Neuropeptide F-immunoreactivity in the monogenean parasiteDiclidophora merlangi

The localisation and distribution of neuropeptide F (NPF)-immunoreactivity (IR) in the monogenean fish-gill parasite,Diclidophora merlangi, have been investigated by whole-mount immunocytochemistry interfaced with confocal scanning laser microscopy and, at the ultrastructural level, by indirect immunogold labeling. Using antisera directed to intact synthetic NPF (Moniezia expansa, residues 1–39) or to the C-terminal decapeptide (residues 30–39) of synthetic NPF (M. expansa), immunostaining was found throughout the central (CNS) and peripheral nervous systems (PNS), including the innervation of the reproductive system. Immunoreactivity was found to be more intense using the antiserum to the C-terminal decapeptide fragment of NPF. At the subcellular level, gold labeling of NPF-IR was found exclusively over the contents of dense-cored vesicles that occupied nerve axons of both the CNS and the PNS. The distribution pattern of immunostaining for NPF mirrored exactly that previously documented for the vertebrate pancreatic polypeptide (PP) family of peptides and for FMRFamide. This finding and the results of preabsorption experiments strongly suggest that NPF is the predominant native neuropeptide inD. merlangi and that it accounts for most of the immunostaining previously obtained with PP and FMRFamide antisera.     

Serotoninergic and peptidergic nerve elements in the protoscolex ofEchinococcus 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 organismEchinococcus 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 (PHI) 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.     

Electron immunogold labeling of regulatory peptide immunoreactivity in the nervous system ofMoniezia expansa (Cestoda: Cyclophyllidea)

An electron immunogold-labeling technique was used in conjunction with a post-embedding procedure to demonstrate for the first time the ultrastructural distribution of the parasitic platyhelminth neuropeptide, neuropeptide F (NPF(, in the nervous system of the cestodeMoniezia expansa. Two axon types, distinguished by their populations of different-sized electron-dense vesicles, were identified. Immunogold labeling demonstrated an apparent homogeneity of PP, FMR Famide and NPF (M. expansa) antigenic sites throughout the larger dense-cored vesicles within the central nervous system. Triple labeling clearly demonstrated the co-localisation of immunoreactivities (IR) for NPF, PP and FMR Famide within the same dense-cored vesicles. The presence of NPF-IR within the vesicles occupying the perikaryon of the neuronal cell body indicated that the peptides had undergone post-translational C-terminal amidation prior to entering the axon. Antigen pre-absorption experiments using NPF prevented labeling with either PP or FMR famide antisera, and the failure of these antisera to block NPF-IR supports the view that some, if not all, of the PP/FMR Famide-IR is due to NPF-like peptides.     

Immunocytochemical demonstration of neuropeptides in the peripheral nervous system of the roundwormAscaris suum (Nematoda,ascaroidea)

The localisation and distribution of neuropeptides in the peripheral nervous system of the pig roundwormAscaris suum have been determined by an indirect immunofluoresence technique in conjunction with confocal microscopy. Of the 31 antisera tested, immunostaining was obtained only with antisera to peptide YY (PYY), pancreatic polypeptide (PP) and FMRFamide. Immunostaining for PYY and FMRFamide was evident in the amphidial and papillary ganglia associated with the anterior nerve ring and in the nerves from these ganglia that terminated in sensory receptors within the buccal lips of the parasite. The only peptide immunoreactivity (IR) observed in the reproductive system of either sex was that evident in the nerve supply to the distal region of the vagina in the female worm. It took the form of a well-developed plexus of parallel nerve fibres, cross-connectives and looped commissures. The nerve net diminished in the more proximal region of the vagina. PP-IR was less intense than that for PYY and FMRFamide and was more restricted in distribution, being confined to a small number of nerve fibres in the nerve supply to the vagina; it did not occur in the nerves supplying the anterior sensory receptors. The possible roles of neuropeptides in the sensory and reproductive biology of nematodes are discussed.     

Occurrence and distribution of putative neurotransmitters in the frog-lung parasiteHaplometra cylindracea (Trematoda: Digenea)

The localistion and distribution of the cholinergic, serotoninergic and peptidergic components of the nervous system of the frog-lung flukeHaplometra cylindracea have been determined by the application of standard enzyme cytochemical and immunocytochemical techniques to cryostat sections and whole-mount preparations. Cholinesterase activity (ChE), as indicative of acetylcholine, has been demonstrated cytochemically in the CNS and PNS; however, the anterior ganglia were notably unreactive. The occurrence of serotonin was examined by an indirect immunofluorescence technique, and immunoreactivity (IR) was demonstrable in small, paired anterior ganglia and in fine nerve fibres associated with the somatic muscle, cirrus and gonopore. The peptidergic protion of the nervous system was investigated using antisera to 17 mammalian regulatory peptides and the invertebrate peptide FMRFamide, and was visualised by both indirect immunofluorescence and confocal scanning laser microscopy. Positive immunostaining occurred with antisera raised against pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), substance P (SP), peptide histidine isoleucine (PHI) and FMRFamide. Immunoreactivity to PP, PYY and FMRFamide was widespread throughout the nervous system and was evident in large, paired anterior ganglia, the dorsal commissure, main nerve tracts and the extensive array of small fibres that constitute the PNS. In contrast, the distribution of nerves immunoreactive to SP and PHI was less apparent, with PHI-IR occurring exclusively within the fibrous neuropile of the ganglia and in fibres of the ventral nerve cord. Results are discussed with respect to the distribution of the various neurochemical elements and their roles as putative neurotransmitters and/or regulatory molecules.     

Neuropeptide F-immunoreactivity in the tetrathyridium ofMesocestoides corti (Cestoda: Cyclophyllidea)

The distribution pattern and subcellular localisation of neuropeptide F (NPF) immunoreactivity (IR) in the tetrathyridium stage ofMesocestoides 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.     

Comparative studies on the productivity ofFasciola gigantica andF. hepatica sporocysts inLymnaea tomentosa that died after a cercarial shedding or without emission

A histology study was performed onFasciola gigantica- orF. hepatica-infectedLymnaea tomentosa that died after a cercarial shedding or without emission to compare the parasite productivity of each trematode. Degenerated rediae increased in number throughout the experiment. Their number rose rapidly after day 79 in snails that died after shedding in theF. gigantica group; they increased more slowly in snails that died without shedding. In theF. hepatica group the number rose after day 63 in the former snails and after day 79 in the latter. The contents of normal rediae evolved inversely. The number of morulae, for example, dropped rapidly after day 79 in theF. gigantica group. In theF. hepatica group it dropped after day 63 in snails that died after shedding and decreased after day 79 in the other dead snails. Free and degenerated cercariae were more numerous in snails that died without emission than in those that died at shedding. They rapidly increased in number after day 77. The numbers of rediae ofF. gigantica were substantially greater than those ofF. hepatica. In each group considered separately, it was likewise higher in snails that died without shedding than in those that died after the shedding of cercariae.     

Immunocytochemical demonstration of 5-hydroxytryptamine (serotonin) and vertebrate neuropeptides in the nervous system of excysted cysticercoid larvae of the rat tapeworm,Hymenolepis diminuta (Cestoda,Cyclophyllidea)

The localisation and distribution of 5-hydroxytryptamine (5-HT, or serotonin) and a number of vertebrate neuropeptides in the nervous system of excysted (0–24 h) cysticercoid larvae of Hymenolepis diminuta were determined by an indirect immunofluorescence technique in wholemount preparations. In the central nervous system, cell bodies and nerve fibres immunoreactive to 5-HT are present in the main commissure, lateral and rostellar ganglia, and the longitudinal nerve cords and their connectives. In the peripheral nervous system, immunoreactive nerve fibres occur in a poorly developed nerve plexus within each sucker. Among the vertebrate peptides tested, antisera to pancreatic polypeptide (PP), polypeptide YY (PYY), peptide histidine isoleucine (PHI) and gastrin-releasing peptide (GRP) gave positive results. Immunoreactivity to PP and PYY paralleled that of 5-HT, with greater numbers of cell bodies present in the different locations within the scolex nervous system, and the sucker plexus being more prominent. The number of PP-reactive cells in the lateral ganglia and main lateral, longitudinal nerve cords increased over the 24-h period in culture. Results with antisera of different specificities to PP and PYY suggest that the immunoreactivity may be due to a peptide with closer structural affinity to PYY than to PP. Immunoreactivity to PHI is restricted to the main lateral nerve cords in the body of 0-h worms, extending into the median nerve cords by 12 h and 24 h. Immunoreactivity to GRP became evident after 12 h in culture and was confined to the longitudinal nerve cords, in particular the median nerve cords. The results are discussed in relation to the proposed transmitter and regulatory roles of 5-hydroxytryptamine and the neuropeptides.     

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 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. Received: 3 April 1995 / Accepted: 16 June 1995     

The neuro-muscular system in cercaria with different patterns of locomotion

The neuro-muscular system (NMS) of cercariae with different swimming patterns was studied with immunocytochemical methods and confocal scanning laser microscopy. Specimens of the continuously swimming Cercaria parvicaudata, Maritrema subdolum and Himasthla elongata were compared with specimens of the intermittently swimming Cryptocotyle lingua and the attached Podocotyle atomon. The patterns of F-actin in the musculature, 5-HT immunoreactive (-IR), FMRFamide-IR neuronal elements, α-tubulin-IR elements in the nervous and sensory systems and DAPI-stained nuclei were investigated. The general plan of the NMS was similar in all cercariae studied. No major structural differences in the patterns of muscle fibres were observed. However, in the tail of C. lingua, transverse muscle fibres connecting the bands of longitudinal muscles were found. No major structural differences in the 5-HT- or FMRFamide-IR nervous systems were observed. The number of 5-HT-IR neurones in the cercarial bodies varied between 12 and 14. The number and distribution of the α-tubulin-IR processes on the cercarial bodies and tails differed from each other. The relation between the number and structure of the α-tubulin-IR processes and the host finding strategy of the cercariae is discussed. A detailed schematic picture of the NMS in the tails of C. lingua and M. subdolum is presented.     

The spatial relationship between the musculature and the 5-HT and FMRFamide immunoreactivities in cercaria,metacercaria and adult <Emphasis Type="Italic">Opisthorchis felineus</Emphasis> (Digenea)

The organisation of the neuromuscular system in cercariae, metacercariae and adult Opisthorchis felineus was studied. The patterns of nerves immunoreactive (IR) to antibodies towards serotonin (5-HT) and FMRFamide are described in relation to the musculature, stained with TRITC-conjugated phalloidin. The general organisation of the musculature in the body wall, suckers, pharynx, intestine and sphincter of the excretory pore remains the same from the larval stages to the adult worms. However, the diameter of the individual muscle fibres increases distinctly in the adult worms. The general pattern of 5-HT IR fibres in cercariae, metacercariae and adult O. felineus remains the same. Despite the large increase in body size, the number of 5-HT IR neurones remains almost the same in the cercariae and metacercariae and only a modest increase in number of neurones was observed in the adult worms. Thus the proportion of 5-HT IR neurones/body mass is greatest in the actively moving cercariae. Anti-FMRFamide stains the nervous system strongly.     

Fasciola hepatica: an unusual case of adaptation to a Moroccan population of Galba truncatula

Parent snails, F1, and F2, originating from a Moroccan population of Galba truncatula, were experimentally subjected to sympatric miracidia of Fasciola hepatica at 20°C to determine the mode of adaptation of this parasite to the local intermediate host via the study of redial burdens and by following cercarial shedding. In spite of an increase in the global prevalences of infections, the frequencies recorded for immature or mature infections in dissected snails did not show any significant variations from parents to F2. If the redial burdens in mature infections were mainly composed of first-generation rediae in parent snails, the numbers of rediae belonging to subsequent generations significantly increased in the F1 and F2 generations. In mature infections, the lengths of first-generation rediae and their contents significantly decreased from parent snails to F2. Conversely, in the first redial cohort of the second generation (R2a rediae), only the intraredial contents increased significantly with snail generation. In mature infections, daughter redia production was ensured by all mother rediae of the first generation in parent snails, whereas it was progressively effected by the first mother redia in the F1 and F2 infections. The numbers of cercariae shed by snails increased significantly from parents to F2. If the cercariae produced by first-generation rediae decreased with snail generation, an inverse finding was noted in the case of R2a rediae. We suggest that snails impose a bottleneck on the development of the first mother redia, so that other mother rediae of the first generation react with greater growth (until 3.9 mm in parent snails), an important daughter redia production and a high cercarial production. As the first mother redia remained alive until day 49 post-exposure but without growth after week 2, this mode of development must be considered as a new developmental pattern of redial generations, and it is useful to determine its frequency in snails, as it has never been found in naturally or experimentally infected snails in central France.     

Ultrastructural localisation of FMRFamide- and pancreatic polypeptide-immunoreactivities within the central nervous system of the liver fluke,Fasciola hepatica (Trematoda,Digenea)

A post-embedding immunogold technique was used to examine the subcellular distribution of immunoreactivities to the invertebrate peptide, FMR-Famide, and to vertebrate pancreatic polypeptide (PP) within the central nervous system of the trematode,Fasciola hepatica. Gold labeling of peptide was localised exclusively over both dense-cored and ellipsoidal electron-dense vesicles (with a homogeneous matrix) present within nerve cell bodies, small and giant nerve processes of the neuropile in the cerebral ganglia and transverse commissure, as well as in the main longitudinal nerve cords. Double labeling demonstrated an apparent co-localisation of FMRFamide and PP immunoreactivities in the same dense-cored vesicles, although populations of ellipsoidal electron-dense vesicles that labeled solely for FMRFamide were also evident. Antigen pre-absorption studies indicated little, if any, cross-reactivity of the two antisera.     

Research into the life history of Philophthalmus sp., recovered from geese in Bulgaria. II

Summary The development of Philophthalmus sp. in an experimentally infected intermediate host, the fresh-water snail Fagotia (Microcolpia) acicularis Fer. is studied.The parthenogenesis of Philophthalmus sp. in the intermediate host involve 3 generations of rediae and cercariae. The first and second generations (maternal and daughter) of rediae develop in the cardiac ventricle, while the third generation rediae (granddaughter) and the cercariae develop in the liver. Third generation rediae do not develop simultaneously and this prolongs the period during which cercariae leave the intermediate host. The earliest invasive cercariae are formed by the end of the third month following the infection of snails. The rediae and cercariae overwinter in the intermediate host, so that the latter contribute to the year-to-year transmission of infection. The adolescariae of Philophthalmus sp. are comparatively less resistant to the environment. They perish comparatively quickly when desiccated, frozen or kept in rotting media. They survive best in running water (up to 15 days).     

The musculature and associated innervation of adult and intramolluscan stages of<Emphasis Type="Italic"> Echinostoma caproni</Emphasis> (Trematoda) visualised by confocal microscopy

Gross anatomy of muscle and sensory/motor innervation of adult and intramolluscan developmental stages of Echinostoma caproni have been investigated to ascertain the organisation and the functional correlates of any stage-specific patterns of staining. Using indirect immunocytochemistry to demonstrate neuroactive substances and the phalloidin-fluorescence technique for staining myofibril F-actin, the muscle systems and aminergic and peptidergic innervation of daughter rediae, cercariae, metacercariae, and pre- and post-ovigerous adults were examined and compared using confocal scanning laser microscopy. A complex arrangement of specific muscle fibre systems occurs within the body wall (composed of circular, longitudinal and diagonal fibres), suckers (radial, equatorial, meridional), pharynx (radial, circular), gut caeca (mainly circular), cercarial tail (circular, pseudo-striated longitudinal), and ducts of the reproductive system (circular, longitudinal), presumed to serve locomotor, adhesive, alimentary and reproductive functions. Immunostaining for serotonin (5-HT) and FMRFamide-related peptides (FaRPs) was evident throughout the central (CNS) and peripheral (PNS) nervous systems of all stages, and use of dual-labelling techniques demonstrated separate neuronal pathways for 5-HT and FaRP in both CNS and PNS. FaRP expression in the innervation of the ootype wall was demonstrated only in post-ovigerous worms and not in pre-ovigerous worms, suggesting an involvement of FaRP neuropeptides in the process of egg assembly. Comparison of the present findings with those recorded for other digeneans suggests that muscle organisation and innervation patterns in trematodes are highly conserved.     

An immunocytochemistry study comparing the occurrence of neuroactive substances in the nervous system of cercariae and metacercariae of the eye flukeDiplostomum spathaceum

The nervous system of two larval stages (cercariae, metacercariae) of eye flukeDiplostomum spathaceum was investigated immunocytochemically by the application of antisera to the amino acid glutamate and to neuropeptides isolated from invertebrates (Mollusca) and from vertebrates to whole-mount preparations. In cercariae, positive immunoreactivity (IR) was observed with antisera raised against Catch-relaxing peptide (CARP), FMRFamide, -caudodorsal cell peptide (-CDCP), substance P, vasotocin, and vasopressin. In metacercariae, in addition to positive staining with these antisera, the ones raised against glutamate, APGWamide, caudodorsal cell hormone I (CDCH-I), and small cardiac peptide B (SCP_B) also gave positive IR in the nervous system. In the two larval stages the most extensive pattern of IR was observed with anti-FMRFamide and anti-CARP. In the nervous system of metacercariae the same immunoreactive neurosubstances appeared to be present as in that of cercariae. The increase in the variety of immunoreactive neurosubstances in the more complex nervous system of metacercariae is discussed in relation to parasite development and to host adaptation.     

The neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) can activate a ligand-gated ion channel in Helix neurons

This report presents evidence that the molluscan neuropeptide FMRFamide can directly activate a ligand-gated ion channel in Helix neurones. Using the patch-clamp technique we have observed unitary currents activated by the application of FMRFamide onto outside-out patches. As for the whole-cell response, Na⁺ ions are the main charge carriers. We conclude that FMRFamide may act as a fast depolarising neurotransmitter in the Helix nervous system.     

Temporal variation in prevalence and cercarial development of <Emphasis Type="Italic">Echinostephilla patellae</Emphasis> (Digenea,Philophthalmidae) in the intertidal gastropod <Emphasis Type="Italic">Patella vulgata</Emphasis>

The trematode Echinostephilla patellae is an abundant but scarcely investigated parasite found in coastal ecosystems of the British Isles. Redial and cercarial stages of the digenean occur in the digestive gland and gonads of common limpets Patella vulgata. Here, we present data on the temporal distribution of E. patellae infections in P. vulgata from an intertidal site on the Irish south coast as well as on the intramolluscan development of the cercariae over a period of one year. Prevalence of infection showed temporal variation with a distinct peak in September, possibly related to an increase in the abundance of bird final hosts coinciding with comparatively high temperatures at the study locality during the summer months. Maturation of cercarial stages was strongly correlated with water temperature. Whilst fully developed cercariae were present in the rediae from May to November, large numbers of infective stages occurred in the limpets’ mantle blood vessels — where they accumulate prior to release — between June and September, suggesting this period of time to be the main transmission window for E. patellae cercariae.     

Cercarial production of Fascioloides magna in the snail Galba truncatula (Gastropoda: Lymnaeidae)

Single-miracidium infections of Fascioloides magna in two populations of Galba truncatula were carried out under laboratory conditions to count free rediae and cercariae in snail cadavers just after death. Cercaria-shedding snails were in low numbers, and their shell height at day 60 p.e. was significantly greater than that of numerous infected snails that died without cercarial shedding. In snails that died between days 44 and 60 p.e. (at 20°C), the numbers of second-generation rediae significantly increased with increasing shell heights of infected snails. First-generation rediae showed insignificant, quantitative variations, while scarce rediae of the third generation were only found in the highest snails. Cercariae were only produced by the second redial generation. In both groups of snails, free cercariae appeared from 6 mm of shell height, and their numbers increased in the upper classes up to 32.9 per snail. Metacercariae were only found from 9 mm of shell height and were in low numbers. The global cercarial production ranged from 163.5 to 210.0 in the highest classes of snail size from both groups and was limited, whereas the mean burdens of free rediae fluctuated from 39.5 to 43.9. The death of numerous infected snails without cercarial shedding might be explained by the presence of a very high number of second-generation rediae simultaneously growing within the body of these snails.