Insect Olfactory System Research Articles

Effects of Atmospheric Pollutants on Volatile-Mediated Insect Ecosystem Services.

Primary and secondary atmospheric pollutants, including carbon monoxide (CO), carbon dioxide (CO2), nitrogen oxides (NOx), ozone (O3), sulphur dioxide (SO2) and particulate matter (PM2.5/PM10) with associated heavy metals (HMs) and micro- and nanoplastics (MPs/NPs), have the potential to influence and alter interspecific interactions involving insects that are responsible for providing essential ecosystem services (ESs). Given that insects rely on olfactory cues for vital processes such as locating mates, food sources and oviposition sites, volatile organic compounds (VOCs) are of paramount importance in interactions involving insects. While gaseous pollutants reduce the lifespan of individual compounds that act as olfactory cues, gaseous and particulate pollutants can alter their biosynthesis and emission and exert a direct effect on the olfactory system of insects. Consequently, air pollutants can affect ecosystem functioning and the services regulated by plant-insect interactions. This review examines the already identified and potential impacts of air pollutants on different aspects of VOC-mediated plant-insect interactions underlying a range of insect ES. Furthermore, we investigate the potential susceptibility of insects to future environmental changes and the adaptive mechanisms they may employ to efficiently detect odours. The current body of knowledge on the effects of air pollutants on key interspecific interactions is biased towards and limited to a few pollinators, herbivores and parasitoids on model plants. There is a notable absence of research on decomposers and seed dispersers. With exception of O3 and NOx, the effects of some widespread and emerging environmental pollutants, such as secondary organic aerosols (SOAs), SO2, HMs, PM and MPs/NPs, remain largely unexplored. It is recommended that the identified knowledge gaps be addressed in future research, with the aim of designing effective mitigation strategies for the adverse effects in question and developing robust conservation frameworks.

Screening, validation and functional characterization of genes encoding proteins that interact with sensory neuron membrane protein 1b (SNMP1b) from Cyrtotrachelus buqueti (Coleoptera: Curculionidae)

Sensory neuron membrane proteins (SNMPs) play critical roles in insect olfactory system. However, functional studies outside of Drosophila remain limited, especially in Coleoptera species. In our previous study, a SNMP1 (CbuqSNMP1b) was identified from Cyrtotrachelus buqueti (Coleoptera: Curculionidae), an insect pest that seriously influence the development of the bamboo industry. Here in a membrane protein yeast two-hybrid system, protein interactions between CbuqSNMP1b as a bait protein and a cDNA library of antenna of male C. buqueti adults as prey protein were assessed. Of 29 proteins identified as putative interactors, the Minus-C odorant-binding protein (CbuqOBP1) was selected for further analysis. The interaction between CbuqSNMP1b and CbuqOBP1 was further confirmed by both the in vivo yeast spotting analysis and the in vitro glutathione-S-transferase pull-down assay. Fluorescence binding assays indicated that the interaction between CbuqSNMP1b and CbuqOBP1 could enhance the binding abilities of CbuqOBP1 to four adult C. buqueti biologically active volatiles. The knockdown of CbuqSNMP1b + CbuqOBP1 expression by RNA interference significantly reduced the behavior responses of male adults to ethyl hexanoate and trans,trans-2,4-Nonadienal. These results increase our understanding of insect SNMP1 and will aid in exploring the underlying mechanisms of CbuqSNMP1b functions in the future.

Key amino acids in odorant-binding protein OBP7 enable Bradysia odoriphaga to recognize host plant volatiles

Bradysia odoriphaga (Diptera: Sciaridae) is a devastating underground pest that can cause serious economic losses. Odorant binding proteins (OBPs) are crucial components of the insect olfactory system, playing key roles in locating host plants, oviposition sites, and mates. Therefore, they are considered potential targets for pest control. Here, we obtained one OBP gene (BodoOBP7) from the antennal transcriptome of B. odoriphaga, and observed that the expression level of BodoOBP7 was primarily in the antennae of both sexes, with significantly higher expression level in females than in males. Fluorescence competitive binding assays indicated that BodoOBP7 exhibited strong binding affinities for the six host plant volatiles, including propyl disulfide, dipropyl trisulfide, dimethyl trisulfide, 2-tridecanone, 2-undecanone and alpha-ionone. Subsequently, homology modeling, molecular docking and site-directed mutagenesis revealed that four key amino acid residues (Phe79, Phe99, Ile96, Leu100) participate in the binding of BodoOBP7 with six host plant volatiles. Our results demonstrate that BodoOBP7 is involved in olfactory recognition in B. odoriphaga. These findings may enhance our understanding of the interaction mechanisms between host plants and B. odoriphaga, potentially offering new perspectives for the development of effective green control strategies.

Identification of miRNAs Involved in Olfactory Regulation in Antennae of Beet Webworm, Loxostege sticticalis (Lepidoptera: Pyralidae).

The beet webworm, Loxostege sticticalis, is a typical migratory pest. Although miRNAs participate in many physiological functions, little is known about the functions of miRNAs in olfactory regulation. In this study, 1120 (869 known and 251 novel) miRNAs were identified in the antennae of L. sticticalis by using high-throughput sequencing technology. Among the known miRNAs, 189 from 49 families were insect-specific, indicating that these miRNAs might play unique roles in insects. Furthermore, based on the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, we found that 3647 and 1393 miRNAs were associated with localization and the regulation of localization, respectively, and 80 miRNAs were enriched in the neuroactive ligand-receptor interaction pathway. These miRNAs might be involved in the olfactory system of L. sticticalis. Notably, qRT-PCR showed that most of the tested miRNAs presented similar expression patterns compared with the RNA-seq data and that miR-87-3, novel-miR-78, and novel-miR-142 were significantly differentially expressed in the antennae of males and females. In addition, 21 miRNAs were predicted to target 23 olfactory genes, including 10 odorant-binding proteins (OBPs), 3 chemosensory proteins (CSPs), 4 odorant receptors (ORs), 1 ionotropic receptor (IR), and 5 gustatory receptors (GRs). The olfactory-related miRNAs exhibited low-abundance transcripts, except undef-miR-55 and undef-miR-523, and gender-biased expression was not observed for olfactory-related miRNAs. Our findings provide an overview of the potential miRNAs involved in olfactory regulation, which may provide important information on the function of miRNAs in the insect olfactory system.

Rhythms in insect olfactory systems: underlying mechanisms and outstanding questions.

Olfaction is a critical sensory modality for invertebrates, and it mediates a wide range of behaviors and physiological processes. Like most living organisms, insects live in rhythmic environments: the succession of nights and days is accompanied by cyclic variations in light intensity and temperature, as well as in the availability of resources and the activity of predators. Responding to olfactory cues in the proper temporal context is thus highly adaptive and allows for the efficient allocation of energy resources. Given the agricultural or epidemiological importance of some insect species, understanding olfactory rhythms is critical for the development of effective control strategies. Although the vinegar fly Drosophila melanogaster has been a classical model for the study of olfaction and circadian rhythms, recent studies focusing on non-model species have expanded our understanding of insect olfactory rhythms. Additionally, recent evidence revealing receptor co-expression by sensory neurons has brought about an ongoing paradigm shift in our understanding of insect olfaction, making it timely to review the state of our knowledge on olfactory rhythms and identify critical future directions for the field. In this Review, we discuss the multiple biological scales at which insect olfactory rhythms are being analyzed, and identify outstanding questions.

Odorant receptors tuned to isothiocyanates in Drosophila melanogaster and their evolutionary expansion in herbivorous relatives.

Plants release complex volatile compounds to attract mutualists, deter herbivores, and deceive pollinators. Here, we used herbivorous specialist flies that feed on mustard plants ( Scaptomyza spp.) and microbe-feeding species ( Drosophila melanogaster and Scaptomyza spp.) to investigate how plant-derived electrophilic toxins such as isothiocyanates (ITCs) affect insects, and how flies detect these compounds through olfaction. In survival assays, D. melanogaster exposed to volatile allyl isothiocyanate (AITC), a toxin derived from many Brassicales plants, were acutely intoxicated, demonstrating the high toxicity of this volatile compound to non-specialized insects. Through single sensillum recordings (SSR) from olfactory organs and behavioral assays, we found that the Odorant receptor 42a (Or42a) is necessary for AITC detection and behavioral aversion. Comparative transcriptome and RNA FISH studies across the drosophilid genus Scaptomyza revealed lineage-specific triplication of Or42a in the Brassicales specialists and a doubling of Or42a -positive-olfactory sensory neurons. Heterologous expression experiments showed that Or42a paralogs in Brassicales-specialists exhibited broadened sensitivity to ITCs in a paralog-specific manner. Finally, AlphaFold2 modeling followed by site-directed mutagenesis and SSR identified two critical amino acid substitutions that conferred Or42a heighten sensitivity to Brassicales-derived ITCs. Our findings suggest that ITCs, which are toxic to most insects, can be detected and avoided by non-specialists like D. melanogaster through olfaction. In Brassicales specialists, these same Ors experienced gene duplication events that resulted in an expanded sensitivity to ITC compounds. Thus, the insect's olfactory system can rapidly adapt to toxic ecological niches provided by chemically-defended host plants through co-option of chemosensory capabilities already present in their ancestors.

Two Structural Analogs of Kairomones are Detected by an Odorant Receptor HvarOR28 in the Coccinellid Hippodamia variegata.

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.

Molecular Characterization of Chemosensory Protein (CSP) Genes and the Involvement of AgifCSP5 in the Perception of Host Location in the Aphid Parasitoid Aphidius gifuensis.

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.

Molecular Characterization of Plant Volatile Compound Interactions with Cnaphalocrocis medinalis Odorant-Binding Proteins.

Odorant-binding proteins (OBPs) play important roles in the insect olfactory system since they bind external odor molecules to trigger insect olfactory responses. Previous studies have identified some plant-derived volatiles that attract the pervasive insect pest Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), such as phenylacetaldehyde, benzyl acetate, 1-heptanol, and hexanal. To characterize the roles of CmedOBPs in the recognition of these four volatiles, we analyzed the binding abilities of selected CmedOBPs to each of the four compounds, as well as the expression patterns of CmedOBPs in different developmental stages of C. medinalis adult. Antennaes of C. medinalis adults were sensitive to the studied plant volatile combinations. Expression levels of multiple CmedOBPs were significantly increased in the antennae of 2-day-old adults after exposure to volatiles. CmedOBP1, CmedOBP6, CmedPBP1, CmedPBP2, and CmedGOBP2 were significantly up-regulated in the antennae of volatile-stimulated female and male adults when compared to untreated controls. Fluorescence competition assays confirmed that CmedOBP1 could strongly bind 1-heptanol, hexanal, and phenylacetaldehyde; CmedOBP15 strongly bound benzyl acetate and phenylacetaldehyde; and CmedOBP26 could weakly bind 1-heptanol. This study lays a theoretical foundation for further analysis of the mechanisms by which plant volatiles can attract C. medinalis. It also provides a technical basis for the future development of efficient plant volatile attractants of C. medinalis.

Associative Learning of Quantitative Mechanosensory Stimuli in Honeybees.

The proboscis extension response (PER) has been widely used to evaluate honeybees' (Apis mellifera) learning and memory abilities, typically by using odors and visual cues for the conditioned stimuli. Here we asked whether honeybees could learn to distinguish between different magnitudes of the same type of stimulus, given as two speeds of air flux. By taking advantage of a novel automated system for administering PER experiments, we determined that the bees were highly successful when the lower air flux was rewarded and less successful when the higher flux was rewarded. Importantly, since our method includes AI-assisted analysis, we were able to consider subthreshold responses at a high temporal resolution; this analysis revealed patterns of rapid generalization and slowly acquired discrimination between the rewarded and unrewarded stimuli, as well as indications that the high air flux may have been mildly aversive. The learning curve for these mechanosensory stimuli, at least when the lower flux is rewarded, more closely mimics prior data from olfactory PER studies rather than visual ones, possibly in agreement with recent findings that the insect olfactory system is also sensitive to mechanosensory information. This work demonstrates a new modality to be used in PER experiments and lays the foundation for deeper exploration of honeybee cognitive processes when posed with complex learning challenges.

Augmenting insect olfaction performance through nano-neuromodulation.

Biological olfactory systems are highly sensitive and selective, often outperforming engineered chemical sensors in highly complex and dynamic environments. As a result, there is much interest in using biological systems to build sensors. However, approaches to read-out information from biological systems, especially neural signals, tend to be suboptimal due to the number of electrodes that can be used and where these can be placed. Here we aim to overcome this suboptimality in neural information read-out by using a nano-enabled neuromodulation strategy to augment insect olfaction-based chemical sensors. By harnessing the photothermal properties of nanostructures and releasing a select neuromodulator on demand, we show that the odour-evoked response from the interrogated regions of the insect olfactory system can not only be enhanced but can also improve odour identification.

Genome-Wide Identification of the Genes of the Odorant-Binding Protein Family Reveal Their Role in the Olfactory Response of the Tomato Leaf Miner (Tuta absoluta) to a Repellent Plant

The remarkable biological and evolutionary adaptations of insects to plants are largely attributed to the powerful chemosensory systems of insects. The tomato leaf miner (Tuta absoluta) is a destructive invasive pest with a global distribution that poses a serious threat to the production of nightshade crops, especially tomatoes. Functional plants can attract or repel insect pests by releasing volatiles that interact with the olfactory system of insects, thereby reducing the damage of insect pests to target crops. However, there is limited research on the interaction between T. absoluta olfactory genes and functional plants. In this study, 97 members of the putative odorant-binding protein (OBP) family have been identified in the whole genome of T. absoluta. Phylogenetic analysis involving various Lepidopteran and Dipteran species, including D. melanogaster, revealed that OBP gene families present conserved clustering patterns. Furthermore, the Plus-C subfamily of OBP showed extremely significant expansion. Moreover, the expression levels of the OBP genes varied significantly between different developmental stages; that is, the highest number of OBP genes were expressed in the adult stage, followed by the larval stage, and fewer genes were expressed in high abundance in the egg stage. On the other hand, through a Y-tube olfactometer, we identified a functional plant—Plectranthus tomentosa—that significantly repels adult and larval T. absoluta. Finally, we screened the OBP genes in response to tomato and P. tomentosa volatiles at the genomic level of T. absoluta using RT-qPCR. These results laid a good foundation for controlling T. absoluta with functional plants and further studying olfactory genes.

Females smell differently: characteristics and significance of the most common olfactory sensilla of female silkmoths.

In the silkmoth Bombyx mori, the role of male sensilla trichodea in pheromone detection is well established. Here we study the corresponding female sensilla, which contain two olfactory sensory neurons (OSNs) and come in two lengths, each representing a single physiological type. Only OSNs in medium trichoids respond to the scent of mulberry, the silkworm's exclusive host plant, and are more sensitive in mated females, suggesting a role in oviposition. In long trichoids, one OSN is tuned to (+)-linalool and the other to benzaldehyde and isovaleric acid, both odours emitted by silkworm faeces. While the significance of (+)-linalool detection remains unclear, isovaleric acid repels mated females and may therefore play a role in avoiding crowded oviposition sites. When we examined the underlying molecular components of neurons in female trichoids, we found non-canonical co-expression of Ir8a, the co-receptor for acid responses, and ORco, the co-receptor of odorant receptors, in long trichoids, and the unexpected expression of a specific odorant receptor in both trichoid sensillum types. In addition to elucidating the function of female trichoids, our results suggest that some accepted organizational principles of the insect olfactory system may not apply to the predominant sensilla on the antenna of female B. mori.

The Plant Volatile-Sensing Mechanism of Insects and Its Utilization.

Plants and insects are engaged in a tight relationship, with phytophagous insects often utilizing volatile organic substances released by host plants to find food and egg-laying sites. Using plant volatiles as attractants for integrated pest management is vital due to its high efficacy and low environmental toxicity. Using naturally occurring plant volatiles combined with insect olfactory mechanisms to select volatile molecules for screening has proved an effective method for developing plant volatile-based attractant technologies. However, the widespread adoption of this technique is still limited by the lack of a complete understanding of molecular insect olfactory pathways. This paper first describes the nature of plant volatiles and the mechanisms of plant volatile perception by insects. Then, the attraction mechanism of plant volatiles to insects is introduced with the example of Cnaphalocrocis medinalis. Next, the progress of the development and utilization of plant volatiles to manage pests is presented. Finally, the functions played by the olfactory system of insects in recognizing plant volatiles and the application prospects of utilizing volatiles for green pest control are discussed. Understanding the sensing mechanism of insects to plant volatiles and its utilization will be critical for pest management in agriculture.

Non-canonical odor representation and learning in Dipteran brains

For decades, the vinegar fly Drosophila melanogaster has provided a window into the structure and function of the insect olfactory system, revealing a remarkable organizational correspondence between insects and vertebrates. In both clades, olfactory sensory neurons expressing the same class of sensory receptor proteins exclusively target a dedicated neuropil known as a glomerulus. Here, we review recent evidence from Drosophila and other Dipteran taxa that challenges this canonical view, showing widespread co-expression of olfactory sensory proteins within the same neurons. We discuss the potential consequences of co-expression for early sensory processing in the insect olfactory system. As well, we situate these findings within the broader framework of olfactory learning, highlighting recent findings that suggest a wider importance of the antennal lobe than has been previously appreciated.

The olfactory system of Pieris brassicae caterpillars: from receptors to glomeruli.

The olfactory system of adult lepidopterans is among the best described neuronal circuits. However, comparatively little is known about the organization of the olfactory system in the larval stage of these insects. Here, we explore the expression of olfactory receptors and the organization of olfactory sensory neurons in caterpillars of Pieris brassicae, a significant pest species in Europe and a well-studied species for its chemical ecology. To describe the larval olfactory system in this species, we first analyzed the head transcriptome of third-instar larvae (L3) and identified 16 odorant receptors (ORs) including the OR coreceptor (Orco), 13 ionotropic receptors (IRs), and 8 gustatory receptors (GRs). We then quantified the expression of these 16 ORs in different life stages, using qPCR, and found that the majority of ORs had significantly higher expression in the L4 stage than in the L3 and L5 stages, indicating that the larval olfactory system is not static throughout caterpillar development. Using an Orco-specific antibody, we identified all olfactory receptor neurons (ORNs) expressing the Orco protein in L3, L4, and L5 caterpillars and found a total of 34 Orco-positive ORNs, distributed among three sensilla on the antenna. The number of Orco-positive ORNs did not differ among the three larval instars. Finally, we used retrograde axon tracing of the antennal nerve and identified a mean of 15 glomeruli in the larval antennal center (LAC), suggesting that the caterpillar olfactory system follows a similar design as the adult olfactory system, although with a lower numerical redundancy. Taken together, our results provide a detailed analysis of the larval olfactory neurons in P. brassicae, highlighting both the differences as well as the commonalities with the adult olfactory system. These findings contribute to a better understanding of the development of the olfactory system in insects and its life-stage-specific adaptations.

The Orco gene involved in recognition of host plant volatiles and sex pheromone in the chive maggot Bradysia odoriphaga

The insect olfactory recognition system plays a crucial role in the feeding and reproductive behaviors of insects. The odorant receptor co-receptor (Orco), as an obligatory chaperone, is critical for odorant recognition by way of forming heteromeric complexes with conventional odorant receptors (ORs). To investigate the biological functions of Orco in perceiving host plant volatiles and sex pheromone, the Orco gene was identified from the chive maggot Bradysia odoriphaga transcriptome data. Multiple sequence alignment reveals that BodoOrco exhibits an extremely high sequence identity with Orcos from other dipteran insects. The expression of BodoOrco is significantly higher in adults than in larvae and pupae, and the BodoOrco gene is primarily expressed in the antennae of both sexes. Furthermore, the Y-tube assay indicated that knockdown of BodoOrco leads to significant reductions in B. odoriphaga adults' response to all tested host plant volatiles. The dsOrco-treated unmated male adults show less attraction to unmated females and responded slowly compared with dsGFP control group. These results indicated that BodoOrco is involved in recognition of sex pheromone and host plant volatiles in B. odoriphaga and has the potential to be used as a target for the design of novel active compounds for developing ecofriendly pest control strategies.

Transcription factor CncC regulates the expression of antennal CYP6MU1 gene responsible for trans-2-hexen-1-al and nonanal recognition in Locusta migratoria

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.

Molecular analysis of NlugCSP3 with behaviorally active ligands of the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)

The insect olfactory system is sensitive to the complicated chemical environment. Insect’s chemosensory proteins (CSPs) supposedly act as transport of plant volatiles across the sensillar lymph and competitive fluorescent binding assay was commonly used to test the binding affinities with various plant volatiles. However, extensive research to determine the physiological role of CSPs is necessary and helpful through binding interaction of a protein with the plant volatiles. In this comparative study, we employed phylogenetic analysis, fluorescence spectra, quenching mode, and thermodynamic force to characterize Nilaparvata lugens CSP3 (NlugCSP3). The phylogenetic tree revealed that amino acid sequence of NlugCSP3 showed extremely close similarities with Laodelphax striatella (LstrCSP10 & LstrCSP11) and Sogatella furcifera (SurCSP3). The Stern-Volmer (SV) curve of NlugCSP3 fluorescence quenching indicated that nonadecane and 2-tridecanone clearly quenched NlugCSP3 fluorescence as a stable static quenching mode. Meanwhile, α-terpinene and farnesene collided with NlugCSP3, instead of forming stable complexes. The thermodynamic analysis of NlugCSP3 revealed that spontaneous binding interaction occurred in nonadecane and 2-tridecanone and is driven primarily by hydrophobic interactions. This study not only provides the information about the binding interaction of protein with the volatiles, but also improves the efficient recognition of behaviorally elicited volatiles that could be used in the management of brown planthopper.

Gut microbiota influences feeding behavior via changes in olfactory receptor gene expression in Colorado potato beetles.

The Colorado potato beetle (CPB) is an internationally recognized plant quarantine pest that causes serious losses to potato agricultural production. The gut microbiota plays an important role in its growth and development, and the olfactory system plays an important role in insect feeding behavior. The gut microbiota is known to be capable of inducing changes in the olfactory systems of insects. However, the way these associated gut microbes influence the feeding-related behaviors of CPBs remains unclear. To explore the relationship between them, fresh potato leaves immersed in a mixture of five antibiotics (tetracycline, penicillin, ofloxacin, ciprofloxacin, and ampicillin) at specific concentrations for 1 h were fed to adult CPBs to reduce the abundance of gut microbes. We found that the feeding behavior of CPBs was significantly affected by the gut microbiota and that Pseudomonas was significantly higher in abundance in the control group than in the antibiotic group. We then used transcriptome sequencing to explore the differences in olfactory receptor genes in the heads of non-treatment and antibiotic-fed CPBs. Through Illumina Hiseq™ sequencing and screening of differential genes, we found that the olfactory receptor gene LdecOR9 was significantly upregulated and LdecOR17 was significantly downregulated after antibiotic feeding. A real-time polymerase chain reaction was used to verify the changes in olfactory receptor gene expression in the non-treatment groups and antibiotic-treated groups. The feeding behavior was partially rescued after CPBs were re-fed with intestinal bacteria. These results indicate that a certain amount of gut microbiota can result in the loss of the olfactory discrimination ability of CPBs to host plants. In summary, this study investigated the relationship between gut microbiota and olfactory genes, providing a reference for research on microbial control.