Peptides (v.28, #1)
Editorial Board (CO2).
Contents List (v-vi).
Invertebrate neuropeptides VII by Ronald J. Nachman (1-2).
Corazonin in insects by Reinhard Predel; Susanne Neupert; William K. Russell; Olaf Scheibner; Ronald J. Nachman (3-10).
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 [Arg7]-corazonin, originally isolated from cockroaches, are known, namely [His7]-corazonin which is expressed in certain locusts and the stick insect Carausius morosus, and [Thr4, His7]-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. ([Gln10]-corazonin). [Arg7]-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, [Thr4, His7]-corazonin, seems to be restricted to bees (Apidae); whereas wasps (Vespidae) and a bumble bee (Apidae) express other corazonins, specifically [His7]-corazonin and [Tyr3, Gln7, Gln10]-corazonin, respectively. A novel corazonin form, [His4, Gln7]-corazonin, was also detected in all South African members of the newly described insect order Mantophasmatodea. The [His4, Gln7]-corazonin separates these species from the Namibian Mantophasmatodea which express [Arg7]-corazonin and can be used as a distinct character to distinguish these morphologically similar insects.
Keywords: Mass spectrometry; Insect neuropeptide; Hymenoptera; Diptera; Mantophasmatodea;
Mass spectrometric analysis of FMRFamide-like immunoreactive neurons in the prothoracic and subesophageal ganglion of Periplaneta americana by Susanne Neupert; Matthias Gundel (11-17).
MALDI-TOF mass spectrometry combined with immunocytochemistry and retrograde labeling, was used to study the expression pattern and morphology of Pea-FMRFamide-related peptides in single neurons of the prothoracic ganglion and the subesophageal ganglion (SEG) of the American cockroach Periplaneta americana. In contrast to the postero-lateral cells (PLCs) of the meta- and mesothoracic ganglion, the prothoracic FMRFamide-related peptides expressing neurons not only extend in the posterior median nerve but also in an anterior median nerve, which is described herein. The peptidome of the prothoracic PLCs is identical with that of the meso- and metathoracic neurons, respectively. In this study, we identified a truncated form of Pea-FMRFa-24 which was found to be more abundant than the peptide originally designated as Pea-FMRF-24. FMRFamide-related peptides expressing postero-lateral cells were also detected in the labial neuromere of the SEG. Although their projection could not be solved, mass spectrometric analyses revealed the same peptide complement in these neurons as found in the thoracic postero-lateral cells. In all neurons which we studied no co-localized peptides of other peptide families were observed.
Keywords: Peptidomics; Single cell analysis; Mass spectrometry; FMRFamide; Neuropeptide; Insect;
I-conotoxins in vermivorous species of the West Atlantic: Peptide sr11a from Conus spurius by Manuel B. Aguilar; Estuardo López-Vera; Edgar P. Heimer de la Cotera; Andrés Falcón; Baldomero M. Olivera; María Maillo (18-23).
Peptide sr11a was purified from the venom of Conus spurius, a vermivorous cone snail collected in the Yucatan Channel, in the Western Atlantic. Its primary structure was determined by automatic Edman degradation after reduction and alkylation. Its molecular mass, as determined by MALDI-TOF mass spectrometry (average mass 3650.77 Da), confirmed the chemical data (calculated average mass, 3651.13 Da). The sequence of peptide sr11a (CRTEGMSCγγNQQCCWRSCCRGECEAPCRFGP&; γ, gamma-carboxy-Glu; &, amidated C-terminus) shows eight Cys residues arranged in the pattern that defines the I-superfamily of conotoxins. Peptide sr11a contains two gamma-carboxy-Glu residues, a post-translational modification that has been found in other I-conotoxins from species that live in the West Pacific: r11e from the piscivorous Conus radiatus, and κ-BtX from the vermivorous Conus betulinus. Peptide sr11a is the eighth I-conotoxin isolated from a Conus venom and the first I-conotoxin from a species from the Western Atlantic. Peptide sr11a produced stiffening of body, limbs and tail when injected intracranially into mice.
Keywords: Conidae; Cone snail; Western Atlantic; Conus spurius; Conotoxin; I-conotoxin;
An O-conotoxin from the vermivorous Conus spurius active on mice and mollusks by Karen S. Luna-Ramírez; Manuel B. Aguilar; Andrés Falcón; Edgar P. Heimer de la Cotera; Baldomero M. Olivera; María Maillo (24-30).
Here, we report the purification, amino acid sequence and a preliminary biological characterization of a peptide, sr7a, from the venom of Conus spurius, a vermivorous species collected in the Yucatan Channel, Mexico. The peptide consists of 32 amino acid residues (CLQFGSTCFLGDDDICCSGECFYSGGTFGICS&; &, amidated C-terminus) and contains six cysteines arranged in the pattern (C–C–CC–C–C) that characterizes the O-superfamily of conotoxins. This superfamily includes several pharmacological families (ω-, κ-, μO-, δ- and γ-conotoxins) that target Ca2+, K+, Na+ and pacemaker voltage-gated ion channels. Compared with other O-conotoxins that were purified from venoms, this peptide displays sequence similarity with ω-SVIA (from Conus striatus), δ-TxVIA/B (from Conus textile), ω-CVID (from Conus catus) and κ-PVIIA (from Conus purpurascens). At a dose of 250 pmol, peptide sr7a elicited hyperactivity when injected intracranially into mice and produced paralysis when injected into the pedal muscle of freshwater snails, Pomacea paludosa, but it had no apparent effect after intramuscular injection into the limpet Patella opea or the freshwater fish Lebistes reticulatus.
Keywords: Conidae; Cone snail; Western Atlantic; Conus spurius; Conotoxin; O-conotoxin;
Wide phylogenetic distribution of Scorpine and long-chain β-KTx-like peptides in scorpion venoms: Identification of “orphan” components by Elia Diego-García; Elisabeth F. Schwartz; Gina D'Suze; Sergio A. Román González; Cesar V.F. Batista; Blanca I. García; Ricardo C. Rodríguez de la Vega; Lourival D. Possani (31-37).
Scorpine and toxins specific for potassium channels of the family beta (β-Ktx) are two types of structurally related scorpion venom components, characterized by an unusually long extended N-terminal segment, followed by a Cys-rich domain with some resemblance to other scorpion toxins. In this communication, we report evidence supporting the ubiquitous presence of Scorpine and β-KTx-like polypeptides and their precursors in scorpions of the genus Tityus of the family Buthidae, but also included is the first example of such peptides in scorpions from the family Iuridae. Seven new β-KTxs or Scorpine-like peptides and precursors are reported: five from the genus Tityus (T. costatus, T. discrepans and T. trivittatus) and two from Hadrurus gertschi. The cDNA precursors for all of these peptides were obtained by molecular cloning and their presence in the venoms were confirmed for various peptides. Analysis of the sequences revealed the existence of at least three distinct groups: (1) β-KTx-like peptides from buthids; (2) Scorpine-like peptides from scorpionid and iurid scorpions; (3) heterogeneous peptides similar to BmTXKβ of buthids and iurids. The biological function for most of these peptides is not well known; that is why they are here considered “orphan” peptides.
Keywords: Amino acid sequence determination; cDNA cloning; Scorpion toxins;
Development of a real-time PCR assay for measurement of yellow protein mRNA transcription in the desert locust Schistocerca gregaria: A basis for isolation of a peptidergic regulatory factor by Filip Sas; Murshida Begum; Tim Vandersmissen; Marisa Geens; Ilse Claeys; Sofie Van Soest; Jurgen Huybrechts; Roger Huybrechts; Arnold De Loof (38-43).
A major unresolved issue in insect endocrinology concerns the question of whether or not insects have sex hormones. Conclusive evidence in favor of the presence of such hormones awaits the establishment of appropriate bioassays in males. The cuticle of sexually mature males of the desert locust Schistocerca gregaria turns yellow in gregarious conditions only. Neither females nor isolated males ever turn yellow. The yellowing is due to the deposition in the cuticle of a male-specific Yellow Protein (YP), of which the amino acid sequence is known. In this paper, we describe the partial cloning of the cDNA encoding this Yellow Protein. The tissue distribution and temporal expression of the YP-mRNA is studied in detail using RT-PCR. Furthermore, an RT-PCR based bioassay was developed, which may serve as a reliable tool to help identify the hormones controlling the yellowing process. In addition to juvenile hormone, we have shown that a factor present in the brain–corpora cardiaca is involved in the yellow coloration, as injection of an extract induces the expression of YP-mRNA in isolated gregarious males.
Keywords: Yellow protein; Phase transition; Insect endocrinology; Cuticle; Juvenile hormone; Neuropeptides;
Both prothoracicotropic hormone and an autocrine factor are involved in control of prothoracic gland ecdysteroidogenesis in Locusta migratoria and Schistocerca gregaria by Tim Vandersmissen; Arnold De Loof; Shi-Hong Gu (44-50).
In Bombyx mori, ecdysteroidogenesis by the prothoracic glands (PGs) is controlled by both prothoracicotropic hormone (PTTH) and a factor secreted by the glands themselves. This factor, which is active both in vitro and in vivo, has been named ‘autocrine factor’ (AF). To find out whether or not this dual control also exists in other species, in particular in hemimetabolous ones, we applied similar methods as were used to discover AF in Bombyx to the locusts Locusta migratoria and Schistocerca gregaria. Our results unequivocally show that locust PGs also secrete an as yet unidentified autocrine factor. Possible roles of AF are discussed.
Keywords: Autocrine activation; PTTH; Ecdysone; Insect endocrinology; Metamorphosis; Prothoracic gland;
A model genetic system for testing the in vivo function of peptide toxins by Hugo W. Tedford; Francesco Maggio; Robert A. Reenan; Glenn King (51-56).
We have developed a model genetic system for analyzing the function of peptide toxins from animal venoms. We engineered and propagated strains of Drosophila melanogaster expressing heat-inducible transgenes encoding either κ-ACTX-Hv1c or ω-ACTX-Hv1a, two insect-specific neurotoxic peptides found in the venom of the Australian funnel-web spider Hadronyche versuta. Heat induction of transgene expression for 20 min was sufficient to kill all transgenic flies, indicating that the ion channels targeted by these toxins are viable insecticide targets. The unusual phenotype of flies induced to express ω-ACTX-Hv1a recapitulates that of a hypomorphic allele of the high-voltage-activated calcium channel Dmca1D, suggesting that this is likely to be the target of ω-ACTX-Hv1a.
Keywords: Peptide toxin; Atracotoxin; Drosophila melanogaster; Transgenesis;
Structure-activity relationships for in vitro diuretic activity of CAP2b in the housefly by Ronald J. Nachman; Geoffrey M. Coast (57-61).
A series of truncated and Ala-replacement analogs of the peptide Manse-CAP2b (pELYAFPRV-NH2) were assayed for diuretic activity on Malpighian tubules of the housefly Musca domestica (M. domestica). The C-terminal hexapeptide proved to be the active core, the minimum sequence required to retain significant diuretic activity. However, full activity required the C-terminal heptapeptide, which was equipotent with the most active of the native housefly CAP2b peptides. Replacement of Arg7 and Val8 with Ala led to inactivity and a large 70-fold drop in potency, respectively, indicating that these were critical residues. The Leu2 was semicritical, where a six-fold loss in potency was observed. Conversely, the replacement of all other residues with Ala led to much smaller effects on potency and these positions were considered to be noncritical. This structure-activity relationship data can aid in the design of mimetic agonist/antagonist analogs of this diuretic peptide family with enhanced biostability and bioavailability, as tools for arthropod endocrinologists and as potential pest management agents capable of disrupting the water balance in pest flies.
Keywords: CAPA gene; Periviscerokinin; CAP2b; Perisympathetic organs; Insect neuropeptide; Malpighian tubule;
Evolutionary selective trends of insect/mosquito antimicrobial defensin peptides containing cysteine-stabilized α/β motifs by R.S. Dassanayake; Y.I.N. Silva Gunawardene; S.S. Tobe (62-75).
Insect defensins containing cysteine-stabilized α/β motifs (Cs-α/β defensin) are cationic, inducible antibacterial peptides involved in humoral defence against pathogens. To examine trends in molecular evolution of these antimicrobial peptides, sequences similar to the well-characterized Cs-α/β defensin peptide of Anopheles gambiae, using six cysteine residues as landmarks, were retrieved from genomic and protein databases. These sequences were derived from different orders of insects. Genes of insect Cs-α/β defensin appear to constitute a multigene family in which the copy number varies between insect species. Phylogenetic analysis of these sequences revealed two main lineages, one group comprising mainly lepidopteran insects and a second, comprising Hemiptera, Coleoptera, Diptera and Hymenoptera insects. Moreover, the topology of the phylogram indicated dipteran Cs-α/β defensins are diverse, suggesting diversity in immune mechanisms in this order of insects. Overall evolutionary analysis indicated marked diversification and expansion of mature defensin isoforms within the species of mosquitoes relative to non-mosquito defensins, implying the presence of finely tuned immune responses to counter pathogens. The observed higher synonymous substitution rate relative to the nonsynonymous rate in almost all the regions of Cs-α/β defensin of mosquitoes suggests that these peptides are predominately under purifying selection. The maximum-likelihood models of codon substitution indicated selective pressure at different amino acid sites in mosquito mature Cs-α/β defensins is differ and are undergoing adaptive evolution in comparison to non-mosquito Cs-α/β defensins, for which such selection was inconspicuous; this suggests the acquisition of selective advantage of the Cs-α/β defensins in the former group. Finally, this study represents the most detailed report on the evolutionary strategies of Cs-α/β defensins of mosquitoes in particular and insects in general, and indicates that insect Cs-α/β defensins have evolved by duplication followed by divergence, to produce a diverse set of paralogues.
Keywords: Cs-α/β defensin; Mosquito; Insect; Phylogenetic analysis; Evolution;
Loss of DNA: A plausible molecular level explanation for crustacean neuropeptide gene evolution by Francisco Martínez-Pérez; Darinka Durán-Gutiérrez; Luis Delaye; Arturo Becerra; Guadalupe Aguilar; Samuel Zinker (76-82).
Alignment of nucleotides of APGWamide, RPCH and AKH genes gives region stretches (common regions) present in all family member variants. Common regions were separated by gap sections in the larger variants of family members. Consensus sequences for single polynucleotides from virtual hybrid molecules of DNA were obtained by joining the common regions of DNA and deleting the extra DNA nucleotides. Conceptual translation of these virtual hybrids resulted in polypeptides similar to APGWamide, RPCH and the AKH pre-pro-peptide. Virtual polypeptides were also similar to LWamide and RFamide along hydras to mammals. DNA loss probably explains the origin of neuropeptides.
Keywords: Neurohormone origin; Crustacean neuropeptides; RPCH; AKH; APGWamide; Gene evolution; Gene origin;
Comparative genomic analysis of allatostatin-encoding (Ast) genes in Drosophila species and prediction of regulatory elements by phylogenetic footprinting by P.R.F. Bowser; S.S. Tobe (83-93).
The role of the YXFGLa family of allatostatin (AST) peptides in dipterans is not well-established. The recent completion of sequencing of genomes for multiple Drosophila species provides an opportunity to study the evolutionary variation of the allatostatins and to examine regulatory elements that control gene expression. We performed comparative analyses of Ast genes from seven Drosophila species (Drosophila melanogaster, Drosophila simulans, Drosophila ananassae, Drosophila yakuba, Drosophila pseudoobscura, Drosophila mojavensis, and Drosophila grimshawi) and used phylogenetic footprinting methods to identify conserved noncoding motifs, which are candidates for regulatory regions. The peptides encoded by the Ast precursor are nearly identical across species with the exception of AST-1, in which the leading residue may be either methionine or valine. Phylogenetic footprinting predicts as few as 3, to as many as 17 potential regulatory sites depending on the parameters used during analysis. These include a Hunchback motif approximately 1.2 kb upstream of the open reading frame (ORF), overlapping motifs for two Broad-complex isoforms in the first intron, and a CF2-II motif located in the 3′-UTR. Understanding the regulatory elements involved in Ast expression may provide insight into the function of this neuropeptide family.
Keywords: Allatostatin-encoding genes; Drosophila species; Phylogenetic footprinting;
Recombinant production and structural studies of the Aplysia water-borne protein pheromone enticin indicates it has a novel disulfide stabilized fold by Scott F. Cummins; Fang Xie; Milind Misra; Andinet Amare; Jennifer A. Jakubowski; Melissa R. de Vries; Jonathan V. Sweedler; Gregg T. Nagle; Catherine H. Schein (94-102).
Enticin is one of three Aplysia proteins released during egg laying that act in concert with the pheromone attractin to attract other Aplysia and stimulate mating behavior. Whereas the enticin cDNA predicts a 69-residue mature protein, enticin isolated from the albumen gland was found to be posttranslationally processed in vivo by cleavage at Arg50 residue to generate a smaller 49-residue mature peptide. The Arg50 cleavage site is conserved in enticin from both Aplysia californica and Aplysia brasiliana. In order to generate sufficient enticin for structural studies, recombinant full-length protein was produced in a soluble form in Escherichia coli using a cold shock promoter-based protein expression system. The enticin cDNA was cloned into the bacterial vector pCold III, and efficiently expressed, as determined by amino acid microsequence and immunoblot analyses. Recombinant enticin, which contained an additional N-terminal 13-residue translation-enhancing element, was purified by reversed-phase HPLC and compared to enticin isolated from the albumen gland. The three disulfide bonds in enticin were characterized by endoproteinase Glu-C proteolysis followed by mass spectrometric characterization of the fragments. The cysteine pairing, for both recombinant and native enticin, was I–II, III–IV, and V–VI, confirming that the protein produced in the bacterial system was correctly folded. The circular dichroism spectrum of the recombinant protein indicated it was predominantly α-helical. While this was consistent with fold recognition server results indicating a fold for enticin similar to that of attractin, the disulfide bonding pattern differs. A model for enticin was prepared based on its helical structure and these disulfide constraints.
Keywords: Aplysia; pCold III; Enticin; Temptin; Attractin; Pheromone;
Functional comparison of two evolutionary conserved insect neurokinin-like receptors by Jeroen Poels; Heleen Verlinden; Jakub Fichna; Tom Van Loy; Vanessa Franssens; Kazimierz Studzian; Anna Janecka; Ronald J. Nachman; Jozef Vanden Broeck (103-108).
Tachykinins are multifunctional neuropeptides that have been identified in vertebrates as well as invertebrates. The C-terminal FXGXRa-motif constitutes the consensus active core region of invertebrate tachykinins. In Drosophila, two putative G protein-coupled tachykinin receptors have been cloned: DTKR and NKD. This study focuses on the functional characterization of DTKR, the Drosophila ortholog of the stable fly's tachykinin receptor (STKR). Tachykinins containing an alanine residue instead of the highly conserved glycine (FXAXRa) display partial agonism on STKR-mediated Ca2+-responses, but not on cAMP-responses. STKR therefore seems to differentiate between a number of tachykinins. Gly- and Ala-containing tachykinins are both encoded in the Drosophila tachykinin precursor, thus raising the question of whether DTKR can also distinguish between these two tachykinin types. DTKR was activated by all Drosophila tachykinins and inhibited by tachykinin antagonists. Ala-containing analogs did not produce the remarkable activation behavior previously observed with STKR, suggesting different mechanisms of discerning ligands and/or activating effector pathways for STKR and DTKR.
Keywords: Agonist; Antagonist; Drosophila; G protein; Neuropeptide; Tachykinin; Spantide;
Characterization and expression of the short neuropeptide F receptor in the African malaria mosquito, Anopheles gambiae by Stephen F. Garczynski; Joe W. Crim; Mark R. Brown (109-118).
A short neuropeptide F (sNPF) precursor and a sNPF receptor (sNPFR) were characterized for the mosquito, Anopheles gambiae. The sNPFR was expressed in CHO-K1 cells, and it exhibited high affinity binding, IC50 ∼3–5 nM, for specific sNPFs. sNPF1 potently inhibited forskolin-stimulated cAMP production by transfected cells, suggesting sNPFR acts via Gi/o. Transcripts for sNPF and sNPFR were present in all body regions of larvae, pupae, and adults, and immunoblots for sNPFR confirmed this distribution in females. Membranes from female heads and thoraces exhibited prototypical high affinity binding for radiolabeled sNPF, indicating sNPFR is a bona fide endogenous receptor.
Keywords: Insect; Drosophila; Aedes aegypti; G protein-coupled receptor; Neuropeptide F; Neuropeptide Y;
Presence of angiotensin converting enzyme isoforms in larval lepidoptera (Spodoptera littoralis) by E. Lemeire; J. Van Camp; G. Smagghe (119-126).
In this research the presence of angiotensin converting enzyme (ACE) in larvae of the lepidopteran Spodoptera littoralis was evaluated. Making use of the substrate Abz-FRK-(Dnp)P-OH and the specific inhibitor captopril at 10 μM, ACE activity was determined in a fluorescence assay for intact larvae, hemolymph, head, midgut and dorsal tissue. In dorsal tissue and hemolymph, ACE activity was highest. These data are consistent with a possible role for ACE in contractions of the dorsal vessel and metabolism of circulating peptide hormones in the hemolymph. After the presence of ACE was confirmed, a sequential procedure of anion exchange and size exclusion chromatography was applied to purify ACE from whole wandering larvae (last stage). With this procedure, three different ACE pools were collected that cleaved the fluorogenic substrate Abz-FRK-(Dnp)P-OH. Activity could be inhibited by a final concentration of 2.5 μM captopril. In addition, two out of three samples eluted at different salt concentration and thus ACE 1, 2 and 3 represent at least two different ACE isoforms. These data reveal that ACE is present in S. littoralis and that at least two out of three isolated ACE forms are truly isoforms.
Keywords: Angiotensin converting enzyme; Lepidoptera; Tissue; ACE activity; Isoform;
Expression of NEP2, a soluble neprilysin-like endopeptidase, during embryogenesis in Drosophila melanogaster by Nicholas D. Bland; Josie E. Thomas; Neil Audsley; Alan.D. Shirras; Anthony J. Turner; R. Elwyn Isaac (127-135).
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 (Gly7-Leu8), 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.
Keywords: Peptidase; Neprilysin; Tachykinin; Stellate cell; Malpighian tubules; Embryogenesis; Drosophila melanogaster;
In vitro transport of an allatostatin across the foregut of Manduca sexta larvae and metabolism by the gut and hemolymph by Neil Audsley; Robert J. Weaver (136-145).
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 41-6, due to cleavage of the C-terminal di-peptide GL-amide, and cydiastatin 42-8, 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 41-6 and cydiastatin 42-8. 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.
Keywords: Neuropeptide; Metabolism; MALDI-TOF; Mass spectrometry; Insect; Protease inhibitor;
A C-terminal aldehyde analog of the insect kinins inhibits diuresis in the housefly by Ronald J. Nachman; Jean-Alain Fehrentz; Jean Martinez; Krzyztof Kaczmarek; Janusz Zabrocki; Geoffrey M. Coast (146-152).
The insect kinins are present in a wide variety of insects and function as potent diuretic peptides in flies. A C-terminal aldehyde insect kinin analog, Fmoc-RFFPWG-H (R-LK-CHO), demonstrates stimulation of Malpighian tubule fluid secretion in crickets, but shows inhibition of both in vitro and in vivo diuresis in the housefly. R-LK-CHO reduced the total amount of urine voided over 3 h from flies injected with 1 μL of distilled water by almost 50%. The analog not only inhibits stimulation of housefly fluid secretion by the native kinin Musdo-K, but also by thapsigargin, a SERCA inhibitor, and by ionomycin, a calcium ionophore. The activity of R-LK-CHO is selective, however, as related C-terminal aldehyde analogs do not demonstrate an inhibitory response on housefly fluid secretion. The selective inhibitory activity of R-LK-CHO on housefly tubules represents an important lead in the development of environmentally friendly insect management agents based on the insect kinins.
Keywords: Insect kinins; Pest control; Musca domestica;
Angiotensin-converting enzyme as a target for the development of novel insect growth regulators by R. Elwyn Isaac; Nazarius S. Lamango; Uma Ekbote; Christine A. Taylor; Debra Hurst; Robert J. Weaver; Ahmet Carhan; Susan Burnham; Alan D. Shirras (153-162).
Insect angiotensin converting enzyme (ACE) is a zinc metallopeptidase capable of inactivating a variety of small to medium size peptide hormones by cleavage of C-terminal dipeptides and dipeptideamides. High levels of ACE activity are found in the hemolymph and in reproductive tissues of insects, where the enzyme is considered to have an important role in the metabolism of bioactive peptides. Therefore, inhibiting ACE activity is expected to interfere with the peptidergic endocrine system and to have detrimental effects on growth, development and reproduction. We will review the studies showing that ACE inhibitors do indeed disrupt growth and reproduction in various insect species. We will also present some new genetic and pharmacological data that strengthens our conclusion that ACE should be considered as a potential target for the development of new insect growth regulators.
Keywords: Insect control; Angiotensin-converting enzyme; Reproduction; Peptidase; ANCE; ACE inhibitors;
Neuropeptidergic control of Octopus oviducal gland by Carlo Di Cristo; Anna Di Cosmo (163-168).
The oviducal gland of the female of Octopus vulgaris lies about halfway along the oviduct. Progesterone and 17β-estradiol receptors have been immunolocalized in the nuclei of the cells of the glandular compartment of previtellogenic glands. We also have evidence of FMRFamide-like and cGnRH-I-like immunoreactivity in the nerve endings that reach the oviducal gland. Moreover, we have recently shown APGWamide immunoreactivity in the glandular cells of the inner part of the oviducal gland. Here we report a review on these findings as well as our latest studies on the effect that neuropeptides may exert on the secretory activity of the oviducal gland. cAMP seems to be a possible second messenger involved in such a process. We discuss the findings of a neuropeptidergic action on the glandular cells of oviducal gland in a more complex frame of molecules, such as steroids, biogenic amines and neuromodulators, controlling the activity of the gland.
Keywords: Cephalopods; Oviducal gland; Neuropeptides; Steroids; Fusiform ganglion; Forskolin; cAMP;
Role of receptor interaction in the mode of action of insecticidal Cry and Cyt toxins produced by Bacillus thuringiensis by I. Gómez; L. Pardo-López; C. Muñoz-Garay; L.E. Fernandez; C. Pérez; J. Sánchez; M. Soberón; A. Bravo (169-173).
Cry toxins from Bacillus thuringiensis are used for insect control. Their primary action is to lyse midgut epithelial cells. In this review we will summarize recent findings on the Cry toxin–receptor interaction and the role of receptor recognition in their mode of action. Cry toxins interact sequentially with multiple receptors. In lepidopteran insects, Cry1A monomeric toxins interact with the first receptor and this interaction triggers oligomerization of the toxins. The oligomer then interacts with second receptor inducing insertion into membrane microdomains and larval death. In the case of mosquitocidal toxins, Cry and Cyt toxins play a part. These toxins have a synergistic effect and Cyt1Aa overcomes Cry toxin resistance. Recently, it was proposed that Cyt1Aa synergizes or suppresses resistance to Cry toxins by functioning as a membrane-bound receptor for Cry toxin.
Keywords: Cry toxins; Cyt toxins; Bacillus thuringiensis; Receptor; Binding epitopes; Mode of action;
Neural and hormonal control of muscular activity of the spermatheca in the locust, Locusta migratoria by Angela B. Lange; Rosa da Silva (174-184).
The spermatheca in insects is a tubular structure within the female that acts as a repository for spermatozoa deposited by the male during copulation. The spermatozoa remain viable within the spermatheca for extended periods of time, and are then delivered to the site of fertilization during oviposition (egg-laying). Thus, the production of viable offspring is dependent upon the coordination of events associated with fertilization, including the passage of the egg through the lateral and common oviducts and the passage of spermatozoa along the spermathecal duct. The egg and the spermatozoa are propelled along their respective tracts by contractions of the visceral muscles intrinsic to the oviduct and spermatheca. The neural and hormonal control of muscular activity of the locust oviducts has been well reviewed, with more recent studies examining the control over the spermatheca. This review highlights more recent literature, including new data, for neural and hormonal control of muscular activity of the spermatheca of the locust, Locusta migratoria, making reference to examples in other insects where relevant. A variety of neuronal types project to the spermatheca in L. migratoria, and a variety of neuroactive chemicals, including neuropeptides and amines, influence contraction. A comparison is made between the control of oviducts and spermatheca in L. migratoria with regard to their neural substrate and the composition of neuroactive chemicals.
Keywords: Reproductive system; Neuropeptides; Amines; Oviduct; Oviposition;
The role of neuropeptides in caterpillar nutritional ecology by Jacqueline C. Bede; Jeremy N. McNeil; Stephen S. Tobe (185-196).
Plant diet strongly impacts the fitness of insect herbivores. Immediately, we think of plant defensive compounds that may act as feeding deterrents or toxins. We are, probably, less aware that plants also influence insect growth and fecundity through their nutritional quality. However, most herbivores respond to their environment and select the diet which optimizes their growth and development. This regulation of nutritional balance may occur on many levels: through selecting and ingesting appropriate plant tissue and nutrient digestion, absorption and utilization. Here, we review evidence of how nutritional requirements, particularly leaf protein to digestible carbohydrate ratios, affect caterpillar herbivores. We propose a model where midgut endocrine cells assess and integrate hemolymph nutritional status and gut content and release peptides which influence digestive processes. Understanding the effects of diet on the insect herbivore is essential for the rational design and implementation of sustainable pest management practices.
Keywords: Neuropeptides; Nutritional ecology; Nutrient requirements;