Development in vitro of the neuromusculature of two strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus

International Journal for Parasitology 33 (2003) 413–424 www.parasitology-online.com

Development in vitro of the neuromusculature of two strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus Michael T. Stewarta, Angela Mousleya, Bozena Koubkova´b, Sˇa´rka Sˇebelova´a, Nikki J. Marksa, David W. Haltona,* a

Parasitology Research Group, School of Biology and Biochemistry, Medical Biology Centre, Queen’s University Belfast, Belfast BT9 7BL, UK b Department of Zoology and Ecology, Faculty of Science, Masaryk University, 611 37 Brno, Czech Republic Received 4 November 2002; received in revised form 2 January 2003; accepted 2 January 2003

Abstract Confocal microscopy interfaced with cytochemical procedures has been used to monitor development of the major muscle systems and associated serotoninergic (5-HT, 5-hydroxytryptamine) and peptidergic (FaRP, FMRFamide-related peptide) innervation of the strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus during cultivation in vitro. Sexually undifferentiated metacercariae were successfully grown to ovigerous adults using tissue culture medium NCTC 135, chicken serum and egg albumen. Eggs were produced after 5 days in culture but had abnormal shells and failed to embryonate. 5-HT and FaRP (the flatworm FaRP, GYIRFamide) were localised immunocytochemically in both central and peripheral nervous systems of developing worms. During cultivation, the central serotoninergic and FaRPergic neuronal pathways of the forebody became more extensive, but retained the same basic orthogonal arrangement as found in the excysted metacercaria. Longitudinal extensor and flexor muscles of the hindbody provide support for the developing reproductive complex. The male reproductive tracts were established in advance (day 3) of those of the female system (day 4); completion of the latter was marked by the appearance of the ootype/egg chamber. The inner longitudinal muscle fibres of the female tract appeared prior to the outer and more densely arranged circular muscles. Circular fibres dominate the muscle complement of both alimentary and reproductive tracts. 5-HTand GYIRFamide-immunoreactivities were demonstrable in the central nervous system (CNS) and subtegumental parasympathetic nervous system (PNS) throughout the culture period, but innervation of the developing reproductive structures was reactive just for 5-HT. Only at the onset of egg production was FaRP-IR observed in the reproductive system and was expressed only in the innervation of the ootype, a finding consistent with the view that FaRPs may regulate egg assembly in platyhelminths. q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Strigeid trematodes; Apatemon; Cotylurus; In vitro cultivation; Neuromusculature; Reproductive system; Confocal microscopy

1. Introduction Numerous confocal microscopic investigations have provided often striking imagery of the cytochemical nature and gross anatomical arrangement of the nerve and muscle systems of flatworm parasites (see, reviews by Halton and Gustafsson, 1996; Day and Maule, 1999), with a fewer but no less significant number of motility studies furnishing baseline information on their physiology (Day et al., 1997; Pax et al., 1996; Marks et al., 1996, 1997; Day and Maule, 1999). Collectively these data indicate that muscle function in flatworm parasites is influenced by an interactive set of * Corresponding author. Tel.: þ 44-28-90-335792; fax: þ 44-28-90236505. E-mail address: [email protected] (D.W. Halton).

neuronal substances derived from an extensive and chemically complex nervous system, comprising cholinergic, aminergic and peptidergic components. The most widely researched peptidergic components of helminth nervous systems are those of the neuropeptide family of FaRPs (FMRFamide-related peptides). FaRPergic nerves are abundant and widely distributed in both central and peripheral nervous systems of all species examined. Four FaRP sequences have been isolated from flatworms to date and have been shown to be bioactive, promoting myoexcitatory effects on intact worms or muscle-strip preparations (Maule et al., 2002); there is also some evidence that their signalling mechanism involves a G-protein coupled receptor (Graham et al., 2000). An area of great interest in the FaRP signalling

0020-7519/03/$30.00 q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0020-7519(03)00011-0

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mechanism of helminths is its utility as a potential drug target and the possibility that FaRPs may regulate reproductive function in these parasites (Maule et al., 2002). There is, for example, extensive experimental data to show endogenous FaRPs potently modulate the activity of the ovijector in the pig nematode, Ascaris suum, implicating their involvement in the mechanism of egg release in vivo (Fellowes et al., 1998, 2000). Far less is known about the role of FaRPs in reproduction in flatworm parasites, largely because of the anatomical and technical difficulties in isolating their reproductive structures for experimentation. However, support for a role for FaRPs in coordinating reproductive function in flatworm parasites comes from an immunocytochemical study of the tree-frog parasite, Polystoma nearcticum by Armstrong et al. (1997). They showed that FaRPergic innervation to the parasite’s ootype/egg chamber was only demonstrable during host spawning, suggesting environmental cues triggered expression and processing of FaRP precursor genes within the parasite’s reproductive system. In contrast, positive immunostaining for 5-HT in the ootype nerves was unaffected by the reproductive state of the worm. In view of the aforementioned technical problems, one alternative approach to providing material for monitoring neuromuscular involvement in reproductive function in flatworm parasites is to employ the methodology of in vitro cultivation, in conjunction with cytochemistry and confocal microscopy. Trematode metacercariae provide valuable material for experimental work in vitro since many occur in sterile environments within the intermediate host, especially in fish and often in large numbers, and can be readily cultured (Smyth and Halton, 1983). Strigeid metacercariae, in particular, can be grown to sexual maturity within a matter of a few days in fairly simple media, since they are relatively undemanding nutritionally. Moreover, they are usually non-progenetic insofar as they are reproductively undifferentiated as metacercariae, thus offering potential as model material for examining aspects of growth and differentiation in trematode parasites. In the present study, 5-HT and FaRP-immunocytochemistry have been employed, together with fluorescentlylabelled phalloidin as a probe for F-actin, to monitor development of the strigeid nervous system and associated musculature from the undifferentiated metacercarial stage to ovigerous adult. Particular attention has been paid to development of the egg chamber/ootype and its innervation for evidence of differential expression of FaRP neuropeptides with respect to egg assembly. Two species of strigeid have been used in the study: Apatemon cobitidis proterorhini and Cotylurus erraticus, whose metacercarial stages encyst in tubenose goby and rainbow trout, respectively. While in vitro cultivation of Apatemon has not been previously described, it is known that Cotylurus can be grown in vitro from metacercarial stage to ovigerous adult in 6 days, a rate comparable with that in vivo in its natural

host, the black headed gull (Larus ridibundis) (Mitchell et al., 1978).

2. Materials and methods

2.1. Parasite recovery and culture Encysted metacercariae of Apatemon were dissected from the body cavity of tubenose goby, Proterorhinus marmoratus caught from the Musov reservoir on the river Dyje, Czech Republic and transferred to sterile Hanks’ saline containing antibiotics (streptomycin 100 mg/ml, penicillin 100 IU/ml) for air mail to Belfast. Metacercarial cysts of Cotylurus were removed aseptically from the pericardium and heart muscle of rainbow trout, Oncorhynchus mykiss reared in fish farms in Co. Antrim, Northern Ireland and stored in sterile Hanks’ saline at 48C. Apatemon metacercariae were excysted in sterilised excystment media as described by Kearn et al. (1989) by first placing cysts for 30 min in 0.1% pepsin pH 2 at 418C, followed by brief washing in Hanks’ saline and exposure to 0.1% trypsin and 0.1% bile salt solution, resulting in excystment within 1 min. Cotylurus metacercariae were excysted (Mitchell et al., 1978) by pre-treatment of cysts with 0.5% pepsin at pH 2 for 20 min followed by short washing in sterile Hanks’ saline and transfer into sterilised excystment media containing 0.5% trypsin and 0.2% sodium taurocholate (Sigma – Aldrich Company Limited) at pH 7.4. Excystment occurred after approximately 30 min incubation at 418C. Samples ðn ¼ 10Þ of freshly excysted metacercariae were fixed and stained as described below and used for comparison with worms grown in culture. Both species were cultured to ovigerous adults as follows. Excysted metacercariae were placed in 15-ml sized falcon tubes containing 5 ml of sterile culture medium, plus antibiotic (as Hanks’ saline), and incubated under air at 418C in a stationary water bath for up to 5 days. The medium consisted of 50% chicken serum (Sigma – Aldrich, heat-treated at 578C for 45 min to destroy complement) and 25% NCTC 135 (Sigma – Aldrich). The medium was supplemented with 25% hen egg albumen removed aseptically and heat-treated as described above. By trial, a maximum of five metacercariae were used per tube since larger numbers resulted in increased risk of contamination and reduced growth rates due to nutrient depletion. The tubes were agitated manually three times per day and media changed daily. Samples of worms ðn ¼ 30Þ were recovered for each of the 5 days in culture and washed vigorously three times in Hanks’ saline and flat-fixed for 1 h in 4% paraformaldehyde (w/v) in 0.1 M phosphate-buffered saline (PBS; 0.145 M NaCl; 0.025 M NaH2PO4 2H2O; 0.075 M Na2HPO4; pH 7.4) and then transferred to fresh fixative for a further 3 h.

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2.2. Phalloidin staining Following an overnight wash in antibody diluent (PBS containing 0.35% [v/v] Triton X-100; 0.1% [w/v] sodium azide; 0.1% [w/v] BSA), specimens were incubated in 200 ng/ml phalloidin fluorescein isothiocyanate (FITC) for 24 h, washed overnight in antibody diluent and mounted in glycerol/PBS (1:9, containing 2.5% [w/v] 1,4 diazabicyclo[2.2.2]octane) and viewed using a Leica TCS-NT confocal scanning laser microscope (Leica Microsystems). 2.3. Double-staining of muscle and nerve Worms were separately incubated in antisera to the turbellarian (Bdelloura candida, Girardia tigrina) FMRFamide-related neuropeptide (FaRP), GYIRFamide (working dilution 1/500) and to serotonin (5-hydroxytryptamine, 5HT, 1/300). Specimens were incubated in primary antisera for 5 days, washed in antibody diluent for 24 h and subsequently immersed in secondary rabbit anti-guinea pig immunoglobin (IgG; for GYIRFamide) or swine antirabbit IgG immunosera (for 5-HT), with either the fluorophore tetramethylrhodamine isothiocyanate (TRITC) or FITC for 5 days. Specimens were washed again in antibody diluent, incubated for 24 h in phalloidin FITC or TRITC and, after a final overnight wash in antibody diluent, were mounted in PBS/glycerol for confocal microscopic examination. Immunocytochemical controls were: (a) omission of primary antiserum; (b) substitution of primary antiserum with non-immune serum; and (c) liquid-phase preadsorption of primary antisera by addition of appropriate antigen (50 – 200 ng antigen/ml of diluted antiserum; GYIRFamide was purchased from Sigma-Genosys Biotechnologies [Europe] Limited). Samples of worms in culture ðn ¼ 20Þ were also processed for scanning electron microscopy (SEM), according to the method outlined by Halton (1979), to provide general information on the surface topography of the developing worm.

3. Results During culture, quite considerable variation in size of worms was found within each tube and some (5 – 10%) showed little or no evidence of growth. For this reason, observations were made and results recorded for the bestdeveloped specimens in each case. The developmental sequence observed within the hindbody was comparable for the two genera, although growth rates of the hindbody during the 5 days in culture were different. Thus, outgrowth of the hindbody of Apatemon was more or less linear and reached approximately 600 mm in length whereas for Cotylurus the rate was more exponential in pattern, attaining some 990 mm in length. However, in both genera, oviposition occurred within 5 days of culture (Table 1).

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The body of the metacercaria is divided by a constriction into a conspicuous cup-shaped forebody (Fig. 1A), which acts essentially as an attachment and feeding organ and an inconspicuous cylindrical hindbody that undergoes considerable development to accommodate the reproductive complex and extensions of the intestinal caeca of the adult worm. Phalloidin staining for muscle revealed the two worms to be comparable in gross anatomical structure. Pseudosuckers or lappets lie on either side of the oral sucker and behind the ventral sucker is a protrusible, bilobed holdfast organ (Fig. 1A). The male reproductive system consists of paired testes arranged in tandem, together with vas deferens, seminal vesicle and ejaculatory pouch, the latter opening posteriorly into the genital atrium or bursa (Fig. 1B), which in Cotylurus contains within its cavity the genital bulb. This genital bulb is a muscular, sucker-like structure that everts and inverts intermittently in culture; in its inverted state it lies within the genital atrium but protrudes as a bulb from the end of the worm when everted (Fig. 1C). The female system comprises a single anterior ovary and oviduct, Laurer’s canal, extensive bilateral vitellaria, paired vitelline ducts and reservoir, egg chamber or ootype with surrounding Mehlis’ gland, and from which a short uterus loops anteriorly before descending to the posterior genital atrium. All of the developmental stages exhibited serotoninergic and peptidergic immunoreactivities, following the immunocytochemical procedures employed, and were subsequently counterstained with phalloidin and examined by confocal scanning laser microscopy to establish anatomical detail of the developing neuromusculature. The distribution of serotoninergic and peptidergic elements and disposition of muscle in Cotylurus approximated that demonstrable in Apatemon and so a single description is given for both genera. 3.1. Day 1 Within 1 h of cultivation, the forebody had enlarged quite significantly and revealed an almost identical staining pattern for FaRP neuropeptide and 5-HT to that of the freshly excysted metacercaria, with a pair of strongly immunoreactive cerebral ganglia and three pairs of associated longitudinal nerve cords comprising the central nervous system (CNS). Within 24 h in culture, there was an increase in the number of cross-connectives and nerve fibres associated with the muscle and penetration glands of the lappets; there was also increased innervation around the holdfast provided by projections from a commissure that connects the ventral nerve cords (Fig. 2A). The holdfast was frequently seen to actively protrude from the ventral surface of worms in culture. Serotoninergic and peptidergic immunoreactive fibres innervate large extensor and flexor muscles in the forebody and these appear to interdigitate with the circular and thinner longitudinal muscle fibres of the holdfast (Fig. 2A). Forebody wall muscle was organised

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Table 1 Development changes in the reproductive apparatus and associated innervation of Apatemon cobitidis proterorhini and Cotylurus erraticus cultured in vitro from metacercaria to ovigerous adult Day

Developmental stages

Length of hindbody (mm) Apatemon

0

Genital analgen present. 5-HT- and FaRP-IRs in longitudinal nerve cords (CNS) and sub-tegumental plexuses (PNS) Early testes recognisable. 5-HT-IR in innervation of developing male reproductive system. No FaRP-IR in hindbody observed beyond that of nerve cords and sub-tegumental plexus Muscle of male reproductive system completed; terminal copulatory complex formed with associated 5-HT-IR Testes fully formed. Uterus recognisable in places with associated 5-HT-IR Ootype formed with associated 5-HT-IR; oocytes in ovary; proliferation of vitelline follicles. Weak or no FaRP-IR evident in proximity to ootype Eggs in uterus. 5-HT-IR of ootype unchanged. Strong FaRP-IR in innervation to ootype. NB: 5-HT-IR and FaRP-IR persists in CNS and subtegumental plexuses throughout development

1

2 3 4 5

Cotylurus

100

70

220

190

300

250

410 550

690 870

600

990

n ¼ 20.

into outer circular, intermediate longitudinal and inner diagonal fibres in contrast to that of the hindbody where the diagonal fibres were replaced by an extra layer of more widely spaced bands of circular muscle. This arrangement of somatic muscle remained the same throughout development of the hindbody. In approximately 50% of specimens examined ðn ¼ 30Þ during the first 24 h of culture, the genital primordia were identified as clusters of undifferentiated cells within the outgrowing stump of hindbody. The cells increased in number up to the end of the day at which time early testes, posterior genital atrium and genital bulb were discernable in some specimens (Cotylurus). During this time, the CNS had extended from the forebody into the growing hindbody and remained consistently immunoreactive for both 5-HT and FaRP for the remaining 5-day culture period. Development was also accompanied by extensive 5-HT and FaRPimmunoreactivities in the peripheral nervous system that traced a sub-tegumental network of fine fibres from the forebody into the hindbody, and which ended close by the genital atrium and in tissue encircling the gonopore (Fig. 2B). Posterior extensions of the two gut caeca also followed outgrowth of the hindbody, terminating just proximal to the genital atrium (Fig. 2F).

differentiated (Fig. 2E insert). At the end of the second day, a S-shaped ejaculatory pouch appeared in Apatemon, composed of densely packed inner longitudinal and outer circular myofibres (Fig. 2F). 3.3. Day 3 By day 3, the hindbody had elongated considerably (Fig. 3A) and could be seen to accommodate fully formed testes and accessory ducts, together with the early stages of the developing female reproductive system, most notably the uterus (Fig. 3A, B). The vas deferens, developing seminal vesicle and ejaculatory pouch run alongside the uterus and enter the genital atrium. Here, a subsurface network of 5HT-immunoreactive nerve fibres was evident around the common genital opening (Fig. 3C, D). Posteriorly, the wall of the uterus at this stage comprised longitudinal and circular fibres, but more anteriorly, it was incomplete, comprising loose fibres encircling longitudinal muscle or consisting merely of portions of longitudinal muscle. 5-HT immunoreactivity (IR) was consistently observed in nerves running in close proximity to all observed sections of uterus independent of its stage of development. 3.4. Day 4

3.2. Day 2 The second day of culture was characterised by continued outgrowth of the hindbody (Fig. 2C) and differentiation of a portion of the genital anlagen into paired testes. Extensor and flexor muscles continued to extend posteriorly from the forebody and either connected to the body wall or intestine (Fig. 2D), or ended blindly in the hindbody. Branches of these large bands of muscle ran to the terminal region of the developing hindbody (Fig. 2E) to support the genital bulb which in Cotylurus was now well

The female reproductive system became fully developed over the fourth day in culture (Fig. 4A), with oocytes recognisable in the small spherical ovary. Branches of the extensor-flexor muscle system run in close apposition to the various ducts of the developing female system and presumably provide support (Fig. 4B –D). As with the uterus, the walls of the oviduct develop fine longitudinal muscle fibres prior to gaining an outer layer of dense circular fibres. The oviduct is joined by Laurer’s canal, a duct that opens to the dorsal surface and consists of very

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dense bands of circular muscle, before it links with the vitelline duct to form the ovovitelline duct (Fig. 4B). A distinct proliferation of vitelline follicles was apparent along the dorsal side of the hindbody. The ootype/egg chamber is the last component of the reproductive apparatus to have its longitudinal and circular muscles established, and these are in continuity with the muscle bands of the ascending limb of the uterus (Fig. 4C). No eggs were present in the uterus at this stage and only weak expression of FaRP was detected in the innervation of the developing ootype/ egg assembly apparatus of one or two specimens. In contrast, consistently strong 5-HT-IR was seen in the nerve fibres and associated cell bodies innervating the ootype, uterus, ejaculatory duct and genital atrium (Fig. 4D). 3.5. Day 5 Three important developments occurred during the fifth day of culture: the extensor and flexor muscles of the hindbody no longer appeared in close association with the female reproductive tract (Fig. 5A); the uterus now contained small numbers of eggs (n ¼ 1 – 5, Fig. 5B) and there was strong expression of FaRP neuropeptide in the innervation of the ootype/egg chamber (Fig. 5D). Thus, a fine network of nerve fibres, immunoreactive for FaRP, emanated from the ventral nerve cords to innervate the egg assembly apparatus which was now located centrally in the hindbody. Strong FaRP-immunostaining was present in the cell bodies and associated plexus of fibres that innervated the predominately circular muscle of the oviduct, ootype and uterus (Fig. 5C –E). Most of this innervation was varicose in nature, imparting a distinct beaded pattern of peptide immunoreactivity. 5-HT-immunostaining was comparable to that observed in 4-day-old worms, although the number of cross-connectives in the PNS had increased. Structurally, the ootype wall at this stage, consisted of dense bands of circular muscle and to a lesser extent, thinner longitudinal fibres (Fig. 5D); where the ootype merged with the uterus, the muscle bands were less tightly packed (Fig. 5E). All of the eggs examined ðn ¼ 40Þ from cultured worms had abnormal shells and failed to embryonate. A schematic of the gross anatomical arrangement of serotoninergic (green) and FaRPergic (red) nerve elements and their relationship with reproductivce structures in the hindbody of C. erraticus is presented in Fig. 6. Fig. 1. (A –C) Scanning electron micrographs of Cotylurus erraticus: (A) forebody showing oral sucker (os), ventral sucker (vs), paired pseudosuckers (ps) and holdfast organ (hf, inverted); (B) posterior of Cotylurus hindbody with genital bulb contracted. A Y-shaped slit marks the opening of the genital atrium (ga) through which spermatozoa (sp) can be seen. The excretory pore (ep) lies ventral to the genital atrium and the end of the hindbody bears uniciliated sense endings (arrow). (C) Posterior of Cotylurus hindbody showing the everted genital bulb (gb) covered by small protuberances (arrow). The ejaculatory duct emerges on the dorsal surface of the bulb when spermatozoa (sp) are being released.

4. Discussion Development in vitro of the sexually undifferentiated strigeid metacercariae of Apatemon cobitidis proterorhini and Cotylurus erraticus to ovigerous adults was achieved within 5 days at 418C, using a semi-solid culture medium of NCTC135, chicken serum and egg albumen. The inclusion of albumen as a rich source of amino acids appeared to be of

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nutritional benefit, insofar as it reduced the time to egg production by 1 – 2 days compared to that obtained by Mitchell et al. (1978) for C. erraticus. The provision of a semi-solid culture medium has been linked to reduced maturation time in the trematodes, Diplostomum spatha-

ceum and Bucephaloides gracilescens in that, it provides substratum for attachment and apparently prompts a more rapid rate of ingestion of food (Kannangara and Smyth, 1974; Halton and Johnston, 1983). However, increased contamination was also noted in those tubes containing

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Fig. 3. (A –D) Confocal images of specimens after 3 days in culture. (A) Hindbody of Cotylurus has grown out and ducting of the female reproductive system is recognisable. u, uterus; v, vas deferens. (B) Apatemon showing an incomplete uterus (ut) with sections absent or consisting of only longitudinal fibres. The distal portion of the reproductive complex, consisting of S-shaped ejaculatory pouch (ej) and uterus (ut) opening into the genital atrium (ga), appears fully formed. (C) The ejaculatory pouch (ej) in Cotylurus opens onto the surface of the inverted genital bulb (gb). ut, uterus. (D) 5-HT-IR in cell bodies (cb) associated with the longitudinal and circular muscle of the uterus (ut) and common genital opening in Apatemon.

Fig. 2. (A –B) Confocal images of strigeids after 1 day in culture: (A) Cotylurus (muscles stained green by FITC-phalloidin) showing one lobe of the everted holdfast (hf) projecting from the cup-shaped forebody (fb). Arrows indicate the opening of cup. Note extensor and flexor muscles (ex). Insert shows holdfast muscle (red, TRITC-phalloidin) and associated FaRPergic nerves (green). (B) 5-HT immunoreactivity (IR, red) in hindbody of Apatemon showing sub-surface plexus of fine nerves derived from ventral nerve cords (vnc) that fuse posteriorly around the excretory pore (ep). Insert. Hindbody wall muscle of Cotylurus. Arrows indicate accumulations of fluorophore. ep, excretory pore. (C –F) Confocal images of phalloidin-FITC stained specimens after 2 days in vitro. (C) Apatemon showing extensor and flexor muscles (arrows) extending from forebody (fb) into hindbody (hb). (D) Extensor muscle (ex) attaches to inner circular muscle (icm) of hindbody wall of Cotylurus while sending a process posteriorly (unlabelled arrow) to intestine (it). (E) Extensor/ flexor muscles (ex) extend to the posterior of Apatemon where the genital atrium (ga) is formed. ej, ejaculatory pouch. Insert. Hindbody of Cotylurus with genital bulb (gb) within genital atrium. ep, excretory pore (ep). (F) In Apatemon the ejaculatory pouch (ej), supported by extensors (ex) and composed of longitudinal and circular muscles, opens into the genital atrium. Note circular (cm) and longitudinal muscle (lm) of gut caeca.

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Fig. 4. (A–D) Confocal images of muscle and associated innervation in Cotylurus (A,B) and Apatemon (C,D) after 4 days in culture. (A) Structurally the female reproductive apparatus appears fully formed. ot, ootype; ut, uterus. (B) The ovary (ov) opens to the oviduct (ovd) which is joined by Laurer’s canal (Lc). The vitelline duct (vd) joins the oviduct forming the ovovitelline duct (oid) before running into the ootype (ot), which opens into the ascending limb of the uterus (aut). (C) Extensor/flexor muscles (ex) run closely associated with the muscle of the female system and attach to the circular muscle (cm) around the genital opening. ej, ejaculatory pouch; ot, ootype; ut, uterus. (D) 5-HT-IR in cell bodies (cb) and nerve fibres (unlabelled arrows) innervating ootype (ot), uterus (ut), ejaculatory pouch (ej) and genital atrium (ga).

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Fig. 5. Confocal images of muscle (A,B) and associated GYIRFamide-IR (C–E) in nerves of the egg assembly apparatus of ovigerous Cotylurus (5 days in culture). (A) Gross anatomy of musculature of mature adult. Note everted genital bulb (gb). Extensor/flexor muscles (ex) run to the posterior of the worm but no longer connect with uterus (ut), ootype (ot), or vitelline duct (vd). ej, ejaculatory pouch; ov, ovary. (B) Abnormally-shaped eggs (eg) autofluoresce in uterus (ut). (C) FaRP-IR in nerves around the mainly circular muscle of oviduct (ovd). ov, ovary. (D) Pulses of FaRP staining (unlabelled arrows) in ootype innervation (ot). cm, circular muscle; longitudinal muscle (lm). (E) The circular muscle (cm) of the uterus (ut) is more loosely arranged. Note GYIRFamide-IR in associated cell bodies (cb) and fibres.

albumen despite heat treatment prior to use. Although the rate of development achieved in vitro was comparable to that found for Cotylurus raised naturally in gull chicks, the eggs produced were abnormal in appearance and failed to embryonate, indicating imperfect physicochemical conditions and/or possible lack of one or more essential amino acids or growth factors. Smyth (1959) believed the failure to produce viable eggs in culture was due to an inability of the medium to satisfy the synthetic requirements of the vitellaria. Fertile eggs capable of embryonation have been recorded from cultured specimens of the related

strigeid, Cotylurus lutzi by incorporation of chick intestinal mucosal extracts into the medium (Basch et al., 1973). However, the essential factor(s) responsible has never been defined and all subsequent attempts to repeat this success with C. erraticus, Cotylurus variaegatus and Cotylurus strigeoides have met with failure (Fried et al., 1978; Halton, personal observations). While there was some enlargement of the forebody in the initial stages of culture, the anatomical arrangement of somatic muscle in this region remained fairly constant throughout development, with outer circular, intermediate

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Fig. 6. Schematic showing the anatomical arrangement of serotoninergic (green) and FaRPergic (red) innervation and its relationship with reproductive structures in Cotylurus hindbody following 5 days in culture from the excysted metacercarial stage. Key to abbreviations: ats, site of anterior testis; cb, cell body; ej, ejaculatory pouch; ex, extensor muscle; fb, forebody; ga, genital atrium; gb, genital bulb; gm, ganglionated mass of cells; it, intestine; Lc, Laurer’s canal; lnc, lateral nerve cord; ot, ootype; ov, ovary; ovd, oviduct; pts, site of posterior testis, stp, sub-tegumental plexus; sv, seminal vesicle; ut, uterus; v, vas deferens; vi, vitelline follicle; vnc, ventral nerve cord; vr, vitelline reservoir.

longitudinal and inner diagonal fibres as described by Stewart et al. (2003). A quite different arrangement of muscle appears in the hindbody wall reflecting its role in accommodating the developing reproductive complex. Here diagonal fibres are absent, being replaced by an extra layer of more widely spaced circular muscle. This provides insertion for branches of the extensor and flexor muscles that presumably not only serve in movement of the outgrowing hindbody but also seem to support the developing reproductive apparatus.

Significant changes occurred in nerves supplying some of the adhesive organs during culture. For example, FaRPergic innervation of the pseudosuckers or lappets and, in particular, the holdfast became more extensive over the first 24 h in culture, and this elaboration of peptide was accompanied by an increase in the muscular activities of these organs. Thus, the two elongated lobes of the holdfast were frequently seen protruding from the cupped forebody in an exploratory manner. Their eversion involves simultaneous contraction of well-developed longitudinal and circular muscles, aided by the rhythmical pulsatory action of the forebody and distension of the fluid-filled excretory system which partly extends into the holdfast organ itself ¨ hman, 1965). The holdfast is a structurally complex (O musculo-glandular organ believed to function mainly in extracorporeal digestion and absorption of nutrients (Erasmus, 1969a), and the observed activity could be indicative of worms beginning to feed on the culture medium. On the other hand, the lappets appear to be the primary organs of attachment in strigeids (Erasmus, 1969b). Evidence from the present study supported this in that most worms from days 1 or 2 in culture were seen attached to clumps of egg albumen by their lappets. A grid-like arrangement of circular and longitudinal fibres makes up the muscle of the intestine, unlike the situation in either Schistosoma mansoni (Mair et al., 2000), where longitudinal fibres are absent, or in Fasciola hepatica where the fibres have a loosely organised arrangement (Mair et al., 1998). Peristaltic contraction of circular fibres presumably ensure efficient movement of ingesta through the intestine while a well-organised array of longitudinal fibres enables rapid shortening of the gut during periods of vigorous hindbody contractions, such as has been observed during oviposition. Novel details of the developing muscle of the reproductive apparatus were demonstrated in both species. The ducting of the female system is composed mainly of circular muscle; longitudinal fibres are present to a lesser extent but appear in the duct walls before the larger bands of circular muscle develop around them. The male reproductive tracts are established in advance of those of the female system, with the ootype/egg chamber being the last to appear. Interestingly, the muscle of Laurer’s canal is particularly well developed consistent with view that this dorsally opening duct represents a vestigial vagina. In at least two trematodes, Lialope copulans and Corryriclum faba, insemination via Laurer’s canal has been observed; however, there is also evidence albeit indirect that the canal serves as a conduit for excess shell material which might otherwise disrupt the precision with which eggs are assembled in the ootype (Dawes, 1956). Observations on specimens in culture revealed instances where worms were attached to one another by means of their posterior ends and in apparent copula. It is known that strigeids mate tail-to-tail, being attached to host mucosa by the lappets of the forebody such that their hindbodies are

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free to locate partner worms, mediated it is assumed via the numerous sensory endings that adorn the distal hindbody. With respect to this, the fine deposits of phalloidin-FITC localised in discrete pockets around the posterior of the hindbody may mark filaments of actin associated with these receptors. Although there are no published data on the strigeid genital bulb, studies on flatworms such as the acoel turbellarian, Otocelis luteola by Grae and Kozloff (1999) have ascribed a role for similar structures in providing temporary storage for sperm following cross-insemination. In Cotylurus, it would appear that the genital bulb of one worm grasps that of its partner by muscular contraction such that sperm can be released through the opening of the dorsally situated ejaculatory duct. While immunostaining patterns for 5-HT and FaRP were comparable in the forebody throughout development, these two neuroactive substances occupied separate neuronal pathways, a finding consistent with other flatworms examined (Halton and Gustafsson, 1996). FaRPergic neurons were smaller in size (approximately 16 £ 8 mm) and more numerous and dominated the innervation of the attachment apparatus, most notably the lappets and holdfast. Most had beaded axons reflecting sites of FaRP accumulation, some of which may be for paracrine release into adjacent muscle tissue, during its passage down the axon (Maule et al., 1990a). On the other hand, serotoninergic neurons were larger (approximately 22 £ 12 mm) and less frequent in number but, in contrast to the FaRP innervation, their axons serving the muscle of both male and female systems stained consistently strong for 5-HT from early development. 5-HT has been implicated in basic developmental processes, such as cell proliferation and morphogenesis in many of the invertebrate phyla examined (Weiger, 1997) and as such, may have a role in neuroand myogenesis in flatworms. Serotonin-induced neuroplasticity appears to be conserved across a broad range of invertebrate phyla (Mitchell et al., 2001), offering an explanation as to why this substance is so prominent throughout the developing nervous tissue of these strigeids. When examined experimentally, 5-HT has been implicated as an excitatory neurotransmitter (Pax et al., 1996), but can also modulate the effect of FaRPs on isolated muscle fibres of trematodes (Day et al., 1994). The FaRP- and 5-HT-IRs associated with the muscle of the developing reproductive tracts displayed a mixed pattern. 5-HT-immunoreactive neurons supplied the only observable innervation to the copulatory apparatus and terminal portion of the uterus during development, whereas both 5-HT and FaRPergic fibres served the ootype/egg assembly apparatus but with temporal variation. Thus, while 5-HT was a consistent feature of the innervation along the developing female reproductive tract, FaRP-IR was demonstrable in the innervation of the ootype/egg assembly apparatus only when the worm was producing eggs. In contrast, FaRP-immunostaining in the CNS fibres innervating the attachment apparatus and sub-tegumental plexus in

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the hindbody was independent of the reproductive state of the worm and was evident throughout development. Peptidergic innervation of the ootype muscle has been demonstrated in a number of other trematodes, namely, the monogeneans, Diclidophora merlangi (Maule et al., 1990b) and Eudiplozoon nipponicum (Zurawski et al., 2001) and the trematodes, Fasciola hepatica (Magee et al., 1989; Marks et al., 1995) and Schistosoma mansoni (Marks et al., 1995). However, the strongest evidence to support FaRP involvement in the neuronal control of egg production comes from the study by Armstrong et al. (1997) of the monogenean, Polystoma nearcticum in which FaRP expression in the ootype innervation appeared to be switched on and off to coincide with egg production. The present study corroborates these findings and gives further credence to the view that FaRPs may regulate contraction of ootype musculature during egg assembly.

Acknowledgements We would like to thank the European Social Fund for Postgraduate Awards (M.T.S., A.M.), the Grant Agency of the Czech Republic #524/00/0844 (D.W.H B.K.) and Queen’s University Belfast for a Visiting Postdoctoral Fellowship (Sˇ .Sˇ .).

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