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Bumblebees are crucial pollinators, providing essential ecosystem services and global food production. The success of pollination services relies on the interaction between sensory organs and the environment. The antenna functions as a versatile multi-sensory organ, pivotal in mediating chemosensory/olfactory information, and governs adaptive responses to environmental changes. Despite an increasing number of RNA-sequencing studies on insect antenna, comprehensive antennal transcriptome studies at the different life stages were not elucidated systematically. Here, we quantified the expression profile and dynamics of coding/microRNA genes of larval head and antennal tissues from early- and late-stage pupa to the adult of Bombus terrestris as suitable model organism among pollinators. We further performed Pearson correlation analyses on the gene expression profiles of the antennal transcriptome from larval head tissue to adult stages, exploring both positive and negative expression trends. The positively correlated coding genes were primarily enriched in sensory perception of chemical stimuli, ion transport, transmembrane transport processes and olfactory receptor activity. Negatively correlated genes were mainly enriched in organic substance biosynthesis and regulatory mechanisms underlying larval body patterning and the formation of juvenile antennal structures. As post-transcriptional regulators, miR-1000-5p, miR-13b-3p, miR-263-5p and miR-252-5p showed positive correlations, whereas miR-315-5p, miR-92b-3p, miR-137-3p, miR-11-3p and miR-10-3p exhibited negative correlations in antennal tissue. Notably, based on the inverse expression relationship, positively and negatively correlated microRNA (miRNA)-mRNA target pairs revealed that differentially expressed miRNAs predictively targeted genes involved in antennal development, shaping antennal structures and regulating antenna-specific functions. Our data serve as a foundation for understanding stage-specific antennal transcriptomes and large-scale comparative analysis of transcriptomes in different insects., (© 2024 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Background: Insects detect odours using odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs) in the antennae. Ecologically important odours are often detected by selective and abundant OSNs; hence, ORs with high antennal expression. However, little is known about the function of highly expressed ORs in beetles, since few ORs have been functionally characterized. Here, we functionally characterized the most highly expressed OR (ItypOR36) in the bark beetle Ips typographus L. (Coleoptera, Curculionidae, Scolytinae), a major pest of spruce. We hypothesized that this OR would detect a compound important to beetle fitness, such as a pheromone component. We next investigated the antennal distribution of this OR using single sensillum recordings (SSR) and in situ hybridization, followed by field- and laboratory experiments to evaluate the behavioural effects of the discovered ligand., Results: We expressed ItypOR36 in HEK293 cells and challenged it with 64 ecologically relevant odours. The OR responded exclusively to the monoterpene-derived ketone lanierone with high sensitivity. Lanierone is used in chemical communication in North American Ips species, but it has never been shown to be produced by I. typographus, nor has it been studied in relation to this species' sensory physiology. Single sensillum recordings revealed a novel and abundant lanierone-responsive OSN class with the same specific response as ItypOR36. Strikingly, these OSNs were co-localized in sensilla together with seven different previously described OSN classes. Field experiments revealed that low release rates of lanierone inhibited beetle attraction to traps baited with aggregation pheromone, with strongest effects on males. Female beetles were attracted to lanierone in laboratory walking bioassays., Conclusions: Our study highlights the importance of the so-called 'reverse chemical ecology' approach to identify novel semiochemicals for ecologically important insect species. Our discovery of the co-localization pattern involving the lanierone OSN class suggests organizational differences in the peripheral olfactory sense between insect orders. Our behavioural experiments show that lanierone elicits different responses in the two sexes, which also depend on whether beetles are walking in the laboratory or flying in the field. Unravelling the source of lanierone in the natural environment of I. typographus is required to understand these context-dependent behaviours., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests., (© 2024. The Author(s).)

Bactrocera dorsalis is one of the major invasive pests worldwide. The acetic acid-enriched sweet bait trapping is an important method for monitoring and controlling this fly. Several studies showed that acetic acid is perceived by ionotropic receptors (IRs). Thus, we annotated 65 IR genes in the B. dorsalis genome. We also investigated the IRs involved in acetic acid perception in this fly by behavioral, electrophysiological, and molecular methods. As the results indicated, the antennae are the main olfactory organs to sense acetic acid. Among the antennal IRs showed acetic acid-induced expression profiles, IR8a was proven to perceive acetic acid by CRISPR/Cas9-mediated mutagenesis. Additionally, calcium imaging showed that IR64a and IR75a are potential acetic acid receptors respectively co-expressed with IR76b and IR8a. This study represents the first comprehensive characterization of IRs in B. dorsalis at the whole-genome level, revealing the significant role of IRs in acetic acid perception.

The olfactory sense is crucial for organisms, facilitating environmental recognition and interindividual communication. Ithomiini butterflies exemplify this importance not only because they rely strongly on olfactory cues for both inter- and intra-sexual behaviors, but also because they show convergent evolution of specialized structures within the antennal lobe, called macroglomerular complexes (MGCs). These structures, widely absent in butterflies, are present in moths where they enable heightened sensitivity to, and integration of, information from various types of pheromones. In this study, we investigate chemosensory evolution across six Ithomiini species and identify possible links between expression profiles and neuroanatomical. To enable this, we sequenced four new high-quality genome assemblies and six sex-specific antennal transcriptomes for three of these species with different MGC morphologies. With extensive genomic analyses, we found that the expression of antennal transcriptomes across species exhibit profound divergence, and identified highly expressed ORs, which we hypothesize may be associated to MGCs, as highly expressed ORs are absent in Methona, an Ithomiini lineage which also lacks MGCs. More broadly, we show how antennal sexual dimorphism is prevalent in both chemosensory genes and non-chemosensory genes, with possible relevance for behavior. As an example, we show how lipid-related genes exhibit consistent sexual dimorphism, potentially linked to lipid transport or host selection. In this study, we investigate the antennal chemosensory adaptations, suggesting a link between genetic diversity, ecological specialization, and sensory perception with the convergent evolution of MCGs. Insights into chemosensory gene evolution, expression patterns, and potential functional implications enhance our knowledge of sensory adaptations and sexual dimorphisms in butterflies, laying the foundation for future investigations into the genetic drivers of insect behavior, adaptation, and speciation., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

In natural environments, general plant volatiles and herbivore-induced plant volatiles (HIPVs) serve as critical clues for predatory natural enemies in the search for prey. The insect olfactory system plays a vital role in perceiving plant volatiles including HIPVs. In this study, we found that HIPV ( E , E )-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) and the plant volatile geranyl acetate (GA), two structurally similar chemicals, displayed electrophysiological activities on the antennae of the ladybird Hippodamia variegata , but were only attractive to adult females in behavior. Moreover, mated female ladybirds laid a significantly higher number of eggs on TMTT-treated and GA-treated cotton leaves compared to controls. Screening of female-biased odorant receptors (ORs) from the antennal transcriptomes, performing Xenopus oocytes expression coupled with two-electrode voltage clamp recordings, suggested that HvarOR28 specifically tuned to TMTT and GA. Molecular docking and site-directed mutagenesis revealed that the amino acid residues Tyr143 and Phe81 of HvarOR28 are the key site for binding with TMTT and GA. Furthermore, RNA interference (RNAi) assay demonstrated that HvarOR28 -silenced individuals demonstrated a notable decrease in electrophysiological responses, even female adults almost lost behavioral preference for the two compounds. Thus, it could be concluded that HvarOR28 in H. variegata contributes to facilitating egg laying through the perception of TMTT and GA. These findings may help to develop new olfactory modulators based on the behaviorally active ligands of HvarOR28.

Winged parthenogenetic aphids are mainly responsible for migration and dispersal. Aphid alarm pheromone (E)-β-Farnesene (EBF) has dual effects on repelling and stimulating wing differentiation in aphids. Previous studies have shown that the odorant coreceptor SmisOrco is involved in the perception of EBF by S. miscanthi; however, its EBF-specific odorant receptor (OR) and the difference between winged and wingless aphids remain unclear. In this study, the Xenopus oocyte expression system and RNAi technology were used to detect the transmission of EBF signals, and it was found that the olfactory receptor SmisOR5 is an EBF-specific OR in S. miscanthi and is specifically highly expressed in the antennae of winged aphids. Furthermore, when OR5 was silenced with dsRNA, the repellent effect of EBF was weakened, and aphids showed more active aimless movements. Therefore, as a specific OR for EBF, the high expression level of SmisOR5 in winged aphids suggests a molecular basis for its high sensitivity to EBF. This study advances our understanding of the molecular mechanisms of aphid EBF perception and provides novel ideas for effective management and prevention of the migration of winged aphids., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is the most destructive pest, causing severe damage to mulberry production in China's sericulture industry. The insecticide application in mulberry orchards poses a significant risk of poisoning to Bombyx mori. Shifting from insecticides to odor attractants is a beneficial alternative, but not much data is available on the olfactory system of G. pyloalis. We identified 114 chemosensory genes from the antennal transcriptome database of G. pyloalis, with 18 odorant-binding protein (OBP) and 17 chemosensory protein (CSP) genes significantly expressed in the antennae. Ligand-binding assays for two antennae-biased expressed general odorant-binding proteins (GOBPs) showed high binding affinities of GOBP1 to hexadecanal, β-ionone, and 2-ethylhexyl acrylate, while GOBP2 exhibited binding to 4-tert-octylphenol, benzyl benzoate, β-ionone, and farnesol. Computational simulations indicated that van der Waal forces predominantly contributed to the binding free energy in the binding processes of complexes. Among them, Phe12 of GOBP1 and Phe19 of GOBP2 were demonstrated to play crucial roles in their bindings to plant volatiles using site-directed mutagenesis experiments. Moreover, hexadecanal and β-ionone attracted G. pyloalis male moths in the behavioral assays, while none of the candidate plant volatiles significantly affected female moths. Our findings provide a comprehensive understanding of the molecular mechanisms underlying olfactory recognition in G. pyloalis, setting the groundwork for novel mulberry pests control strategies based on insect olfaction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling affect social aggregation, mood and psychiatric disorders, nociceptive and depressive behaviors. Olfactory dysfunction is one of the distinct symptoms of these behaviors, but function and mechanism of JAK and STAT in modulating olfaction remain largely unknown. Migratory locusts show olfactory preference for their own volatiles. We thus use this animal model to explore functions and mechanisms of JAK and STAT5B in mediating olfaction response to their own volatiles. Tissue distribution study shows that JAK and STAT5B express in antennae and brains, especially in antennal lobes and mushroom bodies in locust brains, and knockdown of these two genes by RNA interference (RNAi) in antennae and brains results in the loss of olfactory preference for locust volatiles, including chemical odorants indole and β-ionone. RNA-seq analysis reveals that JAK and STAT5B RNAi knockdown downregulates a functional class of transcripts in nucleoprotein complex, including heterogeneous nuclear ribonucleoprotein C (hnRNPC) and small nuclear ribonucleoprotein polypeptide F (SNRPF). HnRNPC and SNRPF mRNAs and proteins are also expressed in antennae and brains, and RNAi knockdown of these two genes reduces the percentage of locusts preferring volatiles, including chemical odorants indole and β-ionone. Furthermore, RNAi knockdown of dopamine receptor 1 (DopR1) results in the decrease of JAK mRNA level in antennae, and JAK/STAT5B, hnRNPC and SNRPF are required for dopamine receptor 1 (DopR1) to modulate olfactory preference for their own volatiles. This study confirms that JAK/STAT5B signaling modulates olfaction by affecting expression levels of hnRNPC and SNRPF, and this pathway is also required for DopR1 to modulate olfactory preference for their own volatiles. These findings highlight novel roles of JAK and STAT5B in modulating olfactory preference. This study provides novel insights into functional links among JAK/STAT5B signaling, RNA binding proteins and DopR1 underlying the modulation of olfactory behaviors., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Insulin receptors (InR) are an integral component of the insulin/insulin-like growth factor signaling pathway, which plays a vital role in insect development, lifespan, reproduction, and olfactory sensitivity. However, whether InR participate in the peripheral olfactory system of insects remains unclear. Recently, we found that 2-heptanone (2-HT) affects AcerInR expression, the gene for an InR protein, in Apis cerana cerana. We then examined the spatiotemporal expression profile of the gene in A. cerana cerana. The mRNA of AcerInR was primarily expressed in the antennae, wings, and legs of forager bees, which are probable chemosensory tissues. The results of fluorescence competitive binding assays, combined with site-directed mutagenesis, demonstrated that AcerOBP6 and AcerOBP14 exhibit strong binding affinities to 2-HT. Furthermore, after foragers were fed with double-stranded AcerInR, the expression levels of AcerOBP6 and AcerOBP14 decreased significantly, as did the electroantennogram responsiveness to 2-HT and some other odorants. In conclusion, our findings provide a foundation for understanding the involvement of AcerInR in the odor perception of A. cerana cerana. Moreover, they offer novel insights into the olfactory recognition mechanism in insects., (© 2024 Institute of Zoology, Chinese Academy of Sciences.)

Aethina tumida (small hive beetle, SHB) is a rapidly spreading invasive parasite of bee colonies. The olfactory system plays a key role in insect behavior, and odorant-binding proteins (OBPs) are involved in the first step of the olfactory signal transduction pathway and the detection of host volatiles. However, the olfactory mechanism of OBPs in SHB-localized bee colonies is unclear. In this study, electroantennogram (EAG) and behavioral bioassay showed that only three compounds (2-heptanone, ocimene, and ethyl palmitate) from bee colonies triggered high electrophysiological and behavioral responses. Three antenna-specific OBP genes (OBP6, OBP11, and OBP19) were identified, and they were significantly expressed on adult days 6-7. Furthermore, by combining RNA interference (RNAi) with EAG, olfactometer bioassay, competitive fluorescence binding assays, and molecular docking, we found that these three OBP genes were involved in the recognition of 2-heptanone and ethyl palmitate, and AtumOBP6 is also involved in the recognition of ocimene. These data indicate that AtumOBP6, AtumOBP11, and AtumOBP19 play an important role in the olfactory response to bee colony volatiles. Our results provide new insights into the functions of the OBP families in A. tumida and help to explore more potential target genes for environmentally friendly pest control strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Odor detection in insects is largely mediated by structures on antennae called sensilla, which feature a strongly conserved architecture and repertoire of olfactory sensory neurons (OSNs) and various support cell types. In Drosophila, OSNs are tightly apposed to supporting cells, whose connection with neurons and functional roles in odor detection remain unclear. Coupling mechanisms between these neuronal and non-neuronal cell types have been suggested based on morphological observations, concomitant physiological activity during odor stimulation, and known interactions that occur in other chemosensory systems. For instance, it is not known whether cell-cell coupling via gap junctions between OSNs and neighboring cells exists, or whether hemichannels interconnect cellular and extracellular sensillum compartments. Here, we show that innexins, which form hemichannels and gap junctions in invertebrates, are abundantly expressed in adult drosophilid antennae. By surveying antennal transcriptomes and performing various immunohistochemical stainings in antennal tissues, we discover innexin-specific patterns of expression and localization, with a majority of innexins strongly localizing to glial and non-neuronal cells, likely support and epithelial cells. Finally, by injecting gap junction-permeable dye into a pre-identified sensillum, we observe no dye coupling between neuronal and non-neuronal cells. Together with evidence of non-neuronal innexin localization, we conclude that innexins likely do not conjoin neurons to support cells, but that junctions and hemichannels may instead couple support cells among each other or to their shared sensillum lymph to achieve synchronous activity. We discuss how coupling of sensillum microenvironments or compartments may potentially contribute to facilitate chemosensory functions of odor sensing and sensillum homeostasis., (© 2024. The Author(s).)

Background: Hygienic behavior, a specialized form of immune response evolved in social insects, plays a crucial role in safeguarding colonies from disease spread. In honeybee colonies, such behavior typically entails the dual steps of uncapping and removal of unhealthy and deceased brood. Although in recent years, numerous studies have examined the development of hygienic behavior, the mechanisms underlying the division in the performance of uncapping and removal have yet to be sufficiently elucidated. In this regard, long non-coding RNAs (lncRNAs) have been evidenced to be engaged in regulating the physiological activities of honeybees; however, whether lncRNAs are likewise involved in the uncapping and removal tasks has not been clarified., Results: In this study, the strong hygienic Apis cerana worker bees were used and the processes of uncapping and removal behaviors in three colonies were assayed with freeze-killed brood in the field. We then sequenced the antennal RNAs of honeybees to identify differentially expressed lncRNAs and performed lncRNA-mRNA association analysis to establish the differences between uncapping and removal. We detected 1,323 differentially expressed lncRNAs in the antennae, and the findings of lncRNA-mRNA association analyses revealed that the target genes of differentially expressed lncRNAs between uncapping and removal worker bees were predominantly linked to response to stimulus, receptor activity, and synapse. Notably, among the lncRNAs enriched in cellular response to stimulus, XR_001766094.2 was exclusively expressed in the uncapping worker bees. Based on these findings, we hypothesize that XR_001766094.2 plays a key role in distinguishing uncapping from removal behaviors by responding to external stimulus, thereby suggesting that the division of hygienic behaviors is governed by differential thresholds of responsiveness to environmental cues., Conclusion: We characterized differences in the uncapping and removal behaviors of worker bees from a perspective of lncRNAs. Uncapping bees may be equipped with a more rapid stimulatory response and more acute olfactory sensitivity, contributing to the rapid hygienic behavior in honeybee colonies. Our results thus establish a foundation for potential lncRNA-mediated gene expression regulation in hygienic behavior., (© 2024. The Author(s).)

Nestmate recognition in ants is regulated through the detection of cuticular hydrocarbons by odorant receptors (ORs) in the antennae. These ORs are crucial for maintaining colony cohesion that allows invasive ant species to dominate colonized environments. In the invasive Argentine ant, Linepithema humile, ORs regulating nestmate recognition are thought to be present in a clade of nine-exon odorant receptors, but the identity of the specific genes remains unknown. We sought to narrow down the list of candidate genes using transcriptomics and phylostratigraphy. Comparative transcriptomic analyses were conducted on the antennae, head, thorax, and legs of Argentine ant workers. We have identified a set of twenty-one nine-exon odorant receptors enriched in the antennae compared to the other tissues, allowing for downstream verification of whether they can detect Argentine ant cuticular hydrocarbons. Further investigation of these ORs could allow us to further understand the mechanisms underlying nestmate recognition and colony cohesion in ants., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Dittmann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Riptortus pedestris (Hemiptera: Alydidae), a common agricultural pest, is the major causative agent of "soybean staygreen." However, the interactions between chemosensory proteins (CSPs) in R. pedestris and host plant volatiles have yet to be comprehensively studied. In this study, we performed real-time fluorescence quantitative polymerase chain reaction (PCR) to analyze the antennal expression of RpedCSP22 and subsequently analyzed the interactions between 21 soybean volatiles, five aggregation pheromones, and RpedCSP22 protein in vitro using a protein expression system, molecular docking, site-directed mutagenesis, and fluorescence competitive binding experiments. The RpedCSP22 protein showed binding affinity to three soybean volatiles (benzaldehyde, 4-ethylbenzaldehyde, and 1-octene-3-ol), with optimal binding observed under neutral pH conditions, and lost binding ability after site-directed mutagenesis. In subsequent RNA interference (RNAi) studies, gene silencing was more than 90 %, and in silenced insects, electroantennographic responses were reduced by more than 75 % compared to non-silenced insects. Moreover, Y-tube olfactory behavioral assessments revealed that the attraction of R. pedestris to the three soybean volatiles was significantly attenuated. These findings suggest that RpedCSP22 plays an important role in the recognition of host plant volatiles by R. pedestris andprovides a theoretical basis for the development of novel inhibitors targeting pest behavior., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Riptortus pedestris (Hemiptera: Alydidae) is a notable soybean pest, with diapause and non-diapause individuals showing different sensitivities to aggregation pheromones. This study aimed to investigate how R. pedestris detects aggregation pheromones through electroantennogram (EAG) and behavioral experiments, transcriptome sequencing and qRT-PCR, as well as competitive fluorescence-binding assay. Results indicated that diapausing females and males of R. pedestris exhibited a heightened EAG response and were more attracted to the aggregation pheromone components compared to their non-diapause counterparts. Transcriptome sequencing and qRT-PCR analyses revealed significantly higher expression of RpedOBP1 in the antennae of diapause females and males compared to non-diapausing R. pedestris. The competitive fluorescence-binding assay demonstrated that RpedOBP1 displayed the strongest binding affinity to E2HE2H, suggesting its crucial role in recognizing the aggregation pheromone. These findings have the potential to inform the development of integrated pest management strategies utilizing behavioral approaches for bean bug control., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The northern bark beetle, Ips duplicatus , is an emerging economic pest, reportedly infesting various species of spruce ( Picea spp.), pine ( Pinus spp.), and larch ( Larix spp.) in Central Europe. Recent climate changes and inconsistent forest management practices have led to the rapid spread of this species, leaving the current monitoring strategies inefficient. As understanding the molecular components of pheromone detection is key to developing novel control strategies, we generated antennal transcriptomes from males and females of this species and annotated the chemosensory proteins. We identified putative candidates for 69 odorant receptors (ORs), 50 ionotropic receptors (IRs), 25 gustatory receptors (GRs), 27 odorant-binding proteins (OBPs), including a tetramer-OBP, 9 chemosensory proteins (CSPs), and 6 sensory neuron membrane proteins (SNMPs). However, no sex-specific chemosensory genes were detected. The phylogenetic analysis revealed conserved orthology in bark beetle chemosensory proteins, especially with a major forest pest and co-habitant, Ips typographus . Recent large-scale functional studies in I. typographus chemoreceptors add greater significance to the orthologous sequences reported here. Nevertheless, identifying chemosensory genes in I. duplicatus is valuable to understanding the chemosensory system and its evolution in bark beetles (Coleoptera) and, generally, insects.

The identification of odorant-binding proteins (OBPs) involved in host location by Oides leucomelaena ( O. leucomelaena Weise, 1922, Coleoptera, Galerucinae) is significant for its biological control. Tools in the NCBI database were used to compare and analyze the transcriptome sequences of O. leucomelaena with OBP and other chemosensory-related proteins of other Coleoptera insects. Subsequently, MEGA7 was utilized for OBP sequence alignment and the construction of a phylogenetic tree, combined with expression profiling to screen for candidate antennae-specific OBPs. In addition, fumigation experiments with star anise volatiles were conducted to assess the antennae specificity of the candidate OBPs. Finally, molecular docking was employed to speculate on the binding potential of antennae-specific OBPs with star anise volatiles. The study identified 42 candidate OBPs, 8 chemosensory proteins and 27 receptors. OleuOBP3, OleuOBP5, and OleuOBP6 were identified as classic OBP family members specific to the antennae, which was confirmed by volatile fumigation experiments. Molecular docking ultimately clarified that OleuOBP3, OleuOBP5, and OleuOBP6 all exhibit a high affinity for β-caryophyllene among the star anise volatiles. We successfully obtained three antennae-specific OBPs from O. leucomelaena and determined their high-affinity volatiles, providing a theoretical basis for the development of attractants in subsequent stages.

Insects rely on olfaction for mating, finding oviposition sites, and locating hosts. Hyphantria cunea is a serious pest that severely damages forests. Differential expression analysis of olfactory-related genes between males and females is the basis for elucidating the functions of olfactory-related proteins in H. cunea . In this study, Illumina HiSeqTM 4000 high-throughput sequencing technology was used to perform transcriptome sequencing of the antennal tissues of adult male and female H. cunea . Functional annotation was conducted using the NR, Swiss-Prot, KOG, KEGG, and GO databases, and the results showed that the antennal transcriptome of adult H. cunea contained 50,158 unigenes. Differential expression analysis identified 3923 genes that were significantly differentially expressed between male and female antennae. A total of 221 olfactory-related genes were annotated, and 96 sex-biased genes were identified, including 13 odorant receptors (ORs), 48 odorant binding proteins (OBPs), 7 chemosensory proteins (CSPs), 10 ionotropic receptors (IRs), 10 sensory neuron membrane proteins (SNMPs), 2 gustatory receptors (GRs), and 6 odorant-degrading enzymes (ODEs), indicating that there were differences in olfaction between male and female H. cunea . Quantitative real-time PCR was used to verify the expression levels of 21 putative general odorant receptor genes in male and female antennae. HcunOR4 and HcunOR5 showed female-biased expression; HcunOR48 , HcunOR49 and HcunOR50 showed male-biased expression. The results were consistent with the transcriptome differential analysis. The screening of male-biased odorant receptor genes might provide a theoretical basis for the functional characterization of odorant receptors for recognizing sex pheromones in H. cunea .

Graphosoma rubrolineatum (Hemiptera: Pentatomidae) is an important pest of vegetables and herbs (e.g., Umbelliferae and Cruciferae) in China, Siberia, Korea, and Japan. Insects are highly dependent on their olfactory system to detect odorants. However, no molecular-mediated olfactory genes in G. rubrolineatum have yet been identified. In this study, we first established the antennal transcriptome of G. rubrolineatum and identified 189 candidate olfactory genes, including 31 odorant-binding proteins (OBPs), 15 chemosensory proteins (CSPs), four sensory neuron membrane proteins (SNMPs),94 odorant receptors (ORs), 23 ionotropic receptors (IRs), and 22 gustatory receptors (GRs). Additionally, phylogenetic trees were constructed for olfactory genes between G. rubrolineatum and other hemipteran insects. We also detected the expression profiles of ten OBPs, five CSPs, two SNMPs, five ORs, four IRs, and four GRs by real-time quantitative PCR. The results revealed that most genes (GrubOBP1/11/31, GrubCSP3/8, GrubSNMP1a/1b, GrubOrco/OR9/11/13, GrubGR1/4/22, GrubIR25/75h/76b/GluR1) were highly expressed in the antennae, GrubOBP13/31 and GrubCSP4/11/12 were highly expressed in the legs, while GrubOBP20 and GrubGR19 were highly expressed in the wings. Our results will enrich the gene inventory of G. rubrolineatum and provide further insight into the molecular chemosensory mechanisms of G. rubrolineatum., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Guo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Recognition of sex pheromones released by heterosexual moths via sex pheromone receptors is key for establishing mating connections in moths. The day-flying moth Phauda flammans is an oligophagous pest in southern cities of China and Southeast Asian countries. Our previous study reported that male P. flammans can be attracted to two sex pheromone compounds [Z-9-hexadecenal and (Z, Z, Z)-9,12,15-octadecadienal] released by females in the field; however, the mechanism of olfactory recognition is not clear. In this study, two sex pheromone receptor genes (PflaOR29 and PflaOR44) were cloned. Among the different tissues, both PflaOR29 and PflaOR44 were highly expressed in the antennae of mated male adults. At different developmental stages, the expression levels of PflaOR29 and PflaOR44 were significantly greater in mated male adults than other stages. The fluorescence signals of PflaOR29 and PflaOR44 were mostly distributed on the dorsal side of the antennae, with a large number of trichoid sensilla. The results of the gene function of PflaOR29 and PflaOR44 based on a Drosophila empty neuron heterologous expression system indicated that PflaOR29 strongly responded to (Z, Z, Z)-9,12,15-octadecadienal but not to Z-9-hexadecenal, whereas PflaOR44 did not respond to the two sex pheromones. Our findings clarify the sex pheromone receptor gene corresponding to (Z, Z, Z)-9,12,15-octadecatrienal. These results provide essential information for analyzing the mechanism of sexual communication in diurnal moths and for identifying target genes for the development of efficient attractants., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of financial interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Odorant-binding proteins (OBPs) play key roles in host plant location by insects, and can accordingly serve as important targets for the development of attractants. In this study, we detected the high expression of SlitOBP34 in male antennae of Spodoptera litura. Subsequently, the fluorescence competitive binding experiments displayed that the SlitOBP34 protein has binding affinity for different ligands. Then, protein-ligand interaction analyses found the presence of six amino acid residues may serve as key recognition sites. Further electroantennographic and biobehavioral assessments revealed that the electrophysiological responses of male antennae were evoked in response to stimulation with the six identified host volatiles, and that these volatiles attracted male moths to varying extents. Notably, low concentrations of benzaldehyde, 1-hexanol, and cis-3-hexenyl acetate were found to have significant attractant effects on male moths, thereby identifying these three host volatiles as potential candidates for the development of male attractants. These findings advance our current understanding of the olfactory-encoded mechanisms of host plants selection in S. litura and have enabled us to develop novel adult attractants for controlling the pest in the future., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: The important wood-boring pest Batocera horsfieldi has evolved a sensitive olfactory system to locate host plants. Odorant-binding proteins (OBPs) are thought to play key roles in olfactory recognition. Therefore, exploring the physiological function of OBPs could facilitate a better understanding of insect chemical communications., Results: In this research, 36 BhorOBPs genes were identified via transcriptome sequencing of adults' antennae from B. horsfieldi, and most BhorOBPs were predominantly expressed in chemosensory body parts. Through fluorescence competitive binding and fluorescence quenching assays, the antenna-specific BhorOBP28 was investigated and displayed strong binding affinities forming stable complexes with five volatiles, including (+)-α-Pinene, (+)-Limonene, β-Pinene, (-)-Limonene, and (+)-Longifolene, which could also elicit conformation changes when they were interacting with BhorOBP28. Batocera horsfieldi females exhibited a preference for (-)-Limonene, and a repellent response to (+)-Longifolene. Feeding dsOBP19 produced by a bacteria-expressed system with a newly constructed vector could lead to the knockdown of BhorOBP28, and could further impair B. horsfieldi attraction to (-)-Limonene and repellent activity of (+)-Longifolene. The analysis of site-directed mutagenesis revealed that Leu7, Leu72, and Phe121 play a vital role in selectively binding properties of BhorOBP28., Conclusion: By modeling the molecular mechanism of olfactory recognition, these results demonstrate that BhorOBP28 is involved in the chemoreception of B. horsfieldi. The bacterial-expressed dsRNA delivery system gains new insights into potential population management strategies. Through the olfactory process concluded that discovering novel behavioral regulation and environmentally friendly control options for B. horsfieldi in the future. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Background: Lucilia cuprina (Wiedemann, 1830) (Diptera: Calliphoridae) is the main causative agent of flystrike of sheep in Australia and New Zealand. Female flies lay eggs in an open wound or natural orifice, and the developing larvae eat the host's tissues, a condition called myiasis. To improve our understanding of host-seeking behavior, we quantified gene expression in male and female antennae based on their behavior., Methods: A spatial olfactometer was used to evaluate the olfactory response of L. cuprina mated males and gravid females to fresh or rotting beef. Antennal RNA-Seq analysis was used to identify sensory receptors differentially expressed between groups., Results: Lucilia cuprina females were more attracted to rotten compared to fresh beef (> fivefold increase). However, males and some females did not respond to either type of beef. RNA-Seq analysis was performed on antennae dissected from attracted females, non-attracted females and males. Transcripts encoding sensory receptors from 11 gene families were identified above a threshold (≥ 5 transcript per million) including 49 ATP-binding cassette transporters (ABCs), two ammonium transporters (AMTs), 37 odorant receptors (ORs), 16 ionotropic receptors (IRs), 5 gustatory receptors (GRs), 22 odorant-binding proteins (OBPs), 9 CD36-sensory neuron membrane proteins (CD36/SNMPs), 4 chemosensory proteins (CSPs), 4 myeloid lipid-recognition (ML) and Niemann-Pick C2 disease proteins (ML/NPC2), 2 pickpocket receptors (PPKs) and 3 transient receptor potential channels (TRPs). Differential expression analyses identified sex-biased sensory receptors., Conclusions: We identified sensory receptors that were differentially expressed between the antennae of both sexes and hence may be associated with host detection by female flies. The most promising for future investigations were as follows: an odorant receptor (LcupOR46) which is female-biased in L. cuprina and Cochliomyia hominivorax Coquerel, 1858; an ABC transporter (ABC G23.1) that was the sole sensory receptor upregulated in the antennae of females attracted to rotting beef compared to non-attracted females; a female-biased ammonia transporter (AMT_Rh50), which was previously associated with ammonium detection in Drosophila melanogaster Meigen, 1830. This is the first report suggesting a possible role for ABC transporters in L. cuprina olfaction and potentially in other insects., (© 2024. The Author(s).)

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).)

Volatile sex pheromones are vital for sexual communication between males and females. Females of the American cockroach, Periplaneta americana, produce and emit two sex pheromone components, periplanone-A (PA) and periplanone-B (PB). Although PB is the major sex attractant and can attract males, how it interacts with PA in regulating sexual behaviors is still unknown. In this study, we found that in male cockroaches, PA counteracted PB attraction. We identified two odorant receptors (ORs), OR53 and OR100, as PB/PA and PA receptors, respectively. OR53 and OR100 were predominantly expressed in the antennae of sexually mature males, and their expression levels were regulated by the sex differentiation pathway and nutrition-responsive signals. Cellular localization of OR53 and OR100 in male antennae further revealed that two types of sensilla coordinate a complex two-pheromone-two-receptor pathway in regulating cockroach sexual behaviors. These findings indicate distinct functions of the two sex pheromone components, identify their receptors and possible regulatory mechanisms underlying the male-specific and age-dependent sexual behaviors, and can guide novel strategies for pest management., (© 2024. Science China Press.)

Behavioural regulation in insect societies remains a fundamental question in sociobiology. In hymenopteran societies, the queen plays a crucial role in regulating group behaviour by affecting individual behaviour and physiology through modulation of worker gene expression. Honey bee (Apis mellifera) queens signal their presence via queen mandibular pheromone (QMP). While QMP has been shown to influence behaviour and gene expression of young workers, we know little about how these changes translate in older workers. The effects of the queen pheromone could have prolonged molecular impacts on workers that depend on an early sensitive period. We demonstrate that removal of QMP impacts long-term gene expression in the brain and antennae in foragers that were treated early in life (1 day post emergence), but not when treated later in life. Genes important for division of labour, learning, chemosensory perception and ageing were among those differentially expressed in the antennae and brain tissues, suggesting that QMP influences diverse physiological and behavioural processes in workers. Surprisingly, removal of QMP did not have an impact on foraging behaviour. Overall, our study suggests a sensitive period early in the life of workers, where the presence or absence of a queen has potentially life-long effects on transcriptional activity., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Aphidius gifuensis is the dominant parasitic natural enemy of aphids. Elucidating the molecular mechanism of host recognition of A. gifuensis would improve its biological control effect. Chemosensory proteins (CSPs) play a crucial role in insect olfactory systems and are mainly involved in host localization. In this study, a total of nine CSPs of A. gifuensis with complete open reading frames were identified based on antennal transcriptome data. Phylogenetic analysis revealed that AgifCSPs were mainly clustered into three subgroups (AgifCSP1/2/7/8, AgifCSP3/9, and AgifCSP4/5/6). AgifCSP2/5 showed high expression in the antennae of both sexes. Moreover, AgifCSP5 was found to be specifically expressed in the antennae. In addition, fluorescent binding assays revealed that AifCSP5 had greater affinities for 7 of 32 volatile odor molecules from various sources. Molecular docking and site-directed mutagenesis results revealed that the residue at which AgifCSP5 binds to these seven plant volatiles is Tyr75. Behavior tests further confirmed that trans -2-nonenal, one of the seven active volatiles in the ligand binding test, significantly attracted female adults at a relatively low concentration of 10 mg/mL. In conclusion, AgifCSP5 may be involved in locating aphid-infested crops from long distances by detecting and binding trans -2-nonenal. These findings provide a theoretical foundation for further understanding the olfactory recognition mechanisms and indirect aphid localization behavior of A. gifuensis from long distances by first identifying the host plant of aphids.

In the polyphagous insect Monolepta signata (M. signata) (Coleoptera: Chrysomelidae), antennae are important for olfactory reception used during feeding, mating, and finding a suitable oviposition site. Based on NextSeq 6000 Illumina sequencing, we assembled the antennal transcriptome of mated M. signata and described the first chemosensory gene repertoire expressed in this species. The relative expression levels of some significant chemosensory genes were conducted by quantitative real-time PCR. We identified 114 olfactory-related genes based on the antennal transcriptome database of M. signata, including 21 odorant binding proteins (OBPs), six chemosensory proteins (CSPs), 46 odorant receptors (ORs), 15 ionotropic receptors (IRs), 23 gustatory receptors (GRs) and three sensory neuron membrane proteins (SNMPs). Blastp best hit and phylogenetic analyses showed that most of the chemosensory genes had a close relationship with orthologs from other Coleoptera species. Overall, this study provides a foundation for elucidating the molecular mechanism of olfactory recognition in M. signata as well as a reference for the study of chemosensory genes in other species of Coleoptera., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 He et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The Asian palm weevil, Rhynchophorus ferrugineus, is a tremendously important agricultural pest primarily adapted to palm trees and causes severe destruction, threatening sustainable palm cultivation worldwide. The host plant selection of this weevil is mainly attributed to the functional specialization of odorant receptors (ORs) that detect palm-derived volatiles. Yet, ligands are known for only two ORs of R. ferrugineus, and we still lack information on the mechanisms of palm tree detection. This study identified a highly expressed antennal R. ferrugineus OR, RferOR2, thanks to newly generated transcriptomic data. The phylogenetic analysis revealed that RferOR2 belongs to the major coleopteran OR group 2A and is closely related to a sister clade containing an R. ferrugineus OR (RferOR41) tuned to the non-host plant volatile and antagonist, α-pinene. Functional characterization of RferOR2 via heterologous expression in Drosophila olfactory neurons revealed that this receptor is tuned to several ecologically relevant palm-emitted odors, most notably ethyl and methyl ester compounds, but not to any of the pheromone compounds tested, including the R. ferrugineus aggregation pheromone. We did not evidence any differential expression of RferOR2 in the antennae of both sexes, suggesting males and females detect these compounds equally. Next, we used the newly identified RferOR2 ligands to demonstrate that including synthetic palm ester volatiles as single compounds and in combinations in pheromone-based mass trapping has a synergistic attractiveness effect to R. ferrugineus aggregation pheromone, resulting in significantly increased weevil catches. Our study identified a key OR from a palm weevil species tuned to several ecologically relevant palm volatiles and represents a significant step forward in understanding the chemosensory mechanisms of host detection in palm weevils. Our study also defines RferOR2 as an essential model for exploring the molecular basis of host detection in other palm weevil species. Finally, our work showed that insect OR deorphanization could aid in identifying novel behaviorally active volatiles that can interfere with weevil host-searching behavior in sustainable pest management applications., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The longhorned beetles are key players for the maintenance of biodiversity in the terrestrial ecosystem. As xylophagous cerambycid insects in Coleoptera, the beetles have evolved specialized olfactory and gustatory systems to recognize chemical cues in the surrounding habitats. Despite over 36,000 described species in the Cerambycidae family including a wood-boring pest Pharsalia antennata, only a limited number of them (<1 %) have been characterized regarding their chemical ecology at the molecular level. Here, we surveyed four membrane protein gene families in P. antennata related to chemoreception through transcriptomics, phylogenetics and expression profiling analyses. In total, 144 genes encoding 72 odorant receptors (ORs), 33 gustatory receptors (GRs), 23 ionotropic receptors (IRs), four sensory neuron membrane proteins (SNMPs) and 12 ionotropic glutamate receptors (iGluRs) were harvested from the transcriptome of multiple tissues including antennae and legs of both sexes. The lineage-specific expansion of PantORs possibly implied a diverse range of host plants in this beetle, supporting this correlation between the host range and olfactory receptor repertoire sizes across cerambycid species. Further phylogenetic analysis revealed that Group 2 was contributed mainly to the large OR gene repertoire in P. antennata, representing 18 genes in Group 2A and eight in Group 2B. On the other hand, some key chemosensory genes were identified by applying a phylogenetics approach, such as PantOR21 close to the 2-phenylethanol receptor in Megacyllene caryae, three carbon dioxide GRs and seven Antennal IRs (A-IRs) clades. We also determined sex- and tissue-specific expression profiles of 69 chemosensory genes, revealing the high expression of most PantORs in antennae. Noticeably, 10 sex-biased genes (six PantORs, three PantIRs and PantSNMP1a) were presented in antennae, five sex-biased PantGRs in legs and 39 sex-biased genes (15 PantORs, 13 PantGRs, eight PantIRs and three PantSNMPs) in abdomens. These findings have greatly enhanced our knowledge about the chemical ecology of P. antennata and identify candidate molecular targets for mediating smell and taste of this beetle., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Atherigona orientalis Schiner (1868) is an acknowledged agricultural pest owing to its feeding habits and breeding locations. This insect is a tropical and subtropical pest in fruits and vegetables, in which >50 varieties of fruits and vegetables in 26 families, such as Capsicum annuum, Lycopersicon esculentum, and Cucumis melo have been attacked. Moreover, A. orientalis may also develop in rotten crops and feces or insect carcasses, which are also considered one kind of sanitary pest and medical insect. At present, the invasion ranges of A. orientalis are still increasing and more preventive and management measures are to be processed. To gain a better understanding of the molecular mechanisms involved in olfactory reception in A. orientalis, the transcriptome of male and female antennae and legs was systematically analyzed. In total, 131 chemosensory-related genes, including 63 odorant receptors (ORs), 20 gustatory receptors (GRs), 18 ionotropic receptors (IRs), 27 odorant binding proteins (OBPs), 1 chemosensory protein (CSP), and 2 sensory neuron membrane proteins (SNMPs), were identified. The analysis focused on obtaining expression information of candidate olfactory genes at the transcriptomic level by examining the differentially expressed genes (DEGs) in all samples. Totally, 41 DEGs were identified between male antennae (MA) and female antennae (FA), including 32 ORs, 5 OBPs, 1 IR, 2 GRs and 1 SNMP. In MA versus male legs (ML), 78 DEGs were identified (45 ORs, 18 OBPs, 6 GRs, 6 IRs, 1 CSP and 2 SNMPs). In FA and female legs (FL), 96 DEGs were identified (51 ORs, 21 OBPs, 9 GRs, 12 IRs, 1 CSP and 2 SNMPs). For ML and FL, 3 DEGs were identified, including 2 ORs and 1 SNMP. Our results supplement valuable insights for future research on the chemoreception mechanisms in A. orientalis., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

The Japanese pine sawyer Monochamus alternatus serves as the primary vector for pine wilt disease, a devastating pine disease that poses a significant threat to the sustainable development of forestry in the Eurasian region. Currently, trap devices based on informational compounds have played a crucial role in monitoring and controlling the M. alternatus population. However, the specific proteins within M. alternatus involved in recognizing the aforementioned informational compounds remain largely unclear. To elucidate the spatiotemporal distribution of M. alternatus chemosensory-related genes, this study conducted neural transcriptome analyses to investigate gene expression patterns in different body parts during the feeding and mating stages of both male and female beetles. The results revealed that 15 genes in the gustatory receptor (GR) gene family exhibited high expression in the mouthparts, most genes in the odorant binding protein (OBP) gene family exhibited high expression across all body parts, 22 genes in the odorant receptor (OR) gene family exhibited high expression in the antennae, a significant number of genes in the chemosensory protein (CSP) and sensory neuron membrane protein (SNMP) gene families exhibited high expression in both the mouthparts and antennae, and 30 genes in the ionotropic receptors (IR) gene family were expressed in the antennae. Through co-expression analyses, it was observed that 34 genes in the IR gene family were co-expressed across the four developmental stages. The Antenna IR subfamily and IR8a/Ir25a subfamily exhibited relatively high expression levels in the antennae, while the Kainate subfamily, NMDA subfamily, and Divergent subfamily exhibited predominantly high expression in the facial region. MalIR33 is expressed only during the feeding stage of M. alternatus , the MalIR37 gene exhibits specific expression in male beetles, the MalIR34 gene exhibits specific expression during the feeding stage in male beetles, the MalIR8 and MalIR39 genes exhibit specific expression during the feeding stage in female beetles, and MalIR8 is expressed only during two developmental stages in male beetles and during the mating stage in female beetles. The IR gene family exhibits gene-specific expression in different spatiotemporal contexts, laying the foundation for the subsequent selection of functional genes and facilitating the full utilization of host plant volatiles and insect sex pheromones, thereby enabling the development of more efficient attractants.

Phyllotreta striolata , the striped flea beetle, is one of the most destructive pests in Brassicaceae plants worldwide. Given the drawbacks associated with long-term use of chemical insecticides, green strategies based on chemical ecology are an effective alternative for beetle control. However, the lack of information on beetle ecology has hindered the development of effective biocontrol strategies. In this report, we identified two odorants, (S)-cis-verbenol and (-)-verbenone, which displayed significant attraction for P. striolata ( p < 0.05), indicating their great potential for P. striolata management. Using the Drosophila "empty neuron" system, an antenna-biased odorant receptor, PstrOR17, was identified as responsible for the detection of (-)-verbenone and (S)-cis-verbenol. Furthermore, the interactions between PstrOR17 and (-)-verbenone or (S)-cis-verbenol were predicted via modeling and molecular docking. Finally, we used RNAi to confirm that PstrOR17 is essential for the detection of (-)-verbenone and (S)-cis-verbenol to elicit an attraction effect. Our results not only lay a foundation for the development of new and effective nonchemical insecticide strategies based on (S)-cis-verbenol and (-)-verbenone, but also provide new insight into the molecular basis of odorant recognition in P. striolata .

Chemoreception through odorant receptors (ORs), ionotropic receptors (IRs) and gustatory receptors (GRs) represents the functions of key proteins in the chemical ecology of insects. Recent studies have identified chemoreceptors in coleopterans, facilitating the evolutionary analysis of not only ORs but also IRs and GRs. Thus, Cerambycidae, Tenebrionidae and Curculionidae have received increased attention. However, knowledge of the chemoreceptors from Scarabaeidae is still limited, particularly for those that are sympatric. Considering the roles of chemoreceptors, this analysis could shed light on evolutionary processes in the context of sympatry. Therefore, the aim of this study was to identify and compare the repertoires of ORs, GRs and IRs between two sympatric scarab beetles, Hylamorpha elegans and Brachysternus prasinus. Here, construction of the antennal transcriptomes of both scarab beetle species and analyses of their phylogeny, molecular evolution and relative expression were performed. Thus, 119 new candidate chemoreceptors were identified for the first time, including 17 transcripts for B. prasinus (1 GR, 3 IRs and 13 ORs) and 102 for H. elegans (22 GRs, 14 IRs and 66 ORs). Orthologs between the two scarab beetle species were found, revealing specific expansions as well as absence in some clades. Purifying selection appears to have occurred on H. elegans and B. prasinus ORs. Further efforts will be focused on target identification to characterize kairomone and/or pheromone receptors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Diachasmimorpha longicaudata is a hymenopteran fruit fly endoparasitoid. Females of this species find their hosts for oviposition by using complex sensorial mechanisms in response to physical and chemical stimuli associated with the host and host habitat. Ecological and behavioral aspects related to host-seeking behavior for oviposition have been extensively studied in D. longicaudata, including the identification of volatile organic compounds acting as attractants to females. In this sense, molecular mechanisms of chemoreception have been explored in this species, including a preliminary characterization of odorant-binding proteins (OBPs), chemosensory proteins (CSPs) and odorant receptors (ORs), among other proteins. Functional assays on OBP and CSP have been conducted as a first approach to identify molecular mechanisms associated with the female host-seeking behavior for oviposition. The aims of the present study were to identify the D. longicaudata sensory gene repertoire expressed in the antenna of sexually mature and mated individuals of both sexes, and subsequently, characterize transcripts differentially expressed in the antennae of females to identify candidate genes associated with the female host-seeking behavior for oviposition., Results: A total of 33,745 predicted protein-coding sequences were obtained from a de novo antennal transcriptome assembly. Ten sensory-related gene families were annotated as follows: 222 ORs, 44 ionotropic receptors (IRs), 25 gustatory receptors (GRs), 9 CSPs, 13 OBPs, 2 ammonium transporters (AMTs), 8 pickpocket (PPKs) receptors, 16 transient receptor potential (TRP) channels, 12 CD36/SNMPs and 3 Niemann-Pick type C2 like proteins (NPC2-like). The differential expression analysis revealed 237 and 151 transcripts up- and downregulated, respectively, between the female and male antennae. Ninety-seven differentially expressed transcripts corresponded to sensory-related genes including 88 transcripts being upregulated (87 ORs and one TRP) and nine downregulated (six ORs, two CSPs and one OBP) in females compared to males., Conclusions: The sensory gene repertoire of D. longicaudata was similar to that of other taxonomically related parasitoid wasps. We identified a high number of ORs upregulated in the female antenna. These results may indicate that this gene family has a central role in the chemoreception of sexually mature females during the search for hosts and host habitats for reproductive purposes., (© 2024. The Author(s).)

Antennae and legs (primarily the tarsal segments) of insects are the foremost sensory organs that contact a diverse range of toxic chemicals including insecticides. Binding proteins expressed in the two tissues are potential molecular candidates serving as the binding and sequestering of insecticides, like chemosensory proteins (CSPs). Insect CSPs endowed with multiple roles have been suggested to participate in insecticide resistance, focusing mainly on moths, aphids and mosquitos. Yet, the molecular underpinnings underlying the interactions of cerambycid CSPs and insecticides remain unexplored. Here, we present binding properties of three antenna- and tarsus-enriched RhorCSPs (RhorCSP1, CSP2 and CSP3) in Rhaphuma horsfieldi to eight insecticide classes totaling 15 chemicals. From the transcriptome of this beetle, totally 16 CSP-coding genes were found, with seven full-length sequences. In phylogeny, these RhorCSPs were distributed dispersedly in different clades. Expression profiles revealed the abundant expression of RhorCSP1, CSP2 and CSP3 in antennae and tarsi, thus as representatives for studying the protein-insecticide interactions. Binding assays showed that the three RhorCSPs were tuned differentially to insecticides but exhibited the highest affinities with hexaflumuron, chlorpyrifos and rotenone (dissociation constants <13 μM). In particular, RhorCSP3 could interact strongly with 10 of tested insecticides, of which four residues (Tyr25, Phe42, Val65 and Phe68) contributed significantly to the binding of six, four, three and four ligands, respectively. Of these, the binding of four mutated RhorCSP3s to a botanical insecticide rotenone was significantly weakened compared to the wildtype protein. Furthermore, we also evidenced that RhorCSP3 was a broadly-tuned carrier protein in response to a wide variety of plant odorants outside insecticides. Altogether, our findings shed light on different binding mechanisms and odorant-tuning profiles of three RhorCSPs in R. horsfieldi and identify key residues of the RhorCSP3-insecticide interactions., (Copyright © 2023. Published by Elsevier Inc.)

Background: Odorant-binding proteins (OBPs) are essential in insect's daily behaviors mediated by olfactory perception. Megachile saussurei Radoszkowski (Hymenoptera, Megachilidae) is a principal insect pollinating alfalfa (Medicago sativa) in Northwestern China. The olfactory function have been less conducted, which provides a lot of possibilities for our research., Results: Our results showed that 20 OBPs were identified in total. Multiple sequence alignment analysis indicated MsauOBPs were highly conserved with a 6-cysteine motif pattern and all belonged to the classic subfamily, coding 113-196 amino acids and sharing 41.32%-99.12% amino acid identity with known OBPs of other bees. Phylogenetic analysis indicated there were certain homologies existed among MsauOBPs and most sequences were clustered with that of Osmia cornuta (Hymenoptera, Megachilidae). Expression analysis showed the identified OBPs were mostly enriched in antennae instead of other four body parts, especially the MsauOBP2, MsauOBP3, MsauOBP4, MsauOBP8, MsauOBP11 and MsauOBP17, in which the MsauOBP2, MsauOBP4 and MsauOBP8 presented obvious tissue-biased expression pattern. Molecular docking results indicated MsauOBP4 might be the most significant protein in recognizing alfalfa flower volatile 3-Octanone, while MsauOBP13 might be the most crucial protein identifying (Z)-3-hexenyl acetate. It was also found the lysine was a momentous hydrophilic amino acid in docking simulations., Conclusion: In this study, we identified and analyzed 20 OBPs of M. saussurei. The certain homology existed among these OBPs, while some degree of divergence could also be noticed, indicating the complex functions that different MsauOBPs performed. Besides, the M. saussurei and Osmia cornuta were very likely to share similar physiological functions as most of their OBPs were clustered together. MsauOBP4 might be the key protein in recognizing 3-Octanone, while MsauOBP13 might be the key protein in binding (Z)-3-hexenyl acetate. These two proteins might contribute to the alfalfa-locating during the pollination process. The relevant results may help determine the highly specific and effective attractants for M. saussurei in alfalfa pollination and reveal the molecular mechanism of odor-evoked pollinating behavior between these two species., (© 2023. The Author(s).)

The sophisticated olfactory system of insects is plays a critical role in detecting chemical signals and guiding insect behaviors, such as selecting mates, finding hosts, evading predators, and discovering oviposition sites. Therefore, exploring and clarifying the molecular processes of this system is crucial for developing new insecticides or efficient pest control methods. Plodia interpunctella (Hübner) is a disruptive insect pest damaging the stored grains over the world. However, the olfactory processes of P. interpunctella remain unclear. Herein, we employed a transcriptome analysis to identify olfactory and differentially expressed genes to characterize their expression patterns in different developmental stages and antennal tissue. Subsequently, a total of 172 potential olfactory-related genes included 42 odorant-binding proteins, 12 chemosensory proteins, 51 odorant receptors, 13 gustatory receptors, three sensory neuron membrane proteins, and 51 ionotropic receptors. Furthermore, phylogenetic analysis and BLASTx best-hit analyses showed that these olfactory genes were closely linked with those identified in other lepidopterans. Transcriptome analysis revealed 49 differentially expressed olfactory-related genes, and a semiquantitative reverse transcription polymerase chain reaction showed that 11 olfactory genes were particularly expressed in the legs and wings of female P. interpunctella. Meanwhile, PintOBP29 was notably expressed in female antennae and legs. Genes with high expression levels in the abdomen showed high expression in the legs, but low expression in the antennae. Our findings provide the candidate genetic factors for analysis of the olfactory processes in P. interpunctella., (© 2023 Wiley Periodicals LLC.)

The flower bug Orius sauteri (Heteroptera: Anthocoridae), is a polyphagous predator and a natural enemy widely used in biological pest control to micro-pests including aphids, spider mites, thrips and so on. In the present study, the transcriptome analysis of adult heads in O. sauteri were performed and identified a total of 38 chemosensory genes including 24 odorant binding proteins (OBPs) and 14 chemosensory proteins (CSPs). Subsequently, we conducted quantitative real-time PCR to detect the tissue expression level of 18 OBPs and 8 CSPs. The results showed that almost all OsauOBPs and OsauCSPs have a high expression level in the adult heads of both sexes. In addition, 5 OsauOBPs (OBP1, OBP2, OBP3, OBP4 and OBP14) have a significantly higher expressed in male heads than female, indicating that these chemosensory proteins might be involved in the male-specific behaviors such as pheromone reception and mate-seeking. This study will provide helpful reference for subsequent understanding of chemoreception mechanism in O. sauteri., Competing Interests: Declaration of competing interest The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

The orientation of the oligophagous cone-feeding moth Dioryctria abietella (Lepidoptera: Pyralidae) to host plants primarily relies on olfactory-related proteins, particularly those candidates highly expressed in antennae. Here, through a combination of expression profile, ligand-binding assay, molecular docking and site-directed mutagenesis strategies, we characterized the chemosensory protein (CSP) gene family in D. abietella. Quantitative real-time PCR (qPCR) analyses revealed the detectable expression of all 22 DabiCSPs in the antennae, of which seven genes were significantly enriched in this tissue. In addition, the majority of the genes (19/22 relatives) had the expression in at least one reproductive tissue. In the interactions of four antenna-dominant DabiCSPs and different chemical classes, DabiCSP1 was broadly tuned to 27 plant-derived odors, three man-made insecticides and one herbicide with high affinities (K i  < 6.60 μM). By contrast, three other DabiCSPs (DabiCSP4, CSP6 and CSP17) exhibited a narrow odor binding spectrum, in response to six compounds for each protein. Our mutation analyses combined with molecular docking simulations and binding assays further identified four key residues (Tyr25, Thr26, Ile65 and Val69) in the interactions of DabiCSP1 and ligands, of which binding abilities of this protein to 12, 15, 16 and three compounds were significantly decreased compared to the wildtype protein, respectively. Our study reveals different odor binding spectra of four DabiCSPs enriched in antennae and identifies key residues responsible for the binding of DabiCSP1 and potentially active compounds for the control of this pest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The order Isopoda contains both aquatic and terrestrial species, among which Hemilepistus reaumurii, which lives in arid environments and is the most adapted to terrestrial life. Olfaction has been deeply investigated in insects while it has received very limited attention in other arthropods, particularly in terrestrial crustaceans. In insects, soluble proteins belonging to two main families, Odorant Binding Proteins (OBPs) and Chemosensory Proteins (CSPs), are contained in the olfactory sensillar lymph and are suggested to act as carriers of hydrophobic semiochemicals to or from membrane-bound olfactory receptors. Other protein families, namely Nieman-Pick type 2 (NPC2) and Lipocalins (LCNs) have been also reported as putative odorant carriers in insects and other arthropod clades. In this study, we have sequenced and analysed the transcriptomes of antennae and of the first pair of legs of H. reaumurii focusing on soluble olfactory proteins. Interestingly, we have found 13 genes encoding CSPs, whose sequences differ from those of the other arthropod clades, including non-isopod crustaceans, for the presence of two additional cysteine residues, besides the four conserved ones. Binding assays on two of these proteins showed strong affinities for fatty acids and long-chain unsaturated esters and aldehydes, putative semiochemicals for this species., Competing Interests: Conflicts of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cytochrome P450 monooxygenases (P450s) are a superfamily of multifunctional heme-containing proteins and could function as odorant-degrading enzymes (ODEs) in insect olfactory systems. In our previous study, we identified a P450 gene from the antennal transcriptome of Locusta migratoria, LmCYP6MU1, which could be induced by a variety of volatiles. However, the regulatory mechanisms of this gene in response to volatiles remain unknown. In current study, we investigated the tissues and development stages expression patterns of LmCYP6MU1 and determined its olfactory function in the recognition of the main host plant volatiles which induced LmCYP6MU1 expression. The results showed that LmCYP6MU1 was antenna-rich and highly expressed throughout the antennal developmental stages of locusts. LmCYP6MU1 played important roles in the recognition of trans-2-hexen-1-al and nonanal. Insect CncC regulates the expression of P450 genes. We tested whether LmCncC regulates LmCYP6MU1 expression. It was found that LmCncC knockdown in the antennae resulted in the downregulation of LmCYP6MU1 and repressed the volatiles-mediated induction of LmCYP6MU1. LmCncC knockdown reduced the electroantennogram (EAG) and behavioral responses of locusts to volatiles. These results suggested that LmCncC could regulate the basal and volatiles-mediated inducible expression of LmCYP6MU1 responsible for the recognition of trans-2-hexen-1-al and nonanal. These findings provide an original basis for understanding the regulation mechanisms of LmCncC on LmCYP6MU1 expression and help us better understand the LmCncC-mediated olfactory plasticity., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Quadrastichus mendeli Kim is one of the most important parasitoids of Leptocybe invasa Fisher et La Salle, which is an invasive gall-making pest in eucalyptus plantations in the world. Gall-inducing insects live within plant tissues and induce tumor-like growths that provide the insects with food, shelter, and protection from natural enemies. Empirical evidences showed that sensory genes play a key role in the host location of parasitoids. So far, what kind of sensory genes regulate parasitoids to locate gall-inducing insects has not been uncovered. In this study, sensory genes in the antenna and abdomen of Q. mendeli were studied using high-throughput sequencing. In total, 181,543 contigs was obtained from the antenna and abdomen transcriptome of Q. mendeli. The major sensory genes (chemosensory proteins, CSPs; gustatory receptors, GRs; ionotropic receptors, IRs; odorant binding proteins, OBPs; odorant receptors, ORs; and sensory neuron membrane proteins, SNMPs) were identified, and phylogenetic analyses were performed with these genes from Q. mendeli and other model insect species. The gene co-expression network constructed by WGCNA method is robust and reliable. There were 10,314 differentially expressed genes (DEGs), and among them, 99 genes were DEGs. A comprehensive sequence resource with desirable quality was built by comparative transcriptome of the antenna and abdomen of Q. mendeli, enriching the genomic platform of Q. mendeli., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

The turnip aphid, Lipaphis erysimi Kaltenbach, inflicts heavy damage on cruciferous crops worldwide. In these insects, olfactory perception is crucial for mating, host location, and oviposition. Both odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are responsible for the delivery of host odorants and pheromones during initial molecular interactions. In this study, antennal and body transcriptomes of L. erysimi were generated through the deep sequencing of RNA libraries. A dataset of 11 LeryOBP and four LeryCSP transcripts was identified among assembled unigenes and subjected to sequence analysis. Phylogenetic analysis found a one-to-one orthologous relationship between LeryOBP/LeryCSP and its corresponding homologs from other aphid species. Further quantitative real-time PCR analyses across developmental stages and tissues showed that five LeryOBP genes (i.e., LeryGOBP, LeryOBP6, LeryOBP7, LeryOBP9, and LeryOBP13) and LeryCSP10 were specifically or significantly elevated in the antennae compared with other tissues. Moreover, two transcripts (i.e., LeryGOBP and LeryOBP6) exhibited remarkably higher expression levels in alate aphids, implying their potentially functional role in the perception of new host plant locations. These results present the identification and expression of OBP/CSP genes in L. erysimi, providing valuable insights into their putative role in olfactory signal transduction., (© 2023 Wiley Periodicals LLC.)

Termites have an elaborate social system that involves cooperation and division of labour among colony members. Although this social system is regulated by chemical signals produced in the colony, it remains unclear how these signals are perceived by other members. Signal transduction is well known to be triggered by the reception of odorant molecules by some binding proteins in the antennae, after which, a signal is transmitted to chemosensory receptors. However, there is insufficient information on the role of chemosensory genes involved in signal transduction in termites. Here, we identified the genes involved in chemosensory reception in the termite Reticulitermes speratus and performed a genome-wide comparative transcriptome analysis of worker and soldier antennae. First, we identified 31 odorant-binding proteins (OBPs), and three chemosensory protein A (CheA) from the genome data. Thereafter, we performed RNA sequencing to compare the expression levels of OBPs, CheAs, and previously identified chemosensory receptor genes between worker and soldier antennae. There were no receptor genes with significant differences in expression between castes. However, the expression levels of three non-receptor odorant-detection/binding proteins (OBP, CheA, and Sensory neuron membrane protein) were significantly different between castes. Real-time qPCR (RT-qPCR) analysis using antennae and other head parts confirmed that these genes were highly expressed in soldier antennae. Finally, independent RT-qPCR analysis showed that the expression patterns of these genes were altered in soldiers from different social contexts. Present results suggest that gene expression levels of some non-receptors are affected by both castes and behavioural interactions among colony members in termites., (© 2023 Royal Entomological Society.)

Odorant-degrading enzymes in insects play a vital role in maintaining olfactory sensitivity. However, the role and molecular mechanism of glutathione S -transferases (GSTs) in odorant inactivation has been rarely studied. In the present study, 31 GSTs were identified from the antennal transcriptome of Holotrichia parallela . HpGSTd1 possesses the highest transcriptome expression level. Recombinant HpGSTd1 showed degradation activity toward various unsaturated aldehyde volatiles. Furthermore, the metabolite of cinnamaldehyde was identified by high-resolution mass spectrometry (HRMS). The molecular docking analysis and site-directed mutagenesis revealed the key residues of HpGSTd1 in degrading odorants. In addition, the unsaturated aldehyde volatiles elicited the behavioral and electrophysiological responses of H. parallela . Taken together, our findings suggest that HpGSTd1 may play an essential role in inactivating odorants in H. parallela , which provides new insights for identifying molecular targets and exploring effective olfactory regulators for this underground pest.

In the forest ecosystem dominated by the Pinaceae plants, this boring pest Dioryctria abietella is subject to a variety of odorants derived from host and nonhost plants, in which olfactory-related proteins enriched in antennae are key behavioral modulators for the orientation of feeding and ovipositing hosts. Here, we addressed the odorant binding protein (OBP) gene family in D. abietella. Expression profiles revealed that the majority of OBPs were abundantly expressed in the antennae at a female-biased level. A male-antenna-biased DabiPBP1 was a strong candidate for detecting type I and type II pheromones of D. abitella female moths. Using a prokaryotic expression system combined with affinity chromatography, we harvested two antenna-dominant DabiOBPs. In the ligand-binding assays, the two DabiOBPs exhibited different odorant response spectra, as DabiOBP17 was tuned to most odorants with higher affinities compared to DabiOBP4. Of these, DabiOBP4 could strongly bind syringaldehyde and citral (dissociation constants (K i ) < 14 μM). A floral volatile, benzyl benzoate (K i  = 4.72 ± 0.20 μM), was the best ligand for DabiOBP17. Remarkably, several green leaf volatiles were found to strongly interact with DabiOBP17 (K i  < 8.5 μM), including Z3-hexenyl acetate, E2-hexenol, Z2-hexenal and E2-hexenal that may mediate a repellent response to D. abietella. Structural analyses of ligands revealed that the binding of the two DabiOBPs to odorants was associated with carbon-chain lengths and functional groups. Molecular simulations identified several key residues involved in the interactions of DabiOBPs and ligands, suggesting specific binding mechanisms. This study highlights olfactory roles of two antennal DabiOBPs in D. abietella, helping the identification of potentially behavioral compounds for the population control of this pest., Competing Interests: Declaration of Competing Interest The authors have declared that no competing interest exists., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: The Rhus gall aphid Schlechtendalia chinensis specially uses the only species Rhus chinensis and certain moss species (Mniaceae) as its primary host plant and secondary host plants, respectively. Rhus galls are formed on the primary host by the sucking of aphids, and used in traditional medicine as well as other various areas due to their high tannin contents. Chemoreception is critical for insect behaviors such as host searching, location and identification of mates and reproductive behavior. The process of chemoreception is mediated by a series of protein gene families, including odorant-binding proteins (OBPs), chemosensory proteins (CSPs), olfactory receptors (ORs), gustatory receptors (GRs), ionotropic receptors (IRs), and sensory neuron membrane proteins (SNMPs). However, there have been no reports on the analysis of molecular components related to the chemoreception system of S. chinensis at the genome level., Results: We examined the genes of eight OBPs, nine CSPs, 24 ORs, 16 GRs, 22 IRs, and five SNMPs in the S. chinensis genome using homological searches, and these chemosensory genes appeared mostly on chromosome 1. Phylogenetic and gene number analysis revealed that the gene families, e.g., ORs, GRs, CSPs and SNMPs in S. chinensis, have experienced major contractions by comparing to Myzus persicae, while the two gene families OBPs and IRs had slight expansion. The current results might be related to the broader host range of M. persicae versus the specialization of S. chinensis on only a host plant. There were 28 gene pairs between genomes of S. chinensis and Acyrthosiphon pisum in the chemoreceptor gene families by collinear comparison. Ka/Ks ratios (< 1) indicated that the genes of S. chinensis were mainly affected by purification selection during evolution. We also found the lower number and expression level of chemoreception genes in S. chinensis than in other 11 aphid species, such as ORs, GRs and IRs, which play an important role in host search., Conclusion: Our study firstly identified the genes of the different chemosensory protein gene families in the S. chinensis genome, and analyzed their general features and expression profile, demonstrating the importance of chemoreception in the aphid and providing new information for further functional research., (© 2023. The Author(s).)

We sequenced and analyzed the transcriptomes from different tissues of the soldier beetle, Podabrus annulatus (Coleoptera: Cantharidae), and obtained 75.74 Gb clean reads which were assembled into 95,274 unigenes. Among these transcripts, 25,484 unigenes of highly quality were annotated. Based on annotation and tBLASTn results, we identified a total of 101 candidate olfactory-related genes for the first time, including 11 putative odorant-binding proteins (OBPs), 6 chemosensory proteins (CSP), 50 olfactory receptors (ORs), 25 gustatory receptors (GRs), 6 ionotropic receptors (IRs), and 3 sensory neuron membrane proteins (SNMPs). BLASTX best-hit results indicated that these chemosensory genes were most identical to their respective orthologs from Photinus pyralis. Phylogenetic analyses also revealed that the ORs, GRs, and IRs of Podabrus annulatus are closely related to those of Photinus pyralis. The fragment per kilobase per million mapped fragments (FPKM) values showed that the PannOBP2, PannOBP3, and PannOBP10 were predominantly expressed in the antennae, PannOBP1 in the abdomen-thorax, while others were not identified to be tissue-specific. These olfactory-related differentially expressed genes (DEGs) demonstrated different roles in the olfactory system of Podabrus annulatus. This study establishes the groundwork for future research into the molecular mechanism of olfactory recognition in Podabrus annulatus., (© 2023 Wiley Periodicals LLC.)

To identify odors in complex environments accurately, insects have evolved multiple olfactory proteins. In our study, various olfactory proteins of Odontothrips loti Haliday, an oligophagous pest that primarily affects Medicago sativa (alfalfa), were explored. Specifically, 47 putative olfactory candidate genes were identified in the antennae transcriptome of O. loti , including seven odorant-binding proteins (OBPs), nine chemosensory proteins (CSPs), seven sensory neuron membrane proteins (SNMPs), eight odorant receptors (ORs), and sixteen ionotropic receptors (IRs). PCR analysis further confirmed that 43 out of 47 genes existed in O. loti adults, and O.lot OBP1, O.lot OBP4, and O.lot OBP6 were specifically expressed in the antennae with a male-biased expression pattern. In addition, both the fluorescence competitive binding assay and molecular docking showed that p -Menth-8-en-2-one, a component of the volatiles of the host, had strong binding ability to the O.lotOBP6 protein. Behavioral experiments showed that this component has a significant attraction to both female and male adults, indicating that O.lotOBP6 plays a role in host location. Furthermore, molecular docking reveals potential active sites in O.lotOBP6 that interact with most of the tested volatiles. Our results provide insights into the mechanism of O. loti odor-evoked behavior and the development of a highly specific and sustainable approach for thrip management.