Peptides (v.24, #10)

Invertebrate neuropeptides IV by R.J. Nachman (1455-1456).

We used a combination of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and immunocytochemistry to investigate the peptides from abdominal perisympathetic organs of Manduca sexta. Altogether three mass peaks, detected in mass spectra from single abdominal perisympathetic organs were identical with already known neuropeptides, namely CAP2b, CCAP, and Manduca-allatotropin. Only CAP2b was found throughout the postembryonic development. In larvae, perisympathetic organs of the abdominal ganglia 1 and 7 do not accumulate neuropeptides. During the metamorphosis, the number of putative hormones stored in the abdominal perisympathetic organs, increases dramatically. Not a single substance, however, obtained in mass spectra of larval perisympathetic organs disappeared in the respective adult neurohemal organs. Peptides from abdominal perisympathetic organs are different from those of thoracic perisympathetic organs and the retrocerebral complex. Manduca-FLRFa-2 and -3 are enriched in thoracic perisympathetic organs; FLRFa-1, corazonin and adipokinetic hormone are abundant peptides of the retrocerebral complex. The majority of ion signals, however, represent unknown substances. An antiserum which recognized CAP2b allowed the morphological characterization of a median neurosecretory system in the abdominal ventral nerve cord of M. sexta, which resembles that of cockroach embryos. Double stainings confirmed that crustacean cardioactive peptide (CCAP) becomes colocalized with CAP2b in median neurosecretory cells during the last larval instar. This colocalization continues in adult insects.
Keywords: Periviscerokinin; CCAP; CAP2b; Neuropeptides; Perisympathetic organ; Immunocytochemistry; Insect nervous system; Manduca sexta; MALDI-TOF mass spectrometry;

The occurrence of neuropeptides in the brain of larvae of the tobacco hawkmoth, Manduca sexta, and tomato moth, Lacanobia oleracea, was investigated using matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and post-source decay (PSD). Methanolic extracts of 100 brains separated by reversed-phase high performance liquid chromatography yielded numerous ion peaks, some of which were common to both species. In M. sexta six [M+H]+ ions were in agreement with peptides previously structurally characterised from M. sexta (FLRF-amides I, II and III, M. sexta allatostatin, CAP2b and myoinhibitory peptide VI), whereas a further five corresponded to other known lepidopteran peptides (cydiastatins 3 and 4, helicostatins 1 and 6 and helicokinin II). Of these the identities of FLRF-amide I, cydiastatins 3 and 4 and CAP2b were confirmed by PSD analysis. Fourteen [M+H]+ ions corresponding to known lepidopteran peptides (FLRF-amide I, cydiastatins 2, 3 and 4, helicostatins 1, 5, 6, 7 and 9, CCAP, CAP2b, M. sexta allatostatin and myoinhibitory peptide VI) were measured in L. oleracea brain extracts. From this insect, cydiastatins 3 and 4, helicostatin 5 and FLRF-amide I were identified by PSD. These peptides had not previously been structurally characterised from L. oleracea.
Keywords: Peptidomics; Insect; Lepidoptera; Allatostatin;

Peptide profiling of a single Locusta migratoria corpus cardiacum by nano-LC tandem mass spectrometry by G. Baggerman; E. Clynen; J. Huybrechts; P. Verleyen; S. Clerens; A. De Loof; L. Schoofs (1475-1485).
The pars intercerebralis–corpora cardiaca complex in insects is the functional equivalent of the vertebrate brain-pituitary axis. During the past few decades more than 40 neuropeptides have been isolated from the locust brain-corpus cardiacum complex. Tedious and time-consuming successive purification rounds of large tissue extracts were necessary to achieve the purification and sequencing of most of these signal molecules. Nowadays, the combination of nanoscale liquid chromatography and the very sensitive tandem mass spectrometry allows us to identify and sequence peptides in very low concentration directly from tissue extracts. In this manuscript, we review previous data on the peptidome analysis of the locust corpora cardiaca, with emphasis on AKH processing. In addition, we report the peptide profiling of a single corpus cardiacum from Locusta migratoria. 23 peptides were isolated and sequenced in a single nano-LC-MS/MS experiment, demonstrating the sensitivity and effectiveness of mass spectrometry in peptide research.
Keywords: Peptidomics; Insect; Q-TOF; Proteomics;

Mass spectrometric analysis of putative capa-gene products in Musca domestica and Neobellieria bullata by Reinhard Predel; William K Russell; Shane E Tichy; David H Russell; Ronald J Nachman (1487-1491).
Neuropeptides of the capa-gene are typical of the abdominal neurosecretory system of insects. In this study, we investigated these peptides in two widely distributed and large pest flies, namely Musca domestica and Neobellieria bullata. Using a combination of MALDI-TOF and ESI-QTOF mass spectrometry, periviscerokinins and a pyrokinin were analyzed from single perisympathetic organ preparations. The species-specific peptide sequences differ remarkably between the related dipteran species. These differences could make it possible to develop peptide-analogs with group- or species-specific efficacy.
Keywords: Musca domestica; Neobellieria bullata; CAP2b ; Periviscerokinin; Perisympathetic organs; Mass spectrometry; Insect neuropeptide;

Peptidomics of the locust corpora allata: identification of novel pyrokinins (-FXPRLamides) by E Clynen; G Baggerman; J Huybrechts; L Vanden Bosch; A De Loof; L Schoofs (1493-1500).
The peptidomes of the corpora allata of Locusta migratoria and Schistocerca gregaria were investigated by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nanoscale liquid chromatography quadrupole time-of-flight tandem mass spectrometry (nanoLC-Q-TOF MSMS). The pyrokinin (-FXPRLamide) family seems to be predominant. In addition to the known pyrokinins, we de novo sequenced four pyrokinins in L. migratoria and five in S. gregaria. In addition, one pyrokinin-like peptide (-PRLamide) was identified in S. gregaria. Besides the -(FX)PRLamides, FLRFamide-1, the allatostatins (A family) and numerous as yet unidentified peptides are also present in the corpora allata.
Keywords: Allatostatins; Endocrinology; Juvenile hormone; Locusta migratoria; MALDI-TOF; Mass spectrometry; Neuropeptides; Peptidome; Pyrokinins; Schistocerca gregaria;

The distribution of FMRFamide immunoreactivity in the brain-retrocerebral complex of adult female Diploptera punctata was examined. Immunoreactivity was observed in the brain and corpus allatum as well as in the corpus cardiacum. Immunoreactivity co-localized with allatostatin immunoreactivity within several lateral neurosecretory cells of the brain and in their endings within the corpus allatum. By in vitro radiochemical assay of juvenile hormone release, the effect of two native D. punctata RFamides, an FLRFamide (Leucomyosuppressin) and an FIRFamide were examined. The latter, for which the sequence (SKPANFIRFamide) is reported here, stimulated juvenile hormone release but acted only on corpora allata from females at the end of vitellogenesis (day 6). The interaction of these two RFamides and three D. punctata allatostatins, Dippu-AST 2, 5, and 7 were similarly examined. Only Dippu-AST 2 stimulated release of RFamides from the corpora allata and only on day 6 whereas both RFamides were able to attenuate the inhibitory activity of Dippu-AST 2.
Keywords: RFamides; Diploptera punctata; Corpora allata; Juvenile hormone; Allatostatins;

The gut tissues and associated nervous system of the African migratory locust, Locusta migratoria, were found to contain FMRFamide-like immunoreactive (FLI) material throughout the five larval instars and 2 weeks into the adult stage in both males and females. FMRFamide-like immunoreactivity associated with the locust gut was described using camera lucida techniques. FMRFamide-like immunoreactivity is observed in the frontal connectives, recurrent nerve, and oesophageal nerves; projections from the ingluvial ganglion onto the anterior midgut, and from the proctodeal nerve onto the hindgut and posterior midgut; in the neuropils of the frontal ganglion, hypocerebral ganglion and ingluvial ganglia; 30 cell bodies in the frontal ganglion; multipolar sensory cells on the foregut; and endocrine-like cells in the gastric caecae and midgut. Radioimmunoassay (RIA) was used to determine the quantities of FLI material in foreguts, gastric caecae, anterior and posterior midguts, and hindgut of first–fifth instar larvae, 1–3- and 14–17-day male and female adult locusts. As expected, as the tissue size (assessed by total protein content) increases, so does the amount of FLI material in each tissue. Normalizing for tissue size reveals significant differences in FLI content among the stages for each tissue tested. Reversed phase-high pressure liquid chromatography (RP-HPLC) followed by RIA has identified four groups of FLI fractions present in the gut, and different members of these groups are present in the various gut tissues.
Keywords: FMRFamides; Insect; Foregut; Midgut; Hindgut; Gastric caecae; RIA; HPLC; Immunohistochemistry; Camera lucida; Development;

Role of FMRFamide in the reproduction of Octopus vulgaris: molecular analysis and effect on visual input by Carlo Di Cristo; Pasquale Delli Bovi; Anna Di Cosmo (1525-1532).
As a part of continuous research on the neurobiology of the cephalopods in general, and the neuroendocrine control of reproduction in Octopus vulgaris in particular, the presence, the molecular analysis and the effect of FMRFamide on the screening-pigment migration in the visual system have been analysed. FMRFamide immunoreactive fibres are present in the outer plexiform layer of the retina as well as in the plexiform zone of the deep retina. These fibres presumably come from optic and olfactory lobes. We isolated an incomplete Octopus FMRFamide cDNA which encodes an amino terminal truncated precursor containing several FMRFamide-related peptides (FaRPs) showing a high degree of identity with the FaRPs encoded in the precursor of Sepia officinalis, except for the presence of an Rpamide related peptide, present only in cnidarians. Finally, stimulation of isolated retina demonstrated that the effect of this tetrapeptide, coupled with dopamine, is the induction of an extreme adaptation of the retina to the light condition. This situation de facto inhibits sexual maturation. Our results on the effect of FMRFamide on the retina confirm the suggested hypothesis that this peptide plays an inhibitory role on the activity of optic gland.
Keywords: Neuropeptides; Photoperiod; Cephalopods; Invertebrate neuroendocrinology; Reproduction; Octopus;

Distribution and function of an Aplysia cardioexcitatory peptide, NdWFamide, in pulmonate snails by F. Morishita; H. Minakata; K. Sasaki; K. Tada; Y. Furukawa; O. Matsushima; S.T. Mukai; A.S.M. Saleuddin (1533-1544).
The distribution and function of an Aplysia cardioexcitatory peptide, NdWFamide, were examined in the nervous system of pulmonate snails. We chemically identified the authentic NdWFamide from a land snail (Euhadra congenita) and a freshwater snail (Lymnaea stagnalis). NdWFamide potentiated the heartbeat of those snails. Immunohistochemistry using anti-NdWFamide antibody demonstrated the distribution of NdWFamide-containing neurons and fibers in the central nervous system, as well as peripheral tissues, such as the cardiovascular region and accessory sex organs. These results suggest that NdWFamide is a neuropeptide mediating the neural regulation of the activity of the cardiovascular and reproductive systems of snails.
Keywords: Neuropeptide; Purification; d-Amino acids; Immunohistochemistry; Lymnaea stagnalis;

Phosphatidylcholine and phosphatidylethanolamine are the major constituents of the phospholipid pool in cockroach (Periplaneta americana) fat body and hemolymph. Both species of phospholipid are significantly decreased 6 h after injecting hypertrehalosemic hormone I (HTH-I) into the hemocoel. Loss of phospholipid is accompanied by an accumulation of the phospholipid degradation products glycerophosphorylcholine and glycerol. HTH-I also increases phospholipase activity in the hemolymph and this is thought to be responsible for the depletion of hemolymph phospholipid. Phospholipase activity peaks approximately 2 h after injection of HTH-I and returns to normal at 6 h. In vitro, total phospholipid in the fat body is decreased by HTH-I whereas the concentration of diacylglycerol displays a corresponding increase. HTH-I elevates free fatty acid levels but has no effect on triacylglycerol. These effects of HTH-I are blocked by the phospholipase inhibitor mepacrine.
Keywords: Fat body; Fatty acids; Hemolymph; Hypertrehalosemic hormone; Lipid metabolism; Neuropeptide; Neutral lipid; Phospholipase A2; Phospholipid;

Using a polyclonal antiserum to Dippu-allatostatin 7 (Dippu-AST 7; formerly AST 1) of the cockroach Diploptera punctata, we have demonstrated the presence of AST-like immunoreactivity (ALI) in cells and processes throughout the nervous system, gut, and peripheral tissues of unfed fifth instar and adult Rhodnius prolixus. ALI in apparent neurosecretory cells of the brain, suboesophageal ganglion, and mesothoracic ganglionic mass, as well as in midgut endocrine cells, suggests that Rhodnius allatostatins may act as neurohormones/hormones. The presence of ALI in possible interneurons and areas of neuropile throughout the CNS also suggests roles as neuromodulators and/or neurotransmitters. Dippu-AST 7 inhibits spontaneous and leucokinin 1 (LK 1)-induced contractions of the Rhodnius hindgut in a dose-dependent manner. The low concentrations capable of inhibiting both spontaneous (10−12  M) and LK 1-induced contractions (10−10 to 10−9  M) suggest that ASTs may be acting as neurohormones/hormones on the hindgut. We have also shown that Dippu-AST 7 influences the muscle activity of the Rhodnius dorsal vessel at concentrations as low as 10−11  M.
Keywords: Insect; Neuropeptides; Allatostatins; Immunoreactivity; Hindgut; Dorsal vessel;

Immunolocalization of allatostatin-like neuropeptides and their putative receptor in eyestalks of the tiger prawn, Penaeus monodon by Nanthika Panchan; William G Bendena; Paul Bowser; Panida Lungchukiet; Stephen S Tobe; Weerawan Sithigorngul; Parin Chaivisuthangkura; Achariya Rangsiruji; Amorn Petsom; Thanit Pewnim; Paisarn Sithigorngul (1563-1570).
Allatostatin (AST)-like immunoreactivity (IR) was localized in the eyestalk of Penaeus monodon by immunohistochemistry using four anti-AST antibodies. Depending on the antisera, AST-like immunoreactivity was detected in neuronal bodies of the lamina ganglionalis, cell bodies anterior to the medulla externa and cell bodies on the anterior and posterior of the medulla terminalis. Neuronal processes in neuropiles of the medulla externa, medulla terminalis, sinus gland and nerve fibers in the optic nerve were also recognized. No IR in cell bodies or in nerve fibers was found in the medulla interna. Strong AST-like immunoreactivity was found in hundreds of cells of the X organ. The localization of AST-like peptides suggests that they function as neurotransmitters and/or neuromodulators. Antiserum to the Drosophila AST receptor (Dar-2) recognized a single protein in P. monodon eyestalk protein extracts that was identical in size to that found in Drosophila protein extracts. Using this antiserum the putative P. monodon AST receptor was localized to the sinus gland in both juvenile and adult eyestalks. To our knowledge this is the first demonstration of a neuropeptide receptor localized to the crustacean sinus gland. This suggests that ASTs may function directly on the sinus gland as a neuromodulator. In juvenile eyestalks, the putative AST receptor was also localized to neuronal X organ cells of the medulla terminalis in males but not in females. The significance of this sex-specific receptor localization is unclear but emphasizes that ASTs function within the nervous system of the eyestalk.
Keywords: Crustacean; Insect; Shrimp; Sinus gland; G-linked protein receptor;

In insects primary urine is produced by the Malpighian tubules under hormonal control. Here we have analysed the effects of the peptide locustatachykinin I (Lom-TK-I) on secretion in isolated Malphigian tubules. We also mapped the distribution of Lom-TK immunoreactivity in the gut in comparison with Locusta diuretic hormone (Lom-DH) and serotonin, two other factors that are active on locust tubules. Lom-TK-I produces an immediate, potent and long-lasting stimulation of fluid secretion. Furthermore, we show that Lom-TK-I acts synergistically with Lom-DH on fluid secretion and demonstrate that Lom-TKs are co-localised with Lom-DH in endocrine cells of the midgut ampullae. Thus, the two peptides might be released together to act synergistically on fluid secretion. Also serotonin and Lom-DH act synergistically and we can demonstrate a plexus of serotonin-containing axon processes over the midgut.
Keywords: Tachykinin-related peptide; Diuretic hormone; Serotonin; Insect hormones; Diuresis; Locust;

Corazonin promotes tegumentary pigment migration in the crayfish Procambarus clarkii by M.G Porras; A De Loof; M Breuer; H Aréchiga (1581-1589).
The undecapeptide corazonin (pGlu-Thr-Phe-Gln-Tyr-Ser-His-Gly-Trp-Thr-AsnNH2) elicits a retraction of erythrophore pigment granules and dispersion of leucophore pigment granules in the crayfish Procambarus clarkii. The effects are dose-dependent from 10−10 to 10−5  M. Influence on erythrophores is lower than that of Red Pigment Concentrating Hormone (RPCH), which is inactive on leucophores. Corazonin effects are partly blocked by an anti-corazonin antibody, and even less by an anti-RPCH antibody. Corazonin effects are completely suppressed by the calcium chelator BAPTA. Immunoreactive somata and fibers were identified in various regions of the eyestalk (medulla terminalis, medulla interna and medulla externa) with the anti-corazonin antibody. These results suggest the possible existence of a corazonin-like peptide in crustaceans.
Keywords: Adipokinetic Hormone; Corazonin; Crayfish; Crustaceans; Insects; Neuropetides; Neurosecretion; Pigments; Red Pigment Concentrating Hormone;

Farnesoic acid O-methyltransferase (FAMeT) catalyzes the conversion of farnesoic acid (FA) to methylfarnesoate (MF) by the mandibular organ (MO) of crustaceans. Here we report the cellular localization of FAMeT and radiochemical assay of endogenous FAMeT activity in shrimp (Metapenaeus ensis) and crayfish (Procambarus clarkii) tissues. As in the eyestalk (ES), FAMeT is concentrated in specific neurosecretory cells of the ventral nerve cord (VNC) whereas only weak FAMeT immunoreactivity was observed in the MO. FAMeT was also detected in the ventral nerve cord, heart (HET), eyestalk, and muscle of the juvenile shrimp. Although the VNC shows the greatest FAMeT immunoreactivity, the heart extract exhibited the highest FAMeT enzymatic activity. These results suggest that FAMeT in the VNC may be inactive or inactivated at the stages of development tested. Contrary to the previous reports in other crustaceans, MO extract in shrimp shows only low FAMeT activity. The eyestalk, epidermis, ovary and testis show appreciable FAMeT activity. The presence of FAMeT in neurosecretory cells of VNC and eyestalk of shrimp and crayfish implies a possible interaction of FAMeT with the eyestalk CHH-family of neuropeptides. The widespread activity of FAMeT suggests that it has a wide spectrum of action in many tissues that contribute to the function and regulation of MF synthesis in shrimp and crayfish.
Keywords: Shrimp; Farnesoic acid O-methyltransferase; Methyl farnesoate;

A nonpeptide provides insight into mechanisms that regulate Drosophila melanogaster heart contractions by Melissa Mispelon; Kiran Thakur; Leslie Chinn; Ryan Owen; Ruthann Nichols (1599-1605).
Here we report the effect of a nonpeptide, benzethonium chloride (bztc), on Drosophila melanogaster larval, pupal, and adult heart rates in vivo. Benzethonium chloride reduced the frequency of spontaneous contractions in the D. melanogaster pupal heart, but not in the larval heart or the adult heart as measured in noninvasive whole animal preparations. When applied directly to the D. melanogaster heart, in the absence of hemolymph, bztc reduced the frequency of spontaneous contractions in larval, pupal, and adult hearts. These findings are consistent with the conclusion that bztc acts through or is regulated by different mechanisms in these three developmental stages. An alternative explanation is that larval hemolymph and adult hemolymph contain a material that interferes with the effect of the nonpeptide on heart contractions. Bztc mimicked the effect of the peptide dromyosuppressin (DMS) on the heart at an equivalent concentration; in contrast, 103-fold more nonpeptide is required to mimic the effect of DMS on fly gut. These findings are consistent with the presence of tissue-specific myosuppressin receptors or mechanisms.
Keywords: Benzethonium chloride; Brain-gut peptide; Dromyosuppressin; Heart;

The synthesis of an analogue of the locust CRF-like diuretic peptide, and the biological activities of this and some C-terminal fragments by Graham J Goldsworthy; J.Sook Chung; Monique S.J Simmonds; Maria Tatari; Sophia Varouni; Constantine P Poulos (1607-1613).
The synthesis is described of an analogue of the locust CRF-like diuretic peptide in which methionine in positions 1,3, and 13 is replaced by isosteric methyl-homoserine residues. This analogue has been tested for biological activity on Malpighian tubules in vitro, and feeding behavior in vivo. It is highly active in stimulating fluid secretion and accumulation of cAMP in tubules, and on increasing the latency to feed and reducing meal duration. A 15 residue fragment from the C-terminus of the CRF-like peptide, Locmi-DP32–46, is fully active in the feeding assay, but has only weak ability to stimulate the accumulation of cAMP in tubules. Two smaller fragments, Locmi-DP32–37 and Locmi-DP41–46, were tested but neither had consistent biological activity in any of the assays used here. None of the peptides tested have any substantive activity in increasing cGMP in tubules.
Keywords: Locust; Diuretic hormone; CRF-like peptide; cAMP; Feeding behavior; Fluid secretion; Malpighian tubules; Methionine replacement;

A C-terminal aldehyde insect kinin analog enhances inhibition of weight gain and induces significant mortality in Helicoverpa zea larvae by Ronald J. Nachman; Geoffrey M. Coast; Céline Douat; Jean-Alain Fehrentz; Krzyztoff Kaczmarek; Janusz Zabrocki; Nan W. Pryor; Jean Martinez (1615-1621).
The first reported examples of C-terminal aldehyde analogs of an insect neuropeptide are described. They are hexapeptide insect kinin analogs Boc-VFFPWG-H and Fmoc-RFFPWG-H. Activity observed for these modified analogs in an in vitro insect diuretic assay confirms that the C-terminal aldehyde group is tolerated by an insect kinin receptor. The two analogs demonstrate greatly enhanced activity over standard C-terminal amide insect kinins in a larval weight gain inhibition assay in the corn earworm Helicoverpa zea. Treatment with Boc-VFFPWG-H led to significant increases in larval mortality at doses of 500 pm (45%) and 5 nm (67%). Boc-VFFPWG-H represents a lead analog in the development of novel, environmentally friendly pest insect management agents based on the insect kinin class of neuropeptides.
Keywords: Insect neuropeptide; Corn earworm; Cricket; Diuresis; Pest management;

Originally named for its ability to stimulate juvenile hormone production by lepidopteran corpora allata, allatotropin has emerged as a neuropeptide with multiple neural, endocrine and myoactive roles. This paper describes the experimental evidence for allatotropin action, its localization in several species of insects, and its multiple effects on a variety of different tissues that lead to increased hemolymph circulation and gut motility. The overall physiological effects may also include species-specific effects such as the regulation of nutrient absorption, modulation of the circadian cycle and migratory preparedness. In addition, we present evidence suggesting that allatotropins are members of a family of myoactive peptides found in several invertebrate phyla. Finally, we speculate that the myoactive properties of allatotropins are basal and it is likely that the stimulatory action of allatotropins on juvenile hormone synthesis evolved secondarily.
Keywords: Allatotropin; Juvenile hormone; Evolution; Neuropeptides; Invertebrate; Insect;

Genomics, evolution and biological functions of the pacifastin peptide family: a conserved serine protease inhibitor family in arthropods by Gert Simonet; Ilse Claeys; Vanessa Franssens; Arnold De Loof; Jozef Vanden Broeck (1633-1644).
The last decade, a new serine protease inhibitor family has been described in arthropods. Eight members were purified from the locusts Locusta migratoria (LMPI-1-2 and HI) and Schistocerca gregaria (SGPI-1-5). The light chain of the heterodimeric protease inhibitor pacifastin, from the freshwater crayfish Pacifastacus leniusculus, was found to be composed of nine consecutive inhibitory domains (PLDs). These domains share a pattern of six conserved cysteine residues (Cys-Xaa9–12-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa2–3-Gly-Xaa3–6-Cys-Thr-Xaa3-Cys) with the locust inhibitors. Via cDNA cloning, eight pacifastin-related precursors have been identified in locusts. Interestingly, additional pacifastin-related precursors have been identified in Diptera, Lepidoptera and Coleoptera utilising an in silico data mining approach.
Keywords: Insect; Trypsin; Phenoloxidase; Innate immunity; Phase polymorphism;