worm, the cercaria has a typical trilaminate surface membrane and associated glycocalyx, just as in the majority of trematodes. However, this is lost within 3 h of host-skin penetration and is replaced by a heptalaminate surface, comprising two closely apposed trilaminate membranes (Hockley and McLaren, 1973). The whole process is a complex one and is accompanied by the transient appearance of tegumental microvilli whose function is believed to be the removal of cercarial surface membrane and glycocalyx (Fig. 8). At the same time, multilaminate bodies, produced in the tegumental cell bodies, migrate through the surface syncytium and fuse with the surface membrane, enabling continuous sloughing and replacement of the cercarial membrane. Experiments in which the outer membrane was labelled with electron-dense cationic tracer molecules, such as ferritin, have suggested a membrane half-life of 2 – 3 h under experimental conditions (Wilson and Barnes, 1977). The speed with which these various surface changes occur highlights the crucial importance of the double outer membrane for parasite survival in the host bloodstream. By splitting the outer and inner lipid bilayers of the two membranes, using freeze-fracture microtomy, the integral proteins/glycoproteins/glycolipids of each membrane are revealed as the intramembranous particles (IMP) which characterise the two external (E1 and E2) and two protoplasmic faces (P1 and P2) (McLaren et al., 1978). The particles can then be counted per unit area and their density and distribution monitored at crucial stages in the schistosome life cycle. Thus, while IMP are present on all four fracture faces, those on the outer leaflet of the outer membrane (E2), that is, the one closest to the host blood, are at their most numerous and of unusually large size by day 5 of laboratory infections in mice. These ultrastructural findings of a marked increase in IMP in the outer bilayer of the double membrane fit beautifully with the immunological findings that as schistosomes enter the bloodstream of a naive or non-immune host their parasite antigens become masked by acquired host blood antigen (McLaren, 1980). In other words, the parasite disguises itself as host tissue by adsorbing host antigen—a proverbial wolf in sheep’s clothing—and so protects itself from host immune attack. The freeze-fracture work suggests the outer bilayer may well have evolved specifically to accommodate the incorporation of masking antigens as a kind of membranous sponge for soaking up host glycolipids.

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Fig. 8. Schematic depicting the changes in tegument ultrastructure during the transformation of the Schistoma mansoni cercaria to schistosomulum in experimentally infected mice (after Hockley and McLaren, 1973).

2.5. Nematode body surface As described, TEM redefined the body surface of parasitic flatworms in the early 1960s as a living protoplasmic layer or tegument delimited by one or more (as in blood flukes) plasma membranes. TEM also revealed, in marked contrast, that the other major group of helminths, the roundworms or nematodes, whether parasitic or not, are bounded by a truly inert cuticle of extracellular material in the form of cross-linked collagens and insoluble proteins synthesised and secreted by an underlying epidermis ( ¼ hypodermis). The structure can be relatively simple or very complex, varying from one genus to another and even displaying regional differences in structure within an individual species. Nematodes are ubiquitous in that species of one sort or another are known to be adapted to every environmental niche on earth, except none has learned to fly! Moreover, it has long been accepted that a key to their success as the most numerous of multicellular animals is the resilient, barrier properties of their highly conserved cuticle. While the electron microscopical findings of its structure were not the revelation that had come from exploring the membranous surfaces of trematodes and cestodes, they nevertheless evoked considerable controversy which continues today as to why the nematode surface, if merely an inert densely cross-linked structure, can be so highly responsive in infection. Again, TEM in combination with immunocytochemical procedures provided some of the answers through detailed analysis of adult nematode cuticle to elucidate the nature of its outermost layer, the epicuticle (see Blaxter et al., 1992; Lee, 2002).

The epicuticle often appears trilaminate in electronmicrographs and while lipophilic when stained nevertheless shows restricted insertion of externally applied probes, arguing against it being organised into conventional lipid bilayers. The likelihood of it been a true delimiting plasma membrane is also refuted by the failure of treatments that dissociate most membranes and by the general absence of IMP following freeze-fracture treatment. It is now evident that the delimiting plasma membrane of a nematode is not at the outer surface of the cuticle but rather at the interface between the cuticle and subjacent epidermis (hypodermis). It was the application of ultrastructural protocols for visualising surface coats, namely cryosectioning followed by indirect immunogold labelling or use of direct gold-labelled lectins, which finally provided the clue as to how some nematodes likely change their surface properties within minutes of entering their mammalian hosts. Cryoimmuno-electronmicroscopy reveals that distal to the nematode epicuticle and distinct from it is a polyanionic, glycan/mucin-rich coat or glycocalyx, some 5–20 nm in thickness depending on the species, which readily binds antibodies and lectins. Standard stains for surface coats such as cationised ferritin, thorotrast and ruthenium red can also enhance its visibility in the electron microscope, following routine fixation with glutaraldehyde and osmium tetroxide. Ultrastructural images by Lee et al. (1993) have also strikingly demonstrated the surface coat being shed by adult Nippostrongylus brasiliensis following fixation in situ and also on freeze-fractures of the surface. While the nematode cuticle is known to be synthesised and secreted by the underlying epidermis (designated the hypodermis), the surface coat per se seems to be derived

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from the products of specialised secretory glands. In Toxocara canis, for example, surface coat glycoproteins are ported to the exterior from excretory and oesophageal glands; amphibial glands are also thought to source the coat in many cases, especially plant parasitic species (Premachandran et al., 1988). In free-living nematodes, the continuous release of excretory/secretory products as a surface coat is seen as providing lubrication and protection against abrasion, dehydration and predation; in parasitic species it is more crucial in helping the worm evade the active immune responses of a stimulated host by shedding surface-bound antibodies. Nematode cuticle as a whole is a labile structure and can be replaced in toto at moults, but usually these moults occur at least 24 h apart. Its outer, disposable surface coat, on the other hand, provides a more immediate and dynamic means of changing the worm’s surface properties within minutes, thereby providing the nematode parasite with a means of evading potent and specific immune responses of the host. While clearly antigenic in nature, the surface coat of the soil stages of plant parasitic nematodes also provides specific attachment sites for microorganisms used in biological control strategies. Phytophagous nematodes, such as root knot and potato cyst nematodes cost in excess of $100 billion per annum in food-crop loss worldwide, and knowledge of the chemical nature of their surface coat is crucial in understanding interactions with the nematophagous fungi and bacteria that are increasingly being used in their control. As already mentioned, surface coat composition seems to be one of mucin-like proteins or glycoproteins with either N-linked or O-linked sugars, glycosylation being detected by a variety of lectin conjugates (Blaxter and Robertson, 1998). Attachment of fungal spores or bacteria appear to be effected by carbohydrate-binding proteins that are speciesspecific, stage-specific or even regionally specific for selective sugar residues in the nematode surface coat (Bird et al., 1989).

3. Scanning electron microscopy Since the 1970s, scanning electron microscopy (SEM) has been used extensively as a valuable investigative tool in taxonomic and structural studies, including evaluation of drug effects, of both parasitic flatworms and nematodes. Its increased depth of focus and resolution over that of the light microscope has enabled details of both external and internal (from tissue sections) surface architecture to be revealed, often strikingly so, and has also provided better understanding of host parasite relationships, particularly with regards attachment. 3.1. Taxonomy The taxonomic value of SEM is patently evident from the images of helminths portrayed in guides and accounts aimed

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at their identification (see Gibbons, 1986, 2002). Nematodes in particular exhibit a relatively low morphological diversity at optical level and for this reason have traditionally been condemned as a phylum of ‘look alikes’ by having uniformity in appearance but which we now know from SEM studies to be erroneous. The enormous resolving power of the scanning electron microscope over the light microscope effectively removed observational constraints on the number of characters available for diagnosis and classification and revealed helminths to be potentially rich in morphological features of value in studying their systematics. Thus, SEM has revealed marked inter- and intra-specific variations and regional differences in surface topography, particularly in exposing previously unimagined microtopographical features and their spatial relationships. In flatworm parasites, systematic studies have involved the fine architectural detail of: the surface openings of alimentary, reproductive, excretory systems and glands; the nature and disposition of putative sense organs; the form and distribution patterns of trematode spines, monogenean hooks and their morphometrics, and the polymorphism of cestode microtriches. Similarly, nematologists made most use of internal structural features and continuously searched for better and more stable taxonomic characters prior to the availability of SEM. For them, SEM introduced better diagnostics, most notably an array of external morphological characters of the head and tail regions, such as lips (Fig. 10), stylets and the teeth of feeding structures; sense organs and body openings; cuticular markings and ornamentations, including: setae, spines, papillae, ridges and cuticular alae; as well as other structures associated with the copulatory system in males and perineal region in females of certain plant parasitic forms. Evidence for a role for the flatworm tegument in sensory processing and signal transduction comes from ultrastructural studies. Presumed sense organs are not only numerous in the tegument of larval and adult flatworm parasites (Fig. 9c – f) but each species examined has several different types to which a variety of functions have been ascribed. Experimental evidence of a sensory function, be it tangoreception, photoreception, rheoreception or chemoreception, is restricted by the small size of the presumed receptor, making electrophysiological and ablation studies technically difficult so that experimental evidence for such is lacking. Nonetheless, baseline data on the fine structure of helminth sense organs, notably photoreceptors, is helping interpret behavioural responses of larval stages and so indirectly providing evidence for the sensory modalities of these structures. In Schistosoma, for example, there is a high concentration of dome-shaped or spherical protuberances, many of which bear cilia or modified cilia (sensilla), distributed over the bodies of both sexes (Fig. 5d). They take the form of ciliated structures, with a single central cilium of 9 þ 2 axonemes arising from a dome-shaped basal elevation; the

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Fig. 9. (a) SEM of a miracidium, the first free-swimming larval stage of a trematode, having hatched from the egg of Fasciola hepatica. Note open lid or operculum (op) of egg capsule (ec). (b) SEM of the ciliary epidermal plates (ep) and ridges (arrows) of the F. hepatica miracidium, exposed following partial removal of cilia. Note apical papilla (p) used in penetration of molluscan host. (c) SEM of a cercaria, the second free-swimming stage of a trematode, Cryptocotyle lingua. Note numerous long sensilla that likely serve as flow receptors, and well-developed tail bearing prominent fin (fn). (d) Detail of sensilla

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cilium may be long or short or retracted into the basal papilla; and non-ciliated structures, appearing as papillae with a wide terminal bulb, as simple dome-shaped elevations, or ring-shaped structures with a central depression (Senft and Gibler, 1977; McLaren, 1980; Dorsey et al., 2002). TEM reveals these structures represent the terminations of fine nerve processes that extend from the peripheral nervous system (see later) to the base of the body surface layer (Fig. 9f). On the basis of ultrastructural evidence, the freeswimming aquatic larval stages of trematodes, e.g. Cryptocotyle, Entobdella have been found to be endowed with a multiplicity of presumed sense organs, including single uniciliated and compound multiciliated nerve terminals on or around the head region and long flexible ‘flow receptors’ (Fig. 9c and d). Many larval stages also possess eyes that can be pigmented, with or without lenses to concentrate light. Twitching movements of the eyes have been observed in Entobdella larvae, and there is ultrastructural evidence of muscle fibres attached to the outer surface of the eye (Kearn and Baker, 1973). An alternative device for concentrating light, unique to flatworms, is a non-pigmented supporting cell characterised by concentric rows of rectangular, membrane-delimited platelets that, collectively, behave as a concave mirror and concentrate light by reflection. TEM has shown that the distance between successive rows of platelets in the eye of Polystoma larva is such that at each interface light interferes with that reflected by the other surfaces so that the reflected waves are in phase and thereby amplified (Fournier and Combes, 1978). Nematode sense organs with a widely varying structure have been described from ultrastructural observations and generally conform to two basic types (Wright, 1980). The most common are cuticular sense organs, the greatest concentration of which is found at the anterior end of the worm (Fig. 10a). These comprise paired amphids, located in pits laterally on the nematode, together with perhaps six inner and six outer labial papilla, and four cephalic papilla arranged radially around the head. Cuticular sense organs are also numerous at the posterior region of male worms where they likely serve in mating. A far more diverse array of structures is shown by the internal sense organs of nematodes which, by and large, serve to detect mechanical stimuli, including those associated with the pharynx in feeding. Amphids are the largest and most elaborate of nematode sense organs, and while most conspicuous in freeliving nematodes are usually reduced in size somewhat in parasitic forms, though highly conserved in structure (Ashton and Schad, 1996; Ashton et al., 1999). The amphidial opening leads into a deep cuticular pit that houses a supportive socket cell, a secretory sheath cell, and

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dendritic processes that arise from sensory neurons as modified cilia. Until TEM discovered these modified cilia in nematode sense organs it was thought nematodes were bereft of cilia. However, these modified cilia differ from normal motile cilia in having no kinetosome and their microtubules diverge from the usual 9 þ 2 configuration to one of, for example, 9 þ 4 or 1 þ 11 þ 4. Amphids serve primarily as chemosensory structures and also have a secretory function which, although likely to be important for chemoreception, also contributes to the surface coat (see earlier). Phasmids comprise another set of chemoreceptors near the posterior end of many parasitic nematodes and resemble amphids in their morphology. As discussed earlier (Section 2), the potential surface area of juvenile and adult cestodes is greatly amplified by a brush border of microtriches and/or microvilli (Fig. 3d and e). SEM has revealed considerable variation in the structure, dimensions and density of these surface extensions. In adult Proteocephalus, for example, systematic scanning of the whole-body surface has shown that the greatest degree of microthrix polymorphism is at the anterior end of the scolex and neck region (Thompson et al., 1980). Here microtriches may be blade-like, peg-like, filamentous, spine-like or strap-like, and this diversity in structure would seem indicative of diversity in function, be it absorption, protection, attachment or locomotion. In developmental stages, specific types of microthrix occur in discrete areas of tegument and have been attributed a role in, for example, budding or evagination. Large palmate microtriches adorn the bothridia of the plerocercoid of the trypanorhynch tapeworm, Grillotia where they serve the excystment of this metacestode (Halton et al., 1994). Details of the origin and development of microtriches, or of their turnover rate, remain obscure. The practical value of SEM in helminth taxonomy was put to good use by Mitchell et al. (1978) who, for the first time, combined it with in vitro cultivation procedures to identify a helminth infection of previously unknown aetiology in farmed rainbow trout in Northern Ireland. Large numbers of cysts were found around the heart of the fish, causing compression and serious cardiac dysfunction. Excystment and SEM revealed the forebody of a strigeid trematode metacercaria, bearing characteristic suckers, lappets and holdfast but whose hindbody was undeveloped (Fig. 11c). The metacercariae were cultured in vitro and within a week the hindbody had grown and differentiated into that of a sexually mature adult worm (Fig. 11a and b). Morphological changes during cultivation were monitored by SEM and revealed development in the hindbody of a papillate genital bulb, a diagnostic feature of the Cotylurus genus of strigeid trematodes (Fig. 11d).

(arrows) seen as uniciliated structures using SEM. (e) SEM of the anterior end of the trematode, Gorgoderina vitelliloba showing distribution of putative aciliated sensory structures (unlabelled arrows) in symmetrical array over the surface of the oral sucker. Note mouth (mo) and the empty socket (ss) that housed the metacercarial stylet. (f) TEM of a putative uniciliated sense structure (arrow) of the monogenean, Diclidophora merlangi showing its fine structural relationship with a sensory neuron (sn) and surrounding tegument (tg).

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Fig. 10. Ascaris suum. (a) SEM of female adult worm showing en face view of the mouth (mo) surrounded by three lips bearing sensory doublet papillae (sp). Note dentigerous ridges on lips (unlabelled arrows). (b) SEM of forebody of worm cut at the level of the triradiate pharynx (ph) showing sections of dorsal nerve (dn), hypodermal cords (hc), somatic muscle (mu) and cuticle (cu). (c) Confocal image of a transverse section through the pharynx of a specimen immunostained for FaRP neuropeptides. Note contractile portions of somatic muscle cells (red, mu) and radial muscles of pharynx (red, ph) and immunoreactive (green) circumpharyngeal nerve ring (cpr). (d) Confocal image of neurones of the ventral ganglion immunostained for FaRP. (e) Transverse section of the pharynx showing pharyngeal nerve ring (green) immunostained for FaRP, with nerves terminating at the periphery (arrows). Note radial muscles (red) of pharynx. (f) TEM of a portion of pharyngeal nerve showing sites of FaRP-immunoreactivity as gold spheres over neuronal vesicles (arrows). Note neurotubles (nt) and mitochondrion (mi).

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Fig. 11. SEM of the strigeid trematode, Cotylurus erraticus. (a) Shows its browsing habit on the mucosa of the gull (Larus ridibundus) intestine. Note damage to villi (p). (b) Feeding involves protrusion of a bilobular, musculo-glandular holdfast (hf) that envelops a host villus for purposes of extracorporeal digestion. (c) Ventral aspect of the worm showing oral sucker (os), paired adhesive lappets (unlabelled arrows), ventral sucker (vs) and opening to the protrusible holdfast (hf). (d) An eversible papillate genital bulb (pb) serves to identify Cotylurus. Note sperm in genital pore (arrow).

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Fig. 12. SEM images of an ectoparasitic monogenean, Gyrodactylus from the stickleback, Gasterosteus showing (a) general morphology (ventral aspect); (b) disposition of parasites over host gill (note ‘footprints’, unlabelled arrows); (c) feeding posture on host epidermis during which adhesive secretions released from the head organ (arrows) secure the forebody to the skin and a protrusible and glandular pharynx digests and sucks in host skin; (d) detail of the marginal hooks of the haptor. an, anchors, he, head organ/adhesive apparatus; mo, mouth; se, sensilla; ha, haptor; mh, marginal hooks (16 in total); ep, host epidermis.

3.2. Organs of attachment As parasites, adult helminths require secure attachment and this is particularly important in ectoparasites of fish

that have to resist being swept off the body of their host by water currents. Their basic adhesive equipment is either a single sucker-like structure usually armed with an array of hooks (for example, Gyrodactylus salaris) or

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Fig. 13. (a,b) SEM of the scolices of tetraphyllidean tapeworms from elasmobranch fishes. (a) Phyllobothrium bearing four foliaceous bothridia (bo) each provided with a supplementary sucker (unlabelled arrow). (b) Pseudanthobothrium provided with four stalked cup-shaped bothridia (bo) and a central myzorhynchus (unlabelled arrow) with histolytic properties. The scolex of each species is adapted to match the patterns of crypts and villi in specific sites on the wall of the spiral valve. (c) A monogenean, Diclidophora merlangi showing its adhesive attitude on the gills of whiting (Merlangius merlangus). The bulk of the worm lies downstream relative to the gill ventilating current and is secured to the secondary gill lamellae (la) of the fish by eight pincer-like clamps, the hinged (arrows) jaws and supporting sclerites (sc) of which are shown in d, e.

a multiple complex of clamps (as in Diclidophora merlangi), both exquisitely designed to fit the appropriate attachment site on the skin or gills, respectively (Figs. 12 and 13). SEM has provided not only highly informative images of

the complex anatomical features of these organs of attachment (haptors) per se but observations of worms fixed in situ have also revealed important detail of the parasite’s adhesive attitude on the host. In Diclidophora species, finger-like

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structures each bearing a pincer-like clamping device secure the worm onto the gills of the fish (Fig. 13d and e). The clamps vary in size, number and distribution such that many of these worms display a species-specific adhesive attitude on the gill arches: D. merlangi occurs most frequently on the first gill of the whiting, whereas D. luscae is found nearly always on the second and third gills of the pout. The worms are maximally streamlined, with the forebody aligned downstream to the gill ventilating water current (Fig. 13c). Some highly modified gill parasites have become asymmetrical having lost the adhesive organs on one side so as to conform to the asymmetrical direction of the water current. Most helminths live as enteric parasites of the vertebrate gut or its diverticula. Trematodes are generally flat and live closely apposed to the mucosal surface by means of suckers; cestodes attach by way of an apical scolex that can vary greatly according to the taxon. In those tetraphyllidean cestodes that parasitise skates and rays the architecture of their attachment organs often matches and complements that of the crypts and villi, thus restricting the worms to specific sites on the wall of the spiral valve (Fig. 13a and b). Where several species of tetraphyllidian cestode inhabit the spiral valve, each may show its own specialised attachment mechanism so enabling them to occupy different tiers. For example, Pseudanthobothrium occupies the first three tiers where its deep cup-shaped bothridia fit neatly over the tops of long villi, while the shallow muscular bothridia in Phyllobothrium are better adapted to the shorter villi of tiers 4 – 6 (Fig. 13a and b). In the latter case, as the parasite grows, each bothridium becomes increasingly foliaceous to the extent that it may embrace as many as 12 villi (Williams, 1968). Such are examples of niche segregation resulting from active site recognition, a situation that favours parasite survival in the host gut through co-existence. 3.3. In vitro screening of drugs In view of the relative ease in preparing specimens for SEM, the method has proved useful for quite rapid in vitro screening of compounds with anthelmintic potential as well as examining the impact of recognised drugs in ascertaining their actions. In the absence of vaccines against any human parasite, chemotherapy will of necessity be the principal means of controlling helminths for the foreseeable future. However, for chemotherapy to be effective knowledge of the sites and modes of action of anthelmintics are critical, not least the molecular events underlying drug resistance and the interrelationship between chemotherapy and the immune response. One of the most susceptible sites of damage to a flatworm parasite following drug treatment is the tegument, and the effects are readily revealed by examining the surface architecture of treated worms by SEM. As described earlier, TEM was instrumental in helping parasitologists discover how the tegument of the human blood fluke, Schistosoma enables the worm to evade the host immune response by adsorbing a mask of host antigen to its surface to prevent host

antibodies binding to it. The drug of choice against schistosomiasis is praziquantel (PZQ) but its full efficacy as an anthelmintic has been shown to require the presence of host antibodies. The combined synergy of drug and antibodies was made visibly evident in experiments where SEM was used to examine tegumental damage of adult schistosomes exposed to PZQ and antischistosome antiserum in vivo (Modha et al., 1990). The antiserum alone induced a membrane repair process in both sexes of the worm, but revealed little other damage; PZQ alone caused the formation of spherical protuberances on the dorsal tubercles of male worms. However, tegumental damage to males after combined PZQ and antiserum treatment was significantly more enhanced than with either treatment alone. In particular, SEM revealed small blebs distributed over the entire surface of the male worm, and surface protuberances of varying size scattered both over and between the dorsal bosses. Many of the protuberances were not only ruptured but broken open to reveal numerous spherical inclusion bodies. Tegumental blebbing is a common stress feature of drugtreated worms and in Schistosoma it is believed to be a Ca2þdependent process by which the parasite attempts to replace damage tegument (Bricker et al., 1983). Thus, SEM has helped confirm that tegumental damage induced by PZQ exposes schistosome surface antigens to which host antibodies can bind, thus triggering a chain of cellular reactions resulting in encapsulation and death of the parasite.

4. Confocal scanning laser microscopy/immunocytochemistry Of the helminth taxa, flatworms are the most primitive extant animals to exhibit bilateral symmetry with attendant cephalisation and condensation of a synaptic nerve net of neurons into a central nervous system with brain. As such, they represent a strategic phylogenetic milestone in the evolution of the nervous system and research indicates that much of the basic neuronal machinery of higher organisms is already present in the lowly flatworm. Historically, however, the nervous system of the parasitic members of the phylum has been difficult to evaluate. Textbooks written as recently as in the 1980s would have us believe the flatworm parasite is largely a sedentary animal whose nervous system is somewhat degenerate and of little relevance to a life-style devoted to feeding and reproducing. In reality, of course, adult and developmental stages of most trematodes and cestodes depend on well developed neuromuscular systems for effective host attachment, invasion and migration, as well as for their not inconsiderable powers of food gathering and reproduction, such that many of these parasites are highly motile and neurochemically quite complex. Indeed, one of the most striking conclusions to emerge from multidisciplinary studies of the neurobiology of helminth parasites in general these past 15 years is that their nervous systems are well-differentiated

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multimessenger signalling systems of considerable versatility, serving not only in motor and behavioural activities essential to their complex and often discontinuous life cycle patterns, but also playing an integrative role in regulating and coordinating events such as development and reproduction. Such revelations began to emerge in the late 1980s as a result of two major developments in the field of cell biology. One was the availability of reliable antisera to vertebrate messenger molecules for use in immunocytochemistry, and the other was the application to immunofluorescence microscopy of the confocal scanning laser microscope. 4.1. Nervous system Before confocal scanning laser microscopy (CSLM) became available, the anatomical arrangement of the nervous systems of helminths had been described from either serial sections or, where size permitted, whole-mount specimens. While use of tissue sections is generally limited in gleaning information on nervous systems owing to the restricted sample volume of each section and the laborious and often inaccurate procedure of 3-dimensional neural mapping, there are exceptional examples where success has been, to say the least, singularly outstanding. One such case is the monumental work of White et al. (1986) who completely reconstructed the nervous system of the free-living nematode, Caenorhabditis elegans from electron micrographs of serial sections of the hermaphrodite. In doing so they identified a total complement of 302 neurons, and provided a wealth of connectivity data based on some 5000 morphologically identifiable chemical synapses, 2000 neuromuscular junctions and 600 gap junctions. Notwithstanding, whole-mount preparations of worms would seem a more ideal medium to envision the helminth nervous system insofar as they have potential to portray the system in toto. Initial success came through the histochemical demonstration of cholinesterase activity, as indirect evidence of the presence of acetylcholine (Halton and Jennings, 1964; Smyth and Halton, 1983) and by fluorescence microscopy for biogenic amines (Shishov, 1991). However, the optical constraints of conventional light microscopy or epi-illumination fluorescence microscopy means that detail is often unclear and obscured by the blur and flare of out-of-focus regions of the specimen, making high definition images of internal anatomy difficult to obtain. In this respect, the confocal principle is particularly valuable in fluorescence microscopy since it confines illumination and detection to the same spot in the specimen at any one time, such that only information within the focal plane of the objective lens is detected and out-of-focus images of the specimen are rejected. As an imaging system, CSLM has been revolutionary, not only in producing fluorescence images of superior clarity and resolution of fine detail but also in enabling non-invasive sections to be obtained from within whole-mount specimens. Thus, by interfacing immunocytochemistry with CSLM, it is possible to optically section

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through whole-mount immunostained preparations of worms, that is, collect images as an extended focus series from a scan in the z-axis and then generate computerised composite images from different levels and so produce accurate spatial resolution of the specimen in three dimensions. In helminths, the technology has been used extensively to map and compare the distribution patterns of neuroactive substances in 3-dimensional reconstructions of nervous systems, as well as to monitor neurochemical changes accompanying parasite ontogeny. For reviews, see Halton and Gustafsson (1996); Maule et al. (2001). The use of well-characterised antibodies in immunochemical procedures, and the discoveries in invertebrates, including flatworms and nematodes, of homologues to recognised vertebrate messenger molecules fostered an upsurge of interest through the 1980/90s in the nervous system of helminths as a target for chemotherapeutic intervention. However, use of heterologous mammalian antisera to identify, for example, neuroactive peptides in helminths by virtue of their cross-reactivity calls for caution since most antibodies used in immunocytochemistry are directed against a relatively small epitope of perhaps 3–8 amino acid residues. The problem was largely resolved by using a range of region-specific antisera raised against several highly conserved sequences of the peptide in question, thereby adding considerable validity to localisation studies in helminths. Antibody specificity notwithstanding, the exploration of invertebrate neurobiology by immunocytochemistry using antisera to vertebrate peptides has been impressive, notably in identifying homologies between helminth and mammalian peptides. Moreover, such studies of parasite nervous systems have often formed the starting point for further investigations of the chemical nature of their neuronal substances, through biochemical isolation, purification and sequencing procedures. 4.2. Neuropeptide F In one remarkable instance of serendipity, this immunocytochemical approach led to the discovery of the structure of the first known flatworm neuropeptide, neuropeptide F (NPF) by virtue of its cross reactivity with antisera directed to the C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP) (i.e. Leu-Tyr-Arg-Pro-ArgTyr·NH2). PP is a member of the superfamily of neuropeptide Y (NPY) vertebrate peptides whose signature tertiary structure is the PP-fold. All members have an amidated Cterminus in which they share certain key amino acids essential for receptor interaction or biological activity. NPF was first isolated and purified from the cestode, Moniezia expansa by Maule et al. (1991) using gel permeation chromatography and reverse-phase HPLC interfaced with a radioimmunoassay employing the PP-antiserum. Because its C-terminus ends in a phenylalaninamide (yF·NH2), compared to a tyrosylamide (yY·NH2) as in vertebrate NPY, the peptide was named NPF. Soon after, similar NPF

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peptides were characterised from the land planarian, Arthurdendyus triangulatus (Curry et al., 1992) and the molluscs, Aplysia californica, Helix aspersa and Lymnaea stagnalis (Rajpara et al., 1992; Lueng et al., 1992; Tensen et al., 1998). To date, NPF is recognised as the most abundant neuropeptide in flatworms, and there is good molecular evidence to suggest it is the ancestral form of the NPY family of vertebrate peptides. Thus, all but one known NPY family genes have a phase 2 intron within the codon for the penultimate arginyl residue, and an intron as such is present in this exact location in the Moniezia gene (Mair et al., 2000). Where investigated, NPF has not been found in any nematode thus far. 4.3. FaRPergic system Historically, the immunocytochemical evidence for the presence of PP-fold peptides in flatworms was thought to be due to non-specific cross-reactivity of a portion of the PP antibodies with the tetrapeptide amide, FMRFamide, a native invertebrate neuropeptide first isolated from the clam, Macrocallista nimbosa by Price and Greenberg (1977). The sequencing of NPF not only confirmed the presence of PP-fold peptides in flatworms but concomitantly called into the question the occurrence of FMRFamide-like peptides (FaRPs) in platyhelminths, particularly since both peptides end C-terminally in an RFamide. However, at least three novel FaRPs have since been isolated and sequenced from platyhelminth species: GNFFRFamide from Moniezia, RYIRFamide from Arthurdendyus and GYIRFamide from the freshwater and marine turbellarians, Girardia tigrina and Bdelloura candida, respectively; a possible fourth FaRP, YIRFamide was also found in extracts of Bdelloura, but may represent a degradation product of GYIRFamide (Maule et al. 1993b, 1994; Johnston et al., 1995, 1996). These biochemical data established unequivocally that FaRPs, albeit relatively few in number, do occur extensively in the nervous systems of flatworms in common with all other major invertebrate taxa notably nematodes (where at least 60 FaRPs are known in some species and where more than half of the neurons show FaRP-immunoreactivity) and arthropods (85 FaRPs identified thus far). Since their isolation, FaRPs have become the most widely researched family of invertebrate peptides in helminth parasites, principally because they are known to be myoactive at nanomolar concentrations and are not present as such in the vertebrate host. Not surprisingly, increasing anthelmintic resistance and the quest for new anti-parasitic drugs has made targeting the FaRPergic signalling system of helminths an attractive proposition in future therapeutic strategies (see Maule et al., 2002). 4.4. Neuronal pathways The topographical simplicity of the helminth nervous system belies a neurochemical complexity more befitting that of higher invertebrates in that a large number of

candidate classical and peptidic neurotransmitters and modulators have been identified directly or indirectly using microscopy and cytochemical methodologies. These include: acetylcholine, 5-hydroxytryptamine, g-aminobutyric acid, glutamate, dopamine, noradrenaline, adrenaline, histamine, nitric oxide, and neuropeptides, most notably FMRFamide-related peptides or FaRPs. Of these methods, the use of antisera for immunolabelling neuroactive substances and their visualisation by confocal microscopy and/or TEM have been particularly revealing and have established much novel detail of the nature of helminth nervous systems (Fig. 14). Moreover, immunolocalization and distribution patterns of neuroactive substances have provided clues as to the putative roles of specific neuromediators. Thus in flatworms, serotoninergic and peptidergic innervations of muscle implicate 5-HT and FaRPs in muscle control, a fact borne out in motility experiments in vitro. In this regard, the combined use of phalloidin, as a specific probe for filamentous actin, has added a further instructive dimension to confocal immunocytochemistry, enabling high resolution, non-invasive optical sectioning of the musculature in helminths and its spatial relationship with nerve (Fig. 15). Serotoninergic fibres are generally much finer in appearance and in the CNS are less likely to be organised into nerve cords than are cholinergic or peptidergic fibres, even allowing for differences in the intensities of staining; often in the more central regions of the nervous system they occur in loose arrays as nerve tracts. Cell bodies that are immunoreactive for 5-HT are usually larger and more distinct than those of peptidergic neurons, and represent a fairly homogeneous population of cells, often exhibiting a marked bilateral symmetry or pairing in their arrangement. Peptide immunoreactive fibres, in common with those reactive for cholinesterase are often closely packed, as in the longitudinal cords, and their somata are somewhat smaller in size and distributed with less symmetry. Mapping the distribution pattern of neuroactive substances in colocalisation studies of helminths, using multiple confocal channels and different fluorophores, has shown overlap of peptidergic and cholinergic neuronal pathways. Results indicate there is every likelihood neuroactive peptides coexist with classical transmitters, inferring a complexity of signalling in helminths akin to that found in higher animals. Using electron immunogold labelling procedures, the subcellular distribution of peptide immunoreactivity has been shown to be localised exclusively to dense-cored vesicles (Fig. 10f), occurring both randomly in axons as well as in concentrations that occupy axonal swellings or varicosities throughout the central and peripheral nervous systems. There is a close anatomical relationship between nerve and muscle and it may be that the varicosities described are synaptoid release sites along the axon from where myotropic and other factors can diffuse across interstitial tissue to interact with their target sites.

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Fig. 14. Confocal fluorescence images of FaRP-immunoreactivity (IR) in the brains of helminth parasites. (a) Whole mount Diclidophora merlangi (monogenean) showing GYIRFamide-IR (green) in the cerebral ganglia (cg), commissure (co) and associated innervation surrounding the pharynx (ph), counterstained with phalloidin for F-actin of the musculature (red). bs, buccal sucker (b) Whole mount Fasciola hepatica (trematode) showing GYIRFamideIR (red) in the cerebral ganglia (cg) and commissure (co) seen through a split in the surface tegument (tg). ph, pharynx. (c) Whole mount Cysticercus tenuicollis (cyclophyllidean metacestode of Taenia hydatigena) showing GNFFRFamide-IR (green) in the brain (cg) and associated elements of the CNS, counterstained with phalloidin (red) for muscle. lnc, longitudinal nerve cord; ac, acetabulum. (d) Longitudinal section Panagrellus redivivus (nematode) showing FMRFamide-IR (green) in the circumpharyngeal nerve ring (cpr) surrounding the pharynx (ph). Note three neurons (p) of the ventral ganglia and elements of the ventral (vnc), dorsal (dnc) and lateral nerve cords (lnc). phb, pharyngeal bulb.

4.5. Neuronal identification and function in nematodes Relatively little work is documented on the constancy or otherwise of neuron populations in flatworms by plotting their numbers and disposition, or by comparing the cellular distribution of particular neuroactive substances in different species. This is in marked contrast to nematodes, where for

some species (e.g. Ascaris suum, C. elegans) the total population of neurons per worm is known, and individual neurons have been identified and their immunoreactivities for particular neuroactive substances categorised (Sithigorngul et al., 1990; Stretton et al., 1978; Schinkmann and Li, 1992). Goldschmidt (1908) began this ground breaking microscopical work nearly 100 years ago,

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Fig. 15. (a) SEM image of adult Schistosoma mansoni pair in permanent copula from human mesenteric veins, where the male worm (p p ) holds the female (p) in the gynaecophoric canal (gc). Note the well-developed oral (os) and ventral suckers (vs). (b) Confocal image of the forebody of a whole-mount of a female schistosome showing immunofluorescence (red) for FaRP neuropeptide in the cerebral ganglion/brain (cg) and associated neurons, including those innervating the oral (os) and ventral (vs) suckers. The muscle fibres of the suckers, oesophagus (oe) and body wall (bw) are stained green using FITC-labelled phalloidin. (c) Confocal image of the neural plexus that innervates the musculature of the oral sucker of a male worm, immunostained for FaRP neuropeptide. (d) Confocal image of trematode body wall musculature revealing a lattice-like arrangement of outer circular (cm) and inner longitudinal fibres (lm), below which are bundles of diagonal muscle fibres (dm) that cross each other at an angle of approximately 1208.

spending many years identifying individual neurons in numerous samples of adult Ascaris (162 in the first 5 mm of worm) and in doing so he established the important concept of the identified neuron. Others, notably Stretton and coworkers, have carried forward this work by adding physiological data about individual neurons using intracellular recording techniques (Walrond et al., 1985; Davis and Stretton, 1989a,b). Today there are extensive anatomical descriptions of neurons and their synaptic connectivities

and these data have enabled predictions to be made as to how the motor system in nematodes controls behaviour (Stretton et al., 1985). The nervous system of adult Ascaris contains 298 neurons including both sensory and motor neurons located in the head and tail. Removal of these two regions of the worm ablates about 200 of the total complement of neurons leaving around 100 neurons concentrated in the dorsal and ventral longitudinal nerve cords. Here they are arranged in seven repetitive segments

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along the body as motorneurons (MNs) and provide innervation to activate the longitudinal somatic muscle of the body wall in locomotory movement. 4.6. Myoneural junctions in nematodes Nematode somatic muscle cells have a very unusual structure in that they make their own cytoplasmic connections with the dorsal and ventral nerve cords, rather than the conventional arrangement of nerves penetrating muscle (Fig. 10c). TEM studies of the somatic muscle cells of Ascaris lumbricoides have revealed a nucleated myocyton ( ¼ muscle bag or belly), rich in glycogen and mitochondria, continuous with a core or spindle of obliquely striated fibres whose actin and myosin filaments are arranged at a more acute angle than in vertebrate skeletal muscle (Rosenbluth, 1965a,b). This oblique arrangement of striations is believed to confer greater extensibility of muscle without compromising velocity of contraction. From the myocyton one or more cytoplasmic arms extend and subdivide into thin fingers of cytoplasm that join with those from other arms to form a functional complex or syncytium that synapses with the MNs in the nerve cord (Rosenbluth, 1965b). Individual processes from the cytoplasmic arms are closely apposed in places marked by tight junctions, thus ensuring electrical coupling between the muscle cells. The anatomical arrangement of the nerve circuits of the motor- and inter-neurons responsible for co-ordinated movement in A. suum was painstakingly deciphered by Stretton et al. (1978) who traced their profiles through light microscope serial sections, augmented by TEM of their synaptic connections. All of the MNs have their cell bodies concentrated in the ventral nerve cord and the arrangement is believed to be similar in other nematode species. Each of the repeated segments has 11 MNs and six longitudinal neurons; together with three right-hand commissures and one left-hand commissure. The MNs are excitatory and release acetylcholine as a fast transmitter (cholinergic) or inhibitory and release GABA as a fast transmitter (GABAergic). The motor nervous system in nematodes is also highly peptidergic and small peptides (FaRPs) are known to be involved and likely operate as co-transmitters and/or modulators of myoactivity (Maule et al., 2001). There is a reciprocal arrangement of the neural circuits such that excitatory impulses to the ventral muscles result in inhibitory impulses being sent to the dorsal muscle and vice versa. This means that as one block of muscle contracts the opposing muscle set relaxes, enabling the worm to move forwards or backwards in a wave-like or sinusoidal movement. Since dorsal muscle cells connect only with the dorsal nerve cord and ventral muscle only with the ventral cord, the alternate waves of contraction and relaxation are generated only along the dorso-ventral plane, in contrast to the situation in snakes and eels where the waves of contraction are of lateral dimension.

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4.7. Neuroactive substances in reproduction As parasites, helminths are typically characterised by a prodigious egg output and an elaborate reproductive system, accounting for the bulk of their metabolic activity and body space. Aminergic and peptidergic neurons have been described that innervate the musculature of the gonoducts and accessory structures of all helminths examined. Most helminths undergo several developmental stages prior to sexual reproduction in the definitive host, and these transformations are likely regulated by a series of endogenous and exogenous cues, the nature of which are largely unknown but may include hormone-like (peptidic) secretions of the nervous system. Microscopic observations have been made on the anatomical and neurochemical development of the nervous system during ontogenesis, facilitating the investigation of potential trigger stimuli on neuronal development and stage-specific expression of neuroactive messenger molecules. In flatworm parasites, the mechanism regulating assembly and production of eggs resides in the egg-forming apparatus or ‘oogenotop’, the focal point of which is a richly innervated and muscularised egg-chamber or ootype that adjoins the proximal uterus and surrounded by Mehlis’ gland. It is to this chamber that oocytes, spermatozoa and shell material are propelled at intervals to form an egg, an event which can occur as frequently as every 3.5 s in the liver fluke, F. hepatica (Happich and Boray, 1969). Egg assembly is the result of a highly ordered series of rhythmical contractions of the muscles in the walls of the ducting system, namely the oviduct, seminal receptacle, ovo-vitelline duct, and ootype itself. How successive events in oviposition in flatworms are initiated and controlled have yet to be determined. However, confocal and electron immunocytochemical studies on a number of flatworm parasites have shown that a neuronal plexus of cell bodies and associated axonal processes contain neuronal vesicles and provide synaptic contacts with the circular and longitudinal muscle fibres and sphincters of the ootype wall and related ducts (Magee et al., 1989; Maule et al., 1990, 1993a; Skuce et al., 1990; Halton et al., 1991; Marks et al., 1994, 1995; Stewart et al., 2003a,b). The cells of these plexuses are quite distinct from the Mehlis’ gland cells and, where examined, have been found to be immunoreactive for NPF and FaRPs, with minor species differences both in their numbers (. 100 in D. merlangi; , 20 in F. hepatica, Schistosoma mansoni, Apatemon cobitidis proterorhini and Cotylurus erraticus) and disposition around the entrance and exit of the ootype (Fig. 16a and b). By analogy with other invertebrates, neurosecretory control of reproductive function in flatworms would seem plausible, and evidence of this comes from confocal studies of peptidergic innervation of the ootype of Polystoma nearcticum. P. nearcticum is a flatworm parasite that undergoes reproductive synchrony with its tree-frog host, Hyla versicolor, becoming reproductively active only in

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Fig. 16. Confocal images of FaRP-immunoreactivity (IR) in helminth reproductive structures. (a) Whole mount female Schistosoma mansoni showing FaRP-IR (red) in the innervation of the muscular wall of the ootype/egg chamber (ec) and associated ducting (uterus, ut). The preparation was counterstained with phalloidin for muscle (green). Note neuronal cell bodies (unlabelled arrows), ventral nerve cord (vnc) of the CNS, and autofluorescense (red) of forming egg capsule (eg). Inset shows formed egg of S. mansoni as seen by SEM, £ 150. (b) Diclidophora merlangi (monogenean) showing immunoreactive neuronal cell bodies (green, unlabelled arrows) and associated innervation of the muscle fibres (red) of the ootype/egg chamber wall. (c) TS vagina vera of Ascaris suum showing IR in the peripheral innervation (green, unlabelled arrows) to the largely circular muscle fibres (red). Note the cuticular lining (p). (d) Portion of muscle (red) from the vagina vera of A. suum showing innervation (unlabelled arrows) along individual fibres.

the short period of host sexual activity during spawning. In this way, it provides a valuable parasite model system for investigating potential trigger stimuli and factors controlling oviposition in flatworm parasites. Confocal immunocytochemical studies of worms collected from frogs during spawning have revealed extensive immunostaining for FaRPs and 5-HT in ootype innervation (Armstrong et al., 1997). In contrast, the ootype innervation of worms recovered from frogs post-spawning showed little or no

demonstrable FaRP staining; levels of 5-HT immunostaining were unaffected by the reproductive state of the worm. Thus, synchronised host – parasite sexual activities appear to be paralleled by neuropeptide expression in the innervation of the egg chamber. Since FaRPs are known to be potently myoactive on flatworm muscle, they may be involved in the coordinated triggering of contractions of the ootype and associated ducts during egg assembly. The recognised ability of 5-HT to stimulate energy metabolism through

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elevation of cAMP, and the finding by Day et al. (1994) that 5-HT enhances the responsiveness of flatworm muscle to excitatory transmitters such as FaRPs and glutamate, may mean the 5-HT of the ootype nerve plexus ensures an energy level sufficient to support ootype contraction, while the FaRP per se triggers actual contraction. Nematodes too are highly fecund. Estimates of egg production in Ascaris, for example, range from 200,000 to 2,000,000 eggs per day. Events are coordinated by an egg laying organ, the ovijector in the form of the proximal saclike vagina uteri and a distal highly muscularised tube the vagina vera. The lumen of the latter is normally kept closed by the high internal pressure from the pseudocoelomic fluid such that the musculature of the vagina vera must act against this pressure in order to receive sperm and release eggs, respectively, during copulation and egg laying. Most myofibrils in the ovijector are circular in orientation but a number of them divide and run for short distances in longitudinal and diagonal directions. The vagina vera is far more muscular that the vagina uteri and there is an abundance of FaRPergic fibres running parallel to the circular and longitudinal muscles, with little innervation in the vagina uteri (Fig. 16c and d). TEM and CSLM interfaced with immunocytochemistry have demonstrated a nerve plexus that extends over the outer surface of the ovijector with varicose fibres displaying accumulations of FaRPimmunoreactive vesicles and associated mitochondria at sites abutting the circular muscle fibres (Fellowes et al., 1999). Functional interpretation of these microscope data suggests that fertilised eggs are forced along the vagina uteri by hydrostatic pressure and muscle contractions such that a bolus of eggs builds up at the vagina uteri-vagina vera junction before being driven into the vagina vera. Once in the vagina vera the peristaltic action of its tightly packed circular muscles forces the eggs out through the gonopore whilst at the same time maintaining the high internal hydrostatic pressure. The myoactivity involved is believed to be regulated by FaRP peptides acting directly or indirectly on stretch receptors to initiate muscle movement. Confirmation of this comes from motility studies on isolated preparations of vagina vera which showed the natural coordinated peristalsis of the organ system could be selectively modulated by Ascaris FaRPs, suggesting multiple receptors are present (Fellowes et al., 1998, 2000). The cuticular ensheathed structure of nematodes is such that the ovijector muscles are one of the few muscle systems exposed to the outside of the worm and as such accessible to drugs. An invagination of body wall cuticle lines the vagina vera portion of the ovijector but is absent from the vagina uteri (Fellowes et al., 1999). Chemical disruption of ovijector function in nematodes would not only reduce their reproductive capacity but would also interfere with control of the internal hydrostatic pressure, a ‘skeletal’ system critical to the worm’s survival.

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5. Future directions Today we are at the theoretical limit of resolution of the light microscope and to a lesser extent the electron microscope. However, exciting new developments continue to be made, especially in the field of imaging cell dynamics, such that microscopy will remain an indispensable technique for studying cellular aspects of helminth parasites. This would seem to be particularly true of research aimed at understanding the functional attributes of the helminth neuropeptide signalling system as a target for antiparasitic drug discovery. One of the constraints of using polyclonal antisera to study neuropeptide signalling systems under the noncompetitive conditions of immunocytochemistry is that they are unable to distinguish clearly between closely related peptides. A notable example is when immunostaining for FaRPs insofar as C-terminally directed anti-FaRP antisera are singularly promiscuous with respect to FaRP recognition and, as such, provide little discriminatory data on patterns of FaRP expression throughout the system. A far more accurate and informative procedure involves the temporal and spatial localisation of gene expression of the FaRPs using whole-mount in situ hybridisation, enabling a greater degree of functional analysis of the peptidergic system in helminths. The method has already been put to good use in characterising the expression patterns of FaRPencoding genes in the potato cyst nematode, Globodera pallida in order to assign FaRP function through comparison with the neuronal map of C. elegans (Kimber et al., 2002). Significant advances have been made both in the numbers of immunocytochemical fluorescent tags available for the microscopist and the confocal imaging technologies by which they can be visualised. While the standard fluorophores mentioned in this review (FITC, TRITC) have to be made outside the cell and then artificially introduced into it, exciting new opportunities are unfolding from the discovery of genes coding for protein molecules that are themselves inherently fluorescent. One of the best known of these autofluoresent proteins is enhanced green fluoresent protein (eGFP) which can be employed as a reporter molecule to monitor the distribution and dynamics of proteins in living systems. Thus, a transgenic organism can be made with the fluorescent protein-coding sequence under the transcriptional control of the promoter belonging to a gene of interest, thereby enabling visible display of the gene’s expression pattern in the living organism. A different fluorescent protein DNA-coding sequence can also be inserted at the beginning or end of the gene for another protein to reveal its location and activities. Numerous research groups are endeavouring to develop systems for the efficient transformation of helminth parasites (or parasite tissues/cells). The development of these techniques would allow the application of fluorescent reporter gene technologies to parasitic worms and could provide a springboard for

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the further exploitation of the burgeoning supply of parasite transcriptome data by the modern microscope. GFP and its analogues are also being used increasingly in analytical experiments aimed at monitoring protein– protein interactions, such as the binding of signalling ligands with their receptors by fluorescence resonance energy transfer (FRET). All of these exciting new developments are beginning to be harnessed using a new breed of spectral confocal microscopes that incorporate high speed multiphoton imaging technology for investigating cell dynamics. Such advances in microscopic methodology bode well for parasitologists being better able to exploit available genomic and proteomic information in their quest to decipher the structure and functional complexities of the helminth parasite.

Acknowledgements Thanks are due to Prof. Aaron G. Maule for helpful comments on the manuscript and to Dr Michael T. Stewart for expert technical assistance with the plates.

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