The neuropeptidome of Rhodnius prolixus brain

We show a sensitive and straightforward off‐line nano‐LC‐MALDI‐MS/MS workflow that allowed the first comprehensive neuropeptidomic analysis of an insect disease vector. This approach was applied to identify neuropeptides in the brain of Rhodnius prolixus, a vector of Chagas disease. This work will contribute to the annotation of genes in the ongoing R. prolixus genome sequence project. Peptides were identified by de novo sequencing and comparisons to known neuropeptides from different organisms by database search. By these means, we were able to identify 42 novel neuropeptides from R. prolixus. The peptides were classified as extended FMRF‐amide‐related peptides, sulfakinins, myosuppressins, short neuropeptide F, long neuropeptide F, SIF‐amide‐related peptides, tachykinins, orcokinins, allatostatins, allatotropins, calcitonin‐like diuretic hormones, corazonin, and pyrokinin. Some of them were detected in multiple isoforms and/or truncated fragments. Interestingly, some of the R. prolixus peptides, as myosuppressin and sulfakinins, are unique in their characteristic C‐terminal domain among insect neuropeptides identified so far.     

Insect peptide hormones, an overview of the present literature.

A comprehensive overview of the recent state of the art of insect peptide hormones with chemical structures is presented. An increased interest in insect neuropeptides and dynamic development of that research area has been influenced by a rapid improvement of instrumentation necessary for isolation and structural characterization. Several research teams have studied the relationships between biological properties of insect and vertebrate peptide hormones. Thus hormones from the AKH family can be considered glucagon counterparts, whereas the myotropic hormones such as proctolin and Lem-PK (LPK) are a substance P equivalent. Insect melanization hormones Bom-MRCH in their structural characteristics and properties resemble those of mammal MSH, and leucosulfakinins Lem-SK-I and -II show some similarities with gastrin II and cholecystokinin. Bombyxin-II (Bom-PTTH-II) reveals a structural homology with human insulin and similar biological properties to adenocorticotropic mammal hormone. Allatostatin (Dip-JHS-I) may be compared to somatostatin as it can be inferred from the observations that this peptide modulates JH secretion in cockroach, Blattella germanica. Determination of the primary structure of eclosion hormones Mas-EH and Bom-EH-II as well as the amino acid sequence of allatotropin and allatostatin is a significant contribution to the understanding of the molecular mechanisms of metamorphosis and insect development.     

Molecular cloning, genomic organization, and expression of a C-type (Manduca sexta-type) allatostatin preprohormone from Drosophila melanogaster

The insect allatostatins are a diverse group of neuropeptides that obtained their names by their inhibitory actions on the corpora allata (two endocrine glands near the insect brain), where they block the biosynthesis of juvenile hormone (a terpenoid important for development and reproduction). Chemically, the allatostatins can be subdivided into three different peptide groups: the large group of A-type (cockroach-type) allatostatins, which have the common C-terminal sequence Y/FXFGLamide; the B-type (cricket-type) allatostatins, which have the C-terminal sequence W(X(6))Wamide in common; and a single allatostatin that we now call C-type allatostatin that was first discovered in the moth Manduca sexta, and which has a nonamidated C terminus, and a structure unrelated to the A- and B-type allatostatins. We have previously cloned the preprohormones for the A- and B-type allatostatins from Drosophila melanogaster. Here we report on the cloning of a Drosophila C-type allatostatin preprohormone (DAP-C). DAP-C is 121 amino acid residues long and contains one copy of a peptide sequence that in its processed form has the sequence Y in position 4) from the Manduca sexta C-type allatostatin. The DAP-C gene has three introns and four exons and is located at position 32D2-3 on the left arm of the second chromosome. Northern blots show that the gene is strongly expressed in larvae and adult flies, but less in pupae and embryos. In situ hybridizations of larvae show that the gene is expressed in various neurons of the brain and abdominal ganglia and in endocrine cells of the midgut. This is the first publication on the structure of a C-type allatostatin from insects other than moths and the first report on the presence of all three types of allatostatins in a representative of the insect order Diptera (flies).     

Myoinhibitory neuropeptides in the American cockroach

A large number of myostimulatory neuropeptides from neurohaemal organs of the American cockroach have been described since 1989. These peptides, isolated from the retrocerebral complex and abdominal perisympathetic organs, are thought to be released as hormones. To study the coordinated action of these neuropeptides in the regulation of visceral muscle activity, it might be necessary to include myoinhibitors as well, however, not a single myoinhibitory neuropeptide of the American cockroach has been described so far. To fill this gap, we describe the isolation of LMS (leucomyosuppressin) and Pea-MIP (myoinhibitory peptide) from neurohaemal organs of the American cockroach. LMS was very effective in inhibiting phasic activity of all visceral muscles tested. It was found in the corpora cardiaca of different species of cockroaches, as well as in related insect groups, including mantids and termites. Pea-MIP which is strongly accumulated in the corpora cardiaca was not detected with a muscle bioassay system but when searching for tryptophane-containing peptides using a diode-array detector. This peptide caused only a moderate inhibition in visceral muscle assays. The distribution of Pea-MIP in neurohaemal organs and cells supplying these organs with Pea-MIP immunoreactive material, is described. Additionally to LMS and Pea-MIP, a member of the allatostatin peptide family, known to exhibit inhibitory properties in other insects, was tested in visceral muscle assays. This allatostatin was highly effective in inhibiting spontaneous activity of the foregut, but not of other tested visceral muscles of the American cockroach.     

Allatostatin C and its paralog allatostatin double C: The arthropod somatostatins

Arthropods do not have one, but two genes encoding an allatostatin C-like peptide. The newly discovered paralog gene was called Ast-CC, and the peptide which it is predicted to make was called allatostatin double C (ASTCC). Genes for both allatostatin C (ASTC) and its paralog were found in the tick Ixodes scapularis as well as dipteran, lepidopteran, coleopteran, aphidoidean and phthirapteran insect species. In addition partial or complete cDNAs derived from Ast-CCs were found in a number of species, including Drosophila melanogaster, Bombyx mori and Rhodnius prolixus. The ASTCC precursors have a second conserved peptide sequence suggesting that they may produce two biologically active peptides. The predicted precursors encoded by the Ast-CCs have some unusual features, particularly in Drosophila, where they lack a signal peptide, and have instead a peptide anchor. These unusual structural features suggest that they are perhaps expressed by cells that are not specialized in neuropeptide synthesis and that in Drosophila ASTCC may be a juxtacrine. Data from the Fly Atlas project show that in Drosophila Ast-CC is little expressed. Nevertheless a P-element insertion in this gene is embryonic lethal, suggesting that it is an essential gene. Similarity between the precursors and receptors of ASTC/ASTCC and somatostatin suggests that ASTC/ASTCC and somatostatin have a common ancestor.     

Odorant receptor phylogeny confirms conserved channels for sex pheromone and host plant signals in tortricid moths

The search for mates and food is mediated by volatile chemicals. Insects sense food odorants and sex pheromones through odorant receptors (ORs) and pheromone receptors (PRs), which are expressed in olfactory sensory neurons. Molecular phylogenetics of ORs, informed by behavioral and functional data, generates sound hypotheses for the identification of semiochemicals driving olfactory behavior. Studying orthologous receptors and their ligands across taxa affords insights into the role of chemical communication in reproductive isolation and phylogenetic divergence. The female sex pheromone of green budworm moth Hedya nubiferana (Lepidoptera, Totricidae) is a blend of two unsaturated acetates, only a blend of both elicits male attraction. Females produce in addition codlemone, which is the sex pheromone of another tortricid, codling moth Cydia pomonella. Codlemone also attracts green budworm moth males. Concomitantly, green budworm and codling moth males are attracted to the host plant volatile pear ester. A congruent behavioral response to the same pheromone and plant volatile in two tortricid species suggests co‐occurrence of dedicated olfactory channels. In codling moth, one PR is tuned to both compounds, the sex pheromone codlemone and the plant volatile pear ester. Our phylogenetic analysis finds that green budworm moth expresses an orthologous PR gene. Shared ancestry, and high levels of amino acid identity and sequence similarity, in codling and green budworm moth PRs offer an explanation for parallel attraction of both species to the same compounds. A conserved olfactory channel for a sex pheromone and a host plant volatile substantiates the alliance of social and habitat signals in insect chemical communication. Field attraction assays confirm that in silico investigations of ORs afford powerful predictions for an efficient identification of behavior‐modifying semiochemicals, for an improved understanding of the mechanisms of host plant attraction in insect herbivores and for the further development of sustainable insect control.     

Peptidomics of the Agriculturally Damaging Larval Stage of the Cabbage Root Fly Delia radicum (Diptera: Anthomyiidae)

The larvae of the cabbage root fly induce serious damage to cultivated crops of the family Brassicaceae. We here report the biochemical characterisation of neuropeptides from the central nervous system and neurohemal organs, as well as regulatory peptides from enteroendocrine midgut cells of the cabbage maggot. By LC-MALDI-TOF/TOF and chemical labelling with 4-sulfophenyl isothiocyanate, 38 peptides could be identified, representing major insect peptide families: allatostatin A, allatostatin C, FMRFamide-like peptides, kinin, CAPA peptides, pyrokinins, sNPF, myosuppressin, corazonin, SIFamide, sulfakinins, tachykinins, NPLP1-peptides, adipokinetic hormone and CCHamide 1. We also report a new peptide (Yamide) which appears to be homolog to an amidated eclosion hormone-associated peptide in several Drosophila species. Immunocytochemical characterisation of the distribution of several classes of peptide-immunoreactive neurons and enteroendocrine cells shows a very similar but not identical peptide distribution to Drosophila. Since peptides regulate many vital physiological and behavioural processes such as moulting or feeding, our data may initiate the pharmacological testing and development of new specific peptide-based protection methods against the cabbage root fly and its larva.     

Rapid assessment of the sex of codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) eggs and larvae

Two different methods were tested to identify the sex of the early developmental stages of the codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) with a WZ/ZZ (female/male) sex chromosome system. First, it was shown that the sex of all larval stages can be easily determined by the presence or absence of sex chromatin, which is formed by the female‐specific W chromosome in interphase nuclei. This trait can also be used to identify the sex of newly hatched larvae but it does require care and accuracy. Secondly, a new sexing technique was developed based on a molecular marker of the codling moth W chromosome. Flanking regions of an earlier described W‐specific sequence (CpW2) were isolated and sequenced and a 2.74 kb sequence (CpW2‐EcoRI), specific for the W chromosome, was obtained. Several PCR tests were conducted, which confirmed that the CpW2‐EcoRI sequence is a reliable marker for the sex identification in codling moth samples of different geographical origin. In addition, a fragment of a codling moth gene, period (Cpper) was isolated and sequenced. Results of southern hybridization of the Cpper probe with female and male genomic DNA suggested that the Cpper gene is located on the Z chromosome. Then a multiplex PCR assay was developed, which co‐amplified the CpW2‐EcoRI sequence to identify the W chromosome and the Z‐linked Cpper sequence, which served as a positive control of accurate processing of tested samples. The multiplex PCR provides an easy and rapid identification of the sex of embryos and early larval instars of the codling moth.     

Mass spectrometric characterization and physiological actions of novel crustacean C-type allatostatins

The crustacean stomatogastric ganglion (STG) is modulated by numerous neuropeptides that are released locally in the neuropil or that reach the STG as neurohormones. Using 1,5-diaminonaphthalene (DAN) as a reductive screening matrix for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric profiling of disulfide bond-containing C-type allatostatin peptides followed by electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) tandem mass spectrometric (MS/MS) analysis, we identified and sequenced a novel C-type allatostatin peptide (CbAST-C1), pQIRYHQCYFNPISCF-COOH, present in the pericardial organs of the crab, Cancer borealis. Another C-type allatostatin (CbAST-C2), SYWKQCAFNAVSCFamide, was discovered using the expressed sequence tag (EST) database search strategy in both C. borealis and the lobster, Homarus americanus, and further confirmed with de novo sequencing using ESI-Q-TOF tandem MS. Electrophysiological experiments demonstrated that both CbAST-C1 and CbAST-C2 inhibited the frequency of the pyloric rhythm of the STG, in a state-dependent manner. At 10⁻⁶ M, both peptides were only modestly effective when initial frequencies of the pyloric rhythm were >0.8 Hz, but almost completely suppressed the pyloric rhythm when applied to preparations with starting frequencies <0.7 Hz. Surprisingly, these state-dependent actions are similar to those of the structurally unrelated allatostatin A and allatostatin B families of peptides.     

γ-Aminobutyric acid immunostaining in the antennal lobe of the moth <Emphasis Type="Italic">Heliothis virescens</Emphasis> and its colocalization with neuropeptides

The antennal lobe is the primary processing center for olfactory information in insects. To understand further the neural circuitry of this brain area, we have investigated the distribution of γ-aminobutyric acid (GABA) and its colocalization with neuropeptides in the antennal lobe of the noctuid moth Heliothis virescens. Immunocytochemical experiments with an antiserum against GABA showed a large number of labeled somata in the antennal lobe; these somata were located exclusively in the lateral cell cluster. Stained neurites innervating all antennal-lobe glomeruli, including the male-specific macroglomerular complex, suggested a prominent role of GABA in processing olfactory information, including signals from pheromones, interspecifically acting odors, and plant odors. Fibers in two antennocerebral tracts (the middle and dorsal antennocerebral tract) exhibited prominent GABA immunoreactivity. Double-labeling experiments revealed that immunostaining for three neuropeptides, viz., A-type allatostatin, Manduca sexta allatotropin, and FMRFamide-related peptides, was largely colocalized with GABA in cell bodies of the lateral cell cluster. The general absence of peptide immunostaining in the antennocerebral tracts strongly indicated that these peptides were colocalized with GABA in local interneurons of the antennal lobe. In contrast, tachykinin-related peptides occurred in a distinct population of local antennal-lobe neurons that did not exhibit GABA immunostaining. Thus, local interneurons that were not GABAergic were present in the moth antennal lobe. This work was supported by the Norwegian University of Science and Technology (project no. 80902101).     

Compatibility of codling moths Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) from South Africa with codling moths shipped from Canada

The sterile insect technique (SIT) has been successfully applied against codling moth Cydia pomonella (Linnaeus) (Lepidoptera; Tortricidae) in British Columbia since 1992 where the mass‐rearing facility produces between 15 and 16 million moths per week. Due to the seasonality of this pest, the facility is only fully utilized for part of the year. The time and expense of implementing SIT against codling moth in South Africa may be substantially reduced if moths from Canada were sexually compatible with those from South Africa. In addition, because the pome fruit‐growing season in both countries is opposite, the programme in Canada might benefit by maintaining moth production year‐round and selling moths to South Africa. Semi‐field studies in small cages and release‐recapture studies were conducted in an unsprayed apple orchard in South Africa to assess mating compatibility of laboratory‐reared codling moth from Canada and wild codling moths from South Africa. The results suggest that Canadian codling moth males were equally attracted to calling Canadian and South African females despite the fact that Canadian moths had been transported (from Canada to South Africa) for 48 h as both pupae and adults. The data also suggest that at lower field temperatures Canadian moths were more active than South African moths. Results from the release‐recapture field trials indicated that Canadian and South African males were equally attracted to Canadian and South African females. These results suggest that codling moths from Canada and South Africa are fully compatible and indicate that Canadian moths can be used for SIT studies in South Africa. As these studies were conducted with moths from two very different climatic and time zones, it is proposed that populations of codling moth in other pome fruit production areas may also be compatible with the Canadian moths.     

Allatostatins from the retrocerebral complex and antennal pulsatile organ of the American cockroach: structural elucidation aided by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

The occurrence of allatostatins in retrocerebral complexes and antennal pulsatile organs of the American cockroach, Periplaneta americana, was investigated. Previously, molecular cloning of the P. americana allatostatin gene had predicted 14 peptides of this family [Ding et al., Comparison of the allatostatin neuropeptide precursors in the distantly related cockroaches Periplaneta americana and Diploptera punctata. Eur J Biochem 1997;234:737-746], however, only two forms had been identified by peptide isolation procedures [Weaver et al., Identification of two allatostatins from the CNS of the cockroach Periplaneta americana: novel members of a family of neuropeptide inhibitors of insect juvenile hormone biosynthesis. Comp Biochem Physiol 1994;107(C):119-127]. Using an extract of only 200 corpora cardiaca/corpora allata, we have found that at least 11 allatostatins occur in the retrocerebral complex. These peptides were already separated from other substances of the crude extract in the first HPLC step with heptafluorobutyric acid as organic modifier, and subsequently identified by MALDI-TOF mass spectrometry. Moreover, we have demonstrated the occurrence of nearly all allatostatins, including the cleavage product of Pea-AST-2 (LPVYNFGL-NH2), in antennal pulsatile organs of males and females. Allatostatins are predominant neuropeptides in these organs. Additionally, only two other known peptides could be identified in these organs by mass screening: proctolin and leucomyosuppressin. The function of allatostatins in antennal pulsatile organs remains unclear. We assume a release into the hemolymph via the ampullac, which could act as neurohemal release sites. The method described for the identification of allatostatins is a very fast method for neuropeptide screening in neurohemal tissues.     

Identification of neuropeptides from brains of larval Manduca sexta and Lacanobia oleracea using MALDI-TOF mass spectrometry and post-source decay

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, CAP(2b) 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 CAP(2b) 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, CAP(2b), 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.     

Quantification of cockroach allatostatin-like peptide and its myotropic effects in males of the earwig Euborellia annulipes

A monoclonal antibody to allatostatin I of the cockroach Diploptera punctata was used to establish a competitive enzyme‐linked immunosorbent assay for quantification of allatostatin‐like peptides in the hindgut of the adult male earwig, Euborellia annulipes. Hindguts of 0‐day males contained significantly more allatostatin‐positive material than those of 8‐day males fed on catfood. However, males starved for the first 8 days of adult life had significantly higher levels of allatostatin‐positive material than those of either 0‐day or of 8‐day fed males. Hindguts from 0‐day old males exhibited lower spontaneous motility in vitro than those from 8‐day males. Hindguts from males at both ages responded to allostatin with reversible, dosage‐dependent decreases in hindgut motility, and responded to proctolin with reversible, dosage‐dependent increases in hindgut motility. When both allatostatin and proctolin were applied to hindgut preparations simultaneously and in equal concentrations, the response varied with the stage of the male. Starvation enhanced hindgut motility and abolished the response to allatostatin, but not to proctolin. These results indicate the presence of material similar to cockroach allatostatins in male earwigs, and that the levels change with age and physiological stage. Furthermore, such peptides may indeed be regulatory neuropeptides and could modulate hindgut contraction. There was an increase in sensitivity to exogenous allatostatin in the hindgut during development from day 0 to day 8 in feeding males, but a loss in sensitivity in response to starvation; sensitivity to exogenous proctolin also increased with age, but such responsiveness was not diminished by starvation.