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.

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.

COMPARATIVE ANALYSES OF THE NEUROPEPTIDE-F (NPF)-RELATED AND FMRFAMIDE-RELATED PEPTIDE (FARP)-IMMUNOREACTIVITIES IN FASCIOLA-HEPATICA AND SCHISTOSOMA SPP

Immunochemical techniques were used to determine the distribution, chemical characteristics and relative abundance of immunoreactivity (IR) to two native platyhelminth neuropeptides, neuropeptide F (NPF) (Moniezia expansa) and the FMRFamide-related peptide (FaRP), GNFFRFamide, in the trematodes, Fasciola hepatica and Schistosoma mansoni; the larger S. margrebowiei was used in the chemical analysis. Extensive immunostaining for the two peptides was demonstrated throughout the nervous systems of both F. hepatica and S. mansoni, with strong IR also in the innervation of muscular structures, including those associated with the egg-forming apparatus. The patterns of immunostaining were similar to those previously described for the vertebrate neuropeptide Y superfamily of peptides and for FMRFamide. Ultrastructurally, gold labelling of NPF- and GNFFRFamide-IRs was localized exclusively to the contents of secretory vesicles in the axons and somatic cytoplasm of neurones. Double-labelling experiments showed an apparent homogeneity of antigenic sites, in all probability due to the demonstrated cross-reactivity of the FaRP antiserum with NPF. Radioimmunoassay of acid-ethanol extracts of the worms detected 8.3 pmol/g and 4.7 pmol/g equivalents of NPF- and FMRFamide-IRs, respectively, for F. hepatica, and corresponding values of 4.9 pmol/g and 4.3 pmol/g equivalents for S. margrebowiei. Gel-permeation chromatography resolved IR to both peptides in discrete peaks and these eluted in similar positions to synthetic NPF (M. expansa) and GNFFRFamide, respectively.

ISOLATION AND PRIMARY STRUCTURE OF A NOVEL AVIAN PANCREATIC-POLYPEPTIDE FROM 5 SPECIES OF EURASIAN CROW

Chicken pancreatic polypeptide is the prototype of the neuropeptide Y (NPY)/PP superfamily of regulatory peptides. This polypeptide was appended the descriptive term avian, despite the presence of some 8600 extant species of bird. Additional primary structures from other avian species, including turkey, goose and ostrich, would suggest that the primary structure of this polypeptide has been highly-conserved during avian evolution. Avian pancreatic polypeptides structurally-characterised to date have distinctive primary structural features unique to this vertebrate group including an N-terminal glycyl residue and a histidyl residue at position 34. The crow family, Corvidae, is representative of the order Passeriformes, generally regarded as the most evolutionarily recent and diverse avian taxon. Pancreatic polypeptide has been isolated from pancreatic tissues from five representative Eurasian species (the magpie, Pica pica; the jay, Garrulus glandarius; the hooded crow, Corvus corone; the rook, Corvus frugilegus; the jackdaw, Corvus monedula) and subjected to structural analyses. Mass spectroscopy estimated the molecular mass of each peptide as 4166 +/- 2 Da. The entire primary structures of 36 amino acid residue peptides were established in single gas-phase sequencing runs. The primary structures of pancreatic polypeptides from all species investigated were identical: APAQPAYPGDDAPVEDLLR-FYNDLQQYLNVVTRPRY. The peptides were deemed to be amidated due to their full molar cross-reactivity with the amide-requiring PP antiserum employed. The molecular mass (4165.6 Da), calculated from the sequences, was in close agreement with mass spectroscopy estimates. The presence of an N-terminal alanyl residue and a prolyl residue at position 34 differentiates crow PP from counterparts in other avian species. These residues are analogous to those found in most mammalian analogues. These data suggest that the term avian, appended to the chicken peptide, is no longer tenable due to the presence of an Ala1, Pro34 peptide in five species from the largest avian order. These data might also suggest that, in keeping with the known structure/activity requirements of this peptide family, crow PP should interact identically to mammalian analogues on mammalian receptors.

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.

NEUROPEPTIDE F-IMMUNOREACTIVITY IN THE MONOGENEAN PARASITE DICLIDOPHORA-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 in D. merlangi and that it accounts for most of the immunostaining previously obtained with PP and FMRFamide antisera.

UROTENSIN II FROM THE RIVER LAMPREY (LAMPETRA-FLUVIATILIS), THE SEA LAMPREY (PETROMYZON-MARINUS), AND THE PADDLEFISH (POLYODON SPATHULA)

Urotensin II was isolated from extracts of the whole brain of the river lamprey (Lampetra fluviatilis) and the sea lamprey (Petromyzon marinus). The primary structure of the peptide from both species is the same (Asn-Asn-Phe-Ser-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val) and this amino acid sequence is identical to that of urotensin II from the dogfish and skate. Consistent with previous morphological studies indicating that the Agnatha lack a caudal neurosecretory system, urotensin II was not detected in an extract of P. marinus spinal cord. The data suggest that the urotensin II may have functioned in the earliest vertebrates as a neurotransmitter/neuromodulator in the central nervous system rather than as a neurohormone of the caudal neurosecretory system. Urotensin II was also isolated from an extract of the spinal cord of a chondrostean fish, the paddlefish (Polyodon spathula). The primary structure of the paddlefish urotensin II (Gly-Ser-Thr-Ser-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val) is the same as that of another chondrostean, the sturgeon (Acipenser ruthenus). The study provides further evidence for a widespread distribution of urotensin II in vertebrate species and suggests that the primary structure of the peptide is better conserved in these phylogenetically ancient fish than in teleosts. (C) 1995 Academic Press, Inc.

TACHYKININS WITH UNUSUAL STRUCTURAL FEATURES FROM A URODELE, THE AMPHIUMA, AN ELASMOBRANCH, THE HAMMERHEAD SHARK, AND AN AGNATHAN, THE RIVER LAMPREY

Tachykinins were purified from extracts of gastrointestinal tissues of the urodele, Amphiuma tridacrylum (three-toed amphiuma), and the elasmobranch Sphyrna lewini (hammerhead shark), and from the brain of the agnathan Lampetra fluviatilis (river lamprey). The amphiuma substance P (SP) (DNPSVGQFYGLM-NH2) contains 12 amino residues compared with 11 for mammalian SP and lacks the Arg/Lys-Pro-Xaa-Pro motif that is characteristic of NK, receptor-selective agonists. Lampetra SP (RKPHPKEFVGLM-NH2) is identical to SP from the sea lamprey and the shark SP-related peptide (AKFDKFYGLM-NH2) is identical to dogfish scyliorhinin L. Amphiuma neurokinin A (NKA) (HKDAFIGLM-NH2) and lamprey NKA (HFDEFVGLM-NH2) contain 9 amino acid residues compared with 10 for mammalian NKA. The shark NKA-related peptide (ASGPTQAGIV(10)GRKRQKGEMF(20)VGLM-NH2) shows limited structural similarity to mammalian neuropeptide gamma and the teleost tachykinin, carassin but contains 24 rather than 21 amino acid residues. The data show that the primary structures of the tachykinins have been very poorly conserved during vertebrate evolution and that pressure has acted only to maintain the functionally important sequence -Phe-Xaa-Gly- Leu-Met-NH2 at the COOH-termini of the peptides.

NOVEL TACHYKININS FROM THE BRAIN OF THE SEA LAMPREY, PETROMYZON-MARINUS, AND THE SKATE, RAJA-RHINA

Using radioimmunoassay for mammalian tachykinins, peptides with substance P-like immunoreactivity and neurokinin A-like immunoreactivity were identified in an extract of the brain of the longnose skate, Raja rhina (elasmobranch) but only a peptide with neurokinin A-like immunoreactivity was identified in the brain of the sea lamprey, Petromyzon marinus (agnathan). The primary structure of the skate peptide with substance P-like immunoreactivity (Ala-Lys-His-Asp-Lys-Phe-Tyr-Gly-Leu-Met-NH2) shows one amino acid substitution (Phe(3) --> His) compared with scyliorhinin I, previously isolated from dogfish brain and gut. The skate neurokinin A-related peptide (His-Lys-Leu-Gly-Ser-Phe-Val-Gly-Leu-Met-NH2) shows tow substitutions (Thr(3) --> Leu and Asp(4) --> Gly) compared with mammalian neurokinin A. Although the COOH-terminus of the lamprey tackhykinin (Arg-Lys-Pro-His-Pro-Lys-Gly-phe-Val-Gly-Leu-Met-NH2) resembles neurokinin A, the presence of the strongly conserved Lys/Arg-Pro-Xaa-Pro motif at the NH2-terminus of the peptide indicates greater structural similarity with substance P. The additional arginine residue at position 1 in the peptide suggests that the lamprey is utilizing a site of postranslational processing in the tachykinin precursor that is different from the equivalent site in mammalian and other lower vertebrate preprotachykinin(s).

Cloning of cDNAs and molecular characterisation of C-type lectin-like proteins from snake venoms

C-type lectin-like proteins (CTLPs) isolated from snake venoms are the largest and most complex non-mammalian vertebrate C-type lectin-like domain family. In the present study, we simultaneously amplified four cDNAs encoding different types of CTLP subunits from the venoms of two different species of snakes by RT-PCR with a single sense primer and a nested universal primer - two CTLP subunit-encoding cDNAs were cloned from Deinagkistrodon acutus venom and two from Agkistrodon halys Pallas venom. All four cloned CTLP subunits exhibited typical motifs in their corresponding domain regions but with relatively-low sequence similarities to each other. Compared with previously-published CTLPs, the four cloned CTLPs subunits showed slight variations in the calcium-binding sites and the disulphide bonding patterns. To our knowledge, these data constitute the first example of co-expression of CTLP platelet glycoprotein Ib-binding subunits and coagulation factors in Agkistrodon halys Pallas venom.

Molecular evolution of proadrenomedullin N-terminal 20 peptide (PAMP):evidence for gene co-option

Posttranslational processing of proadrenomedullin generates two biologically active peptides, adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP). Sequence comparison of homologous proadrenomedullin genes in vertebrate evolution shows a high degree of stability in the reading frame for AM, whereas PAMP sequence changes rapidly. Here we investigate the functional significance of PAMP phylogenetic variation studying two of PAMP's better characterized physiological activities, angiogenic potential and antimicrobial capability, with synthetic peptides carrying the predicted sequence for human, mouse, chicken, and fish PAMP. All tested peptides induced angiogenesis when compared with untreated controls, but chicken and fish PAMP, which lack terminal amidation, were apparently less angiogenic than their human and mouse homologs. Confirming the role of amidation in angiogenesis, Gly-extended and free acid variants of human PAMP produced responses similar to the natural nonamidated peptides. In contrast, antimicrobial activity was restricted to human PAMP, indicating that this function may have been acquired at a late time during the evolution of PAMP. Interestingly, free acid human PAMP retained antimicrobial activity whereas the Gly-extended form did not. This fact may reflect the need for maintaining a tightly defined structural conformation in the pore-forming mechanism proposed for these antimicrobial agents. The evolution of PAMP provides an example of an angiogenic peptide that developed antimicrobial capabilities without losing its original function.

Shock avoidance by discrimination learning in the shore crab (Carcinus maenas) is consistent with a key criterion for pain

Nociception allows for immediate reflex withdrawal whereas pain allows for longer-term protection via rapid learning. We examine here whether shore crabs placed within a brightly lit chamber learn to avoid one of two dark shelters when that shelter consistently results in shock. Crabs were randomly selected to receive shock or not prior to making their first choice and were tested again over 10 trials. Those that received shock in trial 2, irrespective of shock in trial 1, were more likely to switch shelter choice in the next trial and thus showed rapid discrimination. During trial 1, many crabs emerged from the shock shelter and an increasing proportion emerged in later trials, thus avoiding shock by entering a normally avoided light area. In a final test we switched distinctive visual stimuli positioned above each shelter and/or changed the orientation of the crab when placed in the chamber for the test. The visual stimuli had no effect on choice, but crabs with altered orientation now selected the shock shelter, indicating that they had discriminated between the two shelters on the basis of movement direction. These data, and those of other recent experiments, are consistent with key criteria for pain experience and are broadly similar to those from vertebrate studies. © 2013. Published by The Company of Biologists Ltd.

Fasciola hepatica: A light and electron microscope study of sustentacular tissue and heterophagy in the testis

In order to investigate cytolytic activity in the testis of Fasciola hepatica, flukes belonging to several different triclabendazole (TCBZ)-sensitive and TCBZ-resistant isolates, and wildtype flukes from field infections, were studied by light and electron microscopy with a view to identifying sites of heterophagy and macromolecular hydrolysis. At the periphery of the testis tubules in all the flukes examined, large euchromatic nuclei, each bearing a prominent nucleolus, were seen. These were invested with a thin cytoplasmic layer, extensions of which partially enveloped and probably supported the neighbouring spermatogonia. No lateral cell boundaries were identified in this tissue, possibly indicating syncytial organisation. The tissue, considered to be analogous to Sertoli cells in vertebrate testis, was identified as sustentacular tissue. It displayed cytoplasmic features consistent with protein/glycoprotein synthesis (through a granular endoplasmic reticulum-Golgi mediated mechanism) and intracellular digestion/heterophagy (through a lysosomal system). The intracytoplasmic hydrolytic activity of the sustentacular tissue probably serves to scavenge effete cells and cytoplasmic debris, to recycle useful molecules, to promote spermatozoon maturation and possibly to aid osmoregulation within the tubules. Certain protein-containing macromolecules synthesised in the sustentacular tissue may contribute to the seminiferous fluid, or have pheromonal activity. The presence of numerous mitochondria and abundant smooth endoplasmic reticulum is consistent with facilitation of inward and outward movement of micromolecular nutrients, metabolites including excretory products and water. In the sustentacular tissue of certain flukes with dysfunctional spermiogenesis, there was increased heterophagic and cytolytic scavenging activity. The cytoplasmic residual vacuoles remaining after the release of spermatids were also identified as possible sites for lysosome-mediated intracellular digestion and osmoregulation in the testis tubules of F. hepatica. (C) 2012 Elsevier B.V. All rights reserved.