Larval development of the pedunculate barnacles Octolasmis angulata Aurivillius 1894 and Octolasmis cor Aurivillius 1892 (Cirripedia: Thoracica: Poecilasmatidae) from the gills of the mud crab, Scylla tranquebarica Fabricius, 1798.

Detailed studies of larval development of Octolasmis angulata and Octolasmis cor are pivotal in understanding the larval morphological evolution as well as enhancing the functional ecology. Six planktotrophic naupliar stages and one non-feeding cyprid stage are documented in details for the first time for the two species of Octolasmis. Morphologically, the larvae of O. angulata and O. cor are similar in body size, setation patterns on the naupliar appendages, labrum, dorsal setae-pores, frontal horns, cyprid carapace, fronto-lateral gland pores, and lattice organs. Numbers of peculiarities were observed on the gnathobases of the antennae and mandible throughout the naupliar life-cycle. The setation pattern on the naupliar appendages are classified based on the segmentation on the naupliar appendages. The nauplius VI of both species undergoes a conspicuous change before metamorphosis into cyprid stage. The cyprid structures begin to form and modify beneath the naupliar body towards the end of stage VI. This study emphasises the importance of the pedunculate barnacle larval developmental studies not only to comprehend the larval morphological evolution but also to fill in the gaps in understanding the modification of the naupliar structures to adapt into the cyprid life-style.

FMRFamide-like peptides in root knot nematodes and their potential role in nematode physiology.

FMRFamide-like peptides (FLPs) are a diverse group of neuropeptides that are expressed abundantly in nematodes. They exert potent physiological effects on locomotory, feeding and reproductive musculature and also act as neuromodulators. However, little is known about the specific expression patterns and functions of individual peptides. The current study employed rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) to characterize flp genes from infective juveniles of the root knot nematodes, Meloidogyne incognita and Meloidogyne minor. The peptides identified from these transcripts are sequelogs of FLPs from the free-living nematode, Caenorhabditis elegans; the genes have therefore been designated as Mi-flp-1, Mi-flp-7, Mi-flp-12, Mm-flp-12 and Mi-flp-14. Mi-flp-1 encodes five FLPs with the common C-terminal moiety, NFLRFamide. Mi-flp-7 encodes two copies of APLDRSALVRFamide and APLDRAAMVRFamide and one copy of APFDRSSMVRFamide. Mi-flp-12 and Mm-flp-12 encode the novel peptide KNNKFEFIRFamide (a longer version of RNKFEFIRFamide found in C. elegans). Mi-flp-14 encodes a single copy of KHEYLRFamide (commonly known as AF2 and regarded as the most abundant nematode FLP), and a single copy of the novel peptide KHEFVRFamide. These FLPs share a high degree of conservation between Meloidogyne species and nematodes from other clades, including those of humans and animals, perhaps suggesting a common neurophysiological role which may be exploited by novel drugs. FLP immunoreactivity was observed for the first time in Meloidogyne, in the circumpharyngeal nerve ring, pharyngeal nerves and ventral nerve cord. Additionally, in situ hybridization revealed Mi-flp-12 expression in an RIR-like neuron and Mi-flp-14 expression in SMB-like neurons, respectively. These localizations imply physiological roles for FLP-12 and FLP-14 peptides, including locomotion and sensory perception.

Role of calcium influx through voltage-operated calcium channels and of calcium mobilization in the physiology of Schistosoma mansoni muscle contractions.

We tested the hypothesis that voltage-operated Ca2+ channels mediate an extracellular Ca2+ influx in muscle fibres from the human parasite Schistosoma mansoni and, along with Ca2+ mobilization from the sarcoplasmic reticulum, contribute to muscle contraction. Indeed, whole-cell voltage clamp revealed voltage-gated inward currents carried by divalent ions with a peak current elicited by steps to +20 mV (from a holding potential of -70 mV). Depolarization of the fibres by elevated extracellular K+ elicited contractions that were completely dependent on extracellular Ca2+ and inhibited by nicardipine (half inhibition at 4.1 microM). However these contractions were not very sensitive to other classical blockers of voltage-gated Ca2+ channels, indicating that the schistosome muscle channels have an atypical pharmacology when compared to their mammalian counterparts. Futhermore, the contraction induced by 5 mM caffeine was inhibited after depletion of the sarcoplasmic reticulum either with thapsigargin (10 microM) or ryanodine (10 microM). These data suggest that voltage-operated Ca2+ channels do contribute to S. mansoni contraction as does the mobilization of stored Ca2+, despite the small volume of sarcoplasmic reticulum in schistosome smooth muscles.

Inter-phyla studies on neuropeptides: the potential for broad-spectrum anthelmintic and/or endectocide discovery.

Flatworm, nematode and arthropod parasites have proven their ability to develop resistance to currently available chemotherapeutics. The heavy reliance on chemotherapy and the ability of target species to develop resistance has prompted the search for novel drug targets. In view of its importance to parasite/pest survival, the neuromusculature of parasitic helminths and pest arthropod species remains an attractive target for the discovery of novel endectocide targets. Exploitation of the neuropeptidergic system in helminths and arthropods has been hampered by a limited understanding of the functional roles of individual peptides and the structure of endogenous targets, such as receptors. Basic research into these systems has the potential to facilitate target characterization and its offshoots (screen development and drug identification). Of particular interest to parasitologists is the fact that selected neuropeptide families are common to metazoan pest species (nematodes, platyhelminths and arthropods) and fulfil specific roles in the modulation of muscle function in each of the three phyla. This article reviews the inter-phyla activity of two peptide families, the FMRFamide-like peptides and allatostatins, on motor function in helminths and arthropods and discusses the potential of neuropeptide signalling as a target system that could uncover novel endectocidal agents.

Organization of the musculature of schistosome cercariae.

Phalloidin-fluorescein isothiocyanate staining of filamentous actin was used to identify muscle systems within the cercariae of Schistosoma mansoni. Examination of labeled cercariae by confocal scanning laser microscopy revealed distinct organizational levels of myofiber arrangements within the body wall, anterior cone, acetabulum, and esophagus. The body wall throughout showed a typical latticelike arrangement of outer circular and inner longitudinal myofibers, with an additional innermost layer of diagonal fibers in the anterior portion of the body. Circular and longitudinal fibers were also evident in the anterior organ and esophagus and, to some extent, the ventral acetabulum. Most striking was the striation of the cercarial tail musculature.

The ovijector of Ascaris suum: multiple response types revealed by Caenorhabditis elegans FMRFamide-related peptides.

Caenorhabditis elegans possesses 22 FMRFamide-like peptide (flp) genes predicted to encode 60 different FMRFamide-related peptides with a range of C-terminal signatures. Peptides from five flp genes (1, 6, 8, 9 and 14) are known to modulate the ovijector of Ascaris suum in vitro. This study examines the physiological effects of peptides from the remaining 17 flp genes such that the variety of FMRFamide-related peptide-induced ovijector response types can be delineated. Five categories of response were identified according to the pattern of changes in contractile behaviour and baseline tension. Peptides encoded on 16 flp genes (1, 2, 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 20) had qualitatively similar inhibitory (response type 1) actions, with the lowest activity thresholds (1 nM) recorded for peptides with FIRFamide or FLRFamide C-terminal signatures. Peptides encoded on four flp genes (2, 18, 19 and 21), and on the A. suum afp-1 gene, had excitatory actions on the ovijector (response type 2), with PGVLRFamides having the lowest activity threshold (1 nM). An flp-2 peptide (LRGEPIRFamide) induced a transient contraction of the ovijector (activity threshold, 10nM) that was designated response type 3. Response type 4 comprised a transient contraction followed by an extended period of inactivity and was observed with peptides encoded on flp-5 (AGAKFIRFamide, APKPKFIRFamide), flp-8 (KNEFIRFamide) and flp-22 (SPSAKWMRFamide). SPSAKWMRFamide was the most potent peptide tested with an activity threshold of 0.1 nM. A single peptide (AMRNALVRFamide; activity threshold 0.1 microM), encoded on flp-11, induced response type 5, a shortening of the ovijector coupled with an increase in contraction frequency. Although most flp genes encode structurally related peptides that trigger one of the five ovijector response types, flp-2 and flp-11 co-encode FMRFamide-related peptides that induce distinct responses. Within the ovijector of A. suum FaRPs play a complex role involving at least five receptor subtypes or signalling pathways.

Cytochemical studies of the neuromuscular systems of the diporpa and juvenile stages of Eudiplozoon nipponicum (Monogenea: diplozoidae).

Using indirect immuno- and enzyme-cytochemical techniques, interfaced with confocal scanning laser microscopy and standard optical microscopy, neuronal pathways have been demonstrated in whole-mount preparations of the unpaired diporpae and freshly paired juvenile stages of Eudiplozoon nipponicum (Monogenea: Diplozoidae). All 3 main classes of neuronal mediators, cholinergic, aminergic and peptidergic, were identified throughout both central and peripheral elements of a well-differentiated orthogonal nervous system. Neural mapping revealed considerable overlap and similarity in staining of the nervous systems of the diporpa and adult worm. The main differences in the diporpa relate to the innervation of the temporary ventral sucker and dorsal papilla, structures which are unique to the larva and which enable fusion between worms but then disappear. Branches from the longitudinal nerve cords innervate these structures and appear to be involved in the process of somatic fusion, probably giving rise to the inter-specimen connections that later link the 2 central nervous systems in paired adult parasites. In the hindbody, there is extensive haptoral innervation associated with the developing clamps and small central hooks. Reactive neuronal components were found associated with the early stages of clamp development prior to connections being made with the extrinsic adductor muscle bundles. The muscle systems of the diporpa and juvenile stages comprise a lattice-like arrangement of circular, longitudinal and diagonal fibres that make up the body wall, together with buccal suckers, haptoral clamps and associated adductor muscles, and the transient ventral sucker. All have obvious importance to diporpae when they migrate over the gill and undertake body contact, torsion and fusion during the process of pairing. Behaviour during the pairing of diporpae is described.

Characterisation of WE-14 in porcine ocular tissue.

WE-14 is derived from the cell-specific posttranslational processing of chromogranin A (CgA) in subpopulations of neuroendocrine cells and neurons. Region- and site-specific chromogranin A, pancreastatin and WE-14 antisera were employed to study the generation of WE-14 in porcine ocular tissues. No chromogranin A or pancreastatin immunostaining was detected in ocular tissue. Immunohistochemistry detected WE-14 immunostaining in a network of nerve fibre bundles and nerve fibres throughout the limbus, cornea, iris and ciliary body with sparse nerve fibres detected throughout the choroid and sclera. Retinal analysis detected intense WE-14 immunostaining in large ovoid cells in the ganglion cell layer with weak immunostaining in a population of small cells in the inner nuclear layer; weak immunostaining was detected within the fibre layers in the inner plexiform layer. Quantitatively, the highest WE-14 tissue concentration was recorded in aqueous retinal and corneal extracts with lower concentrations in the sclera, choroid and anterior uveal tissues. Chromatographic profiling resolved a minor chromogranin A-like immunoreactant and a predominant immunoreactant co-eluting with synthetic human WE-14. This is the first study to demonstrate that WE-14 is generated in neuronal fibres primarily innervating the anterior chamber and in select cell populations in the retina.

Microscopical evaluation of neural connectivity between paired stages of Eudiplozoon nipponicum (Monogenea: Diplozoidae).

Diplozoidae monogeneans am fish-gill ectoparasites comprising 2 individuals fused in so-called permanent copula. This unique situation occurs when 2 larvae (diporpee) make contact on the host gin, such that their union triggers maturation into an individual adult worm. The present study examined paired stages of Eudiplozoon nipponioun microscopicaily to ascertain whether somatic fusion involves neural connectivity between these 2 heterogenic larvae. Neuronal pathways were demonstrated in whole-mount preparations of the worm, using indirect immunocytochemical techniques interfaced with confocal scanning laser microscopy for peptidergic and serotoninergic innervations and enzyme cytochemical methodology and light microscopy for cholinergic component. Elements of the central nervous systems of paired worms are connected by commissures in the region of fusion so that the 2 systems are in structural continuity. Interindividual connections were mast apparent between corresponding ventral nerve cords. All 3 classes of neuronal mediators were identified throughout both central and peripheral connections of the 2 nervous systems. The anatomical complexity and apparent plasticity of the diplozoon nervous system suggest that it has a pivotal role not only in motility, feeding, and reproductive behavious but also in the events of larval pairing and somatic fusion.

Fasting modifies Aroclor 1254 impact on plasma cortisol, glucose and lactate responses to a handling disturbance in Arctic charr.

Integrated effects of polychlorinated biphenyl (PCB) and nutritional status on responses to handling disturbance were investigated in the Arctic charr (Salvelinus alpinus). The fish were orally contaminated with Aroclor 1254 and held either with or without food for 5 months before they were subjected to a 10-min handling disturbance. Food-deprived fish were given 0, 1, 10 or 100 mg PCB kg(-1) and the fed fish 0 or 100 mg PCB kg(-1). Plasma cortisol, glucose and lactate levels were measured at 0 (pre-handling), 1, 3, 6 and 23 h after the handling disturbance. Food-deprived control fish had elevated plasma cortisol levels compared with fed fish before handling. These basal cortisol levels were suppressed by PCB in food-deprived fish, and elevated by PCB in fed fish. The immediate cortisol and glucose responses to handling disturbance were suppressed by PCB in a dose-dependent way in food-deprived fish. Although these responses were also lowered by PCB in the fed fish, the effect was much less pronounced than in food-deprived fish. There were only minor effects on plasma lactate responses. Our findings suggest that the stress responses of the Arctic charr are compromised by PCB and that the long-term fasting, typical of high-latitude fish, makes these species particularly sensitive to organochlorines such as PCB.

Mesocestoides corti (syn. M. vogae): modulation of larval motility by neuropeptides, serotonin and acetylcholine.

The effect of platyhelminth FaRPs and selected classical neurotransmitters on the motility of intact Mesocestoides corti (syn. M. vogae) tetrathyridial larvae was studied in vitro using a micromotility meter. The effects of the test substances were temperature dependent and these were examined at 4, 23, 30 and 36 degrees C. At 36 degrees C all test substances had concentration-dependent excitatory effects, with thresholds for activity of: 100 nM (GNFFRFamide), 10 microm (YIRFamide), 30 microM (GYIRFamide), 100 nM (serotonin) and 100 microM (acetylcholine). At this temperature significant elevation of motility indices (MI) was recorded within 5 min of the addition of peptide or serotonin. The effect of acetylcholine was slower in onset and appeared 15-20 min post-addition. At 30 degrees C larval motility diminished more rapidly than that recorded at 36 degrees C, following the addition of 1 mM of each test substance. At 23 degrees C only serotonin (1 mM) significantly increased the MI, all other test substances having no apparent effect. Larval movement was completely arrested at 4 degrees C. The results demonstrate for the first time excitatory effects of platyhelminth neuropeptides and acetylcholine on muscle systems in cestode larvae. The fact that the only known cestode FaRP, GNFFRF amide, was more potent than any of the turbellarian FaRPs tested, suggests structural conservation of FaRPs and FaRP receptors within the cestodes.

Isolation and preliminary biological assessment of AADGAPLIRFamide and SVPGVLRFamide from Caenorhabditis elegans.

To date, 9 FMRFamide-related peptides (FaRPs) have been structurally characterised from Caenorhabditis elegans. Radioimmunometrical screening of an ethanolic extract of C. elegans revealed the presence of two additional FaRPs that were purified by reverse-phase HPLC and subjected to Edman degradation analysis and gas-phase sequencing. Unequivocal primary structures for the two FaRPs were determined as Ala-Ala-Asp-Gly-Ala-Pro-Leu-Ile-Arg-Phe-NH(2) and Ser-Val-Pro-Gly-Val-Leu-Arg-Phe-NH(2). Using MALDI-TOF mass spectrometry, the molecular masses of the peptides were found to be 1032 Da (MH) and 875 Da (MH)(+), respectively. Two copies of AADGAPLIRFamide are predicted to be encoded on the precursor gene termed flp-13, while one copy of SVPGVLRFamide is located on flp-18. Synthetic replicates of the peptides were tested on Ascaris suum somatic muscle to assess bioactivity. ADDGAPLIRFamide had inhibitory effects on A. suum muscle strips, which occurred over a range of concentrations from a threshold for activity of 10 nM to 10 microM. SVPGVLRFamide was excitatory on A. suum somatic musculature from a threshold concentration for activity of 1 nM to 10 microM. The inhibitory and excitatory effects of AADGAPLIRFamide and SVPGVLRFamide, respectively, were the same for dorsal and ventral muscle strips as well as innervated and denervated preparations, suggesting that these physiological effects are not nerve cord dependent. Addition of ADDGAPLIRFamide (10 microM) to muscle strips preincubated in high-K(+) and -Ca(2+)-free medium resulted in a normal inhibitory response. Peptide addition to muscle strips preincubated in Cl(-)-free medium showed no inhibitory response, suggesting that the inhibitory response of the peptide may be chloride mediated. A normal excitatory response was noted following the addition of 10 microM SVPGVLRFamide to muscle strips preincubated in high-K(+), Ca(2+)- and Cl(-)-free media.

FMRFamide-related peptides in potato cyst nematodes.

This study presents data demonstrating the presence of FMRFamide-related peptides (FaRPs) in potato cyst nematodes (PCN). Five transcripts of FaRP encoding genes, designated gpflp-1 to gpflp-5, were characterised using RACE. In terms of ORFs, gpflp-1 was 444 base pairs (bp) long and coded for four copies of the FaRP, PF3 (KSAYMRFamide) whilst gpflp-2 was 309 bp long and encoded one copy of the peptide, KNKFEFIRFamide. gpflp-3 (420 bp) Encoded two copies of KHEYLRFamide (AF2) and the genes gpflp-4 and gpflp-5 encoded a total of 11 FaRPs, most of which are novel to PCN. FMRFamide-related peptide (FaRP)-like immunoreactivity was observed in both PCN species, Globodera pallida and Globodera rostochiensis, using an antiserum raised against the invertebrate peptide, FMRFamide. Immunopositive neurones were found throughout the central nervous system in the ventral and dorsal nerve cords and the circumpharyngeal and perianal nerve rings. Reactive neurones were also present peripherally, innervating the highly muscular pharynx with a nerve net and ring-like structures. Positive immunostaining was also observed in neurones running toward the stylet protractor muscles and/or the anterior sensory apparatus. This study implicates a role for FaRPs in feeding, host penetration and sensory function of PCN. This is the first study to characterise FaRP encoding genes from a plant-parasitic nematode using a targeted PCR based RACE approach and further underlines the importance and diversity of this neuropeptide group in the phylum Nematoda.

Organisation of the nervous system in the Acoela: an immunocytochemical study.

In order to broaden the information about the organisation of the nervous system in taxon Acoela, an immunocytochemical study of an undetermined Acoela from Cape Kartesh, Faerlea glomerata, Avagina incola and Paraphanostoma crassum has been performed. Antibodies to 5-HT and the native flatworm neuropeptide GYIRFamide were used. As in earlier studies, the pattern of 5-HT immunoreactivity revealed an anterior structure composed mainly of commissures, a so-called commissural brain. Three types of brain shapes were observed. No regular orthogon was visualised. GYIRFamide immunoreactive cell clusters were observed peripherally to the 5-HT immunoreactive commissural brain. Staining with anti-GYIRFamide revealed more nerve processes than did staining with anti-FMRFamide. As no synapomorphies were found in the organisation of the nervous system of the Acoela and that of the Platyhelminthes, the results support the view that the Acoela is not a member of the Platyhelminthes.

Immunomicroscopical observations on the nervous system of adult Eudiplozoon nipponicum (Monogenea: Diplozoidae).

Neuronal pathways have been examined in adult Eudiplozoon nipponicum (Monogenea: Diplozoidae), using cytochemistry interfaced with confocal scanning laser microscopy, in an attempt to ascertain the status of the nervous system. Peptidergic and serotoninergic innervation was demonstrated by indirect immunocytochemistry and cholinergic components by enzyme cytochemical methodology; post-embedding electron microscopical immunogold labelling revealed neuropeptide immunoreactivity at the subcellular level. All three classes of neuronal mediators were identified throughout both central and peripheral elements of a well-differentiated orthogonal nervous system. There was considerable overlap in the staining patterns for cholinergic and peptidergic components, while dual immunostaining revealed serotonin immunoreactivity to be largely confined to a separate set of neurons. The subcellular distribution of immunoreactivity to the flatworm neuropeptide, GYIRFamide, confirmed neuropeptide localisation in dense-cored vesicles in the majority of the axons and terminal varicosities of both central and peripheral nervous systems. Results reveal an extensive and chemically diverse nervous system and suggest that pairing of individuals involves fusion of central nerve elements; it is likely also that there is continuity between the peripheral nervous systems of the two partner worms.

Physiological effects of FMRFamide-related peptides and classical transmitters on dispersed muscle fibres of the turbellarian, Procerodes littoralis.

The physiological effects of selected classical transmitters and FMRFamide-related peptides (FaRPs) on dispersed muscle fibres from the marine turbellarian, Procerodes littoralis have been examined. Confocal scanning laser microscopy coupled with fluorescein isothiocyanate (FITC) or tetramethylrhodamine (TRITC)-labelled phalloidin revealed a highly developed body wall muscle system with circular, longitudinal and diagonal layers of muscle fibres. Dispersed muscle fibres contracted when depolarized by exposure to extracellular media with elevated K+ (15-100 mM) in a concentration-dependent manner, with a maximal response of 87% achieved at > or = 75 mM. 5-Hydroxytryptamine (5-HT) induced concentration-dependent muscle contraction between 0.01 and 1000 microM, with 10 microM producing a near maximal contraction response of 75%. Acetylcholine (ACh) had less pronounced excitatory effects (0.01-1000 microM), inducing contraction of only 32% of the fibres at 100 microM. The flatworm FMRFamide-related peptides (FaRPs), GYIRFamide, YIRFamide and GNFFRFamide each had concentration-dependent myocontractile effects, indicating the occurrence of at least 1 FaRP receptor on P. littoralis muscle fibres. At 10 microM peptide, GNFFRFamide induced contractions in < 40% of the muscle fibres examined, whereas YIRFamide and GYIRFamide induced contraction in 70 and 75% of muscle fibres, respectively. The order of potency of the peptides was: GYIRFamide > YIRFamide > GNFFRFamide. Pre-incubation of the muscle fibres in 5 microM 5-HT significantly reduced the responses to GYIRFamide, YIRFamide and 5-HT, while the responses to high K+ remained unaltered. Muscle fibres pre-incubated in GYIRFamide (0.1 microM) were also less responsive to 5-HT but not to ACh and high-K+. The GYIRFamide analogue, GYIRDFamide, did not induce muscle contraction (0.01-100 microM) per se, but when co-applied with the myoactive peptides GYIRFamide, YIRFamide or GNFFRFamide, it significantly blocked their ability to elicit contractions. This suggests that the peptides tested may act via a common muscle-based neuropeptide receptor. GYIRDFamide did not alter the contractile effects of high K+, 5-HT or ACh. Collectively, these results indicate that FaRPs, 5-HT and ACh all have the potential to cause muscle contraction in flatworms and that 5-HT and FaRPs alter muscle sensitivity to each other, but do not influence the ability of flatworm muscle fibres to contract.

Comparative study of the spatial relationship between nicotinamide adenine dinucleotide phosphate-diaphorase activity, serotonin immunoreactivity, and GYIRFamide immunoreactivity and the musculature of the adult liver fluke, Fasciola hepatica (Digenea, fasciolidae).

This is the first detailed description of the nitrergic nervous system in a fluke. In this study, the authors analysed the distribution of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity in neuronal and nonneuronal tissues of the adult fluke Fasciola hepatica and compared this with the distribution of the musculature using tetramethylrhodamine isothiocyanate-phalloidin. To assess the correlation between the number of muscle cells in different parts of the fluke and the NADPH-d-stained cells, the nuclei were stained with Hoechst 333 42, which is specific for chromatin. The spatial relation between the NADPH-d-positive nerves and the 5-hydroxytryptamine (serotonin; 5-HT)-immunoreactive (-IR) and GYIRFamide-IR nervous elements was also examined. The methods complement each other. NADPH-d-positive staining occurs in both in neuronal tissue and nonneuronal tissue. Large, NADPH-d-stained neurones were localised in the nervous system. The oral and ventral suckers are innervated with many large NADPH-d-stained neurones. In addition, the NADPH-d staining reaction follows closely the muscle fibres in both the suckers, in the body, and in the ducts of the reproductive organs. The presence of NADPH-d activity along muscle fibres in F. hepatica and in other flatworms supports a possible myoinhibitory role for nitric oxide. Neuronal nitric oxide synthase in flatworms may form a novel drug target, which would facilitate the development of a novel anthelminthic.

Coho salmon Oncorhynchus kisutch strain differences in disease resistance and non-specific immunity, following immersion challenges with Vibrio anguillarum.

Two strains of freshwater-reared coho salmon Oncorhynchus kisutch were compared for differences in the activity of selected non-specific immune factors before and after lethal and non-lethal immersion challenges with the marine bacterial pathogen Vibrio anguillarum (Vang). Two disease challenge experiments were performed. The first experimental challenge resulted in no mortality; however, significant strain and challenge treatment effects were detected at Day 16 post-challenge. Strain differences in plasma lysozyme activity were found in pre-challenge samples. The second challenge experiment compared the same strains of coho salmon following immersion challenges in different doses of Vang. The fish were sampled at Days 0, 2, 7, and 18 post-challenge and mortality, plasma lysozyme, and anterior kidney phagocyte respiratory burst activity were compared. There were significant strain differences in mortality in the high dose group. The more disease-resistant strain was found to have higher levels of plasma lysozyme and anterior kidney phagocyte respiratory burst activity. These strain differences were detected at various times in the lethal (high dose) and non-lethal challenge groups. There was a clear relationship between the enhanced survival of the more disease-resistant strain and a more sustained, elevated non-specific immune response following the experimental disease challenges. The results of this study suggest that the basis for strain differences in innate disease resistance is related to the ability of the fish to respond quickly to the initial infection and to maintain the response until the infection is quelled.

Antibody-producing cells correlated to body weight in juvenile chinook salmon (Oncorhynchus tshawytscha) acclimated to optimal and elevated temperatures.

The immune response of juvenile chinook salmon (Oncorhynchus tshawytscha) ranging in weight from approximately 10 to 55 g was compared when the fish were acclimated to either 13 or 21 degrees C. A haemolytic plaque assay was conducted to determine differences in the number of antibody-producing cells (APC) among fish of a similar age but different body weights. Regression analyses revealed significant increases in the number of APC with increasing body weight when fish were acclimated to either water temperature. These results emphasise the importance of standardising fish weight in immunological studies of salmonids before exploring the possible effects of acclimation temperatures.

Novel transcripts of the estrogen receptor alpha gene in channel catfish.

Complementary DNA libraries from liver and ovary of an immature female channel catfish were screened with a homologous ERalpha cDNA probe. The hepatic library yielded two new channel catfish ER cDNAs that encode N-terminal ERalpha variants of different sizes. Relative to the catfish ERalpha (medium size; 581 residues) previously reported, these new cDNAs encode Long-ERalpha (36 residues longer) and Short-ERalpha (389 residues shorter). The 5'-end of Long-ERalpha cDNA is identical to that of Medium-ERalpha but has an additional 503-bp segment with an upstream, in-frame translation-start codon. Recombinant Long-ERalpha binds estrogen with high affinity (K(d) = 3. 4 nM), similar to that previously reported for Medium-ERalpha but lower than reported for catfish ERbeta. Short-ERalpha cDNA encodes a protein that lacks most of the receptor protein and does not bind estrogen. Northern hybridization confirmed the existence of multiple hepatic ERalpha RNAs that include the size range of the ERalpha cDNAs obtained from the libraries as well as additional sizes. Using primers for RT-PCR that target locations internal to the protein-coding sequence, we also established the presence of several ERalpha cDNA variants with in-frame insertions in the ligand-binding and DNA-binding domains and in-frame or out-of-frame deletions in the ligand-binding domain. These internal variants showed patterns of expression that differed between the ovary and liver. Further, the ovarian library yielded a full-length, ERalpha antisense cDNA containing a poly(A) signal and tail. A limited survey of histological preparations from juvenile catfish by in situ hybridization using directionally synthesized cRNA probes also suggested the expression of ERalpha antisense RNA in a tissue-specific manner. In conclusion, channel catfish seemingly have three broad classes of ERalpha mRNA variants: those encoding N-terminal truncated variants, those encoding internal variants (including C-terminal truncated variants), and antisense mRNA. The sense variants may encode functional ERalpha or related proteins that modulate ERalpha or ERbeta activity. The existence of ER antisense mRNA is reported in this study for the first time. Its role may be to participate in the regulation of ER gene expression.