Peptides (v.32, #3)

Invertebrate neuropeptides XI by Ronald J. Nachman (433).

Neuropeptides associated with the central nervous system of the cabbage root fly, Delia radicum (L) by Neil Audsley; H. June Matthews; Rachel E. Down; Robert J. Weaver (434-440).
▶ First identification of neuropeptides from the cabbage root fly, Delia radicum. ▶ Most peptides distributed throughout the central nervous system. ▶ AKH restricted to the retrocerebral complex. Sulfakinins and FMRFamides present only in brain/SOG and thoracic-abdominal ganglion. ▶ Two novel dipteran FMRFamides and an A-type allatostatin identified.The peptidome of the central nervous system of adult cabbage root fly, Delia radicum (L) was investigated using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Over twenty neuropeptides were identified from three different tissue sources, the combined brain/suboesophageal ganglion (SOG), the retrocerebral complex, and the thoracic-abdominal ganglion (TAG). A number of peptides were identified in all three tissues, including allatostatins, short neuropeptide F-like peptides, corazonin, a pyrokinin, and a myosuppressin. Adipokinetic hormone was restricted to the retrocerebral complex. Other peptides, including FMRFamides and sulfakinins were detected only in the brain/SOG and TAG. Some peptides, notably myoinhibitory peptides and tachykinins, which have been identified in other fly species, were not detected in any tissue sample. This study has structurally characterized for the first time, the neuropeptides from adult D. radicum.
Keywords: Insect; MALDI-TOF mass spectrometry; Corpus cardiacum; Myosuppressin; FMRFamide;

Identification of a cysteine-rich antimicrobial peptide from salivary glands of the tick Rhipicephalus haemaphysaloides by Houshuang Zhang; Wenjie Zhang; Xinzhi Wang; Yongzhi Zhou; Na Wang; Jinlin Zhou (441-446).
The presence of an effective immune response in the hemocoel of ticks is crucial for survival, as it prevents the invasion of pathogens throughout the animal's body. Antimicrobial peptides (AMPs) play an important role in this response by rapidly killing invading microorganisms. In this study, a subtraction hybridization cDNA library was constructed from the salivary glands of the unfed and fed female tick Rhipicephalus haemaphysaloides, and a novel cysteine-rich AMP designated Rhamp (R. haemaphysaloides antimicrobial peptide) was isolated and identified. The Rhamp was encoded by a gene with an open reading frame of 303 bp which encoded a mature peptide with 8 kDa molecular weight. No identity was found by BLAST search to any database entries. The sequence encoding the Rhamp was subcloned into the pGEX-4T vector and expressed in Escherichia coli. The recombinant protein of Rhamp showed chymotrypsin and elastase-inhibitory activity and markedly inhibited the growth of Gram-negative bacteria, including Pseudomonas aeruginosa, Salmonella typhimurium, and E. coli. Moreover, the recombinant protein also exerted low hemolytic activity. These results indicate the Rhamp is a novel antimicrobial peptide with proteinase activity from the tick R. haemaphysaloides.
Keywords: Rhipicephalus haemaphysaloides; Salivary gland; Antimicrobial peptide;

Isolation and characterization of a novel myoactive tetradecapeptide-related peptide isolated from the brain of the squid, Todarodes pacificus by Hye-Jin Go; Eun Hee Jo; Jung-Kil Seo; Yong-Ki Hong; Hyung Ho Lee; Gun Do Kim; Tae Wook Park; Edward J. Noga; Nam Gyu Park (447-453).
A new bioactive tetradecapeptide, GFKDNVSNRIAHGFamide, was isolated from the brain of the squid, Todarodes pacificus. Using isolated T. pacificus esophagus as a bioassay, the peptide was shown to induce potent contraction of smooth muscle. The threshold concentration for contraction was 5 × 10−10  M to 1 × 10−9  M. The peptide was homologous to other molluskan (class Gastropoda) and annelid myoactive tetradecapeptides and to some extent, to arthropodan tridecapeptides. A full-length cDNA encoding the biosynthetic precursor of the active peptide was cloned, revealing that the peptide is probably secreted following processing of a prepropeptide containing a signal peptide and prosequences. This is the first myoactive tetradecapeptide (MATP) to be isolated from any mollusk of the class Cephalopoda and we have named it Todarodes tetradecapeptide (TTP).
Keywords: Tetradecapeptide; Brain; Squid; Esophagus motility; Molecular cloning;

A novel peptide of the adipokinetic hormone (AKH)/red pigment-concentrating hormone (RPCH) family has been elucidated by mass spectrometry from the corpora cardiaca of an African saucer bug species, Laccocoris spurcus. It is the first decapeptide member found in the species-rich taxon Heteroptera, has the primary sequence pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-Gly-Gly amide and is denoted as Lacsp-AKH. The first eight amino acids are identical to the octapeptide Anaim-AKH of the European saucer bug, Ilyocoris cimicoides. The synthetic peptide Lacsp-AKH elevates lipids upon injection into the hemolymph of L. spurcus at a low dose of 3 pmol. Swimming activity in this saucer bug also causes a significant increase in the lipid concentration in the hemolymph. Thus, both results point to an apparent function of the endogenous new decapeptide Lacsp-AKH in L. spurcus, namely, to regulate lipid mobilization. Isolation of an AKH peptide from the corpora cardiaca of the water bug Aphelocheirus aestivalis (Aphelocheiridae) resulted in the assignment of the octapeptide Anaim-AKH, supporting current phylogenies on the infraorder Nepomorpha.
Keywords: Insects; Heteroptera; Laccocoris spurcus; Aphelocheirus aestivalis; AKH/RPCH family; Sequence identification; Mass spectrometry; Exercise swimming;

Distribution and characterization of Corazonin in the central nervous system of Triatoma infestans (Insecta: Heteroptera) by Beatriz P. Settembrini; Daniela de Pasquale; Melissa Postal; Paulo M. Pinto; Célia R. Carlini; Marcelo J. Villar (461-468).
▶ The distribution of corazonin in the central nervous system of the heteropteran insect Triatoma infestans was studied by immunohistochemistry. ▶ The distribution pattern of corazonin in the central nervous system of this species suggests that corazonin may act as a neurohormone. ▶ Mass spectrometric analysis revealed that [Arg7]-corazonin was the only isoform of the neuropeptide present in T. infestans tissue samples.The distribution of corazonin in the central nervous system of the heteropteran insect Triatoma infestans was studied by immunohistochemistry. The presence of corazonin isoforms was investigated using MALDI-TOF mass spectrometry in samples containing the brain, the subesophageal ganglion, the corpora cardiaca–corpus allatum complex and the anterior part of the aorta. Several groups of immunopositive perikarya were detected in the brain, the subesophageal ganglion and the thoracic ganglia. Regarding the brain, three clusters were observed in the protocerebrum. One of these clusters was formed by somata located near the entrance of the ocellar nerves whose fibers supplied the aorta and the corpora cardiaca. The remaining groups of the protocerebrum were located in the lateral soma cortex and at the boundary of the protocerebrum with the optic lobe. The optic lobe housed immunoreactive somata in the medial soma layer of the lobula and at the level of the first optic chiasma. The neuropils of the deutocerebrum and the tritocerebrum were immunostained, but no immunoreactive perikarya were detected. In the subesophageal ganglion, immunostained somata were found in the soma layers of the mandibular and labial neuromeres, whereas in the mesothoracic ganglionic mass, they were observed in the mesothoracic, metathoracic and abdominal neuromeres. Immunostained neurites were also found in the esophageal wall. The distribution pattern of corazonin like immunoreactivity in the central nervous system of this species suggests that corazonin may act as a neurohormone. Mass spectrometric analysis revealed that [Arg7]-corazonin was the only isoform of the neuropeptide present in T. infestans tissue samples.
Keywords: Corazonin; Insect central nervous system; Chagaś disease; Mass spectrometry; Immunohistochemistry;

Identification of kinin-related peptides in the disease vector, Rhodnius prolixus by Victoria Te Brugge; Jean-Paul Paluzzi; Susanne Neupert; Ronald J. Nachman; Ian Orchard (469-474).
We have used an in silico approach to identify a gene from the blood-gorging vector, Rhodnius prolixus, that is predicted to produce an insect kinin prepropeptide. The prepropeptide is 398 amino acids in length and can potentially produce a large number of kinin-related peptides following post-translational processing. A comparison with other insect kinin precursor sequences demonstrates greatest conservation at the C-terminal region of the kinin peptides. Multiple peptides predicted from the kinin gene are phenotypically expressed in R. prolixus, as revealed by MALDI-TOF MS MS, including 12 kinins and one kinin precursor peptide (KPP). Six of these peptides are characterized by the typical insect kinin C-terminal motif FX1X2WGamide and five of these are also found as truncated forms. Five peptides were identified with an atypical, though similar, FX1X2WAamide C-terminus. There is also peptide with a C-terminal DDNGamide motif and a number of non-amidated peptides.
Keywords: In silico analysis; Kinin gene; MALDI-TOF MS MS; Chagas’ disease; Insect;

▶ Rhodnius prolixus is a major vector of Chagas’ Disease. ▶ Crustacean cardioactive peptide (CCAP) is a neuropeptide found throughout the arthropods. ▶ The Rhodnius CCAP gene has been isolated from the central nervous system (CNS). ▶ CCAP gene expression was observed in the CNS using RT-PCR and in situ hybridization.The blood-gorging bug, Rhodnius prolixus, is a major vector of Chagas’ disease in Central and South America. We have cloned and characterized the crustacean cardioactive peptide (CCAP) gene in R. prolixus. The RhoprCCAP gene contains five exons and four introns, and encodes a 129 amino acid prepropeptide, which following post-translation processing, produces CCAP. The predicted RhoprCCAP amino acid sequence is identical to CCAP of crustaceans and other insects, i.e. it is highly conserved. RhoprCCAP mRNA is observed in the central nervous system (CNS) using reverse transcriptase (RT) PCR, but not in the gut and salivary glands. In situ hybridization reveals that the expression of CCAP mRNA is localized to a small number of dorsally situated bilaterally paired neurons within the CNS.
Keywords: Neuropeptide; Central nervous system; Northern blot; In situ hybridization; Insect;

Neuropeptide F and the corn earworm, Helicoverpa zea: A midgut peptide revisited by Yongqin Huang; Joe W. Crim; Andrew B. Nuss; Mark R. Brown (483-492).
The neuropeptide Y family of peptides is implicated in the regulation of feeding across a broad range of animals, including insects. Among vertebrates, neuropeptide Y exerts its actions mainly centrally, whereas peptide YY and pancreatic polypeptide arise from digestive tissues. Among invertebrates, neuropeptide F (NPF) is the sole counterpart of the NPY family. Shared features of NPF sequences derived for Lepidoptera indicate that the midgut peptide (Hez-MP-I) of the corn earworm, Helicoverpa zea, characterized more than a decade ago, is a carboxyl fragment of a full-length NPF. An antibody to Hez-MP-I was used to characterize the peptide's distribution in tissues of larvae, pupae, and adults. Immunostaining demonstrated NPF-related material both in nervous tissues and in abundant endocrine cells of the midgut. Radioimmunoassay of Hez-MP-I in the head, midgut and hemolymph of fifth instar larvae revealed concentration changes corresponding to development and feeding state. As with the vertebrate homologs, NPF may arise both centrally and peripherally to modulate the physiology of feeding and digestion of Lepidoptera.
Keywords: Insect; Lepidoptera; Midgut; Nervous system; Neuropeptide Y;

Spined soldier bugs, Podisus maculiventris, are heteropteran insects that feed voraciously on other insects, particular the soft bodied larval forms of Lepidoptera and Coleoptera. The response of P. maculiventris Malpighian tubules (MTs) to serotonin and known diuretic and antidiuretic peptides has been investigated, and is compared with that of MT from the hematophagous and phytophagous heteropteran bugs Rhodnius prolixus and Acrosternum hilare, respectively. A CRF-related peptide diuretic hormone (DH) from the termite Zootermopsis nevadensis (Zoone-DH) stimulated MT secretion, which was reversed by a member of the CAP2b family of peptides from A. hilare (Acrhi-CAP2b-2), an antidiuretic effect. Serotonin had no effect on secretion, neither did a representative calcitonin-like DH, kinin, tachykinin-related peptide, and an antidiuretic factor from the mealworm Tenebrio molitor (Tenmo-ADFb) in both P. maculiventris or A. hilare. Serotonin is a DH in R. prolixus, and its lack of effect on MT from P. maculiventris and A. hilare suggests this is an adaptation to hematophagy. On the other hand, the antidiuretic activity of members of the CAP2b family in all three bugs is consistent with this being a heteropteran feature rather than a specialism for hematophagy.
Keywords: Heteroptera; Podisus maculiventris; Excretion; Malpighian tubules; Diuretic hormones; Antidiuretic hormones;

Serotonin stimulates secretion by Malpighian tubules (MT) of a number of insects, and functions as a diuretic hormone in Rhodnius prolixus and in larval Aedes aegypti. Serotonin is here shown to be a potent stimulant of secretion by MT of the house cricket, Acheta domesticus, with an apparent EC50 of 9.4 nmol L−1, although its diuretic activity is just 25% of the maximum achievable with either the native CRF-related peptide, Achdo-DH, or a crude extract of the corpora cardiaca. In this respect, the diuretic activity of serotonin is similar to that of the cricket kinin Achdo-KI, and when tested together their actions are not additive, which suggests they target the same transport process. Consistent with this suggestion, the activity of serotonin is chloride-dependent and is associated with a non-selective stimulation of NaCl and KCl transport. In common with Achdo-KI, serotonin has no effect on cAMP production by isolated MT, and both act synergistically with exogenous 8bromo-cAMP in stimulating fluid secretion, most likely by promoting the release of Ca2+ from intracellular stores. A number of serotonin agonists and antagonists were tested to determine the pharmacological profile of receptors on cricket MT. The results are consistent with the diuretic activity of serotonin being mediated through a 5-HT2-like receptor.
Keywords: Acheta domesticus; Malpighian tubule; Serotonin; Diuretic peptide; Second messenger;

Influence of codon usage bias on FGLamide-allatostatin mRNA secondary structure by Francisco Martínez-Pérez; William G. Bendena; Belinda S.W. Chang; Stephen S. Tobe (509-517).
The FGLamide allatostatins (ASTs) are invertebrate neuropeptides which inhibit juvenile hormone biosynthesis in Dictyoptera and related orders. They also show myomodulatory activity. FGLamide AST nucleotide frequencies and codon bias were investigated with respect to possible effects on mRNA secondary structure. 367 putative FGLamide ASTs and their potential endoproteolytic cleavage sites were identified from 40 species of crustaceans, chelicerates and insects. Among these, 55% comprised only 11 amino acids. An FGLamide AST consensus was identified to be (X)1→16Y(S/A/N/G)FGLGKR, with a strong bias for the codons UUU encoding for Phe and AAA for Lys, which can form strong Watson–Crick pairing in all peptides analyzed. The physical distance between these codons favor a loop structure from Ser/Ala-Phe to Lys-Arg. Other loop and hairpin loops were also inferred from the codon frequencies in the N-terminal motif, and the first amino acids from the C-terminal motif, or the dibasic potential endoproteolytic cleavage site. Our results indicate that nucleotide frequencies and codon usage bias in FGLamide ASTs tend to favor mRNA folds in the codon sequence in the C-terminal active peptide core and at the dibasic potential endoproteolytic cleavage site.
Keywords: mRNA; Secondary structure; Allatostatin; Cockroach; Diploptera; Codon bias;

Molecular cloning, characterization and expression analysis of cathepsin A gene in Chinese mitten crab, Eriocheir sinensis by Wei-Wei Li; Lin He; Xing-Kun Jin; Hui Jiang; Li-Li Chen; Ying Wang; Qun Wang (518-525).
Cathepsins, a superfamily of hydrolytic enzymes produced and enclosed within lysosomes, function in immune response in vertebrates; however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of cathepsin A (catA) in Chinese mitten crab (Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The full length catA cDNA (2200 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The catA cDNA contained a 1398 bp open reading frame (ORF) that encoded a putative 465 amino acid (aa) protein. Comparisons with other reported vertebrate cathepsins sequences revealed percent identity range from 48 to 51%. CatA mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in gill and (b) responsive in hemocytes to a Vibrio anguillarum challenge, with peak exposure observed 12 h post-injection. Collectively, data demonstrate the successful isolation of catA from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate.
Keywords: Chinese mitten crab; Eriocheir sinensis; Cathepsin A; Infection; Quantitative real-time PCR;

Antihypertensive effect of insect cells: In vitro and in vivo evaluation by Dorien Staljanssens; John Van Camp; Griet Herregods; Maarten Dhaenens; Dieter Deforce; Johan Van de Voorde; Guy Smagghe (526-530).
In this study, we investigated the in vitro ACE inhibitory and in vivo antihypertensive effect of insect cell extracts. The IC50 of three insect cell lines from different type and insect species origin: S2 (embryo, Drosophila melanogaster), Sf21 (ovary, Spodoptera frugiperda) and Bm5 (ovary, Bombyx mori), were evaluated. Most interesting results were that the IC50 values ranged between 0.4 and 0.9 mg/ml, and that an extra hydrolysis with gastrointestinal enzymes did not increase the ACE inhibitory activity conspicuously. Finally, a single oral administration with a gavage of 150 mg cell extract/kg BW to spontaneous hypertensive rats (SHR) significantly decreased (p  < 0.05) their systolic blood pressure (SBP) with 5–6% (9–12 mm Hg) compared to the controls at 6 h post-administration. Here the undigested and digested insect S2 cell extracts were equal in activity to lower the SBP. To the best of our knowledge, this is the first report of in vivo antihypertensive activity of insect cell extracts and this without an extra digestion requirement.
Keywords: Insect cells; Bioactive peptides; Hydrolysis; ACE inhibition; Blood pressure; SHR;

Insulin-like peptides (ILPs) affect a wide variety of biological events, such as metabolism, lifespan, growth and reproduction. Two ILPs (Spoli-ILP1 and Spoli-ILP2) were identified in the cotton leafworm, Spodoptera littoralis, while the functions and developmental characters are not fully understood. In the present study, we identified the partial sequence of a putative S. littoralis insulin receptor (Spoli-InR) and investigated the stage (age)- and tissue-dependent expression profile of Spoli-InR in addition to Spoli-ILPs during the last larval development and larval-pupal metamorphosis. Spoli-ILP1 and Spoli-ILP2 were specifically expressed in the brain, and their gene expressions were gradually decreased in concert with larval-pupal development. On the other hand, Spoli-InR was expressed in all the selected tissues (brain, testis, fat body, Malpighian tubules, prothoracic glands and midgut), though the gene expression pattern was different among the tissues. Interestingly, the transcript expression pattern of Spoli-InR in the fat body seemed to relate with larval-pupal development. In a parallel experiment, the juvenile hormone mimetic methoprene was able to prolong the larval period when applied before the commitment peak of ecdysteroids titer in the hemolymph, and in this case the expression of Spoli-ILPs and Spoli-InR was affected. These results demonstrated first a relationship between transcript expression of Spoli-ILPs and larval-pupal development, and second they suggested the effect of ILPs may be controlled by not only Spoli-ILPs expression but also Spoli-InR expression.
Keywords: Lepidoptera; Insulin signaling; Metamorphosis; Juvenile hormone;

In vitro activity of pacifastin-like inhibitors in relation to their structural characteristics by Bert Breugelmans; Vincent van Hoef; Gert Simonet; Sofie Van Soest; Guy Smagghe; Jozef Vanden Broeck (539-544).
Information on the structural characteristics and inhibitory activity of the pacifastin family is restricted to a handful of locust pacifastin-related inhibitors. In this report the optimization of a bacterial recombinant expression system is described, resulting in the high yield production of pacifastin-like inhibitors of the desert locust. Subsequently, the relative inhibitory activity of these peptides towards mammalian, locust and caterpillar digestive peptidases has been compared. In general, the enzyme specificity of locust pacifastin-like inhibitors towards trypsin- or chymotrypsin-like peptidases corresponds to the nature of the P1-residue at the reactive site. In addition, other structural characteristics, including specific core interactions, have been reported to result in a different affinity of pacifastin members towards digestive trypsin-like enzymes from mammals and arthropods. One remarkable observation in this study is a specifically designed pacifastin-like peptidase inhibitor, which, unlike other inhibitors of the same family, does not display this specificity and selectivity towards digestive enzymes from different animals.
Keywords: Inhibitor; Pacifastin; Serine peptidase; In vitro; Enzyme specificity; Insect;

▶ Suppression of leucokinin-releasing affects the longevity in Drosophila. ▶ Suppression of leucokinin-releasing affects the resistance to dry starvation. ▶ Leucokinin modulates the olfactory response in Drosophila. ▶ Leucokinin is required for response to trehalose and bitter tastants in Drosophila.Previous studies have revealed leucokinin (LK) expression in the brain and ventral ganglion of Drosophila CNS. One pair of protocerebrum neurons located in the lateral horn area (LHLK) surrounds the peduncles of the mushroom bodies while two pairs of subesophageal neurons (SELKs) project extended processes to the tritocerebrum and through a cervical connection to the ventral ganglion. There, axons of eight or nine pairs of abdominal (ABLK) neurons leave the CNS through the abdominal nerves and processes connecting each other ipsilaterally and contralaterally. The neural functions of LK remain largely unknown, especially those related to Drosophila behavior. Here, we have studied the role of LK in olfactory and gustatory perception by keeping the LK neurons electrically silent through targeted expression of inward rectifier K+ channels. In order to examine the effects of LK failure, we first analyzed the dehydration response, comparing the leucokinin-silent individuals with their parents as a control. Our results showed significant differences that demonstrate the effectiveness of the method. We then tested the olfactory behavioral response to a set of odorants over a range of concentrations in a T-maze paradigm in which flies were allowed to choose between the odorant and solvent compartments. The feeding preference assays were carried out on microplates in which flies were allowed to choose between two colored tastes. Our results show that the blockade of LK release alters both olfactory and gustatory responses, and are therefore evidence that this neuropeptide also modulates chemosensory responses through LHLK and SELK neurons.
Keywords: Leucokinin neuropeptide; Drosophila melanogaster; Chemosensorial modulation;

Open conformation of adipokinetic hormone receptor from the malaria mosquito facilitates hormone binding by Grace Mugumbate; Graham E. Jackson; David van der Spoel (553-559).
Insect flight requires rapid mobilization of energy reserves during flight, which is mediated and regulated by hormonal control via adipokinetic hormones. The structure of the G-protein receptors to which these hormones bind, are crucial in understanding many of the physiological processes in which they play a central role. To date no 3D structure of an insect G-protein coupled receptor (GPCR) is available. Here, the first models of the 3D structures of a GPCR from the malaria mosquito are presented. Homology modeling of the receptor identified from the genome of Anopheles gambiae was used to construct two models of the receptor. The 7 transmembrane helical bundles of these two models are based on the crystal structures of beta2-adrenergic receptor and rhodopsin. The flexible loop regions were modeled using high temperature simulated annealing and constrained molecular dynamic simulations. The two receptor models differ in a number of critical features, the most important of which is that the rhodopsin-based model has a ‘closed’ structure while the beta2-based structure is ‘open’. The ‘open’ conformation provides easy access of the hormone to the binding pocket. Docking calculations with the insect adipokinetic hormones, AKH-1 (pGlu-Leu-Thr-Phe-Thr-Pro-Ala-Trp-NH2) from the malaria mosquito and Del-CC (pGlu-Lys-Asn-Phe-Ser-Pro-Asn-Trp-Gly-Asn-NH2) from the blister beetle showed that while the binding motif of the two is similar, AKH-1 has more than 30 times higher affinity than Del-CC, which strongly suggests that the binding is specific, and that the correct binding site was identified. Using these models it is possible to design antagonists, which block the binding site and are thus species-specific insecticides.
Keywords: GPCR; Anopheles gambiae; Adipokinetic hormones; Homology modeling; Protein docking; Molecular modeling;

Recombinant expression of the toxic peptide ErgTx1 and role of Met35 on its stability and function by J.M. Jimenez-Vargas; R. Restano-Cassulini; V. Quintero-Hernández; G.B. Gurrola; L.D. Possani (560-567).
▶ ErgTx1 is a scorpion toxin that binds preferentially to the outer vestibule of hERG1 channels. ▶ An hydrophobic patch on the surface of this toxin is interacting with the surface of the ion-channel. ▶ Oxidation of the methionine residue in position 35 of the toxin decreases the affinity of ErgTx1 by three order of magnitude. ▶ Here it is shown that Met35 is a fudamental residue in the interaction with the channel.Ergtoxin 1 (ErgTx1) is a 42 amino acid peptide purified from the venom of the Mexican scorpion Centruroides noxius Hoffmann, capable of blocking specifically human potassium channels of the ether-á-go-go-related gene family (hERG). This peptide binds to a partially overlapping site on the channel outer mouth, in which residues of the S5-P linker are critically involved. Here we describe results of site directed mutagenesis of the ErgTx1 gene and its heterologous expression in Escherichia coli. The recombinant products show the fundamental role played by methionine in position 35 (Met35) of the primary structure. Naturally oxidized Met35 decreases by three orders of magnitude the affinity of the peptide for the hERG1 channels. This result is quite relevant, because it shows two possible situations: either Met35 is involved in the proper folding of the molecule or it plays a direct role in the interaction with the channel, i.e., constitutes part of the interacting surfaces. These two situations were evaluated by preparing heterologously expressed ErgTx1 gene and a mutant containing alanine in position 35. Additionally circular dichroism measurements of both native and recombinant peptides were performed. The electrophysiological recordings and the structural values obtained by optical measurements, strongly support the idea that Met35 is indeed a key residue on the interacting surfaces of the toxin with the channels.
Keywords: Alanine scanning; Erg channel; Heterologous expression; K+-channel; Scorpion toxin; 3D-modeling;

Venom peptides from solitary hunting wasps induce feeding disorder in lepidopteran larvae by Ji Hyeong Baek; Yeounjung Ji; Jeon-Soo Shin; Seunghwan Lee; Si Hyeock Lee (568-572).
The cell lytic activity and toxicity against lepidopteran larvae of 13 venom peptides (4 OdVPs and 9 EpVPs) from two solitary hunting wasps, Orancistrocerus drewseni and Eumenes pomiformis, were examined with mastoparan as a reference peptide. Of the 13 peptides, 7 were predicted to have α-helical structures that exhibit the typical character of amphipathic α-helical antimicrobial peptides. The remaining peptides exhibited coil structures; among these, EpVP5 possesses two Cys residues that form an internal disulfide bridge. All the helical peptides including mastoparan showed antimicrobial and insect cell lytic activities, whereas only two of them were hemolytic against human erythrocytes. The helical peptides induced a feeding disorder when injected into the vicinity of the head and thorax of Spodoptera exigua larvae, perhaps because their non-specific neurotoxic or myotoxic action induced cell lysis. At low concentrations, however, these helical peptides increased cell permeability without inducing cell lysis. These findings suggest that the helical venom peptides may function as non-specific neurotoxins or myotoxins and venom-spreading factors at low concentrations, as well as preservatives for long-term storage of the prey via antimicrobial, particularly antifungal, activities.
Keywords: Venom peptide; Solitary wasp; Antimicrobial peptide; Cell lysis;

RNA interference of insulin-related peptide and neuroparsins affects vitellogenesis in the desert locust Schistocerca gregaria by Liesbeth Badisco; Elisabeth Marchal; Pieter Van Wielendaele; Heleen Verlinden; Rut Vleugels; Jozef Vanden Broeck (573-580).
The ‘classic’ insect hormones, juvenile hormone and 20-hydroxyecdysone, can stimulate vitellogenesis and/or ovarian development in adult females of several insect species. Accumulating evidence also indicates a crucial role in female reproductive physiology for peptide hormones, such as insulin-related peptides (IRPs) and neuroparsins (NPs). Especially in dipteran species, IRP signaling has been shown to regulate female reproductive events. The first NP was originally identified from the migratory locust (Locusta migratoria) as an antigonadotropic factor that delayed vitellogenesis. Moreover, NP family members display sequence similarities with the N-terminal domain of vertebrate insulin-like growth factor binding proteins (IGFBPs). In the current study, RNA interference (RNAi) was employed to investigate the possible involvement of IRP and NPs in the control of the female desert locust (Schistocerca gregaria) reproductive system. The cDNAs encoding an IRP (Scg-IRP) and four NPs (Scg-NPs) had previously been cloned from S. gregaria. An RNAi-mediated knock-down of either Scg-NP or Scg-IRP transcript levels was induced in adult female desert locusts and the subsequent effects were analyzed. Knock-down of the Scg-NPs or Scg-IRP affected vitellogenin transcript levels and oocyte growth in a positive and negative way, respectively. The current findings are indicative for a role of Scg-NPs and Scg-IRP in the control of vitellogenin synthesis. A plausible hypothesis is that Scg-IRP may act as a sensor of the nutritional and metabolic status that determines whether vitellogenesis can occur. That the same processes were affected in opposite ways in both RNAi experiments offers an extra argument for antagonizing roles of Scg-NPs and Scg-IRP.
Keywords: Female; Gonadotropin; Hormone; Insect; Ovary; Parsin; Reproduction;

Design, synthesis and biological activity of peptidomimetic analogs of insect allatostatins by Yong Xie; Zhen Peng Kai; Stephen S. Tobe; Xi Le Deng; Yun Ling; Xiao Qin Wu; Juan Huang; Li Zhang; Xin Ling Yang (581-586).
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 (IC50: 0.09 μM) was more active than that of the C-terminal pentapeptide (Tyr–Xaa–Phe–Gly–Leu–NH2, IC50: 0.13 μM) it mimicked and the activity of the analog II (IC50: 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.
Keywords: Allatostatins; Peptide analogs; Peptidomimetics; Juvenile hormone; IGRs;

Biostable multi-Aib analogs of tachykinin-related peptides demonstrate potent oral aphicidal activity in the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae) by Ronald J. Nachman; Kamran Mahdian; Dick R. Nässel; R. Elwyn Isaac; Nan Pryor; Guy Smagghe (587-594).
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-NH2), featured aphicidal activity calculated at an LC50 of 0.0083 nmol/μl (0.0087 μg/μl) and an LT50 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.
Keywords: Aphicide; Hindgut myotropic; Diuresis; Peptidase resistant;

Binding of Bacillus thuringiensis subsp. israelensis Cry4Ba to Cyt1Aa has an important role in synergism by Pablo Emiliano Cantón; Esmeralda Zanicthe Reyes; Iñigo Ruiz de Escudero; Alejandra Bravo; Mario Soberón (595-600).
Bacillus thuringiensis subsp. israelensis (Bti) produces at least four different crystal proteins that are specifically toxic to different mosquito species and that belong to two non-related family of toxins, Cry and Cyt named Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. Cyt1Aa enhances the activity of Cry4Aa, Cry4Ba or Cry11Aa and overcomes resistance of Culex quinquefasciatus populations resistant to Cry11Aa, Cry4Aa or Cry4Ba. Cyt1Aa synergized Cry11Aa by their specific interaction since single point mutants on both Cyt1Aa and Cry11Aa that affected their binding interaction affected their synergistic insecticidal activity. In this work we show that Cyt1Aa loop β6–αE K198A, E204A and β7 K225A mutants affected binding and synergism with Cry4Ba. In addition, site directed mutagenesis showed that Cry4Ba domain II loop α-8 is involved in binding and in synergism with Cyt1Aa since Cry4Ba SI303-304AA double mutant showed decreased binding and synergism with Cyt1Aa. These data suggest that similarly to the synergism between Cry11Aa and Cyt1Aa toxins, the Cyt1Aa also functions as a receptor for Cry4Ba explaining the mechanism of synergism between these two Bti toxins.
Keywords: Bacillus thuringiensis; Cry toxins; Cyt toxins; Synergism;

Role of MAPK p38 in the cellular responses to pore-forming toxins by Helena Porta; Angeles Cancino-Rodezno; Mario Soberón; Alejandra Bravo (601-606).
Understanding the mechanism of action of pore-forming toxins (PFTs) produced by different bacteria, as well as the host responses to toxin action, would provide ways to deal with these pathogenic bacteria. PFTs affect the permeability of target cells by forming pores in their plasma membrane. Target organisms may overcome these effects by triggering intracellular responses that have evolved as defense mechanisms to PFT. Among them it is well documented that stress-activated protein kinases, and specially MAPK p38 pathway, play a crucial role triggering defense responses to several PFTs in different eukaryotic cells. In this review we describe different intracellular effects induced by PFTs in eukaryotic cells and highlight diverse responses activated by p38 pathway.
Keywords: MAPK p38; Pore-forming toxins; Cry toxin; Intracellular responses;

The CCK(-like) receptor in the animal kingdom: Functions, evolution and structures by Dorien Staljanssens; Elnaz Karimian Azari; Olivier Christiaens; Jérôme Beaufays; Laurence Lins; John Van Camp; Guy Smagghe (607-619).
In this review, the cholecystokinin (CCK)(-like) receptors throughout the animal kingdom are compared on the level of physiological functions, evolutionary basis and molecular structure. In vertebrates, the CCK receptor is an important member of the G-protein coupled receptors as it is involved in the regulation of many physiological functions like satiety, gastrointestinal motility, gastric acid secretion, gall bladder contraction, pancreatic secretion, panic, anxiety and memory and learning processes. A homolog for this receptor is also found in nematodes and arthropods, called CK receptor and sulfakinin (SK) receptor, respectively. These receptors seem to have evolved from a common ancestor which is probably still closely related to the nematode CK receptor. The SK receptor is more closely related to the CCK receptor and seems to have similar functions. A molecular 3D-model for the CCK receptor type 1 has been built together with the docking of the natural ligands for the CCK and SK receptors in the CCK receptor type 1. These molecular models can help to study ligand–receptor interactions, that can in turn be useful in the development of new CCK(-like) receptor agonists and antagonists with beneficial health effects in humans or potential for pest control.
Keywords: Cholecystokinin (CCK)-like receptors; GPCR; Evolution; Physiological functions; Satiety; Structures; Ligand–receptor interactions; Agonist/antagonist;

Toward a consensus nomenclature for insect neuropeptides and peptide hormones by Geoffrey M. Coast; David A. Schooley (620-631).
The nomenclature currently in use for insect neuropeptide and peptide hormone families is reviewed and suggestions are made as to how it can be rationalized. Based upon this review, a number of conventions are advanced as a guide to a more rationale nomenclature. The scheme that is put forward builds upon the binomial nomenclature scheme proposed by Raina and Gäde in 1988 , when just over 20 insect neuropeptides had been identified. Known neuropeptides and peptide hormones are assigned to 32 structurally distinct families, frequently with overlapping functions. The names given to these families are those that are currently in use, and describe a biological function, homology to known invertebrate/vertebrate peptides, or a conserved structural motif. Interspecific isoforms are identified using a five-letter code to indicate genus and species names, and intraspecific isoforms are identified by Roman or Arabic numerals, with the latter used to signify the order in which sequences are encoded on a prepropeptide. The proposed scheme is sufficiently flexible to allow the incorporation of novel peptides, and could be extended to other arthropods and non-arthropod invertebrates.
Keywords: Insect; Neuropeptide; Peptide hormone; Classification; Nomenclature;