An Updated Guide to the Identification, Quantitation, and Imaging of the Crustacean Neuropeptidome.

Crustaceans serve as a useful, simplified model for studying peptides and neuromodulation, as they contain numerous neuropeptide homologs to mammals and enable electrophysiological studies at the single-cell and neural circuit levels. Crustaceans contain well-defined neural networks, including the stomatogastric ganglion, oesophageal ganglion, commissural ganglia, and several neuropeptide-rich organs such as the brain, pericardial organs, and sinus glands. As existing mass spectrometry (MS) methods are not readily amenable to neuropeptide studies, there is a great need for optimized sample preparation, data acquisition, and data analysis methods. Herein, we present a general workflow and detailed methods for MS-based neuropeptidomic analysis of crustacean tissue samples and circulating fluids. In conjunction with profiling, quantitation can also be performed with isotopic or isobaric labeling. Information regarding the localization patterns and changes of peptides can be studied via mass spectrometry imaging. Combining these sample preparation strategies and MS analytical techniques allows for a multi-faceted approach to obtaining deep knowledge of crustacean peptidergic signaling pathways.

Distribution, cellular localization, and colocalization of several peptide neurotransmitters in the central nervous system of Aplysia.

Neuropeptides are widely used as neurotransmitters in vertebrates and invertebrates. In vertebrates, a detailed understanding of their functions as transmitters has been hampered by the complexity of the nervous system. The marine mollusk Aplysia, with a simpler nervous system and many large, identified neurons, presents several advantages for addressing this question and has been used to examine the roles of tens of peptides in behavior. To screen for other peptides that might also play roles in behavior, we observed immunoreactivity in individual neurons in the central nervous system of adult Aplysia with antisera raised against the Aplysia peptide FMRFamide and two mammalian peptides that are also found in Aplysia, cholecystokinin (CCK) and neuropeptide Y (NPY), as well as serotonin (5HT). In addition, we observed staining of individual neurons with antisera raised against mammalian somatostatin (SOM) and peptide histidine isoleucine (PHI). However, genomic analysis has shown that these two peptides are not expressed in the Aplysia nervous system, and we have therefore labeled the unknown peptides stained by these two antibodies as XSOM and XPHI There was an area at the anterior end of the cerebral ganglion that had staining by antisera raised against many different transmitters, suggesting that this may be a modulatory region of the nervous system. There was also staining for XSOM and, in some cases, FMRFamide in the bag cell cluster of the abdominal ganglion. In addition, these and other studies have revealed a fairly high degree of colocalization of different neuropeptides in individual neurons, suggesting that the peptides do not just act independently but can also interact in different combinations to produce complex functions. The simple nervous system of Aplysia is advantageous for further testing these ideas.

Anatomy and activity patterns in a multifunctional motor neuron and its surrounding circuits.

Dorsal Excitor motor neuron DE-3 in the medicinal leech plays three very different dynamical roles in three different behaviors. Without rewiring its anatomical connectivity, how can a motor neuron dynamically switch roles to play appropriate roles in various behaviors? We previously used voltage-sensitive dye imaging to record from DE-3 and most other neurons in the leech segmental ganglion during (fictive) swimming, crawling, and local-bend escape (Tomina and Wagenaar, 2017). Here, we repeated that experiment, then re-imaged the same ganglion using serial blockface electron microscopy and traced DE-3's processes. Further, we traced back the processes of DE-3's presynaptic partners to their respective somata. This allowed us to analyze the relationship between circuit anatomy and the activity patterns it sustains. We found that input synapses important for all the behaviors were widely distributed over DE-3's branches, yet that functional clusters were different during (fictive) swimming vs. crawling.

Identification, characterization, and expression analysis of a serotonin receptor involved in the reproductive process of the Pacific abalone, Haliotis discus hannai.

Serotonin receptor (5-HT) is a biogenic amine acting as a neurotransmitter and neuromodulator that mediates various aspects of reproduction and gametogenesis. The full-length nucleotide sequence of Haliotis discus hannai encodes a protein of 417 amino acids with a predicted molecular mass of 46.54 kDa and isoelectric point of 8.94. The structural profile of 5-HTHdh displayed key features of G protein-coupled receptors, including seven hydrophobic transmembrane domains, putative N-linked glycosylation sites, and several phosphorylation consensus motifs. It shares the highest homology of its amino acid sequence with the 5-HT receptor from Haliotis asinina, and to lesser extent of human 5-HT receptor. The cloned sequence possesses two cysteine residues (Cys-115 and Cys-193), which are likely to form a disulfide bond. Phylogenetic comparison with other known 5-HT receptor genes revealed that the 5-HTHdh is most closely related to the 5-HTHa receptor. The three-dimensional structure of the 5-HTHdh showed multiple alpha helices which is separated by a helix-loop-helix (HLH) structure. Quantitative PCR demonstrated that the receptor mRNA was predominantly expressed in the pleuropedal ganglion. Significant differences in the transcriptional activity of the 5-HTHdh gene were observed in the ovary at the ripening stage. An exclusive expression was detected in pleuropedal ganglion, testis, and ovary at higher effective accumulative temperature (1000 °C). In situ hybridization showed that the 5-HTHdh expressing neurosecretory cells were distributed in the cortex of the pleuropedal ganglion. Our results suggest that 5-HTHdh synthesized in the neural ganglia may be involved in oocyte maturation and spawning of H. discus hannai.

Method for Detecting Hyaluronan in Isolated Myenteric Plexus Ganglia of Adult Rat Small Intestine.

The cellular components of the enteric nervous system (ENS), namely enteric neurons and glia, display plasticity and respond to environmental cues deriving from growth factors, extracellular matrix (ECM) molecules, and cell-surface molecules, both in physiological and pathological conditions. ECM, in particular, provides an important framework for the enteric microenvironment and influences the homeostasis of myenteric neuronal circuitries. Isolation of pure myenteric plexus preparations from adult tissue permits to investigate changes in the ENS involving specific ECM, such as hyaluronan. This approach is based upon the possibility to isolate myenteric ganglia from the intestinal wall of either adult animals or humans, after microdissection and subsequent enzymatic digestion of the tissue. Enteric ganglia are free of connective tissue, extracellular collagen, and blood vessels, and thus treatment of intact intestinal segments with highly purified collagenases permits ganglia isolation from the surrounding smooth muscle cells. In this chapter, we describe methods for visualizing HA in isolated primary cultures of adult rat small intestine myenteric ganglia.

Peptide identifications and false discovery rates using different mass spectrometry platforms.

Characterization of endogenous neuropeptides produced from post-translational proteolytic processing of precursor proteins is a demanding task. A variety of complex prohormone processing steps generate molecular diversity from neuropeptide prohormones, making in silico neuropeptide discovery difficult. In addition, the wide range of endogenous peptide concentrations as well as significant peptide complexity further challenge the structural characterization of neuropeptides. Liquid chromatography-mass spectrometry (MS), performed in conjunction with bioinformatics, allows for high-throughput characterization of peptides. Mass analyzers and molecular dissociation techniques render specific characteristics to the acquired data and thus, influence the analysis of the MS data using bioinformatic algorithms for follow-up peptide identification. Here we evaluated the efficacy of several distinct peptidomic workflows using two mass spectrometers, the Thermo Orbitrap Fusion Tribrid and Bruker Impact HD UHR-QqTOF, for confident peptide discovery and characterization. We compared the results in several categories, including the numbers of identified peptides, full-length mature neuropeptides among all identifications, and precursor proteins mapped by the identified peptides. We also characterized the peptide false discovery rate (FDR) based on the occurrence of amidation, a known post-translational modification (PTM) that has been shown to require the presence of a C-terminal glycine. Thus, amidation events without a preceding glycine were considered false-positive amidation assignments. We compared the FDR calculated by the search engine used here to the minimum FDR estimated via false amidation assignments. The search engine severely underestimated the rate of false PTM assignments among the identified peptides, regardless of the specific MS platform used.

Quantification and neurochemical coding of the myenteric plexus in humans: No regional variation between the distal colon and rectum.

BACKGROUND: It remains unclear whether regional variation exists in the human enteric nervous system (ENS) ie, whether intrinsic innervation varies along the gut. Recent classification of gastrointestinal neuropathies has highlighted inadequacies in the quantification of the human ENS. This study used paired wholemounts to accurately quantify and neurochemically code the hindgut myenteric plexus, comparing human distal colon and rectum. METHODS: Paired human descending colonic/rectal specimens were procured from 15 patients undergoing anterior resection. Wholemounts of myenteric plexi were triple-immunostained with anti-Hu/NOS/ChAT antibodies. Images were acquired by motorized epifluorescence microscopy, allowing assessment of ganglionic density/size, ganglionic area density, and neuronal density. 'Stretch-corrected' values were calculated using stretched/relaxed tissue dimensions. KEY RESULTS: Tile-stitching created a collage with average area 99 300 000 µm2 . Stretch-corrected ganglionic densities were similar (colon: median 510 ganglia/100 mm2 [range 386-1170], rectum: 585 [307-923]; P = .99), as were average ganglionic sizes (colon: 57 593 µm2 [40 301-126 579], rectum: 54 901 [38 701-90 211], P = .36). Ganglionic area density (colon: 11.92 mm2 per 100 mm2 [7.53-18.64], rectum: 9.84 [5.80-17.19], P = .10) and stretch-corrected neuronal densities (colon: 189 neurons/mm2 [117-388], rectum: 182 [89-361], P = .31) were also similar, as were the neurochemical profiles of myenteric ganglia, with comparable proportions of NOS+ and ChAT+ neurons (P > .10). CONCLUSIONS AND INFERENCES: This study has revealed similar neuronal and ganglionic densities and neurochemical profiles in human distal colon and rectum. Further investigation of other components of the ENS, incorporating additional immunohistochemical markers are required to confirm that there is no regional variation in the human hindgut ENS.

Top-Down Atmospheric Ionization Mass Spectrometry Microscopy Combined With Proteogenomics.

Mass spectrometry-based protein analysis has become an important methodology for proteogenomic mapping by providing evidence for the existence of proteins predicted at the genomic level. However, screening and identification of proteins directly on tissue samples, where histological information is preserved, remain challenging. Here we demonstrate that the ambient ionization source, nanospray desorption electrospray ionization (nanoDESI), interfaced with light microscopy allows for protein profiling directly on animal tissues at the microscopic scale. Peptide fragments for mass spectrometry analysis were obtained directly on ganglia of the medicinal leech (Hirudo medicinalis) without in-gel digestion. We found that a hypothetical protein, which is predicted by the leech genome, is highly expressed on the specialized neural cells that are uniquely found in adult sex segmental ganglia. Via this top-down analysis, a post-translational modification (PTM) of tyrosine sulfation to this neuropeptide was resolved. This three-in-one platform, including mass spectrometry, microscopy, and genome mining, provides an effective way for mappings of proteomes under the lens of a light microscope.

Changes in the neuropeptide content of Biomphalaria ganglia nervous system following Schistosoma infection.

BACKGROUND: Molluscs, including snails, are prone to parasite infection, which can lead to massive physiological and behavioural changes, yet many of the molecular components involved remain unresolved. Central to this point is the neural system that in snails consists of several ganglia that regulate the animals' physiology and behaviour patterns. The availability of a genomic resource for the freshwater snail Biomphalaria glabrata provides a mean towards the high throughput analysis of changes in the central nervous system (CNS) following infection with Schistosoma miracidia. RESULTS: In this study, we performed a proteomic analysis of the B. glabrata CNS at pre-patent infection, providing a list of proteins that were further used within a protein-protein interaction (PPI) framework against S. mansoni proteins. A hub with most connections for both non-infected and infected Biomphalaria includes leucine aminopeptidase 2 (LAP2), which interacts with numerous miracidia proteins that together belong to the immunoglobulin family of cell adhesion related molecules. We additionally reveal the presence of at least 165 neuropeptides derived from the precursors of buccalin, enterin, FMRF, FVRI, pedal peptide 1, 2, 3 and 4, RYamide, RFamide, pleurin and others. Many of these were present at significantly reduced levels in the snail's CNS post-infection, such as the egg laying hormone, a neuropeptide required to initiate egg laying in gastropod molluscs. CONCLUSIONS: Our analysis demonstrates that LAP2 may be a key component that regulates parasite infection physiology, as well as establishing that parasite-induced reproductive castration may be facilitated by significant reductions in reproduction-associated neuropeptides. This work helps in our understanding of molluscan neuropeptides and further stimulates advances in parasite-host interactions.

Immunohistochemical characteristics of neurons in the ganglia of the greater splanchnic nerve of the pig.

INTRODUCTION: Greater splanchnic nerve (GSN) is by far the largest of the splanchnic nerves and connects the paravertebral and prevertebral ganglia to transmit the majority of nociceptive information from the viscera. Despite its importance, the immunohistochemical features of the porcine GSN neurons have not yet been examined. Therefore, the aim of the study was to investigate the neurochemistry of the porcine GSN neurons and to compare their neurochemical coding with those of the paravertebral and prevertebral ganglia. MATERIAL AND METHODS: Four gilts of Large White Polish breed were examined in this study. Antibodies to tyrosine hydroxylase (TH), dopamine b-hydroxylase (DBH), choline acetyltransferase (ChAT), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), methionine-enkephalin (MET), calcitonin gene-related peptide (CGRP), and substance P (SP) were used for immunohistochemical detection of classical neurotransmitters marker enzymes and neuropeptides in neuronal cell bodies of the GSN. RESULTS: Double-labeling immunofluorescence revealed that virtually all GSN neurons exhibited the presence of catecholamine-synthesizing enzymes (TH/DBH-positive) and subpopulations of neurons contained immunoreactivity to NPY, VIP, SOM, GAL and MET. However, CGRP and SP-immunoreactivity were not observed in neuronal somata. CONCLUSIONS: Our data strongly suggest that the general immunohistochemical characterization of ganglion cells in the porcine greater splanchnic nerve is similar to that of the prevertebral ganglia (e.g. celiacomesenteric ganglion).

Development of Calbindin- and Calretinin-Immunopositive Neurons in the Enteric Ganglia of Rats.

Calbindin D28 K (CB) and calretinin (CR) are the members of the EF-hand family of calcium-binding proteins that are expressed in neurons and nerve fibers of the enteric nervous system. CB and CR are expressed differentially in neuronal subpopulations throughout the central and peripheral nervous systems and their expression has been used to selectively target specific cell types and isolate neuronal networks. The present study presents an immunohistochemical analysis of CB and CR in the enteric ganglia of small intestine in rats of different ages (newborn, 10-day-old, 20-day-old, 30-day-old, 60-day-old, 1-year-old, and 2-year-old). The data obtained suggest a number of age-dependent changes in CB and CR expression in the myenteric and submucous plexuses. In the myenteric plexus, the lowest percentage of CB-immunoreactive (IR) and CR-IR neurons was observed at birth, after which the number of IR cells increased in the first 10 days of life. In the submucous plexus, CB-IR and CR-IR neurons were observed from 10-day-old onwards. The percentage of CR-IR and CB-IR neurons increased in the first 2 months and in the first 20 days, respectively. In all animals, the majority of the IR neurons colocalized CR and CB. From the moment of birth, the mean of the cross-sectional area of the CB-IR and CR-IR neuronal profiles was larger than that of CB- and CR-negative cells.

Individual sympathetic postganglionic neurons coinnervate myenteric ganglia and smooth muscle layers in the gastrointestinal tract of the rat.

A full description of the terminal architecture of sympathetic axons innervating the gastrointestinal (GI) tract has not been available. To label sympathetic fibers projecting to the gut muscle wall, dextran biotin was injected into the celiac and superior mesenteric ganglia (CSMG) of rats. Nine days postinjection, animals were euthanized and stomachs and small intestines were processed as whole mounts (submucosa and mucosa removed) to examine CSMG efferent terminals. Myenteric neurons were counterstained with Cuprolinic Blue; catecholaminergic axons were stained immunohistochemically for tyrosine hydroxylase. Essentially all dextran-labeled axons (135 of 136 sampled) were tyrosine hydroxylase-positive. Complete postganglionic arbors (n = 154) in the muscle wall were digitized and analyzed morphometrically. Individual sympathetic axons formed complex arbors of varicose neurites within myenteric ganglia/primary plexus and, concomitantly, long rectilinear arrays of neurites within circular muscle/secondary plexus or longitudinal muscle/tertiary plexus. Very few CSMG neurons projected exclusively (i.e., ∼100% of an arbor's varicose branches) to myenteric plexus (∼2%) or smooth muscle (∼14%). With less stringent inclusion criteria (i.e., ≥85% of an axon's varicose branches), larger minorities of neurons projected predominantly to either myenteric plexus (∼13%) or smooth muscle (∼27%). The majority (i.e., ∼60%) of all individual CSMG postganglionics formed mixed, heterotypic arbors that coinnervated extensively (>15% of their varicose branches per target) both myenteric ganglia and smooth muscle. The fact that ∼87% of all sympathetics projected either extensively or even predominantly to smooth muscle, while simultaneously contacting myenteric plexus, is consistent with the view that these neurons control GI muscle directly, if not exclusively. J. Comp. Neurol. 524:2577-2603, 2016. © 2016 Wiley Periodicals, Inc.

Mass Spectrometry Imaging and Identification of Peptides Associated with Cephalic Ganglia Regeneration in Schmidtea mediterranea.

Tissue regeneration is a complex process that involves a mosaic of molecules that vary spatially and temporally. Insights into the chemical signaling underlying this process can be achieved with a multiplex and untargeted chemical imaging method such as mass spectrometry imaging (MSI), which can enablede novostudies of nervous system regeneration. A combination of MSI and multivariate statistics was used to differentiate peptide dynamics in the freshwater planarian flatwormSchmidtea mediterraneaat different time points during cephalic ganglia regeneration. A protocol was developed to makeS. mediterraneatissues amenable for MSI. MS ion images of planarian tissue sections allow changes in peptides and unknown compounds to be followed as a function of cephalic ganglia regeneration. In conjunction with fluorescence imaging, our results suggest that even though the cephalic ganglia structure is visible after 6 days of regeneration, the original chemical composition of these regenerated structures is regained only after 12 days. Differences were observed in many peptides, such as those derived from secreted peptide 4 and EYE53-1. Peptidomic analysis further identified multiple peptides from various known prohormones, histone proteins, and DNA- and RNA-binding proteins as being associated with the regeneration process. Mass spectrometry data also facilitated the identification of a new prohormone, which we have named secreted peptide prohormone 20 (SPP-20), and is up-regulated during regeneration in planarians.

Distal Rectal Skip-Segment Hirschsprung Disease and the Potential for False-Negative Diagnosis.

In skip-segment Hirschsprung disease (SS-HSCR), an aganglionic segment of bowel, which extends proximally from the distal rectum, is interrupted by a ganglionated "skip segment." Skip segments are usually located far proximal to the rectum where they do not interfere with initial diagnosis, although the possibility of distal SS-HSCR should be considered during interpretation of intraoperative biopsies or patients with atypical postoperative courses. We report 2 cases of SS-HSCR with skip areas in the distal rectum, 1 of which led to a false-negative diagnosis by suction rectal biopsy. These 2 cases of SS-HSCR, along with others in the literature, highlight the point that ganglionic skip segments can confuse clinicians and lead to inadequate bowel resection, diagnostic delay, or a false-negative diagnosis. The pathogenesis of SS-HSCR is discussed in light of recent discoveries regarding transmesenteric migration of vagal neural crest cells and the role of sacral neural crest cells in hindgut neurodevelopment.

Biologically Active Substances from Marine Hydrobionts with Antibacterial Activity in Composition of New Wound Dressings.

The data on the experimental study of the efficacy of the gel wound dressings based on chitosan and calcium alginate containing bioactive substances from marine hydrobionts with complex therapeutic action (sulfated polysaccharides from brown algae, hydrolyzed bivalves, peptides from nerve ganglia of cephalopods) are described. The model of thermal burns complicated by Staphylococcus aureus infection was used. Planimetric and bacteriological investigations revealed pronounced wound healing and antibacterial effects of the gel coating. The gel containing sulfated polysaccharides from brown algae showed the highest wound healing activity.

An Unexpected Diversity of Photoreceptor Classes in the Longfin Squid, Doryteuthis pealeii.

Cephalopods are famous for their ability to change color and pattern rapidly for signaling and camouflage. They have keen eyes and remarkable vision, made possible by photoreceptors in their retinas. External to the eyes, photoreceptors also exist in parolfactory vesicles and some light organs, where they function using a rhodopsin protein that is identical to that expressed in the retina. Furthermore, dermal chromatophore organs contain rhodopsin and other components of phototransduction (including retinochrome, a photoisomerase first found in the retina), suggesting that they are photoreceptive. In this study, we used a modified whole-mount immunohistochemical technique to explore rhodopsin and retinochrome expression in a number of tissues and organs in the longfin squid, Doryteuthis pealeii. We found that fin central muscles, hair cells (epithelial primary sensory neurons), arm axial ganglia, and sucker peduncle nerves all express rhodopsin and retinochrome proteins. Our findings indicate that these animals possess an unexpected diversity of extraocular photoreceptors and suggest that extraocular photoreception using visual opsins and visual phototransduction machinery is far more widespread throughout cephalopod tissues than previously recognized.

In situ Ca2+ imaging of the enteric nervous system.

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca(2+)) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca(2+) imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca(2+) indicator such as Fluo-4 permits measurements of Ca(2+) responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca(2+) responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca(2+) imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca(2+) responses in enteric neurons and glial cells.

Immunohistochemical detection of GnRH-like peptides in the neural ganglia and testis of Haliotis asinina.

Gonadotropin releasing hormone (GnRH) is a peptide that is conserved in both vertebrate and invertebrate species. In this study, we have demonstrated the distribution pattern of two isoforms of GnRH-like peptides in the neural ganglia and testis of reproductively mature male abalone, H. asinina, by immunohistochemistry and whole mount immunofluorescence. We found octopus (oct) GnRH and tunicate-I (t) GnRH-I immunoreactivities (ir) in type 1 neurosecretory cells (NS1) and they were expressed mostly within the ventral horn of the cerebral ganglion, whereas in pleuropedal ganglia they were localized primarily in the dorsal horn. Furthermore, tGnRH-I-ir were strongly detected in fibers at the caudal part of the cerebral ganglia and both ventral and dorsal horns of the pleuropedal ganglia. In the testis, only octGnRH-ir was found primarily in the granulated cell and central capillaries within the trabeculae. These results suggest that multiple GnRH-like peptides are present in the neural ganglia which could be the principal source of their production, whereas GnRH may also be synthesized locally in the testis and act as the paracrine control of testicular maturation.

Effects of AIBL on Oncomelania hupensis, the intermediate snail host of Schistosoma japonicum: an enzyme histochemical study.

OBJECTIVE: To explore the effect of AIBL on Oncomelania hupensis, the intermediate snail host of Schistosoma japonicum. METHODS: The enzyme histochemical profiles of cholinesterase, cytochrome oxidase, lactate dehydrogenase, nitric oxide synthase, and succinate dehydrogenase in the soft tissues of Oncomelania hupensis, the intermediate host snail of Schistosoma japonicum, were analyzed before and after treatment with the active ingredient of Buddleia lindleyana (AIBL), a potent and safe plant molluscicide. RESULTS: Treatment with AIBL induced a notable decrease in the activities of the five enzymes (P<0.01). CONCLUSIONS: The results indicate that AIBL impairs the activities of the enzymes, thereby influencing the transfer of neurotransmitter and energy supply in Oncomelania hupensis and ultimately harming their various physiological functions, which are considered to cause death of the species.

Schistosome I/Lamides--a new family of bioactive helminth neuropeptides.

Here we report the identification of a new family of helminth neuropeptides with members in both nematodes and flatworms, and include preliminary cell biological and functional characterisation of one of the peptides from the trematode parasite of humans, Schistosoma mansoni. Bioinformatics and Rapid Amplification of cDNA Ends (RACE)-PCR were used to identify the complete S. mansoni neuropeptide precursor gene Sm-npp-1, which encodes three pentapeptides bearing the motif (A/G)FVR(I/L).NH(2). Similar peptides were identified in three other flatworm species and in 15 nematode species. Quantitative PCR (qPCR) and immunocytochemical (ICC) analyses showed that Sm-npp-1 is constitutively expressed in larval and adult worms. ICC and confocal microscopy were employed to localise one of the schistosome NPP-1 peptides (GFVRIamide) in adult worms and schistosomules; antibodies labelled a pair of neurones in the cerebral ganglia that extend posteriorly along the main nerve cords. GFVRIamide displayed no detectable co-localisation with FMRFamide-like peptides (FLPs), nor was it detectable in muscle innervation. Exogenously applied peptide had a significant inhibitory effect on the mobility of whole adult worm pairs at 10(-5)M (n = 9). Finally, we explored Sm-npp-1 function in schistosomules using RNA interference (RNAi); we successfully achieved specific knockdown of the Sm-npp-1 transcript (54.46 ± 10.41% knockdown, n = 3), but did not detect any clear, aberrant mobility or morphological phenotypes. NPP-1-like peptides are a new family of helminth peptides with a cell-specific expression pattern distinct from FLPs and a modulatory effect on schistosome muscular activity.