Odorant Ligands Research Articles

Odorant-binding proteins as recognition elements for smell exploration

The sense of smell in animals enables them to detect and discriminate among many volatile odor molecules in complex environments. This task is accomplished by the family of G-proteins (olfactory receptor proteins, odorant-binding proteins, and chemosensory proteins). Here, we mechanistically elucidate how odors bind to odorant-binding proteins (OBPs) by studying the binding properties of 50 odorant ligands to six OBPs. We confirmed that OBP7 from Ailuropoda melanoleuca and OBP9 from Anopheles gambiae have binding affinities for 16 odors, and further analyzed the structures and protein-ligand interactions by homology modeling and molecular docking. This study found that OBP7 had an affinity for nonanol and camphor, and the docking results indicated that Glu120 was a residue that played a key role in the binding process of OBP7 to odorant ligands. We also explored the binding properties between OBP9 and ligands as well as the binding mechanism, and found that OBP9 bound well to some odorant molecules with repellent efficacy, such as nonanol, camphor, and citral. These findings not only contributed to our understanding of how OBPs were sensitively bound to odorant ligands but also promoted them as suitable targets for applied research in various industrial fields such as pesticides, cosmetics, and pharmaceuticals.

Inundative practice for screening siRNA management candidates against a notorious predatory beetle using olfactory silencing

Calosoma maximoviczi, a predatory pest beetle, poses a significant threat to wild silk farm production due to its predation on wild silkworms. Given the coexistence of this species with beneficial silkworms in the farm orchards, chemical pesticides are not an ideal solution for controlling its population. In this study, we employed a comprehensive multi-target RNA interference (RNAi) approach to disrupt the olfactory perception of C. maximoviczi through independently silencing 16 odorant receptors (ORs) in the respective genders. Specifically, gene-specific siRNAs were designed to target a panel of ORs, allowing us to investigate the specific interactions between odorant receptors and ligands within this species. Our investigation led to identifying four candidate siOR groups that effectively disrupted the beetle's olfactory tracking of various odorant ligands associated with different trophic levels. Furthermore, we observed sex-specific differences in innate RNAi responses reflected by subsequent gene expression, physiological and behavioral consequences, underscoring the complexity of olfactory signaling and emphasizing the significance of considering species/sex-specific traits when implementing pest control measures. These findings advance our understanding of olfactory coding patterns in C. maximoviczi beetles and establish a foundation for future research in the field of pest management strategies.

IORandLigandDB: A Website for Three-Dimensional Structure Prediction of Insect Odorant Receptors and Docking with Odorants.

The use of insect-specific odorants to control the behavior of insects has always been a hot spot in research on "green" control strategies of insects. However, it is generally time-consuming and laborious to explore insect-specific odorants with traditional reverse chemical ecology methods. Here, an insect odorant receptor (OR) and ligand database website (iORandLigandDB) was developed for the specific exploration of insect-specific odorants by using deep learning algorithms. The website provides a range of specific odorants before molecular biology experiments as well as the properties of ORs in closely related insects. At present, the existing three-dimensional structures of ORs in insects and the docking data with related odorants can be retrieved from the database and further analyzed.

Emerging roles of olfactory receptors in glucose metabolism.

Olfactory receptors (ORs) are widely expressed in extra-nasal tissues, where they participate in the regulation of divergent physiological processes. An increasing body of evidence over the past decade has revealed important regulatory roles for extra-nasal ORs in glucose metabolism. Recently, nonodorant endogenous ligands of ORs with metabolic significance have been identified, implying the therapeutic potential of ORs in the treatment of metabolic diseases, such as diabetes and obesity. In this review, we summarize current understanding of the expression patterns and functions of ORs in key tissues involved in glucose metabolism modulation, describe odorant and endogenous OR ligands, explain the biased signaling downstream of ORs, and outline OR therapeutic potential.

Antagonistic interactions between odorants alter human odor perception

The olfactory system uses hundreds of odorant receptors (ORs), the largest group of the G-protein-coupled receptor (GPCR) superfamily, to detect a vast array of odorants. Each OR is activated by specific odorous ligands, and like other GPCRs, antagonism can block activation of ORs. Recent studies suggest that odorant antagonisms in mixtures influence olfactory neuron activities, but it is unclear how this affects perception of odor mixtures. In this study, we identified a set of human ORs activated by methanethiol and hydrogen sulfide, two potent volatile sulfur malodors, through large-scale heterologous expression. Screening odorants that block OR activation in heterologous cells identified a set of antagonists, including β-ionone. Sensory evaluation in humans revealed that β-ionone reduced the odor intensity and unpleasantness of methanethiol. Additionally, suppression was not observed when methanethiol and β-ionone were introduced simultaneously to different nostrils. Our study supports the hypothesis that odor sensation is altered through antagonistic interactions at the OR level.

Transcriptome Profiling of Anhidrotic Eccrine Sweat Glands Reveals that Olfactory Receptors on Eccrine Sweat Glands Regulate Perspiration in a Ligand-Dependent Manner

Sweat maintains systemic homeostasis in humans. Although sweating disorders may cause multifaceted health problems, therapeutic options for sweat disorders have not yet been established. To gain new insight into the mechanism underlying the regulation of perspiration, we compared eccrine sweat gland transcriptomes from hidrotic and anhidrotic lesions from patients with anhidrosis and found out that olfactory receptors were expressed differentially in anhidrotic and hidrotic eccrine sweat glands. We then confirmed OR51A7 and OR51E2 expression in human eccrine sweat glands by in situ hybridization and immunohistochemistry. An alkaline phosphatase-TGFα shedding assay revealed that β-ionone activates G-proteins through OR51A7 or OR51E2. The effect of topically applied β-ionone on sweating was examined with the quantitative sudomotor axon reflex test, which showed that responses to β-ionone differed between sexes. Topical β-ionone attenuated female sweating and augmented male sweating. Taken together, this study suggests that olfactory receptors expressed in eccrine sweat glands may regulate sweating in response to odorous ligands on the basis of sex. These unexpected results indicate that olfactory receptors may modulate sweating and that olfactory receptor modulators may contribute to the management of sweat disorders.

Characterization and Functional Analysis of OcomOBP7 in Ophraella communa Lesage

The olfactory system plays a key role in various insect behaviors, and odorant-binding proteins participate in the first step of the olfactory process. Ophraella communa Lesage is an oligophagous phytophagous insect that is a specific biological control agent for Ambrosia artemisiifolia L. The leaf beetle must identify and locate A. artemisiifolia through olfaction; however, its odorant-binding protein (OBP) function has not yet been reported. In this study, OcomOBP7 was cloned, and its tissue expression profile and binding ability were analyzed using RT-qPCR and fluorescence binding assays, respectively. Sequence analysis demonstrated that OcomOBP7 belongs to the classical OBP family. The RT-qPCR results showed that OcomOBP7 was specifically expressed in the antennae, indicating that OcomOBP7 may be involved in chemical communication. The fluorescence binding assay showed that OcomOBP7 has an extensive binding ability to alkenes. The electroantennography experiments showed that O. communa antennal response to α-pinene and ocimene decreased significantly after interference because the two odors specifically bound to OcomOBP7. In summary, α-pinene and ocimene are odorant ligands corresponding to OcomOBP7, indicating that OcomOBP7 is involved in the chemical recognition of A. artemisiifolia. Our study lays a theoretical foundation for research into O. communa attractants, which is helpful for the better biological control of A. artemisiifolia by O. communa.

Genetic variation in the human olfactory receptor OR5AN1 associates with the perception of musks.

Humans have significant individual variations in odor perception, derived from their experience or sometimes from differences in the olfactory receptor (OR) gene repertoire. In several cases, the genetic variation of a single OR affects the perception of its cognate odor ligand. Musks are widely used for fragrance and are known to demonstrate specific anosmia. It, however, remains to be elucidated whether the OR polymorphism contributes to individual variations in musk odor perception. Previous studies reported that responses of the human musk receptor OR5AN1 to a variety of musks in vitro correlated well with perceptual sensitivity to those odors in humans and that the mouse ortholog, Olfr1440 (MOR215-1), plays a critical role in muscone perception. Here, we took advantage of genetic variation in OR5AN1 to examine how changes in receptor sensitivity are associated with human musk perception. We investigated the functional differences between OR5AN1 variants in an in vitro assay and measured both perceived intensity and detection threshold in human subjects with different OR5AN1 genotypes. Human subjects homozygous for the more sensitive L289F allele had a lower detection threshold for muscone and found macrocyclic musks to be more intense than subjects homozygous for the reference allele. These results demonstrate that the genetic variation in OR5AN1 contributes to perceptual differences for some musks. In addition, we found that the more functional variant of OR5A1, a receptor involved in β-ionone perception, is associated with the less functional variant of OR5AN1, suggesting that the perceived intensities of macrocyclic musks and β-ionone are inversely correlated.

Recording from Fly Olfactory Sensilla.

Olfactory systems detect and discriminate an enormous diversity of volatile environmental stimuli and provide important paradigms to investigate how sensory cues are represented in the brain. Key stimulus-coding events occur in peripheral olfactory sensory neurons, which typically express a single olfactory receptor-from a large repertoire encoded in the genome-with a defined ligand-response profile. These receptors convert odor ligand recognition into spatial and temporal patterns of neural activity that are transmitted to, and interpreted in, central brain regions. Drosophila provides an attractive model to study olfactory coding because it possesses a relatively simple peripheral olfactory system that displays many organizational parallels to those of vertebrates. Moreover, nearly all olfactory sensory neurons have been molecularly characterized and are accessible for physiological analysis, as they are exposed on the surface of sensory organs (antennae and maxillary palps) housed in specialized hairs called sensilla. This protocol describes how to perform recordings of odor-evoked activity from Drosophila olfactory sensilla, covering the basics of sample preparation, setting up the electrophysiology rig, assembling an odor stimulus-delivery device, and data analysis. The methodology can be used to characterize the ligand-recognition properties of most olfactory sensory neurons and the role of olfactory receptors (and other molecular components) in signal transduction.

Olfactory receptors in macrophages and inflammation.

Olfactory receptors (ORs) that bind odorous ligands are the largest family of G-protein-coupled receptors. In the olfactory epithelium, approximately 400 and 1,100 members are expressed in humans and mice, respectively. Growing evidence suggests the extranasal functions of ORs. Here, we review OR expression and function in macrophages, specialized innate immune cells involved in the detection, phagocytosis, and destruction of cellular debris and pathogens as well as the initiation of inflammatory responses. RNA sequencing data in mice suggest that up to 580 ORs may be expressed in macrophages. Macrophage OR expression is increased after treatment with the Toll-like receptor 4 ligand lipopolysaccharide, which also induces the transcription of inflammasome components. Triggering human OR6A2 or its mouse orthologue Olfr2 with their cognate ligand octanal induces inflammasome assembly and the secretion of IL-1β, which exacerbates atherosclerosis. Octanal is positively correlated with blood lipids like low-density lipoprotein –cholesterol in humans. Another OR, Olfr78, is activated by lactate, which promotes the generation of tumor-associated macrophages that dampen the immune response and promote tumor progression. Olfactory receptors in macrophages are a rich source of untapped opportunity for modulating inflammation. It is not known which of the many ORs expressed in macrophages promote or modulate inflammation. Progress in this area also requires deorphanizing more ORs and determining the sources of their ligands.

Novel Anosmia‐Inducing Compounds for Environmentally Friendly Mosquito Vector Control: Structural Determinants of ORco Ligands Antagonizing Odorant Receptor Function

Insect repellents are important means of personal protection against bites from mosquitoes carrying various pathogens. Odorant‐dependent behaviours are triggered by odor ligands bound to odorant receptors. Insect odorant receptors are heterotetrameric ligand‐gated cation channels composed of an obligatory subunit, ORco, and one of many variable subunits, ORx, to which odorant ligands bind. Upon expression ex vivo, ORco forms homomeric channels gated by specific ligands acting as channel agonists. In past studies, we identified ligands of natural origin that bind to the African malaria mosquito vector Anopheles gambiae ORco (AgamORco). Some of these ligands function as specific ORco channel antagonists, orthosteric or allosteric relative to a predicted ORco agonist binding site, and cause severe inhibition of the olfactory function in a variety of mosquito species. Given that ORco is evolutionarily conserved, we hypothesized that it should be feasible to develop an AgamORco‐based screening platform allowing fast identification of new ORco ligands acting as mosquito odorant receptor antagonists causing anosmia‐like deficits to mosquitoes.We are now reporting on the compilation of common structural features of previously identified orthosteric AgamORco antagonists and generation of a ligand‐based pharmacophore. In silico screening of a collection of volatile compounds of natural origin resulted in identification of several AgamORco ligand hits. Parallel cell‐based screening of the same compounds against AgamORco was also employed in order to identify ligands acting as AgamORco antagonists. The screening resulted in identification of several ligands inhibiting AgamORco channel function ex vivo by at least 40% and inducing anosmic behaviors to Aedes albopictus mosquitoes in vivo. To test the pharmacophore model for binding site specificity, binding competition assays were undertaken, which distinguished the identified antagonists into orthosteric and allosteric ones. Direct comparisons of the pharmacophore predictions with the results of the cell‐based screen revealed that the pharmacophore predicted correctly 100% of the orthosteric antagonists and none of the allosteric ones. These results confirm the pharmacophore's value for in silico prediction of AgamORco orthosteric ligands. Upon combination with the AgamORco‐specific cell‐based screening platform, the pharmacophore provides a valuable tool for fast identification of multiple, ecologically friendly vector control agents. Finally, to confirm the predicted AgamORco agonist binding site and determine the binding orientation of orthosteric antagonists in it, we are assessing the interactions of these ligands with specific amino acids in the AgamORco binding pocket by site‐specific mutagenesis.

Identification and expression of odorant binding proteins in the egg-parasitoid Trissolcus basalis (Wollaston) (Hymenoptera, Scelionidae, Telenominae)

Trissolcus basalis (Wollaston) (Hymenoptera, Scelionidae) is an egg-parasitoid of the southern green stink bug, Nezara viridula (Linneaus) (Hemiptera, Pentatomidae). Many behaviors associated with female T. basalis host-finding and acceptance are mediated by chemosensory pathways, for which olfactory, gustatory and ionotropic receptors have been previously identified. Odorant binding proteins (OBPs) are small, globular proteins, one of the functions of which is the transport of odorant ligands through the aqueous lymph of chemosensory sensilla to these receptors. We identified 18 classical OBP sequences in the T. basalis genome and transcriptomes sharing an average 26.8% pairwise identity. Gene tree analyses suggest very limited lineage-specific expansion and identify potential orthologs among other scelionids and Hymenoptera. Transcriptome mapping and qPCR comparison of expression levels in antennae and bodies of both sexes determine that at least five TbOBPs are preferentially expressed in the female antennae. These are, therefore, prime candidates for further study to determine their role in detecting host-produced semiochemicals.

A physicochemical model of odor sampling.

We present a general physicochemical sampling model for olfaction, based on established pharmacological laws, in which arbitrary combinations of odorant ligands and receptors can be generated and their individual and collective effects on odor representations and olfactory performance measured. Individual odor ligands exhibit receptor-specific affinities and efficacies; that is, they may bind strongly or weakly to a given receptor, and can act as strong agonists, weak agonists, partial agonists, or antagonists. Ligands interacting with common receptors compete with one another for dwell time; these competitive interactions appropriately simulate the degeneracy that fundamentally defines the capacities and limitations of odorant sampling. The outcome of these competing ligand-receptor interactions yields a pattern of receptor activation levels, thereafter mapped to glomerular presynaptic activation levels based on the convergence of sensory neuron axons. The metric of greatest interest is the mean discrimination sensitivity, a measure of how effectively the olfactory system at this level is able to recognize a small change in the physicochemical quality of a stimulus. This model presents several significant outcomes, both expected and surprising. First, adding additional receptors reliably improves the system’s discrimination sensitivity. Second, in contrast, adding additional ligands to an odorscene initially can improve discrimination sensitivity, but eventually will reduce it as the number of ligands increases. Third, the presence of antagonistic ligand-receptor interactions produced clear benefits for sensory system performance, generating higher absolute discrimination sensitivities and increasing the numbers of competing ligands that could be present before discrimination sensitivity began to be impaired. Finally, the model correctly reflects and explains the modest reduction in odor discrimination sensitivity exhibited by transgenic mice in which the specificity of glomerular targeting by primary olfactory neurons is partially disrupted.

UV-Irradiation- and Inflammation-Induced Skin Barrier Dysfunction Is Associated with the Expression of Olfactory Receptor Genes in Human Keratinocytes.

Olfactory receptors (ORs) have diverse physiological roles in various cell types, beyond their function as odorant sensors in the olfactory epithelium. These previous findings have suggested that ORs could be diagnostic markers and promising therapeutic targets in several pathological conditions. In the current study, we sought to characterize the changes in the expression of ORs in the HaCaT human keratinocytes cell line exposed to ultraviolet (UV) light or inflammation, well-recognized stimulus for skin barrier disruption. We confirmed that major olfactory signaling components, including ORs, GNAL, Ric8b, and adenylate cyclase type 3, are highly expressed in HaCaT cells. We have also demonstrated that the 12 ectopic ORs detectable in HaCaT cells are more highly expressed in UV-irradiated or inflamed conditions than in normal conditions. We further assessed the specific OR-mediated biological responses of HaCaT cells in the presence of known odorant ligands of ORs and observed that specific ligand-activated ORs downregulate skin barrier genes in HaCaT cells. This study shows the potential of OR as a marker for skin barrier abnormalities. Further research is needed to explore how OR is implicated in the development and progression of barrier dysfunction.

Volatile allosteric antagonists of mosquito odorant receptors inhibit human-host attraction

Odorant-dependent behaviors in insects are triggered by the binding of odorant ligands to the variable subunits of heteromeric olfactory receptors. Previous studies have shown, however, that specific odor binding to ORco, the common subunit of odorant receptor heteromers, may allosterically alter olfactory receptor function and profoundly affect subsequent behavioral responses. Using an insect cell–based screening platform, we identified and characterized several antagonists of the odorant receptor coreceptor of the African malaria vector Anopheles gambiae (AgamORco) in a small collection of natural volatile organic compounds. Because some of the identified antagonists were previously shown to strongly repel Anopheles and Culex mosquitoes, we examined the bioactivities of the identified antagonists against Aedes, the third major genus of the Culicidae family. The tested antagonists inhibited the function of Ae. aegypti ORco ex vivo and repelled adult Asian tiger mosquitoes (Ae. albopictus). Binary mixtures of specific antagonists elicited higher repellency than single antagonists, and binding competition assays suggested that this enhanced repellence is due to antagonist interaction with distinct ORco sites. Our results also suggest that the enhanced mosquito repellency by antagonist mixtures is due to additive rather than synergistic effects of the specific antagonist combinations on ORco function. Taken together, these findings provide novel insights concerning the molecular aspects of odorant receptor function. Moreover, our results demonstrate that a simple screening assay may be used for the identification of allosteric modifiers of olfactory-driven behaviors capable of providing enhanced personal protection against multiple mosquito-borne infectious diseases.

Immobilization of Olfactory Receptors Carried by Nanosomes onto a Gold Sensor Surface.

Mammalian olfactory receptors (ORs) constitute the largest family of G-protein-coupled receptors, with up to about 1000 different genes per species, each having specific odorant ligands. ORs could be used as sensing elements of highly specific and sensitive bioelectronic hybrid devices such as bioelectronic noses. After optimized immobilization onto the device, natural ORs provide molecular recognition of various odors with their intrinsic sensitivity, discrimination, and detection properties. However, the main difficulties are related to the low expression level of recombinant ORs, their stability and potential loss of activity. Such drawbacks can be successfully overcome in bioelectronic noses integrating nanosomes (nanometric membrane vesicles carrying ORs) that are stably immobilized through a specific antibody. The advantages of such a platform rely on the fact that ORs stay in the natural membrane environment, and thus preserve their full activity. Thanks to their small sizes, nanosomes offer potential for micro- and nano-scale sensor development. In this paper, we summarize the key elements regarding nanosomes production and manipulation and provide an example of their immobilization onto a gold sensor surface. Rat ORI7 is used as a representative OR that can be functionally expressed in Saccharomyces cerevisiae. The receptor was not purified but only nanosomes were prepared. Nanosomes were immobilized onto functionalized gold surface using the anti-I7 antibody. Utilization of the antibody provides enrichment of ORI7 on the sensor surface but also uniform and appropriate orientation of the receptors. These features are crucial in optimization of bioelectronic nose' analytical performances.

Conformational Sensing by a Mammalian Olfactory Receptor

To identify odors, the mammalian nose deploys hundreds of olfactory receptors (ORs) from the rhodopsin-like class of the G protein-coupled receptor superfamily. Odorants having multiple rotatable bonds present a problem for the stereochemical shape-based matching process assumed to govern the sense of smell through OR-odorant recognition. We conformationally restricted the carbon chain of the odorant octanal to ask whether an OR can respond differently to different odorant conformations. By using calcium imaging to monitor signal transduction in sensory neurons expressing the mouse aldehyde OR, Olfr2, we found that the spatial position of the C7 and C8 carbon atoms of octanal, in relation to its -CHO group, determines whether an aliphatic aldehyde functions as an agonist, partial agonist or antagonist. Our experiments provide evidence that an odorant can manipulate an OR through its intrinsic conformational repertoire, in unexpected analogy to the photon-controlled aldehyde manipulation observed in rhodopsin.

Odorant ligands for the CO2 receptor in two Anopheles vectors of malaria

Exhaled CO2 is an important host-seeking cue for Anopheles mosquitoes, which is detected by a highly conserved heteromeric receptor consisting of three 7-transmembrane proteins Gr22, Gr23, and Gr24. The CO2 receptor neuron has been shown to also respond sensitively to a variety of odorants in Aedes aegypti. The detection of CO2 is important for upwind navigation and for enhancing the attraction to body heat as well as to skin odorants. The orthologs of the CO2 receptor proteins are present in malaria-transmitting mosquitoes like Anopheles coluzzii and Anopheles sinensis. Activators and inhibitors of the CO2-neuron were tested on the maxillary palps in these two species by single-sensillum electrophysiology. The electrophysiological testing of three prolonged-activator odorants identified originally in Aedes aegypti also showed varying ability to reduce the CO2-ellicited increase in spikes. These findings provide a foundation for comparing the functional conservation with the evolutionary conservation of an important class of odorant receptor. The identification of a suite of natural odorants that can be used to modify the CO2-detection pathway may also contribute to odor-blends that can alter the behavior of these disease transmitting mosquitoes.

Expression and function of the ectopic olfactory receptor OR10G7 in patients with atopic dermatitis

Ectopic olfactory receptors (ORs) are found in the skin, but their expression and biological function in normal skin and skin form patients with atopic dermatitis (AD) are unknown. We sought to characterize the expression of ORs in the skin and assess OR-mediated biological responses of primary human keratinocytes in the presence of odorant ligands. OR expression was examined by using whole-transcriptome sequencing of skin tape strips collected from patients with AD and healthy control (HC) subjects. OR10G7 and filaggrin 1 (FLG-1) expression was analyzed by using RT-PCR and immunostaining in skin biopsy specimens and primary human keratinocytes from patients with AD and HC subjects. ATP and cyclic AMP production by control and OR10G7 small interfering RNA-transfected keratinocytes in response to odorant stimulation with acetophenone and eugenol was assessed. A total of 381 OR gene transcripts were detected in the skin samples, with the greatest OR expression detected in the skin tape strips corresponding to the upper granular layer of the skin. OR10G7 expression was significantly increased in skin biopsy specimens from patients with AD compared with those from HC subjects (P=.01) and inversely correlated with FLG-1 expression (P=.009). OR10G7 expression was greatest in undifferentiated keratinocytes from patients with AD and was downregulated with progressive differentiation. Primary human keratinocytes produced ATP, an essential neurotransmitter in sensory pathways, in response to acetophenone and eugenol, odorants previously identified as potential ligands for this receptor. This response was abolished in OR10G7 small interfering RNA-transfected keratinocytes. OR10G7 is expressed at significantly greater levels in undifferentiated keratinocytes from patients with AD compared with HC subjects. OR10G7 is likely involved in transmission of skin-induced chemosensory responses to odorant stimulation, which might modulate differential nociceptive responses in AD skin.

Expression Profiles and Functional Characterization of Two Odorant-Binding Proteins From the Apple Buprestid Beetle Agrilus mali (Coleoptera: Buprestidae).

The apple buprestid beetle, Agrilus mali Matsumura (Coleoptera: Buprestidae), can respond to various volatiles, but the underlying mechanism of odorant perception for this insect is poorly understood. Here, we cloned A. mali's odorant-binding proteins 3 (AmalOBP3) and 8 (AmalOBP8) and characterized their expression patterns and binding profiles. Sequence and phylogenetic analyses showed that AmalOBP3 and AmalOBP8 were distributed in the classic and minus-C OBP subfamily, respectively. AmalOBP3 was specifically and abundantly expressed in antennae of both sexes. AmalOBP8 displayed high transcript levels in antennae of both sexes, abdomens of males, and wings of both sexes. Both AmalOBPs exhibited much higher expression in male antennae than in female antennae, suggesting that they could be important in perception of male-specific olfactory cues (e.g., some sex pheromones). Out of the 40 odorant ligands tested, AmalOBP3 and AmalOBP8 bound to 15 and 21 different odorants, respectively, indicating a distinct and selective binding profile for them. Both AmalOBPs seemed to have very strong binding affinity to aliphatic alcohols and aldehydes with 12 to 15 carbon atoms. Alcohols, esters, and terpenoids were more likely to be good ligands for both AmalOBPs than aldehydes and alkanes. Together with its broad expression in different tissues, strong binding with higher numbers of putative ligands for AmalOPB8 means that this protein can have more extensive functional roles in chemosensation of A. mali. Our results provide insights into the molecular basis of chemosensation in A. mali, as well as a basis for developing detection, monitoring, and management tools for this serious pest.