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Xylosandrus compactus and Liparthrum colchicum are two Scolytinae recorded for the first time in Italy in 2011 and 2019, respectively. X. compactus is an “ambrosia beetle” causing damages to several plants typical of the Mediterranean maquis through its digging activity on twigs and branches of the host plants. L. colchicum is a “bark beetle”, monophagous on laurel shrubs and trees. During a survey performed in summer of 2020, in “Tenuta Salviati” located in the Migliarino Natural Park (Pisa, Tuscany), adults of X. compactus and L. colchicum were collected from the same branches and apical twigs of Laurus nobilis hedges. The study aimed at characterizing fungi isolated from the external surface of the two scolytids bodies, to evaluate similarities and even their role in fungal spreading in the environment.

The vector‐borne bacterium Xylella fastidiosa(Wells and Raju) causes several serious diseases to plants. Recently, different subspecies of X. fastidiosa were reported in some European countries. The risk of the bacterium’s spread on the entire European territory is very high; therefore, it has been added into the priority pest list (2019/1702/EU Regulation). The main purposes of this work were to verify the presence of potential vectors in areas at a high risk of introduction in Tuscany and to ascertain the presence of X. fastidiosa in these insect vectors. Over 4,000 Auchenorrhyncha were collected and analysed from 2015 to 2019. Among the xylem sap-feeder putative vectors, most of the insects collected belonged to the family Aphrophoridae, but also many species of leafhopper were identified. Overall, in Tuscany four species were the most represented: Philaenus spumarius(L.), Cicadella viridis(L.), Synophropsis lauri (Horvath) and Neophilaenus campestris(Fallen).In 2018 an outbreak of X. fastidiosa subsp. multiplex was reported in Monte Argentario (Grosseto province, Tuscany). In 2019 X. fastidiosa subspecies multiplex ST 87 was detected in seven P. spumarius and three N. campestris collected from the infected area.

The Japanese beetle, Popillia japonica, is a highly polyphagous Scarabeidae native to Japan that colonized North America and Azores in the last century and has recently invaded Italy and Switzerland. Considering its economic impact to the horticulture and turfgrass industries, this species was ranked within the EU priority pests list in 2019. In order to reconstruct the source of introductions of this pest, we investigated the genetic variability of P. japonica in its native and invaded areas worldwide by analyzing 9 microsatellite loci and two mitochondrial genes, COI and CytB. There are two populations of Japanese beetles, from a limited area within central Japan, that are likely the source of the North American population. Moreover, the microsatellite data and resulting phylogeographic reconstruction suggests that the two European populations originated from independent introductions from southeast (Azores) and northeastern (Italy and Switzerland) North America (1). (1) For simplicity, in this paper North America refers to Canada and USA

Several essential oils, including citronella (lemongrass, Cymbopogon sp., Poaceae), are well-known mosquito repellents. A drawback of such products is their limited protection time resulting from the high volatility of their active components. In particular, citronella oil protects for2 h, although formulations with fixatives can increase this time.We synthesized hydroxylated cyclic acetals of citronellal, the main component of citronella, to obtain derivatives with lower volatility and weaker odour. The crude mixture of isomers obtained in the reaction was tested under laboratory conditions for its repellency against two mosquito species, the major malaria vector Anopheles gambiae and the arbovirus vector Aedes albopictus, and found to be endowed with longer protection time with respect to DEET (N,N-diethyl-meta-toluamide) at the same concentration. Formulated products were tested in a latin square human field trial, in an area at a high density of A. albopictus for 8 h from the application. We found that the performance of the citronellal derivatives mixture is comparable (95% protection for ≤3.5 h) with those of the most widespread synthetic repellents DEET and Icaridin, tested at a four-fold higher doses.Modifying the hydrophilicity and volatility of natural repellents is a valuable strategy to design insect repellents with a long-lasting effect. © 2022 The Authors. Pest Management Science published by John WileySons Ltd on behalf of Society of Chemical Industry.

The fig tree weevil Aclees sp. cf. foveatus (Coleoptera: Curculionidae), introduced in Italy in 2005, is currently causing significant economic and environmental losses to fig tree nurseries and orchards in Central Italy. Fig damages are due to the adults feeding on leaves and fruits, and to the galleries dug by the xylophagous larvae in the trunk, which lead the plants to death. To date, no chemical or biological control methods resulted to be effective against this invasive pest. In order to gain information about possible semiochemicals involved in mate recognition and choice, both the volatile organic compounds (VOCs) and the epicuticular lipids of male and female specimens were analysed. VOCs emissions of specimens were characterized essentially by monoterpenes, while epicuticular lipids contained long chained 2-ketones, alkanes, alkenes, including some methyl alkenes, and several fatty acid propyl esters. The attractiveness of reconstituted VOCs blends of the two sexes was tested in electrophysiological and behavioural assays in laboratory conditions. Both the male and the female reconstituted VOCs drove a significant response towards individuals of the opposite sex, thus demonstrating features of sexual attractants. Our results suggest a possible application of VOCs blends as pheromonic attractants in field monitoring and mass trapping of Aclees sp. cf. foveatus.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are regarded as carriers of pheromones and odorants in insect chemoreception. These proteins are typically located in antennae, mouth organs and other chemosensory structures; however, members of both classes of proteins have been detected recently in other parts of the body and various functions have been proposed. The best studied of these non-sensory tasks is performed in pheromone glands, where OBPs and CSPs solubilise hydrophobic semiochemicals and assist their controlled release into the environment. In some cases the same proteins are expressed in antennae and pheromone glands, thus performing a dual role in receiving and broadcasting the same chemical message. Several reports have described OBPs and CSPs in reproductive organs. Some of these proteins are male specific and are transferred to females during mating. They likely carry semiochemicals with different proposed roles, from inhibiting other males from approaching mated females, to marking fertilized eggs, but further experimental evidence is still needed. Before being discovered in insects, the presence of binding proteins in pheromone glands and reproductive organs was widely reported in mammals, where vertebrate OBPs, structurally different from OBPs of insects and belonging to the lipocalin superfamily, are abundant in rodent urine, pig saliva and vaginal discharge of the hamster, as well as in the seminal fluid of rabbits. In at least four cases CSPs have been reported to promote development and regeneration: in embryo maturation in the honeybee, limb regeneration in the cockroach, ecdysis in larvae of fire ants and in promoting phase shift in locusts. Both OBPs and CSPs are also important in nutrition as solubilisers of lipids and other essential components of the diet. Particularly interesting is the affinity for carotenoids of CSPs abundantly secreted in the proboscis of moths and butterflies and the occurrence of the same (or very similar CSPs) in the eyes of the same insects. A role as a carrier of visual pigments for these proteins in insects parallels that of retinol-binding protein in vertebrates, a lipocalin structurally related to OBPs of vertebrates. Other functions of OBPs and CSPs include anti-inflammatory action in haematophagous insects, resistance to insecticides and eggshell formation. Such multiplicity of roles and the high success of both classes of proteins in being adapted to different situations is likely related to their stable scaffolding determining excellent stability to temperature, proteolysis and denaturing agents. The wide versatility of both OBPs and CSPs in nature has suggested several different uses for these proteins in biotechnological applications, from biosensors for odours to scavengers for pollutants and controlled releasers of chemicals in the environment.

In arthropods, the large majority of studies on olfaction have been focused on insects, where most of the proteins involved have been identified. In particular, chemosensing in insects relies on two families of membrane receptors, olfactory/gustatory receptors (ORs/GRs) and ionotropic receptors (IRs), and two classes of soluble proteins, odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). In other arthropods, such as ticks and mites, only IRs have been identified, while genes encoding for OBPs and CSPs are absent. A third class of soluble proteins, called Niemann-Pick C2 (NPC2) has been suggested as potential carrier for semiochemicals both in insects and other arthropods. Here we report the results of a proteomic analysis on olfactory organs (Haller's organ and palps) and control tissues of the tick Ixodes ricinus, and of immunostaining experiments targeting NPC2s. Adopting different extraction and proteomic approaches, we identified a large number of proteins, and highlighted those differentially expressed. None of the 13 NPC2s known for this species was found. On the other hand, using immunocytochemistry, we detected reaction against one NPC2 in the Haller's organ and palp sensilla. We hypothesized that the low concentration of such proteins in the tick's tissues could possibly explain the discrepant results. In ligand-binding assays the corresponding recombinant NPC2 showed good affinity to the fluorescent probe N-phenylnaphthylamine and to few organic compounds, supporting a putative role of NPC2s as odorant carriers.

Chemosensory Proteins (CSPs), a family of small soluble polypeptides, own their name to their highly abundant expression in chemosensory organs of insects. However, CSPs are extremely versatile and perform different tasks in chemical communication, but also in unrelated functions, such as development and insecticide resistance. Their multifunction is certainly linked to their simple structure and easy refolding.

The mite Varroa destructor is the major parasite of the honey bee and is responsible for great economical losses. The biochemical tools used by Varroa to detect semiochemicals produced by the host are still largely unknown. We have performed proteomic analysis on chemosensory organs of this species in order to identify putative soluble carriers for pheromones and other olfactory cues emitted by the host. In particular, we have analysed forelegs, mouthparts (palps, chelicera and hypostome) and the second pair of legs (as control tissue) in reproductive and phoretic stages of the Varroa life cycle. We identified 958 Varroa proteins, most of them common to organs and stages. Sequence analysis shows that four proteins can be assigned to the odorant-binding protein (OBP)-like class, which bear some similarity to insect OBPs, but so far are only reported in some Chelicerata. In addition, we have detected the presence of two proteins belonging to the Niemann-Pick family, type C2 (NPC2), which have been suggested to act as semiochemical carriers. This work contributes to elucidating the chemical communication systems in Varroa with the aim of understanding how detection of semiochemicals has evolved in terrestrial non-hexapod Arthropoda. Data are available via ProteomeXchange with identifier PXD008679.

We applied a “shotgun” approach based on nanoliquid chromatography–high resolution mass spectrometry associated to label free quantification (LFQ) to identify proteins varying with age, independently from the physiological state, in Aedes albopictus, a mosquito species which in the last decades invaded temperate regions in North America and Europe, creating concerns for associated high nuisance and risk of arbovirus transmission. The combined “shotgun” and LFQ approach was shown to be highly suitable to simultaneously compare several biological samples, as needed in a study aimed to analyze different age-groups and physiological states of adult mosquito females. The results obtained represent the first wide-scale analysis of protein expression in Ae. albopictus females: > 1000 and 665 proteins were identified from few micrograms of crude protein extracts of mosquito heads and thoraxes, respectively. Six of these proteins were shown to significantly vary from 2- to 16-day-old females, independently from their physiological state (i.e. virgin, mated, host-seeking, blood-fed, and gravid). Biological significance Mosquito-borne diseases, such as malaria, dengue and other arboviroses, are a persistent cause of global mortality and morbidity, affecting hundreds of thousands of people. Billions of people living in tropical areas are at risk of being bitten every day by an infective mosquito female and the spread of tropical species such as Aedes albopictus to temperate areas is creating alarm in the northern hemisphere. Mosquito longevity is a critical factor affecting mosquito-borne pathogen transmission cycles and the mosquito capacity to transmit pathogens. However, large scale analyses of the age structure of mosquito field populations is hampered by the lack of optimal age-grading approaches. Our findings open new perspectives for the development of reliable, simple and cheap protein-based assays to age-grade Ae. albopictus females and, most likely, other mosquito species of higher medical relevance, such as the main dengue vector, Aedes aegypti, and the major Afrotropical malaria vectors. These assays would greatly contribute to epidemiological studies aimed at defining the actual vectorial capacity of a given mosquito species. Moreover, they would be very valuable in assessing the effectiveness of mosquito control interventions based on the relative ratio between young and old individuals before and after the intervention.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are regarded as carriers of pheromones and odorants in insect chemoreception. These proteins are typically located in antennae, mouth organs and other chemosensory structures; however, members of both classes of proteins have been detected recently in other parts of the body and various functions have been proposed. The best studied of these non-sensory tasks is performed in pheromone glands, where OBPs and CSPs solubilise hydrophobic semiochemicals and assist their controlled release into the environment. In some cases the same proteins are expressed in antennae and pheromone glands, thus performing a dual role in receiving and broadcasting the same chemical message. Several reports have described OBPs and CSPs in reproductive organs. Some of these proteins are male specific and are transferred to females during mating. They likely carry semiochemicals with different proposed roles, from inhibiting other males from approaching mated females, to marking fertilized eggs, but further experimental evidence is still needed. Before being discovered in insects, the presence of binding proteins in pheromone glands and reproductive organs was widely reported in mammals, where vertebrate OBPs, structurally different from OBPs of insects and belonging to the lipocalin superfamily, are abundant in rodent urine, pig saliva and vaginal discharge of the hamster, as well as in the seminal fluid of rabbits. In at least four cases CSPs have been reported to promote development and regeneration: in embryo maturation in the honeybee, limb regeneration in the cockroach, ecdysis in larvae of fire ants and in promoting phase shift in locusts. Both OBPs and CSPs are also important in nutrition as solubilisers of lipids and other essential components of the diet. Particularly interesting is the affinity for carotenoids of CSPs abundantly secreted in the proboscis of moths and butterflies and the occurrence of the same (or very similar CSPs) in the eyes of the same insects. A role as a carrier of visual pigments for these proteins in insects parallels that of retinol-binding protein in vertebrates, a lipocalin structurally related to OBPs of vertebrates. Other functions of OBPs and CSPs include anti-inflammatory action in haematophagous insects, resistance to insecticides and eggshell formation. Such multiplicity of roles and the high success of both classes of proteins in being adapted to different situations is likely related to their stable scaffolding determining excellent stability to temperature, proteolysis and denaturing agents. The wide versatility of both OBPs and CSPs in nature has suggested several different uses for these proteins in biotechnological applications, from biosensors for odours to scavengers for pollutants and controlled releasers of chemicals in the environment.

In mosquitoes females, host-seeking, host-choice and other reproductive behaviours are driven by odorants. Species of the Afrotropical Anopheles gambiae complex display divergent host-preferences associated with significant differences in their efficiency as vectors of human malaria. Odorant perception starts in chemosensilla which are primarily housed in the antennae and maxillary palps. Taking advantage of recently completed genome sequencing efforts for species of the Anopheles gambiae complex, we applied shotgun proteomics to characterize the profile of soluble proteins of antennae and maxillary palps of three different species: An. coluzzii, An. arabiensis and An. quadriannulatus, showing remarkable differences in anthropophilic behaviour Protein profiles with particular focus on soluble proteins involved in the olfactory-based detection of odours were compared. Several differences between species were found in the abundance of Insect Cuticle proteins. Moreover, proteins belonging to Glutathione S-transferase and Odorant binding proteins, the latter of which known to be directly involved in odour recognition, are also different among the three species, with An. coluzzii and An. arabiensis being less different with respect to An. quadriannulatus..

Mosquito repellents represent a cleaner and safer alternative for population control and reduce the diseases they carry in large areas of the world. Recently, research has been focused on repellents of natural origins, both crude essential oils and their main constituents. We have observed that, although a large number of compounds can be efficiently used as mosquito repellents, their efficacy is never higher than those of commercial products DEET and Icaridin. Reasoning that probably specific and exceptionally active repellents might not exist, we focused our research on products that could provide longer protection times with respect to current commercial formulations while being used at lower concentrations. Based on the structure of menthone, a moderate natural repellent, we designed and synthesised some cyclic ketals that, because of their reduced volatility, could be effective for longer periods. In particular, a 1 % solution of one of such derivatives can still reduce mosquito bites by 90 % after 2 h, while DEET provides the same performance only for 15 min, when used at the same concentration. The approach we illustrate can be applied to other compounds and other systems and offers the additional advantage that derivatives of reduced volatility are also endowed with weaker odours.

The recently available genome of Aedes albopictus - the most worldwide-spread human arbovirus vector - has revealed a large genome repertory and a great plasticity which are believed to have contributed to the species success as an invasive species and opened the way to genomic, transcriptomic and proteomic studies. We carried out the first wide-scale quantitative proteomic analysis of Ae. albopictus female head and thorax by means of a 'shotgun' approach based on nano liquid chromatography-high resolution mass spectrometry associated to protein Label Free Quantification (LFQ) which allows to assess differences in protein expression between tissues and different physiological stages. We identified 886 and 721 proteins in heads and thoraxes respectively, 5 of which were exclusively expressed in thoraxes and 170 in heads, consistently with the more complex head physiology. Head-protein expression was found to be highly divergent between virgin and mated females and limited before and after blood-feeding and oviposition. The large repertoire of proteins identified represents an instrumental source of data for genome annotation and gene-expression studies, and may contribute to studies aimed at investigating the molecular bases of physiological processes of this successful invasive species.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are endowed with several different functions besides being carriers for pheromones and odorants. Based on a previous report of a CSP acting as surfactant in the proboscis of the moth Helicoverpa armigera, we revealed the presence of orthologue proteins in two other moths Plutella xylostella and Chilo suppressalis, as well as two butterflies Papilio machaon and Pieris rapae, using immunodetection and proteomic analysis. The unusual conservation of these proteins across large phylogenetic distances indicated a common specific function for these CSPs. This fact prompted us to search for other functions of these proteins and discovered that CSPs are abundantly expressed in the eyes of H. armigera and possibly involved as carriers for carotenoids and visual pigments. This hypothesis is supported by ligand-binding experiments and docking simulations with retinol and β-carotene. This last orange pigment, occurring in many fruits and vegetables, is an antioxidant and the precursor of visual pigments. We propose that structurally related CSPs solubilise nutritionally important carotenoids in the proboscis, while they act as carriers of both β-carotene and its derived products 3-hydroxyretinol and 3-hydroxyretinal in the eye. The use of soluble olfactory proteins, such as CSPs, as carriers for visual pigments in insects, here reported for the first time, parallels the function of retinol-binding protein in vertebrates, a lipocalin structurally related to vertebrate odorant-binding proteins.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) mediate both perception and release of chemical stimuli in insects. The genome of the honey bee contains 21 genes encoding OBPs and 6 encoding CSPs. Using a proteomic approach, we have investigated the expression of OBPs and CSPs in the mandibular glands of adult honey bees in relation to caste and age. OBP13 is mostly expressed in young individuals and in virgin queens, while OBP21 is abundant in older bees and is prevalent in mated queens. OBP14, which had been found in larvae, is produced in hive workers' glands. Quite unexpectedly, the mandibular glands of drones also contain OBPs, mainly OBP18 and OBP21. We have expressed three of the most represented OBPs and studied their binding properties. OBP13 binds with good specificity oleic acid and some structurally related compounds, OBP14 is better tuned to monoterpenoid structures, while OBP21 binds the main components of queen mandibular pheromone as well as farnesol, a compound used as a trail pheromone in the honey bee and other hymenopterans. The high expression of different OBPs in the mandibular glands suggests that such proteins could be involved in solubilization and release of semiochemicals.

Background The Western honey bee (Apis mellifera L.) is a critical component of human agriculture through its pollination activities. For years, beekeepers have controlled deadly pathogens such as Paenibacillus larvae, Nosema spp. and Varroa destructor with antibiotics and pesticides but widespread chemical resistance is appearing and most beekeepers would prefer to eliminate or reduce the use of in-hive chemicals. While such treatments are likely to still be needed, an alternate management strategy is to identify and select bees with heritable traits that allow them to resist mites and diseases. Breeding such bees is difficult as the tests involved to identify disease-resistance are complicated, time-consuming, expensive and can misidentify desirable genotypes. Additionally, we do not yet fully understand the mechanisms behind social immunity. Here we have set out to discover the molecular mechanism behind hygienic behavior (HB), a trait known to confer disease resistance in bees. Results After confirming that HB could be selectively bred for, we correlated measurements of this behavior with protein expression over a period of three years, at two geographically distinct sites, using several hundred bee colonies. By correlating the expression patterns of individual proteins with HB scores, we identified seven putative biomarkers of HB that survived stringent control for multiple hypothesis testing. Intriguingly, these proteins were all involved in semiochemical sensing (odorant binding proteins), nerve signal transmission or signal decay, indicative of the series of events required to respond to an olfactory signal from dead or diseased larvae. We then used recombinant versions of two odorant-binding proteins to identify the classes of ligands that these proteins might be helping bees detect. Conclusions Our data suggest that neurosensory detection of odors emitted by dead or diseased larvae is the likely mechanism behind a complex and important social immunity behavior that allows bees to co-exist with pathogens. Electronic supplementary material The online version of this article (doi:10.1186/s12864-014-1193-6) contains supplementary material, which is available to authorized users.

Detection of chemical signals both in insects and in vertebrates is mediated by soluble proteins, highly concentrated in olfactory organs, which bind semiochemicals and activate, with still largely unknown mechanisms, specific chemoreceptors. The same proteins are often found in structures where pheromones are synthesized and released, where they likely perform a second role in solubilizing and delivering chemical messengers in the environment. A single class of soluble polypeptides, called Odorant-Binding Proteins (OBPs) is known in vertebrates, while two have been identified in insects, OBPs and CSPs (Chemosensory Proteins). Despite their common name, OBPs of vertebrates bear no structural similarity with those of insects. We observed that in arthropods OBPs are strictly limited to insects, while a few members of the CSP family have been found in crustacean and other arthropods, where however, based on their very limited numbers, a function in chemical communication seems unlikely. The question we address in this review is whether another class of soluble proteins may have been adopted by other arthropods to perform the role of OBPs and CSPs in insects. We propose that lipid-transporter proteins of the Niemann-Pick type C2 family could represent likely candidates and report the results of an analysis of their sequences in representative species of different arthropods.

Fluorescence-linked binding assays allow determination of dissociation constants at equilibrium and have recently become increasingly popular, thanks to their ease of operation. Currently used probes, such as 1-aminoanthracene and N-phenyl-1-naphthylamine, are excited and emit in the ultraviolet region, but alternative ligands operating in the visible spectrum would be highly desirable for applications in biosensing devices. Based on the two above structures, we have designed and synthesised six new fluorescent probes to be used in ligand-binding assays. The compounds are derivatives of naphatalene, anthracene and fluoranthene and present two aromatic moieties linked by an amine nitrogen. We have measured the emission spectra of the new probes and their binding to three odorant-binding proteins. The probes bind the tested proteins with different affinities, generally with dissociation constants about one order of magnitude lower than the parent compounds. The extended aromatic systems present in the new compounds produced a shift of both excitation and emission peaks at higher wavelength, close or within the visible spectrum, thus facilitating measurements in biosensors for odorants and small organic molecules using optical devices.

Odorant-binding proteins (OBPs) are small soluble polypeptides found in sensory organs of vertebrates and insects as well as in secretory glands and are dedicated to detection and release of chemical stimuli. OBPs of vertebrates belong to the family of lipocalin proteins, while those of insects are folded into α-helical domains. Both types of architectures are extremely stable to temperature, organic solvents and proteolytic digestion. These characteristics make OBPs suitable elements for fabricating biosensors to be used in the environment, as well as for other biotechnological applications. The affinity of OBPs for small volatile organic compounds is in the micromolar range, and they have broad specificity to a range of ligands. For biotechnological applications, OBPs can be expressed in bacterial systems at low cost and are easily purified. The large amount of information available on their structures and affinities to different molecules should allow the design of specific mutants with desired characteristics and represent a solid base for tailoring OBPs for different applications.

Despite the great economical interest of locusts in agriculture, knowledge on their chemoreception systems is still poor. Phenylacetonitrile is recognised as a pheromone of the desert locust Schistocerca gregaria, triggering gregarization, promoting aggregation and inhibiting courtship. However, in the other major locust species, Locusta migratoria, pheromones have not been reported. We have identified the two isomers of naphthylpropionitrile from the male reproductive organs of L. migratoria. Chemical synthesis has confirmed the identity of the two compounds. Both isomers show significant affinity to CSP91, a protein reported in the testis, but not to three other proteins of the same family (CSP180, CSP540 and CSP884) expressed in female accessory glands. The striking similarity of these compounds with phenylacetonitrile and the unusual nature of such chemicals strongly suggest that naphthylpropionitrile could be pheromones for L. migratoria, while their site of expression and binding activity indicate a role in communication between sexes.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are small soluble polypeptides that bind semiochemicals in the lymph of insect chemosensilla. In the genome of Anopheles gambiae, 66 genes encode OBPs and 8 encode CSPs. Here we monitored their expression through classical proteomics (2D gel-MS analysis) and a shotgun approach. The latter method proved much more sensitive and therefore more suitable for tiny biological samples as mosquitoes antennae and eggs. Females express a larger number and higher quantities of OBPs in their antennae than males (24 vs 19). OBP9 is the most abundant in the antennae of both sexes, as well as in larvae, pupae and eggs. Of the 8 CSPs, 4 were detected in antennae, while SAP3 was the only one expressed in larvae. Our proteomic results are in fairly good agreement with data of RNA expression reported in the literature, except for OBP4 and OBP5, that we could not identify in our analysis, nor could we detect in Western Blot experiments. The relatively limited number of soluble olfactory proteins expressed at relatively high levels in mosquitoes makes further studies on the coding of chemical messages at the OBP level more accessible, providing for few specific targets. Identification of such proteins in Anopheles gambiae might facilitate future studies on host finding behavior in this important disease vector.

Solitary bees are major pollinators but their chemical communication system has been poorly studied. We investigated olfactory coding in Osmia cornuta from two perspectives, chemical and biochemical. We identified (E)-geranyl acetone and 2-hexyl-1,3-dioxolane, specifically secreted by females and males, respectively. A transcriptome analysis of antennae revealed 48 ORs (olfactory receptors), six OBPs (odorant-binding proteins), five CSPs (chemosensory proteins), and a single SNMP (sensory neuron membrane protein). The numbers of ORs and OBPs are much lower than in the honeybee, in particular, C-minus OBPs are lacking in the antennae of O. cornuta. We have expressed all six OBPs of O. cornuta and studied their binding specificities. The best ligands are common terpene plant odorants and both volatiles produced by the bee and identified in this work.

Chemosensory proteins (CSPs) are a class of small proteins expressed only in arthropods and endowed with heterogeneous functions. Some of them are involved in chemical communications, others in development or other physiological roles. The numbers of CSPs in different species of insects range from 4 in Drosophila to at least 70 in locusts, whereas in other arthropods such as crustaceans and millipedes, only 2–3 very similar sequences have been reported in each species. We have expressed, in a bacterial system, 5 of the 8 CSPs predicted by the genome of the malaria mosquito Anopheles gambiae, 4 identified at the protein level (SAP1, SAP2, SAP3, and CSP3) and a fifth annotated as part of this work, obtaining the proteins with high yields and in their soluble forms. Purified CSPs have been used to study their ligand-binding properties, both using competitive binding assays and quenching of intrinsic tryptophan fluorescence, in order to get insights into their physiological functions. The agreement between the 2 sets of data supports the assumptions that the ligands, including the fluorescent reporter, bind within the core of the proteins. Their different affinities toward a set of pure chemicals suggest specific roles in chemical communication.

Chemosensory proteins (CSPs) are small soluble proteins often associated with chemosensory organs in insects but include members involved in other functions, such as pheromone delivery and development. Although the CSPs of the sensory organs have been extensively studied, little is known on their functions in other parts of the body. A first screening of the available databases has identified 70 sequences encoding CSPs in the oriental locust Locusta migratoria manilensis. Applying proteomic analysis, we have identified 17 of them abundantly expressed in the female reproductive organs, but only one (CSP91) in male organs. Bacterially expressed CSP91 binds fatty acids with a specificity for oleic and linoleic acid, as well as medium-length alcohols and esters. The same acids have been detected as the main gas chromatographic peaks in the dichloromethane extracts of reproductive organs of both sexes. The abundance and the number of CSPs in female reproductive organs indicates important roles for these proteins. We cannot exclude that different functions can be associated with each of the 17 CSPs, including delivery of semiochemicals, solubilization of hormones, direct control of development, or other unknown tasks.

BackgroundAphids are agricultural pests of great economical interest. Alternatives to insecticides, using semiochemicals, are of difficult applications. In fact, sex pheromones are of little use as aphids reproduce partenogenetically most of the time. Besides, the alarm pheromone, (E)-ß-farnesene for a great number of species, is difficult to synthesize and unstable in the environment. The search for novel semiochemicals to be used in population control can be efficiently approached through the study of the olfactory system at the biochemical level. Recently odorant-binding proteins (OBPs) have been shown to play a central role in olfactory recognition, thus becoming the target of choice for designing new semiochemicals.Methodology/principal findingsTo address the question of how the alarm message is recognised at the level of OBPs, we have tested 29 compounds, including (E)-ß-farnesene, in binding assays with 6 recombinant proteins and in behaviour experiments. We have found that good repellents bind OBP3 and/or OBP7, while non repellents present different spectra of binding. These results have been verified with two species of aphids, Acyrthosiphon pisum and Myzus persicae, both using (E)-ß-farnesene as the alarm pheromone.ConclusionsOur results represent further support to the idea (so far convincingly demonstrated only in Drosophila) that OBPs are involved in decoding the chemical information of odorants and pheromones, and for the first time provide such evidence in other insect species and using wild-type insects. Moreover, the data offer guidelines and protocols for the discovery of potential alarm pheromones, using ligand-binding assays as a preliminary screening before subjecting selected compounds to behaviour tests.

Apis mellifera (Amel) relies on its olfactory system to detect and identify new-sources of floral food. The Odorant-Binding Proteins (OBPs) are the first proteins involved in odorant recognition and interaction, before activation of the olfactory receptors. The Amel genome possess a set of 21 OBPs, much fewer compared to the 60–70 OBPs found in Diptera genomes. We have undertaken a structural proteomics study of Amel OBPs, alone or in complex with odorant or model compounds. We report here the first 3D structure of a member of the C-minus class OBPs, AmelOBP14, characterized by only two disulfide bridges of the three typical of classical OBPs. We show that AmelOBP14 possesses a core of 6 α-helices comparable to that of classical OBPs, and an extra exposed C-terminal helix. Its binding site is located within this core and is completely closed. Fluorescent experiments using 1-NPN displacement demonstrate that AmelOBP14 is able to bind several compounds with sub micromolar dissociation constants, among which citralva and eugenol exhibit the highest affinities. We have determined the structures of AmelOBP14 in complex with 1-NPN, eugenol and citralva, explaining their strong binding. Finally, by introducing a double cysteine mutant at positions 44 and 97, we show that a third disulfide bridge was formed in the same position as in classical OBPs without disturbing the fold of AmelOBP14.

Chemical communication in insects is mediated by soluble binding proteins, belonging to two large families, Odorant-binding Proteins (OBPs) and Chemosensory Proteins (CSPs). Recently, evidence has been provided that OBPs are involved in recognition of chemical stimuli. We therefore decided to investigate the expression of OBPs and CSPs in the honeybee at the protein level, using a proteomic approach. Our results are in agreement with previous reports of expression at the RNA level and show that 12 of the 21 OBPs predicted in the genome of the honeybee Apis mellifera and 2 of the 6 CSPs are present in the foragers' antennae, while the larvae express only three OBPs and a single CSP. MALDI mass spectrometry on crude antennal extracts and MALDI profiling on sections of antennae demonstrated that these techniques can be applied to investigate individual differences in the expression of abundant proteins, such as OBPs and CSPs, as well as to detect the presence of proteins in different regions of the antenna. Finally, as part of a project aimed at the characterization of all OBPs and CSPs of the honeybee, we expressed 5 OBPs and 4 CSPs in a bacterial system and measured their affinity to a number of ligands. Clear differences in their binding spectra have been observed between OBPs, as well as CSPs.