International Journal for Parasitology 31 (2001) 783±792
www.parasitology-online.com
Immunomicroscopical observations on the nervous system of adult
Eudiplozoon nipponicum (Monogenea: Diplozoidae)
T.H. Zurawski a, A. Mousley b, G.R. Mair b, G.P. Brennan b, A.G. Maule b,
M. Gelnar a, D.W. Halton b,*
a
Department of Zoology and Ecology, Masaryk University, 611 37 Brno, Czech Republic
b
Parasitology Research Group, Queen's University Belfast, Belfast BT7 1NN, UK
Received 22 January 2001; received in revised form 6 March 2001; accepted 6 March 2001
Abstract
Neuronal pathways have been examined in adult Eudiplozoon nipponicum (Monogenea: Diplozoidae), using cytochemistry interfaced
with confocal scanning laser microscopy, in an attempt to ascertain the status of the nervous system. Peptidergic and serotoninergic
innervation was demonstrated by indirect immunocytochemistry and cholinergic components by enzyme cytochemical methodology;
post-embedding electron microscopical immunogold labelling revealed neuropeptide immunoreactivity at the subcellular level. All three
classes of neuronal mediators were identi®ed throughout both central and peripheral elements of a well-differentiated orthogonal nervous
system. There was considerable overlap in the staining patterns for cholinergic and peptidergic components, while dual immunostaining
revealed serotonin immunoreactivity to be largely con®ned to a separate set of neurons. The subcellular distribution of immunoreactivity to
the ¯atworm neuropeptide, GYIRFamide, con®rmed neuropeptide localisation in dense-cored vesicles in the majority of the axons and
terminal varicosities of both central and peripheral nervous systems. Results reveal an extensive and chemically diverse nervous system and
suggest that pairing of individuals involves fusion of central nerve elements; it is likely also that there is continuity between the peripheral
nervous systems of the two partner worms. q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights
reserved.
Keywords: Monogenean neurobiology; Cholinesterase; Serotonin; Neuropeptides; Confocal microscopy; Immunogold labelling
1. Introduction
Eudiplozoon nipponicum (Goto, 1891) is an oviparous
monogenean gill parasite of Far East origin and was ®rst
recorded in Europe on farmed carp (Cyprinus carpio) in
Camarque, France (Denis et al., 1983) and later in the
Czech Republic (Gelnar et al., 1989). Members of the Diplozoidae are unique examples of platyhelminths that display
precocious sexual behaviour insofar as the adult worm is
composed of two fused individuals in permanent copula,
each of which is unable to continue further development
alone. Fusion begins in the larval stage when, on the gills
of the host, the two so-called `diporpae' come into contact,
each grasping the dorsal papillae of the other by means of its
ventral sucker. This initiates a metamorphosis of the joined
pair during which there is reciprocal fusion of the external
openings of the male and female genital ducts, ensuring
cross-fertilisation between the two hermaphroditic partners.
The present study set out to explore the gross neuroanatomy, major neuronal pathways and chemical nature of the
nervous system of this unusual ¯atworm parasite. It is the
®rst account of neuropeptidergic components and the ®rst
demonstration of neuropeptide immunoreactivity at the
ultrastructural level in a member of the Diplozoidae.
There are previous brief descriptions of cholinergic
elements in Diplozoon paradoxum (Halton and Jennings,
1964) and of biogenic amines, as evidenced from glyoxylic
acid-induced ¯uorescence, in E. nipponicum (Lyukshina
and Shishov, 1988).
2. Materials and methods
2.1. Specimen collection and preparation
* Corresponding author. Tel.: 144-2890-335792; fax: 144-2890236505.
E-mail address: [email protected] (D.W. Halton).
Live specimens of E. nipponicum were collected from the
gills of 1-year-old carp (C. carpio) netted in the experimental pond system of the Research Institute of Fisheries and
0020-7519/01/$20.00 q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.
PII: S 0020-751 9(01)00192-8
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Hydrobiology in Vodnany, South Bohemia, Czech Republic
during October and November 1999. The worms were ¯attened and ®xed in 4% (w/v) paraformaldehyde solution in
phosphate-buffered saline (PBS) (pH 7.4) for 4 h at 48C.
2.2. Enzyme cytochemistry
For the demonstration of cholinesterase activity, as indirect evidence of the presence of acetylcholine, adult specimens (n ˆ 10) of the worm were ®xed for 2 h at 48C before
being washed in PBS, rinsed in distilled water, and incubated with 5-bromo-chloro-indolyl acetate (after Pearse,
1960) and examined by light microscopy. Controls
consisted of (i) omission of substrate and (ii) pre-incubation
of specimens in 1 £ 10 25 M eserine sulphate in PBS for 1 h
to inactivate cholinesterase activity.
2.3. Immunocytochemistry
Serotonin (5-HT) and neuropeptide immunoreactivities
were visualised by the indirect immuno¯uorescence technique of Coons et al. (1955). Worms were ¯at-®xed for 4 h
and then washed for 24 h in antibody diluent (PBS containing 0.5% (v/v) Triton X-100, 0.1% (w/v) bovine serum
albumin (BSA) and 0.1% (w/v) sodium azide) prior to
processing. The primary antisera were 5-HT antiserum
(448(1)) raised in a New Zealand White rabbit and used at
a working dilution of 1:500, and neuropeptide antiserum
raised to the ¯atworm FMRFamide-related peptide
(FaRP), GYIRFamide, in a guinea pig at a working dilution
of 1:500. The secondary antisera employed were swine antirabbit IgG conjugated with tetramethylrhodamine isothiocyanate (Dako) for 5-HT (working dilution 1:1000) and
rabbit anti-guinea pig IgG conjugated to ¯uorescein isothiocyanate (Dako) (1:1000) for FaRP. In both cases, wholemount preparations of worms were incubated in the primary
antiserum for 72 h at 48C, washed over 24 h in antibody
diluent, and then incubated in the appropriate secondary
antiserum for 48 h at 48C. Specimens (n ˆ 10) were counterstained with phalloidin-¯uorescein isothiocyanate or
tetramethylrhodamine isothiocyanate (200 ng/ml) in antibody diluent for 48 h, after which they were washed for a
further 24 h in antibody diluent-wash and then mounted in
PBS/glycerol (1:9 (v/v)) and examined using a Leica TCS
NT confocal scanning laser microscope.
For double-labelling of 5-HT and GYIRFamide, ¯at-®xed
and washed adult worm specimens (n ˆ 10) were incubated
®rst with anti-5-HT (48 h), washed overnight in antibody
diluent and then exposed for 24 h to swine anti-rabbit IgGtetramethylrhodamine isothiocyanate (Sigma, 1:500). After
an overnight wash in antibody diluent, specimens were reincubated with anti-5-HT (24 h). They were again washed
overnight in antibody diluent, then incubated with antiGYIRFamide (48 h) (1:500), washed overnight in antibody
diluent, and ®nally treated with ¯uorescein isothiocyanateconjugated rabbit anti-guinea pig IgG (24 h) prior to being
washed, mounted and examined as described.
Controls included (i) omission of primary antisera and (ii)
liquid-phase pre-adsorption of antisera with the appropriate
antigen (50±500 ng ml 21 GYIRFamide).
2.4. Immunoelectron microscopy
For ultrastructural examination, transverse slices (5 mm
in thickness) of worms (n ˆ 5) were obtained from the anterior region to include the cerebral ganglia and associated
commissure, and from the haptor region to localise the
clamp ganglia of the worm. The tissue was ®xed for 6 h at
48C in 8% glutaraldehyde (Agar Scienti®c) in 0.1 M sodium
cacodylate buffer (pH 7.2) containing 3% sucrose and 0.1
mM CaCl2´2H2O. The tissues were washed in several
changes of cold buffer, post-®xed in 1% OsO4 (aqueous)
for 2 h at 48C, dehydrated through graded ethanol to propylene oxide, in®ltrated and embedded in Agar 100 resin
(Agar Scienti®c). Ultrathin sections were cut on a Reichert
Ultracut E ultramicrotome, collected on bare 200-mesh
nickel grids and dried at room temperature.
For electron immunocytochemistry, grids were etched
with 10% hydrogen peroxide for 10 min and rinsed thoroughly with 20 mM Tris±HCl buffer (pH 8.2) containing
0.1% BSA and Tween 20 (1:40 dilution). Following incubation with normal goat serum (1:20 dilution) for 30 min, grids
were transferred to a 20 ml droplet of primary antiserum
(anti-GYIRFamide). GYIRFamide antiserum was diluted
1:70 000 with 0.1% BSA/Tris±HCl buffer and applied to
sections overnight. After thorough washing in BSA/Tris±
HCl, grids were transferred to a 20 ml drop of 10 nm
gold-conjugated goat anti-rabbit IgG (Bio Cell International, 1:100) and incubated for 2 h. All incubations were
at room temperature, after which sections were bufferwashed, lightly ®xed with 2% double-distilled glutaraldehyde (Agar Scienti®c) for 3 min, and ®nally washed with
buffer and rinsed with distilled water. Grids were doublestained with uranyl acetate (4 min) and lead citrate (8 min)
and examined in a JEOL 100CX or Philips CM100 transmission electron microscope, operating at 100 keV.
Controls consisted of (i) incubation of sections with the
gold marker in the absence of primary antibody, (ii) incubation with non-immune rabbit serum (Dako) instead of
primary antiserum, followed by gold marker and secondary
antiserum, and (iii) liquid-phase pre-adsorption of antisera
with GYIRFamide (100±1000 ng ml 21 diluted antiserum).
3. Results
3.1. Gross structure of the nervous system
Based on the staining patterns for the FaRP, GYIRFamide
and cholinesterase, as an indicator of acetylcholine, the
gross structural organisation of the nervous system of Eudiplozoon is summarised schematically in Fig. 1. It is seen to
be typically orthogonal in arrangement. Thus, the central
nervous system essentially takes the form of a pair of cere-
T.H. Zurawski et al. / International Journal for Parasitology 31 (2001) 783±792
Fig. 1. A schematic of adult E. nipponicum illustrating the major neuronal
pathways of the central nervous system, as evidenced by whole-mount
immunostaining for the FaRP neuropeptide, GYIRFamide. b, brain; bs,
buccal sucker; cc, commissure; cl, clamps; dnc, dorsal nerve cord; i, intestine; lnc, longitudinal nerve cord; m, mouth; oo, ootype; ph, pharynx; sne,
caudal surface nerves; tc, transverse connective; vnc, ventral nerve cord.
bral ganglia, connected by a commissure, from which
extend paired anterior and posterior nerve tracts (Fig. 1).
The two cerebral ganglia are each composed of eight to
10 strongly immunoreactive cell bodies and are connected
by a broad commissure of varicose ®bres, the whole
complex being positioned dorsally behind the pharynx
(Fig. 2A,D,E). The neuronal cell bodies measured approximately 12 mm in diameter, but a pair of larger somata,
approximately 24 mm in diameter, extended posteriorly
from the transverse commissure (Fig. 2E). From each ganglion, four main nerves proceed anteriorly, and three nerve
cords (ventral, dorsal and lateral) proceed posteriorly. While
the ventral cords are the best developed, the dorsal cords are
the least developed, extending for only a short distance
down the body. The inner pair of anterior nerves and associated bipolar somata (approximately 15 mm in length)
serve to innervate the pharynx, while the outer pairs of
anterior ®bres extend to the buccal suckers and mouth
region (Fig. 2A,D,E). Of these, the best developed and
785
most immunoreactive for peptide were the middle two
pairs of ®bres that extend between the buccal suckers and
terminate on either side of the mouth in close association
with the paired musculo-glandular organs described by
Khotenovsky (1985) (Fig. 2F). Each of these nerves has a
large cell body, measuring approximately 18 £ 20 mm in
diameter, and an associated smaller (18 £ 9 mm) bipolar
neuron. All three main anterior nerves are varicose in nature
and interconnected by numerous branches on each side of
the pharynx and in the buccal region. They provide a rich,
anastomosing network of ®ne ®bres throughout the muscle
®bres of the buccal suckers (Fig. 2A,D,F).
The ventral nerve cords were strongly FaRP-immunopositive throughout and comprised several (10 or more) individual axons displaying numerous varicosities. Single cell
bodies were positioned at irregular intervals near the surface
of the cords and, in general, measured approximately 12 £ 18
mm in diameter (Fig. 3C). The lateral cords comprised fewer
(®ve or six) component axons and associated cell bodies, but
were strongly immunoreactive for peptide (Fig. 3C). Both
ventral and lateral cords of each partner worm run the full
length of the body, crossing the point of fusion, and extending
into the appropriate haptors (Figs. 1 and 3D). At regular
intervals down the body, transverse connectives cross-link
the lateral and ventral cords, imparting a very distinctive
rectilinear appearance to this part of the central nervous
system (Figs. 1 and 2B); the connectives each comprise
three to four bipolar neurons with beaded axons.
At frequent intervals, nerve ®bres originating in the
ventral and lateral nerve cords extend through bipolar cell
bodies to the subsurface layers where they branch and
anastomose to form part of the peripheral nervous system,
as a network of ®ne ®bres associated with the body-wall
musculature (Fig. 3E). In the forebody region, and located
in each of the lateral nerve cords, was a large and strongly
immunoreactive multipolar cell body (approximately
13 £ 25 mm in size) from which branched ®bres extend
peripherally into the adjacent tegumental region to terminate as swollen and strongly immunoreactive nerve endings.
Other presumed sensory nerves were evident in the mouth
region and in the posterior portion of each haptor.
The ventral nerve cords on each side of the body continue
into the haptor and fuse posteriorly in the form of a neural
loop, giving rise at intervals to unipolar and bipolar axons
that encircle each of the four pairs of clamps and provide a
rich innervation to their musculature (Figs. 1 and 3A,B). At
the level of the haptor a well-developed transverse connective of nerve ®bres and associated unipolar cell bodies joins
the two ventral nerve cords. The much ®ner lateral nerve
cords also extend into the haptor and run around its margins,
with connections extending to each of the clamps (Fig. 2B).
In the posterior portion of the haptor, there is an extensive
plexus of very ®ne nerve ®bres and associated cell bodies
(approximately 6±7 mm in diameter) running from the nerve
complex of the clamp musculature to the posterior tip of the
worm (Figs. 1 and 3B).
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Fig. 2. (A,B) Light micrographs demonstrating cholinergic elements (blue staining) in E. nipponicum. (A) Forebody region showing staining in the paired
cerebral ganglia (cg) and commissure (co), ventral nerve cord (vnc) and innervation of the buccal suckers (bs), pharynx (ph) and paired musculo-glandular
organs (*). (B) Haptor region showing staining of the ventral nerve cords (vnc), transverse commissures (tc) and innervation of clamps (cl). (C±F) Confocal
scanning laser micrographs demonstrating immunostaining for 5-HT and FaRP neuropeptides, and for F-actin following staining with phalloidin, in the
forebody of E. nipponicum. (C) Images showing immunolabelling with ¯uorescein isothiocyanate conjugate (green) for FaRP (i), immunolabelling with
tetramethylrhodamine isothiocyanate conjugate (red) for 5-HT (ii), and composite showing dual labelling with both ¯uorophores (iii). (D) Detail of dual
labelling shown in (C, iii). Note immunostaining in cerebral ganglia (cg) and commissure (co), ventral and lateral nerve cords (vnc, lnc). Note pharynx (ph),
buccal suckers (bs) and musculo-glandular structures (*). (E) Dual stained preparation showing phalloidin staining (red) of muscle of buccal suckers (bs),
pharynx (ph) and musculo-glandular structures (*). Note muscle bundles (arrows) running posteriorly from the pharynx and immunostaining for FaRPs (green)
in the cerebral ganglia (cg), commissure (co) and associated nerves. (F) Dual stained preparation showing the rich FaRPergic innervation (green) of the buccal
suckers (bs) and musculo-glandular organs (*). Note pharynx (ph) and mouth region (mo).
T.H. Zurawski et al. / International Journal for Parasitology 31 (2001) 783±792
787
Fig. 3. (A±F) Confocal scanning laser micrographs demonstrating immunostaining for 5-HT and FaRP neuropeptides, and for F-actin following staining with
phalloidin, in the haptor and body region of E. nipponicum. (A) Dual stained preparation showing serotoninergic (red) and peptidergic (green) innervation to
the four pairs of clamps (1±4) of the haptor. Note serotoninergic cell bodies (scb), peptidergic cell bodies (pcb), ventral nerve cord (vnc) and lateral innervation
(lin). (B) Dual stained preparation showing extensive peptidergic innervation (pin, green) to the four pairs of muscular clamps (1±4) stained red with phalloidin.
Note extrinsic muscle bundles (arrows) and caudal innervation (cin). (C) Mid-body region showing 5-HT (red) and FaRP (green) immunoreactive ®bres in
separate neuronal pathways of the ventral (vnc) and lateral (lnc) nerve cords. Note serotoninergic (scb) and peptidergic (pcb) cell bodies. (D) Image showing
ventral nerve cords (vnc) at the point of fusion of two individual worms. Note the crossover of two separate tracts of peptidergic (green) and serotoninergic
(red) ®bres, and their associated cell bodies (pcb, scb). (E) Plexus of peptidergic cell bodies (pcb) and associated ®bres in the peripheral nervous system
subjacent to the tegument. Note phalloidin staining (red) of muscle ®bres (arrows) of body wall. (F) Image showing peptidergic innervation (red) of the muscle
(green) of the walls of the ootype (ot). Note that it is in the form of a plexus of some 25±30 immunoreactive cells (unlabelled arrows) and associated varicose
®bres. ov, oviduct; ut, uterus.
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A portion of the peripheral nervous system was also associated with the reproductive system where immunoreactivity for GYIRFamide was largely con®ned to the innervation
of the ootype. Here a plexus of some 25±30 cells, each
measuring approximately 11 mm in diameter, with associated varicose ®bres, was found alongside the muscle of
the ootype wall (Fig. 3F). Neuronal cells were also observed
proximal to the walls of the oviduct and uterus, but their
immunoreactivity was weak compared to that of the innervation of the ootype.
Cholinesterase activity was observed in both the central
and peripheral nervous systems, except for the innervation
of the reproductive tracts; otherwise, the general pattern of
staining approximated that described for the neuropeptide
immunoreactivity but without the varicosities (Fig. 2A,B).
In contrast, positive immunoreactivity for 5-HT, while
evident in both central and peripheral nervous systems,
occupied separate neuronal pathways to that observed for
cholinesterase activity and the neuropeptide (Fig. 2C,D).
The strongest staining for 5-HT was recorded in the brain
region where at least three unipolar cell bodies, situated
alongside peptidergic cells and measuring 25±30 £ 15±20
mm in size, occur in each of the ganglia, the latter being
interconnected by a ®ne transverse commissure of bipolar
(approximately 10 mm in size) neurons (Fig. 2C,D). From
one or more of these ganglionic cells, ®bres extend anteriorly to innervate the musculo-glandular organs and mouth
region, while others run posteriorly alongside peptidergic
®bres as part of the longitudinal nerve cords and their
cross-connectives (Fig. 2C,D).
Serotoninergic ®bres were most numerous in the ventral
nerve cords where single 5-HT-immunoreactive cell bodies
(approximately 20 mm in diameter) occurred at intervals in
symmetrical arrangement along the body (Fig. 2C,D). A
large (40 £ 20 mm) 5-HT-immunoreactive multipolar cell
body marked the point on each side of the body at which
the serotoninergic ®bres in the ventral nerve cord divided
and entered the haptor. Here they provided innervation to
the muscles of the clamps on the one hand, and an extensive
plexus of ®ne nerve ®bres around the margins of the haptor
(Fig. 3A) on the other. By far the most intense immunostaining for 5-HT was in the extensive plexus of ®bres
located between the surface tegument and body-wall
musculature. No serotoninergic nerves associated with the
ootype were observed.
Controls for GYIRFamide and 5-HT failed to stain in the
absence of the appropriate primary antisera, and all immunostaining was quenched by pre-incubation of antisera with
500 ng ml 21 of the corresponding antigen. Eserine controls
for cholinesterase activity were at all times negative.
3.2. Immunoelectron microscopy
Ultrastructural examination of neurons in the central
nervous system in both anterior and posterior regions of
Eudiplozoon revealed the presence of occasional somata
and numerous axons, both of which contained numerous,
presumptive neurosecretory vesicles. Each axon was delimited by a plasma membrane and contained dense,
membrane-bound vesicles, microtubules and mitochondria
(Fig. 4A). Two distinct types of vesicles were identi®ed
within neurons. The majority were either large, dense vesicles (mean diameter 100 nm) or small electron-lucent vesicles (mean diameter 50 nm). The larger vesicles were oval
to spherical in pro®le, containing a dense core of material
sometimes separated from the delimiting membrane by a
narrow `halo', thus resembling neurosecretory (peptidergic)
vesicles as described in other invertebrates (Fig. 4B). In
general, both populations of secretory bodies were
randomly dispersed throughout the axons. Accumulations
of dense secretory bodies were also common in axonal swellings or varicosities that occurred at regular intervals along
the nerve tracts, particularly in regions near the cerebral
ganglia and dorsal commissure. Here the numbers of vesicles were often quite large and ®lled the complete crosssectional area of the axon. Possible release sites were
observed where axons containing accumulations of dense
vesicles abutted the extracellular matrix around muscle
®bres, notably those of the clamp musculature (Fig. 4C).
Most of the immunogold labelling for neuropeptide was
speci®c, with the gold probe concentrated over the contents
of the dense secretory bodies, especially in the cerebral
ganglia, dorsal commissure, longitudinal nerve cords (Fig.
4B) and the nerve axons providing innervation to the clamp
musculature. Much of the gold tag was associated with
populations of dense vesicles that occupied neuronal varicosities in the commissure and cerebral ganglia, while smaller amounts of label were found mainly over scattered
dense-cored vesicles in the longitudinal nerve cords and
nerve axons of the clamp musculature. A number of axons
containing dense vesicles that remained untagged were
observed, alongside axons that were immunoreactive for
peptide (Fig. 4A). No labelling was observed outside the
axon. Any small, electron-lucent vesicles or other cell organelles observed were at all times immunonegative.
4. Discussion
The nervous system of Eudiplozoon is typically orthogonal in arrangement with respect to its gross anatomy and
consistent with that in other ¯atworm taxa (Halton and
Gustafsson, 1996; Halton et al., 1998). However, when the
worms are united in pairs in permanent copula, the tracts of
paired longitudinal nerve cords of one individual cross over
those of its partner at the point of fusion. Connections
between elements of the nervous systems of the two partner
worms were apparent, particularly in the area of fusion, and
these will be examined and mapped by confocal microscopy
in a separate study.
The staining observed in this study has revealed a welldifferentiated nervous system in the worm, both structurally
T.H. Zurawski et al. / International Journal for Parasitology 31 (2001) 783±792
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Fig. 4. (A±C) Electron micrographs showing gold labelling for neuropeptide immunoreactivity in the nervous system of E. nipponicum. (A) Portion of a
peptidergic neuron from the brain region, showing large accumulations of immunoreactive dense-cored vesicles (arrows) in the cytoplasm of the soma (SO)
and axon (AX). Note that the axon below contains vesicles (*) that are unreactive for the peptide. NT, neurotubule (scale bar, 0.5 mm). (B) Detail of a portion of
axon from a peptidergic neuron, showing that label is con®ned to the contents of the dense-cored vesicles (scale bar, 0.1 mm). (C) Portion of peptidergic axon in
close apposition to muscle ®bre (MF) and showing a localised accumulation (varicosity) of immunoreactive dense-cored vesicles (arrows). NT, neurotubule
(scale bar, 0.2 mm).
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T.H. Zurawski et al. / International Journal for Parasitology 31 (2001) 783±792
and chemically. Interestingly, the ®rst recorded use of the
indoxyl acetate technique for demonstrating cholinesterase
activity and thereby cholinergic components of the nervous
system in toto in a ¯atworm was with whole-mount preparations of D. paradoxum by Halton and Jennings (1964). Little
or no description of the nervous system in the worm was
given at that time, but use of the technique has since been
applied with great success to numerous other parasitic
species (Halton and Gustafsson, 1996; Halton et al., 1998;
Cable et al., 1996). In Eudiplozoon, there is rich innervation
associated with the forebody, re¯ecting perhaps functional
importance in exploration and in the feeding process. As
expected in a gill parasite, the strongest nerve roots in
terms of size extend into the adhesive haptor where they
support an extensive ganglionic array of neurons innervating the muscle of the clamps.
The patterns of staining have indicated that serotoninergic pathways, both central and peripheral, are separate from
those reactive for peptidergic and cholinergic elements.
Moreover, analysis of anatomical details has shown marked
differences between serotoninergic and peptidergic neurons.
The former are less numerous and have ®ner ®bres and
larger bilaterally-arranged cell bodies; the latter are more
numerous and comprise relatively small cell bodies with
®bres that are generally varicose in nature. The varicosities
most likely represent `pulses' of peptidic secretion in the
axons en route from their site of synthesis in the cell bodies;
however, the possibility that some of these beaded accumulations represent sites of paracrine-like release of neuropeptide cannot be discounted. Similar ®ndings have been
described in studies of other ¯atworms (Halton and Gustafsson, 1996; Halton et al., 1998).
The present demonstration of extensive cholinergic, serotoninergic and peptidergic neuronal pathways in the nervous
system of Eudiplozoon adds to the growing list of ¯atworm
parasites in which a multiplicity of neuroactive messenger
molecules has been found (for details, see reviews in Halton
and Gustafsson, 1996; Halton et al., 1994, 1998; Day and
Maule, 1999). Flatworms are the most primitive extant
animals where there is a cephalic ganglion as an archaic
brain and central nervous system (Reuter and Halton,
2001). The cytochemical discoveries reported thus far
point to a functional complexity to the ¯atworm nervous
system hitherto unimagined and indicate the need for
more neurobiological research into this important basal
group of the Bilateria.
The association of cholinesterase with the ¯atworm
nervous system and its long-established role in cholinergic
transmission point to acetylcholine serving as a neurotransmitter in these organisms. Acetylcholine is a putative transmitter in ¯atworms, and pharmacological studies have shown
both it and cholinomimetics to inhibit ¯atworm muscle,
suppressing its natural, spontaneous rhythm and eventually
inducing ¯accid paralysis. Similarly, 5-HT appears to be of
widespread occurrence in the nervous systems of ¯atworms.
There is good evidence that it plays a role as an excitatory
neurotransmitter (Pax et al., 1996). However, such a dual
mechanism for modulating motility in ¯atworms may be an
oversimpli®cation since there is every likelihood that other
signal molecules are involved, most notably neuropeptides.
A wide variety of peptide immunoreactivities have been
described in parasitic worms, including a number through
cross-reaction with antisera to known vertebrate and invertebrate peptides (Halton et al., 1994; Pax et al., 1996).
However, the immunogens responsible for much of this
staining remain unknown. To date, six authentic ¯atworm
neuropeptides have been isolated and characterised, of
which the best known are the FaRPs. These are as follows:
RYIRFamide isolated from the land turbellarian, Arthurdendyus ( ˆ Artioposthia) triangulatus, by Maule et al.
(1994); GYIRFamide isolated from the aquatic turbellarians, Giardia ( ˆ Dugesia) tigrina and Bdelloura candida,
by Johnston et al. (1995, 1996); YIRFamide isolated from B.
candida by Johnston et al. (1996); and GNFFRFamide
isolated from the cestode, Moniezia expansa, by Maule et
al. (1993). The remaining ¯atworm peptides that have been
structurally characterised are the PP-fold peptides, neuropeptide F, a 39-residue peptide isolated from M. expansa by
Maule et al. (1991), and a 36-residue neuropeptide F analogue isolated from A. triangulatus by Curry et al. (1992) (for
details, see review by Day and Maule, 1999).
The use of antisera generated to these authentic ¯atworm
neuropeptides has revealed, as in the immunocytochemical
part of the present study, that FaRPs are prevalent in the
nervous systems of all ¯atworm species examined thus far
(Halton et al., 1997). That FaRPs are ubiquitous in ¯atworms has led workers to speculate on some of the roles
these neuropeptides may play in ¯atworm biology. These
include neurotransmission and neuromodulation, neuromuscular regulation, thereby in¯uencing activities such as motility and adhesion, and reproductive activity and egg
formation. In physiological studies that have been
conducted so far, FaRPs have been found to be myoexcitatory, inducing contraction of isolated muscle ®bres from
Schistosoma mansoni (Day et al., 1994) and B. candida
(Johnston et al., 1996), and of muscle strips from Fasciola
hepatica (Marks et al., 1996; Graham et al., 1997).
Peptidergic innervation of the muscular wall of the egg
chamber or ootype in Eudiplozoon is consistent with the
®ndings for other monogeneans, namely the gill parasites,
Diclidophora merlangi (Maule et al., 1990) and Discocotyle
sagitatta (Cable et al., 1996), and the polystomatid, Polystoma nearcticum (Armstrong et al., 1997) from the urinary
bladder of a tree frog. In P. nearcticum, immunocytochemistry has shown that FaRPs are expressed in the ootype
innervation only during host spawning when the frog host
is sexually active and when eggs are being produced by the
parasite. These neuropeptides may serve to trigger contraction of ootype musculature during the period of egg assembly.
In the present study, immunoelectron microscopy was
used to demonstrate the subcellular distribution of GYIR-
T.H. Zurawski et al. / International Journal for Parasitology 31 (2001) 783±792
Famide immunoreactivity. Examination of the central and
peripheral nervous systems of Eudiplozoon has revealed
distinct populations of peptide-producing cells, resembling
classical neurosecretory cells and neuronal axons in ultrastructure. Their perikarya and axons contain the usual inclusions of neurotubules, extensive granular cytoplasma,
mitochondria and different sizes and population densities
of secretory vesicles. The peptide content of vesicles has
been revealed by the immunogold tagging of GYIRFamide,
with gold particles occurring selectively over the dense vesicles. The little labelling that did occur between the vesicles
may relate to poor ®xation, allowing some of the peptide to
leak out of the vesicles. Other factors contributing to background labelling have been discussed by Reuter et al.
(1990). While labelled vesicles were observed in perikarya,
nerve processes and synapses, no labelling was evident in
the endoplasmic reticulum cisternae or Golgi saccules. This
may be due to either insuf®cient GYIRFamide concentration or insuf®cient af®nity of the applied antibodies for the
pre-pro-protein of GYIRFamide. A number of electrondense and electron-lucent vesicles were observed to be
free of gold particles, suggesting they may contain peptides
other than GYIRFamide or be non-peptidic in nature.
The GYIRFamide-containing vesicles accumulate in
axon-like processes that extend from the perikarya to
synapse with the muscle. It is possible to speculate that
the peptidergic ®bres subserve a motor function and in¯uence muscle motility. In some places, the peptidergic cell
ducts are swollen with secretion and such varicosities may
represent sites for the release of peptides pre-synaptically.
The released peptides could then diffuse through the intercellular spaces and exert a paracrine-like effect on other
cells and tissues (Maule et al., 1990).
Acknowledgements
This work was supported in part by a grant from The
Grant Agency of the Czech Republic (project #524/00/
0844) to M.G. and D.W.H., by a Research Project grant of
the Masaryk University Brno (project # J07/98: 143100010)
to T.H.Z., and by a European Social Fund grant to A.M. The
authors would like to thank the staff of the Research Institute of Fisheries and Hydrobiology in Vodnany, Czech
Republic, for their kind help with collecting the ®sh.
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