Peptides (v.31, #3)

Contents List (v-vi).

Invertebrate neuropeptides X by Ronald J. Nachman (371).

This is the first report on the structural identity of a neuropeptide of the insect order Grylloblattodea. A peptide was isolated and sequenced from the retrocerebral corpora cardiaca–corpora allata complex of the ice crawler, Galloisiana yuasai. The sequence of the peptide was deduced from the multiple MS N electrospray mass data as that of an octapeptide: pGlu-Val-Asn-Phe-Ser-Pro-Thr-Trp amide. The retention time on reversed-phase HPLC and the CID MS2 mass spectra of a synthetic peptide with the same primary structure were exactly the same as of the natural peptide. The sequence represents a novel peptide of the adipokinetic hormone family which contains presently 50 members. The primary structure differs in only one position to a few previously discovered AKHs. A scenario is outlined that makes it likely that the most recently discovered insect order, the Mantophasmatodea, and the Grylloblattodea are closely related.
Keywords: Insects; Grylloblattodea; Ice crawlers; Mantophasmatodea; Adipokinetic hormone family; Mass spectrometry;

CAPA-peptides of praying mantids (Mantodea) by Rene Koehler; Reinhard Predel (377-383).
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.
Keywords: Mantis; Insect neuropeptide; Periviscerokinins; Perisympathetic organs; Mass spectrometry; Immunocytochemistry; Peptide processing;

cDNA was prepared from the venom duct of a single Conus spurius specimen collected near the coast of Campeche, México. From it, PCR products were generated aiming to clone I-conotoxin precursors. Thirty clones were sequenced and predicted to encode ten distinct precursors: seven of I2-conotoxins and three of I2-like-conotoxins. These precursors contain three different, mature toxins, sr11a, sr11b and sr11c, of which two are novel and one (sr11a) has been previously purified and characterized from the venom of this species. The precursors include a 26- (I2) or 23- residue signal peptide (I2-like), a 31-residue “pro” region (I2-like), and a 32-residue mature toxin region (I2 and I2-like). In addition, all the precursors have a 13-residue “post” region which contains a γ-carboxylation recognition sequence that directs the γ-carboxylation of Glu-9 and Glu-10 of toxin sr11a and, possibly, Glu-13 of toxin sr11b and Glu-9 of toxin sr11c. This is the first time that a “post” region has been found in precursors of I-conotoxins that also contain a “pro” region. The “post” peptide is enzymatically processed to yield the amidated mature toxin sr11a, which implies that γ-carboxylation occurs before amidation. Phylogenetic analysis at the whole precursor level indicates that the I2-like-conotoxins of C. spurius are more related to I2-conotoxins than to I1- and I3-conotoxins from other species, and that they might represent a new subgroup of the I2-superfamily. The three I-conotoxins from C. spurius have charge differences at seven to nine positions, suggesting that they might have different molecular target types or subtypes.
Keywords: Conoidea; Conidae; Conus spurius; I-conotoxin; cDNA cloning; Conotoxin precursor;

Conservation of the egg-laying hormone neuropeptide and attractin pheromone in the spotted sea hare, Aplysia dactylomela by Scott F. Cummins; Parinyaporn Nuurai; Gregg T. Nagle; Bernard M. Degnan (394-401).
In the marine opisthobranch mollusc, Aplysia, secreted peptides and proteins play an essential role in egg laying and mate attraction. Aplysia californica egg laying is initiated by secretion of the egg-laying hormone (ELH) peptide while mate attraction is made possible by protein pheromones, such as attractin, released into the surrounding seawater with the egg cordon. In this study, we investigated the existence of similar egg-laying hormone and attractin products in the spotted sea hare, Aplysia dactylomela, a species that is widely distributed in almost all tropical and temperate oceans, including Australia's Great Barrier Reef. Immunological analysis revealed that an ELH-like transmitter is present within bag cell somata and processes of the abdominal ganglion. A molecular genetic approach found that the ELH precursor mRNA is synthesized in the abdominal ganglia and encodes a 36-residue peptide (dELH) that is cleaved from the prohormone prior to secretion. It is most closely related to A. californica and A. brasiliana ELH (91.7% identical). We also found that A. dactylomela synthesize an attractin pheromone in the albumen gland that is released during egg laying. The gene encodes a 58-residue mature protein that is 74.9% similar to A. californica attractin. We demonstrate that an increase in seawater temperature can disrupt attractins higher order interactions, such as those with the pheromone temptin, and accelerates attractin degradation. Together, these findings further expands our understanding of pheromone intermolecular interactions and presents an opportunity for further study of how increases in sea water temperature may affect this important marine communication system.
Keywords: Aplysia; Egg-laying hormone; Attractin; Pheromone; Temperature;

Evidence for a Phe-Gly-Leu-amide-like allatostatin in the beetle Tenebrio molitor by Karen L. Elliott; Kuen Kuen Chan; Barbara Stay (402-407).
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.
Keywords: A-type allatostatin; Beetle; Tenebrio molitor; Corpora allata;

Two capa-genes are expressed in the neuroendocrine system of Rhodnius prolixus by Susanne Neupert; William K. Russell; David H. Russell; Reinhard Predel (408-411).
CAPA-peptides have been isolated from a broad range of insect species and are typical of the neurosecretory system of the abdominal ventral nerve cord. In Rhodnius prolixus, which is known to transmit Chagas’ disease, CAPA-peptides have potent antidiuretic effects. Recently, two capa-genes were isolated from this hemipteran insect; it was the first description of the occurrence of multiple capa-genes from a single insect species. The expression of peptides from one of these capa-genes was confirmed by mass spectrometry Paluzzi (2008) . In this study, the abdominal neuroendocrine system of R. prolixus was screened for the occurrence of products of the second annotated capa-gene. Single nerve preparations of abdominal segmental nerves 1–3 of R. prolixus were analyzed by MALDI-TOF mass spectrometry and the products of both capa-genes were identified by tandem mass spectrometry. The co-occurrence of the different CAPA-peptides, including CAPA-precursor peptides, in all spectra from abdominal segmental nerves 1 to 3 suggests a common expression of both capa-genes in the same neuroendocrine cells. A comparison of mass spectra obtained from abdominal segmental nerves with those of the subesophageal ganglion (SEG) revealed an obvious differential processing of the CAPA-precursors within the CNS.
Keywords: Neuropeptides; MALDI-TOF mass spectrometry; Hemiptera; Periviscerokinins; Differential processing;

Sea anemone toxins bind to site 3 of the sodium channels, which is partially formed by the extracellular linker connecting S3 and S4 segments of domain IV, slowing down the inactivation process. In this work we have characterized the actions of BcIII, a sea anemone polypeptide toxin isolated from Bunodosoma caissarum, on neuronal sodium currents using the patch clamp technique. Neurons of the dorsal root ganglia of Wistar rats (P5–9) in primary culture were used for this study (n  = 65). The main effects of BcIII were a concentration-dependent increase in the sodium current inactivation time course (IC50  = 2.8 μM) as well as an increase in the current peak amplitude. BcIII did not modify the voltage at which 50% of the channels are activated or inactivated, nor the reversal potential of sodium current. BcIII shows a voltage-dependent action. A progressive acceleration of sodium current fast inactivation with longer conditioning pulses was observed, which was steeper as more depolarizing were the prepulses. The same was observed for other two anemone toxins (CgNa, from Condylactis gigantea and ATX-II, from Anemonia viridis). These results suggest that the binding affinity of sea anemone toxins may be reduced in a voltage-dependent manner, as has been described for α-scorpion toxins.
Keywords: Site-3 toxins; Voltage-gated sodium channels; Fast inactivation; CgNa; ATX-II; Neurotoxins;

Molecular characterization of neuropeptide F from the eastern subterranean termite Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) by Andrew B. Nuss; Brian T. Forschler; Joe W. Crim; Victoria TeBrugge; Jan Pohl; Mark R. Brown (419-428).
Neuropeptide F (NPF)-like immunoreactivity was previously found to be abundant in the eastern subterranean termite, Reticulitermes flavipes. Purification of the NPF from a whole body extract of worker termites was accomplished in the current study by HPLC and heterologous radioimmunoassay for an NPF-related peptide, Helicoverpa zea Midgut Peptide-I. A partial amino acid sequence allowed determination of the corresponding cDNA that encoded an open reading frame deduced for authentic R. flavipes NPF (Ref NPF): KPSDPEQLADTLKYLEELDRFYSQVARPRFa. Effects of synthetic NPFs on muscle contractions were investigated for isolated foreguts and hindguts of workers, with Drm NPF inhibiting spontaneous contractions of hindguts. Phylogenetic analysis of invertebrate NPF sequences reveals two separate groupings, with Ref NPF occurring within a clade composed exclusively of arthropods.
Keywords: Neuropeptide Y; Insect; Gut motility;

Ecdysis triggering hormone signaling in arthropods by Ladislav Roller; Inka Žitňanová; Li Dai; Ladislav Šimo; Yoonseong Park; Honoo Satake; Yoshiaki Tanaka; Michael E. Adams; Dušan Žitňan (429-441).
Ecdysis triggering hormones (ETHs) from endocrine Inka cells initiate the ecdysis sequence through action on central neurons expressing ETH receptors (ETHR) in model moth and dipteran species. We used various biochemical, molecular and BLAST search techniques to detect these signaling molecules in representatives of diverse arthropods. Using peptide isolation from tracheal extracts, cDNA cloning or homology searches, we identified ETHs in a variety of hemimetabolous and holometabolous insects. Most insects produce two related ETHs, but only a single active peptide was isolated from the cricket and one peptide is encoded by the eth gene of the honeybee, parasitic wasp and aphid. Immunohistochemical staining with antiserum to Manduca PETH revealed Inka cells on tracheal surface of diverse insects. In spite of conserved ETH sequences, comparison of natural and the ETH-induced ecdysis sequence in the honeybee and beetle revealed considerable species-specific differences in pre-ecdysis and ecdysis behaviors. DNA sequences coding for putative ETHR were deduced from available genomes of several hemimetabolous and holometabolous insects. In all insects examined, the ethr gene encodes two subtypes of the receptor (ETHR-A and ETHR-B). Phylogenetic analysis showed that these receptors fall into a family of closely related GPCRs. We report for the first time the presence of putative ETHs and ETHRs in genomes of other arthropods, including the tick (Arachnida) and water flea (Crustacea). The possible source of ETH in ticks was detected in paired cells located in all pedal segments. Our results provide further evidence of structural and functional conservation of ETH–ETHR signaling.
Keywords: Ecdysis triggering hormone; ETH receptor; Inka cell; Ecdysis behavior; Insect; Tick; Crustacean;

Allatostatin-C receptors in mosquitoes by Jaime G. Mayoral; Marcela Nouzova; Anne Brockhoff; Marianne Goodwin; Salvador Hernandez-Martinez; Dietmar Richter; Wolfgang Meyerhof; Fernando G. Noriega (442-450).
In the present work we describe the functional and molecular characterization of two Aedes aegypti allatostatin-C receptor paralogs (AeAS-CrA and AeAS-CrB) and provide a detailed quantitative study of the expression of the AS-C receptor genes in an adult insect. The tissue distribution of the two AS-C receptors differed significantly; the mRNA levels of AeAS-CrB in the Malpighian tubules were the highest detected, while transcripts for AeAS-CrA were relatively low in this tissue. In addition, the transcript levels of both receptors were different in the thoracic and abdominal ganglia, corpora allata (CA) and the testis of the male. In the CA, the AeAS-CrB mRNA levels were constant from 0 to 72 h after female emergence, while the AeAS-CrA levels increased at 72 h. To complement the receptor expression studies, we analyzed the tissue specificity for allatostatin-C mRNA in female mosquitoes. Expression was high in abdominal ganglia and brain. Transcript levels of allatostatin-C in the head of females were elevated at eclosion and there were no major changes during the first week of adult life or after blood feeding. Fluorometric Imaging Plate Reader (FLIPR) recordings of calcium transients in HEK293T cells transiently expressing both putative receptors showed that they both responded selectively to allatostatin-C stimulation in the nanomolar concentration range. However, the peptide showed slightly greater affinity for AeAS-CrB than AeAS-CrA. Our studies suggest that some of the pleiotropic effects of allatostatin-C in mosquitoes could be mediated by the different receptor paralogs. Transcriptional regulation of the AS-C receptors may not have a critical role in the changes of CA responsiveness to the peptide that we previously described.
Keywords: Mosquito; Allatostatin; Receptor; GPCR; Juvenile hormone; Aedes;

Evolution and functional divergence of enzymes involved in sesquiterpenoid hormone biosynthesis in crustaceans and insects by Jerome H.L. Hui; Alexander Hayward; William G. Bendena; Tokiharu Takahashi; Stephen S. Tobe (451-455).
Juvenile hormone (JH) and methyl farnesoate (MF) play well-known roles in the development and reproduction of insects and crustaceans. Juvenile hormone acid O-methyltransferase (JHAMT) and farnesoic acid O-methyltransferase (FAMeT) are the enzymes responsible for catalyzing the biosynthesis of JH and MF, respectively. It is not clear whether the genes that encode these enzymes are present in animal lineages outside of the arthropods. Based on DNA sequence similarity, the literature suggests that an FAMeT ortholog is present in humans. However, vertebrates do not appear to produce JH or MF. To help unravel the evolution of hormonal systems in animals we have carried out the first comparative genomic analysis of JHAMT and FAMeT. We identify the first JHAMT ortholog in a crustacean genome, and FAMeT orthologs in annelid and cephalochordate genomes. Moreover, phylogenetic analyses suggest that there is no true homolog of FAMeT in humans contrary to previous hypotheses. Our analyses suggest that the presence of multiple FAMeT isoforms in arthropods may be a consequence of different evolutionary mechanisms. The genes responsible for hormone biosynthesis in extant insects and crustaceans appear to have been present at least in the Pancrustacea. Different selective forces appear to have subsequently acted on the two lineages, leading to the present functional divergence. Our use of comparative genomics and phylogenetic analysis advance knowledge of the relationships of the hormonal enzyme genes in question, and provide new insights into the evolution of hormonal systems in the largest animal phylum, the Arthropoda.
Keywords: Evolution of function; Enzymes; Hormones; Insects; Crustaceans; Comparative genomics;

20-Hydroxyecdyone (20E), an active form of ecdysteroid, is the key hormone in insect growth and development. The biosynthesis of ecdysteroid is triggered and under the control of the neuropeptide, prothoracicotropic hormone (PTTH). To date, five cytochrome P450 enzymes, namely Spook (Spo), Phantom (Phm), Disembodied (Dib), Shadow (Sad) and Shade (Shd) related to ecdysteroid biosynthesis, are identified and the character of last four enzymes is well studied in Drosophila melanogaster, Bombyx mori and Manduca sexta. These genes are called Halloween genes and mediate the biosynthesis of 20E from cholesterol. In this study, we extended these works to a major pest insect in agriculture, the cotton leafworm Spodoptera littoralis (Lepidoptera: Noctuidae). We identified the sequence of five Halloween genes, and the converted amino acid sequences were compared with those of other insects. The phylogenetic analysis clearly showed separated clusters of each gene and the evolutional conservation in insects with a high similarity in Lepidoptera. Spo, phm, dib and sad were predominantly expressed in prothoracic glands, and shd was expressed in fat body and Malpighian tubules at the last instar larvae. Spo expression was kept high level between day 2 and day 4 after ecdysis. The expression of phm and dib peaked at day 2, and sad and shd expressions peaked at day 2 and day 4 after ecdysis. In addition, the hemolymph ecdysteroid titer showed a small peak at day 2 and a large peak at day 4 after ecdysis. These results suggest the importance of Halloween genes in ecdysone biosynthesis by prothoracic glands and conversion of ecdysone into 20E by fat body in larval–pupal metamorphosis.
Keywords: Cytochrome P450; Halloween genes; Ecdysteroid; Metamorphosis; Spodoptera;

Neurohormones implicated in the control of Malpighian tubule secretion in plant sucking heteropterans: The stink bugs Acrosternum hilare and Nezara viridula by Geoffrey M. Coast; Victoria A. TeBrugge; Ronald J. Nachman; Juan Lopez; Jeffrey R. Aldrich; Angela Lange; Ian Orchard (468-473).
Plant sucking heteropteran bugs feed regularly on small amounts of K+-rich plant material, in contrast to their hematophagous relatives which imbibe large volumes of Na+-rich blood. It was anticipated that this would be reflected in the endocrine control of Malpighian tubule (MT) secretion. To explore this, neuroendocrine factors known to influence MT secretion were tested on MT of the pentatomid plant sucking stink bugs, Acrosternum hilare and Nezara viridula, and the results compared with previously published data from Rhodnius prolixus. Serotonin had no effect on N. viridula MT, although it stimulates secretion by R. prolixus MT >1000-fold, and initiates a rapid diuresis to remove excess salt and water from the blood meal. Kinins had no effect on stink bug MT, but secretion was increased by Zoone-DH, a CRF-like peptide, although the response was a modest 2–3-fold acceleration compared with 1000-fold in R. prolixus. Native CAPA peptides, which have diuretic activity in dipteran flies, had antidiuretic activity in MT of the stink bug (Acrhi/Nezvi-CAPA-1 and -2), as previously shown with Rhopr-CAPA-2 in R. prolixus. The antidiuretic activity of Rhopr-CAPA-2 has been linked with terminating the rapid diuresis, but results with stink bugs suggest it is a general feature of heteropteran MT.
Keywords: Heteroptera; Pentatomidae; Diuretic hormones; Serotonin; CRF-like peptides; CAPA peptides; Antidiuretic;

A novel serine protease inhibitor (AmPI) was purified from larval hemolymph of tasar silkworm, Antheraea mylitta by two-step process of trypsin-affinity and gel-filtration (FPLC) chromatography. AmPI was active against larval midgut and commercial bovine trypsin and chymotrypsin. The extent of purification was determined by SDS and Native PAGE. The protease inhibitor had an apparent molecular weight of approximately 14.5 kDa as determined by SDS-PAGE. Its activity was stable over a pH range of 4.5–9 and temperatures range of 4-65 °C. Molecular weight as determined by MALDITOF-MS was between 13241.63 and 13261.66 Da. MS profile of AmPI also suggests two isoforms of AmPI because of glycosylation by heptose (C7H14O7). This confirmed the result of Native PAGE showing two bands. N-terminal amino acid sequence of this protein did not show similarity to any known protease inhibitor. To study the functional implications of AmPI in insect, it was localized in insect body tissue of different larval instars by immunogold labeling technique using GAR-gold conjugate as secondary antibody. The pattern of localization suggests constitutive nature of AmPI, which may have role in insect's defense mechanism.
Keywords: Antheraea mylitta; Cuticle; Epidermis; Immunogold; Localization; Mass spectrum; Protease inhibitor;

Ala-Val-Phe and Val-Phe: ACE inhibitory peptides derived from insect protein with antihypertensive activity in spontaneously hypertensive rats by Lieselot Vercruysse; John Van Camp; Nicole Morel; Pierre Rougé; Griet Herregods; Guy Smagghe (482-488).
In this study, we evaluated the stability/bioavailability and in vivo antihypertensive activity of the tripeptide, Ala-Val-Phe, that was recently purified from insect protein (Spodoptera littoralis; Lepidoptera) and that showed in vitro angiotensin converting enzyme (ACE) inhibitory activity. This tripeptide is partly hydrolyzed by mucosal peptidases to Val-Phe, a more potent in vitro ACE inhibitor. In organ bath experiments using rat aorta, Val-Phe showed ACE inhibition, while Ala-Val-Phe did not. Single oral administration (5 mg/kg body weight) to spontaneously hypertensive rats led to a significant decrease in blood pressure for both peptides. Docking experiments indicated an active character for Val-Phe and an inactive character for Ala-Val-Phe as potential inhibitors of human ACE. From our results, it can be suggested that after oral administration of Ala-Val-Phe, Val-Phe is released by in vivo peptidases and is responsible for in vivo activity of Ala-Val-Phe. To the best of our knowledge this is the first report of in vivo antihypertensive activity of peptides derived from insect protein.
Keywords: Bioactive peptides; In vitro; Organ baths/rat aorta; In vivo; Bioavailability/stability;

The C-type allatostatin, Manduca sexta allatostatin (Manse-AS) and the analog δR3δR5Manse-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 δR3δR5Manse-AS analog had an estimated LC50 of 0.18 μg/μl diet, and was more potent than Manse-AS. At a dose of 0.35 μg δR3δR5Manse-AS/μl 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 δR3δR5Manse-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.
Keywords: Myoinhibitory; Neuropeptide; Cathepsin L cysteine protease; Chymotrypsin-like; Trypsin-like; Carboxypeptidase-like;

The insect kinins are multifunctional neuropeptides found in a variety of arthropod species, including the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae). A series of biostable insect kinin analogs based on the shared C-terminal pentapeptide core region were fed in solutions of artificial diet to the pea aphid over a period of 3 days and evaluated for antifeedant and aphicidal activity. The analogs contained either α,α-disubstituted or β-amino acids in key positions to enhance resistance to tissue-bound peptidases and retain activity in a number of insect kinin bioassays and/or on expressed receptors. Three of the biostable analogs demonstrated antifeedant activity, with a marked reduction in honeydew formation observed after 1 day, and very high mortality. In contrast, an unmodified, parent insect kinin and two other analogs containing some of the same structural components that promote biostability are inactive. The most active analog, double Aib analog K-Aib-1 ([Aib]FF[Aib]WGa), featured aphicidal activity calculated at an LC50 of 0.063 nmol/μl (0.048 μg/μl) and an LT50 of 1.68 days, matching the potency of some commercially available aphicides. The mechanism of this activity has yet to be established. The aphicidal activity of the biostable insect kinin analogs may result from different potential mechanisms as disruption of digestive processes by interfering with gut motility patterns, digestive enzyme release, and/or with fluid cycling in the gut, and also nutrient transport across the gut itself; all processes shown to be regulated by the insect kinins in other insects. However the mechanism(s) is(are) not yet known. The active insect kinin analogs represent potential leads in the development of selective, environmentally friendly pest aphid control agents.
Keywords: Kinin analog; Antifeedant; Aphicide; Acyrthosiphon pisum; Insect; Gut; Myotropic; Diuresis; Peptidase resistant;

Control of ecdysteroidogenesis in prothoracic glands of insects: A review by Elisabeth Marchal; Hans Peter Vandersmissen; Liesbeth Badisco; Sandrien Van de Velde; Heleen Verlinden; Masatoshi Iga; Pieter Van Wielendaele; Roger Huybrechts; Gert Simonet; Guy Smagghe; Jozef Vanden Broeck (506-519).
The very first step in the study of the endocrine control of insect molting was taken in 1922. Stefan Kopec characterized a factor in the brain of the gypsy moth, Lymantria dispar which appeared to be essential for metamorphosis. This factor was later identified as the neuropeptide prothoracicotropic hormone (PTTH), the first discovery of a series of factors involved in the regulation of ecdysteroid biosynthesis in insects. It is now accepted that PTTH is the most important regulator of prothoracic gland (PG) ecdysteroidogenesis. The periodic increases in ecdysteroid titer necessary for insect development can basically be explained by the episodic activation of the PGs by PTTH. However, since the characterization of the prothoracicostatic hormone (PTSH), it has become clear that in addition to ‘tropic factors’, also ‘static factors’, which are responsible for the ‘fine-tuning’ of the hemolymph ecdysteroid titer, are at play. Many of these regulatory factors are peptides originating from the brain, but also other, extracerebral factors both of peptidic and non-peptidic nature are able to affect PG ecdysteroidogenesis, such as the ‘classic’ insect hormones, juvenile hormone (JH) and the molting hormone (20E) itself. The complex secretory pattern of ecdysteroids as observed in vivo is the result of the delicate balance and interplay between these ecdysiotropic and ecdysiostatic factors.
Keywords: Ecdysteroid; Halloween genes; Hormone; Insect; Juvenile hormone; Neuropeptide; Peptide; Prothoracic gland;

Tachykinin-related peptides and their receptors in invertebrates: A current view by Tom Van Loy; Hans Peter Vandersmissen; Jeroen Poels; Matthias B. Van Hiel; Heleen Verlinden; Jozef Vanden Broeck (520-524).
Members of the tachykinin peptide family have been well conserved during evolution and are mainly expressed in the central nervous system and in the intestine of both vertebrates and invertebrates. In these animals, they act as multifunctional messengers that exert their biological effects by specifically interacting with a subfamily of structurally related G protein-coupled receptors. Despite the identification of multiple tachykinin-related peptides (TKRPs) in species belonging to the insects, crustaceans, mollusks and echiuroid worms, only five invertebrate receptors harboring profound sequence similarities to mammalian receptors for tachykinins have been functionally characterized to date. Three of these have been cloned from dipteran insect species, i.e. NKD (neurokinin receptor from Drosophila), DTKR (Drosophila tachykinin receptor) and STKR (tachykinin-related peptide receptor from the stable fly, Stomoxys calcitrans). In addition, two receptors from non-insect species, present in echiuroid worms and mollusks, respectively have been identified as well. In this brief review, we will survey some recent findings and insights into the signaling properties of invertebrate tachykinin-related peptides via their respective receptors. In this context, we will also point out the necessity to take into account differences in signaling mechanisms induced by distinct TKRP isoforms in insects.
Keywords: Tachykinin; Invertebrate; G protein-coupled receptor;