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Purification of small peptide components in the venoms of the solitary sphecid wasps, Sphex argentatus argentatus and Isodontia harmandi, led to the isolation of several major peptides. Analysis of MS/MS spectra by MALDI-TOF/TOF revealed the sequence of a new peptide Sa112 (EDVDHVFLRF-NH2), which is structurally very similar to leucomyosupressin (pQDVDHVFLRF-NH2) and SchistoFLRFamide (PDVDHVFLRF-NH2), the FMRFamide-like peptides from cockroach and locust, respectively. Indeed, this new peptide, like SchistoFLRFamide, inhibited the frequency and amplitude of spontaneous contractions of the locust oviduct in a dose-dependent manner. A non-amidated peptide Sa12b (EDVDHVFLRF) was also isolated, but this peptide had no effect on spontaneous locust oviduct contraction. This is the first example of a FMRF-like peptide to be found in solitary wasp venom. Additionally, a truncated form of the myosuppressins, which has previously been synthesized and tested for biological activity, DVDHVFLRF-NH2 (Sh5b), was found for the first time as a natural product. Four other novel peptides were isolated and characterized as Sa81 (EDDLEDFNPTVS), Sa10 (EDDLEDFNPTIA), Sh41 (DDLSDFNPKV), and Sh42 (EDDLSDFNPKV). They are structurally related to each other, having a high content of acidic amino acids, but no structural similarity to any known peptides. Ion channel associated activities of Sh41 and Sh42 were tested, but did not show any activity for Na+, K+, Ca2+ channels.

The scorpion Tityus serrulatus venom comprises a complex mixture of molecules that paralyzes and kills preys, especially insects. However, venom components also interact with molecules in humans, causing clinic envenomation. This cross-interaction may result from homologous molecular targets in mammalians and insects, such as (NEP)-like enzymes. In face of these similarities, we searched for peptides in Tityus serrulatus venom using human NEP as a screening tool. We found a NEP-inhibiting peptide with the primary sequence YLPT, which is very similar to that of the insect neuropeptide proctolin (RYLPT). Thus, we named the new peptide [des-Arg(1)]-proctolin. Comparative NEP activity assays using natural substrates demonstrated that [des-Arg(1)]-proctolin has high specificity for NEP and better inhibitory activity than proctolin. To test the initial hypothesis that molecular homologies allow Tityus serrulatus venom to act on both mammal and insect targets, we investigated the presence of a NEP-like in cockroaches, the main scorpion prey, that could be likewise inhibited by [des-Arg(1)]-proctolin. Indeed, we detected a possible NEP-like in a homogenate of cockroach heads whose activity was blocked by thiorphan and also by [des-Arg(1)]-proctolin. Western blot analysis using a human NEP monoclonal antibody suggested a NEP-like enzyme in the homogenate of cockroach heads. Our study describes for the first time a proctolin-like peptide, named [des-Arg(1)]-proctolin, isolated from Tityus serrulatus venom. The tetrapeptide inhibits human NEP activity and a NEP-like activity in a cockroach head homogenate, thus it may play a role in human envenomation as well as in the paralysis and death of scorpion preys.

In insects, posttranslational modifications of neuropeptides are largely restricted to C- and N-terminal amino acids. The most common modifications, N-terminal pyroglutamate formation and C-terminal α-amidation, may prevent a fast degradation of these messenger molecules. This is particularly important for peptide hormones. Other common posttranslational modifications of proteins such as glycosylation and phosphorylation seem to be very rare in insect neuropeptides. To check this assumption, we used a computer algorithm to search an extensive data set of MALDI-TOF mass spectra from cockroach tissues for ion signal patterns indicating peptide phosphorylation. The results verify that phosphorylation is indeed very rare. However, a candidate was found and experimentally verified as phosphorylated CAPA pyrokinin (GGGGpSGETSGMWFGPRL-NH2) in the cockroach Lamproblatta albipalpus (Blattidae, Lamproblattinae). Tandem mass spectrometry revealed the phosphorylation site as Ser(5). Phosphorylated CAPA pyrokinin was then also detected in most other cockroach lineages (e.g. Blaberidae, Polyphagidae) but not in closely related blattid species such as Periplaneta americana. This is remarkable since the sequence of CAPA pyrokinin is identical in Lamproblatta and Periplaneta. A consensus sequence of CAPA pyrokinins of cockroaches revealed a conserved motif that suggests phosphorylation by a Four-jointed/FAM20C related kinase.

Huwentoxin-I (HWTX-I) is a 33-residue peptide isolated from the venom of Ornithoctonus huwena and could inhibit TTX-sensitive voltage-gated sodium channels and N-type calcium channels in mammalian dorsal root ganglion (DRG) neurons. However, the effects of HWTX-I on mammalian central neuronal and insect sodium channel subtypes remain unknown. In this study, we found that HWTX-I potently inhibited sodium channels in rat hippocampal and cockroach dorsal unpaired median (DUM) neurons with the IC(50) values of 66.1±5.2 and 4.80±0.58nM, respectively. Taken together with our previous work on DRG neurons (IC(50)≈55nM), the order of sodium channel sensitivity to HWTX-I inhibition was insect central DUM≫mammalian peripheral>mammalian central neurons. HWTX-I exhibited no effect on the steady-state activation and inactivation of sodium channels in rat hippocampal and cockroach DUM neurons.

Immunohistochemical reactivity against short neuropeptide F (sNPF) was observed in the brain-corpus cardiacum and midgut paraneurons of the American cockroach, Periplaneta americana. Four weeks of starvation increased the number of sNPF-ir cells in the midgut epithelium but the refeeding decreased the number in 3h. Dramatic rises in sNPF contents in the midgut epithelium and hemolymph of roaches starved for 4 weeks were confirmed by ELISA. Starvation for 4 weeks reduced α-amylase, protease and lipase activities in the midgut of P. americana but refeeding restored these to high levels. Co-incubation of dissected midgut with sNPF at physiological concentrations inhibited α-amylase, protease and lipase activities. sNPF injection into the hemocoel led to a decrease in α-amylase, protease and lipase activities, whereas PBS injection had no effects. The injection of d-(+)-trehalose and l-proline into the hemocoel of decapitated adult male cockroaches that had been starved for 4 weeks had no effect on these digestive enzymes. However, injection into the hemocoel of head-intact starved cockroaches stimulated digestive activity. Injection of d-(+)-trehalose and l-proline into the lumen of decapitated cockroaches that had been starved for 4 weeks increased enzymes activities and suppressed sNPF in the midgut. Our data indicate that sNPF from the midgut paraneurons suppresses α-amylase, protease and lipase activities during starvation. Injection of d-(+)-trehalose/l-proline into the hemocoel of head-intact starved cockroach decreased the hemolymph sNPF content, which suggests that sNPF could be one of the brain factors, demonstrating brain-midgut interplay in the regulation of digestive activities and possibly nutrition-associated behavioral modifications.

Allatostatins (ASTs) comprise a family of insect neuropeptides isolated from cockroaches and found to inhibit the production of juvenile hormone (JH) by the corpora allata (CA). For this reason, the ASTs can be regarded as possible IGR candidates for pest control. Six peptidomimetic analogs according to the C-terminal pentapeptide of ASTs were prepared by solid-phase organic synthetic methods in an attempt to obtain new simple substitution agents. Assays of inhibition of JH biosynthesis in vitro by corpora allata from the cockroach Diploptera punctata showed that the activity of analog I (IC(50): 0.09 μM) was more active than that of the C-terminal pentapeptide (Tyr-Xaa-Phe-Gly-Leu-NH(2), IC(50): 0.13 μM) it mimicked and the activity of the analog II (IC(50): 0.13 μM) proved roughly equivalent to the C-terminal pentapeptide. The results indicate that a new simple mimicry for Tyr-Xaa-Phe-Gly has been discovered; analog I may be a novel compound candidate for potential IGRs. This study will be useful for the design of new AST analogs for insect management.

Dictyoptera which consist of cockroaches, termites, and praying mantids are among the oldest pterygote insects known. Whereas the localization and sequences of neuropeptides from a number of cockroaches are very well known, nearly nothing is known about the neuropeptides typical of praying mantids. In this study, the neuroanatomy of the median neuroendocrine system in the abdominal ventral nerve cord and the sequences of the CAPA-peptides which are expressed in the respective neuroendocrine cells were analyzed. Altogether, 40 species belonging to different families of Mantodea were included. In contrast to cockroaches, the mantids mostly express two CAPA-periviscerokinins (PVKs), only in Mantis religiosa a third PVK was identified. These PVKs are sequence-related to the PVKs of basal cockroaches (Polyphagidae). In a group of closely related Mantodea (Paramantinae), extended forms of PVK-2 were observed. As shown, these forms are possibly the result of substitutions in the N-terminal cleavage sites of the respective PVKs. No trace of a CAPA-pyrokinin was found in any of the praying mantids.

The allatostatins (ASTs) with Phe-Gly-Leu-amide C-terminal sequence are multifunctional neuropeptides discovered as inhibitors of juvenile hormone (JH) synthesis by corpora allata (CA) of cockroaches. Although these ASTs inhibit JH synthesis only in cockroaches, crickets, termites and locusts, isolation of peptides or of cDNA/genomic DNA or analysis of genomes indicates their occurrence in many orders of insects with the exception of coleopterans. The gene for these ASTs has not been found in the genome of the red flour beetle Tribolium castaneum (Family Tenebrionidae). Yet, in view of widespread occurrence of these peptides in insects, crustaceans and nematodes, they would be expected to occur in beetles. This study provides evidence for the presence of FGLa-like ASTs in the tenebrionid beetle, Tenebrio molitor, and scarabid beetle, Popillia japonica. Extract of brain from both beetles inhibited JH synthesis by cockroach CA dose dependently and reversibly. 20 brain equivalents of T. molitor and P. japonica extracts inhibited JH synthesis 64+/-5 and 65+/-0.6% respectively. Antibody against cockroach allatostatin (Diploptera punctata AST-7) used in an enzyme-linked immunosorbent assay reacted with brain extract of these beetles. Antibody against D. punctata AST-5 localized FGLa-like ASTs in the brain and subesophageal ganglion of T. molitor and P. japonica. In addition, pretreatment of T. molitor brain extract with anti-D. punctata AST-5 reduced the inhibition of JH synthesis and pretreatment of anti-D. punctata AST-5 with D. punctata AST-5 diminished the immunoreactivity of the antibody. Thus we predict that FGLa-like allatostatins will be found in beetles.

The first insect allatotropin-related peptide (ATRP) was isolated from head extracts of the adult sphinx moth Manduca sexta [Kataoka H, Toschi A, Li JP, Carney RL, Schooley DA, Kramer SJ. Identification of an allatotropin from adult Manduca sexta. Science 1989;243:1481-3.]. Meanwhile ATRPs are known from different holometabolous insects but only a single ATRP could be identified from hemimetabolous insects [Paemen L, Tips A, Schoofs L, Proost P, Van Damme J, De Loof A. Lom-AG-myotropin: a novel myotropic peptide from the male accessory glands of Locusta migratoria. Peptides 1991;12:7-10.]. This means that the extensive analysis of neuropeptides from Leucophaea maderae and Periplaneta americana, which led to the discovery of many novel insect neuropeptides, did not result in the detection of any ATRP. In this study, we used another approach to find a cockroach ATRP by first identifying Manse-AT immunoreactive neurons in the terminal ganglion that can be stained by retrograde labeling and are suitable for dissection and subsequent mass spectrometric analysis. The peptidomic analysis of these putative ATRP neurons paved the way for the identification of the first cockroach ATRP. MALDI-TOF/TOF tandem mass spectrometry revealed a sequence identity with Locmi-AG-MT-1 which classifies this ATRP as a highly conserved neuropeptide. A mass spectrometric screening of the nervous system allowed the detection of ATRP-ion signals in different parts of the CNS of P. americana as well as L. maderae. The data obtained in this study will be incorporated in a map of peptidergic neurons from the CNS of the American cockroach, P. americana.

Injection of 0.1 pmol of the octapeptide Peram-AKH II (pGlu-Leu-Thr-Phe-Thr-Pro-Asn-TrpNH(2)) elicits a significant hypertrehalosemic response in the American cockroach, Periplaneta americana; a maximal effect is obtained with 1pmol. The latter amount also lowers the level of neutral lipid (NL) and phospholipid (PL) in the hemolymph. The evidence supports the idea that Peram-AKH II promotes the liberation of fatty acids from hemolymph phospholipid, and indirectly diacylglycerol in the same compartment. The fatty acids are then transported into the fat body where they are converted into triacylglycerol for storge. Because lipolysis and trehalose synthesis are initiated by a common concentration of Peram-AKH II it is reasonable to suggest that the physiological function of Peram-AKH II involves the participation of both metabolic pathways.

The midgut plays a major role in digestion and absorption of nutrients in insects, and contains endocrine cells throughout the epithelial layer that express neuropeptides, including crustacean cardioactive peptide (CCAP). In the present study, we demonstrate regulation of digestive enzyme activities by CCAP in response to nutrient ingestion in the cockroach, Periplaneta americana. The midgut of the cockroach exhibits maximal alpha-amylase and protease activities 3 h after intake of either starch or casein, but not of non-nutrients. Similar time-dependent responses of CCAP expression in midgut endocrine cells were observed after feeding starch and casein, but not after non-nutrients. We also show that incubation of the dissected midgut with CCAP leads to an increase in alpha-amylase and protease activity in a time-dependent manner, with the maximal activity at 2 h. Taken together, our data indicate the existence of an inducible mechanism where endocrine cells in the midgut are stimulated to synthesize and secrete CCAP by nutrients, and CCAP then up-regulates the activity of digestive enzymes.

The aliphatic amino diacid alpha-aminosuberic acid can function as an effective, stable mimic of the hydrolysis-susceptible Tyr(SO3H) group in sulfakinin neuropeptide analogs for both hindgut contractile activity in cockroach and food intake-inhibition activity in the desert locust. In the analog, the acidic sulfate group is replaced with an acidic carboxyl group. The degree of activity of sulfakinin analogs is correlated with the carboxyl/alpha-carbon distance in the cockroach hindgut contractile assay. The results represent an important step in the design and synthesis of biostable, sulfakinin analogs that could potentially suppress the feeding behavior of destructive insect pests of agricultural importance.

The primary structures and molecular mass data of neuropeptides that are released from major neuroendocrine sites of the American cockroach, Periplaneta americana, are well known. In the current study we extensively surveyed neuropeptides of the corpora cardiaca, the corpora allata and the abdominal perisympathetic organs, from 14 related cockroach species belonging to the family Blattidae. Mainly, this survey was executed by using mass spectrometric methods (MALDI-TOF MS). Peptides which appeared to be modified, as judged from their mass data and comparison with known data from P. americana, were fragmented by means of the post-source decay technique to determine their amino acid sequence. Single organ preparations sufficed to reveal the peptide pattern in neurohemal organs and to identify species-specific modifications, making it possible to glean information also from tissues of less abundant species that were caught during field trips. The peptide inventory described in this study is very typical of cockroaches of the family Blattidae and clearly separates this taxon from other taxa of cockroaches. The majority of neuropeptides was identical in all investigated species. Some peptides, however, displayed remarkable variations in their sequence which hint to a differential rate of modification among peptides of the same neuropeptide family. These modifications serve to distinguish further relatedness of taxa within the Blattidae.

Myosuppressins are a group of 10-residues FMRFamide-related peptides reported in Dictyoptera, Orthoptera, Lepidoptera and Diptera. Myosuppressins inhibit visceral muscle contractions and, in the cockroach Blattella germanica, inhibit food intake. In B. germanica, the cDNA of leucomyosuppressin (LMS) has been cloned and sequenced. The deduced precursor is 96 amino acids long and contains a single copy of LMS. Brain mRNA levels remain constant during the first reproductive cycle of adult females, whereas those in the gut show a slight decline during the time of maximal food intake. Comparison of myosuppressin precursors of different species reveals that all have the same organization. Phylogenetic analysis suggests that the precursor experienced an accelerated evolution in Lepidoptera and Diptera with respect to Dictyoptera, whereas only Lepidoptera has radical changes in the bioactive peptide.

Corpora allata (CA) of embryos of Diploptera punctata have been previously shown to produce JH III. We have re-examined sesquiterpenoid biosynthesis throughout embryonic development and have found that early embryos produce both methyl farnesoate (MF) and JH III; as development proceeds, less MF and more JH is produced. The cockroach allatostatin peptide Dippu-allatostatin (AST) 7 inhibits sesquiterpenoid production by CA of mid to late embryos whereas it exerts a dose-dependent stimulatory effect in early embryos. This stimulatory effect is particularly apparent on MF biosynthesis. CA become innervated by allatostatin-containing nerves in early embryos (35% development). Shortly thereafter, the allatostatin-containing innervation of the CA appears complete.

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.

Several cardioactive peptides have been identified in insects and most of them are likely to act on the heart as neurohormones. Here we have investigated the cardioactive properties of members of a family of insect tachykinin-related peptides (TRPs) in heterologous bioassays with two coleopteran insects, Tenebrio molitor and Zophobas atratus . Their effects were compared with the action of the pentapeptide proctolin. We tested the cardiotropic activity of LemTRP-4 isolated from the midgut of the cockroach Leucophaea maderae , CavTK-I and CavTK-II isolated from the blowfly Calliphora vomitoria . The semi-isolated hearts of the two coleopteran species were strongly stimulated by proctolin. We observed a dose dependent increase in heartbeat frequency (a positive chronotropic effect) and a decrease in amplitude of contractions (a negative inotropic effect). In both beetles the TRPs are less potent cardiostimulators and exert lower maximal frequency responses than proctolin. LemTRP-4 applied at 10 −9 –10 −6 M was cardiostimulatory in both species inducing an increase of heart beat frequency. The amplitude of contractions was stimulated only in Z. atratus . CavTK-I and CavTK-II also exerted cardiostimulatory effects in Z. atratus at 10 −9 –10 −6 M. Both peptides stimulated the frequency, but only CavTK-II increased the amplitude of the heart beat. In T. molitor , however, the CavTKs induced no significant effect on the heart. Immunocytochemistry with antisera to the locust TRPs LomTK-I and LomTK-II was employed to identify the source of TRPs acting on the heart. No innervation of the heart by TRP immunoreactive axons could detected, instead it is possible that TRPs reach the heart by route of the circulation. The likely sources of circulating TRPs in these insects are TRP-immunoreactive neurosecretory cells of the median neurosecretory cell group in the brain with terminations in the corpora cardiaca and endocrine cells in the midgut. In conclusion, LemTRP-4, CavTK-I and CavTK-II are less potent cardiostimulators than proctolin and also exert stimulatory rather than inhibitory action on amplitude of contractions. The differences in the responses to proctolin and TRPs suggest that the peptides regulate heart activity by different mechanisms.

Neuropeptides of the cockroach allatostatin (AST) family are known for their ability to inhibit the production of juvenile hormone by the corpora allata of cockroaches. Since their discovery, they have also been shown to modulate myotropic activity in a range of insect species as well as to act as neurotransmitters in Crustaceans and possibly in insects. The midgut of cockroaches contains numerous endocrine cells, some of which produce AST whereas others produce the FMRFamide-related peptide, leucomyosuppressin (LMS). We have determined if ASTs and LMS are also able to influence carbohydrate-metabolizing enzyme activity in the midgut of the cockroach, Diploptera punctata. Dippu-AST 7 stimulates activity of both invertase and alpha-amylase in a dose-dependent fashion in the lumen contents of ligatured midguts in vitro, but not in midgut tissue, whereas the AST analog AST(b)phi2, a cyclopropyl-ala, hydrocinnamic acid analog of Dippu-AST 6, has no effect. Leucomyosuppressin also stimulates enzyme activity in lumen contents only, although the EC50 is considerably greater than for Dippu-AST. Dippu-AST is also able to inhibit proctolin-induced contractions of midgut muscle, and this action had already been described for LMS [18]. Thus, in this organ, AST and LMS have at least two distinct physiological effects.

Muren, J. E. and D. R. NASsel. Seven tachykinin-related peptides isolated from the brain of the Madeira cockroach: Evidence for tissue-specific expression of isoforms. Peptides 18(1) 7–15, 1997.—We have isolated seven tachykinin-related peptides (TRPs) from an acidic extract of 1000 brains of the cockroach Leucophaea maderae. Four different reversed-phase high performance liquid chromatography (RP-HPLC) column systems were required to obtain pure peptides. During the purification the fractions were monitored in a radioimmunoassay (RIA) with an antiserum to the locust TRP locustatachykinin I (LomTK I) and a cockroach hindgut muscle contraction bioassay. The sequences of the seven isolated LomTK immunoreactive and myostimulatory peptides were determined by Edman degradation. Six of these were confirmed by mass spectrometry and chemical synthesis as: APSGFLGVRamide, APAMGFQGVRamide, APAAGFFGMRamide, VPASGFFGMRamide, GPSMGFHGMRamide, and APSMGFQGMRamide. The seventh peptide, APEESPKRAPSGFLGVRamide, was confirmed only by mass spectrometry. These peptides were designated Leucophaea maderae tachykinin-related peptides 1, 2, 5–9 (LemTRP 1, 2, 5–9). Two more peptides were isolated using the same assays: SGLDSLSGATFGGNR and ALFEESTVSAEPR. The first shares the C-terminus FX1GX2R with the LemTRPs, whereas the second one is not related to the TRPs (none of these peptides were synthesized and thus putative C-terminal amidation was not confirmed). Three of the brain peptides, LemTRP 1, 2, and 5, have previously been isolated from the midgut of L. maderae, whereas the others appear to be brain specific. Because the LemTRPs 3 and 4 appear to be unique for the midgut, we have indications for tissue-specific expression of TRPs. All seven confirmed LemTRPs of the brain are myotropic and induce increases in the amplitude and frequency of spontaneous contractions and tonus of hindgut muscle in L. maderae.

A series of insect kinin analogues, AFFPWG-X, modified at the C -terminal group, were evaluated in a cricket Malpighian tubule secretion bioassay. The results were compared with activity profiles observed in a cockroach hindgut myotropic bioassay for these analogues. Although the replacement of the C -terminal amide group with a negatively charged acid leads to a precipitous drop in diuretic activity, it can be partially restored with the introduction of ester groups such as methyl or benzyl. The presence of branched chain character in the C -terminal group or a C -terminal α-carbon-amide distance spanning five methylene group spacers is incompatible with the receptor interaction required for biological activity. Significant diuretic activity is retained with four or fewer methylene groups in this region. C -terminal group analogues containing -SCH 3 , -NHCH 2 CH 2 OCH 3 , or -OCH 2 (C 6 H 5 ) offered the greatest retention of diuretic activity while providing increased hydrophobicity and/or steric bulk. The data are of potential value in the development of mimetic analogues of this insect neuropeptide family. Mimetic analogues are potentially valuable tools to insect neuroendocrinologists studying diuresis and/or engaged in the development of future pest management strategies.

A new member of the AKH/RPCH family was isolated from the corpora cardiaca of the firebug Pyrrhocoris apterus. It is the second adipokinetic peptide identified in this species. The peptide was characterized and its structure was deduced from the multiple MSN electrospray mass spectra as that of an octapeptide with the sequence pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH2. The peptide differs from the original P. apterus AKH (Pya-AKH) by one amino acid in position 3. Topical application and/or injection of the peptide induced lipid mobilization, but was inactive in mobilization of carbohydrates.

A family of myotropic neuropeptides sharing the common C- terminal pentapeptide Phe-Xxx-Pro-Arg-Leu-NH2 (Xxx = Ser, Thr, Val), known as the pyrokinins, has been isolated from the cockroach Leucophaea maderae and locust Locusta migratoria of the order Orthoptera. A hormone (Bom-DH) that elicits diapause induction in the silkworm Bombyx mori (order Lepidoptera) also contains this C- terminal pentapeptide (Xxx = Gly). The orthopteran pyrokinin neuropeptides elicit significant diapause-inducing activity in the lepidopteran silkworm. Despite containing the sterically bulky, inflexible Val residue in the variable Xxx position, the locust pyrokinin Lom-PK is threefold more active than native Bom-DH as a diapause induction agent. The C- terminally truncated cockroach leucopyrokinin (LPK) fragment. Thr-Ser-Phe-Thr-Pro-Arg-NH2 [LPK(2–7)], proved virtually inactive in the silkworm assay, demonstrating the importance of an intact C- terminal pentapeptide sequence to diapause induction activity. Bom-DH also elicits significant myostimulatory activity in a cockroach hindgut assay, although at a level several orders of magnitude less than the native myotropic peptide LPK. However, the C- terminal pentapeptide of Bom-DH (Xxx = Gly) is equipotent with the LPK C- terminal pentapeptide (Xxx = Thr) as a myostimulatory agent. The cross-activity observed for the various pyrokinins suggests that the receptors that mediate the disparate physiological processes of diapause in the silkworm and hindgut contraction in the cockroach share homologous features. Previous data suggest that pyrokinins demonstrate cross-activity in silkworm pheromone production, locust/cockroach oviduct myotropic assays, and in armyworm cuticular melanization. These results, combined with the present work, increase the known spectrum of cross-activity for this insect neuropeptide family to include five different physiological processes.

An amidated decapeptide, exhibiting strong inhibitory activity of spontaneous visceral muscle movements, was isolated from 9000 brain-corpora cardiaca-corpora allata-subesophageal ganglion complexes of the migratory locust, Locusta migratoria. During the process of HPLC purifications, the biological activity of the fractions was monitored using the isolated hindgut of the cockroach Leucophaea maderae. The primary structure of this myotropic peptide is Pro-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-NH2 and is identical to SchistoFLRFamide isolated from the grasshopper, Schistocerca gregaria. It shares the carboxy-terminal sequence FLRFamide with several identified peptides from different phyla. At this moment, six decapeptides isolated from different insect species are identical at 7 of the 10 amino acid residues (X-D-V-X-H-X-FLRFamide). The cockroach, fly, and locust peptides differ only by the N-terminal amino acid residue. Synthetic SchistoFLRFamide showed biological as well as chemical characteristics indistinguishable from the native peptide. It provoked a decrease in frequency and amplitude of contractions of the locust oviduct. By means of a polyclonal antiserum directed against the carboxy terminal of SchistoFLRFamide, we demonstrated that the male accessory glands, the heart, the oviduct, and the salivary glands were innervated by axons containing SchistoFLRFamide-like immunoreactivity. Administration of SchistoFLRFamide elicited an immediate effect on the basal membrane potential of the opalescent tubule gland cells.

The tachykinin-related peptides (TRPs) are multifunctional neuropeptides found in a variety of arthropod species, including the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae). Two new biostable TRP analogs containing multiple, sterically hindered Aib residues were synthesized and found to exhibit significantly enhanced resistance to hydrolysis by angiotensin converting enzyme and neprilysin, membrane-bound enzymes that degrade and inactivate natural TRPs. The two biostable analogs were also found to retain significant myostimulatory activity in an isolated cockroach hindgut preparation, the bioassay used to isolate and identify the first members of the TRP family. Indeed one of the analogs (Leuma-TRP-Aib-1) matched the potency and efficacy of the natural, parent TRP peptide in this myotropic bioassay. The two biostable TRP analogs were further fed in solutions of artificial diet to the pea aphid over a period of 3 days and evaluated for antifeedant and aphicidal activity and compared with the effect of treatment with three natural, unmodified TRPs. The two biostable multi-Aib TRP analogs were observed to elicit aphicidal effects within the first 24 h. In contrast natural, unmodified TRPs, including two that are native to the pea aphid, demonstrated little or no activity. The most active analog, double-Aib analog Leuma-TRP-Aib-1 (pEA[Aib]SGFL[Aib]VR-NH(2)), featured aphicidal activity calculated at an LC(50) of 0.0083 nmol/μl (0.0087 μg/μl) and an LT(50) of 1.4 days, matching or exceeding the potency of commercially available aphicides. The mechanism of this activity has yet to be established. The aphicidal activity of the biostable TRP analogs may result from disruption of digestive processes by interfering with gut motility patterns and/or with fluid cycling in the gut; processes shown to be regulated by the TRPs in other insects. These active TRP analogs and/or second generation analogs offer potential as environmentally friendly pest aphid control agents.

The pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family plays a multifunctional role in an array of important physiological processes in a variety of insects. An active core analog containing an (E)-alkene, trans-Pro isosteric component was evaluated in four disparate PK/PBAN bioassays in four different insect species. These bioassays include pheromone biosynthesis in the moth Heliothis peltigera, melanization in the larval Spodoptera littoralis, pupariation acceleration in the larval fly Neobellieria bullata, and hindgut contraction in the cockroach Leucophaea maderae. The conformationally constrained analog demonstrated activity equivalent to parent PK/PBAN peptides of equal length in all four PK/PBAN bioassays, and matched and/or approached the activity of peptides of natural length in three of them. In the melanization bioassay, the constrained analog exceeded the efficacy (maximal response) of the natural PBAN1-33 by a factor of 2 (at 1 nmol). The results provide strong evidence for the orientation of Pro and the core conformation adopted by PK/PBAN neuropeptides during interaction with receptors associated with a range of disparate PK/PBAN bioassays. The work further identifies a scaffold with which to design mimetic PK/PBAN analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting PK/PBAN-regulated systems.

Insect growth regulators play an important role in Integrated Pest Management systems. Cockroach-type allatostatins (FGLamides) (ASTs), which are a family of basic peptides first isolated from brains of Diploptera punctata were originally discovered on the basis of their ability to inhibit the production of juvenile hormone by the corpora allata. For this reason, the ASTs can be regarded as possible IGR candidates for pest control although the absence of effect in vivo, rapid degradation and high production costs of the natural peptides preclude their use in pest management. However, we have synthesized a new AST mimic, H17 , from the pentapeptide C-terminal active core of the AST. This mimic is able to significantly inhibit the biosynthesis of JH by cockroach CA in vitro (IC 50 value: 12 nM) and in vivo following injection (IC 50 value: 33 nM). H17 also shows a highly significant inhibition of JH production in topical cuticular assays in vivo. Our results suggest that H17 has potential as an IGR for cockroach control.

An ELISA for corazonin, a cardioactive neuropeptide from the American cockroach, Periplaneta americana, was developed. It was used to isolate corazonin from the cockroach Nauphoeta cinerea, the locust Schistocerca americana, and the hawkmoth Manduca sexta. The peptides from Nauphoeta and Manduca had the same retention times as Periplaneta corazonin, and their amino acid compositions also suggested that these peptides are identical with corazonin. The corazonin-immunoreactive peptide from Schistocerca eluted slightly earlier on HPLC than corazonin, and its structure was determined to be [His7]corazonin, or pGlu-Thr-Phe-Gln-Tyr-Ser-His-Gly-Trp-Thr-Asn-amide. These results indicate that corazonin is generally present in insects and that its structure has been well conserved.

Allatostatins (ASTs), with a C-terminal sequence Tyr/Phe-Xaa-Phe-Gly-Leu/Ile-amide, are multifunctional neuropeptides that were first discovered by their ability to inhibit juvenile hormone (JH) synthesis by the corpora allata (CA) in cockroaches. These A-type ASTs have since been demonstrated to inhibit JH synthesis in crickets, termites and more recently locusts. The gene for the precursor of A-type ASTs has been identified in several species of cockroaches, in crickets and in locusts, but not yet in termites, although 5 AST peptides were isolated from the lower termite Reticulitermes flavipes that are identical to known cockroach ASTs. In this study, primers designed from AST amino acid sequences of cockroaches are used to identify the gene for the preproAST peptides in R. flavipes. In addition, the expression of the gene in brain tissues is demonstrated for egg-laying and non-egg-laying neotenic reproductives. The gene codes for 14 individual peptides and its sequence is closer to that of cockroaches and the cricket than to that of other insect orders in which these peptides do not act as allatostatins. Among the known cockroach AST genes, the termite AST gene is most similar to that of Periplaneta americana, a species belonging to the primitive family Blattidae.

The allatostatins (ASTs), with a Tyr/Phe-Xaa-Phe-Gly-Leu/Ile-amide C-terminus, are neuropeptides that occur in many orders of insects, but are known to inhibit juvenile hormone (JH) synthesis by corpora allata (CA) only in cockroaches, crickets, and termites. 5 AST peptides with similar sequences to those of 6 species of cockroaches have been isolated and sequenced from extract of brain tissue of the termite Reticulitermes flavipes. The amino acid sequence of a 6th peptide, R. flavipes AST-7, determined by LC–MS/MS following HPLC fractionation of brain extract, is S-P-S-S-G-N-Q-R-L-Y-G-F-G-L-NH2. The 8 terminal amino acids are identical to AST-7 of the cockroach Diploptera punctata. R. flavipes and D. punctata AST-7s inhibited JH synthesis by CA of both species equally and their affinity for antibody against D. punctata AST-7 is similar. Immunoreactivity of termite tissue with this antibody indicates neuro- and myomodulatory activity of the peptide in addition to its demonstrated allatostatic function. The density of AST immunostaining in axons within the CA of R. flavipes and the rate of JH synthesis by similar glands were negatively correlated. This is evidence that when AST is abundant in the glands it is being released in vivo to limit JH production.

A number of evidences suggest that tachykinin-related peptides (TRPs) of insects can stimulate food consumption after being released from the midgut to the hemolymph. The idea of the present work has been to test this hypothesis in the anautogenous cockroach Blattella germanica. First, we have identified the peptide LemTRP-1 (APSGFLGVR-NH2) from brain extracts, by means of an ELISA developed with a polyclonal antibody against this peptide. ELISA studies have also shown that, whereas brain LemTRP-1 levels were fairly constant, midgut levels increase to a maximum on day 3 after adult emergence, falling thereafter until the end of the gonadotrophic cycle. Interestingly, maximum values of food consumption are concomitant with the decrease of LemTRP-1 immunoreactivity in the midgut. Furthermore, starvation decreases LemTRP-1 immunoreactivity in midgut, whereas in the hemolymph it increases. Finally, injection of synthetic LemTRP-1 to adult females significantly stimulates food consumption. The whole observations suggest that LemTRP-1 is released from the midgut to the hemolymph when sustained food consumption is required to maintain vitellogenesis at the highest levels, and that LemTRP-1 in the hemolymph stimulates food consumption in these days. © 2007 Elsevier Inc. All rights reserved., Financial support from the Spanish Ministry of Science and Technology (projects AGL2002-01169 and AGL2005-00773 (X.B.) and BFU2006-01090 (J.L.M.)) and the Generalitat de Catalunya (2005 SGR 00053) are gratefully acknowledged.

Allatostatins are a family of related neuropeptides that play an important role in development, reproduction, and digestion in insects. The cockroach Diploptera punctata has 13 allatostatin neuropeptides, with pleiotropic functions, two of which are: inhibition of juvenile hormone (JH) production and inhibition of gut muscle contraction. In this study, the conformation and dynamics of D. punctata allatostatin 5 (Dippu-AST 5) and allatostatin 8 (Dippu-AST 8) are investigated by CD, NMR, and molecular dynamics simulations. These peptides contain eight and nine residues, respectively, and the identical six-residue C-terminal motif. Yet Dippu-AST 5 and Dippu-AST 8 affect juvenile hormone production and hindgut contraction with different potencies. Dippu-AST 5 is one of the most potent inhibitors of juvenile hormone production and one of the least potent inhibitors of gut contraction, whereas Dippu-AST 8 has the opposite potencies with respect to these tissues. From the NMR structure, it is clear that Dippu-AST 5 has a 3(10) helix involving three of its residues and a "gamma" turn at the end of its C-terminal motif. In contrast Dippu-AST 8 has an open "pi" turn among five of its central residues. In addition, the orientation preferences within the membrane of the two peptides were simulated. Our simulation results show that the C-terminal segment of Dippu-AST 5 orients in the membrane surface with an average angle of 17.5 degrees, whereas Dippu-AST 8 orients with an average angle of 5.1 degrees. Taken together, from the structures and orientation preferences of these peptides within the membrane, it appears that these peptides may interact with the receptor very differently.

Two Drosophila receptors (AlstR/DAR-1 and DAR-2) with sequence similarity to mammalian galanin receptors have been previously identified. These receptors have been shown to form specific interactions with neuropeptides that resemble cockroach allatostatins (ASTs), which have a characteristic Tyr/Phe-Xaa-Phe-Gly-Leu-NH 2 carboxyl-terminus. We hypothesized that similar allatostatin receptors exist in the cockroach Diploptera punctata that may regulate the numerous effects that this family of peptides exerts on a range of target tissues. The polymerase chain reaction (PCR) was used, with primer design based on the Drosophila allatostatin receptor (AlstR). Using these primers, a putative allatostatin-like receptor cDNA was isolated from a λZAP-cDNA library prepared from the corpora allata of the D. punctata . As an approach to testing the function of this receptor in vivo , the technique of double-stranded RNA (dsRNA) gene interference was tested. Initial experiments suggest that the putative inhibition of receptor RNA expression may increase juvenile hormone (JH) production.

Corazonin is a peptidergic neurohormone of insects that is expressed in neurosecretory neurons of the pars lateralis of the protocerebrum and transported via nervi corporis cardiaci to the storage lobes of the corpora cardiaca. This peptide occurs with a single isoform in all insects studied so far, with the exception of the Coleoptera in which no corazonin form could be detected. Very few modifications of [Arg(7)]-corazonin, originally isolated from cockroaches, are known, namely [His(7)]-corazonin which is expressed in certain locusts and the stick insect Carausius morosus, and [Thr(4), His(7)]-corazonin recently described from the honey bee Apis mellifera. In this study, we performed a comprehensive screening for corazonin in the different insect groups after detecting of a fourth isoform in a crane fly, Tipula sp. ([Gln(10)]-corazonin). [Arg(7)]-corazonin is distributed in most major lineages of insects, and is thus the ancient form which was present at the time the phylum Insecta evolved. The replacement of Arg with His at position 7 from the N-terminus occurred several times in the evolution of insects. The third isoform, [Thr(4), His(7)]-corazonin, seems to be restricted to bees (Apidae); whereas wasps (Vespidae) and a bumble bee (Apidae) express other corazonins, specifically [His(7)]-corazonin and [Tyr(3), Gln(7), Gln(10)]-corazonin, respectively. A novel corazonin form, [His(4), Gln(7)]-corazonin, was also detected in all South African members of the newly described insect order Mantophasmatodea. The [His(4), Gln(7)]-corazonin separates these species from the Namibian Mantophasmatodea which express [Arg(7)]-corazonin and can be used as a distinct character to distinguish these morphologically similar insects.

The corpus cardiacum (CC) and corpus allatum (CA) of the locust, Locusta migratoria, contain intense proctolin-like immunoreactivity (PLI) within processes and varicosities. In contrast, in the cockroach, Diploptera punctata, although a similar staining pattern occurs within the CC, PLI appears absent within the CA. The possible role of proctolin as a releasing factor for adipokinetic hormone (AKH) and juvenile hormone (JH) was investigated in the locust. Proctolin caused a dose-dependent increase in AKH I release (determined by RP-HPLC) from the locust CC over a range of doses with threshold above 10(-8)M and maximal release at about 10(-7)M proctolin. Isolated glandular lobes of the CC released greater amounts of AKH I following treatment with proctolin and in these studies AKH II was also released. Confirmation of AKH I release was obtained by injecting perfusate from incubated CCs into locusts and measuring hemolymph lipid concentration. Perfusate from CC incubated in proctolin contained material with similar biological activity to AKH. Proctolin was also found to significantly increase the synthesis and release of JH from locust CA, with the increase being greatest from CAs that had a relatively low basal rate of JH biosynthesis (

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

The pentapeptide proctolin, originally identified in the cockroach, has been shown to be widely distributed in many insects and to have a broad range of physiological functions. In the oviduct of the locust, Locusta migratoria , proctolin’s role as a neurotransmitter/neuromodulator has been well documented; however, a neurohormonal role in the locust is less certain. This review will examine the various roles of proctolin in locust oviduct contraction and will present evidence that a substance chromatographically, immunologically and physiologically indistinguishable from proctolin is present in the hemolymph of the locust, L. migratoria . This material is concentrated in the plasma, rather than the hemocytes, and is present at concentrations ranging from 0.1 to 0.2 nM. This review extends the role of proctolin in insects, and suggests that proctolin may play a neurohormonal role in the locust.

The first member of the periviscerokinin peptide family in Locusta migratoria was identified by post-source decay fragmentation on a MALDI-TOF mass spectrometer using a single neurohemal organ only. The primary sequence of this decapeptide, code-named Lom-PVK, is Ala-Ala-Gly-Leu-Phe-Gln-Phe-Pro-Arg-Val-NH 2 . Unlike the situation in cockroaches, Lom-PVK is the only abundant periviscerokinin in L. migratoria . It is present in abdominal perisympathetic organs of various species of locusts and grasshoppers. Its myotropic properties, namely to increase the frequency of the contraction of the heart in L. migratoria and stimulate amplitude and tonus of the locust foregut, is reminiscent of the action of Periplaneta -PVKs.

Allatostatins (ASTs) of the Tyr/Phe-Xaa-Phe-Gly Leu/Ile-NH2 family are a group of insect neuropeptides that inhibit juvenile hormone biosynthesis by the corpora allata. We have obtained genomic DNA sequences that specify the preproallatostatin precursor for the cockroaches, Blatta orientalis, Blattella germanica, Blaberus craniifer and Supella longipalpa. The sequences obtained are similar to those of Diploptera punctata and Periplaneta americana reported previously. The precursors of all these cockroach species are similar in size, and the organization of the ASTs that they contain (there are 13 or 14, depending on the species) have been conserved. With the sequences of these precursors, and using the homologous sequence in the orthopteran Schistocerca gregaria as an outgroup, a phylogenetic analysis using parsimony was carried out. The dendrograms obtained from these analyses, using the amino acid as well as the nucleotide sequences, are comparable with current models for cockroach phylogeny. Parsimony analysis was also used to study the genealogy of the different ASTs within the same precursor. Results suggest that the AST sequences were generated through a process of internal gene duplication which occurred before these species diverged from each other in evolutionary time.

While neuropeptides of the allatostatin family inhibit in vitro production of juvenile hormone, which modulates aspects of development and reproduction in the cockroach, Diploptera punctata, they are susceptible to inactivation by peptidases in the hemolymph, gut, and bound to internal tissues. Patterns of peptidase cleavage were investigated in two allatostatin analogs in which sterically bulky components were incorporated into the active core region to block peptidase attack. The results were used to design and synthesize the first pseudopeptide analog of an insect neuropeptide resistant to degradation by both hemolymph and tissue-bound peptidases. This pseudotetrapeptide allatostatin mimetic analog represents a valuable tool to neuroendocrinologists studying mechanisms by which the natural peptides operate and the physiological consequences of challenging an insect with an allatostatin that is not readily degraded via peptidase enzymes. Disruption of critical physiological processes modulated by neuropeptides such as the allatostatins via peptidase-resistant mimetic analogs could form the basis for novel pest insect management strategies in the future.