IMMUNOELECTRON MICROSCOPIC STUDIES OF REGULATORY PEPTIDES IN THE NERVOUS-SYSTEM OF THE MONOGENEAN PARASITE, DICLIDOPHORA-MERLANGI

Specific antisera, directed against the highly conserved C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP) and the invertebrate peptide FMRFamide, have been used in conjunction with post-embedding, IgG-conjugated colloidal gold immunostaining to demonstrate peptide immunoreactivity at subcellular level in the nervous system of adult Diclidophora merlangi. Gold labelling revealed that immunoreactivity for PP and FMRFamide was localized exclusively in dense-cored vesicles occupying the majority of axons in the central nervous system. Double-labelling demonstrated an apparent co-localization of PP and FMRFamide in the same dense-cored vesicles. Antigen preabsorption experiments indicated cross-reactivity of the two antisera as unlikely, and that some if not all of the PP/FMRFamide immunostaining in the parasite was due to a neuropeptide F-like peptide.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF NEUROPEPTIDES IN THE CENTRAL-NERVOUS-SYSTEM OF THE ROUNDWORM, ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

The localization and distribution of neuropeptides in the central nervous system of the pig roundworm, Ascaris suum, have been determined by an indirect immunofluorescence technique in conjunction with confocal microscopy. Antisera to 25 vertebrate peptides and two invertebrate peptides were used to screen the worm for immunoreactivity (IR). Immunostaining was obtained with antisera to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), gastrin, cholecystokinin (CCK), substance P (SP), atrial natriuretic peptide (ANP), salmon gonadotropin-releasing hormone (SGnRH), mammalian gonadotropin-releasing hormone (MGnRH), chromogranin A (CGA) and FMRFamide. The most extensive patterns of IR occurred with antisera to PYY, FMRFamide and gastrin. IR was evident in nerve cells and fibres in the ganglia associated with the anterior nerve ring and in the main nerve cords and their commissures; IR to FMRFamide also occurred in the posterior nerve ring. Immunostaining for the other peptides was confined to the nerve cords, with the number of immunoreactive nerve fibres varying from peptide to peptide.

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.

NEUROPEPTIDE-F - A NOVEL PARASITIC FLATWORM REGULATORY PEPTIDE FROM MONIEZIA-EXPANSA (CESTODA, CYCLOPHYLLIDEA)

Using a C-terminally directed pancreatic polypeptide (PP) antiserum and immunocytochemical methods, PP-immunoreactivity (IR) was localized throughout the central (CNS) and peripheral nervous systems (PNS) of the cestode, Moniezia expansa. In the CNS, immunostaining was evident in the paired cerebral ganglia (primitive brain), connecting commissure, and the paired longitudinal nerve cords that are cross-linked by numerous regular transverse connectives. The PNS was seen to consist of a fine anastomosing nerve-net of immunoreactive fibres, many of which were closely associated with reproductive structures. Radioimmunoassay of this peptide IR in acid-alcohol extracts of the worm measured 192.8 ng/g of PP-IR. HPLC analyses of the M. expansa PP-IR identified a single molecular form which was purified to homogeneity. Plasma desorption mass spectrometry (PDMS) of purified parasite peptide resolved a single peptide with a molecular mass of 4599 +/- 10 Da. Automated gas-phase Edman degradation identified a 39-amino acid peptide with a C-terminal phenylalaninamide. Examination of its primary structure shows that it displays significant sequence homology with the vertebrate neuropeptide Y superfamily, suggesting that this platyhelminth-derived peptide is the phylogenetic precursor. Neuropeptide F (M. expansa) is the first regulatory peptide to be fully sequenced from the phylum Platyhelminthes and may represent a member of an important new class of invertebrate neuropeptide.

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.

‘Invasional meltdown’: evidence for unexpected consequences and cumulative impacts of multispecies invasions

Empirical support for ‘invasional meltdown’, where the presence of one invading species facilitates another and compounds negative impacts on indigenous species, is equivocal with few convincing studies. In Ireland, the bank vole was introduced 80 years ago and now occupies a third of the island. The greater white-toothed shrew arrived more recently within the invasive range of the bank vole. We surveyed the abundance of both invasive species and two indigenous species, the wood mouse and pygmy shrew, throughout their respective ranges. The negative effects of invasive on indigenous species were strong and cumulative bringing about species replacement. The greater white-toothed shrew, the second invader, had a positive and synergistic effect on the abundance of the bank vole, the first invader, but a negative and compounding effect on the abundance of the wood mouse and occurrence of the pygmy shrew. The gradual replacement of the wood mouse by the bank vole decreased with distance from the point of the bank vole’s introduction whilst no pygmy shrews were captured where both invasive species were present. Such interactions may not be unique to invasions but characteristic of all multispecies communities. Small mammals are central in terrestrial food webs and compositional changes to this community in Ireland are likely to reverberate throughout the ecosystem. Vegetation composition and structure, invertebrate communities and the productivity of avian and mammalian predators are likely to be affected. Control of these invasive species may only be effected through landscape and habitat management.

Development of the nervous system in Dugesia tigrina during regeneration after fission and decapitation

In the present study of Dugesia tigrina the development of the nervous system is followed and compared during regeneration after fission and after decapitation. Immunocytochemistry was used, with antisera raised against the biogenic amine, 5-hydroxytryptamine (5-HT) and the two neuropeptides, neuropeptide F (NPF), and FMRF amide. The results indicate that two processes are involved in the formation of the new cerebral ganglion. First, new processes sprouting from the original main longitudinal nerve cords bend transversely, indicating the position of the developing horseshoe-shaped anterior cerebral commissure. Then new nerve cells in front of the commissure differentiate from neoblasts and their growth cones fasciculate with the fibres from the old main longitudinal nerve cords. In the cerebral ganglion, 5-HT-IR cells appear before NPF-IR cells, in contrast to the pharynx where NPF-IR cells differentiate before the 5-HT-IR cells. In the peripheral nervous system, NPF-IR fibres and cells appear at a very early stage and dominate the whole regeneration process. A role for the PNS in early pattern formation is suggested.

The pharmacology of nematode FMRFamide-related peptides

FMRFamide-related peptides (FaRPs) are the largest known family of invertebrate neuropeptides. Immunocytochemical screens of nematode tissues using antisera raised to these peptides have localized extensive FaRP-immunostaining to their nervous systems. Although 21 FaRPs have been isolated and sequenced from extracts of free-living and parasitic nematodes, available evidence indicates that other FaRPs await discovery. While our knowledge of the pharmacology of these native nematode neuropeptides is extremely limited, reports on their physiological activity in nematodes are ever increasing. All the nematode FaRPs examined so far have been found to have potent and varied actions on nematode neuromuscular activity. It is only through the extensive pharmacological and physiological assessment of the tissue, cell and receptor interactions of these peptidic messengers that an understanding of their activity on nematode neuromusculature will be possible. In this review, Aaron Maule and colleagues examine the current understanding of the pharmacology of nematode FaRPs.

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.

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.

THE CHOLINERGIC, SEROTONINERGIC AND PEPTIDERGIC COMPONENTS OF THE NERVOUS-SYSTEM OF MONIEZIA-EXPANSA (CESTODA, CYCLOPHYLLIDEA)

The central (CNS) and peripheral (PNS) nervous systems of the cyclophyllidean tapeworm, Moniezia expansa, were examined for the presence of cholinergic, serotoninergic and peptidergic elements using enzyme cytochemical and immunocytochemical techniques in conjunction with light and confocal scanning laser microscopy. Cholinesterase activity and 5-hydroxytryptamine- and regulatory peptide-immunoreactivities (IRs) were localized to the nerve fibres and cell bodies of all of the major neuronal components in the CNS of the worm, including the cerebral ganglia and connecting commissure, the 10 longitudinal nerve cords and associated transverse ring commissures. Although each of the 3 systems appeared well developed and comprised a significant portion of the nervous system, the serotoninergic constituent was the most highly developed, consisting of a vast array of nerve fibres and cell bodies distributed throughout the strobila of the worm. A close association of cholinesterase reactivity and peptide-IRs was evident throughout the CNS, indicating the possible co-localization of acetylcholine and neuropeptides. Within the PNS, cholinergic activity and serotoninergic- and peptidergic-IRs occurred in the subtegumental network of nerve fibres and somatic musculature. Although all 3 neurochemical elements were present in the acetabula, they were found in different nerve fibres; only cholinergic and peptidergic cell bodies were found. The common genital opening, vagina and ootype regions of the reproductive system displayed a rich innervation of all 3 types of neuronal populations. Within the peptidergic system, immunostaining with antisera raised to the C-terminus of the neuropeptide Y superfamily of peptides and the invertebrate peptides, neuropeptide F (M. expansa) and FMRFamide was the most prevalent. Limited positive-IR for substance P and neurokinin A were also recorded in the CNS of the worm.

A CYTOCHEMICAL STUDY OF THE NERVOUS-SYSTEM OF THE PROTEOCEPHALIDEAN CESTODE, PROTEOCEPHALUS-POLLANICOLA

The localization and distribution of cholinergic, serotoninergic and peptidergic nerve elements in the proteocephalidean tapeworm, Proteocephalus pollanicola, have been investigated by enzyme histochemistry, and by an indirect immunofluorescence technique interfaced with confocal scanning laser microscopy. Cholinesterase (ChE) activity was localized in the major components of the central nervous system (CNS) and the peripheral nervous system (PNS), including the innervation of the reproductive structures of the worm. Serotoninergic (5-HT) nerves were found in the paired cerebral ganglia, transverse commissure and in the 10 longitudinal nerve cords. Antisera to 17 mammalian regulatory peptides and the invertebrate peptide FMRFamide have been used to explore the peptidergic nervous system of the worm. The most extensive immunostaining occurred with antisera raised to members of the neuropeptide Y superfamily, namely neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP). In all cases, intense immunoreactivity was found in numerous cell bodies and fibres of both the CNS and PNS, including the innervation of the reproductive apparatus. FMRFamide antisera stained the same structures to a comparable degree as those raised to the NPY superfamily. Cholinergic and peptidergic elements were much more prevalent within the CNS, while the serotoninergic nerve fibres tended to dominate in the PNS. The overlap obtained in staining patterns for the peptidergic and cholinergic components suggests that there may be a certain amount of co-localization of peptides with small-molecule transmitter substances in the same neurone. Weak staining for the tachykinin, substance P and for calcitonin gene-related peptide(CGRP) was confined to the major longitudinal nerve cords.

The Doñana ecological disaster:contamination of a world heritage estuarine marsh ecosystem with acidified pyrite mine waste

One of the most important bird breeding and over wintering sites in the west of Europe, the Coto Doñana, was severely impacted by the release of 5 million cubic meters of acid waste from the processing of pyrite ore. The waste entered ecologically sensitive areas of the park (including breeding areas for internationally endangered bird species) causing sustained pH decreases from pH 8.5 to 4.5 and resulting in massive metal contamination of the impacted ecosystem. The contaminating sludge waste contained arsenic at 0.6%, lead at 1.2% and zinc at 0.8% on a dry weight basis. The acid conditions facilitated the solubilization of these metals, leading to water concentrations lethal for aquatic wildlife. The accident caused considerable fish and invertebrate kills and has severe consequences for the protected bird species dependent on the impacted habitats and adjacent areas.