allatostatin-1 and proctolin

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

The genome of the spider mite was prospected for the presence of genes coding neuropeptides, neurohormones and their putative G-protein coupled receptors. Fifty one candidate genes were found to encode neuropeptides or neurohormones. These include all known insect neuropeptides and neurohormones, with the exception of sulfakinin, corazonin, neuroparsin and PTTH. True orthologs of adipokinetic hormone (AKH) were neither found, but there are three genes encoding peptides similar in structure to both AKH and the AKH-corazonin-related peptide. We were also unable to identify the precursors for pigment dispersing factor (PDF) or the recently discovered trissin. However, the spider mite probably does have such genes, as we found their putative receptors. A novel arthropod neuropeptide gene was identified that shows similarity to previously described molluscan neuropeptide genes and was called EFLamide. A total of 65 putative neuropeptide GPCR genes were also identified, of these 58 belong to the A-family and 7 to the B-family. Phylogenetic analysis showed that 50 of them are closely related to insect GPCRs, which allowed the identification of their putative ligand in 39 cases with varying degrees of certainty. Other spider mite GPCRs however have no identifiable orthologs in the genomes of the four holometabolous insect species best analyzed. Whereas some of the latter have orthologs in hemimetabolous insect species, crustaceans or ticks, for others such arthropod homologs are currently unknown.

Topics: Amino Acid Sequence; Animals; Arthropod Proteins; Insect Hormones; Insulins; Invertebrate Hormones; Molecular Sequence Data; Nerve Tissue Proteins; Neuropeptides; Neurotransmitter Agents; Oligopeptides; Receptors, G-Protein-Coupled; Tetranychidae

Genome mining has provided a valuable tool for peptide discovery in many species, yet no crustacean has undergone this analysis. Currently, the only crustacean with a sequenced genome is the cladoceran Daphnia pulex, a model organism in many fields of biology. Here, we have mined the D. pulex genome for peptide-encoding genes. For each gene identified, the encoded precursor protein was deduced, and its mature peptides predicted. Twenty-four peptide-encoding genes were identified, including ones predicted to produce members of the A-type allatostatin, B-type allatostatin, C-type allatostatin, allatotropin (ATR), bursicon α, bursicon β, calcitonin-like diuretic hormone, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone, ecdysis-triggering hormone, eclosion hormone (EH), insulin-like peptide (ILP), molt-inhibiting hormone, neuropeptide F, orcokinin (two genes), pigment-dispersing hormone, proctolin, red pigment concentrating hormone/adipokinetic hormone (RPCH/AKH), short neuropeptide F, SIFamide, sulfakinin, and tachykinin-related peptide (TRP) families/subfamilies. In total, 96 peptides were predicted from these genes. Our identification of isoforms of corazonin, EH, ILP, proctolin, RPCH/AKH, sulfakinin and TRP are the first for D. pulex, while our prediction of ATR from this species is the first from any crustacean. The number of peptides predicted in our study shows the power of genome mining for peptide discovery, and provides a model for future genomic analyses of the peptidomes of other crustaceans. In addition, the data presented in our study provide foundations for future molecular, biochemical, anatomical, and physiological investigation of peptidergic signaling in D. pulex and other cladoceran species.

Topics: Animals; Daphnia; Genome; Invertebrate Hormones; Neuropeptides; Oligopeptides

Here we describe the neuronal organization of the arcuate body in the brain of the wandering spider Cupiennius salei. The internal anatomy of this major brain center is analyzed in detail based on allatostatin-, proctolin-, and crustacean cardioactive peptide (CCAP)-immunohistochemistry. Prominent neuronal features are demonstrated in graphic reconstructions. The stainings revealed that the neuroarchitecture of the arcuate body is characterized by several distinct layers some of which comprise nerve terminals that are organized in columnar, palisade-like arrays. The anatomy of the spider's arcuate body exhibits similarities as well as differences when compared to the central complex in the protocerebrum of the Tetraconata. Arguments for and against a possible homology of the arcuate body of the Chelicerata and the central complex of the Tetraconata and their consequences for the understanding of arthropod brain evolution are discussed.

Topics: Animals; Biological Evolution; Brain; Calcitonin; Gene Expression Regulation; Immunohistochemistry; Neuropeptides; Oligopeptides; Peptide Fragments; Spiders; Staining and Labeling

The Lottia gigantea genome was prospected for the presence of genes coding neuropeptides and neurohormones. Four genes code insulin-related peptides: two genes code molluscan insulin-like growth hormones, one gene an insulin very similar to vertebrate insulin, and the fourth a peptide related to drosophila insulin-like peptide 7. Four other genes encode the cysteine-knot proteins GPA2/GPB5 and bursicon/parabursicon. Another 37 genes code for precursors of the following neuropeptides: achatin, APGWamide, allatostatin C, allatotropin, buccalin (perhaps an allatostatin A homolog), cerebrin, CCAP, conopressin, elevenin (the predicted neuropeptide made by abdominal neuron 11 in Aplysia), egg laying hormone (two genes), enterin, feeding circuit activating neuropeptide (FCAP), FFamide, FMRFamide, GGNG, a GnRH-like peptide, the newly discovered LASGLVamide, LFRFamide, LFRYamide, LRNFVamide, luqin, lymnokinin, myomodulin (two genes), the newly discovered NKY, NPY, pedal peptide (three genes), PKYMDT, pleurin, PXFVamide, small cardioactive peptides, tachykinins (two genes) and WWamide (an allatostatin B homolog). One gene was found to encode FWISamide, while about 20 closely related genes were found to encode WWFamide. These small neuropeptides appear homologous to the NdWFamide, which contains d-Trp; these genes are similar to the Aplysia gene encoding NWFamide. Some of these peptides had not been previously identified from mollusks, such as the predicted hormones similar to Drosophila and vertebrate insulins, bursicon, the putative proctolin homolog PKYMDT and allatostatin C. Together with neuropeptides which are likely homologs of other insect neuropeptides, such as cerebrin and WWamide, this shows that despite significant differences the molluscan and arthropod neuropeptidomes are more similar than generally recognized.

Topics: Amino Acid Sequence; Animals; Genome; Insect Hormones; Insecta; Insulin; Invertebrate Hormones; Molecular Sequence Data; Mollusca; Neuropeptides; Neurotransmitter Agents; Oligopeptides; Sequence Homology, Amino Acid

In earwigs, the male reproductive system is complex, comprising accessory glands and long dual intromittent organs for transfer of materials to the female and for removal of rival sperm. We investigated potential factors altering contractions of the male reproductive tracts in vitro. Tracts from 0-day (newly emerged) males displayed relatively little motility in vitro; however, those from 5-day (intermediate stage of sexual maturity) and 8-day (fully mature) males pulsed vigorously. Both 1 and 100 nM proctolin (RYLPT-OH) stimulated the rate of contraction of reproductive tracts from both 5-day and 8-day males. In contrast, 1 nM and 100 nM FGLa AST (cockroach allatostatin) did not affect pulsations. However, 10 microM FGLa AST decreased activity of reproductive tracts. Mating decreased motility of tracts from 5-day old males, but did not alter motility of tracts from 8-day old males. Castration of larvae significantly suppressed reproductive tract motility in subsequent 8-day old adults compared with those of intact or sham-operated adults. Castration also suppressed seminal vesicle size. Lastly, we assessed the presence and distribution of proctolin-like and allatostatin-like immunoreactivity in tissues. Immunoreactivity to FGLa AST and proctolin was widespread, occurring in the brain and ventral ganglia. Surprisingly, we did not detect immunoreactivity to either FGLa AST or proctolin within the reproductive system; however, proctolin immunoreactivity was evident in nerves extending from the terminal ganglion of 8-day, but not 0-day, males. Collectively, these experiments demonstrate that the male earwig reproductive system is an appropriate model for use in addressing sexual maturation and activities in male insects.

Topics: Animals; Castration; Central Nervous System; Cockroaches; Female; Ganglia; Immunohistochemistry; Insecta; Male; Microscopy, Confocal; Movement; Neuropeptides; Oligopeptides; Sexual Behavior, Animal; Urogenital System

The Aphidoidea is an insect superfamily comprising most of the known aphid species. While small in size, these animals are of considerable economic importance as many members of this taxon are serious agricultural pests, inflicting physical damage upon crop plants and serving as vectors in the transmission of viral plant diseases. In terms of identifying the paracrines/hormones used to modulate behavior, particularly peptides, members of the Aphidoidea have largely been ignored, as it is not tractable to isolate the large pools of tissue needed for standard biochemical investigations. Here, a bioinformatics approach to peptide discovery has been used to overcome this limitation of scale. Specifically, in silico searches of publicly accessible aphidoidean ESTs were conducted to identify transcripts encoding putative peptides precursors, with the mature peptides contained within them deduced using peptide processing software and homology to known arthropod sequences. In total, 39 ESTs encoding putative peptides precursors were identified from four aphid species: Acyrthosiphon pisum (14 ESTs), Aphis gossypii (four ESTs), Myzus persicae (20 ESTs) and Toxoptera citricida (one EST). These precursors included ones predicted to encode isoforms of B-type allatostatin, crustacean cardioactive peptide, FMRFamide-related peptide (both myosuppressin and short neuropeptide F subfamilies), insect kinin, orcokinin, proctolin, pyrokinin/periviscerokinin/pheromone biosynthesis activating neuropeptide, SIFamide and tachykinin-related peptide. In total, 83 peptides were characterized from the identified precursors, most novel, including two B-type allatostatins possessing the variant -WX(7)Wamide motif, two N-terminally extended proctolin isoforms and an N-terminally truncated and substituted SIFamide. Collectively, these results expand greatly the number of known/predicted aphid peptide paracrines/hormones, and provide a strong foundation for future molecular and physiological investigations of peptidergic control in this insect group.

Topics: Amino Acid Sequence; Animals; Aphids; Computational Biology; Expressed Sequence Tags; FMRFamide; Insect Hormones; Molecular Sequence Data; Neuropeptides; Oligopeptides; Peptide Hormones; Sequence Homology, Amino Acid; Tachykinins

Dippu-allatostatins (ASTs) have pleiotropic effects in Locusta migratoria. Dippu-ASTs act as releasing factors for adipokinetic hormone I (AKH I) from the corpus cardiacum (CC) and also alter juvenile hormone (JH) biosynthesis and release from the corpus allatum (CA). Dippu-AST-like immunoreactivity is found within lateral neurosecretory cells (LNCs) of the brain and axons within the paired nervi corporis cardiaci II (NCC II) to the CC and the CA, where there are extensive processes and nerve endings over both of these neuroendocrine organs. There was co-localization of Dippu-AST-like and proctolin-like immunoreactivity within these regions. Dippu-ASTs increase the release of AKH I in a dose-dependent manner, with thresholds below 10(-11)M (Dippu-AST 7) and between 10(-13) and 10(-12)M (Dippu-AST 2). Both proctolin and Dippu-AST 2 caused an increase in the cAMP content of the glandular lobe of the CC. Dippu-AST 2 also altered the release of JH from the locust CA, but this effect depended on the concentration of peptide and the basal release rates of the CA. These physiological effects for Dippu-ASTs in Locusta have not been shown previously.

Topics: Animals; Brain; Corpora Allata; Cyclic AMP; Female; Immunohistochemistry; Insect Hormones; Juvenile Hormones; Locusta migratoria; Neuropeptides; Oligopeptides; Pyrrolidonecarboxylic Acid

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

The pericardial organs (POs) are a pair of neurosecretory organs that surround the crustacean heart and release neuromodulators into the hemolymph. In adult crustaceans, the POs are known to contain a wide array of peptide and amine modulators. However, little is known about the modulatory content of POs early in development. We characterize the morphology and modulatory content of pericardial organs in the embryonic lobster, Homarus americanus. The POs are well developed by midway through embryonic (E50) life and contain a wide array of neuromodulatory substances. Immunoreactivities to orcokinin, extended FLRFamide peptides, tyrosine hydroxylase, proctolin, allatostatin, serotonin, Cancer borealis tachykinin-related peptide, cholecystokinin, and crustacean cardioactive peptide are present in the POs by approximately midway through embryonic life. There are two classes of projection patterns to the POs. Immunoreactivities to orcokinin, extended FLRFamide peptides, and tyrosine hydroxylase project solely from the subesophageal ganglion (SEG), whereas the remaining modulators project from the SEG as well as from the thoracic ganglia. Double-labeling experiments with a subset of modulators did not reveal any colocalized peptides in the POs. These results suggest that the POs could be a major source of neuromodulators early in development.

Topics: Animals; Heart; Nephropidae; Nervous System; Neural Pathways; Neuropeptides; Neurosecretory Systems; Neurotransmitter Agents; Oligopeptides; Serotonin; Tyrosine 3-Monooxygenase

Diploptera punctata allatostatins are brain neuropeptides that inhibit juvenile hormone synthesis by corpora allata. They also occur in nerves of many organs other than corpora allata. The distribution of allatostatins in, and the effect of allatostatins on two other organs, antennal pulsatile organ and hindgut, are demonstrated here. Allatostatin I-like immunoreactive material is present in cells of subesophageal and terminal abdominal ganglia; these ganglia are known to contain the cells that project to antennal heart nerve and proctodeal nerve, respectively. Electron micrographs of both organs show nerve terminals with allatostatic immunoreactive granules along with terminals containing nonimmunoreactive granules. Immunoreactive neurosecretory granules are about 200 nm in largest dimension. In the antennal pulsatile organ, profiles of the nerve terminals are larger in the ampullar wall than in the muscle; in hindgut the terminals with immunoreactive granules are associated with the muscle net below the circular muscle. Hindgut responded to allatostatins I and IV with a dose-dependent decrease in amplitude and frequency of contraction that was reversible, with the threshold concentration for response between 10(-8) and 10(-7) M. In contrast, pulsatile organ muscle showed no such change with either allatostatin at 10(-7)-10(-4) M. However, both organs responded to proctolin with increased amplitude and frequency of contractions. Allatostatins I and IV inhibited the proctolin-induced increase of hindgut contraction, whereas no such effect was seen in antennal pulsatile organ muscle. Extract of antennal pulsatile organ muscle showed proctolin-like bioactivity that comigrated with authentic proctolin on three sequential HPLC systems.

Topics: Animals; Cockroaches; Digestive System; Digestive System Physiological Phenomena; In Vitro Techniques; Juvenile Hormones; Microscopy, Electron; Muscle Contraction; Muscle, Smooth; Nervous System; Nervous System Physiological Phenomena; Neuropeptides; Neurotransmitter Agents; Oligopeptides

The antennal heart of Periplaneta americana, a small accessory circulatory pump in the head, shows a rhythmicity with myogenic automatism. The muscle fibres extending throughout of the dilator muscle are electrically coupled. Among the peptides, proctolin causes a dose-dependent strong excitation, but allatostatin does not affect heart rhythm. However, allatostatin applied before proctolin antagonized the proctolin effect. In contrast to this, an immediate heart block produced by octopamine is similar to that produced by electrical stimulation of the nervus cardioantennalis. This inhibition is caused by a K(+)-dependent hyperpolarization. The second effect of octopamine is a delayed increase of the cAMP level. Because octopamine is present at the antennal heart, a physiological role is assumed.

Topics: Action Potentials; Animals; Cyclic AMP; Electric Stimulation; Heart; Heart Rate; Neuropeptides; Neurotransmitter Agents; Octopamine; Oligopeptides; Periplaneta; Potassium