Odorant receptor orthologues in conifer-feeding beetles display conserved responses to ecologically relevant odours.

Insects are able to detect a plethora of olfactory cues using a divergent family of odorant receptors (ORs). Despite the divergent nature of this family, related species frequently express several evolutionarily conserved OR orthologues. In the largest order of insects, Coleoptera, it remains unknown whether OR orthologues have conserved or divergent functions in different species. Using HEK293 cells, we addressed this question through functional characterization of two groups of OR orthologues in three species of the Curculionidae (weevil) family, the conifer-feeding bark beetles Ips typographus L. ("Ityp") and Dendroctonus ponderosae Hopkins ("Dpon") (Scolytinae), and the pine weevil Hylobius abietis L. ("Habi"; Molytinae). The ORs of H. abietis were annotated from antennal transcriptomes. The results show highly conserved response specificities, with one group of orthologues (HabiOR3/DponOR8/ItypOR6) responding exclusively to 2-phenylethanol (2-PE), and the other group (HabiOR4/DponOR9/ItypOR5) responding to angiosperm green leaf volatiles (GLVs). Both groups of orthologues belong to the coleopteran OR subfamily 2B, and share a common ancestor with OR5 in the cerambycid Megacyllene caryae, also tuned to 2-PE, suggesting a shared evolutionary history of 2-PE receptors across two beetle superfamilies. The detected compounds are ecologically relevant for conifer-feeding curculionids, and are probably linked to fitness, with GLVs being used to avoid angiosperm nonhost plants, and 2-PE being important for intraspecific communication and/or playing a putative role in beetle-microbe symbioses. To our knowledge, this study is the first to reveal evolutionary conservation of OR functions across several beetle species and hence sheds new light on the functional evolution of insect ORs.

The olfactory coreceptor IR8a governs larval feces-mediated competition avoidance in a hawkmoth.

Finding a suitable oviposition site is a challenging task for a gravid female moth. At the same time, it is of paramount importance considering the limited capability of most caterpillars to relocate to alternative host plants. The hawkmoth, Manduca sexta (Sphingidae), oviposits on solanaceous plants. Larvae hatching on a plant that is already attacked by conspecific caterpillars can face food competition, as well as an increased exposure to predators and induced plant defenses. Here, we show that feces from conspecific caterpillars are sufficient to deter a female M. sexta from ovipositing on a plant and that this deterrence is based on the feces-emitted carboxylic acids 3-methylpentanoic acid and hexanoic acid. Using a combination of genome editing (CRISPR-Cas9), electrophysiological recordings, calcium imaging, and behavioral analyses, we demonstrate that ionotropic receptor 8a (IR8a) is essential for acid-mediated feces avoidance in ovipositing hawkmoths.

Mutagenesis of odorant coreceptor Orco fully disrupts foraging but not oviposition behaviors in the hawkmoth Manduca sexta.

The hawkmoth Manduca sexta and one of its preferred hosts in the North American Southwest, Datura wrightii, share a model insect-plant relationship based on mutualistic and antagonistic life-history traits. D. wrightii is the innately preferred nectar source and oviposition host for M. sexta Hence, the hawkmoth is an important pollinator while the M. sexta larvae are specialized herbivores of the plant. Olfactory detection of plant volatiles plays a crucial role in the behavior of the hawkmoth. In vivo, the odorant receptor coreceptor (Orco) is an obligatory component for the function of odorant receptors (ORs), a major receptor family involved in insect olfaction. We used CRISPR-Cas9 targeted mutagenesis to knock out (KO) the MsexOrco gene to test the consequences of a loss of OR-mediated olfaction in an insect-plant relationship. Neurophysiological characterization revealed severely reduced antennal and antennal lobe responses to representative odorants emitted by D. wrightii In a wind-tunnel setting with a flowering plant, Orco KO hawkmoths showed disrupted flight orientation and an ablated proboscis extension response to the natural stimulus. The Orco KO gravid female displayed reduced attraction toward a nonflowering plant. However, more than half of hawkmoths were able to use characteristic odor-directed flight orientation and oviposit on the host plant. Overall, OR-mediated olfaction is essential for foraging and pollination behaviors, but plant-seeking and oviposition behaviors are sustained through additional OR-independent sensory cues.

Putative ligand binding sites of two functionally characterized bark beetle odorant receptors.

BACKGROUND: Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. RESULTS: We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. CONCLUSIONS: The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.

Identification and Characterization of Two "Sensory Neuron Membrane Proteins" (SNMPs) of the Desert Locust, Schistocerca gregaria (Orthoptera: Acrididae).

Pheromone-responsive neurons of insects not only require specific receptors but in addition several auxiliary components, including the "sensory neuron membrane protein," SNMP. Accordingly, SNMP is considered as a marker for neurons responding to pheromones. For the desert locust Schistocerca gregaria, it is known that the behavior, including aggregation behavior and courtship inhibition, is largely controlled by pheromones. However, little is known about pheromones, their receptors, and the pheromone-responsive cells in locusts. In this study, we have identified two SNMP subtypes, SNMP1 and SNMP2, and compared their phylogenetic relationship and primary structure motifs with SNMPs from other species. Both SNMPs were found in chemosensory tissues, especially the antennae. Employing double in situ hybridization, we identified and localized the SNMP-expressing cells in the antennae. Cells expressing SNMP1 were localized to sensilla trichodea but also to sensilla basiconica, which in locust respond to pheromones. One or a few cells express SNMP1 within the multineuron clusters from sensilla basiconica, whereas the SNMP2 subtype was expressed in cells surrounding the neuron clusters, possibly supporting cells. Based on the finding that SNMP1 is expressed in distinct neurons under chemosensory sensilla, it is conceivable that these cells may represent pheromone-responsive neurons of the desert locust.

Identification of Odorant Binding Proteins and Chemosensory Proteins in Antennal Transcriptomes of the Jumping Bristletail Lepismachilis y-signata and the Firebrat Thermobia domestica: Evidence for an Independent OBP-OR Origin.

Chemosensory protein (CSP) and gustatory receptor genes have been identified in all major arthropod groups. However, odorant binding proteins (OBP) and olfactory receptor genes are insect specific, suggesting that both gene families originated after the Hexapoda-Crustacea split (~470 million years ago). The seemingly parallel diversification of OBP and olfactory receptors has been suggested as coevolution between these genes after insect terrestrialization. To test this hypothesis we used the recently published transcriptomes of the jumping bristletail Lepismachilis y-signata and the firebrat Thermobia domestica to search for putative OBP and CSP sequences and analyzed their relationship to binding proteins of other insects and crustaceans. Our results suggest an evolution and expansion of OBPs as an adaptation to a terrestrial insect lifestyle, independently from the emergence of olfactory receptors.

Antennal transcriptome of Manduca sexta.

In recent years, considerable progress has been made in understanding the molecular mechanisms underlying olfaction in insects. Because of the diverse nature of the gene families involved, this process has largely relied on genomic data. As a consequence, studies have focused on a small subset of species with extensive genomic information. For Lepidoptera, a large order historically crucial to olfactory research, this circumstance has mostly limited advances to the domesticated species Bombyx mori, with some progress in the noctuid Heliothis virescens based on a nonpublic partial genome database. Because of the limited behavioral repertoire and nonexistent ecological importance of Bombyx, molecular data on the tobacco hornworm Manduca sexta are of utmost importance, especially with regards to its position as a classical olfactory model and its complex natural behavior. Here we present the use of transcriptomic and microarray data to identify members of the main olfactory gene families of Manduca. To assess the quality of our data, we correlate information on expressed receptor genes with detailed morphological data on the antennal lobe. Finally, we compare the expression of the near-complete transcript sets in male and female antennae.

Antennal transcriptome analysis of the chemosensory gene families in the tree killing bark beetles, Ips typographus and Dendroctonus ponderosae (Coleoptera: Curculionidae: Scolytinae).

BACKGROUND: The European spruce bark beetle, Ips typographus, and the North American mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Curculionidae: Scolytinae), are severe pests of coniferous forests. Both bark beetle species utilize aggregation pheromones to coordinate mass-attacks on host trees, while odorants from host and non-host trees modulate the pheromone response. Thus, the bark beetle olfactory sense is of utmost importance for fitness. However, information on the genes underlying olfactory detection has been lacking in bark beetles and is limited in Coleoptera. We assembled antennal transcriptomes from next-generation sequencing of I. typographus and D. ponderosae to identify members of the major chemosensory multi-gene families. RESULTS: Gene ontology (GO) annotation indicated that the relative abundance of transcripts associated with specific GO terms was highly similar in the two species. Transcripts with terms related to olfactory function were found in both species. Focusing on the chemosensory gene families, we identified 15 putative odorant binding proteins (OBP), 6 chemosensory proteins (CSP), 3 sensory neuron membrane proteins (SNMP), 43 odorant receptors (OR), 6 gustatory receptors (GR), and 7 ionotropic receptors (IR) in I. typographus; and 31 putative OBPs, 11 CSPs, 3 SNMPs, 49 ORs, 2 GRs, and 15 IRs in D. ponderosae. Predicted protein sequences were compared with counterparts in the flour beetle, Tribolium castaneum, the cerambycid beetle, Megacyllene caryae, and the fruit fly, Drosophila melanogaster. The most notable result was found among the ORs, for which large bark beetle-specific expansions were found. However, some clades contained receptors from all four beetle species, indicating a degree of conservation among some coleopteran OR lineages. Putative GRs for carbon dioxide and orthologues for the conserved antennal IRs were included in the identified receptor sets. CONCLUSIONS: The protein families important for chemoreception have now been identified in three coleopteran species (four species for the ORs). Thus, this study allows for improved evolutionary analyses of coleopteran olfaction. Identification of these proteins in two of the most destructive forest pests, sharing many semiochemicals, is especially important as they might represent novel targets for population control.

Microscopic morphology and distribution of the antennal sensilla in the double-spined bark beetle, Ips duplicatus (Coleoptera: Curculionidae).

The double-spined spruce bark beetle, Ips duplicatus, has become an infamous secondary pest of Norway spruce, causing extensive ecological and economic destruction in many Central European countries. Antennae are the primary olfactory organs that play a fundamental role in insect-host chemical communication; therefore, understanding morphology is crucial before conducting electrophysiological investigations. Here, we present our analysis of sensilla types on the antennal surface of I. duplicatus for the first time, using high-resolution-scanning electron microscopy. We studied the external morphological characteristics of antennae and the types, numbers, and distribution of the antennal sensilla in males and females. Our results revealed the presence of five different types of morphologically distinct sensilla: sensilla chaetica, sensilla basiconica, sensilla trichodea, sensilla coeloconica, and Böhm's sensilla. We observed two subtypes of sensilla chaetica (SChI and SChII), four subtypes of sensilla basiconica (SBI, SBII, SBIII, and SBIV), three subtypes of sensilla trichodea (STrII, STrIII, and STrIV) and two subtypes of sensilla coeloconica (SCoI and SCoII), respectively in I. duplicatus males and females. Minor differences in length and numbers between the sexes for some sensilla types were found. Distribution maps for different sensillar types were constructed, and specific areas for the respective sensilla were found. Possible functions of observed sensilla types are discussed. The present study provides a basis for future electrophysiological studies to understand how I. duplicatus detects ecologically important olfactory cues. RESEARCH HIGHLIGHTS: • The first report of morphology and distribution pattern of the antennal sensilla in Ips duplicatus is discussed. • A total of 6 main types and 11 antennal sensilla subtypes were observed in male and female Ips duplicatus. • Minor sex-specific differences were seen in the length and numbers in several sensilla types.

Cross-generation pheromonal communication drives Drosophila oviposition site choice.

In a heterogeneous and changing environment, oviposition site selection strongly affects the survival and fitness of the offspring.1,2 Similarly, competition between larvae affects their prospects.3 However, little is known about the involvement of pheromones in regulating these processes.4,5,6,7,8 Here, we show that mated females of Drosophila melanogaster prefer to lay eggs on substrates containing extracts of conspecific larvae. After analyzing these extracts chemically, we test each compound in an oviposition assay and find that mated females display a dose-dependent preference to lay eggs on substrates spiked with (Z)-9-octadecenoic acid ethyl ester (OE). This egg-laying preference relies on gustatory receptor Gr32a and tarsal sensory neurons expressing this receptor. The concentration of OE also regulates larval place choice in a dose-dependent manner. Physiologically, OE activates female tarsal Gr32a+ neurons. In conclusion, our results reveal a cross-generation communication strategy essential for oviposition site selection and regulation of larval density.

A salivary GMC oxidoreductase of Manduca sexta re-arranges the green leaf volatile profile of its host plant.

Green leaf volatiles (GLVs) are short-chain oxylipins that are emitted from plants in response to stress. Previous studies have shown that oral secretions (OS) of the tobacco hornworm Manduca sexta, introduced into plant wounds during feeding, catalyze the re-arrangement of GLVs from Z-3- to E-2-isomers. This change in the volatile signal however is bittersweet for the insect as it can be used by their natural enemies, as a prey location cue. Here we show that (3Z):(2E)-hexenal isomerase (Hi-1) in M. sexta's OS catalyzes the conversion of the GLV Z-3-hexenal to E-2-hexenal. Hi-1 mutants that were raised on a GLV-free diet showed developmental disorders, indicating that Hi-1 also metabolizes other substrates important for the insect's development. Phylogenetic analysis placed Hi-1 within the GMCß-subfamily and showed that Hi-1 homologs from other lepidopterans could catalyze similar reactions. Our results indicate that Hi-1 not only modulates the plant's GLV-bouquet but also functions in insect development.

Competing beetles attract egg laying in a hawkmoth.

In nature, plant-insect interactions occur in complex settings involving multiple trophic levels, often with multiple species at each level.1 Herbivore attack of a host plant typically dramatically alters the plant's odor emission in terms of concentration and composition.2,3 Therefore, a well-adapted herbivore should be able to predict whether a plant is still suitable as a host by judging these changes in the emitted bouquet. Although studies have demonstrated that oviposition preferences of successive insects were affected by previous infestations,4,5 the underlying molecular and olfactory mechanisms remain unknown. Here, we report that tobacco hawkmoths (Manduca sexta) preferentially oviposit on Jimson weed (Datura wrightii) that is already infested by a specialist, the three-lined potato beetle (Lema daturaphila). Interestingly, the moths' offspring do not benefit directly, as larvae develop more slowly when feeding together with Lema beetles. However, one of M. sexta's main enemies, the parasitoid wasp Cotesia congregata, prefers the headspace of M. sexta-infested plants to that of plants infested by both herbivores. Hence, we conclude that female M. sexta ignore the interspecific competition with beetles and oviposit deliberately on beetle-infested plants to provide their offspring with an enemy-reduced space, thus providing a trade-off that generates a net benefit to the survival and fitness of the subsequent generation. We identify that α-copaene, emitted by beetle-infested Datura, plays a role in this preference. By performing heterologous expression and single-sensillum recordings, we show that odorant receptor (Or35) is involved in α-copaene detection.

Changes in antennal gene expression underlying sensory system maturation in Rhodnius prolixus.

Triatomine bugs are the blood feeding insect vectors transmitting Chagas disease to humans, a neglected tropical disease that affects over 8 million people, mainly in Latin America. The behavioral responses to host cues and bug signals in Rhodnius prolixus are state dependent, i.e., they vary as a function of post-ecdysis age. At the molecular level, these changes in behavior are probably due to a modulation of peripheral and central processes. In the present study, we report a significant modulation of the expression of a large set of sensory-related genes. Results were generated by means of antennal transcriptomes of 5th instar larvae along the first week (days 0, 2, 4, 6 and 8) after ecdysis sequenced using the Illumina HiSeq platform. Significant age-induced changes in transcript abundance were established for more than 6120 genes (54,7% of 11,186 genes expressed) in the antenna of R. prolixus. This was especially true between the first two days after ecdysis when more than 2500 genes had their expression significantly altered. In contrast, expression profiles were almost identical between day 6 and 8, with only a few genes showing significant modulation of their expression. A total of 86 sensory receptors, odorant carriers and odorant degrading enzymes were significantly modulated across age points and clustered into three distinct expression profiles. The set of sensory genes whose expression increased with age (profile 3) may include candidates underlying the increased responsiveness to host cues shown by R. prolixus during the first days after molting. For the first time, we describe the maturation process undergone at the molecular level by the peripheral sensory system of a hemimetabolous insect.

Chemoreceptor Diversity in Apoid Wasps and Its Reduction during the Evolution of the Pollen-Collecting Lifestyle of Bees (Hymenoptera: Apoidea).

Chemoreceptors help insects to interact with their environment, to detect and assess food sources and oviposition sites, and to aid in intra- and interspecific communication. In Hymenoptera, species of eusocial lineages possess large chemoreceptor gene repertoires compared with solitary species, possibly because of their additional need to recognize nest-mates and caste. However, a critical piece of information missing so far has been the size of chemoreceptor gene repertoires of solitary apoid wasps. Apoid wasps are a paraphyletic group of almost exclusively solitary Hymenoptera phylogenetically positioned between ant and bee, both of which include eusocial species. We report the chemosensory-related gene repertoire sizes of three apoid wasps: Ampulex compressa, Cerceris arenaria, and Psenulus fuscipennis. We annotated genes encoding odorant (ORs), gustatory, and ionotropic receptors and chemosensory soluble proteins and odorant-binding proteins in transcriptomes of chemosensory tissues of the above three species and in early draft genomes of two species, A. compressa and C. arenaria. Our analyses revealed that apoid wasps possess larger OR repertoires than any bee lineage, that the last common ancestor of Apoidea possessed a considerably larger OR repertoire (∼160) than previously estimated (73), and that the expansion of OR genes in eusocial bees was less extensive than previously assumed. Intriguingly, the evolution of pollen-collecting behavior in the stem lineage of bees was associated with a notable loss of OR gene diversity. Thus, our results support the view that herbivorous Hymenoptera tend to possess smaller OR repertoires than carnivorous, parasitoid, or kleptoparasitic species.

A highly-contiguous genome assembly of the Eurasian spruce bark beetle, Ips typographus, provides insight into a major forest pest.

Conifer-feeding bark beetles are important herbivores and decomposers in forest ecosystems. These species complete their life cycle in nutritionally poor substrates and some can kill enormous numbers of trees during population outbreaks. The Eurasian spruce bark beetle (Ips typographus) can destroy >100 million m3 of spruce in a single year. We report a 236.8 Mb I. typographus genome assembly using PacBio long-read sequencing. The final phased assembly has a contig N50 of 6.65 Mb in 272 contigs and is predicted to contain 23,923 protein-coding genes. We reveal expanded gene families associated with plant cell wall degradation, including pectinases, aspartyl proteases, and glycosyl hydrolases. This genome sequence from the genus Ips provides timely resources to address questions about the evolutionary biology of the true weevils (Curculionidae), one of the most species-rich animal families. In forests of today, increasingly stressed by global warming, this draft genome may assist in developing pest control strategies to mitigate outbreaks.

Developmental and sexual divergence in the olfactory system of the marine insect Clunio marinus.

An animal's fitness strongly depends on successful feeding, avoidance of predators and reproduction. All of these behaviours commonly involve chemosensation. As a consequence, when species' ecological niches and life histories differ, their chemosensory abilities need to be adapted accordingly. The intertidal insect Clunio marinus (Diptera: Chironomidae) has tuned its olfactory system to two highly divergent niches. The long-lived larvae forage in a marine environment. During the few hours of terrestrial adult life, males have to find the female pupae floating on the water surface, free the cryptic females from their pupal skin, copulate and carry the females to the oviposition sites. In order to explore the possibility for divergent olfactory adaptations within the same species, we investigated the chemosensory system of C. marinus larvae, adult males and adult females at the morphological and molecular level. The larvae have a well-developed olfactory system, but olfactory gene expression only partially overlaps with that of adults, likely reflecting their marine vs. terrestrial lifestyles. The olfactory system of the short-lived adults is simple, displaying no glomeruli in the antennal lobes. There is strong sexual dimorphism, the female olfactory system being particularly reduced in terms of number of antennal annuli and sensilla, olfactory brain centre size and gene expression. We found hints for a pheromone detection system in males, including large trichoid sensilla and expression of specific olfactory receptors and odorant binding proteins. Taken together, this makes C. marinus an excellent model to study within-species evolution and adaptation of chemosensory systems.

Sawfly Genomes Reveal Evolutionary Acquisitions That Fostered the Mega-Radiation of Parasitoid and Eusocial Hymenoptera.

The tremendous diversity of Hymenoptera is commonly attributed to the evolution of parasitoidism in the last common ancestor of parasitoid sawflies (Orussidae) and wasp-waisted Hymenoptera (Apocrita). However, Apocrita and Orussidae differ dramatically in their species richness, indicating that the diversification of Apocrita was promoted by additional traits. These traits have remained elusive due to a paucity of sawfly genome sequences, in particular those of parasitoid sawflies. Here, we present comparative analyses of draft genomes of the primarily phytophagous sawfly Athalia rosae and the parasitoid sawfly Orussus abietinus. Our analyses revealed that the ancestral hymenopteran genome exhibited traits that were previously considered unique to eusocial Apocrita (e.g., low transposable element content and activity) and a wider gene repertoire than previously thought (e.g., genes for CO2 detection). Moreover, we discovered that Apocrita evolved a significantly larger array of odorant receptors than sawflies, which could be relevant to the remarkable diversification of Apocrita by enabling efficient detection and reliable identification of hosts.

Evaluation of the DREAM Technique for a High-Throughput Deorphanization of Chemosensory Receptors in Drosophila.

In the vinegar fly Drosophila melanogaster, the majority of olfactory receptors mediating the detection of volatile chemicals found in their natural habitat have been functionally characterized (deorphanized) in vivo. In this process, receptors have been assigned ligands leading to either excitation or inhibition in the olfactory sensory neuron where they are expressed. In other, non-drosophilid insect species, scientists have not yet been able to compile datasets about ligand-receptor interactions anywhere near as extensive as in the model organism D. melanogaster, as genetic tools necessary for receptor deorphanization are still missing. Recently, it was discovered that exposure to artificially high concentrations of odorants leads to reliable alterations in mRNA levels of interacting odorant receptors in mammals. Analyzing receptor expression after odorant exposure can, therefore, help to identify ligand-receptor interactions in vivo without the need for other genetic tools. Transfer of the same methodology from mice to a small number of receptors in D. melanogaster resulted in a similar trend, indicating that odorant exposure induced alterations in mRNA levels are generally applicable for deorphanization of interacting chemosensory receptors. Here, we evaluated the potential of the DREAM (Deorphanization of receptors based on expression alterations in mRNA levels) technique for high-throughput deorphanization of chemosensory receptors in insect species using D. melanogaster as a model. We confirmed that in some cases the exposure of a chemosensory receptor to high concentration of its best ligand leads to measureable alterations in mRNA levels. However, unlike in mammals, we found several cases where either confirmed ligands did not induce alterations in mRNA levels of the corresponding chemosensory receptors, or where gene transcript-levels were altered even though there is no evidence for a ligand-receptor interaction. Hence, there are severe limitations to the suitability of the DREAM technique for deorphanization as a general tool to characterize olfactory receptors in insects.

Sensilla Morphology and Complex Expression Pattern of Odorant Binding Proteins in the Vetch Aphid Megoura viciae (Hemiptera: Aphididae).

Chemoreception in insects is mediated by several components interacting at different levels and including odorant-binding proteins (OBPs). Although recent studies demonstrate that the function of OBPs cannot be restricted to an exclusively olfactory role, and that OBPs have been found also in organs generally not related to chemoreception, their feature of binding molecules remains undisputed. Studying the vetch aphid Megoura viciae (Buckton), we used a transcriptomic approach to identify ten OBPs in the antennae and we examined the ultrastructural morphology of sensilla and their distribution on the antennae, legs, mouthparts and cauda of wingless and winged adults by scanning electron microscopy (SEM). Three types of sensilla, trichoid, coeloconic and placoid, differently localized and distributed on antennae, mouthparts, legs and cauda, were described. The expression analysis of the ten OBPs was performed by RT-qPCR in the antennae and other body parts of the wingless adults and at different developmental stages and morphs. Five of the ten OBPs (MvicOBP1, MvicOBP3, MvicOBP6, MvicOBP7, and MvicOBP8), whose antibodies were already available, were selected for experiments of whole-mount immunolocalization on antennae, mouthparts, cornicles and cauda of adult aphids. Most of the ten OBPs were more expressed in antennae than in other body parts; MvicOBP1, MvicOBP3, MvicOBP6, MvicOBP7 were also immunolocalized in the sensilla on the antennae, suggesting a possible involvement of these proteins in chemoreception. MvicOBP6, MvicOBP7, MvicOBP8, MvicOBP9 were highly expressed in the heads and three of them (MvicOBP6, MvicOBP7, MvicOBP8) were immunolocalized in the sensilla on the mouthparts, supporting the hypothesis that also mouthparts may be involved in chemoreception. MvicOBP2, MvicOBP3, MvicOBP5, MvicOBP8 were highly expressed in the cornicles-cauda and two of them (MvicOBP3, MvicOBP8) were immunolocalized in cornicles and in cauda, suggesting a possible new function not related to chemoreception. Moreover, the response of M. viciae to different components of the alarm pheromone was assessed by behavioral assays on wingless adult morph; (-)-α-pinene and (+)-limonene were found to be the components mainly eliciting an alarm response. Taken together, our results represent a road map for subsequent in-depth analyses of the OBPs involved in several physiological functions in M. viciae, including chemoreception.

In Search for Pheromone Receptors: Certain Members of the Odorant Receptor Family in the Desert Locust Schistocerca gregaria (Orthoptera: Acrididae) Are Co-expressed with SNMP1.

Under given environmental conditions, the desert locust (Schistocera gregaria) forms destructive migratory swarms of billions of animals, leading to enormous crop losses in invaded regions. Swarm formation requires massive reproduction as well as aggregation of the animals. Pheromones that are detected via the olfactory system have been reported to control both reproductive and aggregation behavior. However, the molecular basis of pheromone detection in the antennae of Schistocerca gregaria is unknown. As an initial step to disclose pheromone receptors, we sequenced the antennal transcriptome of the desert locust. By subsequent bioinformatical approaches, 119 distinct nucleotide sequences encoding candidate odorant receptors (ORs) were identified. Phylogenetic analyses employing the identified ORs from Schistocerca gregaria (SgreORs) and OR sequences from the related species Locusta migratoria revealed a group of locust ORs positioned close to the root, i.e. at a basal site in a phylogenetic tree. Within this particular OR group (termed basal or b-OR group), the locust OR sequences were strictly orthologous, a trait reminiscent of pheromone receptors from lepidopteran species. In situ hybridization experiments with antennal tissue demonstrated expression of b-OR types from Schistocerca gregaria in olfactory sensory neurons (OSNs) of either sensilla trichodea or sensilla basiconica, both of which have been reported to respond to pheromonal substances. More importantly, two-color fluorescent in situ hybridization experiments showed that most b-OR types were expressed in cells co-expressing the "sensory neuron membrane protein 1" (SNMP1), a marker indicative of pheromone-sensitive OSNs in insects. Analyzing the expression of a larger number of SgreOR types outside the b-OR group revealed that only a few of them were co-expressed with SNMP1. In summary, we have identified several candidate pheromone receptors from Schistocerca gregaria that could mediate responses to pheromones implicated in controlling reproduction and aggregation behavior.