Cytochemical studies of the neuromuscular systems of the diporpa and juvenile stages of Eudiplozoon nipponicum (Monogenea : Diplozoidae) T. H. ZURAWSKI 1 , A. MOUSLEY 2 , A. G. MAULE 2 , M. GELNAR 1 and D. W. HALTON 2 * 1 Department of Zoology and Ecology, Masaryk University, 611 37 Brno, Czech Republic 2 Parasitology Research Group, Queen’s University Belfast, Belfast BT9 7BL, UK (Received 17 September 2002; revised 29 October 2002; accepted 29 October 2002) SUMMARY Using indirect immuno- and enzyme-cytochemical techniques, interfaced with confocal scanning laser microscopy and standard optical microscopy, neuronal pathways have been demonstrated in whole-mount preparations of the unpaired diporpae and freshly paired juvenile stages of Eudiplozoon nipponicum (Monogenea : Diplozoidae). All 3 main classes of neuronal mediators, cholinergic, aminergic and peptidergic, were identified throughout both central and peripheral elements of a well-differentiated orthogonal nervous system. Neural mapping revealed considerable overlap and similarity in staining of the nervous systems of the diporpa and adult worm. The main differences in the diporpa relate to the inner- vation of the temporary ventral sucker and dorsal papilla, structures which are unique to the larva and which enable fusion between worms but then disappear. Branches from the longitudinal nerve cords innervate these structures and appear to be involved in the process of somatic fusion, probably giving rise to the inter-specimen connections that later link the 2 central nervous systems in paired adult parasites. In the hindbody, there is extensive haptoral innervation associated with the developing clamps and small central hooks. Reactive neuronal components were found associated with the early stages of clamp development prior to connections being made with the extrinsic adductor muscle bundles. The muscle systems of the diporpa and juvenile stages comprise a lattice-like arrangement of circular, longitudinal and diagonal fibres that make up the body wall, together with buccal suckers, haptoral clamps and associated adductor muscles, and the transient ventral sucker. All have obvious importance to diporpae when they migrate over the gill and undertake body contact, torsion and fusion during the process of pairing. Behaviour during the pairing of diporpae is described. Key words : Eudiplozoon nipponicum, monogenean neurobiology, diporpa, cholinesterase, serotonin, neuropeptide, confocal microscopy. INTRODUCTION Monogeneans belonging to the family Diplozoidae are common blood-feeding ectoparasites of the gills of cyprinid fish. The group is unusual in that it comprises individual worms that become fused to one another in a typical cross fashion and remain as such throughout life. Not surprisingly, development of diplozoons is unique. The ciliated oncomiracidium hatches from the egg in water and has rhabdomeric eyes, a well-developed pharynx and a blind-ending intestinal caecum. The posterior haptor is well dif- ferentiated and at this stage is usually armed with a pair of small hooks and a single pair of clamps. While attached to the gills of the fish, each larva undergoes marked changes in morphology, in that it loses its eyespots and surface cilia, and develops a branched intestine; a small muscular sucker develops on the mid-ventral surface and a small conical projection or papilla appears on the dorsal surface. This post-oncomiracidium stage is termed the diporpa, originally named and described by Dujardin (1845). Diporpae feed on blood and are probably suf- ficiently mobile to migrate over the gill surface, grip- ping the filaments alternately with mouth and then hooks and clamps. In this way, when two diporpae are on the same gill arch they are likely to meet. When in pairs, the two become intimately associated such that the ventral sucker of one diporpa secures itself to the dorsal papilla of its partner. Through a twisting of the two bodies, the free sucker of one individual embraces the free papilla of its mate. As such the two worms become permanently fused at the point of contact to produce a double animal, the diplozoon. This juvenile stage now undergoes development, with the appearance of successive pairs of clamps on the posterior haptor ; 4 pairs characterize the fully formed attachment apparatus of the sexually mature adult. Unpaired diporpae can also develop up to 4 pairs of clamps, but without fusion with a part- ner they fail to mature. Once united, the two worms undergo a period of reproductive development dur- ing which time gonads appear and the male and fe- male genital ducts become fused so that the two individuals are in ‘permanent copulation’. * Corresponding author : Parasitology Research Group, Queen’s University Belfast, Belfast BT9 7BL, UK. Tel: +44 2890 335792. Fax : +44 2890 236505. E-mail : d.halton@qub.ac.uk 349 Parasitology (2003), 126, 349–357. f 2003 Cambridge University Press DOI: 10.1017/S0031182002002871 Printed in the United Kingdom