Comparative Morphology of the Body Wall in Flatworms (Platyhelminthes)1

194

REVIEW / SYNTHÈSE

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. Résumé : Le corps mou des plathelminthes s’explique en grande partie par la structure de leur paroi corporelle et l’absence d’éléments sclérotiques, tels que la cuticule. Les groupes libres, c’est-à-dire la majorité des turbellariés, sont très variés, mais la structure de base de leur paroi consiste en un simple épithélium cilié superposé à un réseau de muscles. Nous illustrons cette structure de la paroi chez un rhabditophore typhloplanoïde typique, et nous commentons les variations observées chez des représentants des acoeles, des caténulides et d’autres rhabditophores libres. Le groupe principal de platheminthes parasites, les rhapditophores néodermates, ont une séquence de développement au stade lar- vaire pendant laquelle un épiderme cilié de même type que celui des turbellariés libres est remplacé par le néoderme, un épiderme spécialisé qui assure, croit-on, leur succès comme parasites. Ce néoderme forme un syncytium qui re- couvre le corps en une couche continue qui est rattachée à des péricaryons situés sous la musculature de la paroi. Le néoderme peut être considéré comme une adaptation particulière d’un mécanisme de développement commun à tous les plathelminthes dans lequel la croissance et le renouvellement de l’épiderme s’accomplissent par des cellules de re- change 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 néoblaste dont la présence peut être le caractère apomorphe qui unit tous les groupes taxonomiques de plathelminthes.

[Traduit par la Rédaction] Tyler and Hooge 210

Introduction The soft-bodied nature arises largely from the relative sim- plicity of the body wall: a simple epithelium (i.e., single lay- Flatworms are soft-bodied bilaterians with a body struc- ered) overlying a network of muscles and lacking cuticle or ture that has inspired some evolutionary biologists to use dermal skeletal elements (Fig. 1). them as models for the ancestor of the rest of the Bilateria. The free-living flatworms, known as turbellarians (class Turbellaria in non-cladistic classifications), have, for the Received 25 February 2003. Accepted 6 August 2003. most part, a ciliated, cellular epidermis (Fig. 1A), whereas Published on the NRC Research Press Web site at the parasitic flatworms, the Neodermata (encompassing the http://cjz.nrc.ca on 16 April 2004. classes Trematoda, Monogenea, and Cestoda), have a syn- S. Tyler2 and M. Hooge. Department of Biological Sciences, cytial, nonciliated epidermis whose nuclei-bearing parts lie University of Maine, 5751 Murray Hall, Orono, ME 04469- sunken below the musculature (Fig. 1B). Turbellarians show 5751, USA. considerable variety, however: special regions of the epider- 1This review is one of a series dealing with aspects of the mis may have a syncytial structure or sunken cell bodies, or biology of the phylum Platyhelminthes. This series is one of may lack cilia. Reflecting their origins from a turbellarian- several virtual symposia on the biology of neglected groups like ancestor, the Neodermata have a larval stage bearing a that will be published in the Journal from time to time. ciliated, cellular epidermis, much like that of turbellarians, 2Corresponding author (e-mail: [email protected]). and this is replaced by the syncytial epidermis, which is then

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 cells, also arise from such neoblasts in flatworms; no differ- course of infecting a new host. entiated somatic cells in the body are capable of dividing The mode of replacement of the epidermis, whether as (Peter et al. 2001). part of growth or general maintenance of the epidermis or Without an autapomorphy such as the neoblast, there is the development of the neodermis, appears to be unique to some justification for questioning the monophyletic nature the Platyhelminthes. Rather than arising from dividing cells of the phylum Platyhelminthes (Smith et al. 1986) and, in within the epidermis, as appears to be the case for other fact, recent proposals based on molecular data are to place metazoans, new epidermal cells arise from stem cells, the two orders of platyhelminths, the Acoela and Nemerto- neoblasts, that reside below the body wall and then migrate dermatida, into separate phyla outside the Platyhelminthes into the epidermis as they differentiate. This developmental (Ruiz-Trillo et al. 1999; Jondelius et al. 2002; Ruiz-Trillo et mode applies even in embryogenesis of the turbellarians al. 2002). Other morphological data support the monophy- (i.e., embryonic and definitive epidermises grow by immi- letic nature of at least three clades within the Platyhelmin- gration of stem cells from underlying layers) and is the way thes: the Acoelomorpha, Catenulida, and Rhabditophora. in which each new generation of neodermis arises as larval The Acoelomorpha, comprising the Acoela and Nemertoder- stages develop in sequence in the more complex parasitic matida, share features of epidermal ultrastructure (the form life cycles of neodermatans (Tyler and Tyler 1997). The of the cilia and their rootlets), as well as of certain glands neoblast stem cell system, and particularly its role in re- (Tyler and Rieger 1977; Smith and Tyler 1985; Smith et al. newal of the epidermis, may in fact be an autapomorphic 1986; Ehlers 1992a). The acoels, which have often figured trait for the Platyhelminthes (Rieger and Ladurner 2001). in phylogenetic hypotheses of the origin of the Bilateria, are Muscles of the body wall, as well as all other differentiated mostly small marine worms with a digestive system consist-

© 2004 NRC Canada 196 Can. J. Zool. Vol. 82, 2004 ing of a syncytium (and so lacking a lumen, hence the name Two belt-form junctions join each cell to its neighbors: an Acoela). The Catenulida, a small group containing a strange apical-most zonula adherens and, just basal to this, a septate primitive marine family but mostly freshwater species, share junction (Fig. 2D). Spot-form junctions, namely hemiadher- a special orientation and position of the male reproductive ens junctions, link the cells to the underlying basal lamina and excretory organs (Smith et al. 1986). The rest of the (Figs. 2A and 2C). The zonula adherens links prominently platyhelminths, including the parasitic classes and such well- with the actin filaments of the terminal web. Actin filaments known free-living groups as the planarians (Tricladida) and also compose stress-fiber bundles that anchor basally to the polyclads, form the monophyletic group Rhapditophora and hemiadherens junctions. share features (at least primitively) including adhesive or- All general epidermal cells are multiciliated and their cilia gans and rhabdite glands. anchor into the cytoplasm by two rootlets, a tubular rostral In all three clades, neoblasts residing below the body wall rootlet extending from the rostral side of the ciliary basal give rise to other cells, including those involved in growth body and a solid-cored vertical rootlet extending from its and replacement of the epidermis. The body wall and its re- basal end and penetrating the terminal web (Fig. 2A). lation to these stem cells may provide the best characters for Microvilli between the cilia bear a glycocalyx that forms a reuniting these three clades into the single phylum Platyhel- diffuse mucous coat over the epithelial surface. minthes. Other mucoid coverings are formed by secretions of gland cells that penetrate the epidermis between the epidermal The epidermis of turbellarians cells; their cell bodies lie below the body wall in the parenchyma. Among these glands are prominent rhabdite glands A rhabditophoran model whose secretions, the rhabdites, have a characteristic mor- A starting point for understanding the structure of the phology with a multilayered lamellate cortex and a granular body wall of turbellarian platyhelminths is consideration of medula. The terminal necks of the gland cells, as well as the the body wall in a microturbellarian member of the rhab- dendrites of sensory cells in the epidermis, are linked api- ditophoran order Rhabocoela, the largest of the turbellarian cally to the epithelial cells by the typical epithelial junctions, orders containing species commonly encountered in both the zonula adherens and septate junctions. freshwater and marine habitats. A typhloplanoid rhabdocoel3 The basement membrane of the epidermis is, as usual for serves as a good model (Fig. 2), having a simple cellular, basement membranes (Pedersen 1991), bilayered; the basal ciliated epidermis with a well-developed basement mem- lamina (lamina densa) appears as a dense sheet in immediate brane overlying a typical muscle network of circular, longi- contact with the basal plasma membrane, and the reticular tudinal, and diagonal fibers — in short, a body wall fitting a lamina (lamina fibroreticularis), appearing more diffuse and model we might expect for bilaterians in general, apart from fibrous, faces the underlying muscle cells (Fig. 2C). Lying the lack of cuticle or other skeletal elements. on the inner side of the basement membrane are cell pro- Epidermal cells of the typhloplanoid are cuboidal to cesses of pigment cells bearing dense granules that give the squamous and form a simple epithelium (Fig. 2A). Rather animal its brown color. than being strictly boxlike, they interdigitate with one an- Beneath the basement membrane and pigment cell pro- other along their lateral sides and send processes along the cesses are the muscles of the body wall comprising three ori- basement membrane under their neighbors. They also ac- entations: an outermost circular muscle layer, an innermost commodate the necks of glands and dendrites of sensory longitudinal muscle layer, and between them, diagonal mus- cells that penetrate the epidermis through tubular channels in cles whose fibers form a network of two crossing orienta- the cells themselves. tions (Figs. 2C and 3). Elements of the submuscular nerve The cells are typically polarized, with an apical surface plexus are also evident in sections of the musculature bearing cilia and microvilli, a basal surface resting on the (Figs. 2A and 2B). basement membrane, and loosely defined layers in the cyto- In general, this model of body wall structure (i.e., a cellu- plasm between. Most apical in the cytoplasm, immediately lar, ciliated, simple epithelium overlying a three-layered body beneath the plasma membrane, is a layer of small secretory wall musculature) applies to many other turbellarians as vesicles called epitheliosomes or ultrarhabdites, which ap- well. There is significant variety, however, which has been pear to contribute to the mucous covering of the epidermis the subject of more comprehensive reviews of epidermal upon their release (Fig. 2B). Just basal to these vesicles is a ultrastructure (Bedini and Papi 1974; Tyler 1984; Ehlers terminal web of actin filaments; basal to these filaments is a 1985; Rieger et al. 1991b; and Tyler and Tyler 1997). We layer of mitochondria, and then the nucleus, which is more summarize aspects of this variety in the following sections, or less centrally positioned (Figs. 2A and 2B). Around the starting with other turbellarian rhabditophorans (i.e., the nucleus and basal to it are elements of the endomembrane bulk of the free-living platyhelminth species) and comparing system, the Golgi apparatus, and endoplasmic reticulum, as them with the other two clades of platyhelminths, the well as ribosomes free in the cytoplasm. The lobulated shape Acoelomorpha and the Catenulida. Because the neodermatan of the nucleus produces multiple profiles in given sections rhabditophorans are so distinctive in the structure of the (Fig. 2A). body wall, they are treated in a separate section.

3 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 mid- tide level. The specimen was fixed in phosphate-buffered 3% glutaraldehyde, post-fixed in phosphate-buffered 1% osmium tetroxide, and embedded in Epon-Araldite.

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.

Fig. 3. Projection of confocal images of a whole mount of the uous with the extracellular matrix surrounding the muscles typhloplanoid rhabdocoel Jensenia sp. stained with Alexa-488- and parenchymal cells. As would be expected, the reticular phalloidin to reveal musculature. Pharyngeal muscles appear as lamina is thicker in the larger bodied flatworms such as the bright concentric fibers near the center, and muscles of the polyclads and triclads (Pedersen 1966; Ehlers 1985; Rieger gonopore behind it are similarly strongly stained. The remaining et al. 1991b). Fibers in the reticular lamina are helicoidally fibers are muscles of the body wall arranged in longitudinal, cir- packed in some rhabditophorans (species of the genera cular, and diagonal orientations. 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., spine- like 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 Varso- viella 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). Other rhabditophorans Ciliary rootlets in rhabditophorans fairly uniformly have A cellular, ciliated epidermis like that of the typhloplanoid the same structure as that described for the typhloplanoid; species is typical of many rhabditophorans, but considerable i.e., a tubular rostral and a solid-cored vertical rootlet on variation appears in the development of certain components each ciliary basal body. In macrostomorph turbellarians, the in various taxa. The basal matrix underlying the epidermis, vertical rootlet may be slanted more or less in the anterior for instance, is not sharply set off as a basement membrane direction (Tyler 1984). Proseriates have well-developed among the lower rhabditophorans (the Macrostomorpha) as rostral rootlets that converge in a rostral projection of the it is in other rhabditophorans. Instead, it appears as an exten- epidermal cell bearing them (Bedini and Papi 1974; Ehlers sion of a general intercellular matrix that lies between mus- 1985; Rohde and Watson 1995). Some species, however, cle and other subepidermal cells (Rieger et al. 1991b, p. 21). have fewer rootlets; for instance, some parasitic turbellarians It is even discontinuous under the epidermis, i.e., punctate, such as Kronborgia amphipodicola and Seritia stichopi have in some macrostomidans such as Bradynectes spp. or mesh- only a single rostral rootlet (Bresciani and Koie 1970; Rohde like in Haplopharynx spp. and dolichomacrostomids. The re- et al. 1988). maining rhabditophorans, like the typhlopanoid, have a well- Microvilli are a virtually constant feature of all epidermal developed basement membrane with a typical bilayered cells in turbellarians but are absent in some regions of epi- structure: a basal lamina (lamina densa) underlying the epidermis (which then appear smooth surfaced) in such para- dermal cells and a reticular lamina (lamina fibroreticularis) sites as Triloborhynchus sp. and S. stichopi (see Jondelius extending basally and providing the anchor points for the 1988; Rohde et al. 1988). Microvilli show some variation in body wall musculature. The reticular lamina is often contin- shape, being usually conventionally cylindrical as in the typ-

© 2004 NRC Canada Tyler and Hooge 199 hloplanoid (above) but tubercle-like in the Temnocephalida desmosomes of vertebrate epithelia (i.e., as small spot welds and lancet-like in the proseriate Dicoelandropora sp. (Ehlers between cells), but neither desmosomes nor hemidesmoso- 1977; see Tyler and Tyler 1997 for other variations). All mes occur because no turbellarians (and probably no inver- show evidence of a core of actin filaments, as is typical for tebrates; cf. Erber et al. 1999; Orii et al. 2002) have microvilli, and this is more or less well developed in some intermediate filaments in the cytoplasm of their epithelial species, forming, for example, a distinctly dense core in cells. Instead of intermediate filaments, the hemiadherens Cleistogamia longicirrus (Rohde et al. 1988). and spot adherens junctions rely on actin bundles for cyto- Something that could be characterized as an apical skeletal linkage. (Desmosome and hemidesmosome junc- extracellular matrix, i.e., a cuticle, occurs only rarely in the tions previously identified in flatworms, as in Tyler (1984), Platyhelminthes. A cuticle-like covering on nonciliated re- Rieger et al. (1991b), and literature therein, are probably gions of the body of the dalyelliod parasite Hypoblepharina really spot adherens and hemiadherens junctions. Identifica- boehmigi appears to arise by accretion of glandular secretion of even tight junctions, as well as desmosomes in tions (Karling and Nilsson 1974). A true cuticle would be S. stichopi, by Rohde et al. (1988) contradicts the general- expected to appear as a collagen- or chitin-reinforced matrix ization that these are not known in non-chordates (Tyler between the microvilli of the epidermal cells, and while a 2003) and needs further study.) Gap junctions have also not mucoid glycocalyx occupies this position in many platy- been unequivocally identified in the epidermis of platyhel- helminths (Rieger and Rieger 1976), no platyhelminth has a minths. cuticle like that in other spiralians or nematodes. Something The shape of the epidermal cells, especially in smaller tur- comparable to setae and, therefore, cuticle-like, is seen in bellarians, is cuboidal to squamous, with adjacent cells inter- the temnocephalid Notodactylus handschini: scale-like cov- digitating as described for the typhloplanoid. Some groups erings on the epidermal cells are produced in a fashion that with larger bodies, such as the Polycladida, have tall colum- is indistinguishable from that by which setae in annelids and nar cells that may even appear pseudostratified by virtue of lophophorates are produced (Jennings et al. 1992; Rieger rich development of glands crowding the cells. The epider- 1998); just as in setae, the scales have longitudinal channels mis of the parasitic rhabdocoel Anoplodium sp. also appears into which microvilli of the generative cells protrude. pseudostratified because of its gland-rich structure. In such In general, rather than being formed of cuticle, sclerotic turbellarians (e.g., Anoplodium sp. and the macrostomorphs hard parts in platyhelminths consist of either specialized Haplopharynx rostratus and Cylindromacrostomum sp.), the thickenings of the basement membrane (as mentioned glands are entirely epidermal; i.e., their cell bodies, as well above) or of dense accumulations of intracellular actin (e.g., as the necks, lie within the epidermis (Karling 1965; Rieger actin-reinforced, hook-forming microvilli in the proboscises et al. 1991b; Tyler and Tyler 1997). Most species have the of some turbellarians (Doe 1976)). gland bodies below the body wall, and only their necks The cell web shows different degrees of development in reach through the epidermis to the surface. various rhaditophorans, presumably in correlation with func- A more pronounced modification of epidermal cell shape tional requirements for cytoskeletal support. Particularly occurs in insunk epithelia, where the nucleated portion of among the macrostomorphs, with their less-developed basal the cell lies below the body wall musculature, connected to matrix, the terminal web can be quite thick and even multi- the surface portion of the cell by a necklike bridge. Such layered, with helicoidal packing (e.g., Bradynectes sp.), or it cells appear in the epidermis of lecithoepitheliates and can form a complete enclosing layer along all sides of the bdellourid triclads, for instance, and are restricted to the cell, basal and lateral as well as apical (e.g., Microstomum creepsole epithelium of proseriates and terrestrial triclads sp.). Terminal webs that are nearly as well developed appear (Bresslau 1928–1933; Hyman 1951; Bedini and Papi 1974; in some proseriates and rhabdocoels. A basal sheet of cell Curtis et al. 1983; Tyler 1984; Ehlers 1985). web is best developed in proseriates (Bedini and Papi 1974; A syncytial, rather than cellular, epidermis is developed in Ehlers 1985). Tall epidermal cells (e.g., the columnar cells certain groups of rhabditophoran turbellarians, appearing as of polyclads, or other cells bearing stress) often are seen fields or as a mosaic of plates, sometimes among cellular with vertical stress-fiber bundles, which anchor to hemi- regions. Such mosaics are especially characteristic of the adherens junctions in the basal plasma membrane. Temnocephalida, where patterns of plate arrangements are As in the typhloplanoid, the cell junctions that link to specific to given taxa (Joffe and Cannon 1998; Cannon and these cytoskeletal elements and join the epidermal cells to- Joffe 2001). In most temnocephalans, the plates have multi- gether and to the basement membrane are probably universal ple intraepidermal nuclei, but in Didymorchis sp., the plates among platyhelminths and, in fact, among invertebrate have insunk nuclei. Rhabdocoels such as the kalyptorhynchs epithelia in general (see Tyler 2003). Apical edges of the also appear to have syncytia, typically in belt-shaped fields epidermal cells are linked by zonulae adherentes and septate (Rieger et al. 1991b; De Vocht and Schockaert 1999 and lit- junctions, and the bases of the cells attach to the basement erature therein). In lecithoepitheliates and prolecithophorans, membrane by hemiadherens junctions. While the zonula a syncytial epidermis is suspected because of the abundance adherens may be inconspicuous in the epidermis of some of infolding plasma membranes and the lack of clear cell rhabditophorans such as polyclads, lecithoepitheliates, junctions over extended portions of the epidermis (Rieger et proseriates, and triclads, it is probably functionally present al. 1991b), but the true nature of these epidermises needs nonetheless; its inconspicuous nature can be attributed to the further investigation. Most like the syncytial, insunk epider- poor development of the terminal web in these species, but mis of the major groups of parasitic flatworms, the Neo- traces of this junction are visible anyway. Small spot dermata (see below), is the epidermis of the ectocommensal adherens junctions may play the role associated with the turbellarian Genostoma sp. It has well-delimited nonciliated

© 2004 NRC Canada 200 Can. J. Zool. Vol. 82, 2004 and ciliated regions connected by multiple necklike bridges Fig. 4. Ultrastructure of the body wall of the acoel Convoluta that span the basement membrane to the insunk perikarya ly- pulchra. (A) Portions of two epidermal cells and underlying ing below the body wall musculature (Tyler and Tyler 1997). musculature in a near-longitudinal section of the animal showing The polarized distribution of cell organelles varies among interconnections between rootlets (rt) of adjacent cilia and the rhabditophorans. Mitochondria appear in basal positions in paucity of basal matrix between the epidermis and musculature the prolecithophoran Acanthiella sp. and in temnocephalids, (mu). (B) Junctions among muscle and epidermal cells; arrow rather than being mostly restricted to an apical layer beneath marks a special junction between an epidermal cell process and the terminal web, as described for the typhloplanoid. Vacu- a longitudinal muscle showing extracellular material and an oles or secretory inclusions such as epitheliosomes may ap- intercellular gap that is larger between these two cells than that pear throughout the height of the cell instead of being in the spot adherens (sa) junctions between muscle cells. c, cir- limited to the apical-most layer (e.g., in larvae of a polyclad) cular muscle; e, epitheliosome; l, longitudinal muscle. (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 granule- bearing 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 deliv- ered by the glands as packets that traverse the cell or indi- vidually 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 pecially dorsoventral fibers in flat-bodied species), that al. 1991b). The submuscular plexus is part of, or continuous determines the shape. with, the main longitudinal cords of the central nervous system; the other two plexi appear as thin neural processes on Acoelomorpha either side of the basement membrane, between the base- Features of the body wall most clearly link the two ment membrane and the muscles, and among the bases of turbellarian orders Acoela and Nemertodermatida together the epidermal cells. Because of their thin, tenuous nature, into the clade Acoelomorpha (Tyler and Rieger 1977; Ehlers these processes are more easily visualized in special light 1985). While epidermal cilia in rhabditophorans appear to be microscopic preparations (Chien and Koopowitz 1977; relatively independent of one another, even if tightly packed, Ladurner et al. 1997), including immunocytochemical prepa- those in acoelomorphs are interconnected in rows by junc- rations of whole mounts using antibodies to neurotransmit- tures of their rootlets (Fig. 4A). Each of the rostrally point- ters, and are easily overlooked or confused with gland necks ing main rootlets of the epidermal cilia are connected by and other cell processes in electron micrographs. lateral rootlets or by lateral fiber bundles (see summaries by In the absence of other skeletal supports to set body Rieger et al. 1991b; Lundin 1997). In the Acoela, the two shape, it must be the basement membrane, together with the lateral rootlets arise from the main rootlet and join the tips arrangement of muscles including body wall muscles and of main rootlets of adjacent cilia; in the Nemertodermatida muscles crossing the parenchyma between the body wall (es- and some acoels, the lateral fiber bundles link the tips of the

© 2004 NRC Canada Tyler and Hooge 201 main rootlets with posterior rootlets of adjacent cilia. An- An epidermal nerve net appears to be the only nerve net other similarity in cilia is the shape of the ciliary tips, which in certain members of the Acoelomorpha (e.g., Tetraposthia have a distinct shelf (as opposed to the simple, tapering tips colymbetes), and this even incorporates the anterior gangli- seen in the Rhabditophora). onic concentration (brain) and the longitudinal cords (An der Acoels and nemertodermatids are also noteworthy for the Lan 1936). The epidermal net is reactive to serotonin (5- paucity of basal matrix under the epidermis (Fig. 4A). A hydroxytryptamine) immunoreactive tests in acoels (see typical basement membrane is absent, but small islands of Raikova et al. 1998, 2001 for literature; called a “surface some sort of extracellular material can be seen between the net” in these studies). Such a condition illustrates the primi- epidermal cells and underlying muscle. In nemertodermatids tive nature of the Acoela (Raikova et al. 2001). such as Flagellophora sp. and Sterreria sp., this material not only appears spotlike between epidermal cells and muscles Catenulida but also extends between muscles as a kind of general The epidermis of catenulidans is distinct from that of intercellular matrix (Smith and Tyler 1985), something like other platyhelminths in the nature of its ciliation and the that seen in macrostomorphs. In acoels, existence of such a form of the microvilli (Rieger 1981). The cilia are sparser matrix is evident in the enlarged and irregular-width gap in and more widely scattered (less than half the density of cilia the junctions between epidermal and muscle cells (Fig. 4B; in other platyhelminths), and their rootlets are directly op- Tyler and Rieger 1999). These junctions resemble fasciae posing rostral and caudal, virtually horizontal, as opposed to adherentes (so called by Tyler 1984 and Tyler and Rieger rostral and vertical. The rostral rootlets may converge in a 1999) but are probably better called hemiadherens junctions rostral projection of the cell bearing them (Ehlers 1985). given the probability that the gap material is extracellular Cilia of the marine species (family Retronectidae) each sit matrix; the intermuscular junctions are spot adherens junc- recessed slightly in a small, microvilli-ringed pit, which is tions rather than desmosomes (cf. Tyler 2003). not seen in other platyhelminths. The epidermal microvilli in The cell web is particularly well developed in nemerto- catenulidans are distinctive in having a central dense core dermatidans, with a striking terminal web appearing as a (Rieger 1981). The glycocalyx between microvilli may be dense, sometimes multilayered, fibrous sheet beneath the cil- especially dense and cuticle-like in some catenulidans. iary rootlets (in Sterreria sp., for example; Tyler and Rieger Like the acoelomorphs and some macrostomorph rhab- 1977). Prominent stress fibers (actin bundles), oriented verti- ditophorans, some catenulidans have a discontinuous basal cally in the epidermis and linking the terminal web with matrix that appears as small islands between the epidermal junctions to muscles underlying the epidermis, appear in cells and underlying muscle (Doe 1981; Rieger 1981; Ott et thicker epidermises of some Nemertoderma species (Tyler al. 1982; Tyler 1984). A more continuous subepidermal ma- and Rieger 1977). The terminal web may be developed to trix resembling a lamina densa is seen in Stenostomum sp., compensate for the less-developed basement membrane. Pre- while Xenostenostomum sp. has a pronounced lamina sumably also as a consequence of the less-developed base- (Pedersen 1983; Ehlers 1985). The glycocalyx may also be ment membrane, the epidermal cells of many acoels are well developed in retronectid catenulidans (Rieger and insunk, i.e., the nucleated portion of the cell bulges into or Rieger 1976). even through the underlying muscle layer (Tyler 1984). The glycocalyx between microvilli may be dense and The epidermis of neodermatan cuticle-like in some acoelomorphs, especially nemertoderm- rhabditophorans atids, where it may even appear bilayered (Rieger and Rieger 1976; Tyler and Rieger 1977), but in most acoels it is rather Ultrastructure tenuous, no more developed than that of the rhabditophorans. All members of the Neodermata have a special kind of Layering of organelles in the epidermal cells in the polarized syncytial epidermis as adults: a nonciliated epidermis with fashion described for the typhloplanoid is also characteristic its nuclei in so-called cytons or perikarya that lie below the of the acoelomorphs, but some acoels have mitochondria, for body wall musculature, connected to the surface layer by instance, relatively uniformly scattered throughout the cell multiple branching, necklike bridges (Fig. 1B). The surface body. layer itself is a continuous sheet that entirely covers the Like other turbellarians, acoelomorphs have richly glandu- body, uninterrupted by any kind of cell junction except at lar epidermises. Especially prominent are glands with rhab- point-like penetrations of sensory or gland cell processes. doid secretions that discharge onto the epidermal surface to Ehlers (1985) coined the term “neodermis” for this epider- produce a mucous coating (Smith et al. 1982). Other glands mis because it arises new at metamorphosis of the larva. As producing mucoid secretions have granules of more globular a larva infects the first host in the life cycle, it casts off its shape (Rieger et al. 1991b). A variety of forms of granules epidermis and replaces it with a new epidermis. This new produced by other glands appear at special sites such as epidermis arises from cells lying below the epidermal base- around the frontal pore and genital pores (Smith and Tyler ment membrane, which send processes over the basement 1985, 1986). In all acoelomorphs, glands and their necks lie membrane that spread and fuse together to form the defini- between the epidermal cells rather than, as in rhabdocoels, tive surface syncytial sheet. These Anlage cells remain as in channels through the cells. Mucous glands are especially the perikarya of the syncytial epidermis, the neodermis. voluminous in the thick epidermis of species of Nemerto- It is logical to assume that this special kind of epidermis, derma, causing the epidermal cells themselves to appear pseudo- which forms the interface between these parasitic worms and stratified; i.e., epidermal cell nuclei are displaced to a variety their hosts, is the key to their evolutionary success. The of positions by the adjacent glands (Tyler and Rieger 1977). neodermatans make up three quarters of all known

© 2004 NRC Canada 202 Can. J. Zool. Vol. 82, 2004 platyhelminth species, and it appears that something about squamous-epithelial layer can be interpreted as a layer sup- this epidermis that they all share is highly adaptive for a par- plied by the sporocyst in which this larval stage develops. asitic lifestyle. The success may be attributed to the way in The epidermis in adult monogeneans has irregular micro- which such an interface allows these parasites to resist the villi or folds, sometimes limited to specific regions of the defenses of hosts they infest, and it may rest — certainly at body. Some have rounder tubercles in circumscribed least for the gutless tapeworms — on the ability of this epi- cushion-like areas (Ramasamy et al. 1987; Fried and Haseeb dermis to absorb nutrients from the host. 1991; Xylander 1996). Although having a syncytial, insunk epidermis of this spe- Most distinctive is the epidermis of the Cestoda, where cial kind is the rule in the Neodermata, differences in the na- the microvilli are of a slender regular shape with a cylindri- ture of the outermost surface — whether it bears microvilli cal hollow core; i.e., a dense-walled support, presumably of distinctive shape, for instance, or microridges, or pits — actin-based, surrounding a lightly staining central cytoplasm distinguish various neodermatan taxa (Fig. 1B). The epider- (Ehlers 1985; Xylander 2001). The supporting wall material mis also varies in thickness and in the form of its microvilli converges at the tip to make a dense cap. Such microvilli are at different developmental stages, and there are some differ- found in all but the posterior body region of Gyrocotylidea ences among regions of the body surface even within a sin- and most body regions of those stages of the Cestoidea liv- gle organism. This variety has been reviewed by Threadgold ing in the intestine of their host. Although it is often as- (1984), Fried and Haseeb (1991), Coil (1991), and Xylander sumed that such microvilli provide expanded surface for (1996, 2001), but we give a short summary here, largely nutrient absorption (by comparison with the role of the drawn from those sources. microvilli in the host intestine), their function instead ap- Distinctive of the epidermis in the aspidobothrean pears to be to maintain a high-pH boundary layer at the epi- Trematoda are microvilli in the shape of minute, rounded tu- dermal surface, thus preventing enzymes of the host intestine bercles, both in the adult and in the synctial parts of the epi- from digesting it (Uglem and Just 1983; Xylander 2001). dermis in the cotylicidium larva. (The neodermis is already The dense cap is especially developed in certain body re- well developed at hatching of the cotylicidium.) A well- gions of the Cestoidea as a sharp-pointed, elongate projec- developed glycocalyx covers the microvilli, which are tion sitting at an angle to the axis of the base; such underlaid by a fairly well-developed terminal web. Some microvilli are called microtrichs. At the juncture of the cotylocidia have more filiform microvilli with cores of fila- sharp-pointed cap with the base is a trilaminate plate that ments (Rohde 1972). Ciliated cotylocidia (e.g., of Cotylo- links microfilaments in the core with the cap. In the gaster occidentalis; see Fredericksen 1978) have limited amphilinidean cestodes, which live in the coelomic cavity ciliated regions in an otherwise nonciliated, syncytial epider- rather than the intestine of their host, microvilli over the mis, while others lack the ciliated cells, having only the general body surface are shaped like small rounded tubercles syncytial epidermis with tuberculate microvilli (Fried and without the dense points seen in other cestodes (Xylander Haseeb 1991, p. 145; Xylander 1996, p. 234). 2001) and reminiscent of those of aspidogastrean trematodes. The fully developed epidermis of digenean trematodes The epidermis at the anterior end of amphilinideans has (i.e., the neodermis of the adult and cercarial stages) has long, slender microvilli more like those of other tapeworms. short, irregularly shaped microvilli, often a well-developed The calcareous corpuscles of Amphilinidea and Cestoidea glycocalyx, and spines formed of intracellular crystalloids of and the spines of Gyrocotylidea are also distinctive of the actin anchored to the basal membrane and projecting the api- epidermis of cestodes; both are multilayered secretions that cal membrane as thornlike extensions. However, instead of seem to lie in extracellular pockets within the epidermis, and bearing microvilli, the surface may be relatively smooth, they may be excretory products (Xylander 2001). with minute folds and canals developed to different degrees in given regions of the body in species-specific patterns. In Development of the neodermis the sporocyst and redia larval stages, the epidermis may bear Each successive stage in the life cycle of neodermatans small, irregular, microvilli-like folds that may be branched. forms its epidermis by protrusion of cell processes from be- A peculiar variation on epidermal structure in Digenea is low the musculature. These processes fuse above the base- seen in certain sporocysts without a birth pore. In some (e.g., ment membrane into the continuous sheet that covers the Cercaria bucephalopsis), the epidermis is layered into a body and remain connected to the deeper lying perikarya thicker outer, smooth-surfaced, multinucleated part separated that gave rise to them, forming a syncytium. The first and from a thin, inner, anucleated part by a space into which the most obvious of these productions of new epidermis takes anucleate layer projects irregular microvilli; the two layers place as a newly hatched larva invades its first host; how- may or may not be connected by cytoplasmic strands ever, transitions to other larval stages, where those occur (in (Threadgold 1984). In some other cases (e.g., Microphallus the Digenea and Cestoda), also involve development of a spp.), a similar outer nucleated layer eventually becomes new epidermis from underlying cells. In transition through subdivided into squamous-like cells by infolding of the digenean larval stages (e.g., from sporocyst to daughter plasma membrane, while the inner layer becomes attached to sporocysts or redia, or from redia to cercaria), the germinal perikarya lying below the musculature. The microvilli- cells that form the next stage are surrounded by a so-called bearing layer becomes the outer surface at selected regions “primitive epithelium” that is replaced by a new syncytial in sporocysts such as those of Cercaria littorinae (Popiel insunk epidermis as that stage develops. 1978) and looks much like the typical digenean epidermis, The epidermis of free-swimming larvae of monogeneans so it is logical to interpret only this layer as the definitive and trematodes is reminiscent of the turbellarian epidermis, epidermis of this stage. The outer multinucleated layer or being a cellular epidermis one cell thick, completely ciliated

© 2004 NRC Canada Tyler and Hooge 203 in many monogenean oncomiracidia and digenean miracidia, Fig. 5. Projections of confocal images of the dorsal (left) and for example; only partially ciliated or aciliate in other onco- ventral (right) body wall of a specimen of the acoel Haplogon- miracidia; or restricted to small, discrete, tuft-like, ciliated aria phyllospadicis stained with Alexa-488-phalloidin to show patches in aspidogastrean cotylocidia (Threadgold 1984). musculature. Besides the longitudinal and circular muscle fibers, Those cestode larvae with cilia have a syncytial epidermis U-shaped and longitudinal crossover muscles loop posterior to bearing the cilia (Xylander 1987, 2001). Unlike the tur- the mouth (central pore) in the ventral body wall, and a second bellarian epidermis, however, the ciliated cells have interpo- set of longitudinal crossover fibers occupies a corresponding po- lated between, around, or under them ridges of unciliated sition in the dorsal body wall. Diagonal muscles are also visible processes reaching into the epidermis from perikarya lying in the dorsal body wall in the anterior half of the body. The below the musculature; this syncytial tissue comprises the strong muscles of the male copulatory organ appear as a bright presumptive adult epidermis that eventually completely re- sphere, and those of the female pore form a bright ring just ante- places the ciliated cells. The cilia differ from those of most rior to it. Peg-like projections along margins are sensory recep- turbellarians — except, perhaps significantly, those of some tors with actin-reinforced microvilli. (From Hooge and Tyler parasitic turbellarians like Kronborgia sp. and Seritia sp., as 2003, reproduced with permission from Zootaxa, Vol. 131, mentioned above — in having only a single rostral rootlet © 2003 Magnolia Press.) 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 ex- pansion of projections from a syncytium that lies below the epidermis of the miracidium larva. The projections sit ridge- like 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 in- cipient 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 protec- tive coatings from both maternal and the embryo’s own tissues, as well as epidermis-like layers. The living layer first musculature in whole mounts, namely fluorescent markers that covering the embryo is a syncytium arising from subsurface bind specifically to the actin of the muscles (Figs. 3 and 5), it cellular projections of macromeres, and it delaminates into can be seen that only a few groups of flatworms have a sim- three layers, only the innermost of which is retained as the ple network of circular and longitudinal muscles, a pattern larva’s epidermis (see Threadgold 1984; Coil 1991). once thought to apply generally to worm-shaped animals. Most species of the Catenulida and some species of the Musculature Acoela have such a simple network, but most platyhelminths also have diagonally oriented muscles in at least portions of The body wall musculature of platyhelminths is arranged the body wall (von Graff 1904–1908; Luther 1943, 1955; in patterns that are specific to given taxonomic groups. Rieger et al. 1994; Hooge 2001). Circular muscles constitute Using techniques that allow visualization of the complete the outermost layer, the one that sits directly under the epi-

© 2004 NRC Canada 204 Can. J. Zool. Vol. 82, 2004 dermis, and longitudinal muscles lie to the inside of these; body shapes that can be achieved by the contraction of the only exceptions to this generalization are members of the circular, longitudinal, and diagonal musculature are well acoel family Childiidae, in which this layering is reversed, documented (e.g., Ruppert and Barnes 1994). It is possible with longitudinal muscles outermost (Westblad 1942; Hooge that the more complicated musculature patterns of the 2001). In most turbellarian members of the Rhabditophora, Acoela relate to specific feeding motions whereby food is diagonal muscles lie in a layer roughly between the circular stuffed into a simple mouth (without pharynx and pharyn- and longitudinal fiber layers and comprise a paired set of fi- geal musculature) by bending of the body margins (Tyler bers that cross over each other along the ventral and dorsal and Rieger 1999). Food that is positioned in the ventral food midlines (Rieger et al. 1991a; Rieger et al. 1994; Hooge and groove at the anterior end of the body of Convoluta pulchra, Tyler 1999; Hooge 2001). By contrast, in the few specimens for example, may be forced into the midventral mouth by of the neodermatan Rhabditophora that have been examined, pulling the mouth and the posterior end of the body forward the diagonal muscles lie innermost, to the inside of both lon- by contraction of the longitudinal crossover muscles. Body gitudinal and circular muscles (Mair et al. 1998, 2003). movements such as the turning movements of C. pulchra are Kotikova et al. (2002) report the same innermost orientation likely perfomed by the well-developed muscles crossing the for the turbellarian dalyellioid rhabditophoran Castrella parenchyma as well as by longitudinal crossover muscles. truncata. Layering of the longitudinal and diagonal fibers Body wall muscles of platyhelminths are, for the most may not be so distinct, in any case, in that fibers of these part, of a smooth type typical of invertebrates, but some two orientations seem to lie in nearly the same plane in elec- pseudostriated fibers occur in structures that contract rapidly tron micrographs of the body wall (Fig. 2C). Underscoring (Rieger et al. 1991b). The muscles are entirely mesenchymal the relatedness of these fibers, Orii et al. (2002) found that and subepithelial; i.e., none is an epitheliomuscular cell, as diagonal and longitudinal muscles in the triclad Dugesia ja- was assumed before the advent of electron microscopy. Mus- ponica have the same type of myosin, which is different cles anchor to the basement membrane of the epidermis by from that of the circular fibers. This corroborates Westblad’s hemiadherens junctions, specifically at junctures with the (1949) proposal that diagonal fibers may be derived from dense bars of the contractile elements (Fig. 2C). They are longitudinal ones. The thickness and spacing of diagonal fi- also surrounded, at least partially, by the extracellular matrix bers varies among taxa more than that of the other layers. (ECM) that extends from this basement membrane. As men- Large rhabditophorans that use muscles for locomotion, tioned above, catenulidans and nemertodermatidans have a such as triclads and polyclads, have body wall muscle fibers thin, discontinuous, subepithelial ECM, and it may serve that are more numerous and tightly spaced than smaller spe- only to anchor muscles to the epidermis. Similarly, the small cies that move by ciliary gliding. Many, too, have additional islands of material at junctions between muscles and epider- layers of muscles beyond the three conventional layers. The mal cells in the Acoela may be reduced ECM, essentially planarian Girardia tigrina, for example, has an additional remnants of the basement membrane (Tyler and Rieger layer of longitudinal muscles so that the diagonal fibers are 1999). sandwiched between two longitudinal layers, the inner one of which appears stronger (Cebrià et al. 1997); however, the Development of the body wall planarian D. japonica, also large bodied, has no such additional muscle layers (Orii et al. 2002). Large polyclads have Embryonic development as many as five or six layers in total, the six-layered forms Macrostomorph and polyclad turbellarians develop having the layers ordered as follows (from outermost to in- through quartet spiral cleavage in which cell lineages can be nermost): circular, longitudinal, diagonal, circular, diagonal, fairly readily determined, and here the epidermis arises from and longitudinal (Prudhoe 1985). Quite distinct from these micromere quartets 1–3. At gastrulation in Imogene mc- relatively conservative patterns of circular, longitudinal, and grathi, for instance, descendants of these cells form the diagonal muscle fibers in Rhabditophora are those in the ectoderm from which the epidermis and brain arise body wall musculature of the Acoelomorpha, which show (Younossi-Hartenstein and Hartenstein 2000). As would be eight distinguishable arrangements (Hooge 2001). The ma- expected for spiralian development, progeny of the 4d cell, jority of acoelomorphs studied have circular and longitudinal along with micromere 2b, form an inner cell mass at gastru- muscles, as well as U-shaped muscles that extend longitudi- lation from which body wall muscles arise (Younossi- nally from the anterior tip of the animal before wrapping Hartenstein and Hartenstein 2000); pharyngeal musculature, around the posterior rim of the mouth. Many species of gland cells, and the excretory system also arise from this acoels also have so-called longitudinal crossover fibers that mass, and 4d descendants also give rise to endoderm (Boyer are longitudinally oriented in the anterior portion of the et al. 1996). In the polyclad Hoploplana inquilina, the body but then bend medially to cross over each other diago- endomesoderm-producing 4d micromere gives rise to the in- nally along the dorsal and ventral sides (Fig. 5). Other ner longitudinal muscles, while the ectoderm-producing 2b differences arise in the distribution of certain sets of longitu- micromere gives rise to the outer circular muscles (Reiter et dinal and diagonal fibers. In addition to the typical body al. 1996; Boyer et al. 1998). In the more modified spiral wall muscles, the acoel Anaperus tvaerminnensis has quartet cleavage in the Lecithoepitheliata (Reisinger et al. intraepidermal longitudinal muscles, the cell bodies of which 1974), some of the micromere quartets form a Hüll mem- are positioned among the body wall muscles (Ehlers 1994). brane at gastrulation, which constitutes an extra-embryonic The pattern of body wall musculature in flatworms ex- ectoderm. The embryonic ectoderm arises from remaining cluding the Acoela is similar to that found in other soft- micromeres, which spread under the Hüll membrane. Simi- bodied metazoans such as annelids (Hooge 2001), and the larly, in Macrostomum hystricinum, a Hüll membrane or

© 2004 NRC Canada Tyler and Hooge 205 yolk mantle forms an extra-embryonic ectoderm, or primary embryonic cells, namely yolk cells from the parent, rather epidermis, from large, yolky blastomeres, and the later de- than from early blastomeres as in macrostomids or pro- veloping definitive epidermis pushes this aside (Tyler 1981; seriates. It is not clear that it grows by any mechanism Tyler and Tyler 1997). other than simple spreading over the embryo from the A unique duet spiral cleavage pattern is characteristic of mesenchyme-like mass. the Acoela, and here the mesodermal body wall musculature is derived from the endoderm-producing third-duet macro- Implications for origin of the neodermis meres (Henry et al. 2000; Rieger and Ladurner 2003). In all of the major taxa of the Neodermata, there are at Muscle development progresses similarly in C. pulchra least some representatives that have larvae with a ciliated (Ladurner and Rieger 2000) and Macrostomum hystricinum epidermis that is replaced by neodermis at metamorphosis. marinum (Reiter et al. 1996), but somewhat differently from This larval epidermis is cellular in representatives of the the polyclad H. inquilina (Reiter et al. 1996), which passes Trematoda and Monogenea but syncytial in the Cestoda; in through a Müller’s larva before achieving the adult form. In either case, comparison can be drawn with the epidermis in C. pulchra, the first elements of body wall musculature ap- turbellarians. It should not be assumed, however, that only pear after ~20 h (50% of developmental time), when short the larval epidermis is homologous to that of turbellarians. It muscle fibers appear at evenly spaced latitudes of the em- is not that, in evolving from a ciliated ancestor, the Neo- bryo and expand to form circular fibers. At ~23 h, the first dermata have invented a new epidermis or that its larvae re- longitudinal fibers appear. By ~35 h, a fairly complete or- capitulate an ancestral epidermis; rather, the developmental thogonal grid is established, and the final pattern of muscu- mechanism by which the epidermis is replaced is common to lature is completed with the formation of U-shaped and all platyhelminths, and the Neodermata have specifically crossover muscles. adapted this mechanism to a parasitic lifestyle. By replacing Other neoophoran turbellarians besides the lecithoepithe- their epidermis as they attack hosts in the life cycle, the liates have eggs with small oocytes and numerous yolk cells neodermatans presumably gain some advantage, immunolog- and follow highly modified, irregular cleavage without going ical or physiological, in dealing with host defenses. through what could be identified as gastrulation (Hartenstein Lyons (1973) first drew parallels between this epidermal and Ehlers 2000); triclads, in fact, have what is called development at metamorphosis in parasitic flatworms and “blastomere anarchy”. Tissues such as the epidermis appear epidermal development in triclad turbellarian embryos, as to differentiate in situ out of a mass of blastomeres in neo- well as epidermal replacement in turbellarians in general. ophoran embryos (Hartenstein and Ehlers 2000; Younossi- Tyler and Tyler (1997) expanded on this theme with illustra- Hartenstein and Hartenstein 2001). tion of epidermal development in other turbellarians and Many of the turbellarian embryos that have been studied provided evidence that developing embryos in other turbel- seem to produce successive generations of epidermis as larian groups, namely the Acoela, Macrostomida, and Poly- development proceeds. For instance, polyclad turbellarians cladida, have cells immigrating into epidermal positions form a Hüll membrane over the embryo, which is later re- from the parenchyma. In any case, it appears that no epider- placed by an epidermis that differentiates under it from a mis in these embryos, embryonic or definitive (see also mass of micromeres in the center of the embryo (Younossi- below), grows by division of epidermal cells. Such a mecha- Hartenstein and Hartenstein 2000). Triclad turbellarians have nism of epidermal growth and replacement may have pre- three distinct generations of epidermis (Skaer 1965; Benazzi adapted the platyhelminths to parasitism. If host invasion and Gremigni 1982; Baguñà and Boyer 1990). The primary depends on presentation of a new epidermis to host de- epidermis in the embryo is a syncytium (Sakurai and Ishii fenses, the kind of epidermal shedding and replacement that 1995) that encloses a yolk mass; it is replaced by the sec- characterizes free-living platyhelminths may provide a key ondary epidermis, which originates from outwardly migrat- element to this invasion. The ability of free-living platy- ing cells that interpolate themselves into the primary helminths to form syncytial and insunk epidermises may epidermis. The tertiary epidermis, which is the definitive also be key to the parasites’ presentation of a continuous, un- epidermis of the hatched young worm, arises from similarly broken surface to host tissues. migrating cells; like the secondary epidermal cells, these are ciliated but are readily distinguished by their bearing the Growth of epidermis and muscle rhabdoids characteristic of the adult epidermis. Proseriate turbellarians also produce three generations of Differentiated cells in platyhelminths do not divide, so the epidermis (Giesa 1966; Reisinger et al. 1974; Tyler and Ty- epidermis and musculature must be maintained and grow ler 1997). The definitive or tertiary epidermis of the hatch- through immigration of replacement cells from the underly- ling arises from cells in the parenchyma that send processes ing parenchyma (Luther 1904; Hein 1928; Skaer 1965; to the surface to produce a ciliated surface layer connected Ehlers 1985; Smith et al. 1986; Peter et al. 2001). The stem to insunk cell bodies (i.e., it is a cellular epidermis with cells that give rise to these replacement cells are of a kind insunk nuclei). unique to the Platyhelminthes: neoblasts (Baguñà 1981; Embryos of rhabdocoel turbellarians may not show such Ehlers 1985; Ladurner et al. 2000). Neoblasts are relatively obvious generations of epithelia. The epidermis arises (along small cells with prominent nuclei and scant cytoplasm with other organs) out of blastomeres forming a solid containing only free ribosomes and a few mitochondria mesenchyme-like mass (Hartenstein and Ehlers 2000 and lit- (Palmberg 1990; Hori 1997; Gschwentner et al. 2001). They erature therein). Although it does develop under a covering are the only cells capable of dividing in adult flatworms layer, or Hüll membrane, this layer is derived from non- (Baguñà et al. 1989; Ladurner et al. 2000; Newmark and

Sánchez Alvarado 2002) and so must be responsible for the acoel C. pulchra has been shown, through experiments renewal of all cell types during growth, regeneration, and with continuous labeling with BrdU and immunogold cyto- normal maintenance (Baguñà 1981; Ehlers 1985; Palmberg chemistry, to have epidermal cells replaced by S-phase 1990; Baguñà et al. 1994; Hori 1989; Rieger et al. 1999; neoblasts (Thaler et al. 2002). Conventional electron micros- Gschwentner et al. 2001). copy of embryos of C. pulchra indicates that neoblasts mi- Differentiating from neoblasts, epidermal replacement grate into the epidermis as it grows in early developmental cells are first recognizable by their production of a cluster of stages (Tyler and Tyler 1997). centrioles that are destined to be the basal bodies of the epi- Epidermal regeneration in the Neodermata may be simpli- dermal cilia. This differentiation may be evident even before fied by the syncytial nature of the epidermis. Here, any the cells migrate into the epidermis, where further differenti- wound can be repaired by simple spreading of the remaining ation of the cilia and components such as the epitheliosomes syncytial surface layer of the epidermis over the damaged proceeds. Immigrating centriole-bearing cells like these have area, since this remains connected to the many perikarya in been found in catenulidans, macrostomorphs, proseriates, the parent piece. Popiel et al. (1985) have documented such and triclads (Koie and Bresciani 1973; Moraczewski 1977; spreading during wound healing in the trematode Schisto- Hori 1978; Doe 1981; Pedersen 1983; Ehlers 1985; Palmberg soma mansoni; curiously, the newly spread epidermis does 1990). Epidermal replacement cells of macrostomids may not regenerate a new basement membrane in the area it cov- migrate into the epidermis even before they differentiate ers. centrioles (Rieger et al. 1999). Growth and regeneration of muscles is similarly depend- Stem cells still capable of mitosis also appear within the ent on neoblasts. Myoblasts first appear as small rounded epidermis and in basiepithelial positions in certain species cells, eventually produce muscle-specific myosin, and then among the Acoela and Catenulida. Clusters of basiepidermal gradually elongate and differentiate myofilaments (Cebrià et stem cells, some of which can be seen in mitosis, appear in al. 1997). In both a macrostomorph (Macrostomum sp.) and the freshwater catenulidans Rhynchoscolex sp., Catenula sp., a triclad (Schmidtea mediterranea), experimental wounding and Stenostomum sp. (Reisinger 1924; Borkott 1970; Ehlers causes first rearrangement and outgrowth of existing longitu- 1992b). Mitotic and S-phase stem cells similarly appear dinal muscles in the body wall to compensate for extirpated in the epidermis of the acoel Convolutriloba longifissura muscles, then differentiation of new circular muscles from (Haszprunar 1996; R. Gschwentner, personal communica- neoblasts (Cebrià and Romero 2001; Salvenmoser et al. tion). Whether these stem cells are a separate population 2001). from the bulk of neoblasts that lie beneath the body wall or have migrated from that population into the epidermis while Conclusions still uncommitted to the differentiated state has not been determined. Despite its lack of defining sclerotic structures such as cu- Stem cells can be identified by immunocytochemical reac- ticle and dermal skeleton, the body wall of platyhelminths is tion to 5-bromo-2-deoxyuridine (BrdU), which, when sup- structurally complex and shows considerable variety in the plied to animals in culture, is incorporated into the nuclei of arrangement of epidermal and muscle cells and in compo- cells in S-phase, i.e., those synthesizing DNA. Such stem nents such as its basal matrix, cell web, ciliary rootlets, and cells are distributed over the whole body in the acoel secretory inclusions and its relationship to gland, sensory, C. longifissura, for instance (Gschwentner et al. 2001, 2003), and nerve cells. The variety is likely indicative of the basal but are localized along the two main longitudinal nerves nature of these worms. Overlap among body wall characters, along the lateral sides of the body in the rhabditophoran such as the netlike or spotty distribution of basal matrix in Macrostomum sp. (Ladurner et al. 2000). Differentiated lower rhabditophorans such as the macrostomorphs and in cells, presumably including epidermal cells, arise from these the Acoela, Nemertodermatida, and Catenulida, provide sup- labeled cells within 3 days in the acoel (Gschwentner et al. port for the monophyly of the platyhelminths despite chal- 2001) and within 14 days in the macrostomid (Ladurner et lenges to this concept of monophyly from phylogenetic al. 2000). Palmberg (1990) used tritiated thymidine to iden- analysis of some molecular sequences. tify neoblasts and their progeny in the macrostomorph Most critical for monophyly is the common mechanism Microstomum lineare and the catenulidan Stenostomum all platyhelminths seem to share for growth and maintenance leucops and found labeled and differentiated epithelial, ex- of the body wall. As Rieger and his collaborators have pro- cretory, nerve, and sensory cells. Also using tritiated thy- posed (Gschwentner et al. 2001; Rieger and Ladurner 2001), midine, Drobysheva (1988) found labeled epidermal cells in only members of this phylum produce, grow, and maintain polyclads. the epidermis through neoblasts. All animals other than So-called pulsatile bodies in the epidermis and parenchy- platyhelminths are presumed to have stem cells within the ma of acoels and nemertodermatids have been interpreted by epidermis to account for its growth and maintenance — some early workers as epidermal replacement cells (i.e., so-called interstitial cells of cnidarians, for example, or basal cells moving into the epidermis), but electron microscopy re- cells in other bilaterians — or to renew through dediffer- veals them to be degenerating epidermal cells in the process entiation and redifferentiation of other cells (Sánchez of being recycled (see literature in Lundin 2001). Though Alvarado 2000; Galliot and Schmid 2002). The basiepider- epidermal replacement cells have not been identified by his- mal stem cells identified in certain acoels and catenulidans tology in acoels, Gschwentner et al. (2001) have confirmed may correspond to interstitial and basal cells, but the bulk of their presence using immunocytochemistry for S-phase cells the neoblasts in even these species lie within the paren- (through BrdU incorporation) in C. longifissura. Also, the chyma, below the body wall (Gschwentner et al. 2001; R.

Gschwentner, personal communication), and it is likely, Vol. II, Part I. Edited by W. Kükenthal and T. Krumbach. Wal- though by no means certain, that this population supplies ter de Gruyter, Berlin. pp. 52–304. those epidermal neoblasts. While we are far from a compre- Cannon, L.R.G., and Joffe, B.I. 2001. The Temnocephalida. In hensive view of the neoblast in all platyhelminth taxa or Interrelationships of the Platyhelminthes. Edited by D.T.J. from knowing whether no other animal phyla have such Littlewood and R.A. Bray. Taylor and Francis, London. pp. 83– cells, current indications are that the stem cell system of 91. neoblasts in platyhelminths is unique to these worms. If so, Cebrià, F., and Romero, R. 2001. Body-wall muscle restoration dy- it is the first identified autapomorphic trait of the Platy- namics are different in dorsal and ventral blastemas during helminthes and thus the first to validate the monophyly of planarian anterior regeneration. Belg. J. Zool. 131(Suppl. 1): the phylum (cf. Smith et al. 1986). Its occurrence may ex- 111–115. Cebrià, F., Vispo, M., Newmark, P., Bueno, D., and Romero, R. plain other morphological similarities among flatworms. 1997. Myocyte differentiation and body wall muscle regeneration in the planarian Girardia tigrina. Dev. Genes Evol. 207: Acknowledgments 306–316. Chien, P.K., and Koopowitz, H. 1977. Ultrastructure of nerve We thank Reinhard Rieger and Robert Gschwentner for plexus in flatworms. III. The infra-epithelial nervous system. valuable suggestions and discussion of their work on stem Cell Tissue Res. 176: 335–347. cells. This material is based upon work supported by the Na- Coil, W.H. 1991. Platyhelminthes: Cestoidea. In Microscopic anat- tional Science Foundation under Grant Nos. 0118804 and omy of the invertebrates. Vol. 3. Platyhelminthes and Nemer- DBI-9977643. tinea. Edited by F.W. Harrison and B.J. Bogitsh. Wiley-Liss Inc., New York. pp. 211–283. 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