The Neuromuscular System in Flatworms: Serotonin and Fmrfamide Immunoreactivities, and Musculature in Prodistomum Alaskense (Dig

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

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trematode, Prodistomum alaskense, nervous system, serotonin, Keyword: FMRF-related peptides, musculature, tegumental receptors

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

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

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,

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 indirect immunocytochemical methods (Coons et al.

1955). The samples (whole mounts) were incubated during five days either in a primary rabbit anti-5-HT

(1:500) (Immunostar, USA, Product ID: 20080, RRID: AB_572263) or with anti-FMRFamide

(Immunostar, USA, Product ID: 20091, RRID: AB_572232) primary antibodies diluted 1:500 in PBST solution containing 0.1 M PBS and 1% (v/v) Triton X-100 (Sigma, USA). The pre-incubation in BSA was not performed. After a 12-hour wash in PBST, samples were incubated with the secondary fluorescently- labeled AlexaFluor488 immunoglobulines (goat anti-rabbit IgG (H+L), Abcam, USA, Cat# ab150077,

RRID: AB_2630356) for next five days. All procedures were performed in the dark room at 4°C. For microscopic analysis 7–10 replicas (samples) have been used.

Controls included: (1) incubation of samples with only secondary immunoglobulines without

primary antibodies or (2) using of non-immune rabbit serum instead of the primary antiserum. The

negative controls demonstrated the absence of neurotransmitters staining outside the nervous system.

Staining of musculature with TRITC-conjugated phalloidin

For musculature staining the TRITC(tetramethylrhodamine B isothiocyanate)-conjugated

phalloidin (Sigma Aldrich, USA, Cat# P1951), in dilution of 1:200, is used, the samples were kept in the

phalloidin solution in dark for 18-24 hrs at 4°C according to the method described by Wahlberg (1998)

identifying actin filaments of muscle fibres.

Fluorescent and confocal laser scanning microscopy Specimens were investigated underDraft the fluorescent microscope Leica DM1000 (Leica Microsystems, Germany) in the Center of Parasitology of the А.N. Severtsov Institute of Ecology and

Evolution of the Russian Academy of Sciences (Moscow), and with the microscope Leica DM6000B

equipped with a digital camera DC300F (Leica Microsystems, Germany) and confocal laser scanning

microscope Leica TCS SP5 (Leica Microsystem, Germany) at the Institute of Cell Biophysics of

Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences (Moscow

Region).

A fluorescent filter I3 (excitation spectrum of 450–490 nm, emission spectrum of 515 nm) was

used for the identification of Alexa488 fluorophore, while TRITC fluorophore was detected with a N2.1

filter (excitation spectrum of 515–560 nm; emission spectrum of 590 nm). The microphotographs

presented are either a maximal projection of a total of 16 to 64 consequent optical sections reconstructed

at maximum fluorescence intensity, or a single optical section (or a snapshot), obtained by scanning

through 32-80 μm sample thickness. The morphological measurements were carried out on 5fixed whole

mounts of P. alaskense with a computer program provided with the CLSM microscope (Leica LAS AF

Lite, version 2.4.6384.1; Leica Microsystems CMS GmbH). The presented morphological parameters are

given as approximate values measured on fixed tissue.

Scanning electron microscopy

For SEM observations, the PFA preserved worms were further fixed using 5% glutaraldehyde (Spi-Chem,

USA) in 0.1 M sodium cacodylate buffer (pH 7.2) (Electron Microscopy Sciences) for six days at 5° C.

The fixed worms were dehydrated in a graded ethanol series, with a final change in absolute ethanol and then critical-point-dried with liquid CO2 using a Leica EM-CPD300 station. The specimens were mounted on stubs, sputter-coated with 10 nm gold-palladium and examined using a JSM-6510LV scanning electron microscope (JEOL, Japan) operating at 30 kV at the I.D. Papanin Institute for Biology of Inland

Waters of RAS (Borok, Russia).

Results

The length of the body of PFA-fixed Prodistomum alaskense (obtained from prowfish, Z. silenus) was about 1.6 mm, its width in the middle partDraft of the body was about 600 µm. The samples obtained from lumpfish, A. ventricosus, were slightly smaller (the length about 1.4 mm, the width about 420 µm).

Tegumental topography of P. alaskense

The worm has an elongated body (Fig.1e). Under SEM, the ventral and dorsal body surfaces are spinous and possess transverse ridges (Fig. 1a, g – j). The anterior end of the body bears a subterminal oral sucker (Fig. 1a, g). The rim of the oral sucker is armed with rows of multipointed spines each bearing

3-5 teeth that resemble forebody spines (Fig. 1g). At the anterior extremity, dorsally to the oral sucker, the distinct elongated frontal pit exists (Fig. 1a, g), its role is unknown. On the dorsal side of the frontal pit of ciliated receptors are solitary or arranged as groups encompassing up to three papillae (Fig. 1g). The tegumental spines are distributed throughout the body surface (Fig. 1h – j). Gradual transformation of the tegument spines from multipointed encircled by alternating rows on the forebody surface to the simple spines on the hindbody surface have been observed (Fig. 1h – j). The spines have a highest density in the sublateral ventral areas of the forebody and hindbody whereas their mid-ventral areas possess the spines with a lower density (Fig. 1h, i, j). Irregularly distributed ciliated receptors are scattered over the entire body surface and grouped by 3 - 5 on the forebody and hindbody surfaces (Fig. 1f, h).

The ventral sucker is located in the posterior part of the anterior third of the body (Fig. 1a, e). The

rim of both the ventral and oral suckers lacks radially arranged corrugations (Fig. 1b, c, g). A consistent

pattern of nine non-ciliated, dome-shaped papillae are regularly distributed and arranged in a single ring

on the rim of the ventral sucker (Fig. 1b, c). In addition, the rings of multipointed spines are present on

the surface rim of the ventral sucker (Fig 1b, c). Ciliated receptors are scattered irregularly on the surface

of the ventral sucker (Fig. 1d). The common genital pore is situated in close proximity to the ventral

sucker (Fig. 1b, c). The body surface around the genital pore lacks spines (Fig. 1c). Single ciliated

receptors are observed near the genital pore (Fig. 1c).

Musculature

The muscle system elements of the anterior, central and posterior body parts of P. alaskense. are represented on Fig. 2. The body wall musculatureDraft consists of the circular (Fig. 2a-f, j), longitudinal (Fig. 2b-f) and diagonal muscle fibres (Fig. 2b-e, j). The diagonal muscle fibres are localized more rarely in the

posterior body region (Fig. 2e) and absent near the excretory opening (Fig. 2f). The circular muscle fibres

in anterior and middle body regions (Fig. 2b, c) are single and arranged evenly, whereas in the posterior

part of the worm’s body they are paired (Fig. 2e). The radial muscles are well developed in the ventral

and oral suckers (Fig. 2g, m). The musculature of the pharynx is well-defined and represented by radial

muscle fibres (Fig. 2h). The radial muscle fibres are deeply packed in the ventral sucker (Fig. 2b, g, i, m).

Several muscle bundles are running from the base of the ventral sucker into a dorsal direction (Fig. 2i).

In the posterior part of body, a group of muscle fibres is running from the body wall towards the

testes (Fig. 2j, k). The distal part of the reproductive system corresponding to the location of the uterus is

intensively supplied with the longitudinal and circular muscle fibres (Fig. 2l). Densely packed

longitudinal fibres together with the circular ones are detected also in the ducts of the reproductive system

nearby the reproductive opening (Fig. 2m). The excretory pore is surrounded by muscles forming a

sphincter (Fig. 2f).

5НТ– and FMRFamide–immunopositive staining

Serotonergic nerve elements in P. alaskense

5-НТ–IP neurons and their fibers are detected in the central and peripheral nervous system of P.

alaskense (Fig. 3a-h):

- 5-HT–IP cells (four to five cells) measuring of 5-8 µm are located in the area of each head ganglion

(data from 11 specimens). On each side of the oral sucker above the head ganglia there is another

pair of small neurons, from which the neurites run towards the oral sucker. From the head ganglia

two branched 5-HT–IP neurites extend towards the oral sucker (Fig. 3a-d);

- 5-HT–IP staining is detected in the ventral nerve cords, which are traced to the end of the worm’s body.

5-HT–IP staining is also observed in several neurons, located in the ventral nerve cords, as well as

in the commissures connecting them (Fig. 3a-e); - the neurites are extending towards the ventralDraft sucker from three pairs of 5-HT–IP neurons situated in the ventral nerve cords at the level of the ventral sucker (Fig. 3b, d, e). Moreover, 5-HT–IP fibers are

visible among the muscle filaments of the ventral sucker;

- nearby the posterior part of the well-developed muscular pharynx two serotonergic neurons are observed

(Fig. 3a, b, d);

- near the anterior edge of the ventral sucker there are two pairs of small 5-HT–IP neurons (Fig. 3d);

- several small 5-HT–IP neurons, one of which is larger than the others, were detected nearby the genital

pore located to the right upper edge of the ventral sucker (Fig. 3b, d);

- the innervation of the genital pore by the serotonergic neurites extending from the ventral nerve cord are

observed (Fig. 3e);

- near the posterior edge of the ventral sucker the positive staining to serotonin has been found (Fig. 3b,

d). It can be assumed that this staining is related to the nervous elements of distal part of the

reproductive system located in this body region;

- 5-HT–IP neurites are visible along the ducts of the distal part of the reproductive system (Fig. 3f);

- at the level of the ovary localizing before the testes, four bipolar 5-HT–IP neurons (9-14 µm) are

revealed (Fig. 3g, h);

- 5-HT–IP staining is detected in two small cells near the excretory pore (see a schematic drawing Fig.

3i).

A schematic drawing of 5-HT–IP neurons location and 5-HT–IP staining in the area of distal reproductive

system in P. alaskense is presented in Figure 3i.

FMRFаmide–immunopositive staining in P. alaskense

- FMRFa–IP staining is revealed in several neurons located in the paired head ganglia, and in the

brain commissure, comprising the nerve fibres and in two FMRFa–IP neurons (Fig. 4а, b, c);

- FMRFa–IP staining is observed in the ventral nerve cords including several neurons (size of 10-14

µm), as well as in the dorsal and lateral longitudinal nerve cords and in commissures connecting

them (data from 9 specimens) (Fig. 4 а-c). Commissures are located more often in the anterior body region than in the posterior one;Draft - FMRFa–IP staining is detected in two pairs of neurite bundles running from the brain ganglia to

both sides of the oral sucker (Fig. 4b). These neurite bundles are connected with each other at the

posterior edge of the oral sucker (Fig. 4, d).

- two FMRFa–IP neurons are situated before the intestinal bifurcation (Fig. 4f);

- anti-FMRFa stained fibers are also found in the posterior part of the pharynx and in the region of

the intestine before its bifurcation (Fig. 4d inset, 4f); a few neurites are running to the ventral

sucker from several small FMRFa–IP neurons (size of 10-12 µm) located in the ventral nerve cord

at the level of the ventral sucker (Fig. 4e);

- behind the ventral sucker, the longitudinal FMRFa–IP fibers are observed in the distal part of the

uterus wall (Fig. 4c, f);

- at the level of the ovary, the intensive FMRFa–IP staining is observed in several small cell bodies

being distinguished around duct of the reproductive system, the exact compartment of which could

not be clarified (Fig. 4g).

The 5-HT–IP and FMRFa–IP neurons are found mainly in the anterior part of the worm’s body which

include the oral and ventral suckers. A total of 27-29 serotonin–IP neurons are revealed in this region. In

© The Author(s) or their Institution(s) Canadian Journal of Zoology Page 10 of 32 10 the posterior part of the body (behind the ventral sucker) only six 5-HT–IP neurons are detected, four of which are located on the level of the ovary, and two are near the excretory pore. The sizes of cell bodies of 5-HT–IP neurons in brain ganglion are about 5-8 µm, in the ventral nerve cords – 8-9 µm, near pharynx – 12-14 µm. The neurons located on the level of the ovary have the sizes of 9-14 µm.

About five to seven FMRFa–IP neurons we observed in each ventral cord in the anterior body part and two neurons are situated in the area before the intestinal bifurcation. Three peptidergic neurons are observed in each ventral nerve cord of the posterior part of the worm, totally estimated in about 18-22

FMRFa–IP neurons in the whole body of the worm. FMRFa–IP commissures connecting the longitudinal nerve cords are arranged more often in the anterior body region, where about 15 commissures are observed while in the posterior one about 17 commissures are seen. The distance between FMRFa–IP commissures in the anterior body region is about 20-40 µm, whereas this distance in the posterior part of the worm is about 80-90 µm. Draft

Discussion

In the study we described for the first time the organization of the muscular system, the serotonin- and FMRFamide–IP components in the nervous system of the lepocreadid trematode Prodistomum alaskense, an intestinal parasite of –the prowfish, Z. silenus and the lumpfish, A. ventricosus of the north- western Pacific.

The present SEM observation of adult species of P. alaskense revealed for the first time the body surface features of plagiorchiid species belonging to the family Lepocreadiidae. As shown in previous

SEM studies of other plagiorchiid digeneans (order Plagiorchiida), a consistent pattern of the number and arrangement of non-ciliated papillae has proved useful as an indicator of the inter-specific differences; for example, in the cases of the allocreadiids (Caira 1989; Petkevičiūtė et al. 2018) and gorgoderids (Bakke and Žďárská 1985; Stunžėnas et al. 2017). In P. alaskense nine non-ciliated papillae were detected along the surface of the ventral sucker rim. Based on the available data, we can assume that such number of uniformly-sized, evenly spaced papillae has not been previously recorded for any plagiorchiid species by

SEM (Maldonado et al. 2001; Mata-López and Leon-Regagnon 2006; Fillippi et al. 2013). Thus, in some

gorgoderids and allocreadiids (Plagiorchiida), six characteristic papillae have been found on the rim of the

ventral sucker (Bakke and Bailay 1987; Stunžėnas et al. 2017; Petkevičiūtė et al. 2018). In P. alaskense

predominated type of ciliated receptors lacks any distinctive pattern of their arrangement. Thus, the

greatest accumulation of ciliated receptors on the dorsal side of the frontal pit can be considered as a

specific feature of these worms.

Between various morphological features of the digeneans the pattern of the spines of their

tegument is an important taxonomic criterion. The differences in the shape and distribution of the spines

over the body may be significant in taxonomy of the order Plagiorchiida. Particular arrangements and

variations in the spine morphology were also observed in P. alaskense. First, multipointed spines usually

possessing 4 – 5 digitations have been observed along the oral sucker, forebody surface and ventral

sucker. Second, simple spines have been localized on the hindbody surface. Third, the highest density of both types of the spines has been revealedDraft in the sublateral ventral areas of the forebody and hindbody of P. alskense. Abovementioned microtopographical features may be considered as taxonomic significant

aspects of adult P. alaskense. Future studies on related species of the family Lepocreadiidae might show

the common topographical features such as an arrangement of papillae and spines of lepocreadid

digeneans.

Phalloidin staining revealed a well developing muscular system in P. alaskense. The body wall of

the worm consists of three muscle layers: outer circular, intermediate longitudinal and inner diagonal. The

occurrence of the radial muscle fibres in the attachment organs (oral and ventral suckers) and in the

pharynx of P. alaskense is also shown. The muscle fibres running from the ventral sucker in the dorsal

direction are found. The presence of the circular and longitudinal muscle fibres is detected in the distal

part of the reproductive system of parasite. The muscular sphincter is revealed around the excretory

opening. All these data are consistent with available information related to the other trematodes, which

indicate that the muscle system in trematodes is well developed and plays an important role in body

movement, host attachment and function of reproductive organs (Stewart et al. 2003 a, b; Halton and

Maule 2004; Ŝebelová et al. 2004; Petrov and Podvyaznaya 2016; Krupenko and Dobrovolskij 2018;

Krupenko 2019).

At the same time, some unknown details were revealed in the organization of the muscle system in

P. alaskense, not previously described in other trematodes. These include, for example, the observation of the muscle fibers that run from the body wall to the testes in posterior part of the worm’s body. Of particular interest is a finding of the paired arrangement of circular muscle fibers in the wall of the posterior body region of P. alaskense. There is also a rarer arrangement of the diagonal muscle fibers in the posterior part of the body of P. alaskense and their absence near the excretory pore. These data indicate some diversity in the organization of the muscle system in different trematoda species the significance of which in the functioning of the musculature have to be investigated.

In our work the localization of the serotonin and FMRFamide-related neuropeptides in P. alaskense has been studied to show the organization of their nervous system. The presence of these neurotransmitters in the nervous system of trematodes had been reported earlier (Halton and Maule 2004, Terenina and Gustafsson 2014). Draft Serotonin (5-HT) is a widely distributed neurotransmitter in the animal kingdom and found in all investigated flatworms so far (Halton and Maule 2004; Ribeiro et al. 2005; Quiroga et al. 2015). The presence of serotonin in trematodes had been shown by biochemical methods (Catto and Ottesen 1979;

Terenina and Gustafsson 2003). It was found that larval and adult stages of flatworms are capable of active absorption of serotonin from their host through a highly specialized serotonin-transporting system

(Bennett et al. 1973; Catto and Ottesen 1979; Osloobi and Webb 1999). For example, in Schistosoma mansoni Sambon, 1907 sporocysts the absorption of serotonin can be substantial reducing the host’s neurotransmitter level (Boyle et al. 2003, Manger et al. 1996). Serotonin synthesis pathways have been also identified in a number of flatworms (Ribeiro and Webb 1984; Hamdan and Ribeiro 1999). It has been shown experimentally that 5-HT is an excitatory neurotransmitter in parasitic flatworms – cestodes, trematodes and monogenea, causing the contraction of their muscles (Mansour 1984; Pax et al. 1984;

McKay et al. 1989; Thompson and Mettrick 1989; Patocka et al. 2014).

Neuropeptides are widespread signaling molecules in the nervous system of flatworms including the representatives of the family of FMRFamide-related peptides (Gustafsson et al. 2002; Halton and

Maule 2004; McVeigh et al. 2005; Quiroga et al. 2015; Kreshchenko et al. 2018). Four FaRPs were

biochemically identified in flatworms: YIRFamide in Bdelloura candida Girard, 1850, GYIRFamide in B.

candida and Girardia tigrina Girard, 1850, RYIRFamide in Artioposthia triangulata Dendy, 1895 and

GNFFRFamide from Moniezia expansa Rudolphi, 1805 (Maule et al. 1993, 1994; Johnston et al. 1995,

1996), but many more have been predicted bioinformatically (McVeigh et al. 2009; Koziol et al. 2016).

However, the whole functional significance of these neuropeptides in flatworms still remains poorly

understood. The available data indicates that the serotonergic and peptidergic elements of the nervous

system are widely distributed not only in adults, but also in larvae of trematodes (Terenina and

Gustafsson 2014).

Our data show the presence of 5-HT– and FMRFa–IP nerve elements in the central and peripheral

parts of the nervous system of P. alaskense. The studied neurotransmitters are detected in cells and fibers

of paired head ganglia, the longitudinal nerve cords, neurons located along the nerve cords, in fibres comprising the connective commissures. Draft We have identified the serotonergic and FMRFa–IP neurites, running towards the oral and ventral

suckers. These data indicate that 5-HT–IP and FMRFa–IP neurites could be involved in the control of the

attachment organs function in trematode P. alaskense. Innervation of the attachment organs (the oral and

ventral suckers) with 5-HT–IP or FMRFa–IP neurites was also shown before in a number of adult and

larvae trematodes (Barton et al. 1993; Stewart et al. 2003a, b; Šebelova et al. 2004; Lecsanboon et al.

2012; Terenina et al. 2018).

The digestive system of trematodes has the well-developed musculature (Ŝebelová et al. 2004;

Tolstenkov et al. 2010; Krupenko 2014; Petrov and Podvjasnaja 2016; Terenina et al. 2020). In the larval

and adult stages of the life cycle the different compartments of the digestive tract (pharynx, oesophagus,

intestine) of the trematodes are intensively supplied with the serotonergic and peptidergic neurons and

neurites (Tolstenkov et al. 2007; Petrov et al. 2019, Terenina et al. 2015; 2020). In P. alaskense near the

muscular pharynx we identified two 5-HT–IR cells, the presence of which was also found in other

trematodes (Tolstenkov et al. 2010; Terenina et al. 2015). In the distal part of the pharynx of P. alaskense

we found FMRFa–IP nerve fibers. Thus, our data indicate that in P. alaskense the muscle activity of the

© The Author(s) or their Institution(s) Canadian Journal of Zoology Page 14 of 32 14 different compartments of the digestive system participating in the food promotion, is controlled by serotonergic and peptidergic nerve elements.

A well differentiated musculature of the reproductive system of trematodes plays an important role in the process of fertilization and eggs laying (Halton and Maule 2004;). 5-HT– and FMRFa–IP elements were found in the reproductive system of trematodes Cryptocotyle lingua (Creplin, 1825) Fischoeder,

1903, Opisthorchis felineus (Rivolta, 1884) Blanchard, 1895, Allocreadium isoporum (Looss, 1894)

Looss, 1902, Paramphistomum cervi Zeder, 1790, Fasciola hepatica Linnaeus, 1758, Haplometra cylindracea Zeder, 1800, B. gracilescens, Echinostoma caproni Richard, 1964, Opisthioglyphe ranae

(Frölich, 1791) Looss, 1907 and others (Fairweather et al. 1987; McKay et al. 1990; Stewart et al. 2003a, b; Ŝebelová et al. 2004; Tolstenkov et al. 2007; 2010; Terenina et al. 2010; 2015).

Our data show the presence of 5-HT–IP cells near the P. alaskense genital pore. On the whole mount preparations of P. alaskense the positiveDraft staining to 5-HT and FMRFamide are detected in different departments of the worm’s reproductive system. Four 5-HT–IP cells are observed in the area of the ovary location. The positive FMRFamide immunoreactivity is detected in the region corresponding to the location of the proximal part of the uterus. Numerous thin longitudinal FMRFa–IP neurites are observed in the distal uterus of P. alaskense. Our data can suggest the role of the serotonergic and

FMRFa–IP nerve components in the functioning of the reproductive system. Near the excretory pore in P. alaskense we detected two small 5-HT–IP cells, probably taking a part in regulation of the muscle activity of the final sections of the excretory system. The presence of the serotonergic and peptidergic elements near the excretory opening in trematodes was also found in several studies (Terenina et al. 2006;

Tolstenkov et al. 2010).

Thus, our results on P. alaskense (family Lepocreadiidae), from endemic deep-sea fishes, are consistent with the published data for other trematodes on the discovery of the neurotransmitters studied in the central and peripheral compartments of their nervous systems. Our findings confirm the thesis that the presence of the serotonergic and peptidergic (FMRFa-IP) components in central and peripheral regions of the nervous system of trematode is a conservative feature which revealed in parasitic worms

(Terenina and Gustafsson 2003; 2014). The differences in the organization of the nervous system in

different trematoda species are related to the number of the 5-HT–IP and FMRFa–IP neurons. Further

detailed studies to clarify the musculature morphology in the pharynx, oesophagus, intestine and

reproductive system, and their innervations by serotonergic and FMRFamide-like nerve components in P.

alaskense are needed.

Conclusions

The muscular system and the serotonergic and FMRFa–IP components of the nervous system are

described for the first time using immunocytochemical and histochemical methods and confocal scanning

laser microscopy (CLSM) in trematode, P. alaskense (Lepocreadiidae), an intestinal parasite of deepwater

fishes of the north-western Pacific.

The musculature of the body wall of P. alaskense has three layers of muscle fibres – the outer circular, intermediate longitudinal and innerDraft diagonal muscle fibres. The muscles of the attachment, digestive and reproductive organs as well as the excretory sphincter are a well-developed.3. The 5-

НТ–IP and FMRFa–IP neurons and their fibres are detected in the central nervous system of P. alaskense,

in the head ganglia, brain commissure, three pairs of longitudinal nerve cords and connective

commissures. In the periphery, they provide innervations to the oral and ventral suckers, pharynx,

intestine, the proximal and distal parts of the reproductive system and suggest a role in host attachment,

the digestive system functioning and reproduction.

The data obtained on the morphological organization of P. alaskense can be significant not only

for expanding our knowledge on the structure of the nervous and muscle systems of trematodes of various

taxonomic groups but also in future development of new anthelminthic drugs affecting the nervous

systems of parasites.

Acknowledgements:

Authors wish to thank Yashin V.A. for technical assistance at the Optical Microscopy and

Spectrophotometry Core Facilities of Federal Research Center “Pushchino Scientific Center for

Biological Research of the Russian Academy of Sciences” (Pushchino, Moscow Region). The study was partly supported by the Russian Foundation for Basic Research (RU) grant No. 18-04-00349a.

Conflict of Interest: The authors declare that they have no conflict of interest.

Sampling and field studies: All necessary permits for sampling and observational field studies have been obtained by the authors from the competent authorities.

Compliance with ethical standards: All procedures involving animals were performed in accordance with the European Communities Council Directive (November 24, 1986; 86/609/EEC) and the

Declaration on humane treatment of animals. The Protocol of experiments was approved by the bioethics committee of the Institute of Cell Biophysics, RAS and, in accordance with the ethical standards of the

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Figure legends:

Fig. 1. SEM micrographs of the surface topography of Prodistomum alaskense from lumpfish, A. ventricosus. (a) Forebody, the ventral view showing the highest density of tegument spines in sublateral areas; (b) ventral sucker, note nine non-ciliated papillae on the sucker rim; (c) portion of ventral sucker rim and genital pore, note the ventral sucker papillae and the rings of the spines; (d) multipointed spines and ciliated receptors near the ventral sucker; (e) ventral view of the mature worm; (f) ciliated receptor;

(g) oral sucker showing the sucker rim armed with the rings of multipointed spines, frontal pit, and ciliated receptors; (h) multipointed spines and a group of ciliated receptors on lateral forebody surface; (i) low density of multiponted spines on mid-ventral surface of the forebody; (j) simple spines on mid- ventral surface of the hind body. Abbreviations: cr, ciliated receptors; fp, frontal pit; gp, genital pore; mps, multipointed spine; os, oral sucker; pe, posterior end; sa, sucker aperture; ss, simple spine; tr, transverse tegumental ridges; vs, ventral sucker;Draft vsp, ventral sucker papilla. Scale bars are 100 μm (a); 20 μm (b); 10 μm (c, g); 2 μm (d); 200 μm (e); 1 μm (f); 5 μm (h, i, j).

Fig. 2. Musculature of Prodistomum alaskense (from lumpfish, A. ventricosus) stained with TRITC- phalloidin (in red), whole mount preparations; the maxprojections (a-d, l), the snapshots (e-f, g-k, m). (a)

Surface spines and circular muscle fibers (arrows) of the body wall of anterior body region (ventral view), inset – the larger magnification of spinouse tegument; (b) the circular (thin short arrows), longitudinal

(short arrows) and diagonal muscle fibers (arrowheads) in body wall of anterior body region, the ventral sucker and genital pore are visible (ventral view); (c) the circular (short arrows), longitudinal (long arrows) and diagonal (arrowheads) muscle fibers of body wall in the middle body region (dorsal view);

(d) the circular (short arrows), longitudinal (long thin arrows) and diagonal (arrowheads) muscle fibers of body wall in posterior body region, excretory opening is visible, ventral view; (e) the paired circular muscle fibres (short arrows) comprising of body wall of the posterior body part, longitudinal (long thin arrows) and diagonal fibres (arrowheads) are indicated, ventral view; (f) the circular (short arrows) and longitudinal (long thin arrows) muscle filaments of posterior body region and muscular sphincter surrounding the excretory pore (arrows), ventral view; (g) the oral sucker with the radial muscle fibers

(arrows) and the pharynx, ventral view; (h) the radial muscle fibres (arrows) in the pharynx; (i) the dorso-

ventral muscle fibers running from the ventral sucker are indicated (arrows), the genital pore; (j) the

group of the muscles fibers in the body wall running to the testes (circle, arrow), the circular and diagonal

muscle fibres are indicated in this area, ventral view; (к) a higher magnification of the group of muscle

fibers, running from the body wall towards the testes (arrows); (l) the circular and longitudinal muscle

fibres in the ducts of the distal compartments of reproductive system (arrows); (m) the longitudinal

(arrow) muscle fibres in reproductive system nearby the genital pore, the radial muscles (short arrows) in

the ventral sucker and the diagonal muscles (arrowheads) of the body wall are indicated, ventral view.

Abbreviations: cf, circular muscle fibers; df, diagonal muscle fibres; eo, excretory opening; gp, genital

pore; lf, longitudinal fibres; os, oral sucker; ph, pharynx; rf, radial muscle fibres; vs, ventral sucker. Scale

bars are 30 μm (a, b, i); 100 μm (c, d, h); 50 μm (d, f, j, k, l, m); 25 μm (g, and inset on a). Draft Fig. 3. 5-НТ-immunopositive staining (in green) in the nervous system of Prodistomum alaskense from

prowfish, Z. silenus (a, b, d, g) and from lumpfish, A. ventricosus (c, e, f, h); CLSM snapshots (c, e, f) and

maxprojection (h); fluorescent microscopy (g), light microscopy and schematic drawing (i). (a) Neurons

of the brain ganglia, the neurons, located near the oral sucker, the ventral nerve cord, the neurons in the

vicinity of the ventral nerve cord, the neuron near the pharynx, the innervations of the oral sucker with 5-

HT–IP fibers is shown by arrows; (b) the neurons of brain ganglia, the neurons located near the oral

sucker, the neurons in the vicinity of the ventral nerve cord, the neuron near the pharynx, neurons in front

of the ventral sucker, neurons situated near the genital pore, the 5-HT–IP staining near the bottom of the

ventral sucker; (c) a larger magnification of the neurons in the brain ganglia (arrows), the pharynx; (d) the

neurons of the brain ganglia, the neurons, located near the oral sucker, the neurons in the vicinity of the

ventral nerve cord, the neuron near the pharynx, the neurons located in front of the ventral sucker, the

neurons near the genital pore, the 5-HT–IP staining near the bottom of the ventral sucker; innervations of

the ventral sucker are visible (arrows); (e) the innervations of end part of reproductive system (long

arrow) and the ventral sucker (short arrow) with 5-HT–IP neurites (view from the dorsal side of the

body), the neurons near the genital pore; (f) the longitudinal 5-HT–IP neurites in the distal part of

© The Author(s) or their Institution(s) Canadian Journal of Zoology Page 26 of 32 26 reproductive system (arrows), view from the dorsal side, the neurons near the genital pore; (g) the neurons located on the level of the oviduct (arrows), neuron of the ventral nerve cord; (h) a larger magnification of the neurons located on the level of the oviduct (arrows); (i) a schematic drawing of 5-

HT–IP neurons (circle and short arrows) and 5-HT–IP staining (long arrow, encircled) near the posterior edge of the ventral sucker in P. alaskense body, anterior end to the left. Abbreviations: gp, genital pore; os, oral sucker; ov, ovary; nbg, the neurons of brain ganglia; nep, the neurons near the excretory pore; nos, the neurons, located near the oral sucker; nov, the neurons located on the level of ovary; nph, the neurons near the pharynx; nrp, the neurons near the genital pore; nrs, the 5-HT-IR staining near the posterior edge of the ventral sucker; nvnc, the neurons in the ventral nerve cord; nvs, the neurons near the ventral sucker; ph, pharynx; t, testes; v, vitellaria; vnc, ventral nerve cord; vs, ventral sucker; ut, uterus.

Scale bars are 100 μm (a, b, d, g, i); 35 μm (c); 50 μm (e, f, h). Draft Fig. 4. FMRFamide-immunopositive staining (in green) in the nervous system of Prodistomum alaskense from prowfish, Z. silenus (a, b, c, d) and from lumpfish, A. ventricosus (e, f, g), CLSM snapshots (a-f), light and fluorescent microscopy (g), the anterior end of body – to the upper right corner. (a) Neurons of the brain ganglion (arrow), the ventral nerve cords, neurons near ventral nerve cords, the oral and ventral suckers, inset – larger magnification of neuron located in the ventral cord; (b) the neurons of the brain ganglion, neurons in the brain commissure, the dorsal nerve cord and the commissures between them, the neurites running to the oral sucker, the dorsal view; (с) the dorsal nerve cord and commissures between the dorsal cords, FMRFa–IP staining in proximal and distal uterus is visible (view from the dorsal side);

(d) the neurites running to the oral sucker from the brain ganglia (arrows) and the commissure between them, the pharynx, ventral view, inset – the FMRFa–IP neurites near the pharynx (arrows); (e) the neurites running to the ventral sucker (arrows) from the ventral nerve cords; (f) FMRFa–IP staining in the neurites of the distal part of uterus (short arrows) and of the intestine, two neurons in the region of intestine bifurcation indicated by long arrows (view from the dorsal side), the dorsal nerve cords and commissures between the cords, the neurons of the brain commissure are visible; (g) FMRFa–IP staining in the proximal part of the uterus (arrows), view from the dorsal side, the dorsal nerve cords, the ventral

nerve cords, the neuron in the vicinity of the ventral nerve cord are visible, inset a – larger magnification

of FMRFa–IP staining in the proximal uterus, inset b – the position of that FMRFa–IP staining in the

proximal region of the uterus (short arrows). Abbreviations: c, commissures between the nerve cords; cos,

the commissure of the oral sucker; dnc, the dorsal nerve cord; drs, distal uterus; os, oral sucker; nbc,

neurons in the brain commissure; nbg, the neurons of brain ganglia; nfi, neuritis in the intestine; nfos,

neurites of the oral sucker, nvnc, the neurons in the ventral nerve cord; ph, pharynx; prs, proximal uterus;

vnc, ventral nerve cord; vs, ventral sucker. Scale bars are 80 μm (a); 110 μm (b, c); 30 μm (d); 51 μm (e);

50 μm (f); 200 μm (g).

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Figure 1a-j

169x220mm (500 x 500 DPI)

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Figure 2(a-f)

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Figure2 (g-m)

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Figure 3(a-i)

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Figure 4