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Syst Parasitol (2009) 74:81–93 DOI 10.1007/s11230-009-9195-5

The unique ultrastructure of the uterus of the Gyrocotylidea Poche, 1926 (Cestoda) and its phylogenetic implications Larisa G. Poddubnaya Æ Roman Kuchta Æ Ce´line Levron Æ David I. Gibson Æ Toma´sˇ Scholz

Received: 27 February 2009 / Accepted: 19 May 2009 Ó Springer Science+Business Media B.V. 2009

Abstract The members of the order Gyrocotylidea are monozoic tapeworms and generally considered to be the most primitive group of the Cestoda in terms of the evolution of this platyhelminth class. As part of a series of ultrastructural studies on Gyrocotyle urna (Wagener, 1852), three regions of the uterus were distinguished. The proximal region of the uterus is characterised by underlying perikarya, the presence of septate junctions within the epithelial wall and two types of specialised outer coverings, lamellae and cilia. The middle, syncytial region of the uterus is covered by short lamellae and marked by a concentration of sunken glandular perikarya (uterine glands). Glandular spheroidal granules (c.0.45 lm in diameter) of moderately dense content and a fine fibrillar texture L. G. Poddubnaya (&) Institute of Biology of Inland Waters, Russian Academy of Sciences, 152742 Borok, Yaroslavl Province, Russia e-mail: [email protected] R. Kuchta C. Levron T. Scholz Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branisˇovska´ 31, 370 05 Cˇeske´ Budeˇjovice, Czech Republic R. Kuchta T. Scholz Faculty of Science, University of South Bohemia, Branisˇovska´ 31, 370 05 Cˇeske´ Budeˇjovice, Czech Republic D. I. Gibson Department of Zoology, Natural History Museum, Cromwell Road, London SW7-5BD, UK

are liberated by migration through the luminal membrane. The epithelium of the sac-shaped, distal region of the uterine duct is interrupted by cytoplasmic processes of sunken epithelial bodies, covered with lamellae and contains septate junctions. A muscular sphincter surrounds the narrow, terminal region of the distal uterine duct. The ultrastructural pattern of the uterus of the Gyrocotylidea has important discriminating traits (i.e. the presence of sunken perikarya along its entire length, septate junctions within the uterine epithelial cytoplasm of the proximal and distal regions, and cilia on the surface of its proximal region) unique among the Neodermata and which may represent autapomorphic character states for the group.

Introduction The monozoic tapeworms of the order Gyrocotylidea Poche, 1926 (Cestoda) occupy, in evolutionary terms, an ancient, but controversial position in relation to their phylogenetic links with other groups within the Neodermata, and especially with the Cestoda (Olson et al., 2001, 2008; Lockyer et al., 2003; Waeschenbach et al., 2007). Recently, the unique ultrastructural characters of the surface structures and the adhesive glands of the attachment posterior rosette organ of the Gyrocotylidea have been revealed by the work by Poddubnaya et al. (2006, 2008). The cellular organisation of the female reproductive system represents a

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very important character in the evolutionary history of the Platyhelminthes (Karling, 1940; S´widerski & Xylander, 2000). To date, detailed ultrastructural studies of the female system of the Gyrocotylidea are limited to those of Xylander (1987) on vitellogenesis; such investigations of the uterus are therefore lacking. The light microscopical observations of Watson (1911), Lynch (1945) and Bandoni and Brooks (1987) show that two parts of the uterus of gyrocotylideans can be distinguished, i.e. a proximal, spirally coiled portion and a terminal saccate portion. The latter, termed the ‘uterine sac’, is large and occupies a quarter or more of the total uterine extent. The separate uterine pore is located on the ventral surface of the body close to its anterior end. Gulyaev (2002), on the basis of the arrangement and topographical interrelationships of the genital pores of the Gyrocotylidea and the lower cestode orders, has considered that the retention of an independent genital pore and its anterior position, as occurs in the Gyrocotylidea, as the most primitive situation in terms of cestode evolution. The aim of this study is to investigate the ultrastructure of the uterus of a member of the Gyrocotylidea, Gyrocotyle urna (Wagener, 1852). The uterine characters of this parasite are compared with similar structures of other neodermatan groups in search of features useful for phylogenetic investigations of the relationships of the gyrocotylideans with other members of the Neodermata.

Syst Parasitol (2009) 74:81–93 Fig. 1 Proximal region of the uterus of Gyrocotyle urna: a. c low magnification of the uterine epithelium with sunken perikarya; b. portion of the egg-shell bounded by cilia embedded in the external coat, with the epithelial lining showing the luminal structures (cilia and lamellae)—note the epithelium resting on the extracellular basal matrix and surrounded by muscle fibres; c. epithelium with two kinds of luminal covering and an underlying perikaryon; d. epithelial lining showing a cross-section of the basal bodies of the cilia, the extracellular matrix under the epithelium and a septate junction within the epithelial lining; e. septate junction; f. cross-section of a cilium showing peripheral and central microtubule doublets; g. longitudinal section of a cilium showing the embedded basal body and basal plate—note the apical lamellae. Abbreviations: BB, basal body of a cilium; BM, basement matrix; BP, basal plate of a cilium; Cl, cilium; EC, external coat on the luminal surface of the proximal region; EL, epithelial lining; Lm, lamellae; MF, muscle fibres; Mt, microtubules of a cilium; N, nucleus; PC, cytoplasm of a perikaryon; SJ, septate junction. Scale-bars: a, 10 lm; b, c, 2 lm; e–f, 0.5 lm; g, 1 lm

dehydrated through an ethanol series and embedded in paraffin. Light microscope 12 lm sections were stained with haematoxylin and eosin, then mounted in Canada balsam and examined using an Olympus BX-51 microscope. Results We distinguished three regions of the uterus of Gyrocotyle urna based on our TEM observations (Fig. 4a). Proximal region of the uterus

Materials and methods Specimens of Gyrocotyle urna were removed from the spiral valve of a rabbit fish, Chimaera monstrosa L. (Holocephali), caught in the North Sea off Bergen, Norway. The worms were fixed entire using 5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 40 days at 5°C, rinsed 4 times for 20 min periods in the same buffer and postfixed in 1% osmium tetroxide for 1 h. The material was then dehydrated in a graded series of alcohol and acetone, and embedded in mixture of Araldite and Epon. Ultrathin sections (70–90 nm in thickness) were stained with uranyl acetate and lead citrate, and examined using JEM 1011 transmission electron microscopes operating at 80 kV. For histological procedures G. urna specimens were fixed with hot 4% formaldehyde solution,

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The proximal region of the uterus, arising from the oo¨type, includes the first few coils of the uterine duct and is characterised by underlying perikarya, the presence of septate junctions within the epithelial wall and two types of specialised outer covering (Fig. 1). The uterine epithelial lining is surrounded by an extracellular (basement) matrix and muscle fibres (Fig. 1a–d). The perikarya lie deep beneath the basement matrix, possess irregulary shaped nuclei and are connected to the uterine lining by cytoplasmic processes (Fig. 1a, c). Both the perinuclear cytoplasm and these cytoplasmic processes contain the same types of bodies produced by the perikarya and which fill the epithelial lining of the uterus (Fig. 1c–d). The thin epithelial lining of the proximal uterine region (c.0.3 lm in thickness) is delimited by septate junctions (Fig. 1d, e).

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The luminal uterine surface exhibits two types of the projections into the lumen, lamellae and cilia (Fig. 1a–c, g). The lamellae (c.1 lm in the length) project into the lumen (Fig. 1b, c, g), and the basal bodies of the cilia are embedded in the uterine epithelial lining (Figs. 1b, d, g). The wall of the cylindrical basal body of each cilium consists of electron-dense material (Fig. 1d, g). In longitudinal sections of the cilium (Fig. 1b, g), a basal plate can be seen between the basal body and the proximal region of the cilium. Each cilium contains nine peripheral microtubule doublets that are located at an equal distance from the two microtubules of the central doublet (Fig. 1f). The cilia are c.8 lm in length and directed towards the eggs within the uterine lumen (Fig. 1b). Along most of the length of the proximal uterus, there is a loose external coat on its luminal surface within which the surface structures are embedded (Fig. 1a, b). The thickness of the coat is comparable with the length of the surface cilia (Fig. 1a, b). Luminal eggs that are localised along the epithelial lining are bounded by cilia that arise from the external coat (Fig. 1b). Middle region of the uterus The middle region of the uterus is demarcated distally by the sac-shaped uterus (‘uterine sac’), and it is marked by a dense concentration of glandular perikarya, which are localised immediately beneath the uterine wall (Fig. 2a, c, e). The uterine epithelium of this region is covered by short lamellae (Fig. 2d) and possesses a layer of cytoplasm, resting on the thin extracellular matrix, and muscular fibres (Fig. 2c, d). No specialised contacts were seen within the epithelial wall of this middle region. The gland-cells are characterised by numerous spheroidal granules present in various locations in their cytoplasm (Fig. 2c). Different staining intensities of the granule matrix sometimes occur between different granules within the same cell (Fig. 2c). The granules are c.0.45 lm in diameter and usually have a moderately electron-dense content with a fine fibrillar texture (Fig. 2b–d). The relatively dense perinuclear cytoplasm of the uterine glands exhibits enlarged cisternae of rough endoplasmic reticulum, which is fairly extensive and distributed throughout the cytoplasm, and Golgi complexes associated with secretory granules (Fig. 2b, c).

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Syst Parasitol (2009) 74:81–93 Fig. 2 Middle glandular region of the uterus of Gyrocotyle c urna: a. concentration of glandular perikarya below the epithelial lining; b. formation of uterine secretory granules within the cytoplasm of the perikarion; c. sunken uterine gland with a great number of granules within its cytoplasm and a cytoplasmic bridge to the uterine epithelial lining; d. epithelial lining of the uterus with granules migrating through the luminal membrane; e. luminal eggs adjacent to the uterine epithelial lining; f. part of the dense egg-shell close to the uterine lining and bounded by a heterogeneous covering. Abbreviations: C, coat around the eggs; CP, cytoplasmic process; GC, Golgi complex; GP, glandular perikaryon; E.g, egg; EL, epithelial lining; Lm, lamellae; MF, muscle fibres; N, nucleus; RER, rough endoplasmic reticulum; SG, secretory granules. Scalebars: a, 10 lm; b, 0.5 lm; c, 5 lm; d, f, 1 lm; e, 50 lm

Cytoplasmic processes connect the perikarya with the epithelial lining (Fig. 2c). The granules are transported through these cell processes into the uterine lining. Numerous granules are visible along the whole length of the middle region of the uterine epithelium (Fig. 2c, d). Different degrees of discharge of the secretory granules through the uterine epithelium are shown in Fig. 2d. The eggs within the uterine lumen are adjacent to the uterine epithelial lining (Fig. 2e, f). The discharged granular secretion is concentrated around the eggs to form a coat that may reach c.2 lm in thickness and has a heterogeneous internal structure (Fig. 2f). Distal region of the uterus The distal region of the uterus forms a large sac-shaped uterine duct (the ‘uterine sac’), which terminates in the form of a narrow duct strongly supported by a muscular sphincter containing numerous close-packed muscle fibres (Fig. 3a, b, f). These muscles are embedded in the extracellular matrix (Fig. 3f) and form the muscle complex on both sides of the narrow duct (Fig. 3a). Nerve fibres, containing large dense vesicles, are interspersed within these complexes (Fig. 3f). The epithelial lining of this region of the uterus is attached to a basal extracellular matrix, which is surrounded by muscle layers (Fig. 3c, d). The epithelium is interrupted by cytoplasmic processes arising from sunken epithelial cytons (Fig. 3d). Three epithelial components (epithelial lining, cytoplasmic processes and cell bodies) contain a dense cytoplasm filled with numerous vesicles of various density and c.0.1 lm in diameter (Fig. 3c–e). A number of septate junctions were observed within the uterine


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epithelial lining (Fig. 3e).The epithelial surface of the uterine sac bears luminal lamellae (c.0.75 lm in length), which are shorter than are those in proximal uterine region (Fig. 3c–e).

Discussion Ultrastructural features of the uterus of the Gyrocotylidea The fine structure of the uterus, as described above for Gyrocotyle urna, represents another step toward answering questions on the phylogeny of the Gyrocotylidea. The important discriminating traits of the ultrastructural pattern of the uterus of this group are the presence of sunken perikarya along the entire length of the uterus, septate junctions within the uterine epithelial cytoplasm, excluding its middle, glandular region, and cilia on the surface of its proximal region. Several interesting observations can be made based on comparative analyses of the uterine ultrastructure of Gyrocotyle with that of other neodermatan groups for which information is available in the literature (Table 1). Existing knowledge of the relationships between gyrocotylideans and other neodermatans In the past, the kinship of gyrocotylideans with other members of the Neodermata, including the ‘‘true’’ tapeworms (Eucestoda), has been a controversial topic. They were recognised as a class with affiliation to both monogeneans and cestodes by Bychowsky (1957), Ginetsinskaya & Dobrovolskii (1978) and Galkin (1999), whereas other authors have considered gyrocotylideans to be members of the Monogenea (Lynch, 1945; Llewellyn, 1965, 1986) or, together with Amphilinidea, as the Cestodaria (Wardle & McLeod, 1952; Joyeux & Baer, 1961), a sister group to the Eucestoda. Historically, most authors have considered gyrocotylideans to be cestodes (Brooks et al., 1985; Ehlers, 1985; Gibson, 1994; Rohde, 1994; Xylander, 2001). Authors, such as Ivanov (1991) and Rohde et al. (1995), have considered that the Gyrocotylidea, Amphilinidea and Eucestoda form a monophylum. In recent years, molecular data have

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Syst Parasitol (2009) 74:81–93 Fig. 3 Distal uterine region of Gyrocotyle urna: a. histological c sections showing part of the sac-shaped uterine duct and its terminal region, a narrow duct supported by a dense muscular sphincter; b. muscular complex with numerous close-packed muscle fibres; c. epithelial lining covered with lamellae and surrounded by an extracellular basement matrix and muscle fibres; d. sunken epithelial cytons connected to the epithelium by cytoplasmic processes; e. septate junction within the epithelial lining; f. part of the muscular complex. Abbreviations: BM, basement matrix; CP, cytoplasmic process; EL, epithelial lining; EP, epithelial perikaryon; Lm, lamellae; MC, muscular complex; MF, muscle fibres; MS, muscular sphincter; NC, nerve cell; NF, nerve fibres; SJ, septate junction; SU, sacshaped uterine duct. Scale-bars: a, 500 lm; b, 20 lm; c, e, 1 lm; d, f, 2 lm

confirmed that the Gyrocotylidea is the most basal order of tapeworms (Olson et al., 2001, 2008; Lockyer et al., 2003; Waeschenbach et al., 2007). Comparative ultrastructure of the uterus of the Gyrocotylidea and members of the Eucestoda The localisation of the nuclei The members of the Eucestoda possess nuclei within the epithelial layer of the uterus rather than having sunken perikarya along the entire length of the uterus (i.e. along all three morphologically distinct regions of the uterus of G. urna). A nucleated epithelial lining has been observed within the uterus of all eucestodes whose fine structure has been examined, e.g. in caryophyllideans (Davydov et al., 1994; Poddubnaya, 2003b), spathebothriideans (Poddubnaya et al., 2005), bothriocephalideans (Korneva, 2002; Poddubnaya, 2003a), diphyllobothriideans (Poddubnaya, 2002), proteocephalideans (Korneva & Davydov, 2001; Korneva, 2001), nippotaeniideans (Davydov & Korneva, 2000), and also in linstowiid (Conn & Etges, 1984) and hymenolepidid (Conn,1993; Conn & Forman, 1993) cyclophyllideans. On the other hand, it should be noted that the distal uterus lining the paruterine organ of the tapeworm Mesocestoides lineatus (Goeze, 1782) differs from all of these by having nucleated cytons withdrawn into the parenchyma and connected to the anucleated cytoplasm layer via processes (Conn, 1987). The tegumental type of the architecture of the uterine epithelium has been revealed for the distal region of the uterus of the spathebothriidean Cyathocephalus truncatus (Pallas, 1781) (see Poddubnaya et al., 2005)


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Table 1 Ultrastructural features of the uterus of the parasitic platyhelminth groups Traits

Gyrocotylidea

Amphilinidea

Eucestoda

Monogenea

Digenea

Aspidogastrea

Intraepithelial location of nuclei

No

No

Yes

Yes

Yes

Yes

Sunken perikarya

Yes

Yes

No1

No

No

No

Syncytial epithelial lining Presence of septate junctions

No2 Yes

Yes No

Yes No

Yes No

No Yes

No Yes

Apical lamellae

Yes

No

Yes

Yes

Yes

Yes

Apical cilia

Yes

No

No

No

No

No

1

Exceptions are the uterine glands of the: middle uterus of the Caryophyllidea and Spathebothriidea; distal uterus of Cyathocephalus truncatus (Spathebothriidea) and Diphyllobothrium latum (Diphyllobothriidea); and distal uterus (paruterine organ) of Mesocestoides lineatus (Cyclophyllidea)

2

Exception: the uterine glands of the middle uterus of Gyrocotyle urna

and the diphyllobothriidean Diphyllobothrium latum (Linnaeus, 1758) (see Poddubnaya, 2002), in which the distal uterus is continuous with the tegumental lining of main body and is covered with microtriches. Underlying uterine glands of the middle uterus Another important feature of the uterine structure of Gyrocotyle is the presence of uterine glands in the middle region of the uterus, as described above. Cytoplasmic bridges that unite with the uterine lining (the secretion is liberated by an apocrine mechanism) pass down the secretory granules from these numerous gland-cells. The presence of such uterine glands is rare in the Platyhelminthes and has only been described in the middle uterus of two basal orders of the tapeworms, the Caryophyllidea (Davydov & Poddubnaya, 1988; Davydov et al., 1994; Poddubnaya, 2003b; Poddubnaya et al., 2003) and the Spathebothriidea (Davydov et al., 1997; Poddubnaya et al., 2005). Despite the basic similarity between the middle uterine epithelium of these three groups of worms, they differ in other ultrastructural characteristics. The epithelial lining in the Gyrocotylidea and the Caryophyllidea is anucleate, whereas that of the Spathebothriidea is nucleate. The functional significance of these glands in monozoic gyrocotylideans may be the same as in the monozoic caryophyllideans and the polyzoic, but nonsegmented spathebothriideans. The chemical nature of the secretory products of uterine glands has been studied by histochemical methods for the caryophyllidean Caryophyllaus laticeps (Pallas, 1781) (see Davydov & Poddubnaya, 1988) and the spathebothriidean Diplocotyle olrikii Krabbe, 1874 (see Davydov

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et al., 1997). These authors have suggested that the material secreted by the uterine glands produces a protective, lipoproteinaceous fibrous coat on the egg surface, as has also been described for the caryophyllidean Archigetes sieboldi Leuckart, 1878 (see Poddubnaya et al., 2003b), the spathebothriidean Cyathocephalus truncatus (see Poddubnaya et al., 2005) and the gyrocotylidean G. urna (present study). It is interesting to note that a nucleate surface layer of the gravid uterus of hymenolepidid tapeworms (Cyclophyllidea) also produces material that contributes to the protection of the eggs (Conn & Etges, 1984; Conn & Forman, 1993). Luminal surface structures of the uterus The proximal region of the uterine tube of Gyrocotyle is characterised by a specialised outer covering with two types of the surface structures, lamellae and cilia, whereas the middle and distal regions of the uterus are covered with lamellar processes. The apical plasma membrane of the uterine epithelium of most members of the Eucestoda protrudes into the uterine lumen in the form of thin lamellae (Conn & Etges, 1984; Davydov & Poddubnaya, 1988; Conn, 1993; Conn & Forman, 1993; Davydov et al., 1994; Davydov et al., 1997; S´widerski & Tkach, 1997; Davydov & Korneva, 2000; Korneva, 2001, 2002; Korneva & Davydov 2001; Poddubnaya 2002, 2003a, b; Poddubnaya et al., 2005). Parallel with apical uterine lamellae, which are a consistent feature of reproductive epithelia of platyhelminths (Conn & Forman, 1993), microtriches have been shown to line the distal region of the uterus of two tapeworms, the diphyllobothriidean Diphyllobothrium latum (see Poddubnaya, 2002) and the


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spathebothriidean Cyathocephalus truncatus (see Poddubnaya et al., 2005). Nevertheless, judging from the limited information available, it appears that the presence of cilia on the luminal surface of the proximal uterine region of Gyrocotyle may be unique as a surface feature of the uterine epithelium for the members of the Neodermata. Moreover, most previous workers (e.g. Watson, 1911) have supported the view, based on light microscopy, that the lining of the uterus of Gyrocotyle is covered with cilia for the first five or six coils.

(Rodgers & Kuntz, 1940) and Neodiplorchis scaphiopodis (Rodgers, 1941) is a thin, nucleated syncytium resting on the basal matrix. In G. kobayashii the uterine epithelium may be ornamented by lamellar projections, but such elaborations may be absent in the same species (Jones et al., 1998). The luminal structures of the uterine epithelium of P. americanus and N. scaphiopodis are numerous fine cytoplasmic strands (Cable & Tinsley, 1991). Septate junctions were only noted between the uterine epithelium and the oviduct in G. kobayashii (see Jones et al., 1998).

The specialised contacts within the uterine epithelium The marked similarities of the uterine epithelium of members of the Eucestoda include its syncytial structure, with the nuclei occupying a syncytial uterine epithelium, and the total lack of septate junctions (Conn & Etges, 1984; Davydov & Poddubnaya, 1988; Conn, 1993; Conn & Forman, 1993; Davydov et al., 1994; Davydov et al., 1997; S´widerski & Tkach, 1997; Jones, 1998; Davydov & Korneva, 2000; Korneva, 2001, 2002; Korneva & Davydov, 2001; Poddubnaya, 2002, 2003a, b; Poddubnaya et al., 2005). Consequently, the presence of septate junctions within the proximal and distal regions of the uterine epithelium of Gyrocotyle is completely different to the situation in the Eucestoda.

The ultrastructural traits of the uterus of the members of the Digenea

The ultrastructural traits of the uterus of the members of the Amphilinidea No investigations at the electron microscope level appear to have been made on the uterus in amphilinideans, and, in this connection, we use our own unpublished data on the uterine structure of Amphilina foliacea (Rudolphi, 1819). The uterus of this species is recognised by the presence of a syncytial epithelial lining with underlying syncytial epithelial bodies and luminal projections of different kinds. The ultrastructural traits of the uterus of the members of the Monogenea From the limited information on the fine morphology of the monogenean uterus (Cable & Tinsley, 1991; Jones et al., 1998), it appears that the uterine epithelium of a monopisthocotylidean, Gyrodactylus kobayashii Hukuda, 1940, and the polyopisthocotylidean polystomatids Pseudodiplorchis americanus

Characteristics of the digenean uterus have been demonstrated in various families that have been studied using transmission electron microscopy. It seems that apical lamellae of the uterine epithelium are a common ultrastructural trait of this group. With regard to the position of the uterine epithelial cytons, they can either be incorporated within the epithelial lining or withdrawn into the parenchyma. So, in Schistosoma margrebowiei Le Roux, 1933 and S. mansoni Sambon, 1907, the uterine epithelium incorporates sunken cell bodies and has been considered as a modified invagination of the tegument (Erasmus, 1973; Awad & Probert, 1990). Conversely, in other digenean species from other and unrelated families, such as Cryptocotyle lingua (Creplin, 1825) (see Rees, 1979), Quinqueserialis quinqueserialis Barker & Laughlin, 1911 (see Wittrock, 1982), Prosthodendrium ascidia (van Beneden, 1873) (see Podvyaznaya, 1990) and Allassogonoporus amphoraeformis (Modlinger, 1930) (see Podvyaznaya, 2003), the nucleated, not syncytial, uterine epithelium is a continuation of the oviduct/oo¨type. However, a syncytial uterine epithelium has been demonstrated for Philophthalmus rhionica Tchomirov, 1976 (see Galaktionov & Dobrovolskii, 1987). In some digenean groups, the proximal region of the uterus forms a sperm store (uterine seminal receptacle) (Gibson & Bray, 1979; Thulin, 1982), and in most groups the distal extremity of the uterus forms a muscular metraterm (Galaktionov & Dobrovolskii, 1987). In the distal uterus close to the metraterm of A. amphoraeformi, Podvyaznaya (2003) showed the presence of desmosomes. There appear to be no other records of contact between the cells of the uterine

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Fig. 4 Diagrammatic representation of the arrangement of the uterus of the neodermatan groups: a. Gyrocotylidea (I—proximal region, II—middle region, III—distal region); b. Eucestoda and Monogenea; c. Aspidogastrea and Digenea; d. Amphilinidea. Abbreviations: Cl, cilium; CP, cytoplasmic process; EL, epithelial lining; EP, epithelial perikaryon; Lm, lamellae; LP, luminal projections; N, nucleus; SJ, septate junction

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epithelium, although Rees (1979) mentioned the attachment zone between two component cells of 38 day-old metacercariae of C. lingua, and diagrammatic representations of the uterine epithelial lining of some digeneans clearly show cellular junctions between uterine epithelial cells (Podvyaznaya, 1990, 2003). The ultrastructural traits of the uterus of the members of the Aspidogastrea With regard to the uterus of members of the Aspidogastrea, a sister group of the Digenea and presumably basal group of the Trematoda (Gibson, 1987; Roberts & Janovy, 2005), no ultrastructural data on the uterus are available. Therefore, we compare our own unpublished data on the fine structure of the uterus of Aspidogaster limacoides Diesing, 1835, the uterine wall of which is characterised by the luminal lamellae, intraepithelial nuclei and the presence of septate junctions conjoining epithelial cells. The distal end of the uterus of aspidogastreans has powerful muscles in its wall, as does the metraterm (Roberts & Janovy, 2005). The ultrastructural arrangements of the uterus of the neodermatan groups At the electron microscopic level, the organisation of the uterus of the neodermatan groups follows four main patterns: (1)

(2)

(3)

(4)

epithelial lining with an intraepithelial localisation of the nucleus and the presence of septate junctions conjoining epithelial cells (Aspidogastrea and most Digenea) (Fig. 4c); syncytial epithelial lining with an intraepithelial localisation of the cell bodies (Eucestoda and Monogenea) (Fig. 4b); cell bodies withdrawn into the parenchyma and septate junctions present within the uterine epithelial lining (Gyrocotylidea) (Fig. 4a); syncytial epithelial wall with sunken perikarya (Amphilinidea) (Fig. 4d).

The different ultrastructural characters of the uterus may have a potential value useful for the assessment of the phylogenetic relationships of the various groups of parasitic Platyhelminthes.

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Phylogenetic implications The uterine pattern of the Gyrocotylidea is unique among the Neodermata and may represent an autapomorphic character state of the former group. Molecular data have provided strong evidence that the Gyrocotylidea is the most ancient group of cestodes (Olson et al., 2001, 2008). This group has developed, during its long evolutionary history, several unique features present neither in other tapeworms nor in other groups of free-living or parasitic flatworms (Platyhelminthes). The structural pattern of the uterine glands of the middle region of the uterus of Gyrocotyle, as observed in the present study, may be considered as a synapomorphy of three of the four most basal cestode orders, i.e. the Gyrocotylidea, Caryophyllidea and Spathebothriidea. The presence of surface lamellae is a common feature of cestodes, digeneans, monogeneans and gyrocotylideans, but the presence of apical cilia on the uterine epithelium appears to be an autapomorphy of the Gyrocotylidea. The presence of intercellular contacts within the uterine epithelium is a common feature of digeneans, aspidogastreans and gyrocotylideans, but does not occur in other cestode groups examined. The above-mentioned distribution of presumably homologous features in different neodermatan groups tends to support the idea that gyrocotylideans really are basal to the Cestoda and evolved much earlier than other groups of cestodes (i.e. Amphilinidea and Eucestoda). They are certainly features worthy of consideration in any future realignment of the relationships and status of this group. These data also add weight to the somewhat surprising suggestion that trematodes may be more closely related to cestodes than monogeneans, a hypothesis recently suggested by data on the mitochondrial genome of parasitic flatworms (Park et al., 2007). Acknowledgments The present study was supported by the Russian Foundation for Fundamental research project no.0904-00342a and by the American Society of Parasitologists Boris Kuperman Memorial Fund to L.P. during 2007. It was also partly supported by the Grant Agency of the Czech Republic (Projects Nos 524/08/0885 and 524/07/P039) and the Institute of Parasitology, Czech Academy of Sciences (Projects Nos Z60220518 and LC522). The authors would like to thanks the staff of the Centre of Electron Microscopy, Institute of Biology of Inland Waters, Borok, Russia.

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