Effects of different dietary conditions on the expression of trypsin- and chymotrypsin-like protease genes in the digestive system of the migratory locust, Locusta migratoria.

While technological advancements have recently led to a steep increase in genomic and transcriptomic data, and large numbers of protease sequences are being discovered in diverse insect species, little information is available about the expression of digestive enzymes in Orthoptera. Here we describe the identification of Locusta migratoria serine protease transcripts (cDNAs) involved in digestion, which might serve as possible targets for pest control management. A total of 5 putative trypsin and 15 putative chymotrypsin gene sequences were characterized.

Growth-inhibition effects of pacifastin-like peptides on a pest insect: the desert locust, Schistocerca gregaria.

The main reason for the varying degrees of success of peptidase inhibitors (PI) as biological insecticides is the existence of a poorly understood mechanism, which allows pest insects to compensate for PI present in their diet. To challenge this highly flexible physiological mechanism and to prolong the inhibitory effect of PI on insect growth, a number of measures were taken into account before and during experiments with a notorious pest insect, the desert locust, Schistocerca gregaria: (i) non-plant PI (pacifastin-related inhibitors) were used to reduce the risk of a specific co-evolutionary adaptation of the pest insect, (ii) based on the main types of digestive enzymes present in the midgut, mixtures of multiple PI with different enzyme specificity were selected, allowing for a maximal inhibition of the proteolytic activity and (iii) digestive peptidase samples were taken during oral administration experiments to study compensatory mechanisms. Contrary to larvae fed on a diet containing plant-derived PI, a significant growth impediment was observed in larvae that were fed a mixture of different pacifastin-like PI.

Functional analysis of a pancreatic secretory trypsin inhibitor-like protein in insects: silencing effects resemble the human pancreatic autodigestion phenotype.

Introduction: In mammalian pancreatic cells, the pancreatic secretory trypsin inhibitor (PSTI) prevents the premature activation of digestive enzymes and thus plays an important role in a protective mechanism against tissue destruction by autophagy, a process which may ultimately cause diseases such as pancreatitis and pancreatic cancer. Insects, however, lack a pancreas and so far no PSTI-like peptides are functionally characterized.

Results: In several insect species protease inhibitors that structurally resemble the mammalian PSTI were predicted in silico.

In vitro activity of pacifastin-like inhibitors in relation to their structural characteristics.

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.

Identification, distribution and molecular evolution of the pacifastin gene family in Metazoa.

Background: Members of the pacifastin family are serine peptidase inhibitors, most of which are produced as multi domain precursor proteins. Structural and biochemical characteristics of insect pacifastin-like peptides have been studied intensively, but only one inhibitor has been functionally characterised. Recent sequencing projects of metazoan genomes have created an unprecedented opportunity to explore the distribution, evolution and functional diversification of pacifastin genes in the animal kingdom.

A lepidopteran pacifastin member: cloning, gene structure, recombinant production, transcript profiling and in vitro activity.

Members of the pacifastin family have been characterized as serine peptidase inhibitors (PI), but their target enzyme(s) are unknown in insects. So far, the structural and biochemical characteristics of pacifastin-like PI have only been studied in locusts. Here we report the molecular identification and functional characterization of a pacifastin-like precursor in a lepidopteran insect, i.

Pacifastin-related peptides: structural and functional characteristics of a family of serine peptidase inhibitors.

Members of the pacifastin family are serine peptidase inhibitors, found in arthropods and have many members within different insect orders. Based on their structural characteristics, inhibitors of this peptide family are divided into two groups (I and II). Members of both groups exhibit specificity towards different types of serine peptidases.

Quantitative RT-PCR analysis of pacifastin-related precursor transcripts during the reproductive cycle of solitarious and gregarious desert locusts.

In locusts, little is known about the physiological and biochemical mechanisms regulating complex processes, such as reproduction and phase transition. The pacifastin family constitutes a family of peptidic inhibitors of serine proteases that are considered to be important regulators of several physiological processes in arthropods. We have performed a detailed transcript profiling analysis of two pacifastin-related peptide precursors, SGPP-2 and SGPP-4, during the reproductive cycle of adult desert locusts (Schistocerca gregaria).

Purification and characterization of an insulin-related peptide in the desert locust, Schistocerca gregaria: immunolocalization, cDNA cloning, transcript profiling and interaction with neuroparsin.

Members of the insulin superfamily are not restricted to vertebrates, but have also been identified in invertebrate species. In the current report, we present the characterization of Scg-insulin-related peptide (IRP), an insulin-related peptide in the desert locust, Schistocerca gregaria. This peptide was isolated from corpora cardiaca (CC) extracts by means of a high-performance liquid chromatography (HPLC)-based purification strategy.

Comparative genomics of leucine-rich repeats containing G protein-coupled receptors and their ligands.

Leucine-rich repeats containing G protein-coupled receptors (LGRs) constitute a unique cluster of transmembrane proteins sharing a large leucine-rich extracellular domain for hormone binding. In mammals, LGRs steer important developmental, metabolic and reproductive processes as receptors for glycoprotein hormones and insulin/relaxin-related proteins. In insects, a receptor structurally related to human LGRs mediates the activity of the neurohormone bursicon thereby regulating wing expansion behaviour and remodelling of the newly synthesized exoskeleton.

Insect neuropeptide and peptide hormone receptors: current knowledge and future directions.

Peptides form a very versatile class of extracellular messenger molecules that function as chemical communication signals between the cells of an organism. Molecular diversity is created at different levels of the peptide synthesis scheme. Peptide messengers exert their biological functions via specific signal-transducing membrane receptors.

Recombinant aequorin as a reporter for receptor-mediated changes of intracellular Ca2+ -levels in Drosophila S2 cells.

The bioluminescent Ca(2+)-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca(2)(+)-concentrations. Drosophila Schneider 2 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TK III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells.

Functional analysis of synthetic insectatachykinin analogs on recombinant neurokinin receptor expressing cell lines.

The activity of a series of synthetic tachykinin-like peptide analogs was studied by means of microscopic calcium imaging on recombinant neurokinin receptor expressing cell lines. A C-terminal pentapeptide (FTGMRa) is sufficient for activation of the stomoxytachykinin receptor (STKR) expressed in Schneider 2 cells. Replacement of amino acid residues at the position of the conserved phenylalanine (F) or arginine (R) residues by alanine (A) results in inactive peptides (when tested at 1microM), whereas A-replacements at other positions do not abolish the biological activity of the resulting insectatachykinin-like analogs.

Analysis of C-terminally substituted tachykinin-like peptide agonists by means of aequorin-based luminescent assays for human and insect neurokinin receptors.

Aequorin-based assays for stable fly, Stomoxys calcitrans, (STKR) and human (neurokinin receptor 1 (NK1), neurokinin receptor 2 (NK2)) neurokinin-like receptors were employed to investigate the impact of a C-terminal amino acid exchange in synthetic vertebrate ('FXGLMa') and invertebrate ('FX1GX2Ra') tachykinin-like peptides. C-terminally (Arg to Met) substituted analogs of the insect tachykinin-related peptide, Lom-TK I, displayed increased agonistic potencies in luminescent assays for human NK1 and NK2 receptors, whereas they showed reduced potencies in the STKR-assay. The opposite effects were observed when C-terminally (Met to Arg) substituted analogs of substance P were analysed.

Phenolamine-dependent adenylyl cyclase activation in Drosophila Schneider 2 cells.

Drosophila Schneider 2 (S2) cells are often employed as host cells for non-lytic, stable expression and functional characterization of mammalian and insect G-protein-coupled receptors (GPCRs), such as biogenic amine receptors. In order to avoid cross-reactions, it is extremely important to know which endogenous receptors are already present in the non-transfected S2 cells. Therefore, we analyzed cellular levels of cyclic AMP and Ca2+, important second messengers for intracellular signal transduction via GPCRs, in response to a variety of naturally occurring biogenic amines, such as octopamine, tyramine, serotonin, histamine, dopamine and melatonin.

Cloning of the cDNA encoding Scg-SPRP, an unusual Ser-protease-related protein from vitellogenic female desert locusts (Schistocerca gregaria).

The cDNA coding for a Ser-protease-related protein (Scg-SPRP) was cloned from desert locust (Schistocerca gregaria) midgut. The derived amino acid sequence consists of 260 residues and shows strong sequence similarity to insect trypsin-like molecules. It is, however, likely that Scg-SPRP is not a proteolytically active enzyme and that it plays another physiologically relevant role, since two out of three residues which are indispensable for catalytic activity of Ser-proteases are replaced.

Biological activity of structural analogs and effect of oil as a carrier of trypsin modulating oostatic factor of the gray fleshfly Neobellieria bullata.

The trypsin modulating oostatic factor from the gray fleshfly Neobellieria bullata (Neb-TMOF) is released from the ovary at the end of vitellogenesis and inhibits trypsin biosynthesis in the midgut. This inhibition indirectly results in an arrest of oocyte growth. Additional experiments with N.

Isolation and characterization of an adipokinetic hormone release-inducing factor in locusts: the crustacean cardioactive peptide.

A methanolic extract of 7000 desert locust (Schistocerca gregaria) brains contains several factors that stimulate the in vitro release of adipokinetic hormone (AKH) by glandular cells of locust (Locusta migratoria and Schistocerca gregaria) corpora cardiaca. The most potent one has now been fully identified. Matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis revealed a mass of 954.

G-protein-coupled receptors in insect cells.

The main classes of transmembrane signaling receptor proteins are well conserved during evolution and are encountered in vertebrates as well as in invertebrates. All members of the G-protein-coupled receptor superfamily share a number of basic structural and functional characteristics. In both insects and mammals, this receptor class is involved in the perception and transduction of many important extracellular signals, including a great deal of paracrine, endocrine, and neuronal messengers and visual, olfactory and gustatory stimuli.