The red flour beetle's large nose: an expanded odorant receptor gene family in Tribolium castaneum

The Tribolium castaneum genome sequence reveals a large number of odorant receptor (Or) genes compared to those found in other insects whose olfactory genomes have been studied-341 Or genes and pseudogenes, encoding 259 intact odorant receptor proteins. An RT-PCR study of larvae and adults revealed that only 145 (64%) of 233 genes with successful genomic DNA amplifications were expressed. No expression of the other 87 genes was detected at any age, suggesting either that these genes are not expressed in this particular strain, or that they are induced only in certain environmental or developmental conditions. TcOR1, the ortholog of the Drosophila Or83b (DmOr83b) gene, which is required for the function of olfactory receptor proteins in Drosophila, was expressed in extracts from adult and larval heads and in extracts from adult bodies. Expression of 41 TcOr genes was detected in extracts from larval head tissue and 111 in extracts from adult head tissue (both figures exclude TcOr1). Twenty-eight TcOrs were detected only in adult bodies. Beetle pupae were injected with TcOr1 dsRNA; unlike sham-injected and control beetles, these knock-down beetles showed no significant response to the Tribolium aggregation pheromone, supporting the hypothesis that TcOr1 plays a similar decisive role in olfaction to DmOr83b. The substantial number of Ors poses the question of why Tribolium has such a large olfactory receptor repertoire, and underlines the need for more studies of the natural history of this species.     

Identification of multiple odorant receptors essential for pyrethrum repellency in Drosophila melanogaster

Pyrethrum extract from dry flowers of Tanacetum cinerariifolium (formally Chrysanthemum cinerariifolium) has been used globally as a popular insect repellent against arthropod pests for thousands of years. However, the mechanistic basis of pyrethrum repellency remains unknown. In this study, we found that pyrethrum spatially repels and activates olfactory responses in Drosophila melanogaster, a genetically tractable model insect, and the closely-related D. suzukii which is a serious invasive fruit crop pest. The discovery of spatial pyrethrum repellency and olfactory response to pyrethrum in D. melanogaster facilitated our identification of four odorant receptors, Or7a, Or42b, Or59b and Or98a that are responsive to pyrethrum. Further analysis showed that the first three Ors are activated by pyrethrins, the major insecticidal components in pyrethrum, whereas Or98a is activated by (E)-β-farnesene (EBF), a sesquiterpene and a minor component in pyrethrum. Importantly, knockout of Or7a, Or59b or Or98a individually abolished fly avoidance to pyrethrum, while knockout of Or42b had no effect, demonstrating that simultaneous activation of Or7a, Or59b and Or98a is required for pyrethrum repellency in D. melanogaster. Our study provides insights into the molecular basis of repellency of one of the most ancient and globally used insect repellents. Identification of pyrethrum-responsive Ors opens the door to develop new synthetic insect repellent mixtures that are highly effective and broad-spectrum.     

Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction

Fruit flies are attracted by a diversity of odors that signal the presence of food, potential mates, or attractive egg-laying sites. Most Drosophila olfactory neurons express two types of odorant receptor genes: Or83b, a broadly expressed receptor of unknown function, and one or more members of a family of 61 selectively expressed receptors. While the conventional odorant receptors are highly divergent, Or83b is remarkably conserved between insect species. Two models could account for Or83b function: it could interact with specific odor stimuli independent of conventional odorant receptors, or it could act in concert with these receptors to mediate responses to all odors. Our results support the second model. Dendritic localization of conventional odorant receptors is abolished in Or83b mutants. Consistent with this cellular defect, the Or83b mutation disrupts behavioral and electrophysiological responses to many odorants. Or83b therefore encodes an atypical odorant receptor that plays an essential general role in olfaction.     

Cloning and expression pattern of odorant receptor 11 in Asian honeybee drones,Apis cerana (Hymenoptera,Apidae)

Odorant receptors play a crucial role in the special recognition of scent molecules in the honeybee olfaction system. The odorant receptor 11 (AmOR11) in western honeybee drones (Apis mellifera) has been demonstrated to specifically bind to 9-oxo-2-decenoic acid (9-ODA) of queens. However, little is known regarding the functions of OR11 Asian honeybee drones (Apis cerana) in the context of their mating activities. In this study, the odorant receptor 11 gene (AcOr11) from A. cerana was cloned, and its expression profiles were examined during two developmental stages (immature and sexually mature) and different physiological statuses (flying and crawling). The cDNA sequence of AcOr11 was highly similar to that of AmOr11, and encoded a membrane-coupled protein of 384 amino acids. The results of qRT-PCR indicated that AcOr11 was expressed at higher levels in drone antennae compared to brains, and the expression was significantly up-regulated in sexually mature drone brains compared to immature brains. Interestingly, AcOr11 expression in brains of mature flying drones was dramatically higher than those of mature crawling drones. To our knowledge, this study demonstrate a link between AcOr11 gene expression in the brain of honeybee drones and behavior associated with sexual maturity and mating flight.     

Expression of odorant receptor family, type 2 OR in the aquatic olfactory cavity of amphibian frog Xenopus tropicalis

Recent genome wide in silico analyses discovered a new family (type 2 or family H) of odorant receptors (ORs) in teleost fish and frogs. However, since there is no evidence of the expression of these novel OR genes in olfactory sensory neurons (OSN), it remains unknown if type 2 ORs (OR2) function as odorant receptors. In this study, we examined expression of OR2 genes in the frog Xenopus tropicalis. The overall gene expression pattern is highly complex and differs depending on the gene and developmental stage. RT-PCR analysis in larvae showed that all of the OR2η genes we identified were expressed in the peripheral olfactory system and some were detected in the brain and skin. Whole mount in situ hybridization of the larval olfactory cavity confirmed that at least two OR2η genes so far tested are expressed in the OSN. Because tadpoles are aquatic animals, OR2η genes are probably involved in aquatic olfaction. In adults, OR2η genes are expressed in the nose, brain, and testes to different degrees depending on the genes. OR2η expression in the olfactory system is restricted to the medium cavity, which participates in the detection of water-soluble odorants, suggesting that OR2ηs function as receptors for water-soluble odorants. Moreover, the fact that several OR2ηs are significantly expressed in non-olfactory organs suggests unknown roles in a range of biological processes other than putative odorant receptor functions.     

Direct nuclear magnetic resonance observation of odorant binding to mouse odorant receptor MOR244-3

Mammals are able to perceive and differentiate a great number of structurally diverse odorants through the odorant's interaction with odorant receptors (ORs), proteins found within the cell membrane of olfactory sensory neurons. The natural gas industry has used human olfactory sensitivity to sulfur compounds (thiols, sulfides, etc.) to increase the safety of fuel gas transport, storage, and use through the odorization of this product. In the United States, mixtures of sulfur compounds are used, but the major constituent of odorant packages is 2-methylpropane-2-thiol, also known as tert-butyl mercaptan. It has been fundamentally challenging to understand olfaction and odorization due to the low affinity of odorous ligands to the ORs and the difficulty in expressing a sufficient number of OR proteins. Here, we directly observed the binding of tert-butyl mercaptan and another odiferous compound, cis-cyclooctene, to mouse OR MOR244-3 on living cells by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. This effort lays the groundwork for resolving molecular mechanisms responsible for ligand binding and resulting signaling, which in turn will lead to a clearer understanding of odorant recognition and competition.     

Genetic variation in odorant receptors contributes to variation in olfactory behavior in a natural population of Drosophila melanogaster

Chemoreception is a principle modality by which organisms gain information from their environment, and extensive variation in odor-mediated behavior has been documented within and among species. To examine the mechanisms by which sensory systems mediate these responses, we ask to what extent variation in Drosophila melanogaster odorant receptor genes contributes to variation in odor-mediated behavior. Significant differences in behavioral responses to structurally similar odorants, methyl hexanoate and ethyl hexanoate, were found in a natural population. Polymorphisms in 3 genomic regions (Or22a/Or22b, Or35a, and Or47a) were identified and associated with variation in behavior to these esters. Overall similarity in association profiles for both odorants was observed, except for Or47a in which polymorphisms were associated solely with variation in responses to ethyl hexanoate. Our analyses were then extended to examine polymorphisms in 3 odorant receptors previously reported to contribute to variation in olfactory behavior for the chemically distinct odorants benzaldehyde and acetophenone. Two Or10a polymorphisms were associated with variation in response to ethyl hexanoate. Finally, differences in Or35a and Or47a expression were associated with variation in responses to ethyl hexanoate. These results demonstrate that the genetic variation at the peripheral sensory stage plays a role in mediating differences in odor-mediated behavior.     

Allosteric antagonism of insect odorant receptor ion channels

Background

At a molecular level, insects utilize members of several highly divergent and unrelated families of cell-surface chemosensory receptors for detection of volatile odorants. Most odors are detected via a family of odorant receptors (ORs), which form heteromeric complexes consisting of a well-conserved OR co-receptor (Orco) ion channel and a non-conserved tuning OR that provides coding specificity to each complex. Orco functions as a non-selective cation channel and is expressed in the majority of olfactory receptor neurons (ORNs). As the destructive behaviors of many insects are principally driven by olfaction, Orco represents a novel target for behavior-based control strategies. While many natural and synthetic odorants have been shown to agonize Orco/Or complexes, only a single direct Orco modulator, VUAA1, has been described. In an effort to identify additional Orco modulators, we have investigated the structure/activity relationships around VUAA1.

Results

A search of our compound library identified several VUAA1 analogs that were selected for evaluation against HEK cells expressing Orco from the malaria vector Anopheles gambiae (AgOrco). While the majority of compounds displayed no activity, many of these analogs possess no intrinsic efficacy, but instead, act as competitive VUAA1 antagonists. Using calcium mobilization assays, patch clamp electrophysiology, and single sensillum in vivo recording, we demonstrate that one such candidate, VU0183254, is a specific allosteric modulator of OR signaling, capable of broadly inhibiting odor-mediated OR complex activation.

Conclusions

We have described and characterized the first Orco antagonist, that is capable of non-competitively inhibiting odorant-evoked activation of OR complexes, thereby providing additional insight into the structure/function of this unique family of ligand-gated ion channels. While Orco antagonists are likely to have limited utility in insect control programs, they represent important pharmacological tools that will facilitate the investigation of the molecular mechanisms underlying insect olfactory signal transduction.     

Insights into structural features determining odorant affinities to honey bee odorant binding protein 14

Molecular interactions between odorants and odorant binding proteins (OBPs) are of major importance for understanding the principles of selectivity of OBPs towards the wide range of semiochemicals. It is largely unknown on a structural basis, how an OBP binds and discriminates between odorant molecules. Here we examine this aspect in greater detail by comparing the C-minus OBP14 of the honey bee (Apis mellifera L.) to a mutant form of the protein that comprises the third disulfide bond lacking in C-minus OBPs. Affinities of structurally analogous odorants featuring an aromatic phenol group with different side chains were assessed based on changes of the thermal stability of the protein upon odorant binding monitored by circular dichroism spectroscopy. Our results indicate a tendency that odorants show higher affinity to the wild-type OBP suggesting that the introduced rigidity in the mutant protein has a negative effect on odorant binding. Furthermore, we show that OBP14 stability is very sensitive to the position and type of functional groups in the odorant.     

A broadly tuned odorant receptor in neurons of trichoid sensilla in locust,Locusta migratoria

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. Odorant receptors (ORs) on the membrane of chemosensory neurons are believed to be key molecules in sensing exogenous chemical cues. ORs in different species of insects are diverse and should tune a species to its own specific semiochemicals relevant to their survival. The orthopteran insect, locust (Locusta migratoria), is a model hemimetabolous insect. There is very limited knowledge on the functions of locust ORs although many locust OR genes have been identified in genomic sequencing experiments. In this paper, a locust OR, LmigOR3 was localized to neurons housed in trichoid sensilla by in situ hybridization. LmigOR3 was expressed as a transgene in Drosophila trichoid olfactory neurons (aT1) lacking the endogenous receptor Or67d and the olfactory tuning curve and dose-response curves were established for this locust receptor. The results show that LmigOR3 sensitizes neurons to ketones, esters and heterocyclic compounds, indicating that LmigOR3 is a broadly tuned receptor. LmigOR3 is the first odorant receptor from Orthoptera that has been functionally analyzed in the Drosophila aT1 system. This work demonstrates the utility of the Drosophila aT1 system for functional analysis of locust odorant receptors and suggests that LmigOR3 may be involved in detecting food odorants, or perhaps locust body volatiles that may help us to develop new control methods for locusts.     

Molecular cloning,expression profile,odorant affinity,and stability of two odorant‐binding proteins in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae)

The polyembryonic endoparasitoid wasp Macrocentrus cingulum Brischke (Hymenoptera: Braconidae) is deployed successfully as a biocontrol agent for corn pest insects from the Lepidopteran genus Ostrinia in Europe and throughout Asia, including Japan, Korea, and China. The odorants are recognized, bound, and solubilized by odorant‐binding protein (OBP) in the initial biochemical recognition steps in olfaction that transport them across the sensillum lymph to initiate behavioral response. In the present study, we examine the odorant‐binding effects on thermal stability of McinOBP2, McinOBP3, and their mutant form that lacks the third disulfide bonds. Real‐time PCR experiments indicate that these two are expressed mainly in adult antennae, with expression levels differing by sex. Odorant‐binding affinities of aldehydes, terpenoids, and aliphatic alcohols were measured with circular dichroism spectroscopy based on changes in the thermal stability of the proteins upon their affinities to odorants. The obtained results reveal higher affinity of trans‐caryophelle, farnesene, and cis‐3‐Hexen‐1‐ol exhibits to both wild and mutant McinOBP2 and McinOBP3. Although conformational flexibility of the mutants and shape of binding cavity make differences in odorant affinity between the wild‐type and mutant, it suggested that lacking the third disulfide bond in mutant proteins may have chance to incorrect folded structures that reduced the affinity to these odorants. In addition, CD spectra clearly indicate proteins enriched with α‐helical content.     

Distinct Olfactory Signaling Mechanisms in the Malaria Vector Mosquito Anopheles gambiae

Anopheles gambiae is the principal Afrotropical vector for human malaria, in which olfaction mediates a wide range of both adult and larval behaviors. Indeed, mosquitoes depend on the ability to respond to chemical cues for feeding, host preference, and mate location/selection. Building upon previous work that has characterized a large family of An. gambiae odorant receptors (AgORs), we now use behavioral analyses and gene silencing to examine directly the role of AgORs, as well as a newly identified family of candidate chemosensory genes, the An. gambiae variant ionotropic receptors (AgIRs), in the larval olfactory system. Our results validate previous studies that directly implicate specific AgORs in behavioral responses to DEET as well as other odorants and reveal the existence of at least two distinct olfactory signaling pathways that are active in An. gambiae. One system depends directly on AgORs; the other is AgOR-independent and requires the expression and activity of AgIRs. In addition to clarifying the mechanistic basis for olfaction in this system, these advances may ultimately enhance the development of vector control strategies, targeting olfactory pathways in mosquitoes to reduce the catastrophic effects of malaria and other mosquito-borne diseases.     

Drosophila odorant receptors are novel seven transmembrane domain proteins that can signal independently of heterotrimeric G proteins

Olfaction in Drosophila is mediated by a large family of membrane-bound odorant receptor proteins (Ors). In heterologous cells, we investigated whether the structural features and signalling mechanisms of ligand-binding Drosophila Ors are consistent with them being G protein-coupled receptors (GPCRs). The detailed membrane topology of Or22a was determined by inserting epitope tags into the termini and predicted loop regions. Immunocytochemistry experiments in Drosophila S2 cells imply that Or22a has seven transmembrane domains but that its membrane topology is opposite to that of GPCRs, with a cytoplasmic N-terminus and extracellular C-terminus. To investigate Or signalling mechanisms, we expressed Or43b in Sf9 and HEK293 cells, and show that inhibitors of heterotrimeric G proteins (GDP-beta-S), adenylate cyclase (SQ22536), guanylyl cyclase (ODQ), cyclic nucleotide phosphodiesterases (IBMX) and phospholipase C (U73122) have negligible impact on Or43b responses. Whole cell patching of Or43b/Or83b-transfected HEK293 cells revealed the opening of plasma membrane cation channels on addition of ligand. The response was blocked by lanthanum and by 2-APB, but not by Ruthenium red or SKF96365. Based on these data, we conclude that Drosophila Ors comprise a novel family of seven transmembrane receptors that in HEK293 cells signal by opening cation channels, through a mechanism that is largely independent of G proteins.     

Honey bee odorant-binding protein 14: effects on thermal stability upon odorant binding revealed by FT-IR spectroscopy and CD measurements

In the present work, we study the effect of odorant binding on the thermal stability of honey bee (Apis mellifera L.) odorant-binding protein 14. Thermal denaturation of the protein in the absence and presence of different odorant molecules was monitored by Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD). FT-IR spectra show characteristic bands for intermolecular aggregation through the formation of intermolecular β-sheets during the heating process. Transition temperatures in the FT-IR spectra were evaluated using moving-window 2D correlation maps and confirmed by CD measurements. The obtained results reveal an increase of the denaturation temperature of the protein when bound to an odorant molecule. We could also discriminate between high- and low-affinity odorants by determining transition temperatures, as demonstrated independently by the two applied methodologies. The increased thermal stability in the presence of ligands is attributed to a stabilizing effect of non-covalent interactions between odorant-binding protein 14 and the odorant molecule.     

Molecular determinants of odorant receptor function in insects

The olfactory system of Drosophila melanogaster provides a powerful model to study molecular and cellular mechanisms underlying function of a sensory system. In the 1970s Siddiqi and colleagues pioneered the application of genetics to olfactory research and isolated several mutant Drosophila with odorant-specific defects in olfactory behaviour, suggesting that odorants are detected differentially by the olfactory system. Since then basic principles of olfactory system function and development have emerged using Drosophila as a model. Nearly four decades later we can add computational methods to further our understanding of how specific odorants are detected by receptors. Using a comparative approach we identify two categories of short amino acid sequence motifs: ones that are conserved family-wide predominantly in the C-terminal half of most receptors, and ones that are present in receptors that detect a specific odorant, 4-methylphenol, found predominantly in the N-terminal half. The odorant-specific sequence motifs are predictors of phenol detection in Anopheles gambiae and other insects, suggesting they are likely to participate in odorant binding. Conversely, the family-wide motifs are expected to participate in shared functions across all receptors and a mutation in the most conserved motif leads to a reduction in odor response. These findings lay a foundation for investigating functional domains within odorant receptors that can lead to a molecular understanding of odor detection.     

Identification of air phase retronasal and orthonasal odorant pairs

Identifications (IDs) of paired retronasal and orthonasal odorants were studied, with stimuli limited to air phase. Odorants were liquid extracts of plant materials, sold as food flavorings, matched by each subject both for retronasal-only and orthonasal-only air phase intensities and then learned to 100% correct veridical name retronasal-only and orthonasal-only IDs. Subjects were tested for ID of (a) retronasal-only and orthonasal-only odorants, (b) homogeneously paired odorant (the same odorant in retronasal and orthonasal locations), and (c) heterogeneously paired odorants (different odorants in retronasal and orthonasal locations). Paired odorants were presented in two different sequences: retronasal location odorant smelled first or orthonasal location odorant smelled first. IDs were reported after odorants were removed. Results were as follows: (a) no significant differences between correct ID of odorants when in retronasal-only versus orthonasal-only locations, although percent correct IDs were lower for half the retronasal-only location odorants; (b) correct ID of a homogeneously paired odorant equaled or exceeded its unpaired ID, with two successive, identical IDs reported on the majority of its trials; (c) with heterogeneous pairs, for all odorants when in the orthonasal location of a pair, correct ID occurred less often than when these odorants were presented orthonasal-only, but for odorants in the retronasal location, correct ID equaled or exceeded retronasal-only correct ID; and (d) perceived order of presentation of heterogeneous pairs was the reverse of the physically presented sequence for both retronasal-first and orthonasal-first conditions. The heterogeneous odorant ID outcome supports the concept that processing of retronasal and orthonasal odorants differ, and the perceived reversal of the presented sequence is in agreement with the importance of recency in odorant memory.