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The red palm weevil (RPW), Rhynchophorus ferrugineus (Olivier), also known as the Asian palm weevil, is an invasive pest that causes widespread damage to palm trees around the globe. As pheromone communication is crucial for their mass attack and survival on palm trees, the olfactory concept of pest control strategies has been widely explored recently. We aim to understand the molecular basis of olfaction in RPW by studying one of the key olfactory proteins in insect pheromone communication, sensory neuron membrane proteins (SNMPs). SNMPs belong to the CD36 (cluster of differentiation 36) family that perform two distinct olfactory roles in insects, either in pheromone (odorant) transfer to the odorant receptors (SNMP1) or in the pheromone clearing process (SNMP2). In this study, we performed antennal transcriptomic screening and identified six SNMPs, mapping them on the R. ferrugineus genome, and confirmed four distinct SNMPs. Both SNMP1 proteins in RPW, viz., RferSNMPu1 and RferSNMPu2, were mapped onto the same scaffold in different loci in the RPW genome. To further understand the function of these proteins, we first classified them using phylogenetic analysis and checked their tissue-specific expression patterns. Further, we measured the relative transcript abundance of SNMPs in laboratory-reared, field-collected adults and pheromone-exposure experiments, ultimately identifying RferSNMPu1 as a potential candidate for functional analysis. We mapped RferSNMPu1 expression in the antennae and found that expression patterns were similar in both sexes. We used RNAi-based gene silencing to knockdown RferSNMPu1 and tested the changes in the RPW responses to aggregation pheromone compounds, 4-methyl-5-nonanol (ferrugineol) and 4-methyl-5-nonanone (ferrugineone), and a kairomone, ethyl acetate using electroantennogram (EAG) recordings. We found a significant reduction in the EAG recordings in the RferSNMPu1 knockdown strain of adult RPWs, confirming its potential role in pheromone detection. The structural modelling revealed the key domains in the RferSNMPu1 structure, which could likely be involved in pheromone detection based on the identified ectodomain tunnels. Our studies on RferSNMPu1 with a putative role in pheromone detection provide valuable insight into understanding the olfaction in R. ferrugineus as well as in other Curculionids, as SNMPs are under-explored in terms of its functional role in insect olfaction. Most importantly, RferSNMPu1 can be used as a potential target for the olfactory communication disruption in the R. ferrugineus control strategies., (© 2024. The Author(s).)

Locusts depend upon their sense of smell and provide useful models for understanding olfaction. Extending this understanding requires knowledge of the molecular and structural organization of the olfactory system. Odor sensing begins with olfactory receptor neurons (ORNs), which express odorant receptors (ORs). In insects, ORNs are housed, in varying numbers, in olfactory sensilla. Because the organization of ORs within sensilla affects their function, it is essential to identify the ORs they contain. Here, using RNA sequencing, we identified 179 putative ORs in the transcriptomes of the two main olfactory organs, antenna and palp, of the locust Schistocerca americana. Quantitative expression analysis showed most putative ORs (140) are expressed in antennae while only 31 are in the palps. Further, our analysis identified one OR detected only in the palps and seven ORs that are expressed differentially by sex. An in situ analysis of OR expression suggested ORs are organized in non-random combinations within antennal sensilla. A phylogenetic comparison of OR predicted protein sequences revealed homologous relationships among two other Acrididae species. Our results provide a foundation for understanding the organization of the first stage of the olfactory system in S. americana, a well-studied model for olfactory processing., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Odorant-binding proteins (OBPs) are prevalent in the antennal transcriptomes of different orders of insects. Studies on OBPs have focused on their role in the insect chemosensory system, but knowledge of their functions in the insect testis is limited. We sequenced the transcriptomes of the Athetis dissimilis reproductive organs and analyzed the expression of AdisOBP genes in different tissues. We identified 23 OBPs in the testis and ovaries and 31 OBPs in antennal transcriptomes. The results of real-time quantitative PCR revealed that 23 of the 54 OBP genes were highly expressed in both female and male antennae, including three that exhibited male-biased expression and 15 that exhibited female-biased expression. A total of 24 OBPs were highly expressed in the testis of A. dissimilis, while expression of OBPs in the ovaries was very low. These findings highlight the functional diversity of OBPs in insects and can facilitate further studies on the OBPs in A. dissimilis and lepidopteran species.

Locusts are advantageous organisms to elucidate mechanisms of olfactory coding at the systems level. Sensory input is provided by the olfactory receptor neurons of the antenna, which send their axons into the antennal lobe. So far, cellular properties of neurons isolated from the circuitry of the olfactory system, such as transmitter-induced calcium responses, have not been studied. Biochemical and immunocytochemical investigations have provided evidence for acetylcholine as classical transmitter of olfactory receptor neurons. Here, we characterize cell cultured projection and local interneurons of the antennal lobe by cytosolic calcium imaging to cholinergic stimulation. We bulk loaded the indicator dye Cal-520 AM in dissociated culture and recorded calcium transients after applying cholinergic agonists and antagonists. The majority of projection and local neurons respond with increases in calcium levels to activation of both nicotinic and muscarinic receptors. In local interneurons, we reveal interactions lasting over minutes between intracellular signaling pathways, mediated by muscarinic and nicotinic receptor stimulation. The present investigation is pioneer in showing that Cal-520 AM readily loads Locusta migratoria neurons, making it a valuable tool for future research in locust neurophysiology, neuropharmacology, and neurodevelopment.

Sensory genes play a key role in the host location of parasitoids. To date, the sensory genes that regulate parasitoids to locate gall-inducing insects have not been uncovered. An obligate ectoparasitoid, Quadrastichus mendeli Kim & La Salle (Hymenoptera: Eulophidae: Tetrastichinae), is one of the most important parasitoids of Leptocybe invasa, which is a global gall-making pest in eucalyptus plantations. Interestingly, Q. mendeli can precisely locate the larva of L. invasa, which induces tumor-like growth on the eucalyptus leaves and stems. Therefore, Q. mendeli-L. invasa provides an ideal system to study the way that parasitoids use sensory genes in gall-making pests. In this study, we present the transcriptome of Q. mendeli using high-throughput sequencing. In total, 31,820 transcripts were obtained and assembled into 26,925 unigenes in Q. mendeli. Then, the major sensory genes were identified, and phylogenetic analyses were performed with these genes from Q. mendeli and other model insect species. Three chemosensory proteins (CSPs), 10 gustatory receptors (GRs), 21 ionotropic receptors (IRs), 58 odorant binding proteins (OBPs), 30 odorant receptors (ORs) and 2 sensory neuron membrane proteins (SNMPs) were identified in Q. mendeli by bioinformatics analysis. Our report is the first to obtain abundant biological information on the transcriptome of Q. mendeli that provided valuable information regarding the molecular basis of Q. mendeli perception, and it may help to understand the host location of parasitoids of gall-making pests.

The round-headed pine beetle Dendroctonus adjunctus, whose dispersion and colonization behaviors are linked to a communication system mediated by semiochemicals, is one of the five most critical primary pests in forest ecosystems in Mexico. This study provides the first head transcriptome analysis of D. adjunctus and the identification of the nonreceptor olfactory genes involved in the perception of odors. De novo assembly yielded 44,420 unigenes, and GO annotations were similar to those of antennal transcriptomes of other beetle species, which reflect metabolic processes related to smell and signal transduction. A total of 36 new transcripts of nonreceptor olfactory genes were identified, of which 27 encode OBPs, 7 encode CSPs, and 2 encode SNMP candidates, which were subsequently compared to homologous proteins from other bark beetles and Coleoptera species by searching for sequence motifs and performing phylogenetic analyses. Our study provides information on genes encoding nonreceptor proteins in D. adjunctus and broadens the knowledge of olfactory genes in Coleoptera and bark beetle species, and will help to understand colonization and aggregation behaviors for the development of tools that complement management strategies.

Many insects possess the ability to detect fine fluctuations in the environmental CO 2 concentration. In herbivorous species, plant-emitted CO 2 , in combination with other sensory cues, affect many behaviors including foraging and oviposition. In contrast to the comprehensive knowledge obtained on the insect olfactory pathway in recent years, we still know little about the central CO 2 system. By utilizing intracellular labeling and mass staining, we report the neuroanatomy of projection neurons connected with the CO 2 sensitive antennal-lobe glomerulus, the labial pit organ glomerulus (LPOG), in the noctuid moth, Helicoverpa armigera. We identified 15 individual LPOG projection neurons passing along different tracts. Most of these uniglomerular neurons terminated in the lateral horn, a previously well-described target area of plant-odor projection neurons originating from the numerous ordinary antennal-lobe glomeruli. The other higher-order processing area for odor information, the calyces, on the other hand, was weakly innervated by the LPOG neurons. The overlapping LPOG terminals in the lateral horn, which is considered important for innate behavior in insects, suggests the biological importance of integrating the CO 2 input with plant odor information while the weak innervation of the calyces indicates the insignificance of this ubiquitous cue for learning mechanisms.

Olfaction plays key roles in insect survival and reproduction, such as feeding, courtship, mating, and oviposition. The olfactory-based control strategies have been developed an important means for pest management. Streltzoviella insularis is a destructive insect pest of many street tree species, and characterization of its olfactory proteins could provide targets for the disruption of their odour recognition processes and for urban forestry protection. In this study, we assembled the antennal transcriptome of S. insularis by next-generation sequencing and annotated the main olfactory multi-gene families, including 28 odorant-binding proteins (OBPs), 12 chemosensory proteins (CSPs), 56 odorant receptors (ORs), 11 ionotropic receptors (IRs), two sensory neuron membrane proteins (SNMPs), and 101 odorant-degrading enzymes (ODEs). Sequence and phylogenetic analyses confirmed the characteristics of these proteins. We further detected tissue- and sex-specific expression patterns of OBPs, CSPs and SNMPs by quantitative real time-PCR. Most OBPs were highly and differentially expressed in the antennae of both sexes. SinsCSP10 was expressed more highly in male antennae than in other tissues. Two SNMPs were highly expressed in the antennae, with no significant difference in expression between the sexes. Our results lay a solid foundation for understanding the precise molecular mechanisms underlying S. insularis odour recognition.

Antennal olfaction plays a key role in insect survival, which mediates important behaviors like host search, mate choice, and oviposition site selection. As an oligophagous insect, olfaction is extremely important for Ophraella communa to locate host plants. However, information on the olfactory genes has been lacking in O. communa. Using next generation sequencing, we assembled the antennal transcriptome of O. communa and first reported the major chemosensory genes necessary for olfaction in this species. In this study, a total 105 candidate chemosensory genes were identified in O. communa antennae, including 25 odorant-binding proteins (OBPs), 11 chemosensory proteins (CSPs), four sensory neuron membrane proteins (SNMPs), 30 odorant receptors (ORs), 18 ionotropic receptors (IRs), and 17 gustatory receptors (GRs). We also identified full-length sequences of the highly conserved ORco and IR8a/25a family in O. communa. In addition, the expression profile of 15 ORs and four OBPs were validated by quantitative real-time polymerase chain reaction (qPCR). We found that OcomOR2/4/19 and OcomOBP19/20 had a biased expression in male antennae, and OcomOR8 had a biased expression in the female antennae. This large number of chemosensory genes handled by homology analysis and qPCR results will provide the first insights into molecular basis for the olfactory systems of O. communa as well as advance our understanding of olfactory mechanisms in Coleoptera.

The greater wax moth, Galleria mellonella Linnaeus (Lepidoptera: Galleriinae), is a ubiquitous pest of the honeybee, and poses a serious threat to the global honeybee industry. G. mellonella pheromone system is unusual compared to other lepidopterans and provides a unique olfactory model for pheromone perception. To better understand the olfactory mechanisms in G. mellonella, we conducted a transcriptomic analysis on the antennae of both male and female adults of G. mellonella using high-throughput sequencing and annotated gene families potentially involved in chemoreception. We annotated 46 unigenes coding for odorant receptors, 25 for ionotropic receptors, two for sensory neuron membrane proteins, 22 for odorant binding proteins and 20 for chemosensory proteins. Expressed primarily in antennae were all the 46 odorant receptor unigenes, nine of the 14 ionotropic receptor unigenes, and two of the 22 unigenes coding for odorant binding proteins, suggesting their putative roles in olfaction. The expression of some of the identified unigenes were sex-specific, suggesting that they may have important functions in the reproductive behavior of the insect. Identification of the candidate unigenes and initial analyses on their expression profiles should facilitate functional studies in the future on chemoreception mechanisms in this species and related lepidopteran moths.

Cyrtorhinus lividipennis Reuter (Hemiptera: Miridae) is an important egg predator of planthoppers which are destructive rice pests. The chemosensory genes in the mirid antennae play important roles in mating and prey-seeking behaviors. To gain a better understanding of the olfaction of C. lividipennis, we sequenced the antennal transcriptomes of the predator to identify the key olfaction genes. We identified 18 odorant binding proteins (OBPs), 12 chemosensory proteins (CSPs), 1 Niemann-Pick C2 protein (NPC2), 15 odorant receptors (ORs), 6 ionotropic receptors (IRs), 3 gustatory receptors (GRs) and 3 sensory neuron membrane proteins (SNMPs). Quantitative real-time PCR results showed that the relative transcript levels of three ClivORs (ClivOR6, 7 and 14) in the female antennae were 3 to 6 folds higher than that in the male antennae, indicating these genes were more related to oviposition site selection. The relative transcript levels of ClivCSP8 and ClivOR11 were 2.6 and 2.7 times higher in the male antennae than that of the female, respectively, indicating that these genes might be involved in mate searching. Moreover, the responses of dsorco treated predators to volatiles emitted from infested rice were significantly reduced, indicating these volatiles might serve as crucial cues in the host searching of C. lividipennis.

Tropidothorax elegans Distant (Hemiptera: Lygaeidae) is an insect pest that inflicts damage to vegetables and flowering plants across China. The olfactory system regulates insect behavior, such as feeding, mating, oviposition and predator avoidance. Odorant-binding proteins (OBPs) and the chemosensory proteins (CSPs) are two groups of small soluble proteins that initiate olfactory signal transduction in insects. In this study, we generated antennal transcriptomes of male and female T. elegans, and identified 19 putative OBP (14 classic OBPs and five plus-C OBPs) and seven CSP genes. Through real-time quantitative PCR analysis, we found that 14 of the 19 OBP genes were highly expressed in the antennae of both adult females and males, and 3 OBP genes were expressed higher in the antennae of males than females. Some OBP genes were also highly expressed in the legs or wings. Three CSP genes were highly expressed in the antennae of both sexes, and TeleCSP7 showed higher expression in male antennae compare to females. Interestingly, one CSP gene, TeleCSP2, was expressed in all olfactory tissues. Our results provide molecular insights into further investigating of the olfactory system of an important plant pest, T. elegans.

Constitutive expression of Odorant-Binding Proteins (OBPs) in antennae and other body parts has been examined mainly to infer their involvement in insect olfaction, while their regulation in response to semiochemical stimuli has remained poorly known. Previous studies of semiochemical response were basically done using electrophysiology, which integrates the response of the set of OBPs present in an antenna or sensillum, without revealing the regulation of OBPs or which ones might be involved. In this study we used boll weevil as a model and mined its OBPs by RNA-Seq to study their simultaneous antennal expression by qPCR under controlled semiochemical stimuli with aggregation pheromone and plant volatiles. In the absence of a semiochemical stimulus, 23 of 24 OBPs were constitutively expressed in the antenna in both sexes. Semiochemicals changed systemically the expression of OBPs in both sexes. There were different patterns of up- and down-regulation in female antennae for each semiochemical stimulus, consistent with female chemical ecology. On the other hand, the only response in males was down-regulation of some OBPs. We suggest that these systemic changes in OBP expression might be related to enhancing detection of the semiochemical stimuli and/or priming the olfactory system to detect other environmental chemicals.

Bactrocera dorsalis (Hendel) is a fruit-eating pest that causes substantial economic damage to the fresh produce industry in tropical and sub-tropical countries. Methyl eugenol (ME) is a powerful attractant for mature males of B. dorsalis, and has been widely used for detecting, luring and eradicating B. dorsalis populations worldwide. However, the molecular mechanism underlying the olfactory perception of ME remains largely unknown. Here, we analyzed the differential proteomics profiling of the antennae between ME-responsive and ME-non-responsive males by using isobaric tags for relative and absolute quantitation (iTRAQ). In total, 4622 proteins were identified, of which 277 proteins were significant differentially expressed, with 192 up-regulated and 85 down-regulated in responsive male antennae. Quantitative real-time PCR (qRT-PCR) analysis confirmed the authenticity and accuracy of the proteomic analysis. Based on the iTRAQ and qRT-PCR results, we found that the odorant-binding protein 2 (BdorOBP2) was abundantly expressed in responsive male antennae. Moreover, BdorOBP2 was significantly up-regulated by ME in male antennae. Mature males showed significantly greater taxis toward ME than did mature females. Silencing BdorOBP2 reduced mature males' responsiveness to ME. These results indicate that BdorOBP2 may play an essential role in the molecular mechanism underlying B. dorsalis olfactory perception of ME.

Olfaction in insects is essential for host identification, mating and oviposition, in which olfactory proteins are responsible for chemical signaling. Here, we determined the transcriptomes of male and female adult antennae of Anoplophora chinensis, the citrus longhorned beetle. Among 59,357 unigenes in the antennal assembly, we identified 46 odorant-binding proteins, 16 chemosensory proteins (CSPs), 44 odorant receptors, 19 gustatory receptors, 23 ionotropic receptors, and 3 sensory neuron membrane proteins. Among CSPs, AchiCSP10 was predominantly expressed in antennae (compared with legs or maxillary palps), at a significantly higher level in males than in females, suggesting that AchiCSP10 has a role in reception of female sex pheromones. Many highly expressed genes encoding CSPs are orthologue genes of A. chinensis and Anoplophora glabripennis. Notably, AchiPBP1 and AchiPBP2 showed 100% and 96% identity with AglaPBP1 and AglaPBP2 from A. glabripennis, with similar expression profiles in the two species; PBP2 was highly expressed in male antennae, whereas PBP1 was expressed in all three tissues in both males and females. These results provide a basis for further studies on the molecular chemoreception mechanisms of A. chinensis, and suggest novel targets for control of A. chinensis.

Odorant degrading enzymes (ODEs) are thought to be responsible, at least in part, for olfactory signal termination in the chemosensory system by rapid degradation of odorants in the vicinity of the receptors. A carboxylesterase, specifically expressed in Drosophila antennae, called "juvenile hormone esterase duplication (JHEdup)" has been previously reported to hydrolyse different fruit esters in vitro. Here we functionally characterize JHEdup in vivo. We show that the jhedup gene is highly expressed in large basiconic sensilla that have been reported to detect several food esters. An electrophysiological analysis demonstrates that ab1A olfactory neurons of jhedup mutant flies exhibit an increased response to certain food acetates. Furthermore, mutant flies show a higher sensitivity towards the same odorants in behavioural assays. A phylogenetic analysis reveals that jhedup arose as a duplication of the juvenile hormone esterase gene during the evolution of Diptera, most likely in the ancestor of Schizophora, and has been conserved in all the 12 sequenced Drosophila species. Jhedup exhibits also an olfactory-predominant expression pattern in other Drosophila species. Our results support the implication of JHEdup in the degradation of food odorants in D. melanogaster and propose a neofunctionalization of this enzyme as a bona fide ODE in Drosophilids.

Studies on insect olfaction have increased our understanding of insect's chemosensory system and chemical ecology, and have improved pest control strategies based on insect behavior. In this study, we assembled the antennal transcriptomes of the lychee giant stink bug, Tessaratoma papillosa, by using next generation sequencing to identify the major olfaction gene families in this species. In total, 59 odorant receptors, 14 ionotropic receptors (8 antennal IRs), and 33 odorant binding proteins (28 classic OBPs and 5 plus-C OBPs) were identified from the male and female antennal transcriptomes. Analyses of tissue expression profiles revealed that all 59 OR transcripts, 2 of the 8 antennal IRs, and 6 of the 33 OBPs were primarily expressed in the antennae, suggesting their putative role in olfaction. The sex-biased expression patterns of these antenna-predominant genes suggested that they may have important functions in the reproductive behavior of these insects. This is the first report that provides a comprehensive resource to future studies on olfaction in the lychee giant stink bug.

Antennal and abdominal transcriptomes of males and females of the coconut hispine beetle Brontispa longissima were sequenced to identify and compare the expression patterns of genes involved in odorant reception and detection. Representative proteins from the chemosensory gene families likely essential for insect olfaction were identified. These include 48 odorant receptors (ORs), 19 ionotropic receptors (IRs), 4 sensory neuron membrane proteins (SNMPs), 34 odorant binding proteins (OBPs) and 16 chemosensory proteins (CSPs). Phylogenetic analysis revealed the evolutionary relationship of these proteins with homologs from Coleopterans or other insects, and led to the identification of putative aggregation pheromone receptors in B. longissima. Comparative expression analysis performed by calculating FPKM values were also validated using quantitative real time-PCR (qPCR). The results revealed that all ORs and antennal IRs, two IR co-receptors (BlonIR8a and BlonIR25a) and one SNMP (BlonSNMP1a) were predominantly expressed in antennae when compared to abdomens, and approximately half of the OBPs (19) and CSPs (7) were enriched in antennae. These findings for the first time reveal the identification of key molecular components in B. longissima olfaction and provide a valuable resource for future functional analyses of olfaction, and identification of potential targets to control this quarantine pest.

Targeted delivery of nanoscale carriers containing packaged payloads to the central nervous system has potential use in many diagnostic and therapeutic applications. Moreover, understanding of the bio-interactions of the engineered nanoparticles used for tissue-specific delivery by non-invasive delivery approaches are also of paramount interest. Here, we have examined this issue systematically in a relatively simple invertebrate model using insects. We synthesized 5 nm, positively charged gold nanoparticles (AuNPs) and targeted their delivery using the electrospray aerosol generator. Our results revealed that after the exposure of synthesized aerosol to the insect antenna, AuNPs reached the brain within an hour. Nanoparticle accumulation in the brain increased linearly with the exposure time. Notably, electrophysiological recordings from neurons in the insect brain several hours after exposure did not show any significant alterations in their spontaneous and odor-evoked spiking properties. Taken together, our findings reveal that aerosolized delivery of nanoparticles can be an effective non-invasive approach for delivering nanoparticles to the brain, and also presents an approach to monitor the short-term nano-biointeractions.

Drosophila uses 50 different olfactory receptor neuron (ORN) classes that are clustered within distinct sensilla subtypes to decipher their chemical environment. Each sensilla subtype houses 1-4 ORN identities that arise through asymmetric divisions of a single sensory organ precursor (SOP). Despite a number of mutational studies investigating the regulation of ORN development, a majority of the transcriptional programs that lead to the different ORN classes in the developing olfactory system are unknown. Here we use transcriptional profiling across the time series of antennal development to identify novel transcriptional programs governing the differentiation of ORNs. We surveyed four critical developmental stages of the olfactory system: 3rd instar larval (prepatterning), 8 hours after puparium formation (APF, SOP selection), 40 hrs APF (neurogenesis), and adult antennae. We focused on the expression profiles of olfactory receptor genes and transcription factors-the two main classes of genes that regulate the sensory identity of ORNs. We identify distinct clusters of genes that have overlapping temporal expression profiles suggesting they have a key role during olfactory system development. We show that the expression of the transcription factor distal antenna (dan) is highly similar to other prepatterning factors and is required for the expression of a subset of ORs.

Neuromodulation confers flexibility to anatomically-restricted neural networks so that animals are able to properly respond to complex internal and external demands. However, determining the mechanisms underlying neuromodulation is challenging without knowledge of the functional class and spatial organization of neurons that express individual neuromodulatory receptors. Here, we describe the number and functional identities of neurons in the antennal lobe of Drosophila melanogaster that express each of the receptors for one such neuromodulator, serotonin (5-HT). Although 5-HT enhances odor-evoked responses of antennal lobe projection neurons (PNs) and local interneurons (LNs), the receptor basis for this enhancement is unknown. We used endogenous reporters of transcription and translation for each of the five 5-HT receptors (5-HTRs) to identify neurons, based on cell class and transmitter content, that express each receptor. We find that specific receptor types are expressed by distinct combinations of functional neuronal classes. For instance, the excitatory PNs express the excitatory 5-HTRs, while distinct classes of LNs each express different 5-HTRs. This study therefore provides a detailed atlas of 5-HT receptor expression within a well-characterized neural network, and enables future dissection of the role of serotonergic modulation of olfactory processing.

Odorant binding proteins (OBPs) play critical roles in chemical communication of insects, as they recognize and transport environmental chemical signals to receptors. The diving beetle Cybister japonicus Sharp shows a remarkable sexual dimorphism. The foreleg tarsi of males are equipped with large suction cups, believed to help holding the female during underwater courtship and mating. Here, we identified two OBPs highly and specifically expressed in male tarsi, suggesting important functions of these structures in chemical communication. The first protein, CjapOBP1, exhibits the 6 conserved cysteines motif of classic OBPs, while the second, CjapOBP2, contains only four cysteines and can be assigned to the sub-class of C-minus OBPs. Both proteins were expressed in a bacterial system and the purified recombinant proteins were used to for antibodies preparation. Western Blot analysis showed that CjapOBP1 is predominantly expressed in male tarsi and could be also detected in antennae and palpi of both sexes, while CjapOBP2, besides male tarsi, is also present in testis. Ligand-binding experiments showed a good binding affinity between CjapOBP1, CjapOBP2 and citral and coniferyl aldehyde, respectively. These results support a possible function of these two OBPs in the male foreleg tarsi of diving beetles in chemical communication.

Periplaneta americana is a notorious urban pest prevalent in human habitats; very little is known about its chemosensory mechanism. Employing the advanced next-generation sequencing technique, in the present study, we conducted transcriptome sequencing and analysis of the antennae of the adult males and females as well as their mouthparts using an Illumina platform. This resulted in the discovery of a huge number of the members of all major known chemosensory receptor families in P. americana, including 96 odorant receptors (ORs), 53 ionotropic receptors (IRs), and 33 gustatory receptors (GRs). Tissue expression profiles showed most of them mainly expressed in antennae and phylogenetic analysis demonstrated the expansion in the clade distinguishing them from other functionally well-known Lepidoptera species. A high percentage of chemosensory receptor genes (ORs in particular) showing female antenna bias in mRNA expression was observed. Our results provide a basis for further investigations on how P. americana coordinates its chemosensory receptor genes in chemical communication with environments, and for development of novel pest management approaches.

Olfactory proteins form the basis of insect olfactory recognition, which is crucial for host identification, mating, and oviposition. Using transcriptome analysis of Anoplophora glabripennis antenna, we identified 42 odorant-binding proteins (OBPs), 12 chemosensory proteins (CSPs), 14 pheromone-degrading enzymes (PDEs), 1 odorant-degrading enzymes (ODE), 37 odorant receptors (ORs), 11 gustatory receptors (GRs), 2 sensory neuron membrane proteins (SNMPs), and 4 ionotropic receptor (IR). All CSPs and PBPs were expressed in antennae, confirming the authenticity of the transcriptome data. CSP expression profiles showed that AglaCSP3, AglaCSP6, and AglaCSP12 were expressed preferentially in maxillary palps and AglaCSP7 and AglaCSP9 were strongly expressed in antennae. The vast majority of CSPs were highly expressed in multiple chemosensory tissues, suggesting their participation in olfactory recognition in almost all olfactory tissues. Intriguingly, the PBP AglaPBP2 was preferentially expressed in antenna, indicating that it is the main protein involved in efficient and sensitive pheromone recognition. Phylogenetic analysis of olfactory proteins indicated AglaGR1 may detect CO2. This study establishes a foundation for determining the chemoreception molecular mechanisms of A. glabripennis, which would provide a new perspective for controlling pest populations, especially those of borers.

Two sympatric species Helicoverpa armigera and Helicoverpa assulta use (Z)-11-hexadecenal and (Z)-9-hexadecenal as sex pheromone components in reverse ratio. They also share several other pheromone gland components (PGCs). We present a comparative study on the olfactory coding mechanism and behavioral effects of these additional PGCs in pheromone communication of the two species using single sensillum recording, in situ hybridization, calcium imaging, and wind tunnel. We classify antennal sensilla types A, B and C into A, B1, B2, C1, C2 and C3 based on the response profiles, and identify the glomeruli responsible for antagonist detection in both species. The abundance of these sensilla types when compared with the number of OSNs expressing each of six pheromone receptors suggests that HarmOR13 and HassOR13 are expressed in OSNs housed within A type sensilla, HarmOR14b within B and C type sensilla, while HassOR6 and HassOR16 within some of C type sensilla. We find that for H. armigera, (Z)-11-hexadecenol and (Z)-11-hexadecenyl acetate act as behavioral antagonists. For H. assulta, instead, (Z)-11-hexadecenyl acetate acts as an agonist, while (Z)-9-hexadecenol, (Z)-11-hexadecenol and (Z)-9-hexadecenyl acetate are antagonists. The results provide an overall picture of intra- and interspecific olfactory and behavioral responses to all PGCs in two sister species.

Sexual communication in moths offers a simplified scenario to model and investigate insect sensory perception. Both PBPs (pheromone-binding proteins) and PRs (pheromone receptors) are involved in the detection of sex pheromones, but the interplay between them still remains largely unknown. In this study, we have measured the binding affinities of the four recombinant PBPs of Chilo suppressalis (CsupPBPs) to pheromone components and analogs and characterized the six PRs using the Xenopus oocytes expression system. Interestingly, when the responses of PRs were recorded in the presence of PBPs, we measured in several combinations a dramatic increase in signals as well as in sensitivity of such combined systems. Furthermore, the discrimination ability of appropriate combinations of PRs and PBPs was improved compared with the performance of PBPs or PRs alone. Besides further supporting a role of PBPs in the pheromone detection and discrimination, our data shows for the first time that appropriate combinations of PRs and PBPs improved the discrimination ability of PBPs or PRs alone. The variety of responses measured with different pairing of PBPs and PRs indicates the complexity of the olfaction system, which, even for the relatively simple task of detecting sex pheromones, utilises a highly sophisticated combinatorial approach.

Chemical communication is essential for the coordination of complex organisation in ant societies. Recent comparative genomic approaches have revealed that chemosensory genes are diversified in ant lineages, and suggest that this diversification is crucial for social organisation. However, how such diversified genes shape the peripheral chemosensory systems remains unknown. In this study, we annotated and analysed the gene expression profiles of chemosensory proteins (CSPs), which transport lipophilic compounds toward chemosensory receptors in the carpenter ant, Camponotus japonicus. Transcriptome analysis revealed 12 CSP genes and phylogenetic analysis showed that 3 of these are lineage-specifically expanded in the clade of ants. RNA sequencing and real-time quantitative polymerase chain reaction revealed that, among the ant specific CSP genes, two of them (CjapCSP12 and CjapCSP13) were specifically expressed in the chemosensory organs and differentially expressed amongst ant castes. Furthermore, CjapCSP12 and CjapCSP13 had a ratio of divergence at non-synonymous and synonymous sites (dN/dS) greater than 1, and they were co-expressed with CjapCSP1, which is known to bind cuticular hydrocarbons. Our results suggested that CjapCSP12 and CjapCSP13 were functionally differentiated for ant-specific chemosensory events, and that CjapCSP1, CjapCSP12, and CjapCSP13 work cooperatively in the antennal chemosensilla of worker ants.

In honey bees, Varroa sensitive hygiene (VSH) behaviour, which involves the detection and removal of brood parasitised by the mite Varroa destructor, can actively participate in the survival of colonies facing Varroa outbreaks. This study investigated the mechanisms of VSH behaviour, by comparing the antennal transcriptomes of bees that do and do not perform VSH behaviour. Results indicate that antennae likely play a key role in the expression of VSH behaviour. Comparisons with the antennal transcriptome of nurse and forager bees suggest that VSH profile is more similar to that of nurse bees than foragers. Enhanced detection of certain odorants in VSH bees may be predicted from transcriptional patterns, as well as a higher metabolism and antennal motor activity. Interestingly, Deformed wing virus/Varroa destructor virus infections were detected in the antennae, with higher level in non-VSH bees; a putative negative impact of viral infection on bees' ability to display VSH behaviour is proposed. These results bring new perspectives to the understanding of VSH behaviour and the evolution of collective defence by focusing attention on the importance of the peripheral nervous system. In addition, such data might be useful for promoting marker-assisted selection of honey bees that can survive Varroa infestations.

Insect chemosensory proteins (CSPs) are a family of small soluble proteins. To date, their physiological functions in insect olfaction remain largely controversial in comparison to odorant binding proteins (OBPs). In present study, we reported the antenna specific expression of three CSPs (AlinCSP4-6) from Adelphocoris lineolatus, their distinct chemosensillum distribution as well as ligand binding capability thus providing the evidence for the possible roles that they could play in semiochemical detection of the plant bug A. lineolatus. The results of qRT-PCR and western blot assay clearly showed that all of these three CSPs are highly expressed in the adult antennae, the olfactory organ of insects. Further cellular investigation of their immunolocalization revealed their dynamic protein expression profiles among different types of antennal sensilla. In a fluorescence competitive binding assay, the selective ligand binding was observed for AlinCSP4-6. In ad`dition, a cooperative interaction was observed between two co-expressed CSPs resulting in an increase of the binding affinities by a mixture of AlinCSP5 and AlinCSP6 to terpenoids which do not bind to individual CSPs. These findings in combination with our previous data for AlinCSP1-3 indicate a possible functional differentiation of CSPs in the A. lineolatus olfactory system.

Female mosquitoes are major vectors of human disease and the most dangerous are those that preferentially bite humans. A 'domestic' form of the mosquito Aedes aegypti has evolved to specialize in biting humans and is the main worldwide vector of dengue, yellow fever, and chikungunya viruses. The domestic form coexists with an ancestral, 'forest' form that prefers to bite non-human animals and is found along the coast of Kenya. We collected the two forms, established laboratory colonies, and document striking divergence in preference for human versus non-human animal odour. We further show that the evolution of preference for human odour in domestic mosquitoes is tightly linked to increases in the expression and ligand-sensitivity of the odorant receptor AaegOr4, which we found recognizes a compound present at high levels in human odour. Our results provide a rare example of a gene contributing to behavioural evolution and provide insight into how disease-vectoring mosquitoes came to specialize on humans.

There are major impediments to finding improved DEET alternatives because the receptors causing olfactory repellency are unknown, and new chemicals require exorbitant costs to determine safety for human use. Here we identify DEET-sensitive neurons in a pit-like structure in the Drosophila melanogaster antenna called the sacculus. They express a highly conserved receptor, Ir40a, and flies in which these neurons are silenced or Ir40a is knocked down lose avoidance to DEET. We used a computational structure-activity screen of >400,000 compounds that identified >100 natural compounds as candidate repellents. We tested several and found that most activate Ir40a(+) neurons and are repellents for Drosophila. These compounds are also strong repellents for mosquitoes. The candidates contain chemicals that do not dissolve plastic, are affordable and smell mildly like grapes, with three considered safe in human foods. Our findings pave the way to discover new generations of repellents that will help fight deadly insect-borne diseases worldwide.