SEROTONINERGIC AND PEPTIDERGIC NERVE ELEMENTS IN THE PROTOSCOLEX OF ECHINOCOCCUS-GRANULOSUS (CESTODA, CYCLOPHYLLIDEA)

The localisation and distribution of 5-hydroxytryptamine (5-HT, or serotonin) and neuropeptides in the nervous system of the protoscolex of the hydatid organism Echinococcus granulosus were determined by an indirect immunofluorescence technique. Nerve-cell bodies immunoreactive for 5-HT occurred in the lateral ganglia and in association with the lateral longitudinal nerve cords. 5-HT immunostaining was also evident in the central nerve ring, in the rostellar nerves and in the nerve plexus innervating the suckers. Of the antisera used to screen the protoscolex for neuropeptide immunoreactivity (IR), immunostaining was obtained with those raised against pancreatic polypeptide (PP), peptide YY (PYY), substance P (SP), peptide histidine isoleucine (PI-II) and vasoactive intestinal peptide (VIP). The most extensive pattern of IR occurred with antisera to PP and PYY. Immunoreactive nerve elements were evident in the lateral ganglia, central nerve ring, rostellar nerves, rostellar ganglia, sucker plexus and longitudinal nerve cords. The distribution of SP-, PHI- and VIP-IRs was more restricted: SP-IR occurred in the lateral ganglia and sucker nerves, whilst PHI- and VIP-immunoreactive nerve elements were associated with the lateral longitudinal nerve cords. Protoscoleces cultured in vitro for 29 days were also examined and neuroanatomical changes noted. A greater development of the longitudinal nerve cords and their cross-connectives in the body of the worm was evident, and a group of nerve cells were seen to develop at the posterior end of the main lateral nerve cords.

CYTOCHEMICAL OBSERVATIONS ON CHOLINERGIC, SEROTONINERGIC AND PEPTIDERGIC NEURONAL PATHWAYS IN CEPHALOCHLAMYS-NAMAQUENSIS

The localization and distribution of cholinergic, serotoninergic (5-HT, serotonin) and peptidergic components of the nervous system of adult Cephalochlamys namaquensis (Cestoda: Pseudophyllidea) have been determined using enzyme histochemical and immunocytochemical techniques interfaced with light and confocal scanning laser microscopy. All three classes of neuroactive substance showed a similar pattern of staining, occurring extensively throughout the central and peripheral nervous systems of the parasite. There were some minor regional differences in staining, suggesting specific roles for certain classes of neurone, and nerve cell bodies were most evident following immunostaining for serotonin. The general overlap in the distribution of staining may be indicative of som co-localization of neurotransmitter and/or neuromodulatory substances.

IMMUNOCYTOCHEMICAL AND RADIOIMMUNOMETRICAL DEMONSTRATION OF SEROTONIN-IMMUNOREACTIVITIES AND NEUROPEPTIDE-IMMUNOREACTIVITIES IN THE ADULT-RAT TAPEWORM, HYMENOLEPIS-DIMINUTA (CESTODA, CYCLOPHYLLIDEA)

Standard indirect immunocytochemical techniques have been interfaced with confocal scanning laser microscopy (for whole-mount preparations) and epifluorescence microscopy (for cryosections) to investigate the occurrence and distribution of serotoninergic and peptidergic nerve elements in adult H. diminuta. Serotonin (5-HT)-immunoreactivity (IR) was widespread throughout the worm, occurring in the paired cerebral ganglia, transverse commissure, the 10 longitudinal nerve cords and in a plethora of small nerve fibres of the peripheral nervous system. An abundance of serotoninergic nerve cell bodies was found in association with the lateral nerve cords. The genital atrium and accessory reproductive ducts were richly innervated with serotoninergic nerve fibres. Thirty-five antisera to 20 vertebrate regulatory peptides and 1 invertebrate peptide (FMRFamide) were used to screen the worm for neuropeptide IR. Immunostaining was obtained with antisera raised to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), substance P (SP), peptide histidine isoleucine (PHI), xenopsin (XP) and FMRFamide. The most extensive pattern of IR occurred with antisera to PP and PYY, IR being evident in the cerebral ganglia, transverse commissure, longitudinal nerve cords and in small nerve fibres that ramified throughout the parenchyma. A series of bipolar nerve cell bodies between the median nerve cords displayed PP/PYY-IR. The distribution of FMRFamide-IR was reminiscent of the PP/PYY pattern but was less extensive. Comparison of the serotoninergic and peptidergic nervous systems has revealed general similarities and some distinct differences, especially with regard to the distribution of immunoreactive nerve cell bodies. Quantitative data are presented on the levels of PP-, SP-, PHI-, and gastrin-releasing peptide (GRP)-immunoreactivities demonstrable in acid-alcohol extracts of whole worms. The highest level of peptide IR determined was recorded for PP.

CYTOCHEMICAL DEMONSTRATION OF CHOLINERGIC, SEROTONINERGIC AND PEPTIDERGIC NERVE ELEMENTS IN GORGODERINA-VITELLILOBA (TREMATODA, DIGENEA)

Standard enzyme cytochemical and indirect immunocytochemical techniques have been used in conjunction with light and confocal scanning laser microscopy (CSLM) to visualize cholinergic, serotoninergic and peptidergic nerve elements in whole-mount preparations of the amphibian urinary-bladder fluke, Gorgoderina vitelliloba. Cholinesterase (ChE) activity was localized in paired anterior ganglia, a connecting dorsal commissure and in the origins of the ventral nerve cords. Cholinergic ganglia were also evident in shelled embryos in the uterus. Serotonin-immunoreactivity (IR) was more extensive than ChE activity and was identified in both the central and peripheral nervous systems. Serotoninergic nerve fibres were associated with the somatic musculature and female reproductive ducts. Antisera to nine mammalian peptides and one invertebrate (FMRFamide) peptide have been used to investigate the peptidergic nervous system in the parasite. Immunoreactivity was obtained to five peptides, namely pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), substance P (SP) and FMRFamide. Peptidergic nerve fibres were found to be more abundant than demonstrable cholinergic or serotoninergic nerve fibres. NPY-IR was identified only in the main components of the central nervous system. However, PP- and PYY-IR occurred in the anterior ganglia, dorsal commissure, main nerve cords and in numerous small varicose fibres that ramified throughout the worm. Additionally, PP-immunoreactive nerve fibres were found to innervate the musculature of the female reproductive tracts. Six sites of IR were found in the acetabulum, using antisera directed towards the C-terminal end of PP and PYY, and these matched with the distribution of six non-ciliated rosette-like papillae observed by scanning electron microscopy. SP- and FMRFamide-IR were identified in the CNS, and FMRFamide-immunopositive nerve fibres were also evident in association with the gonopore/cirrus region and with the terminal excretory pore. Results are discussed with respect to possible roles for each of the neurochemical types.

Comparative study of the spatial relationship between nicotinamide adenine dinucleotide phosphate-diaphorase activity, serotonin immunoreactivity, and GYIRFamide immunoreactivity and the musculature of the adult liver fluke, Fasciola hepatica (Digenea, Fasciolidae)

This is the first detailed description of the nitrergic nervous system in a fluke. In this study, the authors analysed the distribution of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity in neuronal and nonneuronal tissues of the adult fluke Fasciola hepatica and compared this with the distribution of the musculature using tetramethylrhodamine isothiocyanate-phalloidin. To assess the correlation between the number of muscle cells in different parts of the fluke and the NADPH-d-stained cells, the nuclei were stained with Hoechst 333 42, which is specific for chromatin. The spatial relation between the NADPH-d-positive nerves and the 5-hydroxytryptamine (serotonin; 5-HT)-immunoreactive (-IR) and GYIRFamide-IR nervous elements was also examined. The methods complement each other. NADPH-d-positive staining occurs in both in neuronal tissue and nonneuronal tissue. Large, NADPH-d-stained neurones were localised in the nervous system. The oral and ventral suckers are innervated with many large NADPH-d-stained neurones. Ln addition, the NADPH-d staining reaction follows closely the muscle fibres in both the suckers, in the body, and in the ducts of the reproductive organs. The presence of NADPH-d activity along muscle fibres in F. hepatica and in other flatworms supports a possible myoinhibitory role for nitric oxide. Neuronal nitric oxide synthase in flatworms may form a novel drug target, which would facilitate the development of a novel anthelminthic. (C) 2001 Wiley-Liss, Inc.

Gross anatomy of the muscle systems of Fasciola hepatica as visualized by phalloidin-fluorescence and confocal microscopy

Neuropeptides, biogenic amines and acetylcholine are expressed abundantly within the nervous systems of parasitic flatworms, and are particularly evident in the innervation of the musculature. Such associations have implicated the nervous system in locomotion, host attachment and reproductive co-ordination. Information on the muscle systems of parasitic flatworms is generally sparse, in particular those muscles associated with the reproductive system, intestinal tract and attachment apparatus. Also, the use of sectioned material has left description of the 3-dimensional organization of the musculature largely unrecorded. Using fluorescein isothiocyanate (FITC)-labelled phalloidin as a site-specific probe for filamentous actin, applied to whole-mount preparations of adult Fasciola hepatica and examined by confocal scanning laser microscopy, the present work reports on the organization of the major muscle systems in this trematode parasite. A highly regular array of outer circular, intermediate longitudinal and inner diagonal fibres distinguishes the body wall musculature, which is also involved in the development of both ventral and oral suckers. Circular fibres dominate the duct walls of the male and female reproductive systems, whereas the muscles of the intestinal tract have a somewhat diffuse arrangement of fibres. An understanding of the structural complexity of the muscle systems of parasitic flatworms is considered as fundamental to the interpretation of results from physiological experiments.

Comparative analysis of the distribution of bradykinin-, GYIRFamide- and neuropeptide F-like immunoreactivities in the monogenean, Diclidophora merlangi

An indirect immunocytochemical technique combined with confocal scanning laser microscopy has been used to demonstrate immunoreactivities to the nonapeptide, RPPGFSPFR (bradykinin, BK) and the endogenous flatworm regulatory peptide, GYIRFamide in the nervous system of the monogenean, Diclidophora merlangi. In addition, a simultaneous double-labelling technique was employed to examine possible co-localization of GYIRFamide- and neuropeptide F (NPF) immunoreactivities, using antisera to the C-terminal nonapeptide-amide of NPF (Moniezia expansa, FAIIGRPRF.NH2). BK immunostaining was restricted to a small population of nerve cells and associated fibres within the Ventral nerve cords and to 2 pairs of nerve cells innervating the cirrus and the pharynx, respectively. No immunopositive nerve cells and fibres were identified within the brain or in association with the female reproductive apparatus. In contrast, GYIRFamide staining was abundant throughout the central and peripheral nervous systems, and appeared similar to the staining pattern revealed using an FMRFamide antiserum. GYIRFamide immunoreactivity was localized to nerve cells and fibres within the paired cerebral ganglia and the longitudinal ventral, dorsal and lateral nerve cords and their numerous interconnecting transverse commissures. The plexuses of the buccal suckers, pharynx and clamps of the haptor were strongly immunopositive for GYIRFamide, as were nerve cells innervating the ootype, the oviduct and the vitelline reservoir of the reproductive apparatus. Double-labelling experiments indicated an apparent co-localization of GYIRFamide and NPF immunoreactivities.

Platyhelminth FMRFamide-related peptides

Platyhelminths are the most primitive metazoan phylum to possess a true central nervous system, comprising a brain and longitudinal nerve cords connected by commissures. Additional to the presence of classical neurotransmitters, the nervous systems of all major groups of flatworms examined have widespread and abundant peptidergic components, Decades of research on the major invertebrate phyla, Mollusca and Arthropoda, have revealed the primary structures and putative functions of several families of structurally related peptides, the best studied being the FMRFamide-related peptides (FaRPs). Recently, the first platyhelminth FaRP was isolated from the tapeworm, Moniezia expansa, and was found to be a hexapeptide amide, GNFFRFamide. Two additional PaRPs were isolated from species of turbellarians; these were pentapeptides, RYIRFamide (Artioposthia triangulata) and GYIRFamide (Dugesia tigrina). The primary structure of a monogenean or digenean FaRP has yet to be deduced. Preliminary physiological studies have shown that both of the turbellarian FaRPs elicit dose-dependent contractions of isolated digenean and turbellarian somatic muscle fibres. Unlike the high structural diversity of FaRPs found in molluscs, arthropods and nematodes, the complement of FaRPs in individual species of platyhelminths appears to be restricted to 1 or 2 related molecules. Much remains to be learnt about platyhelminth PaRPs, particularly from peptide isolation, molecular cloning of precursor proteins, receptor localization, and physiological studies. Copyright (C) 1996 Australian Society for Parasitology.

NEUROPEPTIDES AND SEROTONIN IN THE CESTODE, PROTEOCEPHALUS-EXIGUUS - AN IMMUNOCYTOCHEMICAL STUDY

Neuropeptide F (NPF), RFamide and serotonin (5-HT) immunoreactivities have been detected in the nervous system of P. exiguus procercoids and adults, using an indirect immunocytochemical technique in conjunction with confocal scanning laser microscopy. The peptidergic nervous system of the procercoid is well developed, with two brain ganglia, three pairs of longitudinal nerve cords, transverse ring commissures and nerves in the suckers, all showing NPF-immunostaining. Strong NPF- and RF-immunostaining was observed in the CNS and PNS of the adult worm. The distribution patterns of the two neuropeptides were similar. Immunoreactivity for 5-HT was found only in the CNS.

LOCALIZATION OF DIPLOPTERA-PUNCTATA ALLATOSTATIN-LIKE IMMUNOREACTIVITY IN HELMINTHS - AN IMMUNOCYTOCHEMICAL STUDY

The nervous systems of helminths are predominantly peptidergic in nature, although it is likely that the full range of regulatory peptides used by these organisms has yet to be elucidated. Attempts to identify novel helminth neuropeptides are being made using immunocytochemistry with antisera raised against peptides isolated originally from insects. One of these antisera was raised against allatostatin III, a peptide isolated originally from the cockroach, Diploptera punctata, and a member of a family of related peptides found in insects. Allatostatin immunoreactivity was found throughout the nervous systems of Mesocestoides corti tetrathyridia, and adult Moniezia expansa, Diclidophora merlangi, Fasciola hepatica, Schistosoma mansoni, Ascaris suum and Panagrellus redivivus. Immunostaining was observed in the nerve cords and anterior ganglia of all the helminths. It was also apparent in the subtegumental nerves and around the reproductive apparatus of the flatworms, in neurones in the pharynx of D. merlangi, F. hepatica, A. suum and P. redivivus, and in fibres innervating the anterior sense organs in the nematodes. Immunostaining in all species was both reproducible and specific in that it could be abolished by pre-absorption of the antiserum with allatostatins I-IV. These results suggest that molecules related to the D. punctata allatostatins are important components in the nervous systems of a number of helminth parasites, and a free-living nematode. Their distribution within the nervous system suggests they function as neurotransmitters/ neuromodulators with roles in locomotion, feeding, reproduction and sensory perception.

THE GULL-TAPEWORM, DIPHYLLOBOTHRIUM-DENDRITICUM AND NEUROPEPTIDE-F - AN IMMUNOCYTOCHEMICAL STUDY

Neuropeptide F (Moniezia expansa) immunoreactivity (NPF-IR) has been detected in the nervous system of plerocercoid and adult stages of the gull-tapeworm Diphyllobothrium dendriticum, using immunocytochemical methodology. The application of the antiserum for this authentic flatworm neuropeptide to whole-mounts and frozen sections of the worm has resulted in new information about its neuroanatomy. Thus, at regular intervals, transverse nerves extend from the main nerve cords laterally, joining the longitudinal lateral minor cords in the cortical parenchyma. In the adult worm, the transverse nerves are located at the posterior border of each proglottis. The medullary parenchyma lacks NPF-IR. The NPF-immunoreactive cell bodies are bi- to multipolar and preferentially located in the peripheral nervous system, in close association with the holdfast musculature of the scolex and the extensive body musculature. NPF-IR was observed in the innervation to the muscular ducts of the reproductive system. The pattern of NPF-IR was compared with that recorded for RFamide- and 5-HT-IR and double-immunostaining has revealed separate populations of serotoninergic and peptidergic neurones.

GRILLOTIA ERINACEUS (CESTODA, TRYPANORHYNCHA) - LOCALIZATION OF NEUROACTIVE SUBSTANCES IN THE PLEROCERCOID, USING CONFOCAL AND ELECTRON-MICROSCOPIC IMMUNOCYTOCHEMISTRY

Indirect immunocytochemistry, in conjunction with confocal scanning laser microscopy and electron-microscopic immunogold labeling, has been used to localize neuropeptide and 5-hydroxytryptamine (5-HT) immunereactivities (IRs) in the plerocercoid (scolex and surrounding blastocyst) of the trypanorhynch tapeworm, Grillotia erinaceus. Antisera directed to two native cestode neuropeptides, neuropeptide F and the FMRFamide-related peptide, GNFFRFamide, were used to demonstrate the presence of a well-developed and extensive peptide-immunoreactive nervous system of central and peripheral elements in the juvenile scolex. Neuronal connectivity exists between the scolex and the surrounding blastocyst, in which there is a rich innervation of varicose fibers displaying peptide IR. Ultrastructurally, gold labeling of the peptide IR was found exclusively over the contents of dense secretory vesicles in the axons and somatic cytoplasm of neurons. Double-labeling experiments demonstrated an apparent colocalization of peptide IR, although the results of antigen preadsorption procedures indicated substantial cross-reactivity of the two antisera. A separate and well-differentiated 5-HT-immunoreactive nervous system, with a similar anatomical arrangement as the peptide-immunoreactive nervous system, is present in both the scolex and blastocyst (C) 1994 Academic Press, Inc.

PLATYHELMINTH FMRFAMIDE-RELATED PEPTIDES (FARPS) CONTRACT SCHISTOSOMA-MANSONI (TREMATODA, DIGENEA) MUSCLE-FIBERS IN-VITRO

Molluscan FMRFamide and two recently discovered platyhelminth FMRFamide-related peptides (FaRPs), GNFFRFamide from the cestode Moniezia expansa and RYIRFamide from the terrestrial turbellarian Artioposthia triangulata, cause dose-dependent contractions of individual muscle fibres from Schistosoma mansoni in vitro. The most potent FaRP tested was the turbellarian peptide RYIRFamide, which produced a concentration-dependent effect between 10(-9) and 10(-7) M. FMRFamide and GNFFRFamide were less potent, inducing contractions between 10(-8)-10(-6) M and 10(-7)-10(-5) M respectively. The contractile effect of each of these peptides was blocked by the presence of 1 mu M FMR-D-Famide. FMRF free acid did not elicit contraction of the muscle fibres. The FaRP-induced contractions did not occur if the Ca2+ was omitted and 0.5 mu M EGTA. was added to the extracellular medium. The FaRP-induced contractions were not blocked by the Ca2+ channel blockers nicardipine, verapamil or diltiazem, although high Kf-induced contractions of these fibres were blocked by nicardipine. These data indicate the presence of FaRP receptors on schistosome muscle fibres and demonstrate their ability to mediate muscle contraction. The action of these endogenous flatworm peptides on schistosome muscle is the first demonstration of a direct excitatory effect of any putative neurotransmitter on the muscle of a flatworm, and establishes a role for FaRPs in neuromuscular transmission in trematodes. In addition, it provides the first evidence that the peptidergic nervous system is a rational target for chemotherapeutic attack in parasitic platyhelmiths.

PEPTIDES RELATED TO THE DIPLOPTERA-PUNCTATA ALLATOSTATINS IN NONARTHROPOD INVERTEBRATES - AN IMMUNOCYTOCHEMICAL SURVEY

The allatostatins are a family of peptides isolated originally from the cockroach, Diploptera punctata. Related peptides have been identified in Periplaneta americana and the blowfly, Calliphora vomitoria. These peptides have been shown to be potent inhibitors of juvenile hormone synthesis in these species. A peptide inhibitor of juvenile hormone biosynthesis has also been isolated from the moth, Manduca sexta; however, this peptide has no structural homology with the D. punctata-type allatostatins. Investigations of the phylogeny of the D. punctata allatostatin peptide family have been started by examining a number of nonarthropod invertebrates for the presence of allatostatin-like molecules using immunocytochemistry with antisera directed against the conserved C-terminal region of this family. Allatostatin-like immunoreactivity (ALIR) was demonstrated in the nervous systems of Hydra oligactis (Hydrozoa), Moniezia expansa (Cestoda), Schistosoma mansoni (Trematoda), Artioposthia triangulata (Turbellaria), Ascaris suum (Nematoda), Lumbricus terrestris (Oligochaeta), Limax pseudoflavus (Gastropoda), and Eledone cirrhosa (Cephalopoda). ALIR could not be demonstrated in Ciona intestinalis (Ascidiacea). These results suggest that molecules related to the allatostatins may play an important role in nervous system function in many invertebrates as well as in insects and that they also have an ancient evolutionary lineage. (C) 1994 Wiley-Liss, Inc.

SEROTONIN AND NEUROPEPTIDE IMMUNOREACTIVITIES IN THE INTRAMOLLUSCAN STAGES OF 3 MARINE TREMATODE PARASITES

Using an indirect immunofluorescence technique interfaced with confocal scanning laser microscopy, whole-mount preparations of three genera of marine trematode larvae, Cryntocotyle lingua, Cercaria emasculans and Himasthla leptosoma, were screened for 5-hydroxytryptamine (5-HT) and selected neuropeptide immunoreactivities (IRs). IRs for pancreatic polypeptide (PP), peptide YY (PYY) and FMRFamide were found in the central nervous systems of the three species of cercariae, immunostaining the paired ganglia and central commissure and the longitudinal nerve cords, with slight differences in both distribution and intensity of IRs being observed for the different antisera used. PP, PYY and FMRFamide IRs were evident in both central and peripheral components of the nervous system in the rediae of C. lingua. 5-HT IR was confined to the peripheral nervous systems of the cercariae of C. emasculans and the rediae of C. lingua, appearing in the form of a network of immunoreactive fibres and associated large cell bodies. A moderate substance P IR was observed in the nervous system of the cercariae of C. lingua. The patterns of immunostaining described were compared with those obtained using antiserum directed to the C-terminal decapeptide amide of neuropeptide F (NPF), a native parasitic peptide from the cestode Moniezia expansa. Results demonstrated that serotoninergic and peptidergic components were present in the nervous systems of all of the trematode larvae studied and that some, if not all, of the IR for PP. PYY and FMRFamide was due to the presence of a trematode NPF homologue.