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.

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.

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.

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.

G protein coupled receptors as targets for next generation pesticides.

There is an on-going need for the discovery and development of new pesticides due to the loss of existing products through the continuing development of resistance, the desire for products with more favourable environmental and toxicological profiles and the need to implement the principles of integrated pest management. Insect G protein coupled receptors (GPCRs) have important roles in modulating biology, physiology and behaviour, including reproduction, osmoregulation, growth and development. Modifying normal receptor function by blocking or over stimulating its actions may either result in the death of a pest or disrupt its normal fitness or reproductive capacity to reduce pest populations. Hence GPCRs offer potential targets for the development of next generation pesticides providing opportunities to discover new chemistries for invertebrate pest control. Such receptors are important targets for pharmaceutical drugs, but are under-exploited by the agro-chemical industry. The octopamine receptor agonists are the only pesticides with a recognized mode of action, as described in the classification scheme developed by the Insecticide Resistance Action Committee, that act via a GPCR. The availability of sequenced insect genomes has facilitated the characterization of insect GPCRs, but the development and utilization of screening assays to identify lead compounds has been slow. Various studies using knock-down technologies or applying the native ligands and/or neuropeptide analogues to pest insects in vivo, have however demonstrated that modifying normal receptor function can have an insecticidal effect. This review presents examples of potential insect neuropeptide receptors that are potential targets for lead compound development, using case studies from three representative pest species, Tribolium castaneum, Acyrthosiphon pisum, and Drosophila suzukii. Functional analysis studies on T. castaneum suggest that GPCRs involved in growth and development (eclosion hormone, ecdysis triggering hormone and crustacean cardioacceleratory peptide receptors) as well as the dopamine-2 like, latrophilin-like, starry night, frizzled-like, methuselah-like and the smoothened receptors may be suitable pesticide targets. From in vivo studies using native ligands and peptide analogues, receptors which appear to have a role in the regulation of feeding in the pea aphid, such as the PISCF-allatostatin and the various "kinin" receptors, are also potential targets. In Drosophila melanogaster various neuropeptides and their signalling pathways have been studied extensively. This may provide insights into potential pesticide targets that could be exploited in D. suzukii. Examples include the sex peptide receptor, which is involved in reproduction and host seeking behaviours, and those responsible for osmoregulation such as the diuretic hormone receptors. However the neuropeptides and their receptors in insects are often poorly characterized, especially in pest species. Although data from closely related species may be transferable (e.g. D. melanogaster to D. suzukii), peptides and receptors may have different roles in different insects, and hence a target in one insect may not be appropriate in another. Hence fundamental knowledge of the roles and functions of receptors is vital for development to proceed.

Genomic and peptidomic analyses of the neuropeptides from the emerging pest, Drosophila suzukii.

Drosophila suzukii is a highly polyphagous invasive pest which has been recently introduced into Europe and North America, where it is causing severe economic losses through larval infestations of stone and berry fruits. The peptidome of the selected nervous tissues of adult D. suzukii was investigated as a first step in identifying potential targets for the development of novel insecticides. Through in silico analyses of the D. suzukii genome databases 28 neuropeptide families, comprising more than 70 predicted peptides were identified. Using a combination of liquid chromatography and mass spectrometry of tissue extracts, 33 predicted peptides, representing 15 different peptide families were identified by their molecular masses and a total of 17 peptide sequences were confirmed by ion fragmentation. A comparison between the peptides and precursors of D. suzukii and D. melanogaster shows they are highly conserved, with differences only identified in the amino acid sequences of the peptides encoded in the FMRFamide, hugin and ecydysis triggering hormone precursors. All other peptides predicted and identified from D. suzukii appear to be identical to those previously characterized from D. melanogaster. Adipokinetic hormone was only identified in the corpus cardiacum, other peptides present included short neuropeptide F, a pyrokinin and myosuppressin, the latter of which was the only peptide identified from the crop nerve bundle. Peptides present in extracts of the brain and/or thoracico-abdominal ganglion included allatostatins, cardioacceleratory peptide 2b, corazonin, extended FMRFamides, pyrokinins, myoinihibitory peptides, neuropeptide-like precursor 1, SIFamide, short neuropeptide F, kinin, sulfakinins and tachykinin related peptides.

The degradome and the evolution of Drosophila sex peptide as a ligand for the MIP receptor.

The male sex peptide (SP) of Drosophila melanogaster has wide ranging effects on females, including rejection of courting males, increased egg production, changes to the feeding habit, increased synthesis of antimicrobial peptides and elevated locomotor activity during day-time. The peptide activates receptors in sensory neurons of the female reproductive tract and can also traverse into the hemolymph and reach the central nervous system. The SP receptor involved in rejection and egg-laying responses has been shown to be identical to the receptor for the evolutionary conserved myoinhibitory peptides (MIPs) that function as neuropeptides in both males and females. Intriguingly, MIPs cannot substitute for SP when either expressed in the male accessory glands or injected into virgin females. MIPs are linear peptides with an amidated C-terminus which protects them from cleavage by carboxypeptidases, but leaves them exposed to potential attack from aminopeptidase and endopeptidase activities. In contrast, the SP region responsible for eliciting the post-mating response is cyclic and has several hydroxyproline residues N-terminal to the disulfide bridge which is expected to protect the biological activity of SP from peptidases of the male accessory gland and seminal fluid. We now present in vitro data showing that SP is metabolically stable, whereas MIPs are much more susceptible to degradation by peptidases of the male accessory gland and the hemolymph of virgin female D. melanogaster. SP has evolved relatively recently as a MIP receptor ligand that is particularly well adapted to surviving in the hostile degradome of the male accessory gland and seminal fluid.

Signal transduction for Schistocerca gregaria ion transport peptide is mediated via both cyclic AMP and cyclic GMP.

The second messengers involved in the signal transduction for Schistocerca gregaria, ion transport peptide (Schgr-ITP) that regulates ion and fluid transport across the ileum of the desert locust S. gregaria, were measured using competitive enzyme-linked immunosorbent assays (ELISAs). Synthetic Schgr-ITP elevates intracellular levels of both cyclic AMP and cyclic GMP, measured over a 15 min period in the presence of 3-isobutyl-1-methylxanthine, in a dose-dependent manner. Furthermore, crude corpora cardiaca (CC) extracts elevate intracellular cyclic AMP levels 2-fold greater than Schgr-ITP, suggesting that factors present in the CC, other than Schgr-ITP, also act via this second messenger. These results suggest that the interaction of Schgr-ITP with two separate receptors, most likely a G-protein coupled receptor and a membrane bound guanylate cyclase, elevates intracellular levels of cyclic AMP and cyclic GMP to regulate ion and fluid transport across the locust ileum. Cyclic AMP stimulates Cl(-), K(+) and Na(+) reabsorption, whereas secretion of H(+) into the lumen of the ileum is most likely mediated via cyclic GMP. Cyclic GMP also stimulates Cl(-) uptake in a similar manner to cyclic AMP. The measurement of tissue (central nervous system) levels of Schgr-ITP using an indirect ELISA confirms that the peptide is only present in the locust brain and the CC. The amounts present are greatest in the CC, where the peptide is presumably stored for release into the hemolymph when locusts feed.

Adipokinetic hormones (AKHs) of sphingid Lepidoptera, including the identification of a second M. sexta AKH.

The adipokinetic hormones (AKHs) from the corpora cardiaca (CC) of representative species from all three subfamilies of the Sphingidae (hawkmoths) were investigated using matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) and liquid chromatography electrospray ion trap mass spectrometry (LC-ESI MS), including a re-examination of the AKH complement of the tobacco hawkmoth, Manduca sexta. In addition to larvae and adults of M. sexta (subfamily: Sphinginae), adults from the following subfamilies were examined: Macroglossinae (large elephant hawkmoth, Deilephila elpenor), Smerinthinae (poplar hawkmoth, Laothoe populi and eyed hawkmoth, Smerinthus ocellata), and Sphinginae (death's head hawkmoth, Acherontia atropos). All moths are shown to have the nonapeptide Manse-AKH (pELTFTSSWGamide) [corrected] in their CC, together with a second AKH, which, on the basis of mass ions ([M+Na](+), [M+K](+)) and partial sequence analysis is identical in all species examined. The structure of this AKH was extracted from the CC [corrected] of adult M. sexta and shown, by ESI-collision-induced dissociation (CID) tandem mass spectrometry (MS/MS), to be a novel decapeptide AKH with a sequence of pELTFSSWGQamide. [corrected]. The new peptide has been code named Manse-AKH-II. Sequence confirmation was obtained from identical MS studies with synthetic Manse-AKH-II and with the native peptide. Manse-AKH-II has significant lipid-mobilizing activity when injected at low dose (5pmol) into newly emerged adult M. sexta. The potential implications of a second AKH, in M. sexta in particular, are discussed in relation to putative receptor(s).

The host-seeking inhibitory peptide, Aea-HP-1, is made in the male accessory gland and transferred to the female during copulation.

Male accessory glands (MAGs) of insects are responsible for the production of many of the seminal fluid proteins and peptides that elicit physiological and behavioral responses in the post-mated female. In the yellow fever mosquito, Aedes aegypti, seminal fluid components are responsible for stimulating egg production, changing female behavior away from host-seeking toward egg-laying and mating refractoriness, but hitherto no behavior-modifying molecule from the MAGs has been structurally characterized. We now show using mass spectrometry and HPLC/ELISA that the MAG is a major site of synthesis of the biologically active decapeptide, Aea-HP-1 (pERPhPSLKTRFamide) that was first characterized by Matsumoto and colleagues in 1989 from mosquito head extracts and shown to have host-seeking inhibitory properties. The peptide is localized to the anterior portion of the MAG, occurs at high concentrations in the gland and is transferred to the female reproductive tract on copulation. Aea-HP-1 has a pyroglutamic acid at the N-terminus, an amidated carboxyl at the C-terminus and an unusual 4-hydroxyproline in position 4 of the peptide. The structure of the peptide with its blocked N- and C-termini confers resistance to metabolic inactivation by MAG peptidases; however the peptide persists for less than 2h in the female reproductive tract after copulation. Aea-HP-1 is not a ligand for the mosquito sex peptide/myoinhibitory peptide receptor. A. aegypti often mate close to the host and therefore it is possible that male-derived Aea-HP-1 induces short-term changes to female host-seeking behavior to reduce potentially lethal encounters with hosts soon after insemination.

Oral activity of FMRFamide-related peptides on the pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae) and degradation by enzymes from the aphid gut.

Insect myosuppressins and myosuppressin analogues were tested for oral toxicity against the pea aphid Acyrthosiphon pisum (Harris) by incorporation into an artificial diet. Acyrthosiphon pisum myosuppressin (Acypi-MS) and leucomyosuppressin (LMS) had significant dose-dependent effects (0.1-0.5µg peptide/µl diet) on feeding suppression, mortality, reduced growth and fecundity compared with control insects, but Acypi-MS was more potent than LMS. One hundred percent of aphids had died after 10days of feeding on 0.5µg Acypi-MS/µl diet whereas 40% of aphids feeding on 0.5µg LMS/µl diet were still alive after 13days. Myosuppressins were degraded by aphid gut enzymes; degradation was most likely due to a carboxypeptidase-like protease, an aminopeptidase and a cathepsin L cysteine protease. The estimated half-life of Acypi-MS in a gut extract was 30min, whereas LMS was degraded more slowly (t½=54min). No toxicity was observed when the analogues δR(9) LMS and citrolline(9) Acypi-MS or FMRFamide were fed to the pea aphid. These findings not only help to better understand the biological effects of myosuppressins in aphids but also demonstrate the potential use of myosuppressins in a strategy to control aphid pests.

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.

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.

Effects of Manduca sexta allatostatin and an analog on the pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae) and degradation by enzymes from the aphid gut.

The C-type allatostatin, Manduca sexta allatostatin (Manse-AS) and the analog delta R(3)delta R(5)Manse-AS, where R residues were replaced by their d-isomers, were tested for oral toxicity against the pea aphid Acyrthosiphon pisum (Harris) by incorporation into an artificial diet. Both peptides had significant dose-dependent feeding suppression effects, resulting in mortality, reduced growth and fecundity compared with control insects. The delta R(3)delta R(5)Manse-AS analog had an estimated LC(50) of 0.18 microg/microl diet, and was more potent than Manse-AS. At a dose of 0.35 microg delta R(3)delta R(5)Manse-AS/microl diet, 98% of aphids were dead within 3 days, at a rate similar to those aphids that had been starved (no diet controls). On comparison, it required 13 days and three times the dose of Manse-AS fed to aphids to attain 96% mortality. It is possible that the feeding suppression effects of Manse-AS on aphids are due to the inhibition of gut motility. The estimated half-life of Manse-AS when incubated with a gut extract from A. pisum was 54 min. Degradation was most likely due to cathepsin L cysteine and/or trypsin-like proteases, by an unidentified glutamine-specific protease and by a carboxypeptidase-like enzyme. The d-isomers of R in the Manse-AS analog appeared to prevent hydrolysis by cathepsin L cysteine and trypsin-like enzymes, and enhance its half-life (145 min). However delta R(3)delta R(5)Manse-AS was cleaved by enzymes with carboxypeptidase-like and chymotrypsin-like activity. The increased stability of the Manse-AS analog may explain its enhanced feeding suppression effects when continually fed to aphids, and demonstrates the potential use of Manse-AS in a strategy to control aphid pests.

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.

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.

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.

Metabolic inactivation of the circadian transmitter, pigment dispersing factor (PDF), by neprilysin-like peptidases in Drosophila.

Recent studies have firmly established pigment dispersing factor (PDF), a C-terminally amidated octodecapeptide, as a key neurotransmitter regulating rhythmic circadian locomotory behaviours in adult Drosophila melanogaster. The mechanisms by which PDF functions as a circadian peptide transmitter are not fully understood, however; in particular, nothing is known about the role of extracellular peptidases in terminating PDF signalling at synapses. In this study we show that PDF is susceptible to hydrolysis by neprilysin, an endopeptidase that is enriched in synaptic membranes of mammals and insects. Neprilysin cleaves PDF at the internal Ser7-Leu8 peptide bond to generate PDF1-7 and PDF8-18. Neither of these fragments were able to increase intracellular cAMP levels in HEK293 cells cotransfected with the Drosophila PDF receptor cDNA and a firefly luciferase reporter gene, confirming that such cleavage results in PDF inactivation. The Ser7-Leu8 peptide bond was also the principal cleavage site when PDF was incubated with membranes prepared from heads of adult Drosophila. This endopeptidase activity was inhibited by the neprilysin inhibitors phosphoramidon (IC(50,) 0.15 micromol l(-1)) and thiorphan (IC(50,) 1.2 micromol l(-1)). We propose that cleavage by a member of the Drosophila neprilysin family of endopeptidases is the most likely mechanism for inactivating synaptic PDF and that neprilysin might have an important role in regulating PDF signals within circadian neural circuits.

Metabolism of cydiastatin 4 and analogues by enzymes associated with the midgut and haemolymph of Manduca sexta larvae.

The degradation of synthetic cydiastatin 4 (ARPYSFGL-amide) and cydiastatin 4 analogues cydiastatin 4alpha (PPPPPARPYSFGL-amide) and cydiastatin 4beta (PPPPPARPYSF[Acpc]L-amide) by enzymes associated with the midgut and/or haemolymph of the tobacco hawkmoth moth, Manduca sexta was investigated using reversed-phase high performance liquid chromatography (RP-HPLC) combined with matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). Cydiastatin 4 had an estimated half-life of c. 16.5min when incubated with midgut tissue in vitro and c. 2.5min with midgut lumen contents. Two degradation products were identified; cydiastatin(1-6), due to cleavage of the C-terminal di-peptide GL-amide, and cydiastatin(2-8), due to cleavage of the N-terminal A residue. Both cydiastatin 4alpha and cydiastatin 4beta had increased stability to gut and haemolymph enzymes, and full biological activity, but reduced potency compared to cydiastatin 4 when assayed on foregut peristalsis. The P-extended N-terminus of both analogues prevented hydrolysis by aminopeptidases and the replacement of the susceptible G residue with cyclopropylalanine ([Acpc]) counteracted carboxypeptidase activity. However, both analogues were susceptible to amidase-like activity giving an increase in one mass unit presumably due to the conversion of the C-terminal amide group to the free carboxylic acid. No metabolism of cydiastatin 4beta occurred when incubated with larval M. sexta haemolymph over a 90min period.

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.