ULTRASTRUCTURAL-LOCALIZATION OF FMRFAMIDE-IMMUNOREACTIVITY AND PANCREATIC POLYPEPTIDE-IMMUNOREACTIVITY 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, FMRFamide, 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.

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 PERIPHERAL NERVOUS-SYSTEM OF THE ROUNDWORM ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

The localisation and distribution of neuropeptides in the peripheral nervous system of the pig roundworm Ascaris suum have been determined by an indirect immunofluorescence 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.

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.

IMMUNOREACTIVITY TO 2 SPECIFIC REGIONS OF CHROMOGRANIN-A IN THE NERVOUS-SYSTEM OF ASCARIS-SUUM - AN IMMUNOCYTOCHEMICAL STUDY

Antisera to a highly conserved region of chromogranin A (sequence KELTAE) and to a hexapeptide (sequence KGQELE) adjacent to the putative C-terminus of pancreastatin, a peptide whose sequence is found within the chromogranin A molecule, have been used to examine the localisation of immunoreactivity (IR) to these peptides in Ascaris suum. IR to both peptides was found in the nerve rings and nerve cords. In addition, KGQELE-IR was also observed in the pharyngeal neurones and in a network of fibres on the surface of the female gonoduct. The staining was specific in that it could be abolished by preincubation of the antisera with the appropriate antigen. The two antisera appeared to be staining different subsets of neurones, suggesting that (at least) two peptides were being recognised. The widespread distribution of IR to both peptides throughout the nervous system of the parasite suggests that the peptides carrying the epitopes recognised by the antisera are of fundamental importance to the functioning of the parasite's nervous system.

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.

THE ORGANIZATION OF THE NERVOUS-SYSTEM IN PLATHELMINTHES - THE NEUROPEPTIDE F-IMMUNOREACTIVE PATTERN IN CATENULIDA, MACROSTOMIDA, PROSERIATA

The organization of the nervous system of Archilopsis unipunctata Promonotus schultzei and Paramonotus hamatus (Monocelididae, Proseriata) and Stenostomum leucops (Catenulida) and Microstomum lineare (Macrostomida) was studied by immunocytochemistry, using antibodies to the authentic flatworm neuropeptide F (NPF) (Moniezia expansa). The organization of the nervous system of the Monocelididae was compared to that of the nervous system of Bothriomolus balticus (Otoplanidae), a previously studied species of another family of the Proseriata. The results show that the main nerve cords (MCs), independent of lateral or ventral position in the Monocelididae and the Otoplanidae, correspond to each other. The study also confirms the status of the lateral cords as main cords (MCs) in S. leucops and M. lineare. Common for MCs in the members of the investigated taxa are the following features: MCs consist of many fibres, originate from the brain and are adjoined to 5-HT-positive neurons. In Monocelididae and Otoplanidae, the MCs additionally have the same type of contact to the pharyngeal nervous system. Also common for both proseriate families is the organization of the two lateral nerve cords, with weaker connections to the brain, and the pair of dorsal cords running above the brain. The organization of the minor cords differs. The Monocelididae have a pair of thin ventral cords forming a mirror image of the dorsal pair. Furthermore, an unpaired ventral medial cord connecting medial commissural cells was observed in P. schultzei. Marginal nerve cords, observed in Otoplanidae, are absent in Monocelididae. All minor nerve cords are closely connected to the peripheral nerve plexus. The postulated trends of condensation of plexal fibres to cords and/or the flexibility of the peripheral nerve plexus are discussed. In addition, the immunoreactivity (IR) pattern of NPF was compared to the IR patterns of the neuropeptide RFamide and the indoleamine, 5-HT (serotonin). Significant differences between the distribution of IR to NPF and to 5-HT occur. 5-HT-IR dominates in the submuscular and subepidermal plexuses. In the stomatogastric plexus of M. lineare, only peptidergic IR is observed in the intestinal nerve net. The distribution of NPF-IR in fibres and cells of the intestinal wall in M. lineare indicates a regulatory function for this peptide in the gut, while a relationship with ciliary and muscular locomotion is suggested for the 5-HT-IR occurring in the subepidermal and submuscular nerve plexuses. In M. lineare, the study revealed an NPF- and RFamide-positive cell pair, marking the finished development of new zooids. This finding indicates that constancy of these cells is maintained in this asexually reproducing and regenerating species.

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.

ELECTRON IMMUNOGOLD LABELING OF REGULATORY PEPTIDE IMMUNOREACTIVITY IN THE NERVOUS-SYSTEM OF MONIEZIA-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 cestode Moniezia expansa. Two axon types, distinguished by their populations of different-sized electron-dense vesicles, were identified. Immunogold labeling demonstrated an apparent homogeneity of PP, FMRFamide 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 FMRFamide 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 FMRFamide antisera, and the failure of these antisera to block NPF-IR supports the view that some, if not all, of the PP/FMRFamide-IR is due to NPF-like peptides.

TIME-RESOLVED OPTICAL-EMISSION AND ELECTRON-ENERGY DISTRIBUTION FUNCTION MEASUREMENTS IN RF PLASMAS

Comparisons between experimentally measured time-dependent electron energy distribution functions and optical emission intensities are reported for low-frequency (100 and 400 kHz) radio-frequency driven discharges in argon. The electron energy distribution functions were measured with a time-resolved Langmuir probe system. Time-resolved optical emissions of argon resonance lines at 687.1 and 750.4 nm were determined by photon-counting methods. Known ground-state and metastable-state excitation cross sections were used along with the measured electron energy distribution functions to calculate the time dependence of the optical emission intensity. It was found that a calculation using only the ground-state cross sections gave the best agreement with the time dependence of the measured optical emission. Time-dependent electron density, electron temperature, and plasma potential measurements are also reported.

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.

Trait-mediated indirect interactions in a marine intertidal system as quantified by functional responses

Studies of trait-mediated indirect interactions (TMIIs) typically focus on effects higher predators have on per capita consumption by intermediate consumers of a third, basal prey resource. TMIIs are usually evidenced by changes in feeding rates of intermediate consumers and/or differences in densities of this third species. However, understanding and predicting effects of TMIIs on population stability of such basal species requires examination of the type and magnitude of the functional responses exhibited towards them. Here, in a marine intertidal system consisting of a higher-order fish predator, the shanny Lipophrys pholis, an intermediate predator, the amphipod Echinogammarus marinus, and a basal prey resource, the isopod Jaera nordmanni, we detected TMIIs, demonstrating the importance of habitat complexity in such interactions, by deriving functional responses and exploring consequences for prey population stability. Echinogammarus marinus reacted to fish predator diet cues by reducing activity, a typical anti-predator response, but did not alter habitat use. Basal prey, Jaera nordmanni, did not respond to fish diet cues with respect to activity, distribution or aggregation behaviour. Echinogammarus marinus exhibited type II functional responses towards J. nordmanni in simple habitat, but type III functional responses in complex habitat. However, while predator cue decreased the magnitude of the type II functional response in simple habitat, it increased the magnitude of the type III functional response in complex habitat. These findings indicate that, in simple habitats, TMIIs may drive down consumption rates within type II responses, however, this interaction may remain de-stabilising for prey populations. Conversely, in complex habitats, TMIIs may strengthen regulatory influences of intermediate consumers on prey populations, whilst potentially maintaining prey population stability. We thus highlight that TMIIs can have unexpected and complex ramifications throughout communities, but can be unravelled by considering effects on intermediate predator functional response types and magnitudes.

Urotensin II in the central nervous system of the frog Rana ridibunda: Immunohistochemical localization and biochemical characterization

Urotensin II (UII) is traditionally regarded as a product of the neurosecretory cells in the caudal portion of the spinal cord of jawed fishes. A peptide related to UII has been recently isolated from the frog brain, thereby providing the first evidence that UII is also present in the central nervous system of a tetrapod. In the present study, we have investigated the distribution of UII-immunoreactive elements in the brain and spinal cord of the frog Rana ridibunda by immunofluorescence using an antiserum directed against the conserved cyclic region of the peptide. Two distinct populations of UII-immunoreactive perikarya were visualized. The first group of positive neurons was found in the nucleus hypoglossus of the medulla oblongata, which controls two striated muscles of the tongue. The second population of immunoreactive cell bodies was represented by a subset of motoneurons that were particularly abundant in the caudal region of the cord (34% of the motoneuron population). The telencephalon, diencephalon, mesencephalon, and metencephalon were totally devoid of UII-containing cell bodies but displayed dense networks of UII-immunoreactive fibers, notably in the thalamus, the tectum, the tegmentum, and the granular layer of the cerebellum. In addition, a dense bundle of long varicose processes projecting rostrocaudally was observed coursing along the ventral surface of the brain from the midtelencephalon to the medulla oblongata. Reversed-phase high-performance liquid chromatography analysis of frog brain, medulla oblongata, and spinal cord extracts revealed that, in all three regions, UII-immunoreactive material eluted as a single peak which exhibited the same retention time as synthetic frog UII. Taken together, these data indicate that UII, in addition to its neuroendocrine functions in fish, is a potential regulatory peptide in the central nervous system of amphibians. (C) 1996 Wiley-Liss, Inc.

An immunohistochemical study of the distribution of biotin in tissues of pigs and chickens

An optimised indirect peroxidase-anti-peroxidase immunohistochemical technique was used to detect endogenous biotin in frozen tissue sections from biotin-supplemented and biotin-depleted pigs and chickens. A monoclonal anti-biotin antibody was used as primary antibody in this technique. Immunoreactive biotin was detected in many tissues of both species including liver, kidney, pancreas, adipose tissue, adrenal gland, testis, brain, choroid plexus, cardiac and skeletal muscle, epithelium of the respiratory and digestive systems, skin and lymphoid tissues. The specificity of immunostaining for biotin was confirmed by the finding of reduced staining intensities in tissues of biotin-depleted animals compared to those of biotin-supplemented animals. The results of this study suggest that biotin has metabolic functions in a wider range of tissues than previously known. They also indicate that endogenous tissue biotin should be considered as a source of false positive staining when immunohistochemical or histochemical techniques which use avidin or streptavidin reagents or anti-biotin antibodies as components of the detection system, are applied to tissue sections.

Co-operating retrodirective system

Practical demonstration of the operational advantages gained through the use of a co-operating retrodirective array (RDA) basestation and Van Atta node arrangements is discussed. The system exploits a number of inherent RDA features to provide analogue real time multifunctional operation at low physical complexity. An active dual-conversion four element RDA is used as the power distribution source (basestation) while simultaneously achieving a receive sensitivity level of ??109 dBm and 3 dB automatic beam steering angle of ??45??. When mobile units are each equipped with a semi-passive four element Van Atta array, it is shown mobile device orientation issues are mitigated and optimal energy transfer can occur because of automatic beam formation resulting from retrodirective self-pointing action. We show that operation in multipath rich environments with or without line of sight acts to reduce average power density limits in the operating volume with high energy density occurring at mobile nodes sites only. The system described can be used as a full duplex ASK communications link, or, as a means for remote node charging by wireless means, thereby enhancing deployment opportunities between unstabilised moving platforms.