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199 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, 75231 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, 20133 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, 75231 Paris cedex 05, France. Phone: ++ 33 1 40 79 35 03; Fax: ++ 33 1 40 79 34 99; E-mail: [email protected] FOLIA PARASITOLOGICA 46: 199-203, 1999

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Page 1: Ultrastructure of glands in a scutariellid ...folia.paru.cas.cz/pdfs/fol/1999/03/08.pdf · 199 Ultrastructure of glands in a scutariellid (Platyhelminthes) and possible phylogenetic

199

Ultrastructure of glands in a scutariellid (Platyhelminthes) andpossible 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, 75231 Paris cedex 05, France;

2 Present address: Mount Sinai School of Medicine, Department of Cell Biology and Anatomy, 1 Gustave L. Levy Place, Box1007, New York NY 10029, USA;

3 Dipartimento di Biologia, Università di Milano, Via Celoria 26, 20133 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 easternItaly) were observed by transmission electron microscopy. The reservoir and duct of the glands are lined with longitudinalmicrotubules. 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 ofPlatyhelminthes, it is concluded that this structure is known only in this species. Glands with regularly arranged 18 nm fibres, ifcharacteristic for the Scutariellidae, could be considered an autapomorphy of this family, distinguishing it from other membersof the Temnocephalida.

The phylogeny of the Platyhelminthes is currentlythe subject of several conflicting hypotheses. Inaddition to traditional morphology, two methods arecurrently favoured for the research of new characters:molecular systematics, based on sequences of genessuch as 18S and 28S rRNA (see Littlewood et al. 1999,Mollaret et al. 1997, and references therein), andultrastructure of various organs such as spermatozoa(Justine 1991, 1995, Watson and Rohde 1995a) andprotonephridia (Rohde 1991).

The phylogenetic position of the Temnocephalida isa subject of controversy. Traditional classifications (seeWilliams 1981) assigned them various ranks within theTurbellaria, a group now widely recognized asparaphyletic. In a cladistic interpretation, based onmorphological and ultrastructural characters (Brooks1989a,b, Brooks et al. 1985), the Temnocephalidea wasproposed as the sister group for the Neodermata (=Digenea, Monogenea, Cestoda + Udonella). Theposition of Udonella has been recently reconsidered onthe basis of molecular characters and was concluded tobe within the monopisthocotylean monogeneans(Littlewood et al. 1998), as it was in certain previousclassifications (Baer and Euzet 1961). Moreover, theposition of the Temnocephalida as the sister group ofthe 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 attachmentorgans, differences in the protonephridia and DNAsequences. In a recent molecular study of 18S rRNA(Littlewood et al. 1999), the Temnocephalida areconsidered as a member of a large group which is thesister group of the Neodermata; this group also includes

the Typhloplanida, Dalyellida, Kalyptorhynchia,Lecithoepitheliata, Fecampiida, Tricladida andUrastoma.

Within the Temnocephalida, the position of theScutariellidae has been the subject of debate. In mosttraditional classifications, the Temnocephalida (orTemnocephala) are composed of the Temnocephalidaeand other families of the Southern hemisphere, and ofthe Scutariellidae from the Northern hemisphere (Baer1961). The scutariellids are included within theTemnocephalida on the basis of the presence oftentacles, and also because they share their peculiar wayof life, being epibionts on freshwater crustaceans. Asynapomorphy for the Temnocephalidae, based on acharacter of sperm ultrastructure, the presence of anoverlapping row of cortical microtubules arrangedalong a spiral in transverse section, has been proposedfrom the study of two species of Temnocephala (Justineet al. 1987). This character was later found also in otherspecies and proposed as a synapomorphy of theTemnocephalida (Justine 1991). This synapomorphywas then accepted in a large analysis of the parasiticPlatyhelminthes (Brooks and McLennan 1993). Thefinding of scutariellids (Troglocaridicola sp.) in Italy(Gasparo et al. 1984) provided an opportunity to verifythe presence of this synapomorphy in scutariellids.Unexpectedly, this research revealed that thespermatozoon of Troglocaridicola did not show thespiral of microtubules (Iomini et al. 1994), but had acorkscrew structure unknown in other Temnocephalida.Watson and Rohde (1995b) and Watson et al. (1995)described the spermatozoal ultrastructure of severalother Temnocephalida. Watson and Rohde (1995b)

Address for correspondence: J.-L. Justine, Laboratoire de Biologie Parasitaire, Protistologie, Helminthologie, Muséum national d’Histoirenaturelle, 61 rue Buffon, 75231 Paris cedex 05, France. Phone: ++ 33 1 40 79 35 03; Fax: ++ 33 1 40 79 34 99; E-mail: [email protected]

FOLIA PARASITOLOGICA 46: 199-203, 1999

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concluded that the spiral microtubule region wassynapomorphic for the Temnocephalidae, Didym-orchidae and Actinodactylellidae, and that the character“sperm shaft a thin flange or split at the nuclear end”was synapomorphic for the Scutariellidae + these threefamilies. Recently, acetylated tubulin was shown to beabsent from cortical microtubules of Troglocaridicolasperm, as in digeneans, but in contrast to acoels where itis present (Justine et al. 1998); however, nocomparative results are available for temnocephalids.Joffe and Cannon (1998) and Joffe et al. (1998)considered that the Scutariellidae is the sister group tothe other Temnocephalida. Therefore, the description ofany character of possible phylogenetic significance in ascutariellid, such as the subepidermal glands dealt within this paper, is of interest for the understanding of therelationships of the scutariellids and temnocephalids. Itmust be noted, also, that no gene sequence is availableyet for a scutariellid.

MATERIALS AND METHODS

Troglocaridicola sp. specimens were removed from thecarapace and gill cavity of cavernicolous shrimps (Troglocarissp.) 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 acid-glutaraldehyde fixative (Ermak and Eakin 1976) for 24 hr,then rinsed in 0.1 M sodium cacodylate, pH 7.4 buffer, andpostfixed in 1% OsO4 in the same buffer, rinsed again severaltimes in distilled water and stained with saturated aqueousuranyl acetate, for 2 hr in the dark at 4°C, rinsed in distilledwater, dehydrated in an ethanol series, and embedded inSpurr’s resin. Grids were stained with lead citrate and uranylacetate, and observations were performed with a JEOL 100SX and a Philips 201 microscope.

OBSERVATIONS

Several subepidermal glands were seen in the middlebody of the worm, close to the testis. The glands show aduct lined with longitudinal microtubules and areservoir (Fig. 1). The duct passes through theepidermis, which is relatively electron-dense andcontains nuclei; the reservoir is located below the basallamina of the epidermis (Fig. 1). The gland containsgranules, apparently limited by a membrane, which issometimes not perfectly visible (Figs. 2-4). In contrastto many glands in other animals which contain anamorphous material, the granules contain a highlyorganized material. Longitudinal sections show parallelfibres, 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 of60° 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 fibrescan be counted in a 100nm space. Thin links betweenfibres were also seen (Fig. 5). In addition to thesehighly organised fibres, the granules contain anamorphous material (Figs. 2, 4).

DISCUSSION

We reviewed available literature to find if structuresequivalent to the highly organised pattern observed inthe glands of Troglocaridicola had been found in otherspecies. Glands have been described in several speciesof temnocephalids, including Temnocephala novae-zealandiae (Williams 1980, 1988, 1994, Williams andIngerfeld 1988), Temnocephala minor (Cannon andWatson 1996, Xylander 1997), Didymorchis sp. (Joffeet al. 1995a, Rohde and Watson 1990), Notodactylushandschini (Jennings et al. 1992) and Diretocephalaboschmai (Joffe et al. 1995b). The literature on glandsof phylogenetically related groups, such asKalyptorhynchia (e.g. Schizochilus caecus by Ehlers1989) Typhloplanida (e.g. Bothromesostoma byMartínez-Alós et al. 1991, 1994), Fecampiida (e.g.Kronborgia by Williams 1990a,b), Dalyelliida (e.g.Pterastericolidae by Jondelius 1992, Paravortex byNoury-Sraïri et al. 1989) and Urastoma (Noury-Sraïri etal. 1990) was also surveyed. No similar structure wasfound in any of these published papers and a number ofother papers about glands of Platyhelminthes which wesurveyed.

The possibility of a fixation artefact is low in ourmaterial, since the fixation used did not produceaberrant patterns in other structures (Iomini et al. 1994).Therefore, the presence of glands with regularlyarranged 18 nm fibres can be considered as a characterof possible phylogenetic significance and can bepreliminarily proposed as an autapomorphy for theScutariellidae. An autapomorphy is of limited use forunderstanding phylogenetic relationships. It is howeverworthy of description, because such a structure could befound later in another species, thus possibly leading tophylogenetic implications. In the present context ofdiscussion of the relationships between scutariellids andtemnocephalids (Joffe and Cannon 1998) and betweenthese families and other groups (Littlewood et al. 1999),additional data on gland ultrastructure are needed fromother species.Acknowledgements. Fulvio Gasparo collected the animals,and Dr. Stuart Gelder identified the specimens. Dr. TimLittlewood communicated a manuscript in press. Prof. KlausRohde and Prof. Ulrich Ehlers commented on photographs atan early stage of preparation of this paper. Prof. Barrie G. M.Jamieson kindly edited the English.

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Figs. 1-5. Glands in Troglocaridicola sp. (Scutariellidae). Fig. 1. Longitudinal section of gland showing aperture, duct andreservoir. Fig. 2. Transverse section of reservoir; note microtubules lining the periphery. Fig. 3. Parallel fibres, longitudinalsection. Fig. 4. Fibres, transverse section; note presence of amorphous material in the granule. Fig. 5. High magnification offibres, transverse section. Scale bars: Figs. 1, 2 = 1 µm; Figs. 3, 4 = 200 nm; Fig. 5 = 100 nm.

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Received 23 November 1998 Accepted 8 March 1999