Canadian Journal of Zoology The neuromuscular system in flatworms: serotonin and FMRFamide immunoreactivities, and musculature in Prodistomum alaskense (Digenea: Lepocreadiidae), an endemic fish parasite of the north-western Pacific Journal: Canadian Journal of Zoology Manuscript ID cjz-2020-0245.R3 Manuscript Type: Article Date Submitted by the 06-Mar-2021 Author: Complete List of Authors: Nefedova, Darya; A N Severtsov Institute of Ecology and Evolution RAS, Center of Parasitology Terenina, Nadezhda;Draft A N Severtsov Institute of Ecology and Evolution RAS, Centet of Parasitology Mochalova, Natalia; A N Severtsov Institute of Ecology and Evolution RAS, Center of Parasotology Poddubnaya, Larisa; I D Papanin Institute of Biology of Inland Waters RAS Movsesyan, Sergei; A N Severtsov Institute of Ecology and Evolution RAS, Center of Parasitology Gordeev, Ilya I.; Russian Federal Research Institute of Fisheries and Oceanography; Lomonosov Moscow State University Kuchin, Andrei; Institute of Cell Biophysics RAS Kreshchenko, Natalia; Institute of Cell Biophysics RAS Is your manuscript invited for consideration in a Special Not applicable (regular submission) Issue?: trematode, <i>Prodistomum alaskense</i>, nervous system, serotonin, Keyword: FMRF-related peptides, musculature, tegumental receptors © The Author(s) or their Institution(s) Page 1 of 32 Canadian Journal of Zoology 1 The neuromuscular system in flatworms: serotonin and FMRFamide immunoreactivities, and musculature in Prodistomum alaskense (Digenea: Lepocreadiidae), an endemic fish parasite of the north-western Pacific Nefedova D.A.1⃰, Center of Parasitology, А.N. Severtsov Institute of Ecology and Evolution of Russian Academy of Sciences, Leninsky pr., 33, Moscow, Russia, 119071, [email protected] Terenina N.B.1⃰, Center of Parasitology, А.N. Severtsov Institute of Ecology and Evolution of Russian Academy of Sciences, Leninsky pr., 33, Moscow, Russia, 119071, [email protected] *These authors contributed equally to this work and should both be considered as first authors. Mochalova N.V.1, 1Center of Parasitology, А.N. Severtsov Institute of Ecology and Evolution of Russian Academy of Sciences, Leninsky pr., 33, Moscow, Russia, 119071, Moscow, Russia; [email protected] Poddubnaya L.G.2, 2I.D. Papanin InstituteDraft for Biology of Inland Waters of Russian Academy of Sciences, Borok 119, Yaroslavl Province, Russia, 152742, [email protected] Movsesyan S.O.1, 1Center of Parasitology, А.N. Severtsov Institute of Ecology and Evolution of Russian Academy of Sciences, Leninsky pr., 33, Moscow, Russia, 119071, [email protected] Gordeev I.I.3,4, 3Russian Federal Research Institute of Fisheries and Oceanography, Verkhn. Krasnoselskaya Str. 17, Moscow, Russia, 107140; 4Lomonosov Moscow State University, Leninskiye Gory 1, Moscow, Russia, 119234, [email protected] Kuchin A.V.5, Institute of Cell Biophysics of Russian Academy of Sciences, Institutskaya str., 3, Pushchino, Moscow Region, Russia, 142290, [email protected] Kreshchenko N.D.5# Institute of Cell Biophysics of Russian Academy of Sciences, Institutskaya str., 3, Pushchino, Moscow Region, Russia, 142290, [email protected] #Corresponding author: Natalia Kreshchenko, 5Institute of Cell Biophysics of Russian Academy of Sciences, Institutskaya str., 3, Pushchino, Moscow Region, Russia, 142290, [email protected] Phone +78153562952, fax +7 4967330509 © The Author(s) or their Institution(s) Canadian Journal of Zoology Page 2 of 32 2 The neuromuscular system in flatworms: serotonin and FMRFamide immunoreactivities, and musculature in Prodistomum alaskense (Digenea: Lepocreadiidae), an endemic fish parasite of the north-western Pacific Nefedova D.A.1⃰, Terenina N.B.1⃰, Mochalova N.V.1, Poddubnaya L.G.2, Movsesyan S.O.1, Gordeev I.I.3,4, Kuchin A.V.5, Kreshchenko N.D.5 Abstract Using the immunocytochemical method and confocal scanning laser microscopy, the pioneering data are obtained on the muscle system organization and presence and localization of biogenic amine serotonin, and FMRFamide-related peptides in the nervous system of trematode Prodistomum alaskense (Ward and Fillingham, 1934) Bray and Merrett,Draft 1998 (family Lepocreadiidae). This flatworm is an intestinal parasite of endemic representatives of the marine fauna of the north-western Pacific Ocean – the prowfish, Zaprora silenus Jordan, 1896 and the lumpfish, Aptocyclus ventricosus Pallas, 1769. The article provides data of scanning electron microscopy on the tegumental topography of P. alaskense. The body wall musculature of P. alaskense has three layers of muscle fibres – the outer circular, intermediate longitudinal and inner diagonal ones. The muscle system elements are well-developed in the attachment organs, digestive and reproductive systems, in the excretory sphincter. Serotonin- and FMRFamide- immunopositive neurons and neurites are found in the head ganglia, circular commissure, longitudinal nerve cords, and in the transversal connective commissures. The innervation of the oral and ventral suckers, pharynx, and the reproductive system compartments by the serotonergic and FMRFamide- immunopositive neurites is revealed. The results are discussed in connection with the published data on the presence and functional roles of the serotonin and FMRFamide-related peptides in Platyhelminthes. Key words: trematode, Prodistomum alaskense, nervous system, serotonin, FMRFamide-related peptides, musculature, tegumental receptors © The Author(s) or their Institution(s) Page 3 of 32 Canadian Journal of Zoology 3 Introduction The nervous system of trematodes is well differentiated and is essential for integral coordination of somatic functions and movements. Plathelminthes possess an orthogonal type of the nervous system that is comprised of the brain with paired ganglia connected by the brain commissure and a few pairs of longitudinal nerve cords, two ventral cords are the thickest. The nerve cords are connected by transverse commissures. A number of neuronal signaling substances were found in the nervous system of trematodes, including serotonin and neuropeptides, which regulate of various functions of the parasite - nutrition, movement, reproduction, migration, attachment to the host’s organs and tissues (Terenina and Gustafsson 2003; Halton and Maule 2004). Data on the presence and localization of serotonin and neuropeptides is available for different groups of digeneans. However, the information on the digeneans parasitizing fish are scarce and limited to Bucephaloides gracilescens (Rudolphi, 1819) Hopkins, 1954, Helicometra fasciata (Rudolphi, 1819) Odhner,Draft 1902, Apatemon cobitidis proterorhini Vojtek, 1964 and Cotylurus erraticus (Rudolphi, 1809) Shidat, 1928 (Stewart et al. 2003a, b; Terenina and Gustafsson 2014). The digenean Prodistomum alaskense (Ward and Fillingham, 1934) Bray and Merrett, 1998 (Digenea, Lepocreadiidae) is one of the most common intestinal helminths of teleosts in the north-western Pacific including endemics of this area – bathypelagic fishes – prowfish, Zaprora silenus Jordan, 1896 (Actinopterygii, Zaproridae), and lumpfish, Aptocyclus ventricosus Pallas, 1769 (Actinopterygii, Cyclopteridae) (Shvetsova and Pozdnyakov 1999; Gordeev and Sokolov 2020). Z. silenus is the only member of the family Zaproridae; it inhabits depths of up to 675 m (Tuponogov and Kodolov 2014). A. ventricosus is a cyclopterid fish broadly distributed in the north Pacific. It inhabits depths of up to 1700 m and performs significant vertical migrations (Fedorov et al. 2003). The general morphology of the lepocreadids is well described (Gibson 1996; Bray and Merret 1998; Jones et al. 2005). However, data on the nervous system and neuronal signaling substances of these trematodes are absent in the literature. The paper presents data on the organization of the nervous and muscular systems of lepocreadid digenean P. alaskense, an intestinal parasite of deep-sea fish of the north-western Pacific. We provide new information on the distribution of the common neurotransmitters - serotonin (5-hydroxytryptamine, © The Author(s) or their Institution(s) Canadian Journal of Zoology Page 4 of 32 4 5-HT) and FMRFamide-related peptides (FaRPs) in the nervous system of P. alaskense. The data expand our knowledge on the trematodes nervous and muscular systems morphology as well as shed light on the neurochemical mechanisms of the regulation of their vital functions. In addition, this information can facilitate the investigation of the important taxonomic issues. The paper also provides data of scanning electron microscopy (SEM) of the sensory structures on the worm’s surface. Materials and methods A total of 26 specimens of P. alaskense collected from the intestine of four of Z. silenus (15 specimens) and of two of A. ventricosus (11 specimens) were used in this study. Fish specimens were caught with epipelagic trawl at the depth of up to 56 m by the research vessel Professor Kaganovsky during a survey of open waters of north-western part of the Pacific Ocean in June-July of 2018 (Gordeev et al. 2018) and in September-October of Draft2019 in the Bering Sea (Gordeev et al. 2019). The live worms were flat fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PBS, рH 7.4, Sigma, USA) at 4ºС for 12 hours. Then the samples were transferred for storage in PBS buffer with 10% sucrose (Sigma) and kept until stained. Immunocytochemistry The localization of serotonin-immunopositive (5-HT–IP) and FMRFamide-immunopositive (FMRFa–IP) nervous structures is identified using