monogenean neuromusculature: some structural and functional correlates

International Journal for Parasitology 17 "0887# 0598Ð0512

S9919!6408:87:,08[99¦9[99 Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ All rights reservedPII] S9919!6408"87#99952!9

Monogenean neuromusculature]some structural and functional correlates

D[W[ Halton�\ A[G[ Maule\ G[R[ Mair\ C[ ShawComparative Neuroendocrinology Research Group\ The Queen|s University of Belfast\ Belfast BT8 6BL\ Northern Ireland\ U[K[

Received 16 October 0886^ received in revised form 13 November 0886^ accepted 1 March 0887

Abstract

Monogenean neuromuscular systems are structurally and functionally well!di}erentiated\ as evidenced by research onthe _sh!gill parasite\ Diclidophora merlangi[ The nervous system in the worm exhibits a raft of putative intercellularsignalling molecules\ localised in neuronal vesicles[ There is cytochemical evidence of co!localisation of neuropeptides andcholinergic substances\ with aminergic components generally occupying separate neurons[ The phalloidin!~uorescencetechnique for F!actin has enabled the demonstration of muscle organisation in the worm[ Body wall musculature comprisescircular\ longitudinal and diagonal arrays of myo_bres whose contractions are believed to be largely myogenic^ circular_bres predominate in the walls of the reproductive tracts[ The major somatic muscles are longitudinal muscle bundles thattraverse the mesenchyme\ the most extensive of which extend from the pharynx to the clamps of the haptor[ Experimentshave shown that some of these muscles may serve in a withdrawal re~ex in the worm\ which can be evoked by waterturbulence[ These and the muscles of the suckers\ pharynx\ clamps\ male copulatory organ and ootype are provided withextensive synaptic innervation that is strongly immunoreactive for FMRFamide!related peptides "FaRPs#\ suggestingcontractions may be neurogenic[ Examination of the physiological e}ects of known ~atworm FMRFamide!related peptideson muscle contractility in vitro has shown those FMRFamide!related peptides isolated from turbellarians to be the mostexcitatory[ Results are discussed with respect to neuromuscular function in adhesion\ alimentation\ and reproduction inthe worm[ Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ All rights reserved

Keywords] Monogeneans^ Diclidophora^ Neuromusculature^ Immunocytochemistry^ Phalloidin staining^ Muscle physiology^ FMRFa!mide!related peptides^ GYIRFamide

0[ Introduction

The past 09 years have witnessed an upsurgeof information on the neurobiology of ~atwormparasites\ including the monogeneans[ Renewedinterest in the _eld was prompted initially by thediscoveries in invertebrates of homologues to recog!nised vertebrate messenger molecules\ namely regu!

�Corresponding author[ Tel[] ¦33!0121!224681^ fax] ¦33!0121!125494^ e!mail] d[haltonÝqub[ac[uk

latory peptides\ and by the increasing availabilityof well!characterised peptide antisera for use as pro!bes in immunocytochemical studies of ~atwormnervous systems[ More recently\ the isolation andchemical characterisation of endogenous neuro!peptides from ~atworms themselves have enabledin vitro studies to be made on the physiologicalactions of these putative neurotransmitters andmodulators on selected ~atworm muscle systems ð0\1\ 2Ł[ Current data support what has become evidentin many other invertebrate groups\ namely that thenervous system has a multifunctional secretory role

D[W[ Halton et al[ : International Journal for Parasitolo`y 17 "0887# 0598Ð05120509

in mediating not only the parasite|s repertoire ofbehavioural activities but also its growth and repro!ductive development[ This paper attempts to reviewsome of the structural and functional correlates ofthe nerveÐmuscle systems of monogeneans\ draw!ing largely on information that has been gainedfrom work using as a model the _sh!gill parasite\Diclidophora merlangi[

Diclidophora merlangi is one of several speciesof diclidophoridean monogeneans parasitising thegills of mainly gadoid _sh[ The worm measures upto 09mm in length and is distinguished by a large\multiple posterior attachment apparatus or haptorcomprising four pairs of pedunculate\ clamp!likestructures that serve as pincers to grasp the sec!ondary gill lamellae of the host[ Anteriorly\ a sub!terminal mouth leads to the buccal cavity intowhich open a pair of opposable buccal suckers\together with the feeding organ or pharynx^ theworm feeds principally on blood drawn from thehighly vascular lamellae of the _sh gills[

1[ Nervous system

1[0[ Gross structure of the nervous system

Enzyme histochemical staining for cholinesteraseactivity\ based on the indoxyl acetate or the ace!tylthiocholine iodide methods\ provided a break!through in staining the nervous system anddelineating it from the surrounding mesenchymaltissue in small acoelomate ~atworms[ Use of thesemethods on whole!mount preparations of worms\such as Diplozoon paradoxum and Diclidophoramerlangi\ has helped to establish the main ana!tomical details of the monogenean nervous systemin toto ð3\ 4Ł[

In common with other ~atworm taxa\ the mon!ogenean nervous system is di}erentiated into a cen!tral nervous system "CNS# and a peripheral nervoussystem "PNS#[ The CNS consists of paired cerebralganglia and connecting commissure and an orthog!onal arrangement of associated longitudinal nervecords and cross!connectives[ The PNS provides notonly motor innervation to muscle _bres but also acomplexity of presumed sensory nerve endings\both ciliated and non!ciliated\ located mainly on

the body surface[ Of the longitudinal nerve cords\the most prominent are the ventral\ having associ!ated with them more neurons than any of the othercords[ In D[ merlangi\ these main nerve cords arefused posteriorly\ thus forming a kind of neural!ring circuit that\ presumably\ is capable of relayingsignals rapidly between the brain in the forebodyand the posterior haptor[ Within the haptor\ nerveroots extend posteriorly from the main nerve cordat intervals\ each marked by a ganglion of cellbodies\ and run into each of the eight peduncleswhere they divide and anastomose into plexuses of_ne _bres to innervate the muscles of the clamp[Ganglionic collections of nerve cell bodies and _breplexuses are also associated with the buccal suckersand pharynx of the worm\ as well as with the repro!ductive tract where they innervate the muscles ofthe male copulatory organ and the wall of the egg!forming apparatus or ootype[ Thus\ as expected\the major sites of motor innervation in D[ merlangicorrelate with organs of attachment and feedingand with those involved in the reproductive activi!ties of insemination and egg formation[ A schematicof the major anatomical features of the nervoussystem in D[ merlangi is illustrated in Fig[ 0[

1[1[ Fine structure of the nervous system

The ultrastructure of the monogenean nervoussystem is comparable with that described for other~atworms\ the most striking feature being thesecretory nature of the neuron[ In D[ merlangi\ ves!icles of di}erent sizes and densities can be recog!nised\ and\ allowing for some overlap\ at least threetypes of presumptive neurosecretory vesicle can berecognised] small\ electron!lucent vesicles "meandiameter\ 39 nm#\ reminiscent of known cholinergicvesicles in vertebrates^ electron dense!cored vesicles"mean diameter\ 79 nm# of varying core densities\identical in appearance to known aminergic andpeptidergic vesicles^ and occasional large electron!dense vesicles "mean diameter\ 049 nm# resemblingelementary granules of vertebrates[ Immunogoldtagging of neuropeptides at the ultrastructural levelin D[ merlangi has shown that the label most oftenoccurred over electron dense!cored vesicles ð5Ł\making it di.cult to ascribe a function to the vari!ous vesicles solely on the basis of their morphology[

D[W[ Halton et al[ : International Journal for Parasitolo`y 17 "0887# 0598Ð0512 0500

Fig[ 0[ A generalised schematic pattern of the nervous system of Diclidophora merlangi "A#\ with details of the innervation of theforebody "B# and of the clamps of the haptor "C#[ bs\ Buccal sucker^ cg\ cerebral ganglion^ cl\ clamp^ clg\ clamp ganglion^ clm\ clampmuscle^ co\ commissure^ dnc\ dorsal nerve cord^ lnc\ lateral nerve cord^ mo\ mouth^ oo\ ootype^ pe\ male copulatory organ^ ph\ pharynx^tc\ transverse connective^ vnc\ ventral nerve cord[

Neurocrine release sites with all the ultra!structural features of conventional\ asymmetrical"polarised# synapses occur in D[ merlangi and maybe single\ shared "convergent and divergent#\ axo!axonal "en passent#\ or neuromuscular^ typically\many of these synapses show accumulations ofsmall\ electron!lucent vesicles against the pre!synaptic membranes ð6Ł[ In addition to these classicsynaptic structures which provide for direct inputfrom the nervous system\ paracrine!like "synaptoid#release sites have been observed between nerve pro!cesses and the extracellular matrix\ particularlynear muscle _bres and gland cells\ and these have

generally involved accumulations of electron dense!cored vesicles ð5\ 7Ł[

1[2[ Neurochemistry

As already mentioned\ developments in immu!nochemistry in recent years have provided well!characterised antisera for use in localising putativechemical messengers and for exploring the secretorydynamics of the nervous system[ In mammals\ thesmall!molecule transmitters such as acetylcholine"ACh#\ 4!hydroxytryptamine "�4!HT\ serotonin#\noradrenaline\ dopamine\ and g!aminobutyric acid


are synthesised in the nerve terminal and\ whenreleased\ are believed to in~uence post!synapticmembrane potentials through a receptor!mediatedopening and closing of ion channels[

While this rapid intercellular communication sys!tem is the basis of synaptic signalling in all knownmetazoan nervous systems\ a much older "in evol!utionary terms# and somewhat slower system ofintercellular communication is also present and usesas messenger molecules biologically!active peptides"regulatory peptides# of between two to 79 aa resi!dues in length[ In contrast to small molecule trans!mitter substances\ bioactive peptides are derived bysite!speci_c enzymatic excision from larger pre!cursor molecules whose molecular structure is enco!ded by the genome of the cell\ and these are thenprocessed in the endoplasmic reticulum "ER# andpackaged through the Golgi apparatus intosecretory vesicles[ The peptidergic vesicles aremoved along the nerves by axonal transport andtheir contents released by exocytosis\ involving aCa1¦¦!dependent\ stimulus!secretion coupledmechanism[ Today\ scores of short!chain peptidesare known from vertebrate neurons and the list ofthese continues to grow at an amazing pace[ Theirreceptor!mediated actions can bring about not onlyshort! and long!term changes in cellular excit!ability\ but can in~uence events such as cellularmetabolism\ reproductive development and geneexpression[ As already mentioned\ neuropeptidesoccur throughout the Animal Kingdom but are par!ticularly abundant in old "in an evolutionary sense#nervous systems\ such that invertebrates are a par!ticularly rich source[ Not surprisingly\ in view ofthe phylogeny of invertebrates\ examination of thestructure of the genes encoding the precursors ofmany of these peptides often reveals homologieswith certain vertebrate peptides[ For example\ sub!stance P\ insulin and glucagon are found in molluscsand insects\ re~ecting common biosynthetic\ evol!utionary and embryological pathways for peptidicneurotransmitters and hormones[ Some peptidefamilies\ however\ e[g[ FMRFamide!related pep!tides "FaRPs#\ are found only in invertebrates\ withreceptors that are seemingly broadly conserved[ Inview of the likely physiological importance ofFaRPs in ~atworms "see later#\ compounds with ahigh selective a.nity for FaRP receptors could

have value as antiparasitic agents[ For further back!ground information on neuropeptides and theirphylogeny\ reference should be made to the text ofBrown ð8Ł^ for commentary on FaRPs in ~atwormsand their potential in anthelmintic discovery\ seeShaw et al[ ð09Ł and Thompson et al[ ð00Ł\ respec!tively[

1[3[ Cholinergic components of the nervous system

A large proportion of the nervous system of D[merlangi is cholinergic\ as evidenced by strong andextensive staining for eserine!sensitive chol!inesterase "ChE# activity throughout most of thecentral and peripheral nervous components[ Chol!inergic innervation is absent in the muscle of thereproductive tract\ including the egg chamber orootype ð01\ 02Ł[ A generally similar pattern of ChEstaining has been recorded in the freshwater _sh!gillmonogenean\ Discocotyle sagittata ð03Ł\ but with 0notable di}erence[ In D[ merlangi\ there was intenseChE staining of a plexus of interconnecting cellbodies and _bres that innervate the body wall mus!cle in the {{neck|| region of the worm^ comparablestaining has not been observed in D[ sagittata[ Thepossible signi_cance of this {{collar|| of ChE stain!ing in D[ merlangi will be discussed later[

1[4[ Serotoninergic components of the nervous sys!tem

The presence of aminergic transmitter substancesin the nervous system of D[ merlangi is con_ned tothe immunocytochemical demonstration of 4!HTð01Ł[ While elements of both the CNS and PNS wereimmunoreactive for 4!HT\ the overall pattern ofstaining was distinct from that recorded for ChEactivity and for neuropeptides "see below#\ sug!gesting that biogenic amines occupy a separate setof neurons in the worm[ This has been con_rmedin dual!labelling experiments described below[

1[5[ Peptidergic components of the nervous system

The peptidergic portion of the nervous system inD[ merlangi is the most extensive\ accounting forthe bulk of the CNS and PNS ð01\ 02\ 04Ł[ Using arange of antisera to known ~atworm neuropeptides


"e[g[ neuropeptide F and FaRPs#\ mapping theimmunostaining using confocal scanning lasermicroscopy has shown that the distribution patternfor peptidic substances is comparable to that pro!duced with ChE staining\ and in contrast with thatfor aminergic substances[ Moreover\ a similar con!focal study involving the simultaneous incubationof worms with primary antibodies to 4!HT and aFaRP neuropeptide "Halton et al[\ unpublished#has con_rmed that the aminergic elements in thenervous system of D[ merlangi occupy a separateset of neurons[ Two notable exceptions to the over!lap of staining for ChE and neuropeptides in D[merlangi are the innervation of the female repro!ductive tract\ including the egg!forming apparatus"see later#\ which seems to be exclusively pepti!dergic\ and the neuronal plexus associated with thebody!wall muscle in the {{neck|| region of the wormwhich appears to be primarily cholinergic[ The sig!ni_cance of these _ndings have yet to be evaluated[

2[ Muscular system

Basically\ ~atworms possess a well!developedbody!wall musculature consisting essentially of anouter circular and inner longitudinal layer\ withadditional inner layers in many species\ includingdiagonal _bres\ and also dorso!ventral _bres thatcourse throughout the mesenchymal parencyhma[It is assumed that these muscles function antag!onistically\ so that when the circular contract andthe longitudinal and others relax\ the wormbecomes slender and elongate\ and that with thereverse action\ the worm becomes shorter andthicker^ contraction of dorsoventral muscles pre!sumably tend to ~atten the worm and diagonal_bres allow lateral movement of the body[ At inter!vals\ the muscle _bres are attached by attachmentplaques or hemidesmosomes to the basal laminaesurrounding various organs or to _brous interstitialmaterial that rami_es throughout the body as askeletal matrix of the worm^ in this way\ contractionof body!wall muscles in ~atworms is seen as themeans for creating a sti} turgor for body shape andfor e}ecting locomotion[ The musculature of thesuckers\ clamps\ pharynx and male copulatoryorgan in monogeneans is derived from the body

wall layers and consists mainly of radial muscles\but with circular and longitudinal layers next toboth inner and outer walls^ specialised muscles maybe present in some worms\ and those muscles inassociation with hard skeletal parts\ such as hooksor sclerites\ may involve tendon!like connections ofconnective tissue[ Less prominent arrangements ofcircular and fewer longitudinal _bres support thereproductive and\ to a lesser extent\ the alimentarytracts as visceral muscle[

Information on the arrangement of muscles inmonogeneans is generally meagre and is derivedlargely from sections examined by light or electronmicroscopy or from observations\ some datingfrom the end of the last century\ on whole!mountsof worms prepared using conventional dyes\ suchas methylene blue or silver stains[ While some ofthese stained preparations were undoubtedly highlyinformative\ results in general were rarely consistentand were invariably non!speci_c[ More recently\ awhole!mount ~uorescence technique has beendeveloped for muscle staining using the phal!lotoxin\ phalloidin\ as a site!speci_c probe for _la!mentous actin "F!actin#[ When coupled with a~uorophore\ the probe displays the contractile por!tion of the muscle _bres ð05Ł[ For ~atworms\ thetechnique has thus far been applied to a selectednumber of turbellarians ð06Ł and in a preliminarysurvey of muscle organisation in monogeneans\trematodes and cestodes ð07Ł[

The following is an overview of the main musclesystems in D[ merlangi\ illustrated schematically inFig[ 09\ together with any associated innervation[It is based on whole!mount preparations of theworm using the phalloidin technique\ both singlyfor muscle staining per se\ and in tandem with anantibody conjugate to the FaRP\ GYIRFamide\ asa means of gaining insight into the spatial relation!ship of muscle and its innervation[ The observationsare augmented\ where relevant\ by examination ofmuscle ultrastructure\ following the application ofstandard TEM[ Typically\ the bulk of the muscle inD[ merlangi is somatic\ of which there are 1 majorgroupings[

2[0[ Body!wall or subtegumental musculature

Body!wall musculature in D[ merlangi consistsof a thin outer circular layer\ lying immediately


beneath the surface tegument\ an intermediate andmuch thicker layer of longitudinal _bres\ and a well!developed inner group of diagonal _bres crossingperpendicularly and presenting a lattice!likeappearance "Fig[ 3#[ Ultrathin sections show thatthe majority of the muscles are individual myo_bresand that\ collectively\ they form an extensive mus!cular sheath beneath the tegument[ The thick andthin myo_laments in the muscle are arranged ina conservative con_guration\ typical of bilateriansmooth muscle\ with dense bodies irregularly!scat!tered among the _laments[ There is an apparentabsence of any synapses with the nerve processespresent in the body wall region\ suggesting thatcontraction of subtegumental muscle is largelymyogenic[ This is borne out by observations on livespecimens of D[ merlangi\ detached from host gillsand examined in sea water at 09>C\ which reveal aspontaneous rhythmicity of body!wall contractionstypical of invertebrate slow!acting smooth muscle^contractile activity can persist for several days andare generally una}ected in worms that have beendecerebrated ð08Ł[

Observations on live worms also reveal that theforebody is a highly motile structure capable ofextending to almost twice its resting length and ofsweeping from side!to!side in an exploratorymanner[ As already mentioned\ in the {{neck||region of the forebody there is a large concentrationor plexus of cholinergic nerve _bres\ and electronmicroscopy images reveal a close association ofthese nerves with the muscle _bres in this region[The signi_cance of this neuromuscular arrange!ment is unclear\ but it likely enables the parasite toexplore adjacent regions of the host gills withrespect to suitable feeding sites\ as well as to locatepartner worms for purposes of cross insemination\without detachment of the haptor and the risk ofdislodgement from the gills[

2[1[ Longitudinal bands of muscle

The second major somatic muscle system in D[merlangi comprises several extensive and well!developed bundles of longitudinal _bres thatoccupy much of the mesenchymal space betweenthe major organ systems of the worm\ and appearto serve mainly as retractor muscles[ Thus\ in the

forebody\ muscle bands are inserted on the convexsurface of each buccal sucker and run posteriorlyto the pharynx and adjacent body wall\ and pairedmuscle bands connect the basal lamina of the rearportion of the buccal cavity to the pharynx "Figs 1\2 and 4#[ Two large\ pronounced pairs of musclebands extend posteriorly from the pharynx] aninner pair to the musculature of the male copulatoryorgan\ and a much more extensive outer pair thatruns from the pharynx to the haptor "Fig[ 5#[ Dual!labelling for F!actin and the FaRP\ GYIRFamide\has revealed that at the point of insertion on thepharynx\ these longitudinal muscle _bres are con!tinuous with the pharyngeal muscles and con!tiguous with numerous _ne\ varicose nerve _breswhose origin can be traced to neuronal cell bodiesin the adjacent cerebral ganglia and associated com!missure "Fig[ 6#[

The large\ paired muscle bands that run back!ward from the pharynx and forebody comprise bun!dles of up to 39 _bres\ each of composite structure inwhich there are eight or more individual myo_bressurrounded in part by connective tissue[ In longi!tudinal section\ the _bres appear elongate or rib!bon!shaped and branching is evident at their tips[In places\ the distribution of electron!dense bodiesis such that they appear to be aligned in a mannerreminiscent of obliquely!striated muscle\ but with!out forming a Z!disc[ Sarcoplasmic reticulum isscant and limited to small cisternae beneath thesarcolemma^ the bulk of the sarcoplasm is occupiedby numerous\ cristae!rich mitochondria andoccasional glycogen clusters\ re~ecting a tissue ofhigh metabolic activity[

The outer pair of muscle bands runs down eitherside of the seminal vesicle and uterus for some dis!tance "Fig[ 2# before their _bres separate andbecome spread out\ eventually forming eight dis!tinct bundles that enter the eight peduncles of thehaptor and become inserted on the _brous dia!phragm of each clamp[ At the point of insertion\the longitudinal clamp muscles in each peduncleare joined by a second muscle bundle which runsdirectly between adjacent peduncles\ thereby inter!linking all eight clamps "Figs 4\ 7 and 09#[ Theanatomy of the clamps in D[ merlangi and the mech!anism of their operation are well!documented inthe now classic studies of attachment modes in this


Fig[ 1[ Confocal image of the forebody of D[ merlangi\ showing pairs of well!developed longitudinal muscle bundles "arrows# insertedonto the posterior portions of the buccal suckers "bs# and buccal cavity and which run back to the pharynx "ph#[ FITC!phalloidin[Scale bar � 49 mm[ Fig[ 2[ Confocal image of the 1 major groups of longitudinal muscle bundles "arrows# that originate in the pharynxand run down the middle of the worm on either side of the seminal vesicle and uterus "�#[ Approximately one third distance down thebody "point marked by arrows#\ the muscle bundles separate and spread out before entering the haptor[ FITC!phalloidin[ Scalebar � 099 mm[ Fig[ 3[ Confocal image of the body!wall musculature of D[ merlangi\ showing outer circular muscle _bres "runninglaterally# which form a lattice with an intermediate layer of longitudinal muscle _bres "running vertically#\ beneath which are broadbands of diagonal muscle _bres "crossing in both directions#[ FITC!phalloidin[ Scale bar � 49 mm[ Fig[ 4[ Confocal image of thearrangement of the extrinsic musculature of the clamp of D[ merlangi[ One of the longitudinal muscle bundles "lmb# originating in thepharynx is inserted on the _brous diaphragm "fd# of the clamp\ and is ~anked by inter!clamp muscles "icm# that run directly to theadjacent clamps[ FITC!phalloidin[ Scale bar � 49 mm[


)

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Fig[ 5[ Confocal image of the pharynx "ph# and associated elements of the cerebral ganglia "cg# and connecting commissure\ showingan inner pair of longitudinal muscle bundles "lmb0# that runs from the pharynx to the male copulatory organ "pe# and an outer pair"lmb1# that extends from the pharynx to the haptor[ Nerves are stained by indirect immuno~uorescence\ using an antiserum toGYIRFamide and TRITC!coupled secondary antibody "red#^ muscles stained with FITC!phalloidin "green#[ Scale bar�49 mm[ Fig[ 6[Confocal image showing that the rope!like _bres of the longitudinal muscle bundles originate in the pharynx "ph# and are contiguouswith numerous _ne\ varicose _bres "unlabelled arrows# that are immunoreactive for GYIRFamide[ co\ commissure[ Staining as in Fig[ 5[Scale bar � 14 mm[ Fig[ 7[ Confocal image of a peduncle showing a bundle of longitudinal muscle _bres "lmb# inserted on the _brousdiaphragm "fd# of the clamp "cp#[ Note the associated GYIRFamide!immunoreactive innervation "unlabelled arrow#[ Staining as inFig[ 5[ Scale bar � 49 mm[ Fig[ 8[ Confocal image of the ootype "egg chamber# in D[ merlangi\ showing the distinct bands of circularmuscle "cmb# and associated GYIRFamide!immunoreactive innervation "unlabelled arrows#[ Scale bar � 49 mm[


Fig[ 09[ A generalised schematic pattern of the main somatic muscle system and body!wall musculature "inset# of D[ merlangi "A#\ withdetails of the organisation of longitudinal muscle bundles in the forebody "B# and in the clamps of the haptor "C#[ fd\ _brous diaphragm^icm\ inter!clamp muscle^ lmb\ longitudinal muscle band^ see legend to Fig[ 0 for key to other abbreviations[

and other monogeneans by Llewellyn ð19\ 10Ł[ Eachof the clamps bears a pair of opposable hinged jaws\supported by a complex array of skeletal bars orsclerites and operated by intrinsic muscles[ Accord!ing to Llewellyn ð10Ł\ the jaws can be snapped tog!ether rapidly as a result of suction pressure createdin the cavity of the adhesive organ by a {{powerfulextrinsic muscle|| pulling on a _brous diaphragm[Clearly\ this extrinsic muscle is one of eight suchmuscles that extend from the clamps to the pharynxas the bands of longitudinal retractor musclesdescribed above[

2[2[ Clamp musculature and adhesion

In D[ merlangi\ the clamps contain a set of antag!onistic intrinsic muscles whose action involves con!traction of an abductor muscle to open the jawswhen applied to two or three secondary gill lamel!lae\ and contraction of a set of three adductor mus!cles that serve to grasp the lamellae\ prior toclamping ð10Ł[ The _ne structure of these muscleshas not been described\ though preliminary studiesin our laboratory show that the bulk of them aredensely!packed myo_bres orientated in a radial


manner and connected to the outer distal and innerproximal clamp basal laminae by hemidesmosomes[As already mentioned\ there is a rich innervation tothe clamp musculature\ provided by large ganglioncells and nerve bundles that are strongly reactivefor both cholinergic and peptidergic substances"Fig[ 7#[ At the EM level\ there is evidence of syn!aptic terminations between the _ne nerve _bres andthe sarcolemma of the clamp!wall myo_bres\ withpresynaptic collections of presumed electron!lucent\ cholinergic and electron dense!cored\ pep!tidergic vesicles\ together with postsynaptic densit!ies[ Many of the myo_bres are interconnected bygap junctions\ suggesting that they may be coupledfunctionally[ Sarcoplasm is sparse and situated onthe periphery of the myo_bres along with mito!chondria and glycogen deposits[ In transversesection\ the thick and thin myo_laments do notappear to form a regular pattern\ and\ while mostof the thin _laments are arranged around the thick_laments\ many of them are scattered and show nogeometrical association[ Many of the thick _la!ments vary greatly in diameter\ most likely re~ect!ing a gradual tapering along the length\ and havepreviously been shown to be a feature of par!amyosin!containing _laments ð11Ł[

While the clamping action described above reliesinitially on the pull of the extrinsic longitudinalmuscle\ it is unlikely that sustained contraction isrequired to ensure the clamp retains its grip on thegill[ Observations on live D[ merlangi attached tothe gills of freshly!caught _sh show them capableof very extensile\ exploratory movement of the fore!body\ which would not be possible if the longi!tudinal retractor muscles of the worm wereconsistently contracted[ Moreover\ to be e.cient\the longitudinal retractor muscles would berequired to pull against a reasonably solid origin\such as would be provided by the forebody whenattached to the host\ for example\ when feeding[ Itis more likely that sustained attachment to the hostgill is a property of the intrinsic muscles of theclamp\ as described by Llewellyn ð10Ł[ Since par!amyosin is known to have a role in the increasedtension and rigidity of adductor muscles in severalinvertebrate phyla\ including platyhelminths ð12\13Ł\ it is not inconceivable that {{catch|| musclesoperate in the clamping mechanism in D[ merlangi[

2[3[ Adhesive attitude and water turbulence

The adhesive attitude of D[ merlangi on the gillsof whiting is such that its haptor is positionedtowards the gill arch so that the bulk of the wormlies downstream relative to the gill ventilating cur!rent "Fig[ 00#[ In this way\ the extensile forebodyis free to sweep the gill surface in an exploratorymanner\ with respect to feeding sites or neigh!bouring worms as possible partners for copulation[As Llewellyn ð19Ł points out\ the adhesive attitudeof D[ merlangi is obviously well!adapted to themajor environmental problem it faces\ namely min!imising resistance to the ventilatory water currentsof its _sh host[ However\ while these currents aregenerally unidirectional\ considerable water tur!bulence can be generated by pressure changesacross the gills through the opening and closing ofthe _sh|s mouth\ operculum and associated valvesð14Ł[ More signi_cantly perhaps\ is that the normalbreathing rhythm in the _sh can be interrupted bya cough re~ex\ during which the operculum is closedand there is a rapid but transient reversal in the~ow of water across the gills ð15Ł\ posing a notinconsiderable survival problem for the ecto!parasite[ One countermeasure to sudden water tur!bulence would be for the otherwise vulnerableworm to quickly foreshorten its body and so mini!mise the likelihood of dislodgement from the gills^the following experiments have demonstrated thatsuch a withdrawal re~ex exists in D[ merlangi[

Using an isometric muscle transducer system torecord motility in the worm in vitro in arti_cialseawater at 4Ð7>C\ Maule ð08Ł showed that waterturbulence elicited large "up to 1mN force# andrapid\ twitch!like contractions of the bands oflongitudinal muscle[ The evoked contractions\which were comparable in pattern in intact\ decer!ebrated or strip!preparations of the worm "i[e[bereft of lateral margins#\ did not habituate or fati!gue nor were they a}ected by cholinergic or amine!rgic drugs or by tetrodotoxin "Na¦ channelblocker#[ In view of its rapidity\ the withdrawalresponse is likely mediated by the nervous systemof the worm[ Numerous putative sense organsadorn the anterior end of D[ merlangi\ the mostnumerous being long cilia "up to 7mm in length#that terminate nerve elements in the tegument[ A


Fig[ 00[ Scanning electron microscopic image of a specimen of D[ merlangi prepared in situ on the gills of whiting "Merlangius merlangus#to show its adhesive attitude[ Note the 1 clamps secured to secondary gill lamellae "arrows#[ Scale bar � 9[4 mm[

long ~exible cilium would seem an ideal detector"rheoreceptor# for monitoring ~ow!rate changes inthe gill ventilating water current and for providinga sensory input for triggering a rapid withdrawalre~ex to turbulence[

2[4[ Physiology of somatic musculature

Diclidophora merlangi exhibits spontaneousmotor activity in the form of either continuousirregular contractions or bursts of activity withintermittent quiescent periods\ all of which can berecorded as described above[ The motility persistsin vitro for up to 09 h and cut preparations of theworm\ in which some 0Ð1mm of surface tissue aretrimmed from the lateral margins\ perform in asimilar manner\ albeit for shorter periods[ This sug!gests the muscles responsible for the recorded con!tractility are largely the central core of longitudinalretractors described above[ Using such prep!

arations\ a number of classic neurotransmittershave marked e}ects on motor activity ð08Ł[ Thus\the application of biogenic amines\ such as 4!HT\dopamine and noradrenaline all increased the rate\amplitude and basal tension of spontaneous con!tractions and were able to initiate activity in quiesc!ent specimens "Fig[ 01#[ However\ these e}ectscould not be blocked with known antagonists\ indi!cating that the receptors involved likely di}er fromthose that have been characterised from mammals[Acetylcholine is considered to be a putative inhibi!tory neurotransmitter in ~atworms\ including D[merlangi ð16\ 17Ł\ and indeed atropine "an AChinhibitor# stimulates contractility in the worm invitro[ However\ motility studies of the worm\ usinga variety of cholinomimetics and antagonists\ pro!duced a mixture of inhibitory and excitatoryresponses\ highlighting again probable di}erencesin selectivity and speci_city between monogeneanreceptors and those of higher vertebrates[


Fig[ 01[ Recordings showing the myoactive e}ects of ~atworm FaRPs "AÐD# and 4!HT "E# on the spontaneous motility of D[ merlangi"lateral margins trimmed#[ Note in "A# increases in frequency\ amplitude and baseline tension with increasing concentrations ofGYIRFa[ Threshold concentrations for myoactivity were lowest for the turbellarian FaRPs] the tetrapeptide amide\ YIRFa "9[90 mM#"C# and the pentapeptide amides\ RYIRFa "B# and GYIRFa "A# "9[0 mM#\ and highest "×09 mM# for the tapeworm hexapeptide amide\GNFFRFa "D#[ After Moneypenny et al[ ð18Ł[

On the other hand\ the actions of known ~at!worm neuropeptides\ namely three FaRPs fromthree turbellarian species and one from the cestode\Moniezia expansa "neuropeptides have yet to beisolated from a monogenean or a digenean#\ oncontractility in D[ merlangi have been found to beconsistently excitatory in respect of muscle tension\amplitude and frequency "Fig[ 01# ð18Ł[ The mostpotent of the peptides tested were the turbellarianpeptides\ in order\ YIRFamide×GYIRFamide�RYIRFamide^ the least potent being the cestodepeptide\ GNFFRFamide[ These di}erences ine.cacy may re~ect peptide interaction with di}er!ent endogenous receptors in the worm or indicatedi}ering interactions with a single FaRP receptor[

In any case\ the results suggest there is a closerstructural similarity between FaRP receptors inmonogeneans and turbellarians\ than in cyclo!phyllidean cestodes[

2[5[ Forebody musculature and feeding

The bulk of the musculature in the forebody ofD[ merlangi supports the anterior attachmentapparatus\ which is also used in feeding[ There is apair of highly muscular buccal suckers and a larger\spherical muscular pharynx\ together with associ!ated bands of extrinsic longitudinal muscles[ Thebuccal suckers are composed largely of bundles ofradial muscles disposed within a thin layer of cir!


cular and longitudinal _bres and are secured to athick basal lamina by hemidesmosomes^ a band ofcircular muscle lies against the rim of the sucker[Nerve endings are frequent and form synaptic ter!minations with muscle sarcolemmas\ as describedfor the clamps\ suggesting that the opening andclosing of the suckers is controlled neuronally[From observations on the organisation of musclesvisualised by phalloidin staining in whole!mountpreparations of D[ merlangi\ it is possible that dur!ing attachment or feeding\ the mouth and buccalcavity of the worm are opened by contraction ofbands of retractor muscle that run from insertionpoints in the basal lamina surrounding the buccalcavity to their origin in the basal lamina of thepharynx\ and that the buccal suckers are then ~at!tened and ~ared against gill tissue[ Contraction ofthe radial muscles of the suckers produces a vac!uum!_lled concavity\ drawing up a plug of gill tissuewhich is pulled deeper into the buccal cavity andinto contact with the lips of the pharynx\ beingsecured by contraction of the circular muscles atthe sucker rim[ The action of the paired extrinsicsucker muscles\ inserted on the basal lamina behindthe buccal suckers and originating from the sidesof the pharynx capsule\ may serve as retractors todraw the suckers backward and also to assist inreleasing their grip following feeding[

The pharynx in D[ merlangi is presumed to be theprincipal feeding organ\ although feeding per se hasnot been documented[ Nevertheless\ pharyngealstructure in the worm is complex but consistentwith a suctorial organ\ involving well!developedlongitudinal\ radial\ and circular muscles\ and thereare associated nerves and synaptic terminations^the pharyngeal lips are muscular and are believedcapable of expanding into a funnel!like con!_guration ð29Ł[ The paired bands of extrinsic musclethat are inserted on the pharyngeal wall\ asdescribed above\ may act as pharyngeal protrusorsand thereby enhance the e.ciency of feeding in theworm by providing the pharynx musculature witha relatively _rm origin[

2[6[ Musculature of the reproductive tract and itsrole in egg formation

The ducting of both male and female componentsof the hermaphroditic reproductive system of D[

merlangi is invested with a layer of largely circularmuscles\ with much fewer longitudinal _bres[ Themuscle layer is generally thin in the proximal gon!oducts but increases in thickness in the ootype "Fig[8# and uterus of the female system and in the semi!nal vesicle of the male complex^ sphincters alsooccur at strategic points[ The ultrastructural organ!isation of the male copulatory organ is reminiscentof that of the pharynx[ It is a spherical structure ofcompact longitudinal\ radial and circular muscles\invested peripherally with an extensive basal laminafor muscle insertion and for anchorage of the circletof recurved genital hooks^ there is a rich innervationof cholinergic and peptidergic _bres ð02\ 20Ł[ Asmentioned earlier\ extrinsic longitudinal musclesconnect the body of the male copulatory organto the posterior portion of the pharynx\ and sincecopulation in D[ merlangi is believed to involve aform of hypodermic insemination ð21Ł\ these extrin!sic muscles may serve to augment the action of themale copulatory organ as an e.cient armed suckerfor localised attachment and disruption of bodywall tissue in the partner worm[

Egg formation involves a highly!ordered seriesof rhythmical contractions of the muscles in thewalls of the oviduct\ seminal receptacle\ ovo!vit!elline duct\ and the ootype or egg!chamber itself[Sphincter muscles operate to release an oocyte fromthe oviduct and a number of vitelline cells from thevitelline reservoir^ they also regulate the passage ofnewly!formed eggs from the ootype into the uterus[In D[ merlangi\ the muscularised ootype wall andassociated ducts receive a rich peptidergic inner!vation from a surrounding ganglionic plexus ofabout 099 neuronal cells "Fig[ 8#[ The cells are dis!tinct from adjacent gland cells and are immu!noreactive for the invertebrate neuropeptides\neuropeptide F and FaRPs ð7\ 02Ł[ Their strategicposition along the oviduct\ at the exit of the vitellinereservoir\ and around the ootype\ suggests the neu!romusculature in this region has a regulatory rolein egg assembly and reproductive function[ Indirectevidence of this is two!fold[ First\ from the _ndingthat FaRP neuropeptides in the innervation of theegg chamber of the monogenean\ Polystomanearcticum\ are expressed only during the brief per!iod of egg production in the worm\ which is insynchrony with the periodic sexual activity of its


treefrog host at spawning ð22Ł\ and\ second\ fromthe fact that FaRPs are myoexcitatory on mono!genean muscle!strip preparations in vitro ð18Ł[

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monogenean neuromusculature: some structural and functional correlates

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