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REVIEW / SYNTHSE

Comparative morphology of the body wall in flatworms (Platyhelminthes)1

Seth Tyler and Matthew Hooge

Abstract: The soft-bodied nature of the platyhelminths is due largely to the structure of the body wall and its lack of sclerotic elements such as cuticle. Free-living members, i.e., most turbellarians, show considerable variety, but the basic form of the body wall comprises a simple ciliated epithelium overlying a network of muscles. We illustrate this body wall structure in a representative typhloplanoid rhabditophoran and discuss variations in representatives of the Acoela, Catenulida, and other free-living rhabditophorans. The major parasitic groups of platyhelminths, the rhabditophoran Neodermata, follow a developmental pattern that replaces a similar ciliated epidermis in a larval stage with a specialized epidermis called a neodermis, which is assumed to be key to their success as parasites. This neodermis consists of a syncytium that covers the body in a continuous sheet connected to perikarya that lie below the body wall musculature. The neodermis can be seen as a special adaptation of a developmental mechanism common to all platyhelminths, in which epidermal growth and renewal are accomplished by replacement cells originating beneath the body wall. The cell type responsible for all cell renewal, including body wall renewal, in platyhelminths is the neoblast, and its presence may be the one autapomorphic character that unites all taxonomic groups of platyhelminths. Rsum : Le corps mou des plathelminthes sexplique en grande partie par la structure de leur paroi corporelle et labsence dlments sclrotiques, tels que la cuticule. Les groupes libres, cest--dire la majorit des turbellaris, sont trs varis, mais la structure de base de leur paroi consiste en un simple pithlium cili superpos un rseau de muscles. Nous illustrons cette structure de la paroi chez un rhabditophore typhloplanode typique, et nous commentons les variations observes chez des reprsentants des acoeles, des catnulides et dautres rhabditophores libres. Le groupe principal de platheminthes parasites, les rhapditophores nodermates, ont une squence de dveloppement au stade larvaire pendant laquelle un piderme cili de mme type que celui des turbellaris libres est remplac par le noderme, un piderme spcialis qui assure, croit-on, leur succs comme parasites. Ce noderme forme un syncytium qui recouvre le corps en une couche continue qui est rattache des pricaryons situs sous la musculature de la paroi. Le noderme peut tre considr comme une adaptation particulire dun mcanisme de dveloppement commun tous les plathelminthes dans lequel la croissance et le renouvellement de lpiderme saccomplissent par des cellules de rechange provenant de sous la paroi. Le type cellulaire responsable du renouvellement de toutes les cellules, y compris celles de la paroi, chez les plathelminthes est le noblaste dont la prsence peut tre le caractre apomorphe qui unit tous les groupes taxonomiques de plathelminthes. [Traduit par la Rdaction] Tyler and Hooge 210

Introduction
Flatworms are soft-bodied bilaterians with a body structure that has inspired some evolutionary biologists to use them as models for the ancestor of the rest of the Bilateria.
Received 25 February 2003. Accepted 6 August 2003. Published on the NRC Research Press Web site at http://cjz.nrc.ca on 16 April 2004. S. Tyler2 and M. Hooge. Department of Biological Sciences, University of Maine, 5751 Murray Hall, Orono, ME 044695751, USA.
1

This review is one of a series dealing with aspects of the biology of the phylum Platyhelminthes. This series is one of several virtual symposia on the biology of neglected groups that will be published in the Journal from time to time. 2 Corresponding author (e-mail: styler@maine.edu).
Can. J. Zool. 82: 194210 (2004)

The soft-bodied nature arises largely from the relative simplicity of the body wall: a simple epithelium (i.e., single layered) overlying a network of muscles and lacking cuticle or dermal skeletal elements (Fig. 1). The free-living flatworms, known as turbellarians (class Turbellaria in non-cladistic classifications), have, for the most part, a ciliated, cellular epidermis (Fig. 1A), whereas the parasitic flatworms, the Neodermata (encompassing the classes Trematoda, Monogenea, and Cestoda), have a syncytial, nonciliated epidermis whose nuclei-bearing parts lie sunken below the musculature (Fig. 1B). Turbellarians show considerable variety, however: special regions of the epidermis may have a syncytial structure or sunken cell bodies, or may lack cilia. Reflecting their origins from a turbellarianlike ancestor, the Neodermata have a larval stage bearing a ciliated, cellular epidermis, much like that of turbellarians, and this is replaced by the syncytial epidermis, which is then
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doi: 10.1139/Z03-222

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Fig. 1. (A) Schematic representation of the body wall and its layering in turbellarian flatworms as seen in a cut-away view of the body. A typical cellular, ciliated epidermis (with cilia drawn on only some cells) and a layering of diagonal muscle fibers between circular and longitudinal fibers, as would be seen in a rhabdocoel like a typhloplanoid or dalyellioid species, are represented, but many variations on this theme apply to the free-living flatworms in general. The space between the body wall and gut could be filled with parenchymal cells, the cell bodies of epidermal glands, or internal organs such as gonads. The gut could also have its own musculature as indicated in the lower part of the Figure. (Based on a scheme by Rieger and Ladurner 2001.) (B) Schematic representation of the body wall of adult neodermatan flatworms (Neodermata) as seen in a longitudinal section of the body and subdivided to represent typical surface specializations associated with four major groups of the taxon. Diagonal muscle fibers lie innermost, as opposed to between the other two muscle layers as in Fig. 1A.

called a neodermis (for new skin; Ehlers 1985), in the course of infecting a new host. The mode of replacement of the epidermis, whether as part of growth or general maintenance of the epidermis or the development of the neodermis, appears to be unique to the Platyhelminthes. Rather than arising from dividing cells within the epidermis, as appears to be the case for other metazoans, new epidermal cells arise from stem cells, the neoblasts, that reside below the body wall and then migrate into the epidermis as they differentiate. This developmental mode applies even in embryogenesis of the turbellarians (i.e., embryonic and definitive epidermises grow by immigration of stem cells from underlying layers) and is the way in which each new generation of neodermis arises as larval stages develop in sequence in the more complex parasitic life cycles of neodermatans (Tyler and Tyler 1997). The neoblast stem cell system, and particularly its role in renewal of the epidermis, may in fact be an autapomorphic trait for the Platyhelminthes (Rieger and Ladurner 2001). Muscles of the body wall, as well as all other differentiated

cells, also arise from such neoblasts in flatworms; no differentiated somatic cells in the body are capable of dividing (Peter et al. 2001). Without an autapomorphy such as the neoblast, there is some justification for questioning the monophyletic nature of the phylum Platyhelminthes (Smith et al. 1986) and, in fact, recent proposals based on molecular data are to place two orders of platyhelminths, the Acoela and Nemertodermatida, into separate phyla outside the Platyhelminthes (Ruiz-Trillo et al. 1999; Jondelius et al. 2002; Ruiz-Trillo et al. 2002). Other morphological data support the monophyletic nature of at least three clades within the Platyhelminthes: the Acoelomorpha, Catenulida, and Rhabditophora. The Acoelomorpha, comprising the Acoela and Nemertodermatida, share features of epidermal ultrastructure (the form of the cilia and their rootlets), as well as of certain glands (Tyler and Rieger 1977; Smith and Tyler 1985; Smith et al. 1986; Ehlers 1992a). The acoels, which have often figured in phylogenetic hypotheses of the origin of the Bilateria, are mostly small marine worms with a digestive system consist 2004 NRC Canada

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ing of a syncytium (and so lacking a lumen, hence the name Acoela). The Catenulida, a small group containing a strange primitive marine family but mostly freshwater species, share a special orientation and position of the male reproductive and excretory organs (Smith et al. 1986). The rest of the platyhelminths, including the parasitic classes and such wellknown free-living groups as the planarians (Tricladida) and polyclads, form the monophyletic group Rhapditophora and share features (at least primitively) including adhesive organs and rhabdite glands. In all three clades, neoblasts residing below the body wall give rise to other cells, including those involved in growth and replacement of the epidermis. The body wall and its relation to these stem cells may provide the best characters for reuniting these three clades into the single phylum Platyhelminthes.

The epidermis of turbellarians

A rhabditophoran model A starting point for understanding the structure of the body wall of turbellarian platyhelminths is consideration of the body wall in a microturbellarian member of the rhabditophoran order Rhabocoela, the largest of the turbellarian orders containing species commonly encountered in both freshwater and marine habitats. A typhloplanoid rhabdocoel3 serves as a good model (Fig. 2), having a simple cellular, ciliated epidermis with a well-developed basement membrane overlying a typical muscle network of circular, longitudinal, and diagonal fibers in short, a body wall fitting a model we might expect for bilaterians in general, apart from the lack of cuticle or other skeletal elements. Epidermal cells of the typhloplanoid are cuboidal to squamous and form a simple epithelium (Fig. 2A). Rather than being strictly boxlike, they interdigitate with one another along their lateral sides and send processes along the basement membrane under their neighbors. They also accommodate the necks of glands and dendrites of sensory cells that penetrate the epidermis through tubular channels in the cells themselves. The cells are typically polarized, with an apical surface bearing cilia and microvilli, a basal surface resting on the basement membrane, and loosely defined layers in the cytoplasm between. Most apical in the cytoplasm, immediately beneath the plasma membrane, is a layer of small secretory vesicles called epitheliosomes or ultrarhabdites, which appear to contribute to the mucous covering of the epidermis upon their release (Fig. 2B). Just basal to these vesicles is a terminal web of actin filaments; basal to these filaments is a layer of mitochondria, and then the nucleus, which is more or less centrally positioned (Figs. 2A and 2B). Around the nucleus and basal to it are elements of the endomembrane system, the Golgi apparatus, and endoplasmic reticulum, as well as ribosomes free in the cytoplasm. The lobulated shape of the nucleus produces multiple profiles in given sections (Fig. 2A).
3

Two belt-form junctions join each cell to its neighbors: an apical-most zonula adherens and, just basal to this, a septate junction (Fig. 2D). Spot-form junctions, namely hemiadherens junctions, link the cells to the underlying basal lamina (Figs. 2A and 2C). The zonula adherens links prominently with the actin filaments of the terminal web. Actin filaments also compose stress-fiber bundles that anchor basally to the hemiadherens junctions. All general epidermal cells are multiciliated and their cilia anchor into the cytoplasm by two rootlets, a tubular rostral rootlet extending from the rostral side of the ciliary basal body and a solid-cored vertical rootlet extending from its basal end and penetrating the terminal web (Fig. 2A). Microvilli between the cilia bear a glycocalyx that forms a diffuse mucous coat over the epithelial surface. Other mucoid coverings are formed by secretions of gland cells that penetrate the epidermis between the epidermal cells; their cell bodies lie below the body wall in the parenchyma. Among these glands are prominent rhabdite glands whose secretions, the rhabdites, have a characteristic morphology with a multilayered lamellate cortex and a granular medula. The terminal necks of the gland cells, as well as the dendrites of sensory cells in the epidermis, are linked apically to the epithelial cells by the typical epithelial junctions, the zonula adherens and septate junctions. The basement membrane of the epidermis is, as usual for basement membranes (Pedersen 1991), bilayered; the basal lamina (lamina densa) appears as a dense sheet in immediate contact with the basal plasma membrane, and the reticular lamina (lamina fibroreticularis), appearing more diffuse and fibrous, faces the underlying muscle cells (Fig. 2C). Lying on the inner side of the basement membrane are cell processes of pigment cells bearing dense granules that give the animal its brown color. Beneath the basement membrane and pigment cell processes are the muscles of the body wall comprising three orientations: an outermost circular muscle layer, an innermost longitudinal muscle layer, and between them, diagonal muscles whose fibers form a network of two crossing orientations (Figs. 2C and 3). Elements of the submuscular nerve plexus are also evident in sections of the musculature (Figs. 2A and 2B). In general, this model of body wall structure (i.e., a cellular, ciliated, simple epithelium overlying a three-layered body wall musculature) applies to many other turbellarians as well. There is significant variety, however, which has been the subject of more comprehensive reviews of epidermal ultrastructure (Bedini and Papi 1974; Tyler 1984; Ehlers 1985; Rieger et al. 1991b; and Tyler and Tyler 1997). We summarize aspects of this variety in the following sections, starting with other turbellarian rhabditophorans (i.e., the bulk of the free-living platyhelminth species) and comparing them with the other two clades of platyhelminths, the Acoelomorpha and the Catenulida. Because the neodermatan rhabditophorans are so distinctive in the structure of the body wall, they are treated in a separate section.

Specifically, an undescribed genus and species of the family Promesostomidae, subfamily Adenorhynchinae. The specimen, from the US Atlantic coast (Bogue Bank, North Carolina), was found living in medium-grain sand on the exposed beach near Iron Steamer Pier, at midtide level. The specimen was fixed in phosphate-buffered 3% glutaraldehyde, post-fixed in phosphate-buffered 1% osmium tetroxide, and embedded in Epon-Araldite.
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Fig. 2. Ultrastructure of the body wall of a typhloplanoid rhabdocoel flatworm (an undescribed member of the family Promesostomidae, subfamily Adenorhynchinae). (A) Entire thickness of the body wall in a cross section of the animal; two cells in the epidermis with lobulated nuclei, and bearing cilia and microvilli, as well as the underlying basement membrane and musculature, are visible. The rootlet on the cilium at right is the vertical rootlet. (B) Apical surface with microvilli and junctions between two epidermal cells. (C) Basal region of the epidermis and muscle layers. (D) Apical region of an epidermal cell bearing cilia and microvilli and with layers below of terminal web and mitochondria. bm, basement membrane; c, circular muscle; d, diagonal muscle; e, epitheliosome; gl, gland neck; h, hemiadherens junctions; l, longitudinal muscle; m, mitochondrion; n, longitudinal nerve; np, submuscular nerve plexus; p, pigment granule; sj, septate junction; tw, terminal web; za, zonula adherens.

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198 Fig. 3. Projection of confocal images of a whole mount of the typhloplanoid rhabdocoel Jensenia sp. stained with Alexa-488phalloidin to reveal musculature. Pharyngeal muscles appear as bright concentric fibers near the center, and muscles of the gonopore behind it are similarly strongly stained. The remaining fibers are muscles of the body wall arranged in longitudinal, circular, and diagonal orientations.

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Other rhabditophorans A cellular, ciliated epidermis like that of the typhloplanoid species is typical of many rhabditophorans, but considerable variation appears in the development of certain components in various taxa. The basal matrix underlying the epidermis, for instance, is not sharply set off as a basement membrane among the lower rhabditophorans (the Macrostomorpha) as it is in other rhabditophorans. Instead, it appears as an extension of a general intercellular matrix that lies between muscle and other subepidermal cells (Rieger et al. 1991b, p. 21). It is even discontinuous under the epidermis, i.e., punctate, in some macrostomidans such as Bradynectes spp. or meshlike in Haplopharynx spp. and dolichomacrostomids. The remaining rhabditophorans, like the typhlopanoid, have a welldeveloped basement membrane with a typical bilayered structure: a basal lamina (lamina densa) underlying the epidermal cells and a reticular lamina (lamina fibroreticularis) extending basally and providing the anchor points for the body wall musculature. The reticular lamina is often contin-

uous with the extracellular matrix surrounding the muscles and parenchymal cells. As would be expected, the reticular lamina is thicker in the larger bodied flatworms such as the polyclads and triclads (Pedersen 1966; Ehlers 1985; Rieger et al. 1991b). Fibers in the reticular lamina are helicoidally packed in some rhabditophorans (species of the genera Gyratrix, Thysanozoon, and Kytorhynchella) and relatively amorphous in others (species of the genera Castrada, Gieysztoria, Dendrocoelum, and Dugesia). A lamina lucida, appearing as bubble-like, lucent pockets in the interface between the basement membrane and the epidermis, is prominent in dalyellioids, kalyptorhynchs, lecithoepitheliates, and triclads. The basement membrane of a few rhabditophorans has special thickenings that serve as skeletal support (the only instances of dermal skeleton in platyhelminths), e.g., spinelike elaborations in the proboscis epithelium of kalyptorhynchs (Rieger and Doe 1975; Noldt 1988) and calcareous spicules embedded in the basement membrane of some deep-water interstitial turbellarians (Rieger and Sterrer 1975). Ciliation also shows variability. While most rhabditophorans, as well as other turbellarians, are ciliated over the entire epidermis, many have ciliation restricted to specific regions of the body. For example, a ventral creepsole is the only ciliated epidermis in many triclads, the sand-dwelling otoplanids, and some temnocephalans such as Didymorchis spp. The rest of the epidermis usually has only microvilli as the surface projections, which are not necessarily much different from those over the ciliated parts. A ventral creepsole may be evident simply by having denser ciliation on the ventral than the dorsal epidermis, as in the terrestrial triclad Artioposthia triangulata (McGee et al. 1997). Symbiotic turbellarians typically have ciliation restricted in other ways to the anterior third of the body in the temnocephalan Varsoviella sp. (see Cannon and Joffe 2001) and the parasitic rhabdocoel Pterastericola astropectinis (see Jondelius 1989), or to a narrow ventral field in the ectosymbiotic Genostoma sp. (see Hyra 1993), for example. Most temnocephalans are not ciliated at all (Cannon and Joffe 2001), but many other symbiotic turbellarians have full ciliation no different from that of free-living relatives (Tyler and Tyler 1997). Ciliary rootlets in rhabditophorans fairly uniformly have the same structure as that described for the typhloplanoid; i.e., a tubular rostral and a solid-cored vertical rootlet on each ciliary basal body. In macrostomorph turbellarians, the vertical rootlet may be slanted more or less in the anterior direction (Tyler 1984). Proseriates have well-developed rostral rootlets that converge in a rostral projection of the epidermal cell bearing them (Bedini and Papi 1974; Ehlers 1985; Rohde and Watson 1995). Some species, however, have fewer rootlets; for instance, some parasitic turbellarians such as Kronborgia amphipodicola and Seritia stichopi have only a single rostral rootlet (Bresciani and Koie 1970; Rohde et al. 1988). Microvilli are a virtually constant feature of all epidermal cells in turbellarians but are absent in some regions of epidermis (which then appear smooth surfaced) in such parasites as Triloborhynchus sp. and S. stichopi (see Jondelius 1988; Rohde et al. 1988). Microvilli show some variation in shape, being usually conventionally cylindrical as in the typ 2004 NRC Canada

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hloplanoid (above) but tubercle-like in the Temnocephalida and lancet-like in the proseriate Dicoelandropora sp. (Ehlers 1977; see Tyler and Tyler 1997 for other variations). All show evidence of a core of actin filaments, as is typical for microvilli, and this is more or less well developed in some species, forming, for example, a distinctly dense core in Cleistogamia longicirrus (Rohde et al. 1988). Something that could be characterized as an apical extracellular matrix, i.e., a cuticle, occurs only rarely in the Platyhelminthes. A cuticle-like covering on nonciliated regions of the body of the dalyelliod parasite Hypoblepharina boehmigi appears to arise by accretion of glandular secretions (Karling and Nilsson 1974). A true cuticle would be expected to appear as a collagen- or chitin-reinforced matrix between the microvilli of the epidermal cells, and while a mucoid glycocalyx occupies this position in many platyhelminths (Rieger and Rieger 1976), no platyhelminth has a cuticle like that in other spiralians or nematodes. Something comparable to setae and, therefore, cuticle-like, is seen in the temnocephalid Notodactylus handschini: scale-like coverings on the epidermal cells are produced in a fashion that is indistinguishable from that by which setae in annelids and lophophorates are produced (Jennings et al. 1992; Rieger 1998); just as in setae, the scales have longitudinal channels into which microvilli of the generative cells protrude. In general, rather than being formed of cuticle, sclerotic hard parts in platyhelminths consist of either specialized thickenings of the basement membrane (as mentioned above) or of dense accumulations of intracellular actin (e.g., actin-reinforced, hook-forming microvilli in the proboscises of some turbellarians (Doe 1976)). The cell web shows different degrees of development in various rhaditophorans, presumably in correlation with functional requirements for cytoskeletal support. Particularly among the macrostomorphs, with their less-developed basal matrix, the terminal web can be quite thick and even multilayered, with helicoidal packing (e.g., Bradynectes sp.), or it can form a complete enclosing layer along all sides of the cell, basal and lateral as well as apical (e.g., Microstomum sp.). Terminal webs that are nearly as well developed appear in some proseriates and rhabdocoels. A basal sheet of cell web is best developed in proseriates (Bedini and Papi 1974; Ehlers 1985). Tall epidermal cells (e.g., the columnar cells of polyclads, or other cells bearing stress) often are seen with vertical stress-fiber bundles, which anchor to hemiadherens junctions in the basal plasma membrane. As in the typhloplanoid, the cell junctions that link to these cytoskeletal elements and join the epidermal cells together and to the basement membrane are probably universal among platyhelminths and, in fact, among invertebrate epithelia in general (see Tyler 2003). Apical edges of the epidermal cells are linked by zonulae adherentes and septate junctions, and the bases of the cells attach to the basement membrane by hemiadherens junctions. While the zonula adherens may be inconspicuous in the epidermis of some rhabditophorans such as polyclads, lecithoepitheliates, proseriates, and triclads, it is probably functionally present nonetheless; its inconspicuous nature can be attributed to the poor development of the terminal web in these species, but traces of this junction are visible anyway. Small spot adherens junctions may play the role associated with the

desmosomes of vertebrate epithelia (i.e., as small spot welds between cells), but neither desmosomes nor hemidesmosomes occur because no turbellarians (and probably no invertebrates; cf. Erber et al. 1999; Orii et al. 2002) have intermediate filaments in the cytoplasm of their epithelial cells. Instead of intermediate filaments, the hemiadherens and spot adherens junctions rely on actin bundles for cytoskeletal linkage. (Desmosome and hemidesmosome junctions previously identified in flatworms, as in Tyler (1984), Rieger et al. (1991b), and literature therein, are probably really spot adherens and hemiadherens junctions. Identification of even tight junctions, as well as desmosomes in S. stichopi, by Rohde et al. (1988) contradicts the generalization that these are not known in non-chordates (Tyler 2003) and needs further study.) Gap junctions have also not been unequivocally identified in the epidermis of platyhelminths. The shape of the epidermal cells, especially in smaller turbellarians, is cuboidal to squamous, with adjacent cells interdigitating as described for the typhloplanoid. Some groups with larger bodies, such as the Polycladida, have tall columnar cells that may even appear pseudostratified by virtue of rich development of glands crowding the cells. The epidermis of the parasitic rhabdocoel Anoplodium sp. also appears pseudostratified because of its gland-rich structure. In such turbellarians (e.g., Anoplodium sp. and the macrostomorphs Haplopharynx rostratus and Cylindromacrostomum sp.), the glands are entirely epidermal; i.e., their cell bodies, as well as the necks, lie within the epidermis (Karling 1965; Rieger et al. 1991b; Tyler and Tyler 1997). Most species have the gland bodies below the body wall, and only their necks reach through the epidermis to the surface. A more pronounced modification of epidermal cell shape occurs in insunk epithelia, where the nucleated portion of the cell lies below the body wall musculature, connected to the surface portion of the cell by a necklike bridge. Such cells appear in the epidermis of lecithoepitheliates and bdellourid triclads, for instance, and are restricted to the creepsole epithelium of proseriates and terrestrial triclads (Bresslau 19281933; Hyman 1951; Bedini and Papi 1974; Curtis et al. 1983; Tyler 1984; Ehlers 1985). A syncytial, rather than cellular, epidermis is developed in certain groups of rhabditophoran turbellarians, appearing as fields or as a mosaic of plates, sometimes among cellular regions. Such mosaics are especially characteristic of the Temnocephalida, where patterns of plate arrangements are specific to given taxa (Joffe and Cannon 1998; Cannon and Joffe 2001). In most temnocephalans, the plates have multiple intraepidermal nuclei, but in Didymorchis sp., the plates have insunk nuclei. Rhabdocoels such as the kalyptorhynchs also appear to have syncytia, typically in belt-shaped fields (Rieger et al. 1991b; De Vocht and Schockaert 1999 and literature therein). In lecithoepitheliates and prolecithophorans, a syncytial epidermis is suspected because of the abundance of infolding plasma membranes and the lack of clear cell junctions over extended portions of the epidermis (Rieger et al. 1991b), but the true nature of these epidermises needs further investigation. Most like the syncytial, insunk epidermis of the major groups of parasitic flatworms, the Neodermata (see below), is the epidermis of the ectocommensal turbellarian Genostoma sp. It has well-delimited nonciliated
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Can. J. Zool. Vol. 82, 2004 Fig. 4. Ultrastructure of the body wall of the acoel Convoluta pulchra. (A) Portions of two epidermal cells and underlying musculature in a near-longitudinal section of the animal showing interconnections between rootlets (rt) of adjacent cilia and the paucity of basal matrix between the epidermis and musculature (mu). (B) Junctions among muscle and epidermal cells; arrow marks a special junction between an epidermal cell process and a longitudinal muscle showing extracellular material and an intercellular gap that is larger between these two cells than that in the spot adherens (sa) junctions between muscle cells. c, circular muscle; e, epitheliosome; l, longitudinal muscle.

and ciliated regions connected by multiple necklike bridges that span the basement membrane to the insunk perikarya lying below the body wall musculature (Tyler and Tyler 1997). The polarized distribution of cell organelles varies among rhabditophorans. Mitochondria appear in basal positions in the prolecithophoran Acanthiella sp. and in temnocephalids, rather than being mostly restricted to an apical layer beneath the terminal web, as described for the typhloplanoid. Vacuoles or secretory inclusions such as epitheliosomes may appear throughout the height of the cell instead of being limited to the apical-most layer (e.g., in larvae of a polyclad) (Lacalli 1982). Color is often imparted to rhabditophorans by pigment cells in positions like those described for the typhloplanoid (i.e., pigment cell processes along the basement membrane), as, for example, in the triclads (Palladini et al. 1979). These are really parenchymal cells that send pigment granulebearing processes into the body wall along the underside of the basement membrane. In some cases, entirely parenchymal cells may impart color, as in Myozona sp. (Rieger et al. 1991b, p. 64). In others, color may arise from the epitheliosomes or in granules that are positioned throughout the height of the epidermis. The turbellarian epidermis is rich in glands, but little is known about the functions these glands serve. They are typically distinguished in accounts of ultrastructure simply by differences in the appearance of their secretion granules (identified by number, for instance, as type I, type II, etc.) and, in a few studies, in cytochemical staining properties (e.g., Skaer 1961; Pedersen 1963; Tyler 1988; McGee et al. 1997). The glands in most species lie below the body wall, sending their necks to the surface through the epidermis, although, as mentioned above, some species have intraepidermal glands. Gland necks pass between the epidermal cells in macrostomorphs but through the cells in tubular channels in the neoophoran platyhelminths (rhabdocoels, triclads, proseriates, and prolecithophorans). (While some claims have been made that rhabdites in triclads are delivered by the glands as packets that traverse the cell or individually into the cytoplasm of the epidermal cell itself (Pedersen 1963; McGee et al. 1996), neither mechanism appears likely.) Within the body wall are also elements of the nervous system, specifically three nerve plexi: a submuscular plexus, a subepithelial plexus, and an intraepidermal plexus (Rieger et al. 1991b). The submuscular plexus is part of, or continuous with, the main longitudinal cords of the central nervous system; the other two plexi appear as thin neural processes on either side of the basement membrane, between the basement membrane and the muscles, and among the bases of the epidermal cells. Because of their thin, tenuous nature, these processes are more easily visualized in special light microscopic preparations (Chien and Koopowitz 1977; Ladurner et al. 1997), including immunocytochemical preparations of whole mounts using antibodies to neurotransmitters, and are easily overlooked or confused with gland necks and other cell processes in electron micrographs. In the absence of other skeletal supports to set body shape, it must be the basement membrane, together with the arrangement of muscles including body wall muscles and muscles crossing the parenchyma between the body wall (es-

pecially dorsoventral fibers in flat-bodied species), that determines the shape. Acoelomorpha Features of the body wall most clearly link the two turbellarian orders Acoela and Nemertodermatida together into the clade Acoelomorpha (Tyler and Rieger 1977; Ehlers 1985). While epidermal cilia in rhabditophorans appear to be relatively independent of one another, even if tightly packed, those in acoelomorphs are interconnected in rows by junctures of their rootlets (Fig. 4A). Each of the rostrally pointing main rootlets of the epidermal cilia are connected by lateral rootlets or by lateral fiber bundles (see summaries by Rieger et al. 1991b; Lundin 1997). In the Acoela, the two lateral rootlets arise from the main rootlet and join the tips of main rootlets of adjacent cilia; in the Nemertodermatida and some acoels, the lateral fiber bundles link the tips of the
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main rootlets with posterior rootlets of adjacent cilia. Another similarity in cilia is the shape of the ciliary tips, which have a distinct shelf (as opposed to the simple, tapering tips seen in the Rhabditophora). Acoels and nemertodermatids are also noteworthy for the paucity of basal matrix under the epidermis (Fig. 4A). A typical basement membrane is absent, but small islands of some sort of extracellular material can be seen between the epidermal cells and underlying muscle. In nemertodermatids such as Flagellophora sp. and Sterreria sp., this material not only appears spotlike between epidermal cells and muscles but also extends between muscles as a kind of general intercellular matrix (Smith and Tyler 1985), something like that seen in macrostomorphs. In acoels, existence of such a matrix is evident in the enlarged and irregular-width gap in the junctions between epidermal and muscle cells (Fig. 4B; Tyler and Rieger 1999). These junctions resemble fasciae adherentes (so called by Tyler 1984 and Tyler and Rieger 1999) but are probably better called hemiadherens junctions given the probability that the gap material is extracellular matrix; the intermuscular junctions are spot adherens junctions rather than desmosomes (cf. Tyler 2003). The cell web is particularly well developed in nemertodermatidans, with a striking terminal web appearing as a dense, sometimes multilayered, fibrous sheet beneath the ciliary rootlets (in Sterreria sp., for example; Tyler and Rieger 1977). Prominent stress fibers (actin bundles), oriented vertically in the epidermis and linking the terminal web with junctions to muscles underlying the epidermis, appear in thicker epidermises of some Nemertoderma species (Tyler and Rieger 1977). The terminal web may be developed to compensate for the less-developed basement membrane. Presumably also as a consequence of the less-developed basement membrane, the epidermal cells of many acoels are insunk, i.e., the nucleated portion of the cell bulges into or even through the underlying muscle layer (Tyler 1984). The glycocalyx between microvilli may be dense and cuticle-like in some acoelomorphs, especially nemertodermatids, where it may even appear bilayered (Rieger and Rieger 1976; Tyler and Rieger 1977), but in most acoels it is rather tenuous, no more developed than that of the rhabditophorans. Layering of organelles in the epidermal cells in the polarized fashion described for the typhloplanoid is also characteristic of the acoelomorphs, but some acoels have mitochondria, for instance, relatively uniformly scattered throughout the cell body. Like other turbellarians, acoelomorphs have richly glandular epidermises. Especially prominent are glands with rhabdoid secretions that discharge onto the epidermal surface to produce a mucous coating (Smith et al. 1982). Other glands producing mucoid secretions have granules of more globular shape (Rieger et al. 1991b). A variety of forms of granules produced by other glands appear at special sites such as around the frontal pore and genital pores (Smith and Tyler 1985, 1986). In all acoelomorphs, glands and their necks lie between the epidermal cells rather than, as in rhabdocoels, in channels through the cells. Mucous glands are especially voluminous in the thick epidermis of species of Nemertoderma, causing the epidermal cells themselves to appear pseudostratified; i.e., epidermal cell nuclei are displaced to a variety of positions by the adjacent glands (Tyler and Rieger 1977).

An epidermal nerve net appears to be the only nerve net in certain members of the Acoelomorpha (e.g., Tetraposthia colymbetes), and this even incorporates the anterior ganglionic concentration (brain) and the longitudinal cords (An der Lan 1936). The epidermal net is reactive to serotonin (5hydroxytryptamine) immunoreactive tests in acoels (see Raikova et al. 1998, 2001 for literature; called a surface net in these studies). Such a condition illustrates the primitive nature of the Acoela (Raikova et al. 2001). Catenulida The epidermis of catenulidans is distinct from that of other platyhelminths in the nature of its ciliation and the form of the microvilli (Rieger 1981). The cilia are sparser and more widely scattered (less than half the density of cilia in other platyhelminths), and their rootlets are directly opposing rostral and caudal, virtually horizontal, as opposed to rostral and vertical. The rostral rootlets may converge in a rostral projection of the cell bearing them (Ehlers 1985). Cilia of the marine species (family Retronectidae) each sit recessed slightly in a small, microvilli-ringed pit, which is not seen in other platyhelminths. The epidermal microvilli in catenulidans are distinctive in having a central dense core (Rieger 1981). The glycocalyx between microvilli may be especially dense and cuticle-like in some catenulidans. Like the acoelomorphs and some macrostomorph rhabditophorans, some catenulidans have a discontinuous basal matrix that appears as small islands between the epidermal cells and underlying muscle (Doe 1981; Rieger 1981; Ott et al. 1982; Tyler 1984). A more continuous subepidermal matrix resembling a lamina densa is seen in Stenostomum sp., while Xenostenostomum sp. has a pronounced lamina (Pedersen 1983; Ehlers 1985). The glycocalyx may also be well developed in retronectid catenulidans (Rieger and Rieger 1976).

The epidermis of neodermatan rhabditophorans

Ultrastructure All members of the Neodermata have a special kind of syncytial epidermis as adults: a nonciliated epidermis with its nuclei in so-called cytons or perikarya that lie below the body wall musculature, connected to the surface layer by multiple branching, necklike bridges (Fig. 1B). The surface layer itself is a continuous sheet that entirely covers the body, uninterrupted by any kind of cell junction except at point-like penetrations of sensory or gland cell processes. Ehlers (1985) coined the term neodermis for this epidermis because it arises new at metamorphosis of the larva. As a larva infects the first host in the life cycle, it casts off its epidermis and replaces it with a new epidermis. This new epidermis arises from cells lying below the epidermal basement membrane, which send processes over the basement membrane that spread and fuse together to form the definitive surface syncytial sheet. These Anlage cells remain as the perikarya of the syncytial epidermis, the neodermis. It is logical to assume that this special kind of epidermis, which forms the interface between these parasitic worms and their hosts, is the key to their evolutionary success. The neodermatans make up three quarters of all known
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platyhelminth species, and it appears that something about this epidermis that they all share is highly adaptive for a parasitic lifestyle. The success may be attributed to the way in which such an interface allows these parasites to resist the defenses of hosts they infest, and it may rest certainly at least for the gutless tapeworms on the ability of this epidermis to absorb nutrients from the host. Although having a syncytial, insunk epidermis of this special kind is the rule in the Neodermata, differences in the nature of the outermost surface whether it bears microvilli of distinctive shape, for instance, or microridges, or pits distinguish various neodermatan taxa (Fig. 1B). The epidermis also varies in thickness and in the form of its microvilli at different developmental stages, and there are some differences among regions of the body surface even within a single organism. This variety has been reviewed by Threadgold (1984), Fried and Haseeb (1991), Coil (1991), and Xylander (1996, 2001), but we give a short summary here, largely drawn from those sources. Distinctive of the epidermis in the aspidobothrean Trematoda are microvilli in the shape of minute, rounded tubercles, both in the adult and in the synctial parts of the epidermis in the cotylicidium larva. (The neodermis is already well developed at hatching of the cotylicidium.) A welldeveloped glycocalyx covers the microvilli, which are underlaid by a fairly well-developed terminal web. Some cotylocidia have more filiform microvilli with cores of filaments (Rohde 1972). Ciliated cotylocidia (e.g., of Cotylogaster occidentalis; see Fredericksen 1978) have limited ciliated regions in an otherwise nonciliated, syncytial epidermis, while others lack the ciliated cells, having only the syncytial epidermis with tuberculate microvilli (Fried and Haseeb 1991, p. 145; Xylander 1996, p. 234). The fully developed epidermis of digenean trematodes (i.e., the neodermis of the adult and cercarial stages) has short, irregularly shaped microvilli, often a well-developed glycocalyx, and spines formed of intracellular crystalloids of actin anchored to the basal membrane and projecting the apical membrane as thornlike extensions. However, instead of bearing microvilli, the surface may be relatively smooth, with minute folds and canals developed to different degrees in given regions of the body in species-specific patterns. In the sporocyst and redia larval stages, the epidermis may bear small, irregular, microvilli-like folds that may be branched. A peculiar variation on epidermal structure in Digenea is seen in certain sporocysts without a birth pore. In some (e.g., Cercaria bucephalopsis), the epidermis is layered into a thicker outer, smooth-surfaced, multinucleated part separated from a thin, inner, anucleated part by a space into which the anucleate layer projects irregular microvilli; the two layers may or may not be connected by cytoplasmic strands (Threadgold 1984). In some other cases (e.g., Microphallus spp.), a similar outer nucleated layer eventually becomes subdivided into squamous-like cells by infolding of the plasma membrane, while the inner layer becomes attached to perikarya lying below the musculature. The microvillibearing layer becomes the outer surface at selected regions in sporocysts such as those of Cercaria littorinae (Popiel 1978) and looks much like the typical digenean epidermis, so it is logical to interpret only this layer as the definitive epidermis of this stage. The outer multinucleated layer or

squamous-epithelial layer can be interpreted as a layer supplied by the sporocyst in which this larval stage develops. The epidermis in adult monogeneans has irregular microvilli or folds, sometimes limited to specific regions of the body. Some have rounder tubercles in circumscribed cushion-like areas (Ramasamy et al. 1987; Fried and Haseeb 1991; Xylander 1996). Most distinctive is the epidermis of the Cestoda, where the microvilli are of a slender regular shape with a cylindrical hollow core; i.e., a dense-walled support, presumably actin-based, surrounding a lightly staining central cytoplasm (Ehlers 1985; Xylander 2001). The supporting wall material converges at the tip to make a dense cap. Such microvilli are found in all but the posterior body region of Gyrocotylidea and most body regions of those stages of the Cestoidea living in the intestine of their host. Although it is often assumed that such microvilli provide expanded surface for nutrient absorption (by comparison with the role of the microvilli in the host intestine), their function instead appears to be to maintain a high-pH boundary layer at the epidermal surface, thus preventing enzymes of the host intestine from digesting it (Uglem and Just 1983; Xylander 2001). The dense cap is especially developed in certain body regions of the Cestoidea as a sharp-pointed, elongate projection sitting at an angle to the axis of the base; such microvilli are called microtrichs. At the juncture of the sharp-pointed cap with the base is a trilaminate plate that links microfilaments in the core with the cap. In the amphilinidean cestodes, which live in the coelomic cavity rather than the intestine of their host, microvilli over the general body surface are shaped like small rounded tubercles without the dense points seen in other cestodes (Xylander 2001) and reminiscent of those of aspidogastrean trematodes. The epidermis at the anterior end of amphilinideans has long, slender microvilli more like those of other tapeworms. The calcareous corpuscles of Amphilinidea and Cestoidea and the spines of Gyrocotylidea are also distinctive of the epidermis of cestodes; both are multilayered secretions that seem to lie in extracellular pockets within the epidermis, and they may be excretory products (Xylander 2001). Development of the neodermis Each successive stage in the life cycle of neodermatans forms its epidermis by protrusion of cell processes from below the musculature. These processes fuse above the basement membrane into the continuous sheet that covers the body and remain connected to the deeper lying perikarya that gave rise to them, forming a syncytium. The first and most obvious of these productions of new epidermis takes place as a newly hatched larva invades its first host; however, transitions to other larval stages, where those occur (in the Digenea and Cestoda), also involve development of a new epidermis from underlying cells. In transition through digenean larval stages (e.g., from sporocyst to daughter sporocysts or redia, or from redia to cercaria), the germinal cells that form the next stage are surrounded by a so-called primitive epithelium that is replaced by a new syncytial insunk epidermis as that stage develops. The epidermis of free-swimming larvae of monogeneans and trematodes is reminiscent of the turbellarian epidermis, being a cellular epidermis one cell thick, completely ciliated
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203 Fig. 5. Projections of confocal images of the dorsal (left) and ventral (right) body wall of a specimen of the acoel Haplogonaria phyllospadicis stained with Alexa-488-phalloidin to show musculature. Besides the longitudinal and circular muscle fibers, U-shaped and longitudinal crossover muscles loop posterior to the mouth (central pore) in the ventral body wall, and a second set of longitudinal crossover fibers occupies a corresponding position in the dorsal body wall. Diagonal muscles are also visible in the dorsal body wall in the anterior half of the body. The strong muscles of the male copulatory organ appear as a bright sphere, and those of the female pore form a bright ring just anterior to it. Peg-like projections along margins are sensory receptors with actin-reinforced microvilli. (From Hooge and Tyler 2003, reproduced with permission from Zootaxa, Vol. 131, 2003 Magnolia Press.)

in many monogenean oncomiracidia and digenean miracidia, for example; only partially ciliated or aciliate in other oncomiracidia; or restricted to small, discrete, tuft-like, ciliated patches in aspidogastrean cotylocidia (Threadgold 1984). Those cestode larvae with cilia have a syncytial epidermis bearing the cilia (Xylander 1987, 2001). Unlike the turbellarian epidermis, however, the ciliated cells have interpolated between, around, or under them ridges of unciliated processes reaching into the epidermis from perikarya lying below the musculature; this syncytial tissue comprises the presumptive adult epidermis that eventually completely replaces the ciliated cells. The cilia differ from those of most turbellarians except, perhaps significantly, those of some parasitic turbellarians like Kronborgia sp. and Seritia sp., as mentioned above in having only a single rostral rootlet and no vertical rootlet. Small irregular microvilli and mitochondria in an apical layer are features that could be expected of some turbellarian epidermal cells; the cell web, and thus the zonulae adherentes as well, may be inconspicuous, such that the only prominent cell junctions are septate junctions. The development of the neodermis of the digenean trematodes, as first described by Southgate (1970), starts with expansion of projections from a syncytium that lies below the epidermis of the miracidium larva. The projections sit ridgelike between the ciliated epidermal cells of the larva, and as the ciliated cells disintegrate at metamorphosis to the sporocyst stage, the projections expand under them to cover the newly exposed basement membrane. The process appears to be similar in aspidogastrean trematodes (Rohde 1972) whose larvae have ciliated cells overlying the syncytium of the incipient neodermis. The oncomiracidium larvae of the Monogenea have more extensive syncytial bands within the epidermis between the ciliated cells. As Lyons (1973) describes them, these bands have intraepidermal nuclei that are shed as processes from parenchymal cells emerge through the basement membrane to fuse with them and form the presumptive adult epidermis beneath the ciliated cells. In cestodes with aquatic larvae, including gyrocotylideans, amphilinideans, and those cestoidean cestodes having aquatic life cycles, the larva (lycophore or coracidium) sheds its ciliated, syncytial epidermis as it enters its first host (Xylander 1987, 2001). The neodermis expands under this shed layer from processes that reach apically from under the musculature and basement membrane to fuse in a confluent sheet (Lumsden et al. 1974; Xylander 1987). The oncosphere larvae of the cestodes with a terrestrial life cycle appear quite complicated in that they have multiple layers, including protective coatings from both maternal and the embryos own tissues, as well as epidermis-like layers. The living layer first covering the embryo is a syncytium arising from subsurface cellular projections of macromeres, and it delaminates into three layers, only the innermost of which is retained as the larvas epidermis (see Threadgold 1984; Coil 1991).

Musculature
The body wall musculature of platyhelminths is arranged in patterns that are specific to given taxonomic groups. Using techniques that allow visualization of the complete

musculature in whole mounts, namely fluorescent markers that bind specifically to the actin of the muscles (Figs. 3 and 5), it can be seen that only a few groups of flatworms have a simple network of circular and longitudinal muscles, a pattern once thought to apply generally to worm-shaped animals. Most species of the Catenulida and some species of the Acoela have such a simple network, but most platyhelminths also have diagonally oriented muscles in at least portions of the body wall (von Graff 19041908; Luther 1943, 1955; Rieger et al. 1994; Hooge 2001). Circular muscles constitute the outermost layer, the one that sits directly under the epi 2004 NRC Canada

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dermis, and longitudinal muscles lie to the inside of these; the only exceptions to this generalization are members of the acoel family Childiidae, in which this layering is reversed, with longitudinal muscles outermost (Westblad 1942; Hooge 2001). In most turbellarian members of the Rhabditophora, diagonal muscles lie in a layer roughly between the circular and longitudinal fiber layers and comprise a paired set of fibers that cross over each other along the ventral and dorsal midlines (Rieger et al. 1991a; Rieger et al. 1994; Hooge and Tyler 1999; Hooge 2001). By contrast, in the few specimens of the neodermatan Rhabditophora that have been examined, the diagonal muscles lie innermost, to the inside of both longitudinal and circular muscles (Mair et al. 1998, 2003). Kotikova et al. (2002) report the same innermost orientation for the turbellarian dalyellioid rhabditophoran Castrella truncata. Layering of the longitudinal and diagonal fibers may not be so distinct, in any case, in that fibers of these two orientations seem to lie in nearly the same plane in electron micrographs of the body wall (Fig. 2C). Underscoring the relatedness of these fibers, Orii et al. (2002) found that diagonal and longitudinal muscles in the triclad Dugesia japonica have the same type of myosin, which is different from that of the circular fibers. This corroborates Westblads (1949) proposal that diagonal fibers may be derived from longitudinal ones. The thickness and spacing of diagonal fibers varies among taxa more than that of the other layers. Large rhabditophorans that use muscles for locomotion, such as triclads and polyclads, have body wall muscle fibers that are more numerous and tightly spaced than smaller species that move by ciliary gliding. Many, too, have additional layers of muscles beyond the three conventional layers. The planarian Girardia tigrina, for example, has an additional layer of longitudinal muscles so that the diagonal fibers are sandwiched between two longitudinal layers, the inner one of which appears stronger (Cebri et al. 1997); however, the planarian D. japonica, also large bodied, has no such additional muscle layers (Orii et al. 2002). Large polyclads have as many as five or six layers in total, the six-layered forms having the layers ordered as follows (from outermost to innermost): circular, longitudinal, diagonal, circular, diagonal, and longitudinal (Prudhoe 1985). Quite distinct from these relatively conservative patterns of circular, longitudinal, and diagonal muscle fibers in Rhabditophora are those in the body wall musculature of the Acoelomorpha, which show eight distinguishable arrangements (Hooge 2001). The majority of acoelomorphs studied have circular and longitudinal muscles, as well as U-shaped muscles that extend longitudinally from the anterior tip of the animal before wrapping around the posterior rim of the mouth. Many species of acoels also have so-called longitudinal crossover fibers that are longitudinally oriented in the anterior portion of the body but then bend medially to cross over each other diagonally along the dorsal and ventral sides (Fig. 5). Other differences arise in the distribution of certain sets of longitudinal and diagonal fibers. In addition to the typical body wall muscles, the acoel Anaperus tvaerminnensis has intraepidermal longitudinal muscles, the cell bodies of which are positioned among the body wall muscles (Ehlers 1994). The pattern of body wall musculature in flatworms excluding the Acoela is similar to that found in other softbodied metazoans such as annelids (Hooge 2001), and the

body shapes that can be achieved by the contraction of circular, longitudinal, and diagonal musculature are well documented (e.g., Ruppert and Barnes 1994). It is possible that the more complicated musculature patterns of the Acoela relate to specific feeding motions whereby food is stuffed into a simple mouth (without pharynx and pharyngeal musculature) by bending of the body margins (Tyler and Rieger 1999). Food that is positioned in the ventral food groove at the anterior end of the body of Convoluta pulchra, for example, may be forced into the midventral mouth by pulling the mouth and the posterior end of the body forward by contraction of the longitudinal crossover muscles. Body movements such as the turning movements of C. pulchra are likely perfomed by the well-developed muscles crossing the parenchyma as well as by longitudinal crossover muscles. Body wall muscles of platyhelminths are, for the most part, of a smooth type typical of invertebrates, but some pseudostriated fibers occur in structures that contract rapidly (Rieger et al. 1991b). The muscles are entirely mesenchymal and subepithelial; i.e., none is an epitheliomuscular cell, as was assumed before the advent of electron microscopy. Muscles anchor to the basement membrane of the epidermis by hemiadherens junctions, specifically at junctures with the dense bars of the contractile elements (Fig. 2C). They are also surrounded, at least partially, by the extracellular matrix (ECM) that extends from this basement membrane. As mentioned above, catenulidans and nemertodermatidans have a thin, discontinuous, subepithelial ECM, and it may serve only to anchor muscles to the epidermis. Similarly, the small islands of material at junctions between muscles and epidermal cells in the Acoela may be reduced ECM, essentially remnants of the basement membrane (Tyler and Rieger 1999).

Development of the body wall

Embryonic development Macrostomorph and polyclad turbellarians develop through quartet spiral cleavage in which cell lineages can be fairly readily determined, and here the epidermis arises from micromere quartets 13. At gastrulation in Imogene mcgrathi, for instance, descendants of these cells form the ectoderm from which the epidermis and brain arise (Younossi-Hartenstein and Hartenstein 2000). As would be expected for spiralian development, progeny of the 4d cell, along with micromere 2b, form an inner cell mass at gastrulation from which body wall muscles arise (YounossiHartenstein and Hartenstein 2000); pharyngeal musculature, gland cells, and the excretory system also arise from this mass, and 4d descendants also give rise to endoderm (Boyer et al. 1996). In the polyclad Hoploplana inquilina, the endomesoderm-producing 4d micromere gives rise to the inner longitudinal muscles, while the ectoderm-producing 2b micromere gives rise to the outer circular muscles (Reiter et al. 1996; Boyer et al. 1998). In the more modified spiral quartet cleavage in the Lecithoepitheliata (Reisinger et al. 1974), some of the micromere quartets form a Hll membrane at gastrulation, which constitutes an extra-embryonic ectoderm. The embryonic ectoderm arises from remaining micromeres, which spread under the Hll membrane. Similarly, in Macrostomum hystricinum, a Hll membrane or
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yolk mantle forms an extra-embryonic ectoderm, or primary epidermis, from large, yolky blastomeres, and the later developing definitive epidermis pushes this aside (Tyler 1981; Tyler and Tyler 1997). A unique duet spiral cleavage pattern is characteristic of the Acoela, and here the mesodermal body wall musculature is derived from the endoderm-producing third-duet macromeres (Henry et al. 2000; Rieger and Ladurner 2003). Muscle development progresses similarly in C. pulchra (Ladurner and Rieger 2000) and Macrostomum hystricinum marinum (Reiter et al. 1996), but somewhat differently from the polyclad H. inquilina (Reiter et al. 1996), which passes through a Mllers larva before achieving the adult form. In C. pulchra, the first elements of body wall musculature appear after ~20 h (50% of developmental time), when short muscle fibers appear at evenly spaced latitudes of the embryo and expand to form circular fibers. At ~23 h, the first longitudinal fibers appear. By ~35 h, a fairly complete orthogonal grid is established, and the final pattern of musculature is completed with the formation of U-shaped and crossover muscles. Other neoophoran turbellarians besides the lecithoepitheliates have eggs with small oocytes and numerous yolk cells and follow highly modified, irregular cleavage without going through what could be identified as gastrulation (Hartenstein and Ehlers 2000); triclads, in fact, have what is called blastomere anarchy. Tissues such as the epidermis appear to differentiate in situ out of a mass of blastomeres in neoophoran embryos (Hartenstein and Ehlers 2000; YounossiHartenstein and Hartenstein 2001). Many of the turbellarian embryos that have been studied seem to produce successive generations of epidermis as development proceeds. For instance, polyclad turbellarians form a Hll membrane over the embryo, which is later replaced by an epidermis that differentiates under it from a mass of micromeres in the center of the embryo (YounossiHartenstein and Hartenstein 2000). Triclad turbellarians have three distinct generations of epidermis (Skaer 1965; Benazzi and Gremigni 1982; Bagu and Boyer 1990). The primary epidermis in the embryo is a syncytium (Sakurai and Ishii 1995) that encloses a yolk mass; it is replaced by the secondary epidermis, which originates from outwardly migrating cells that interpolate themselves into the primary epidermis. The tertiary epidermis, which is the definitive epidermis of the hatched young worm, arises from similarly migrating cells; like the secondary epidermal cells, these are ciliated but are readily distinguished by their bearing the rhabdoids characteristic of the adult epidermis. Proseriate turbellarians also produce three generations of epidermis (Giesa 1966; Reisinger et al. 1974; Tyler and Tyler 1997). The definitive or tertiary epidermis of the hatchling arises from cells in the parenchyma that send processes to the surface to produce a ciliated surface layer connected to insunk cell bodies (i.e., it is a cellular epidermis with insunk nuclei). Embryos of rhabdocoel turbellarians may not show such obvious generations of epithelia. The epidermis arises (along with other organs) out of blastomeres forming a solid mesenchyme-like mass (Hartenstein and Ehlers 2000 and literature therein). Although it does develop under a covering layer, or Hll membrane, this layer is derived from non-

embryonic cells, namely yolk cells from the parent, rather than from early blastomeres as in macrostomids or proseriates. It is not clear that it grows by any mechanism other than simple spreading over the embryo from the mesenchyme-like mass. Implications for origin of the neodermis In all of the major taxa of the Neodermata, there are at least some representatives that have larvae with a ciliated epidermis that is replaced by neodermis at metamorphosis. This larval epidermis is cellular in representatives of the Trematoda and Monogenea but syncytial in the Cestoda; in either case, comparison can be drawn with the epidermis in turbellarians. It should not be assumed, however, that only the larval epidermis is homologous to that of turbellarians. It is not that, in evolving from a ciliated ancestor, the Neodermata have invented a new epidermis or that its larvae recapitulate an ancestral epidermis; rather, the developmental mechanism by which the epidermis is replaced is common to all platyhelminths, and the Neodermata have specifically adapted this mechanism to a parasitic lifestyle. By replacing their epidermis as they attack hosts in the life cycle, the neodermatans presumably gain some advantage, immunological or physiological, in dealing with host defenses. Lyons (1973) first drew parallels between this epidermal development at metamorphosis in parasitic flatworms and epidermal development in triclad turbellarian embryos, as well as epidermal replacement in turbellarians in general. Tyler and Tyler (1997) expanded on this theme with illustration of epidermal development in other turbellarians and provided evidence that developing embryos in other turbellarian groups, namely the Acoela, Macrostomida, and Polycladida, have cells immigrating into epidermal positions from the parenchyma. In any case, it appears that no epidermis in these embryos, embryonic or definitive (see also below), grows by division of epidermal cells. Such a mechanism of epidermal growth and replacement may have preadapted the platyhelminths to parasitism. If host invasion depends on presentation of a new epidermis to host defenses, the kind of epidermal shedding and replacement that characterizes free-living platyhelminths may provide a key element to this invasion. The ability of free-living platyhelminths to form syncytial and insunk epidermises may also be key to the parasites presentation of a continuous, unbroken surface to host tissues.

Growth of epidermis and muscle

Differentiated cells in platyhelminths do not divide, so the epidermis and musculature must be maintained and grow through immigration of replacement cells from the underlying parenchyma (Luther 1904; Hein 1928; Skaer 1965; Ehlers 1985; Smith et al. 1986; Peter et al. 2001). The stem cells that give rise to these replacement cells are of a kind unique to the Platyhelminthes: neoblasts (Bagu 1981; Ehlers 1985; Ladurner et al. 2000). Neoblasts are relatively small cells with prominent nuclei and scant cytoplasm containing only free ribosomes and a few mitochondria (Palmberg 1990; Hori 1997; Gschwentner et al. 2001). They are the only cells capable of dividing in adult flatworms (Bagu et al. 1989; Ladurner et al. 2000; Newmark and
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Snchez Alvarado 2002) and so must be responsible for the renewal of all cell types during growth, regeneration, and normal maintenance (Bagu 1981; Ehlers 1985; Palmberg 1990; Bagu et al. 1994; Hori 1989; Rieger et al. 1999; Gschwentner et al. 2001). Differentiating from neoblasts, epidermal replacement cells are first recognizable by their production of a cluster of centrioles that are destined to be the basal bodies of the epidermal cilia. This differentiation may be evident even before the cells migrate into the epidermis, where further differentiation of the cilia and components such as the epitheliosomes proceeds. Immigrating centriole-bearing cells like these have been found in catenulidans, macrostomorphs, proseriates, and triclads (Koie and Bresciani 1973; Moraczewski 1977; Hori 1978; Doe 1981; Pedersen 1983; Ehlers 1985; Palmberg 1990). Epidermal replacement cells of macrostomids may migrate into the epidermis even before they differentiate centrioles (Rieger et al. 1999). Stem cells still capable of mitosis also appear within the epidermis and in basiepithelial positions in certain species among the Acoela and Catenulida. Clusters of basiepidermal stem cells, some of which can be seen in mitosis, appear in the freshwater catenulidans Rhynchoscolex sp., Catenula sp., and Stenostomum sp. (Reisinger 1924; Borkott 1970; Ehlers 1992b). Mitotic and S-phase stem cells similarly appear in the epidermis of the acoel Convolutriloba longifissura (Haszprunar 1996; R. Gschwentner, personal communication). Whether these stem cells are a separate population from the bulk of neoblasts that lie beneath the body wall or have migrated from that population into the epidermis while still uncommitted to the differentiated state has not been determined. Stem cells can be identified by immunocytochemical reaction to 5-bromo-2-deoxyuridine (BrdU), which, when supplied to animals in culture, is incorporated into the nuclei of cells in S-phase, i.e., those synthesizing DNA. Such stem cells are distributed over the whole body in the acoel C. longifissura, for instance (Gschwentner et al. 2001, 2003), but are localized along the two main longitudinal nerves along the lateral sides of the body in the rhabditophoran Macrostomum sp. (Ladurner et al. 2000). Differentiated cells, presumably including epidermal cells, arise from these labeled cells within 3 days in the acoel (Gschwentner et al. 2001) and within 14 days in the macrostomid (Ladurner et al. 2000). Palmberg (1990) used tritiated thymidine to identify neoblasts and their progeny in the macrostomorph Microstomum lineare and the catenulidan Stenostomum leucops and found labeled and differentiated epithelial, excretory, nerve, and sensory cells. Also using tritiated thymidine, Drobysheva (1988) found labeled epidermal cells in polyclads. So-called pulsatile bodies in the epidermis and parenchyma of acoels and nemertodermatids have been interpreted by some early workers as epidermal replacement cells (i.e., cells moving into the epidermis), but electron microscopy reveals them to be degenerating epidermal cells in the process of being recycled (see literature in Lundin 2001). Though epidermal replacement cells have not been identified by histology in acoels, Gschwentner et al. (2001) have confirmed their presence using immunocytochemistry for S-phase cells (through BrdU incorporation) in C. longifissura. Also, the

acoel C. pulchra has been shown, through experiments with continuous labeling with BrdU and immunogold cytochemistry, to have epidermal cells replaced by S-phase neoblasts (Thaler et al. 2002). Conventional electron microscopy of embryos of C. pulchra indicates that neoblasts migrate into the epidermis as it grows in early developmental stages (Tyler and Tyler 1997). Epidermal regeneration in the Neodermata may be simplified by the syncytial nature of the epidermis. Here, any wound can be repaired by simple spreading of the remaining syncytial surface layer of the epidermis over the damaged area, since this remains connected to the many perikarya in the parent piece. Popiel et al. (1985) have documented such spreading during wound healing in the trematode Schistosoma mansoni; curiously, the newly spread epidermis does not regenerate a new basement membrane in the area it covers. Growth and regeneration of muscles is similarly dependent on neoblasts. Myoblasts first appear as small rounded cells, eventually produce muscle-specific myosin, and then gradually elongate and differentiate myofilaments (Cebri et al. 1997). In both a macrostomorph (Macrostomum sp.) and a triclad (Schmidtea mediterranea), experimental wounding causes first rearrangement and outgrowth of existing longitudinal muscles in the body wall to compensate for extirpated muscles, then differentiation of new circular muscles from neoblasts (Cebri and Romero 2001; Salvenmoser et al. 2001).

Conclusions
Despite its lack of defining sclerotic structures such as cuticle and dermal skeleton, the body wall of platyhelminths is structurally complex and shows considerable variety in the arrangement of epidermal and muscle cells and in components such as its basal matrix, cell web, ciliary rootlets, and secretory inclusions and its relationship to gland, sensory, and nerve cells. The variety is likely indicative of the basal nature of these worms. Overlap among body wall characters, such as the netlike or spotty distribution of basal matrix in lower rhabditophorans such as the macrostomorphs and in the Acoela, Nemertodermatida, and Catenulida, provide support for the monophyly of the platyhelminths despite challenges to this concept of monophyly from phylogenetic analysis of some molecular sequences. Most critical for monophyly is the common mechanism all platyhelminths seem to share for growth and maintenance of the body wall. As Rieger and his collaborators have proposed (Gschwentner et al. 2001; Rieger and Ladurner 2001), only members of this phylum produce, grow, and maintain the epidermis through neoblasts. All animals other than platyhelminths are presumed to have stem cells within the epidermis to account for its growth and maintenance so-called interstitial cells of cnidarians, for example, or basal cells in other bilaterians or to renew through dedifferentiation and redifferentiation of other cells (Snchez Alvarado 2000; Galliot and Schmid 2002). The basiepidermal stem cells identified in certain acoels and catenulidans may correspond to interstitial and basal cells, but the bulk of the neoblasts in even these species lie within the parenchyma, below the body wall (Gschwentner et al. 2001; R.
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Tyler and Hooge

207 Vol. II, Part I. Edited by W. Kkenthal and T. Krumbach. Walter de Gruyter, Berlin. pp. 52304. Cannon, L.R.G., and Joffe, B.I. 2001. The Temnocephalida. In Interrelationships of the Platyhelminthes. Edited by D.T.J. Littlewood and R.A. Bray. Taylor and Francis, London. pp. 83 91. Cebri, F., and Romero, R. 2001. Body-wall muscle restoration dynamics are different in dorsal and ventral blastemas during planarian anterior regeneration. Belg. J. Zool. 131(Suppl. 1): 111115. Cebri, F., Vispo, M., Newmark, P., Bueno, D., and Romero, R. 1997. Myocyte differentiation and body wall muscle regeneration in the planarian Girardia tigrina. Dev. Genes Evol. 207: 306316. Chien, P.K., and Koopowitz, H. 1977. Ultrastructure of nerve plexus in flatworms. III. The infra-epithelial nervous system. Cell Tissue Res. 176: 335347. Coil, W.H. 1991. Platyhelminthes: Cestoidea. In Microscopic anatomy of the invertebrates. Vol. 3. Platyhelminthes and Nemertinea. Edited by F.W. Harrison and B.J. Bogitsh. Wiley-Liss Inc., New York. pp. 211283. Curtis, S.K., Cowden, R.R., Moore, J.D., and Robertson, J.L. 1983. Histochemical and ultrastructural features of the epidermis of the land planarian Bipalium adventitium. J. Morphol. 175: 171 194. De Vocht, A.J.-P., and Schockaert, E. 1999. The anatomy and ultrastructure of the proboscis in Zonorhynchus-species and implications for phylogenetic relationships within the Eukalyptorhynchia Meixner, 1928 (Platyhelminthes, Rhabdocoela). Belg. J. Zool. 129: 219234. Doe, D.A. 1976. The proboscis hooks in Karkinorhynchidae and Gnathorhynchidae (Turbellaria, Kalyptorhynchia) as basement membrane or intracellular specializations. Zool. Scr. 5: 105 115. Doe, D.A. 1981. Comparative ultrastructure of the pharynx simplex in Turbellaria. Zoomorphology (Berl.), 97: 133193. Drobysheva, I.M. 1988. An autoradiographic study of the replacement of epidermis in polyclad turbellarians. Fortschr. Zool. 36: 97102. Ehlers, B. 1977. Trematoden-artige Epidermisstrukturen bei einem freilebenden proseriaten Struedelwurm. Acta Zool. Fenn. 154: 129136. Ehlers, U. 1985. Das Phylogenetische System der Plathelminthes. Gustav Fischer, Stuttgart, Germany. Ehlers, U. 1992a. On the fine structure of Paratomella rubra Rieger & Ott (Acoela) and the position of the taxon Paratomella Drjes in a phylogenetic system of the Acoelomorpha (Plathelminthes). Microfauna Mar. 7: 265293. Ehlers, U. 1992b. No mitosis of differentiated epidermal cells in the Plathelminthes: mitosis of intraepidermal stem cells in Rhynchoscolex simplex Leidy, 1851 (Catenulida). Microfauna Mar. 7: 311321. Ehlers, U. 1994. Ultrastructure of the unusual body wall musculature of Anaperus tvaerminnensis (Plathelminthes, Acoela). Microfauna Mar. 9: 291300. Erber, A., Riemer, D., Hofemeister, H., Bovenschulte, M.N., Stick, R., Panopoulou, G. et al. 1999. Characterization of the Hydra lamin and its gene: a molecular phylogeny of metazoan lamins. J. Mol. Evol. 49: 260271. Fredericksen, D.W. 1978. The fine structure and phylogenetic position of the cotylocidium larva of Cotylogaster occidentalis Nickerson 1902 (Trematoda: Aspidogastridae). J. Parasitol. 64: 961976. Fried, B., and Haseeb, M.A. 1991. Platyhelminthes: Aspidogastrea,
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Gschwentner, personal communication), and it is likely, though by no means certain, that this population supplies those epidermal neoblasts. While we are far from a comprehensive view of the neoblast in all platyhelminth taxa or from knowing whether no other animal phyla have such cells, current indications are that the stem cell system of neoblasts in platyhelminths is unique to these worms. If so, it is the first identified autapomorphic trait of the Platyhelminthes and thus the first to validate the monophyly of the phylum (cf. Smith et al. 1986). Its occurrence may explain other morphological similarities among flatworms.

Acknowledgments
We thank Reinhard Rieger and Robert Gschwentner for valuable suggestions and discussion of their work on stem cells. This material is based upon work supported by the National Science Foundation under Grant Nos. 0118804 and DBI-9977643.

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