Ultrastructure of glands in a scutariellid (Platyhelminthes) and possible phylogenetic implications

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1 FOLIA PARASITOLOGICA 46: , 1999 Ultrastructure of glands in a scutariellid (Platyhelminthes) and possible phylogenetic implications Carlo Iomini 1,2, Marco Ferraguti 3 and Jean-Lou Justine 1 1 Laboratoire de Biologie Parasitaire, Protistologie, Helminthologie, EP 1790 CNRS Biologie et Évolution des Parasites, Muséum national d Histoire naturelle, 61 rue Buffon, Paris cedex 05, France; 2 Present address: Mount Sinai School of Medicine, Department of Cell Biology and Anatomy, 1 Gustave L. Levy Place, Box 1007, New York NY 10029, USA; 3 Dipartimento di Biologia, Università di Milano, Via Celoria 26, Milano, Italy Key words: Platyhelminthes, Temnocephalida, Scutariellidae, ultrastructure, glands Abstract. Subepidermal glands of the body of Troglocaridicola sp. (from the cavernicolous shrimp Troglocaris sp. in eastern Italy) were observed by transmission electron microscopy. The reservoir and duct of the glands are lined with longitudinal microtubules. Membrane-bound granules inside the gland show a distinctive pattern: they contain fibres, 18 nm in diameter, regularly arranged in bundles with a 5 nm space between fibres. From a survey of the available literature on glands of Platyhelminthes, it is concluded that this structure is known only in this species. Glands with regularly arranged 18 nm fibres, if characteristic for the Scutariellidae, could be considered an autapomorphy of this family, distinguishing it from other members of the Temnocephalida. The phylogeny of the Platyhelminthes is currently the subject of several conflicting hypotheses. In addition to traditional morphology, two methods are currently favoured for the research of new characters: molecular systematics, based on sequences of genes such as 18S and 28S rrna (see Littlewood et al. 1999, Mollaret et al. 1997, and references therein), and ultrastructure of various organs such as spermatozoa (Justine 1991, 1995, Watson and Rohde 1995a) and protonephridia (Rohde 1991). The phylogenetic position of the Temnocephalida is a subject of controversy. Traditional classifications (see Williams 1981) assigned them various ranks within the Turbellaria, a group now widely recognized as paraphyletic. In a cladistic interpretation, based on morphological and ultrastructural characters (Brooks 1989a,b, Brooks et al. 1985), the Temnocephalidea was proposed as the sister group for the Neodermata (= Digenea, Monogenea, Cestoda + Udonella). The position of Udonella has been recently reconsidered on the basis of molecular characters and was concluded to be within the monopisthocotylean monogeneans (Littlewood et al. 1998), as it was in certain previous classifications (Baer and Euzet 1961). Moreover, the position of the Temnocephalida as the sister group of the Neodermata has been denied by Rohde (1990), Rohde et al. (1993) and Ehlers and Sopott-Ehlers (1993) on the basis of non-homology of the attachment organs, differences in the protonephridia and DNA sequences. In a recent molecular study of 18S rrna (Littlewood et al. 1999), the Temnocephalida are considered as a member of a large group which is the sister group of the Neodermata; this group also includes the Typhloplanida, Dalyellida, Kalyptorhynchia, Lecithoepitheliata, Fecampiida, Tricladida and Urastoma. Within the Temnocephalida, the position of the Scutariellidae has been the subject of debate. In most traditional classifications, the Temnocephalida (or Temnocephala) are composed of the Temnocephalidae and other families of the Southern hemisphere, and of the Scutariellidae from the Northern hemisphere (Baer 1961). The scutariellids are included within the Temnocephalida on the basis of the presence of tentacles, and also because they share their peculiar way of life, being epibionts on freshwater crustaceans. A synapomorphy for the Temnocephalidae, based on a character of sperm ultrastructure, the presence of an overlapping row of cortical microtubules arranged along a spiral in transverse section, has been proposed from the study of two species of Temnocephala (Justine et al. 1987). This character was later found also in other species and proposed as a synapomorphy of the Temnocephalida (Justine 1991). This synapomorphy was then accepted in a large analysis of the parasitic Platyhelminthes (Brooks and McLennan 1993). The finding of scutariellids (Troglocaridicola sp.) in Italy (Gasparo et al. 1984) provided an opportunity to verify the presence of this synapomorphy in scutariellids. Unexpectedly, this research revealed that the spermatozoon of Troglocaridicola did not show the spiral of microtubules (Iomini et al. 1994), but had a corkscrew structure unknown in other Temnocephalida. Watson and Rohde (1995b) and Watson et al. (1995) described the spermatozoal ultrastructure of several other Temnocephalida. Watson and Rohde (1995b) Address for correspondence: J.-L. Justine, Laboratoire de Biologie Parasitaire, Protistologie, Helminthologie, Muséum national d Histoire naturelle, 61 rue Buffon, Paris cedex 05, France. Phone: ; Fax: ; justine@mnhn.fr 199

2 concluded that the spiral microtubule region was synapomorphic for the Temnocephalidae, Didymorchidae and Actinodactylellidae, and that the character sperm shaft a thin flange or split at the nuclear end was synapomorphic for the Scutariellidae + these three families. Recently, acetylated tubulin was shown to be absent from cortical microtubules of Troglocaridicola sperm, as in digeneans, but in contrast to acoels where it is present (Justine et al. 1998); however, no comparative results are available for temnocephalids. Joffe and Cannon (1998) and Joffe et al. (1998) considered that the Scutariellidae is the sister group to the other Temnocephalida. Therefore, the description of any character of possible phylogenetic significance in a scutariellid, such as the subepidermal glands dealt with in this paper, is of interest for the understanding of the relationships of the scutariellids and temnocephalids. It must be noted, also, that no gene sequence is available yet for a scutariellid. MATERIALS AND METHODS Troglocaridicola sp. specimens were removed from the carapace and gill cavity of cavernicolous shrimps (Troglocaris sp.) collected from caves in eastern Italy (cave of Comarie, VG 4221, comune di Doberdò del Lago, and cave of Sagrado, VG 4112, comune di Sagrado, Province of Gorizia). Specimens were fixed in paraformaldehyde-picric acidglutaraldehyde fixative (Ermak and Eakin 1976) for 24 hr, then rinsed in 0.1 M sodium cacodylate, ph 7.4 buffer, and postfixed in 1% OsO 4 in the same buffer, rinsed again several times in distilled water and stained with saturated aqueous uranyl acetate, for 2 hr in the dark at 4 C, rinsed in distilled water, dehydrated in an ethanol series, and embedded in Spurr s resin. Grids were stained with lead citrate and uranyl acetate, and observations were performed with a JEOL 100 SX and a Philips 201 microscope. OBSERVATIONS Several subepidermal glands were seen in the middle body of the worm, close to the testis. The glands show a duct lined with longitudinal microtubules and a reservoir (Fig. 1). The duct passes through the epidermis, which is relatively electron-dense and contains nuclei; the reservoir is located below the basal lamina of the epidermis (Fig. 1). The gland contains granules, apparently limited by a membrane, which is sometimes not perfectly visible (Figs. 2-4). In contrast to many glands in other animals which contain an amorphous material, the granules contain a highly organized material. Longitudinal sections show parallel fibres, which are straight (Fig. 3) or curved (Fig. 2). Transverse sections show that fibres, 18 nm in diameter, are arranged along parallel lines, disposed at angles of 60 to each others, with the result that each fibre is in the centre of a hexagon limited by six other fibres (Figs. 4-5). Spaces between fibres are about 5 nm; 4.5 fibres can be counted in a 100nm space. Thin links between fibres were also seen (Fig. 5). In addition to these highly organised fibres, the granules contain an amorphous material (Figs. 2, 4). DISCUSSION We reviewed available literature to find if structures equivalent to the highly organised pattern observed in the glands of Troglocaridicola had been found in other species. Glands have been described in several species of temnocephalids, including Temnocephala novaezealandiae (Williams 1980, 1988, 1994, Williams and Ingerfeld 1988), Temnocephala minor (Cannon and Watson 1996, Xylander 1997), Didymorchis sp. (Joffe et al. 1995a, Rohde and Watson 1990), Notodactylus handschini (Jennings et al. 1992) and Diretocephala boschmai (Joffe et al. 1995b). The literature on glands of phylogenetically related groups, such as Kalyptorhynchia (e.g. Schizochilus caecus by Ehlers 1989) Typhloplanida (e.g. Bothromesostoma by Martínez-Alós et al. 1991, 1994), Fecampiida (e.g. Kronborgia by Williams 1990a,b), Dalyelliida (e.g. Pterastericolidae by Jondelius 1992, Paravortex by Noury-Sraïri et al. 1989) and Urastoma (Noury-Sraïri et al. 1990) was also surveyed. No similar structure was found in any of these published papers and a number of other papers about glands of Platyhelminthes which we surveyed. The possibility of a fixation artefact is low in our material, since the fixation used did not produce aberrant patterns in other structures (Iomini et al. 1994). Therefore, the presence of glands with regularly arranged 18 nm fibres can be considered as a character of possible phylogenetic significance and can be preliminarily proposed as an autapomorphy for the Scutariellidae. An autapomorphy is of limited use for understanding phylogenetic relationships. It is however worthy of description, because such a structure could be found later in another species, thus possibly leading to phylogenetic implications. In the present context of discussion of the relationships between scutariellids and temnocephalids (Joffe and Cannon 1998) and between these families and other groups (Littlewood et al. 1999), additional data on gland ultrastructure are needed from other species. Acknowledgements. Fulvio Gasparo collected the animals, and Dr. Stuart Gelder identified the specimens. Dr. Tim Littlewood communicated a manuscript in press. Prof. Klaus Rohde and Prof. Ulrich Ehlers commented on photographs at an early stage of preparation of this paper. Prof. Barrie G. M. Jamieson kindly edited the English. 200

3 Iomini et al.: Ultrastructure of glands of a scutariellid Figs Glands in Troglocaridicola sp. (Scutariellidae). Fig. 1. Longitudinal section of gland showing aperture, duct and reservoir. Fig. 2. Transverse section of reservoir; note microtubules lining the periphery. Fig. 3. Parallel fibres, longitudinal section. Fig. 4. Fibres, transverse section; note presence of amorphous material in the granule. Fig. 5. High magnification of fibres, transverse section. Scale bars: Figs. 1, 2 = 1 µm; Figs. 3, 4 = 200 nm; Fig. 5 = 100 nm. 201

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