fmrfamide and corazonin-protein--insect

Technological advancements in high-throughput sequencing have resulted in the production/public deposition of an ever-growing number of arthropod transcriptomes. While most sequencing projects have focused on hexapods, transcriptomes have also been generated for members of the Chelicerata. One chelicerate for which a large transcriptome has recently been released is the Western black widow Latrodectus hesperus, a member of the Araneae (true spiders). Here, a neuropeptidome for L. hesperus was predicted using this resource. Thirty-eight peptide-encoding transcripts were mined from the L. hesperus transcriptome, with 216 distinct peptides predicted from the deduced pre/preprohormones. The identified peptides included members of the allatostatin A, allatostatin B, allatostatin C, allatotropin, bursicon α, bursicon β, CAPA/periviscerokinin/pyrokinin, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/ion transport peptide, diuretic hormone 31, diuretic hormone 44, FMRFamide-like peptide (FLP), GSEFLamide, insulin-like peptide, neuropeptide F (NPF), orcokinin, proctolin, short neuropeptide F, SIFamide, sulfakinin and tachykinin-related peptide (TRP) families. Of particular note were the identifications of a carboxyl (C)-terminally extended corazonin, FLPs possessing -IMRFamide, -MMYFamide, and -MIHFamide C-termini, a NPF and a sulfakinin each ending in -RYamide rather than -RFamide, a precursor whose orcokinins include C-terminally amidated isoforms, and a collection of TRPs possessing -FXPXLamide rather than the stereotypical -FXGXLamide C-termini. The L. hesperus peptidome is by far the largest thus far published for any member of the Chelicerata. Taken collectively, these data serve as a reference for future neuropeptide discovery in the Araneae and provide a foundation for future studies of peptidergic control in L. hesperus and other spiders.

Topics: Amino Acid Sequence; Animals; Black Widow Spider; Computer Simulation; FMRFamide; Insect Hormones; Insect Proteins; Invertebrate Hormones; Molecular Sequence Data; Neuropeptides; Oligopeptides; Proteome; Transcriptome

Antisera against a variety of vertebrate and invertebrate neuropeptides were used to characterize neurons with somata in the pars intercerebralis (PI), pars lateralis (PL), and subesophageal ganglion (SEG), designated as PI neurons, PL neurons, and SEG neurons, respectively, all of which project to the retrocerebral complex in the blow fly, Protophormia terraenovae. Immunocytochemistry combined with backfills through the cardiac-recurrent nerve revealed that at least two pairs of PI and SEG neurons for each were FMRFamide-immunoreactive. Immunoreactivity against [Arg7]-corazonin, beta-pigment-dispersing hormone (beta-PDH), cholecystokinin8, or FMRFamide was observed in PL neurons. Immunoreactive colocalization of [Arg7]-corazonin with beta-PDH, [Arg7]-corazonin with cholecystokinin8, or beta-PDH with FMRFamide was found in two to three somata in the PL of a hemisphere. Based on their anatomical and immunocytochemical characteristics, PI neurons were classified into two types, PL neurons into six types, and SEG neurons into two types. Fibers in the retrocerebral complex showed [Arg7]-corazonin, beta-PDH, cholecystokinin8, and FMRFamide immunoreactivity. Cholecystokinin8 immunoreactivity was also detected in intrinsic cells of the corpus cardiacum. The corpus allatum was densely innervated by FMRFamide-immunoreactive varicose fibers. These results suggest that PI, PL, and SEG neurons release [Arg7]-corazonin, beta-PDH, cholecystokinin8, or FMRFamide-like peptides from the corpus cardiacum or corpus allatum into the hemolymph, and that some PL neurons may simultaneously release several neuropeptides.

Topics: Animals; Cholecystokinin; Diptera; Female; FMRFamide; Insect Hormones; Insect Proteins; Neurons; Neuropeptides

A peptidomics approach was applied to determine the peptides in the larval central nervous system of the grey flesh fly, Neobellieria bullata. Fractions obtained by high performance liquid chromatography were analysed by MALDI-TOF and ESI-Q-TOF mass spectrometry. This provided biochemical evidence for the presence of 18 neuropeptides, 11 of which were novel Neobellieria peptides. Most prominently present were the FMRFamide-related peptides: 7 FMRFamides, 1 FIRFamide, and Neb-myosuppressin. The three putative capa-gene products Neb-pyrokinin and the periviscerokinins Neb-PVK-1 and -2 were detected, as well as another pyrokinin. This Neb-PK-2 was also present in the ring gland along with corazonin, Neb-myosuppressin, and Neb-AKH-GK, an intermediate processing product of the adipokinetic hormone. Furthermore, the central nervous system contained Neb-LFamide, proctolin, and FDFHTVamide, designated as Neb-TVamide. With this study, we considerably increased our knowledge of the neuropeptidome of the pest fly N. bullata, which is an important insect model for physiological research.

Topics: Amino Acid Sequence; Animals; Central Nervous System; Chromatography, High Pressure Liquid; Diptera; FMRFamide; Insect Proteins; Larva; Mass Spectrometry; Molecular Sequence Data; Neuropeptides; Peptides; Receptors, Peptide; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Morphological and electrical properties of neurons with somata in the pars intercerebralis (PI) and pars lateralis (PL) were examined by intracellular recording and staining in the adult blow fly, Protophormia terraenovae. According to the location of somata and fiber distribution, two types of PI neurons (PIa and PIb) and two types of PL neurons (PLa and PLb) were identified. PIb neurons were further divided into two subgroups of PIb1 and PIb2 depending on fiber branching patterns in the retrocerebral complex. PIa neurons projected axons to the contralateral nervi corporis cardiaci, whereas PLa and PLb neurons projected axons to the ipsilateral nervi corporis cardiaci. PIb neurons characteristically showed symmetrical morphology with their somata along the midline. PLb neurons had a large branching area in the subesophageal ganglion. In the retrocerebral complex, PIb2 and PLa neurons sent fibers into the corpus allatum. PIa, PIb1 and PLb neurons projected not to the corpus allatum but to the corpus cardiacum-hypocerebral complex or visceral muscles in their vicinity. PIa, PIb and PLa neurons showed long spike durations (3-10 ms). PLb neurons were immunoreactive with antisera against corazonin, FMRFamide, or beta-pigment-dispersing hormone. This is the first report revealing the morphology of individual neurons with somata residing in PI and PL in the adult fly.

Topics: Animals; Biomarkers; Corpora Allata; Diptera; Female; FMRFamide; Insect Hormones; Insect Proteins; Membrane Potentials; Neural Pathways; Neurons; Neuropeptides; Peptides

Activation of G protein-coupled receptors (GPCR) leads to the recruitment of beta-arrestins. By tagging the beta-arrestin molecule with a green fluorescent protein, we can visualize the activation of GPCRs in living cells. We have used this approach to de-orphan and study 11 GPCRs for neuropeptide receptors in Drosophila melanogaster. Here we verify the identities of ligands for several recently de-orphaned receptors, including the receptors for the Drosophila neuropeptides proctolin (CG6986), neuropeptide F (CG1147), corazonin (CG10698), dFMRF-amide (CG2114), and allatostatin C (CG7285 and CG13702). We also de-orphan CG6515 and CG7887 by showing these two suspected tachykinin receptor family members respond specifically to a Drosophila tachykinin neuropeptide. Additionally, the translocation assay was used to de-orphan three Drosophila receptors. We show that CG14484, encoding a receptor related to vertebrate bombesin receptors, responds specifically to allatostatin B. Furthermore, the pair of paralogous receptors CG8985 and CG13803 responds specifically to the FMRF-amide-related peptide dromyosuppressin. To corroborate the findings on orphan receptors obtained by the translocation assay, we show that dromyosuppressin also stimulated GTPgammaS binding and inhibited cAMP by CG8985 and CG13803. Together these observations demonstrate the beta-arrestin-green fluorescent protein translocation assay is an important tool in the repertoire of strategies for ligand identification of novel G protein-coupled receptors.

Topics: Animals; Arrestins; beta-Arrestins; Cell Line; Cloning, Molecular; Cyclic AMP; Dose-Response Relationship, Drug; Drosophila; Drosophila Proteins; FMRFamide; Green Fluorescent Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Insect Hormones; Insect Proteins; Ligands; Luminescent Proteins; Microscopy, Confocal; Neuropeptides; Oligopeptides; Peptides; Protein Transport; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Receptors, Peptide; Receptors, Tachykinin; Transfection