Acta Zoologica (Stockholm), Vol. 71, No. 4, pp. 211-216, 1990 Printed in Great Britain

0001-7272/90$3 .OO+ .OO Pergamon Press plc 0 1990 The Royal Swedish Academy of Sciences

Ultrastructure of the Flame Bulbs of Urastoma cyprinae (Platyhelminthes, 'Prolecithophora', Urastomidae) K. Rohde,l N. N ~ u r y - S r a i ' r i , ~N., ~Watson,' J.-L. Justine4 and L. Euzet2 'Department of Zoology, University of New England, Armidale, New South Wales, 2351, Australia ZLaboratoire de Parasitologie comparte, URA CNRS 698, Universitt de Montpellier 11, F-34095 Montpellier Cedex 2, France 4Laboratoire des Vers, URA CNRS 114, Museum National d'Histoire Naturelle, 61 rue Buffon, F-75231 Paris Cedex 05, France (Accepted for publication 23 February 1990)

Abstract

Rohde, K., Noury-Sraki, N., Watson, N., Justine, J.-L. & Euzet, L. 1990. Ultrastructure of the flame bulbs of Urastoma cyprinae (Platyhelminthes, 'Prolecithophora'. Urastomidae).Acta zool.. Stockh. 71: 211-216. The ultrastructure of the flame bulbs of the turbellarian Urastoma cyprinae from Mytilm galloprovincialis in the Mediterranean is described. The nucleus of the terminal cell is located some distance basal to the rootlets of the cilia forming the flame; the cytoplasm contains numerous tubules approximately 54-66 nm in diameter, and vesicles. Thick walled, densely packed rod-like structures coil around each other with a tendency towards longitudinal orientation close to the flame. The rod-like structures tightly surround the basal part of the flame and the distal cytoplasmic tube in the apical part of the flame. Some of them, including the inner predominantly longitudinally directed ones, are continuous with the cytoplasm of the terminal cell, others are continuous with the cytoplasm of the distal cytoplasmic tube. Internal leptotriches arise from the cytoplasm of the terminal cell and intrude between the basal parts of the cilia of the flame. The distal cytoplasmic tube possesses a septate junction. The flame bulb of Urastoma differs distinctly from those known from other Platyhelminthes; implications for the phylogeny of Platyhelminthes are discussed. Klaus Rohde, Department of Zoology, University of New England, Armidale, New South Wales, 2351, Australia.

Introduction

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The ultrastructure of the protonephridia of Platyhelminthes has received much attention, particularly, though not exclusively, with the aim of clarifying phylogenetic relationships within the phylum (for references see Ehlers 1985; ~ o h d e1986, 1989;-for recent studies see Rohde 1990, and Rohde et al. 1989a, b, in pfess). In spite of the considerable number of studies, several major groups of flatworms have not been examined, and only one study of 'Prolecithophora' has been published (Ehlers 1989). In this paper, we give a description of the ultrastructure of the flame bulb of Urastoma cyprinae, a prolecithophoran from the bivalve Mytilus galloprovincialis in the Mediterranean. Sperm ultrastructure, spermiogenesis, oogenesis and structure of the seminal vesicle, epidermis and subepithelia1 glands of this species and phylogenetic implications were recently discussed by Noury-Srai'ri (1988) and Noury-Srai'ri et al. (1989a, b, 1990). Burt & Bance (1981) and Tyler & Burt (1988) described the ultrastructure of the eye of this species. Materials and Methods Specimens of Urastoma cyprinae (Graff) were collected from the gills of the bivalve Mytilus galloprovincialis Lamarck at Cap d'Agde, French Mediterranean. They were fixed in 2.5% glutaraldehyde in sodium cacodylate buffer (0.1 M, pH 7.2) for 24 h at 4"C, post-fixed in 2% 3Present address: Dkpartment Halieutique, Institut Agronomique et Vktkrinaire Hassan II, HIDAOA, B.P. 6202, Rabat, Morocco.

OsO, for l h in the same buffer and at the same temperature, dehydrated in an alcohol series and propylene oxide, and embedded in Spurr's resin. Sections were stained with uranyl acetate and lead citrate and examined under a Jeol 1200 EX at 60 kV,

Results

The flame bulb contains a bundle of cilia (or 'flagella', a term which might be more appropriate in view of their small number and considerable length but is avoided conforming to the general use of 'cilia', at least in the English literature) with cross-striated rootlets curving towards a common centre (Figs 2 , 7 , 18); the tips of the cilia terminate in different planes, and consequently there are smaller numbers of cilia in cross-sections closer to the tip than to the base of the flame bulb (Figs 10, 11, 18). A typical weir consisting of 'membrane'-connected rods is not present; instead, a system of densely packed rod-like structures coiled around each other extends along the flame (Figs 1-4, 6, 8, 9, 12-14, 18). Some of these structures were seen to be continuous with the cytoplasm of the terminal cell (Fig. l ) , others with the cytoplasm of the distal cytoplasmic-tube (Figs 2-4).coiling of the structures around each other was particularly clear in some oblique sections (Figs 8, 9). Near the base of the flame, parts of the system of rod-like structures bend outwards, away from the flame ( ~ 2-4,i 18), ~ parts ~ extend along the distal cytoplasmic the tube (Figs ll). The is basal to the rootlets of the flame (Figs 7, 18) and the which extends for distance the flame (Figs 1-3, 18), contains an extensive system of 1°7

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Flame Bulbs of Urastoma 213

Figs 8-11, Oblique (Figs 8, 9)*and transverse sections (Figs 10, 11) through flame bulb. Note system of rods coiled around each other (large arrows) tightly surrounding the flame in its basal parts (Figs 8, 9), and separated from it by light cytoplasm of the distal cytoplasmic tube (dt) containing a septate junction (sj) in the more apical parts (Figs 10, 11). c cilia; r tubules in cytoplasm. Scale bars 1 pm.

214 K. Rohde et al.

Figs 12-17. Cross-sections through base of flame bulb, all except Fig. 16 sections through the same flame bulb. Note system of rods (large arrows), internal leptotriches (il) between cilia ( c ) forming the flame, and rootlets ( r ) of cilia. Scale bars 1 pm.

Flame Bulbs of Urastoma 215 Discussion

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Fig. 18. Diagrams of flame bulb.-(A) longitudinal section; (B-D) cross-sections. c cilia of flame; dt distal cytoplasmic tube; g Golgi complex; il internal leptotrich; m mitochondrion; n nucleus; r rootlet of cilium; ro rods (ribs); sj septate junction; t tubules in cytoplasm of terminal cell; v vesicle; ? large body in terminal cell (modified mitochondrion ?); arrowheads indicate open communication of rods with distal cytoplasmic tube and terminal cell (te). b , c and d along left side of A indicate levels of cross-sections B, C and D. Scale bar 1 pm.

tubules (approximately 54-66 nm in diameter), some appearing empty and some filled with a granular material (Figs 1-7, 18). It also contains vesicles (Figs 1-7, 18), mitochondria, Golgi complexes and some large bodies which may be modified mitochondria (Figs 5, 18). Extensions of the terminal cell (internal leptotriches) are scattered between the basal parts of the cilia of the flame (Figs 13-15, 18), but do not extend into the apical part of the flame (Figs 10, 11, 18). The cytoplasm of the distal cytoplasmic tube differs distinctly from that of the terminal cell, consisting of loose cytoplasm with some tubules, mitochondria, vesicles and a septate junction (Figs 10, 11, 18).

The difference in the structure of the cytoplasm of the perikaryon at the base of the flame (numerous tubules and vesicles) and of the 'distal cytoplasmic tube' around the apical part of the flame suggests that both belong to different cells, although a clear boundary between them is obscured by the presence of coiled rod-like structures (see below). The system of rod-like structures of the flame bulb of Urastoma is unique among the Platyhelminthes so far examined. Such structures, coiled tightly around each other in a dense layer, which gradually becomes thinner along the distal cytoplasmic tube, have not been found before. The projections of rod-like structures of this system into the adjacent cytoplasm near the base of the flame are also unique. Continuity of some of the rod-like structures with the distal cytoplasmic tube, and of some of these structures with the terminal cell, indicate that they correspond to the ribs (rods) in the weir of Neodermata and other Platyhelminthes (see below). However, the possibility must be considered that at least some of the coiled rod-like structures do not correspond to ribs but are, in fact, external leptotriches, i.e. outgrowths of ribs. This would explain the lack of 'membranes' between them, which might be present only between the genuine ribs but are covered by the tightly coiled leptotriches. If such 'membranes' are present, filtration-which is likely to occur-would have to proceed either through the coiled rod-like structures themselves or through the contact zones between them, before continuing through the 'membranes'. The numerous tubules in the cytoplasm of the terminal cell are possibly indicative of a particularly high absorptivelexcretory activity, necessary because of the higher energy requirements for such complex filtration. Similar tubules were also observed in caeca1 cells of monogeneans (Rohde 1973, 1975), which are certain to be very actively involved in absorptive andlor secretory processes. The structures described as 'internal leptotriches' arise from the perikaryon of the terminal cell, as they also do, for instance, in the proseriate turbellarian Monocelis (see Rohde et al. 1988), and in the monogenean Dactylogyrus (see Rohde et al. 1989b). In other platyhelminths, they arise from the internal ribs, or from both the internal ribs and the perikaryon of the terminal cell, for instance in the ancyrocephaline monogenean examined by Rohde et al. (19896). Use of the term 'internal leptotriches' does not imply homology of these structures in different taxa. The flame bulb of the only other 'prolecithophoran' examined so far, i.e. of Archimonotresis limophila, differs in some respects from that of Urastoma (see Ehlers 1989). There is a single layer of longitudinal rods which fuses distally to a closed tube (without cell junction). Rods are supported by bundles of microtubules and connected by a 'membrane' (diaphragm in Ehler's terminology) of extracellular material. As in Urastoma, cilia of the flame have single rootlets, but there are no internal leptotriches. The proximal canal cell externally projects over the distal parts of the duct formed by the fused rods of the terminal cell, but there is no system of rods coiled around each

216 K. Rohde et al. other. In spite of these differences, both species correspond in a proximal canal cell close to the perikaryon of the terminal cell and surrounding the tip of the flame. It may well be that the system of coiled rods in Urastoma corresponds to the cytoplasmic projections of the proximal canal cell over the distal half of the terminal cell in A rchimonotresis. The flame bulbs of other Platyhelminthes differ in various ways from that of Urastoma (see discussion by Ehlers 1985; Rohde 1986, 1989) but, in spite of apparent differences, Urastoma resembles the Neodermata, Proseriata, and Macrostomum in the presence of rods continuous with the distal cytoplasmic tube and of rods c~ntinuous with the terminal cell. It also resembles the Proseriata (see Rohde et al. 1988) and the TrematodaIMonogenea among the Neodermata in the presence of a septate junction along the flame bulb (such a junction is also present along part of the duct cells of Archimonotresis). The turbellarian Rhabdocoela, and particularly the 'Dalyellioida', which are often considered to be close to the Neodermata, on the other hand, have a fundamentally different flame bulb, characterized by a single row of longitudinal rods and the lack of a septate junction. This indicates that the Neodermata have evolved from a platyhelminth more 'primitive' than the 'Dalyellioida', to be found among a 'pool' of platyhelminths characterized by a flame bulb formed by two cells, each contributing one row of longitudinal ribs to the weir. The apparent similarities between Neodermata and 'Dalyellioida' may be due to convergent evolution.

Acknowledgements Financial support was given by the University of New England and the Australian Research Grants Commission. We wish to thank Peter Garlick for making facilities at the E. M. Unit, University of New England, available to us, Becky Francis for help with the drawings, and Sandra Higgins for typing the manuscript.

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