Carotid Body Function in Tyrosine Hydroxylase Conditional Olfr78 Knockout Mice.

The Olfr78 gene encodes a G-protein-coupled olfactory receptor that is expressed in several ectopic sites. Olfr78 is one of the most abundant mRNA species in carotid body (CB) glomus cells. These cells are the prototypical oxygen (O2) sensitive arterial chemoreceptors, which, in response to lowered O2 tension (hypoxia), activate the respiratory centers to induce hyperventilation. It has been proposed that Olfr78 is a lactate receptor and that glomus cell activation by the increase in blood lactate mediates the hypoxic ventilatory response (HVR). However, this proposal has been challenged by several groups showing that Olfr78 is not a physiologically relevant lactate receptor and that the O2-based regulation of breathing is not affected in constitutive Olfr78 knockout mice. In another study, constitutive Olfr78 knockout mice were reported to have altered systemic and CB responses to mild hypoxia. To further characterize the functional role of Olfr78 in CB glomus cells, we here generated a conditional Olfr78 knockout mouse strain and then restricted the knockout to glomus cells and other catecholaminergic cells by crossing with a tyrosine hydroxylase-specific Cre driver strain (TH-Olfr78 KO mice). We find that TH-Olfr78 KO mice have a normal HVR. Interestingly, glomus cells of TH-Olfr78 KO mice exhibit molecular and electrophysiological alterations as well as a reduced dopamine content in secretory vesicles and neurosecretory activity. These functional characteristics resemble those of CB neuroblasts in wild-type mice. We suggest that, although Olfr78 is not essential for CB O2 sensing, activation of Olfr78-dependent pathways is required for maturation of glomus cells.

Establishment and maintenance of random monoallelic expression.

This Review elucidates the regulatory principles of random monoallelic expression by focusing on two well-studied examples: the X-chromosome inactivation regulator Xist and the olfactory receptor gene family. Although the choice of a single X chromosome or olfactory receptor occurs in different developmental contexts, common gene regulatory principles guide monoallelic expression in both systems. In both cases, an event breaks the symmetry between genetically and epigenetically identical copies of the gene, leading to the expression of one single random allele, stabilized through negative feedback control. Although many regulatory steps that govern the establishment and maintenance of monoallelic expression have been identified, key pieces of the puzzle are still missing. We provide an overview of the current knowledge and models for the monoallelic expression of Xist and olfactory receptors. We discuss their similarities and differences, and highlight open questions and approaches that could guide the study of other monoallelically expressed genes.

Candidate membrane protein gene families related to chemoreception in a wood-boring beetle, Pharsalia antennata Gahan (Coleoptera: Cerambycidae).

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.

NAD activates olfactory receptor 1386 to regulate type I interferon responses in Plasmodium yoelii YM infection.

Olfactory receptors (Olfr) are G protein-coupled receptors that are normally expressed on olfactory sensory neurons to detect volatile chemicals or odorants. Interestingly, many Olfrs are also expressed in diverse tissues and function in cell-cell recognition, migration, and proliferation as well as immune responses and disease processes. Here, we showed that many Olfr genes were expressed in the mouse spleen, linked to Plasmodium yoelii genetic loci significantly, and/or had genome-wide patterns of LOD scores (GPLSs) similar to those of host Toll-like receptor genes. Expression of specific Olfr genes such as Olfr1386 in HEK293T cells significantly increased luciferase signals driven by IFN-ß and NF-κB promoters, with elevated levels of phosphorylated TBK1, IRF3, P38, and JNK. Mice without Olfr1386 were generated using the CRISPR/Cas9 method, and the Olfr1386-/- mice showed significantly lower IFN-α/ß levels and longer survival than wild-type (WT) littermates after infection with P. yoelii YM parasites. Inhibition of G protein signaling and P38 activity could affect cyclic AMP-responsive element promoter-driven luciferase signals and IFN-ß mRNA levels in HEK293T cells expressing the Olfr1386 gene, respectively. Screening of malaria parasite metabolites identified nicotinamide adenine dinucleotide (NAD) as a potential ligand for Olfr1386, and NAD could stimulate IFN-ß responses and phosphorylation of TBK1 and STAT1/2 in RAW264.7 cells. Additionally, parasite RNA (pRNA) could significantly increase Olfr1386 mRNA levels. This study links multiple Olfrs to host immune response pathways, identifies a candidate ligand for Olfr1386, and demonstrates the important roles of Olfr1386 in regulating type I interferon (IFN-I) responses during malaria parasite infections.

A pattern recognition artificial olfactory system based on human olfactory receptors and organic synaptic devices.

Neuromorphic sensors, designed to emulate natural sensory systems, hold the promise of revolutionizing data extraction by facilitating rapid and energy-efficient analysis of extensive datasets. However, a challenge lies in accurately distinguishing specific analytes within mixtures of chemically similar compounds using existing neuromorphic chemical sensors. In this study, we present an artificial olfactory system (AOS), developed through the integration of human olfactory receptors (hORs) and artificial synapses. This AOS is engineered by interfacing an hOR-functionalized extended gate with an organic synaptic device. The AOS generates distinct patterns for odorants and mixtures thereof, at the molecular chain length level, attributed to specific hOR-odorant binding affinities. This approach enables precise pattern recognition via training and inference simulations. These findings establish a foundation for the development of high-performance sensor platforms and artificial sensory systems, which are ideal for applications in wearable and implantable devices.

Genomic identification and evolutionary analysis of chemosensory receptor gene families in two Phthorimaea pest species: insights into chemical ecology and host adaptation.

BACKGROUND: Insects rely on sophisticated sensitive chemosensory systems to sense their complex chemical environment. This sensory process involves a combination of odorant receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs) in the chemosensory system. This study focused on the identification and characterization of these three types of chemosensory receptor genes in two closely related Phthorimaea pest species, Phthorimaea operculella (potato tuber moth) and Phthorimaea absoluta (tomato leaf miner). RESULTS: Based on manual annotation of the genome, we identified a total of 349 chemoreceptor genes from the genome of P. operculella, including 93 OR, 206 GR and 50 IR genes, while for P. absoluta, we identified 72 OR, 122 GR and 46 IR genes. Through phylogenetic analysis, we observed minimal differences in the number and types of ORs and IRs between the potato tuber moth and tomato leaf miner. In addition, we found that compared with those of tomato leaf miners, the gustatory receptor branch of P. operculella has undergone a large expansion, which may be related to P. absoluta having a narrower host range than P. operculella. Through analysis of differentially expressed genes (DEGs) of male and female antennae, we uncovered 45 DEGs (including 32ORs, 9 GRs, and 4 IRs). CONCLUSIONS: Our research provides a foundation for exploring the chemical ecology of these two pests and offers new insights into the dietary differentiation of lepidopteran insects, while simultaneously providing molecular targets for developing environmentally friendly pest control methods based on insect chemoreception.

Deorphanizing an odorant receptor tuned to palm tree volatile esters in the Asian palm weevil sheds light on the mechanisms of palm tree selection.

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.

Odorant receptors for floral- and plant-derived volatiles in the yellow fever mosquito, Aedes aegypti (Diptera: Culicidae).

Adult mosquitoes require regular sugar meals, including nectar, to survive in natural habitats. Both males and females locate potential sugar sources using sensory proteins called odorant receptors (ORs) activated by plant volatiles to orient toward flowers or honeydew. The yellow fever mosquito, Aedes aegypti (Linnaeus, 1762), possesses a large gene family of ORs, many of which are likely to detect floral odors. In this study, we have uncovered ligand-receptor pairings for a suite of Aedes aegypti ORs using a panel of environmentally relevant, plant-derived volatile chemicals and a heterologous expression system. Our results support the hypothesis that these odors mediate sensory responses to floral odors in the mosquito's central nervous system, thereby influencing appetitive or aversive behaviors. Further, these ORs are well conserved in other mosquitoes, suggesting they function similarly in diverse species. This information can be used to assess mosquito foraging behavior and develop novel control strategies, especially those that incorporate mosquito bait-and-kill technologies.

Spontaneous differentiation of human induced pluripotent stem cells to odorant-responsive olfactory sensory neurons.

Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells (iPSCs), can differentiate into almost all cell types and are anticipated to have significant applications in the field of regenerative medicine. However, there are no reports of successfully directing iPSCs to become functional olfactory sensory neurons (OSNs) capable of selectively receiving odorant compounds. In this study, we employed dual SMAD inhibition and fibroblast growth factor 8 (FGF-8, reported to dictate olfactory fates) along with N-2 and B-27 supplements in the culture medium to efficiently induce the differentiation of iPSCs into neuronal cells with olfactory function through olfactory placode. Temporal gene expression and expression of OSN-specific markers during differentiation indicated that the expression of olfactory marker proteins and various olfactory receptors (ORs), which are markers of mature OSNs, was observed after approximately one month of differentiation culture, irrespective of the differentiation cues, suggesting differentiation into OSNs. Cells that exhibited specific responses to odorant compounds were identified after administering odorant compounds to differentiated iPSC-derived OSNs. This suggests the spontaneous generation of functional OSNs expressing diverse ORs that respond to odorant compounds from iPSCs.

Synchronized Expansion and Contraction of Olfactory, Vomeronasal, and Taste Receptor Gene Families in Hystricomorph Rodents.

Chemical senses, including olfaction, pheromones, and taste, are crucial for the survival of most animals. There has long been a debate about whether different types of senses might influence each other. For instance, primates with a strong sense of vision are thought to have weakened olfactory abilities, although the oversimplified trade-off theory is now being questioned. It is uncertain whether such interactions between different chemical senses occur during evolution. To address this question, we examined four receptor gene families related to olfaction, pheromones, and taste: olfactory receptor (OR), vomeronasal receptor type 1 and type 2 (V1R and V2R), and bitter taste receptor (T2R) genes in Hystricomorpha, which is morphologically and ecologically the most diverse group of rodents. We also sequenced and assembled the genome of the grasscutter, Thryonomys swinderianus. By examining 16 available genome assemblies alongside the grasscutter genome, we identified orthologous gene groups among hystricomorph rodents for these gene families to separate the gene gain and loss events in each phylogenetic branch of the Hystricomorpha evolutionary tree. Our analysis revealed that the expansion or contraction of the four gene families occurred synchronously, indicating that when one chemical sense develops or deteriorates, the others follow suit. The results also showed that V1R/V2R genes underwent the fastest evolution, followed by OR genes, and T2R genes were the most evolutionarily stable. This variation likely reflects the difference in ligands of V1R/V2Rs, ORs, and T2Rs: species-specific pheromones, environment-based scents, and toxic substances common to many animals, respectively.

CD20/MS4A1 is a mammalian olfactory receptor expressed in a subset of olfactory sensory neurons that mediates innate avoidance of predators.

The mammalian olfactory system detects and discriminates between millions of odorants to elicit appropriate behavioral responses. While much has been learned about how olfactory sensory neurons detect odorants and signal their presence, how specific innate, unlearned behaviors are initiated in response to ethologically relevant odors remains poorly understood. Here, we show that the 4-transmembrane protein CD20, also known as MS4A1, is expressed in a previously uncharacterized subpopulation of olfactory sensory neurons in the main olfactory epithelium of the murine nasal cavity and functions as a mammalian olfactory receptor that recognizes compounds produced by mouse predators. While wildtype mice avoid these predator odorants, mice genetically deleted of CD20 do not appropriately respond. Together, this work reveals a CD20-mediated odor-sensing mechanism in the mammalian olfactory system that triggers innate behaviors critical for organismal survival.

Structural basis of ligand specificity and channel activation in an insect gustatory receptor.

Gustatory receptors (GRs) are critical for insect chemosensation and are potential targets for controlling pests and disease vectors, making their structural investigation a vital step toward such applications. We present structures of Bombyx mori Gr9 (BmGr9), a fructose-gated cation channel, in agonist-free and fructose-bound states. BmGr9 forms a tetramer similar to distantly related insect odorant receptors (ORs). Upon fructose binding, BmGr9's channel gate opens through helix S7b movements. In contrast to ORs, BmGr9's ligand-binding pocket, shaped by a kinked helix S4 and a shorter extracellular S3-S4 loop, is larger and solvent accessible in both agonist-free and fructose-bound states. Also, unlike ORs, fructose binding by BmGr9 involves helix S5 and a pocket lined with aromatic and polar residues. Structure-based sequence alignments reveal distinct patterns of ligand-binding pocket residue conservation in GR subfamilies associated with different ligand classes. These data provide insight into the molecular basis of GR ligand specificity and function.

The Olfactory Trail of Neurodegenerative Diseases.

Alterations in olfactory functions are proposed as possible early biomarkers of neurodegenerative diseases. Parkinson's and Alzheimer's diseases manifest olfactory dysfunction as a symptom, which is worth mentioning. The alterations do not occur in all patients, but they can serve to rule out neurodegenerative pathologies that are not associated with small deficits. Several prevalent neurodegenerative conditions, including impaired smell, arise in the early stages of Parkinson's and Alzheimer's diseases, presenting an attractive prospect as a snitch for early diagnosis. This review covers the current knowledge on the link between olfactory deficits and Parkinson's and Alzheimer's diseases. The review also covers the emergence of olfactory receptors as actors in the pathophysiology of these diseases. Olfactory receptors are not exclusively expressed in olfactory sensory neurons. Olfactory receptors are widespread in the human body; they are expressed, among others, in the testicles, lungs, intestines, kidneys, skin, heart, and blood cells. Although information on these ectopically expressed olfactory receptors is limited, they appear to be involved in cell recognition, migration, proliferation, wound healing, apoptosis, and exocytosis. Regarding expression in non-chemosensory regions of the central nervous system (CNS), future research should address the role, in both the glia and neurons, of olfactory receptors. Here, we review the limited but relevant information on the altered expression of olfactory receptor genes in Parkinson's and Alzheimer's diseases. By unraveling how olfactory receptor activation is involved in neurodegeneration and identifying links between olfactory structures and neuronal death, valuable information could be gained for early diagnosis and intervention strategies in neurodegenerative diseases.

A cilia-bound unconventional secretory pathway for Drosophila odorant receptors.

BACKGROUND: Post-translational transport is a vital process which ensures that each protein reaches its site of function. Though most do so via an ordered ER-to-Golgi route, an increasing number of proteins are now shown to bypass this conventional secretory pathway. RESULTS: In the Drosophila olfactory sensory neurons (OSNs), odorant receptors (ORs) are trafficked from the ER towards the cilia. Here, we show that Or22a, a receptor of various esters and alcoholic compounds, reaches the cilia partially through unconventional means. Or22a frequently present as puncta at the somatic cell body exit and within the dendrite prior to the cilia base. These rarely coincide with markers of either the intermediary ER-Golgi-intermediate-compartment (ERGIC) or Golgi structures. ERGIC and Golgi also displayed axonal localization biases, a further indication that at least some measure of OR transport may occur independently of their involvement. Additionally, neither the loss of several COPII genes involved in anterograde trafficking nor ERGIC itself affected puncta formation or Or22a transport to the cilium. Instead, we observed the consistent colocalization of Or22a puncta with Grasp65, the sole Drosophila homolog of mammalian GRASP55/Grh1, a marker of the unconventional pathway. The numbers of both Or22a and Grasp65-positive puncta were furthermore increased upon nutritional starvation, a condition known to enhance Golgi-bypassing secretory activity. CONCLUSIONS: Our results demonstrate an alternative route of Or22a transport, thus expanding the repertoire of unconventional secretion mechanisms in neurons.

Localized Expression of Olfactory Receptor Genes in the Olfactory Organ of Common Minke Whales.

Baleen whales (Mysticeti) possess the necessary anatomical structures and genetic elements for olfaction. Nevertheless, the olfactory receptor gene (OR) repertoire has undergone substantial degeneration in the cetacean lineage following the divergence of the Artiodactyla and Cetacea. The functionality of highly degenerated mysticete ORs within their olfactory epithelium remains unknown. In this study, we extracted total RNA from the nasal mucosae of common minke whales (Balaenoptera acutorostrata) to investigate ORs' localized expression. All three sections of the mucosae examined in the nasal chamber displayed comparable histological structure. However, the posterior portion of the frontoturbinal region exhibited notably high OR expression. Neither the olfactory bulb nor the external skin exhibited the expression of these genes. Although this species possesses four intact non-class-2 ORs, all the ORs expressed in the nasal mucosae belong to class-2, implying the loss of aversion to specific odorants. These anatomical and genomic analyses suggest that ORs are still responsible for olfaction within the nasal region of baleen whales, enabling them to detect desirable scents such as prey and potential mating partners.

Activity-dependent formation of the topographic map and the critical period in the development of mammalian olfactory system.

Neural activity influences every aspect of nervous system development. In olfactory systems, sensory neurons expressing the same odorant receptor project their axons to stereotypically positioned glomeruli, forming a spatial map of odorant receptors in the olfactory bulb. As individual odors activate unique combinations of glomeruli, this map forms the basis for encoding olfactory information. The establishment of this stereotypical olfactory map requires coordinated regulation of axon guidance molecules instructed by spontaneous activity. Recent studies show that sensory experiences also modify innervation patterns in the olfactory bulb, especially during a critical period of the olfactory system development. This review examines evidence in the field to suggest potential mechanisms by which various aspects of neural activity regulate axon targeting. We also discuss the precise functions served by neural plasticity during the critical period.

Epigenetic programming of stochastic olfactory receptor choice.

The mammalian sense of smell relies upon a vast array of receptor proteins to detect odorant compounds present in the environment. The proper deployment of these receptor proteins in olfactory sensory neurons is orchestrated by a suite of epigenetic processes that remodel the olfactory genes in differentiating neuronal progenitors. The goal of this review is to elucidate the central role of gene regulatory processes acting in neuronal progenitors of olfactory sensory neurons that lead to a singular expression of an odorant receptor in mature olfactory sensory neurons. We begin by describing the principal features of odorant receptor gene expression in mature olfactory sensory neurons. Next, we delineate our current understanding of how these features emerge from multiple gene regulatory mechanisms acting in neuronal progenitors. Finally, we close by discussing the key gaps in our understanding of how these regulatory mechanisms work and how they interact with each other over the course of differentiation.

Odorant Receptors Expressing and Antennal Lobes Architecture Are Linked to Caste Dimorphism in Asian Honeybee, Apis cerana (Hymenoptera: Apidae).

Insects heavily rely on the olfactory system for food, mating, and predator evasion. However, the caste-related olfactory differences in Apis cerana, a eusocial insect, remain unclear. To explore the peripheral and primary center of the olfactory system link to the caste dimorphism in A. cerana, transcriptome and immunohistochemistry studies on the odorant receptors (ORs) and architecture of antennal lobes (ALs) were performed on different castes. Through transcriptomesis, we found more olfactory receptor genes in queens and workers than in drones, which were further validated by RT-qPCR, indicating caste dimorphism. Meanwhile, ALs structure, including volume, surface area, and the number of glomeruli, demonstrated a close association with caste dimorphism. Particularly, drones had more macroglomeruli possibly for pheromone recognition. Interestingly, we found that the number of ORs and glomeruli ratio was nearly 1:1. Also, the ORs expression distribution pattern was very similar to the distribution of glomeruli volume. Our results suggest the existence of concurrent plasticity in both the peripheral olfactory system and ALs among different castes of A. cerana, highlighting the role of the olfactory system in labor division in insects.

Odorant Receptor BdorOR49b Mediates Oviposition and Attraction Behavior of Bactrocera dorsalis to Benzothiazole.

The ability to recognize a host plant is crucial for insects to meet their nutritional needs and locate suitable sites for laying eggs. Bactrocera dorsalis is a highly destructive pest in fruit crops. Benzothiazole has been found to induce oviposition behavior in the gravid B. dorsalis. However, the ecological roles and the olfactory receptor responsible for benzothiazole are not yet fully understood. In this study, we found that adults were attracted to benzothiazole, which was an effective oviposition stimulant. In vitro experiments showed that BdorOR49b was narrowly tuned to benzothiazole. The electroantennogram results showed that knocking out BdorOR49b significantly reduced the antennal electrophysiological response to benzothiazole. Compared with wild-type flies, the attractiveness of benzothiazole to BdorOR49b knockout adult was significantly attenuated, and mutant females exhibited a severe decrease in oviposition behavior. Altogether, our work provides valuable insights into chemical communications and potential strategies for the control of this pest.

Different binding properties of odorant-binding protein 8 to insecticides in Orius sauteri.

Chemical sensing systems are vital in the growth and development of insects. Orius sauteri (Poppius) (Hemiptera: Anthocoridae) is an important natural enemy of many pests. The molecular mechanism of odorant binding proteins (OBPs) binding with common insecticides is still unknow in O. sauteri. In this study, we expressed in vitro OsauOBP8 and conducted fluorescence competition binding assay to investigate the function of OsauOBP8 to insecticides. The results showed that OsauOBP8 could bind with four common insecticides (phoxim, fenitrothion, chlorpyrifos, deltamethrin). Subsequently, we used molecular docking to predict and obtained candidate six amino acid residues (K4, K6, K13, R31, K49, K55) and then mutated. The result showed that three key residues (K4, K6, R31) play important role in OsauOBP8 bound to insecticides. Our study identified the key binding sites of OsauOBP8 to insecticides and help to better understand the molecular mechanism of OBPs to insecticides in O. sauteri.