Abstract: Chemical stimuli, generally constituted by small volatile organic molecules, are extremely important for the survival of different insect species. In the course of evolution, insects have developed very sophisticated biochemical systems for the binding and the delivery of specific semiochemicals to their cognate membrane-bound receptors. Chemosensory proteins (CSPs) are a class of small soluble proteins present at high concentration in insect chemosensory organs; they are supposed to be involved in carrying the chemical messages from the environment to the chemosensory receptors. In this paper, we report on the solution structure of CSPsg4, a chemosensory protein from the desert locust Schistocerca gregaria, which is expressed in the antennae and other chemosensory organs. The 3D NMR structure revealed an overall fold consisting of six alpha-helices, spanning residues 13-18, 20-31, 40-54, 62-78, 80-90, and 97-103, connected by loops which in some cases show dihedral angles typical of beta-turns. As in the only other chemosensory protein whose structure has been solved so far, namely, CSP from the moth Mamestra brassicae, four helices are arranged to form a V-shaped motif; another helix runs across the two V's, and the last one is packed against the external face. Analysis of the tertiary structure evidenced multiple hydrophobic cavities which could be involved in ligand binding. In fact, incubation of the protein with a natural ligand, namely, oleamide, produced substantial changes to the NMR spectra, suggesting extensive conformational transitions upon ligand binding.

Abstract: Aroma rice, e.g. Basmati and Jasmine rice, is very popular among rice consumers in South and South-East Asia and is becoming famous in USA and Europe. Thailand is one of the major rice exporters; rice produced from Thailand possesses peculiar properties as aroma, appearance, and texture. One outstanding property of Thai rice, known as �Jasmine rice�, is its fragrance. The characteristic flavour of this rice is principally contributed by 2-acetyl-1-pyrroline. There are many varieties of aroma rice produce in Thailand, among which Khao Dawk Mali 105 (KDLM105) variety is the most famous one. In this experiment, we investigated volatile organic compounds (VOC) of Thai rice (Oryza sativa) by using five varieties: KDLM105, RD15, PTN1, HSP and CNT1. All of them were planted in the experimental field in 2005. The first four varieties are aroma rice, the last one is a rice variety of high yield and good grain. The Jasmine rice bought from the retail store was also used. VOC released to the head space from dry stored and moistured-heated rice were sampled using solid phase microextraction (SPME). In order to optimise the device suitability, three different fibers, Carboxen/PDMS, Carbowax/DVB and PDMS/DVB, were chosen. The resulting compounds were analysed using GC-MS. In parallel, sensory evaluation of rice samples was carried out focussing on odour perception of the panel considering the parameters �variety� and �ageing�. The majority of VOC identified as highly abundant were aldhydes. Hexanal, considered as an off odour, was detected even in recently harvested rice.

Abstract: Trametes versicolor, Gloeophyllum trabeum and Poria placenta are known as wood destructive fungi, causing high economic losses in construction wood and wood composite products. To evaluate the quality of wood as raw-material for the production of high engineering property products, a reliable and sensitive detection of wood rotting fungi is needed. A rapid and non-destructive method for the evaluation of fungal infestation can focus on volatile organic compounds (VOC) released by fungi. Beech wood (Fagus sylvatica) was inoculated either with G. trabeum, P. placenta or T. versicolor and incubated at 22 °C for one week prior to sampling of VOC. Volatiles released to the head space were collected using solid phase microextraction (SPME), 85 µm Carboxen/PDMS StableFlex fiber type, and were analysed by GC-MS. Each fungus growing on beech released a specific pattern of VOC. The detected VOC can be categorised into two broad groups, aliphatic C5-C8 compounds and isoprenoids. Typical fungal odour compounds like 1-octen-3-ol and 3-octanone were produced by all three fungi. Two monoterpenes, alpha-pinene and 3-carene were found. However, dominant components with respect to abundance were sesquiterpenes. Sesquiterpene patterns were characteristic for each species. Moreover, 6-protoilludene was specifically produced by G. trabeum in high amounts. alpha- and beta-barbatene were typical and dominant VOC of T. versicolor. Cis boleti, an insect typically feeding on fruiting bodies of wood rotting fungi like T. versicolor was used for electroantennographic recordings of fungal volatiles. On the basis of these measurements the feasibility of a biosensor for the detection of wood rotting fungi was assessed.

Abstract: Thaumatin-like proteins (TLPs) are polypeptides of about 200 residues synthesized by plants in response to fungal infection. In addition to the exceptionally strong sweet taste exhibited by some members, they are also reported to be endowed with endo-beta-1,3-glucanase activity and alpha-amylase inhibiting properties. However, the detailed mechanism of their antifungal action is not completely understood. So far, TLPs have only been described in plants, with several members of the family expressed in the same species. Here, for the first time in animals, we report the identification of two genes encoding members of the thaumatin-like proteins family in the desert locust Schistocerca gregaria and show their expression in different parts of the body. Southern blot and Western blot experiments revealed the presence of orthologous genes and their expression products in the related species Locusta migratoria. A search through the available genomes yielded similar sequences in the nematode Caenorhabditis but not in Drosophila and other insects. A three-dimensional model of S. gregaria TLP suggests a glucanase function. As in plants, TLPs could play a defense role in insects against pathogens.

Abstract: BACKGROUND: Several sawfly larvae of the Tenthredinidae (Hymenoptera) are called easy bleeders because their whole body integument, except the head capsule, disrupts very easily at a given spot, under a slight mechanical stress at this spot. The exuding haemolymph droplet acts as a feeding deterrent towards invertebrate predators. The present study aimed to describe the cuticle surface, to consider it from a mechanistic point of view, and to discuss potential consequences of the integument surface in the predator-prey relationships. RESULTS: The integument surface of sawfly larvae was investigated by light microscopy (LM) and scanning electron microscopy (SEM) which revealed that the cuticle of easy bleeders was densely covered by what we call "spider-like" microstructures. Such microstructures were not detected in non-easy bleeders. A model by finite elements of the cuticle layer was developed to get an insight into the potential function of the microstructures during easy bleeding. Cuticle parameters (i.e., size of the microstructures and thickness of the epi-versus procuticle) were measured on integument sections and used in the model. A shear force applied on the modelled cuticle surface led to higher stress values when microstructures were present, as compared to a plan surface. Furthermore, by measuring the diameter of a water droplet deposited on sawfly larvae, the integument of several sawfly species was determined as hydrophobic (e.g., more than Teflon(R)), which was related to the sawfly larvae's ability to bleed easily. CONCLUSION: Easy bleeders show spider-like microstructures on their cuticle surface. It is suggested that these microstructures may facilitate integument disruption as well as render the integument hydrophobic. This latter property would allow the exuding haemolymph to be maintained as a droplet at the integument surface.

Abstract: Two different classes of chemosensory proteins (CSPs) in Locusta migratoria have been identified on the basis of the molecular cloning of a series of different cDNAs from the antennae of this insect. Several CSP isoforms have been purified and biochemically characterized from antennal and wing extracts, some of them corresponding to expression products predicted for the identified cDNAs. In wings, the nature of the main endogenous ligand binding to these proteins was determined as oleoamide by a gas chromatography-mass spectrometric approach. One of these isoforms has been expressed in a bacterial system with high yield and used in a fluorescent binding assay. Competitive binding experiments have indicated the presence of long-chain compounds among the best ligands.

Abstract: The aim of this work was to investigate the olfactory system of the walking stick insect, Carausius morosus. Morphological, ultrastructural and immunocytochemical studies of adult female antennae were conducted by scanning and transmission electron microscopy. Extensive cross-section series were made through the last antennal segment to define the cuticular apparatus, wall pore distribution and the number of innervating receptor neurons of each sensillum type. Single-walled wall pore sensilla occur in three subtypes: (i) with 27 or 28 branched receptor neurons, (ii) with two branched neurons and (iii) with one or two unbranched neurons, respectively. Double-walled wall pore sensilla were found in two subtypes with spoke channels, one with four unbranched neurons, the other with two unbranched neurons. One terminal pore sensillum was found, showing two cavities within the hair and being innervated by six sensory cells. Immunocytochemical experiments were performed to show the localization of a 19kDa soluble protein found in the chemosensory organs of C. morosus. This protein shows an amino acid sequence homologous to the family of chemosensory proteins (CSP). The polyclonal antibody raised against the purified protein (CSP-cmA) showed, for the first time in CSPs, a strong labeling in olfactory sensilla, specifically in the sensillum lymph surrounding the dendritic branches of SW-WP sensilla and in the uninnervated lumen between the two concentric walls of DW-WP type 1 sensilla.

Abstract: Chemosensory proteins (CSPs) are a class of small, soluble proteins present at high concentrations in chemosensory organs of different insect species. Several pieces of evidence suggest their involvement in carrying chemical messages from the environment to chemosensory receptors. However, a structural description of the mechanism of delivery has not been reported. In order to provide the first detailed conformational characterization of these molecules, we cloned a specific isoform (CSP-sg4) from Schistocerca gregaria and expressed it in Escherichia coli. The product was obtained with yields of more than 20 mg per L of culture, all in its soluble form. The recombinant protein was identical to the native one with respect to pairing of the disulfide bridges, aggregative state and secondary structure elements. Structural investigations revealed a significantly stable polypeptide with respect to variations in temperature and acidity. CD analysis, preliminary NMR data and secondary structure prediction pointed to a correctly folded structure where helical regions and loops are alternated in a similar fashion as that observed for other classes of odorant- and pheromone-binding proteins presenting no sequence similarity to CSPs.

Abstract: Three related nucleotide sequences, encoding mature proteins of 108-113 amino acids, have been obtained from antennal cDNA of the Phasmid Eurycantha calcarata. Among these, one is also expressed in the tarsi as demonstrated by N-terminal sequence and mass spectrometric analyses of protein samples isolated from both organs. PCR experiments performed with specific primers, showed that this species is also expressed in the mouth organs and in the cuticle, while the other two are antennal specific. All three isoforms are similar to Drosophila OS-D and other proteins reported in several insect orders, but one of them is significantly different from the other two. The best conserved elements are the N-terminal region and the four cysteine residues. Accurate ESMS measurements indicated that all cysteines are involved in two disulphide bonds and ruled out the occurrence of additional post-translational modifications. Polyclonal antibodies, raised against the purified protein, did not react with proteins of the same class expressed in another Phasmid species, Carausius morosus, and in the orthopteran Schistocerca gregaria, nor did antibodies against these proteins recognise those of E. calcarata.

Abstract: Soluble low molecular weight acidic proteins are suspected to transport stimulus molecules to the sensory neurons within insect sensilla. From the antennae of Bombyx mori, we have purified and sequenced a protein (BmorCSP1) bearing sequence similarity to a class of soluble chemosensory proteins recently discovered in several orders of insects. Based on its N-terminal sequence, the cDNA encoding this protein has been amplified and cloned. Differential screening of a B. mori antennal cDNA library led to the identification of a second gene encoding a related protein (BmorCSP2), sharing 35-40% identity to BmorCSP1 and chemosensory proteins from other species. The predicted secondary structures of moth's, chemosensory proteins comprise alpha-helical foldings at conserved positions and a reduced hydrophobicity with respect to this novel family of chemosensory proteins.

Abstract: Pheromone-binding protein (PBP) and general odorant-binding proteins (GOBPs) were purified from the antennae of Bombyx mori and structurally characterised. The amino acid sequence of GOBP-2 has been corrected. The disulphide arrangements of PBP and GOBP-2 have been determined by a combined mass spectrometric/Edman degradation approach. The same cysteine pairings, Cys19-Cys54, Cys50-Cys108, and Cys97-Cys117, were found in both proteins, suggesting that such patterns occur commonly throughout this family of molecules. This arrangement of disulphide bonds indicates that the three-dimensional structure of insect OBPs is defined by three loops, rich in helical content, which can vary in size and charge distribution from one protein to another.

Abstract: Soluble low-molecular-mass protein isoforms were purified from chemosensory organs (antennae, tarsi and labrum) of the desert locust Schistocerca gregaria. Five genes encoding proteins of this group were amplified by PCR from cDNAs of tarsi and sequenced. Their expression products are polypeptide chains of 109 amino acids showing 40-50% sequence identity with putative olfactory proteins from Drosophila melanogaster and Cactoblastis cactorum. Direct structural investigation on isoforms purified from chemosensory organs revealed the presence in the expression products of two of the genes cloned. Two additional protein isoforms were detected and their molecular structure exhaustively characterized. MS analysis of all isoforms demonstrated that the four cysteine residues conserved in the polypeptide chain were involved in disulfide bridges (Cys29-Cys38 and Cys57-Cys60) and indicated the absence of any additional post-translational modifications. Immunocytochemistry experiments, performed with rabbit antiserum raised against the protein isoform mixture, showed selective labelling of the outer lymph in contact sensilla of tarsi, maxillary palps and antennae. Other types of sensilla were not labelled, nor were the cuticle and dendrites of the sensory cells. No binding of radioactively labelled glucose or bicarbonate was detected, in disagreement with the hypothesis that this class of proteins is involved in the CO2-sensing cascade. Our experimental data suggest that the proteins described here could be involved in contact chemoreception in Orthoptera.