Cellular and subcellular localization of SALMFamide (S1)-like immunoreactivity within the central nervous system of the nematode Ascaris suum (Nematoda, Ascaroidea)

The localization and distribution of SALMFamide (S1)-like immunoreactivity (IR), was determined at both the cellular and subcellular level in the central nervous system (CNS) of the nematode roundworm Ascaris suum. The techniques of indirect immunofluorescence in conjunction with confocal scanning laser microscopy and post-embedding, IgG-conjugated colloidal gold immunostaining were used, respectively. Immunostaining was widespread in the CNS of adult A. suum, with immunoreactivity (IR) being localized in nerve cells and fibres in the ganglia associated with the anterior nerve ring and in the main nerve cords and their commissures. At the subcellular level, gold labeling of peptide was localized exclusively over dense-cored vesicles within nerve cell bodies, nerve axons and nerve terminals of the neuropile of the anterior nerve ring, main ganglia and nerve cords in the CNS. Double-labeling demonstrated an apparent co-localization of S1- and FMRFamide-IR-together IR-together with S1- and pancreatic polypeptide (PP)-IR in the same dense-cored vesicles. Antigen preabsorption experiments indicated little cross-reactivity, if any, between the three antisera; indeed, neither FMRFamide nor PP antigens abolished S1 immunostaining.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF A SALMFAMIDE-LIKE NEUROPEPTIDE IN THE NERVOUS-SYSTEM OF ADULT AND LARVAL STAGES OF THE HUMAN BLOOD FLUKE, SCHISTOSOMA-MANSONI

The localization and distribution of SALMFamide immunoreactivity (IR), SI(GFNSALMFamide), in the nervous system of both the adult and larval stages of the trematode Schistosoma mansoni has been determined by an indirect immunofluorescent technique in conjunction with confocal scanning laser microscopy (CSLM). Immunostaining was widespread in the nervous system of adult male and female S. mansoni. In the central nervous system (CNS), IR was evident in nerve cells and fibres in the anterior ganglia, cerebral commissure and dorsal and ventral nerve cords. In the peripheral nervous system (PNS), IR was apparent in nerve plexuses associated with the subtegmental musculature, oral and ventral suckers, the lining of the gynaecophoric canal, and in fine nerve fibres innervating the dorsal tubercles of the male worm. In the reproductive system of male and female worms, S1-IR was only observed around the ootype/Mehlis' gland complex in the female. Immunostaining was also evident in the nervous system of both miracidium and cercarial larval stages. A post-embedding, IgG-conjugated colloidal gold immunostaining technique was employed to examine the subcellular distribution of SALMFamide-IR in the CNS of S. mansoni. Gold labelling of peptide was localized over dense-cored vesicles within nerve cell bodies and fibres constituting the neuropile of the anterior ganglia, cerebral commissure and nerve cords of the CNS. Antigen pre-absorption studies indicated that the results obtained do suggest S1-like immunostaining and not cross-reactivity with other peptides, in particular FMRFamide.

IMMUNOCYTOCHEMICAL LOCALIZATION OF SEROTONIN (5-HT) IN THE NERVOUS-SYSTEM OF THE HYDATID ORGANISM, ECHINOCOCCUS-GRANULOSUS (CESTODA, CYCLOPHYLLIDEA)

The localization and distribution of the serotoninergic components of the nervous system in the hydatid organism, Echinococcus granulosus, were determined by immunocytochemical techniques in conjunction with confocal scanning laser microscopy (CSLM). The distribution of serotonin immunoreactivity (IR) paralleled that previously described for cholinesterase activity, although it was more widespread. Nerve cell bodies and nerve fibres immunoreactive for 5-HT were present throughout the central nervous system (CNS), occurring in the paired lateral, posterior lateral and rostellar ganglia, their connecting commissures and nerve rings in the scolex and in the ten longitudinal nerve cords that run posteriorly throughout the body of the worm. A large population of nerve cell bodies was associated with the lateral nerve cords. In the peripheral nervous system (PNS), immunoreactive nerve fibres occurred in well-developed nerve plexuses innervating the somatic musculature and the musculature of the rostellum and suckers. The genital atrium and associated reproductive ducts were richly innervated with serotoninergic nerve cell bodies and nerve fibres.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF PEPTIDERGIC AND SEROTONINERGIC COMPONENTS IN THE ENTERIC NERVOUS-SYSTEM OF THE ROUNDWORM, ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

The localization and distribution of neuropeptides and an indoleamine (serotonin or 5-hydroxytryptamine) in the enteric nervous system (ENS) of the pig roundworm, Ascaris suum, have been determined by the application of an indirect immunofluorescence technique in conjunction with confocal scanning laser microscopy. Whole-mount preparations of pharyngeal, intestinal and rectal regions were screened with antisera to 23 vertebrate peptides, 2 invertebrate peptides and serotonin(= 5-HT). Positive immunoreactivity (IR) was obtained with antisera to pancreatic polypeptide (PP), peptide YY (PYY), FMRFamide, gastrin and serotonin. The only IR observed in the ENS was that evident in the nerve supply to the pharynx and rectal region; no IR was associated with any region of the intestine. The most extensive patterns of IR occurred with antisera to PW, FMRFamide and serotonin. In the pharyngeal component of the ENS, IR was evident in the lateral and dorsal longitudinal pharyngeal nerves, pharyngeal commissures, nerve plexus, and associated nerve cells and fibres. In contrast, the distribution of IR to the PP and gastrin antisera was more restricted and displayed a lower intensity of immunostaining. The other component of the ENS, the rectal enteric system, only yielded immunostaining to FMRFamide. The possible role of neuropeptides and serotonin in the nutritional biology of nematodes is discussed.

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.

Cholinergic, serotoninergic and peptidergic components of the nervous system of Haematoloechus medioplexus (Trematoda, Digenea), characterised by cytochemistry

Cholinergic, serotoninergic and peptidergic neuronal pathways have been demonstrated in whole-mount preparations of the frog-lung digenean trematode, Haematoloechus medioplexus, using enzyme cytochemical methodologies and indirect immunocytochemical techniques in conjunction with confocal scanning laser microscopy. All 3 classes of neuroactive substance mere found throughout both central and peripheral elements of a well-developed orthogonal nervous system, Peptidergic immunoreactivity was particularly strong, using antisera directed to native flatworm neuropeptides, neuropeptide F, and FMRFamide-related peptides (FaRPs), and there was significant overlap in the staining with that for cholinergic components, The serotoninergic system appeared quite separate, with the staining localised to a different set of neurons. (C) 1997 Australian Society for Parasitology.

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.

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.

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.

5-HYDROXYTRYPTAMINE (SEROTONIN)-IMMUNOREACTIVITY IN THE NERVOUS-SYSTEM OF MESOCESTOIDES-CORTI TETRATHYRIDIA (CESTODA, CYCLOPHYLLIDEA)

An indirect immunocytochemical technique has been interfaced with confocal scanning laser microscopy to investigate the occurrence and distribution of serotoninergic (5-HT) nerve elements in Mesocestoides corti tetrathyridia. Cell bodies and nerve fibers immunoreactive to 5-HT were found concentrated in the innervation around the 4 suckers and associated commissures and in the 5 pairs of longitudinal nerve cords and their cross-connectives. Immunoreactivity was evident also in the extensive, peripheral network of fine fibers of the subtegumental region and in the plexus of varicose fibers that innervate the muscle in each of the suckers. In dividing stages of the tetrathyridium, the immunoreactive lateral nerve cords of adjoining progeny were in continuity around the base of the division cleft.

NEUROPEPTIDE F-IMMUNOREACTIVITY IN THE TETRATHYRIDIUM OF MESOCESTOIDES-CORTI (CESTODA, CYCLOPHYLLIDEA)

The distribution pattern and subcellular localisation of neuropeptide F (NPF) immunoreactivity (IR) in the tetrathyridium stage of Mesocestoides corti were investigated by whole-mount immunocytochemistry in conjunction with confocal scanning laser microscopy (CSLM) and by immunoelectron microscopy using immunogold labeling. Using an antiserum directed to the C-terminal decapeptide amide (residues 30-39) of synthetic NPF (Moniezia expansa), CSLM revealed NPF-IR throughout the central and peripheral nervous systems of parental and dividing tetrathyridia. Ultrastructurally, gold labeling of NPF-IR was confined to the contents of the smaller of the two sizes of electron-dense neuronal vesicle identified.

AN IMMUNOCYTOCHEMICAL STUDY OF PUTATIVE NEUROTRANSMITTERS IN THE METACERCARIAE OF 2 STRIGEOID TREMATODES FROM RAINBOW-TROUT (ONCORHYNCHUS-MYKISS)

Whole mounts of the metacercariae of Diplostomum sp. and Cotylurus erraticus from rainbow trout have been treated cytochemically for the demonstration of cholinergic, serotoninergic (5-hydroxytryptamine) and peptidergic elements in the nervous system. Antisera directed against four vertebrate (pancreatic polypeptide, peptide YY, substance P and peptide histidine isoleucine) and two invertebrate peptides (neuropeptide F and FMRFamide) were used in an indirect immunofluorescence procedure in conjunction with confocal scanning laser microscopy (CSLM). Of the seven antisera tested, all except peptide histidine isoleucine showed significant immunoreactivity. Cholinergic and serotoninergic staining was found primarily in the central nervous system (CNS) and in cell bodies associated with the ventral and dorsal nerve cords in both trematodes. Peptidergic immunoreactivity was localised in the CNS and PNS of both genera, revealing an extensive innervation within the holdfast organ and in and around the oral and ventral suckers.

DISTRIBUTION AND IMMUNOCHEMICAL CHARACTERISTICS OF NEUROPEPTIDE-F (NPF) (MONIEZIA-EXPANSA)-IMMUNOREACTIVITY IN PROTEOCEPHALUS-POLLANICOLA (CESTODA, PROTEOCEPHALIDEA)

1. Using immunocytochemical techniques and confocal scanning laser microscopy, the proteocephalidean cestode, Proteocephalus pollanicola from Lough Neagh pollan (Coregonus autumnalis) was examined for the presence of the native platyhelminth neuropeptide, neuropeptide F (NPF).