Peptidergic control of the crop of the cabbage root fly, Delia radicum (L.) Diptera: Anthomyiidae): A role for myosuppressin.

There is much interest in targeting neuropeptide signaling for the development of new and environmentally friendly insect control chemicals. In this study we have focused attention on the peptidergic control of the adult crop of Delia radicum (cabbage root fly), an important pest of brassicas in European agriculture. The dipteran crop is a muscular organ formed from the foregut of the digestive tract and plays a vital role in the processing of food in adult flies. We have shown using direct tissue profiling by MALDI-TOF mass spectrometry that the decapeptide myosuppressin (TDVDHVFLRFamide) is present in the crop nerve bundle and that application of this peptide to the crop potently inhibits the spontaneous contractions of the muscular lobes with an IC50 of 4.4 × 10-8 M. The delivery of myosuppressin either by oral administration or by injection had no significant detrimental effect on the adult fly. This failure to elicit a response is possibly due to the susceptibility of the peptide to degradative peptidases that cleave the parent peptide to inactive fragments. Indeed, we show that the crop of D. radicum is a source of neuropeptide-degrading endo- and amino-peptidases. In contrast, feeding benzethonium chloride, a non-peptide agonist of myosuppressin, reduced feeding rate and increased the rate of mortality of adult D. radicum. Current results are indicative of a key role for myosuppressin in the regulation of crop physiology and the results achieved during this project provide the basis for subsequent studies aimed at developing insecticidal molecules targeting the peptidergic control of feeding and food digestion in this pest species.

The sexually dimorphic behaviour of adult Drosophila suzukii: elevated female locomotor activity and loss of siesta is a post-mating response.

The polyphagous Drosophila suzukii is a highly invasive species that causes extensive damage to a wide range of berry and stone fruit crops. A better understanding of its biology and especially its behaviour will aid the development of new control strategies. We investigated the locomotor behaviour of D. suzukii in a semi-natural environment resembling a typical summer in northern England and show that adult female D. suzukii are at least 4-fold more active during daylight hours than adult males. This result was reproduced in several laboratory environments and was shown to be a robust feature of mated, but not virgin, female flies. Both males and virgin females kept on a 12 h light:12 h dark (12LD) cycle and constant temperature displayed night-time inactivity (sleep) followed by weak activity in the morning, an afternoon period of quiescence (siesta) and then a prominent evening peak of activity. Both the siesta and the sharp evening peak at lights off were severely reduced in females after mating. Flies of either sex entrained in 12LD displayed a circadian pattern of activity in constant darkness confirming the importance of an endogenous clock in regulating adult activity. This response of females to mating is similar to that elicited in female Drosophila melanogaster by the male sex peptide (SP). We used mass spectrometry to identify a molecular ion (m/z, 5145) corresponding to the poly-hydroxylated SP of D. suzukii and to show that this molecule is transferred to the female reproductive tract during copulation. We propose that the siesta experienced by male and virgin female D. suzukii is an adaptation to avoid unnecessary exposure to the afternoon sun, but that mated females faced with the challenge of obtaining resources for egg production and finding oviposition sites take greater risks, and we suggest that the change in female behaviour is induced by the male SP.

MIPs are ancestral ligands for the sex peptide receptor.

Upon mating, females of many animal species undergo dramatic changes in their behavior. In Drosophila melanogaster, postmating behaviors are triggered by sex peptide (SP), which is produced in the male seminal fluid and transferred to female during copulation. SP modulates female behaviors via sex peptide receptor (SPR) located in a small subset of internal sensory neurons that innervate the female uterus and project to the CNS. Although required for postmating responses only in these female sensory neurons, SPR is expressed broadly in the CNS of both sexes. Moreover, SPR is also encoded in the genomes of insects that lack obvious SP orthologs. These observations suggest that SPR may have additional ligands and functions. Here, we identify myoinhibitory peptides (MIPs) as a second family of SPR ligands that is conserved across a wide range of invertebrate species. MIPs are potent agonists for Drosophila, Aedes, and Aplysia SPRs in vitro, yet are unable to trigger postmating responses in vivo. In contrast to SP, MIPs are not produced in male reproductive organs, and are not required for postmating behaviors in Drosophila females. We conclude that MIPs are evolutionarily conserved ligands for SPR, which are likely to mediate functions other than the regulation of female reproductive behaviors.

Identification and localisation of selected myotropic neuropeptides in the ventral nerve cord of tenebrionid beetles.

MALDI-TOF MS and MS/MS techniques were used for the isolation and identification of neuropeptides from the ventral nerve cord (VNC) of two beetle species Tenebrio molitor and Zophobas atratus. Two peptides, proctolin and myosuppressin (Zopat-MS), with well-established myotropic properties were identified as well as Trica-NVPL-4trunc. The presence of proctolin and myosuppressin was confirmed by immunocytochemical studies in adults and larvae of both beetles. In addition, the myosuppressin gene in Z. atratus was sequenced and expression analyses showed that it is present in all parts of the beetle central nervous system. Results suggest that the identified peptides act as neurotransmitters/neuromodulators in beetles, regulate visceral muscle contractions and indirectly influence important physiological processes such as feeding and reproduction.

New myotropic and metabotropic actions of pyrokinins in tenebrionid beetles.

Pyrokinins are a large family of insect neuropeptides exhibiting pleiotropic activity, but are predominantly myostimulatory hormones. In this study, four pyrokinins Tenmo-PK-1 (HVVNFTPRLa), Tenmo-PK-2 (SPPFAPRLa), Tenmo-PK-3 (HLSPFSPRLa) and Zopat-PK-1 (LPHYPRLa) from the neuro-endocrine system of two tenebrionid beetles, Tenebrio molitor and Zophobas atratus, were tested in homologous bioassays to evaluate their putative myotropic and glycaemic actions. The four investigated bioassays systems (the heart, oviduct, ejaculatory duct and hindgut) revealed species-specific and organ-specific myotropic actions for the pyrokinins tested. In most bioassays with both beetles, the peptides showed myostimulatory properties with different efficacy. However, the T. molitor heart is not sensitive to Tenmo-PK-1, Tenmo-PK-2 and Tenmo-PK-3, and one of the peptides Tenmo-PK-1, is myoinhibitory on the oviduct. Tenmo-PK-2, which is also present in Z. atratus, exerted an inhibitory effect on the contractions of the heart and ejaculatory duct muscles in this beetle. Such myoinhibitory properties of pyrokinins in insects are shown here for the first time. Only one of the peptides tested, Tenmo-PK-2, stimulated a hyperglycaemic response in the haemolymph of larvae of T. molitor and Z. atratus, and this effect suggests a possible additional metabotropic function of this peptide in beetles. The differences in the myotropic and glycaemic responses to pyrokinins suggest that these peptides modulate contractions of muscles from visceral organs and free sugar levels in the haemolymph of the beetles, through complex and species-specific mechanisms.

The Fate of Deroceras reticulatum Following Metaldehyde Poisoning.

The concentration of a pesticide used in agriculture not only has implications for effectiveness of pest control but may also have significant wider environmental consequences. This research explores the acceptability of metaldehyde slug pellets at different concentrations by Deroceras reticulatum (Müller, 1774) (Agriolimacidae), and the changes in the health status of the slug when allowed to recover. The highest metaldehyde concentration (5%) yielded the highest slug mortality; however, it also produced the highest proportion of unpoisoned slugs, suggesting the highest level of pellet rejection. Pellets with 1% metaldehyde were as effective as 3% pellets in paralysing a significant proportion of the population after initial pellet exposure; however, more slugs were able to recover from metaldehyde poisoning at 1% metaldehyde compared with 3%. There was no statistically significant difference between the mortality rate of slugs regardless of metaldehyde concentration, suggesting that a lower concentration of metaldehyde may be as effective as a higher concentration.

Mass spectrometric characterisation of the major peptides of the male ejaculatory duct, including a glycopeptide with an unusual zwitterionic glycosylation.

Peptides present in the seminal fluid of Drosophila melanogaster can function as antimicrobial agents, enzyme inhibitors and as pheromones that elicit physiological and behavioural responses in the post-mated female. Understanding the molecular interactions by which these peptides influence reproduction requires detailed knowledge of their molecular structures. However, this information is often lacking and cannot be gleaned from just gene sequences and standard proteomic data. We now report the native structures of four seminal fluid peptides (andropin, CG42782, Met75C and Acp54A1) from the ejaculatory duct of male D. melanogaster. The mature CG42782, Met75C and Acp54A1 peptides each have a cyclic structure formed by a disulfide bond, which will reduce conformational freedom and enhance metabolic stability. In addition, the presence of a penultimate Pro in CG42782 and Met75C will help prevent degradation by carboxypeptidases. Met75C has undergone more extensive post-translational modifications with the formation of an N-terminal pyroglutamyl residue and the attachment of a mucin-like O-glycan to the side chain of Thr4. Both of these modifications are expected to further enhance the stability of the secreted peptide. The glycan has a rare zwitterionic structure comprising an O-linked N-acetyl hexosamine, a hexose and, unusually, phosphoethanolamine. A survey of various genomes showed that andropin, CG42782, and Acp54A1 are relatively recent genes and are restricted to the melanogaster subgroup. Met75C, however, was also found in members of the obscura species groups and in Scaptodrosophila lebanonensis. Andropin is related to the cecropin gene family and probably arose by tandem gene duplication, whereas CG42782, Met75C and Acp54A1 possibly emerged de novo. We speculate that the post-translational modifications that we report for these gene products will be important not only for a biological function, but also for metabolic stability and might also facilitate transport across tissue barriers, such as the blood-brain barrier of the female insect. BIOLOGICAL SIGNIFICANCE: Seminal fluid peptides of D. melanogaster function as antimicrobials, enzyme inhibitors and as pheromones, eliciting physiological and behavioural responses in the post-mated female. A fuller understanding of how these peptides influence reproduction requires knowledge not only of their primary structure, but also of their post-translational modification. However, this information is often lacking and difficult to glean from standard proteomic data. The reported modifications, including the unusual glycosylation, adds much to our knowledge of this important class of peptides in this model organism, par excellence.

Effects of Manduca sexta allatostatin and an analogue on the peach-potato aphid Myzus persicae (hemiptera: aphididae) and degradation by enzymes in the aphid gut.

The oral toxicity of the C-type allatostatin, Manduca sexta allatostatin (Manse-AS) and the analogue δR³Î´R5Manse-AS, where R residues were replaced by their D-isomers, were tested against the peach-potato aphid Myzus persicae by incorporation into an artificial diet. Both peptides had significant dose-dependent effects on mortality, growth, and fecundity compared with control insects. The analogue, δR³Î´R5Manse-AS, had an estimated LC50 of 0.31 µg/µl diet and was more potent than Manse-AS (estimated LC50 of 0.58 µg/µl diet). At a dose of 0.35 µg δR³Î´R5Manse-AS/µl diet, 76% of the aphids were dead after 6 days and all were dead after 10 days. In comparison, three times the dose of Manse-AS was required to achieve 74% mortality after 8 days and 98% mortality after 16 days. The degradation of both peptides by extracts prepared from the gut of M. persicae was investigated. The estimated half-life of Manse-AS, when incubated with the gut extract from M. persicae, was 31 min. Degradation was due to a cathepsin L-like cysteine protease, carboxypeptidase-like activity, endoprotease activity with glutamine specificity, pyroglutamate aminopeptidase activity, and possibly trypsin-like proteases. The half-life of the δR³Î´R5 Manse-AS analogue was enhanced (73 min) with the D-isomers of R appearing to prevent cleavage around the R residues by cathepsin L-like cysteine proteases or from trypsin-like proteases. The greater stability of the analogue may explain its increased potency in M. persicae. This work demonstrates the potential use of Manse-AS and analogues, with greater resistance to enzymatic attack, in aphid control strategies.

Identification of myotropic neuropeptides from the brain and corpus cardiacum-corpus allatum complex of the beetle, Zophobas atratus.

The neuropeptide profiles of the two major neuro-endocrinological organs, brain and retrocerebral complex corpus cardiacum-corpus allatum (CC/CA) of adult beetles, Zophobas atratus Fabricius (Coleoptera:Tenebrionidae) were analyzed by a combination of high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization time of flight tandem mass spectrometry (MALDI TOF/TOF MS). The homological semi-isolated heart bioassay was used to screen HPLC fractions for myotropic activity in tissues, revealing several cardiostimulatory and cardioinhibitory factors from both the brain and CC/CA. Analysis of HPLC fractions by MALDI-TOF MS identified seven mass ions that could be assigned to other known peptides: leucomyosuppressin (LMS), Tribolium castaneum pyrokinin 2, sulfakinin 1, myoinhibitory peptide 4, a truncated NVP-like peptide, Tenebrio molitor AKH and crustacean cardioactive peptide. In addition, two novel peptides, myosuppressin (pEDVEHVFLRFa), which differs from LMS by one amino acid (E for D at position 4) and pyrokinin-like peptide (LPHYTPRLa) were also identified. To establish cardioactive properties of some of the identified peptides, chemical synthesis was carried out and their activities were tested using the heart bioassay.

The structure of the Drosophila melanogaster sex peptide: Identification of hydroxylated isoleucine and a strain variation in the pattern of amino acid hydroxylation.

In Drosophila melanogaster mating triggers profound changes in the behaviour and reproductive physiology of the female. Many of these post-mating effects are elicited by sex peptide (SP), a 36-mer pheromone made in the male accessory gland and passed to the female in the seminal fluid. The peptide comprises several structurally and functionally distinct domains, one of which consists of five 4-hydroxyprolines and induces a female immune response. The SP gene predicts an isoleucine (Ile14) sandwiched between two of the hydroxyprolines of the mature secreted peptide, but the identity of this residue was not established by peptide sequencing and amino acid analysis, presumably because of modification of the side chain. Here we have used matrix-assisted laser desorption ionisation mass spectrometry together with Fourier-transform ion cyclotron resonance mass spectrometry to show that Ile14 is modified by oxidation of the side chain - a very unusual post-translational modification. Mass spectrometric analysis of glands from different geographical populations of male D. melanogaster show that SP with six hydroxylated side chains is the most common form of the peptide, but that a sub-strain of Canton-S flies held at Leeds only has two or three hydroxylated prolines and an unmodified Ile14. The D. melanogaster genome has remarkably 17 putative hydroxylase genes that are strongly and almost exclusively expressed in the male accessory gland, suggesting that the gland is a powerhouse of protein oxidation. Strain variation in the pattern of sex peptide hydroxylation might be explained by differences in the expression of individual hydroxylase genes.

The insecticidal activity of recombinant garlic lectins towards aphids.

The heterodimeric and homodimeric garlic lectins ASAI and ASAII were produced as recombinant proteins in the yeast Pichia pastoris. The proteins were purified as functional dimeric lectins, but underwent post-translational proteolysis. Recombinant ASAII was a single homogenous polypeptide which had undergone C-terminal processing similar to that occurring in planta. The recombinant ASAI was glycosylated and subject to variable and heterogenous proteolysis. Both lectins showed insecticidal effects when fed to pea aphids (Acyrthosiphon pisum) in artificial diet, ASAII being more toxic than ASAI at the same concentration. Acute toxicity (mortality at < or =48 h exposure; similar timescale to starvation) was only apparent at the highest lectin concentrations tested (2.0 mg ml(-)1), but dose-dependent chronic toxicity (mortality at >3d exposure) was observed over the concentration range 0.125-2.0 mg ml(-1). The recombinant lectins caused mortality in both symbiotic and antibiotic-treated aphids, showing that toxicity is not dependent on the presence of the bacterial symbiont (Buchnera aphidicola), or on interaction with symbiont proteins, such as the previously identified lectin "receptor" symbionin. A pull-down assay coupled with peptide mass fingerprinting identified two abundant membrane-associated aphid gut proteins, alanyl aminopeptidase N and sucrase, as "receptors" for lectin binding.

Neuropeptides of the beetle, Tenebrio molitor identified using MALDI-TOF mass spectrometry and deduced sequences from the Tribolium castaneum genome.

Four neuropeptides were identified from the brain and corpora cardiaca-corpora allata (CC-CA) of the mealworm beetle Tenebrio molitor using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and information derived from the genome of the red flour beetle, Tribolium castaneum. Leucomyosuppressin (a FLRFamide), previously associated with cockroaches, but also subsequently identified from honey bee seen as a prominent peptide in both brain and CC-CA of T.molitor. A coding sequence for this peptide is found in the genome of T. castaneum. In addition, three FXPRLamides (pyrokinins), provisionally Tenmo-PK-1, Tenmo-PK-2 and Tenmo-PK-3 (HVVNFTPRLamide, SPPFAPRLamide, HL(I)SPFSPRLamide) were identified in both CC-CA and brain of T. molitor, again on the basis of predicted occurrence or similarity in T. castaneum. The sequence of Tenmo-PK-2 is the same as the PK-2 of the cockroach, Periplaneta americana. Other peptides readily predicted from the genome of T. castaneum include two AKH/HrTH peptides (Trica-AKH-1; pELNFSTDWamide and Trica-AKH-2; pELNFTPNWamide), the second of which is identical to Pyrap-AKH, an AKH-related peptide (Trica AKH-L; pEVTFSRDWPamide), two CRF-related diuretic factors (Trica-DH 37 and Trica-DH 47), the latter identical to Tenmo-DH 47, a putative antidiuretic factor (Trica-ADFb; LYDDGSYKPHVYGF-OH), two sulfakinin-like peptides (Trica-SK-1; pETSDDY(SO(3))GHLRFamide, and Trica SK-2; GEEPFDDYGHMRFamide), a potential allatostatin-C (Trica-AS; pESRYRQCYFNPISCF-OH), six allatostatin-B/myoinhibitory peptides (Trica-AST-B-1,2,3,4,5 & 6; DWNKDLHIWamide, GWNNLHEGWamide, AWQSLQSGWamide, NWGQFHGGWamide, SKWDNFRGSWamide, EPAWSNLGIWamide), an allatotropin-like peptide (Trica-ATL; GIEALKYHNMDLGTARGYamide), four 'CAPA'-related peptides (Trica-CAPA-1,2,3,4; NKLASVYALTPSLRVamide, RIGKMVSFPRIamide, PGANSGGMWFGPRLamide, SENFTPWAYIILNGEAPIIREVHYSPRLamide), proctolin (RYLPT), a potential SIFamide (Trica-SIFa; TYRKPPFNGSIFamide), an arginine-vasopressin-related peptide (Trica-AVP; CLITNCPRGamide) and an ITP-related peptide (Trica-ITP). No evidence was found for the presence of 'A' allatostatins (Y/FxFGLamides) or corazonin, either in T. molitor, or in the genome of T. castaneum.

Transepithelial flux of an allatostatin and analogs across the anterior midgut of Manduca sexta larvae in vitro.

The transepithelial flux of cydiastatin 4 and analogs across flat sheet preparations of the anterior midgut of larvae of the tobacco hawkmoth moth, Manduca sexta, was investigated using a combination of reversed-phase high-performance liquid chromatography (RP-HPLC), enzyme-linked immunosorbent assay (ELISA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The lumen to hemolymph (L-H) flux of cydiastatin 4 was dose and time-dependent, with a maximum rate of flux of c. 178 pmol/cm2/h) measured after a 60-min incubation with 100 micromol/l of peptide in the lumen bathing fluid. The rates of flux, L-H and H-L, across the isolated gut preparations were not significantly different. These data suggest that uptake across the anterior midgut of larval M. sexta is via a paracellular route. Cydiastatin 4 was modified to incorporate a hexanoic acid (Hex) moiety at the N-terminus, the N-terminus extended with 5 P residues and/or the substitution of G7 with Fmoc-1-amino-cyclopropylcarboxylic acid (Acpc). The incorporation of hexanoic acid enhanced the uptake of these amphiphilic analogs compared to the native peptide. Analogs were also more resistant to enzymes in hemolymph and gut preparations from larval M. sexta. A modified N-terminus gave protection against aminopeptidase-like activity and incorporation of Acpc inhibited endopeptidase-like activity. Although analogs were stable in the hemolymph, they were susceptible to amidase-like activity in the gut, which appears to convert the C-terminal amide group to a free carboxylic acid, identified by an increase in 1 mass unit of the peptide analog.

Predicted versus expressed adipokinetic hormones, and other small peptides from the corpus cardiacum-corpus allatum: a case study with beetles and moths.

This mass spectrometric study confines itself to peptide masses in the range of 500-1500Da. Adipokinetic hormones (AKHs) that are predicted from the genome of the red flour beetle, Tribolium castaneum, and the silk moth, Bombyx mori, are shown to exist as expressed peptides in the corpora cardiaca (CC) of the respective species as evidenced by various mass spectrometric methods. Additionally, some related species were included in this study, such as the tenebrionid beetles Tribolium brevicornis and Tenebrio molitor, as well as the moths Spodoptera frugiperda, Spodoptera littoralis, Mamestra brassicae and Lacanobia oleracea, to investigate whether AKH peptides are structurally conserved in the same genus or family. Interestingly, the AKH peptide of T. brevicornis is identical to that of T. molitor but not to the ones of its close relative T. castaneum. Moreover, other peptides in T. brevicornis, such as various FXPRL amides (=pyrokinins), also match the complement in T. molitor but differ from those in T. castaneum. All the CC of beetles lacked the signal for the mass of the peptide corazonin. All moths have the nonapeptide Manse-AKH expressed in their CC. In addition, whereas the silk moth has the decapeptide Bommo-AKH as a second peptide, all other moths (all noctuids) express the decapeptide Helze-HrTH. In M. brassicae and L. oleracea a novel amidated Gly-extended Manse-AKH is found as a possible third AKH. The noctuid moth species also all express the same FLRF amide-I, corazonin, and a group-specific isoform of a gamma-PGN-(=gamma-SGNP) peptide. In L. oleracea, however, the latter peptide has a novel sequence which is reported for the first time, and the peptide is code-named Lacol-PK.

Evaluation of Chemical Strategies for Improving the Stability and Oral Toxicity of Insecticidal Peptides.

Spider venoms are a rich source of insecticidal peptide toxins. Their development as bioinsecticides has, however, been hampered due to concerns about potential lack of stability and oral bioactivity. We therefore systematically evaluated several synthetic strategies to increase the stability and oral potency of the potent insecticidal spider-venom peptide ω-HXTX-Hv1a (Hv1a). Selective chemical replacement of disulfide bridges with diselenide bonds and N- to C-terminal cyclization were anticipated to improve Hv1a resistance to proteolytic digestion, and thereby its activity when delivered orally. We found that native Hv1a is orally active in blowflies, but 91-fold less potent than when administered by injection. Introduction of a single diselenide bond had no effect on the susceptibility to scrambling or the oral activity of Hv1a. N- to C-terminal cyclization of the peptide backbone did not significantly improve the potency of Hv1a when injected into blowflies and it led to a significant decrease in oral activity. We show that this is likely due to a dramatically reduced rate of translocation of cyclic Hv1a across the insect midgut, highlighting the importance of testing bioavailability in addition to toxin stability.

In vitro transport of an allatostatin across the foregut of Manduca sexta larvae and metabolism by the gut and hemolymph.

The degradation of synthetic cydiastatin 4 by enzymes of the foregut and hemolymph, and transport across the foregut of larvae of the tobacco hawkmoth moth, Manduca sexta, were investigated using reversed-phase high performance liquid chromatography (RP-HPLC) together with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In the hemolymph in vitro, cydiastatin 4 had a half-life of ca. 30 min. Two degradation products were identified; cydiastatin 4(1-6), due to cleavage of the C-terminal di-peptide GL-amide, and cydiastatin 4(2-8), due to cleavage of the N-terminal A residue. This hydrolysis could be inhibited by up to 93% by 1,10-phenanthroline. Other protease inhibitors had lesser effects (<21% inhibition of degradation) including the aminopeptidase inhibitors amastatin and bestatin, and the chelator EDTA. When incubated with foregut extract in vitro, cydiastatin 4 had a half-life of 23 min, and the hydrolysis products detected were also cydiastatin 4(1-6) and cydiastatin 4(2-8). Similarly, 1-10 phenanthroline inhibited foregut enzyme degradation of cydiastatin 4 by ca. 80%, whereas amastatin, bestatin, and EDTA had very little effect (<10% inhibition). Cydiastatin 4 was transported, intact, from the lumen to the hemolymph side of foregut tissues that were mounted as flat sheets in modified Ussing chambers. This trans-epithelial flux of peptide was dose and time-dependent, but was <3% of the amount of cydiastatin 4 present in the lumen bathing saline. In contrast, no trans-epithelial transport of peptide was apparent across everted foregut sac preparations.

Expression of NEP2, a soluble neprilysin-like endopeptidase, during embryogenesis in Drosophila melanogaster.

Members of the neprilysin family of neutral endopeptidases (M13) are typically membrane-bound enzymes known to be involved in the extra-cellular metabolism of signalling peptides and have important roles during mammalian embryogenesis. In this study we show that membranes prepared from embryos of Drosophila melanogaster possess neprilysin-like activity that is inhibited by phosphoramidon and thiorphan, both inhibitors of mammalian neprilysin. Unexpectedly, we also found strong neprilysin-like neutral endopeptidase activity in a soluble embryo fraction, which we identify as NEP2 by Western blot and immunoprecipitation experiments using NEP2 specific antibodies. NEP2 is a soluble secreted member of the neprilysin family that has been shown previously to be expressed in larval and adult Malpighian tubules and in the testes of adult males. In situ hybridization studies reveal expression at stage 10-11 in a pattern similar to that previously described for stellate cell progenitors of the caudal visceral mesoderm. In later stages of embryogenesis, some of these cells appear to migrate into the growing Malpighian tubule. Recombinant NEP2 protein is N-glycosylated and displays optimum endopeptidase activity at neutral pH, consistent with a role as an extracellular peptidase. The recombinant enzyme hydrolyses Drosophila tachykinin peptides (DTK) at peptide bonds N-terminal to hydrophobic residues. DTK2, like Locusta tachykinin-1, was cleaved at the penultimate peptide bond (Gly(7)-Leu(8)), whereas the other Drosophila peptides were cleaved centrally at Xxx-Phe bonds. However, the rates of hydrolysis of the latter substrates were much slower than the hydrolysis rates of DTK2 and Locusta tachykinin-1, suggesting that the interaction of the bulky side-chain of phenylalanine at the S'(1) sub-site is less favorable for peptide bond hydrolysis. The secretion of NEP2 from tissues during embryogenesis suggests a possible developmental role for this endopeptidase in peptide signalling in D. melanogaster.

Peptidergic control in a fruit crop pest: The spotted-wing drosophila, Drosophila suzukii.

Neuropeptides play an important role in the regulation of feeding in insects and offer potential targets for the development of new chemicals to control insect pests. A pest that has attracted much recent attention is the highly invasive Drosophila suzukii, a polyphagous pest that can cause serious economic damage to soft fruits. Previously we showed by mass spectrometry the presence of the neuropeptide myosuppressin (TDVDHVFLRFamide) in the nerve bundle suggesting that this peptide is involved in regulating the function of the crop, which in adult dipteran insects has important roles in the processing of food, the storage of carbohydrates and the movement of food into the midgut for digestion. In the present study antibodies that recognise the C-terminal RFamide epitope of myosuppressin stain axons in the crop nerve bundle and reveal peptidergic fibres covering the surface of the crop. We also show using an in vitro bioassay that the neuropeptide is a potent inhibitor (EC50 of 2.3 nM) of crop contractions and that this inhibition is mimicked by the non-peptide myosuppressin agonist, benzethonium chloride (Bztc). Myosuppressin also inhibited the peristaltic contractions of the adult midgut, but was a much weaker agonist (EC50 = 5.7 µM). The oral administration of Bztc (5 mM) in a sucrose diet to adult female D. suzukii over 4 hours resulted in less feeding and longer exposure to dietary Bztc led to early mortality. We therefore suggest that myosuppressin and its cognate receptors are potential targets for disrupting feeding behaviour of adult D. suzukii.

Functional Characterization and Signaling Systems of Corazonin and Red Pigment Concentrating Hormone in the Green Shore Crab, Carcinus maenas.

Neuropeptides play a central role as neurotransmitters, neuromodulators and hormones in orchestrating arthropod physiology. The post-genomic surge in identified neuropeptides and their putative receptors has not been matched by functional characterization of ligand-receptor pairs. Indeed, until very recently no G protein-coupled receptors (GPCRs) had been functionally defined in any crustacean. Here we explore the structurally-related, functionally-diverse gonadotropin-releasing hormone paralogs, corazonin (CRZ) and red-pigment concentrating hormone (RPCH) and their G-protein coupled receptors (GPCRs) in the crab, Carcinus maenas. Using aequorin luminescence to measure in vitro Ca2+ mobilization we demonstrated receptor-ligand pairings of CRZ and RPCH. CRZR-activated cell signaling in a dose-dependent manner (EC50 0.75 nM) and comparative studies with insect CRZ peptides suggest that the C-terminus of this peptide is important in receptor-ligand interaction. RPCH interacted with RPCHR with extremely high sensitivity (EC50 20 pM). Neither receptor bound GnRH, nor the AKH/CRZ-related peptide. Transcript distributions of both receptors indicate that CRZR expression was, unexpectedly, restricted to the Y-organs (YO). Application of CRZ peptide to YO had no effect on ecdysteroid biosynthesis, excepting a modest stimulation in early post-molt. CRZ had no effect on heart activity, blood glucose levels, lipid mobilization or pigment distribution in chromatophores, a scenario that reflected the distribution of its mRNA. Apart from the well-known activity of RPCH as a chromatophorotropin, it also indirectly elicited hyperglycemia (which was eyestalk-dependent). RPCHR mRNA was also expressed in the ovary, indicating possible roles in reproduction. The anatomy of CRZ and RPCH neurons in the nervous system is described in detail by immunohistochemistry and in situ hybridization. Each peptide has extensive but non-overlapping distribution in the CNS, and neuroanatomy suggests that both are possibly released from the post-commissural organs. This study is one of the first to deorphanize a GPCR in a crustacean and to provide evidence for hitherto unknown and diverse functions of these evolutionarily-related neuropeptides.

Proteomic identification of Drosophila melanogaster male accessory gland proteins, including a pro-cathepsin and a soluble gamma-glutamyl transpeptidase.

BACKGROUND: In Drosophila melanogaster, the male seminal fluid contains proteins that are important for reproductive success. Many of these proteins are synthesised by the male accessory glands and are secreted into the accessory gland lumen, where they are stored until required. Previous studies on the identification of Drosophila accessory gland products have largely focused on characterisation of male-specific accessory gland cDNAs from D. melanogaster and, more recently, Drosophila simulans. In the present study, we have used a proteomics approach without any sex bias to identify proteins in D. melanogaster accessory gland secretions. RESULTS: Thirteen secreted accessory gland proteins, including seven new accessory gland proteins, were identified by 2D-gel electrophoresis combined with mass spectrometry of tryptic fragments. They included protein-folding and stress-response proteins, a hormone, a lipase, a serpin, a cysteine-rich protein and two peptidases, a pro-enzyme form of a cathepsin K-like cysteine peptidase and a gamma-glutamyl transpeptidase. Enzymatic studies established that accessory gland secretions contain a cysteine peptidase zymogen that can be activated at low pH. This peptidase may have a role in the processing of female and other male-derived proteins, but is unlikely to be involved in the processing of the sex peptide. gamma-Glutamyl transpeptidases are type II integral membrane proteins; however, the identified AG gamma-glutamyl transpeptidase (GGT-1) is unusual in that it is predicted to be a soluble secreted protein, a prediction that is supported by biochemical evidence. GGT-1 is possibly involved in maintaining a protective redox environment for sperm. The strong gamma-glutamyl transpeptidase activity found in the secretions provides an explanation for the observation that glutamic acid is the most abundant free amino acid in accessory gland secretions of D. melanogaster. CONCLUSION: We have applied biochemical approaches, not used previously, to characterise prominent D. melanogaster accessory gland products. Of the thirteen accessory gland secreted proteins reported in this study, six were represented in a D. simulans male accessory gland EST library that was biased for male-specific genes. Therefore, the present study has identified seven new secreted accessory gland proteins, including GGT-1, which was not recognised previously as a secreted accessory gland product.