Insect Odorant Receptors Research Articles

Calmodulin modulates insect odorant receptor function

Insect odorant receptors (ORs) are heteromeric complexes of an odor-specific receptor protein (OrX) and a ubiquitous co-receptor protein (Orco). The ORs operate as non-selective cation channels, also conducting Ca2+ ions. The Orco protein contains a conserved putative calmodulin (CaM)-binding motif indicating a role of CaM in its function. Using Ca2+ imaging to monitor OR activity we investigated the effect of CaM inhibition on the function of OR proteins. Ca2+ responses elicited in Drosophila olfactory sensory neurons by stimulation with the synthetic OR agonist VUAA1 were reduced and prolonged by CaM inhibition with the potent antagonist W7 but not with the weak antagonist W5. A similar effect was observed for Orco proteins heterologously expressed in CHO cells when CaM was inhibited with W7, trifluoperazine or chlorpromazine, or upon overexpression of CaM-EF-hand mutants. With the Orco CaM mutant bearing a point mutation in the putative CaM site (K339N) the Ca2+ responses were akin to those obtained for wild type Orco in the presence of W7. There was no uniform effect of W7 on Ca2+ responses in CHO cells expressing complete ORs (Or22a/Orco, Or47a/Orco, Or33a/Orco, Or56a/Orco). For Or33a and Or47a we observed no significant effect of W7, while it caused a reduced response in cells expressing Or22a and a shortened response for Or56a.

Role of Go/i subgroup of G proteins in olfactory signaling of Drosophila melanogaster

Intracellular signaling in insect olfactory receptor neurons remains unclear, with both metabotropic and ionotropic components being discussed. Here, we investigated the role of heterotrimeric Go and Gi proteins using a combined behavioral, in vivo and in vitro approach. Specifically, we show that inhibiting Go in sensory neurons by pertussis toxin leads to behavioral deficits. We heterologously expressed the olfactory receptor dOr22a in human embryonic kidney cells (HEK293T). Stimulation with an odor led to calcium influx, which was amplified via calcium release from intracellular stores. Subsequent experiments indicated that the signaling was mediated by the Gβγ subunits of the heterotrimeric Go/i proteins. Finally, using in vivo calcium imaging, we show that Go and Gi contribute to odor responses both for the fast (phasic) as for the slow (tonic) response component. We propose a transduction cascade model involving several parallel processes, in which the metabotropic component is activated by Go and Gi, and uses Gβγ.

More than apples and oranges - Detecting cancer with a fruit fly's antenna

Cancer cells and non-cancer cells differ in their metabolism and they emit distinct volatile compound profiles, allowing to recognise cancer cells by their scent. Insect odorant receptors are excellent chemosensors with high sensitivity and a broad receptive range unmatched by current gas sensors. We thus investigated the potential of utilising the fruit fly's olfactory system to detect cancer cells. Using in vivo calcium imaging, we recorded an array of olfactory receptor neurons on the fruit fly's antenna. We performed multidimensional analysis of antenna responses, finding that cell volatiles from different cell types lead to characteristic response vectors. The distances between these response vectors are conserved across flies and can be used to discriminate healthy mammary epithelial cells from different types of breast cancer cells. This may expand the repertoire of clinical diagnostics, and it is the first step towards electronic noses equipped with biological sensors, integrating artificial and biological olfaction.

Analysis of an Insect's Olfactory Receptor Neuron Response by NMF Method for Odor Approximation

Analysis of an Insect's Olfactory Receptor Neuron Response by NMF Method for Odor Approximation

Insect Olfactory Receptors as Essential Detectors for Volatile Chemicals in Biomimetic Odorant Sensors

Insect has a highly sensitive and accurate discrimination capacity for sensing odorants in the environment, though its olfactory system is much simpler than mammalians. Insect olfactory receptor (OR) proteins play key roles in odor detection and discrimination. Unlike the complex olfactory signaling in mammalian, the insect ORs can be directly gated by odorants, and can function without the co-expression of downstream signaling molecules. Therefore, insect ORs hold great promise as detectors in a biosensor for detection of volatile chemicals. Several important OR genes in Drosophila, which respond to explosive vapor or related precursors such as PTEN and 2-ehtyl-hexanol, have been reported. The present work screened and selected OR genes which are sensitive to explosive vapor and related precursors by calcium imaging and electrophysiological techniques. Furthermore, a biomimetic odorant sensor which co-expresses these OR genes and a companion receptor Or83b in living cells is being constructed, in order to classify unknown volatile chemicals and detect specific types of illicit substances. This study provides an alternative way for the construction of odorant sensors, comparing to the traditional electronic noses which are not as sensitive and accurate as natural noses, and would promote the development and application of biomimetic odorant sensors in a range of applications such as security and environmental monitoring.

A Conserved Aspartic Acid Is Important for Agonist (VUAA1) and Odorant/Tuning Receptor-Dependent Activation of the Insect Odorant Co-Receptor (Orco)

Insect odorant receptors function as heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor, and a conserved co-receptor (Orco). An Orco agonist, VUAA1, is able to activate both heteromeric and homomeric Orco-containing channels. Very little is known about specific residues in Orco that contribute to cation permeability and gating. We investigated the importance of two conserved Asp residues, one in each of transmembrane domains 5 and 7, for channel function by mutagenesis. Drosophila melanogaster Orco and its substitution mutants were expressed in HEK cells and VUAA1-stimulated channel activity was determined by Ca2+ influx and whole-cell patch clamp electrophysiology. Substitution of D466 in transmembrane 7 with amino acids other than glutamic acid resulted in a substantial reduction in channel activity. The D466E Orco substitution mutant was ∼2 times more sensitive to VUAA1. The permeability of the D466E Orco mutant to cations was unchanged relative to wild-type Orco. When D466E Orco is co-expressed with a conventional tuning odorant receptor, the heteromeric complex also shows increased sensitivity to an odorant. Thus, the effect of the D466E mutation is not specific to VUAA1 agonism or dependent on homomeric Orco assembly. We suggest the gain-of-activation characteristic of the D466E mutant identifies an amino acid that is likely to be important for activation of both heteromeric and homomeric insect odorant receptor channels.

Female Behaviour Drives Expression and Evolution of Gustatory Receptors in Butterflies

Secondary plant compounds are strong deterrents of insect oviposition and feeding, but may also be attractants for specialist herbivores. These insect-plant interactions are mediated by insect gustatory receptors (Grs) and olfactory receptors (Ors). An analysis of the reference genome of the butterfly Heliconius melpomene, which feeds on passion-flower vines (Passiflora spp.), together with whole-genome sequencing within the species and across the Heliconius phylogeny has permitted an unprecedented opportunity to study the patterns of gene duplication and copy-number variation (CNV) among these key sensory genes. We report in silico gene predictions of 73 Gr genes in the H. melpomene reference genome, including putative CO2, sugar, sugar alcohol, fructose, and bitter receptors. The majority of these Grs are the result of gene duplications since Heliconius shared a common ancestor with the monarch butterfly or the silkmoth. Among Grs but not Ors, CNVs are more common within species in those gene lineages that have also duplicated over this evolutionary time-scale, suggesting ongoing rapid gene family evolution. Deep sequencing (∼1 billion reads) of transcriptomes from proboscis and labial palps, antennae, and legs of adult H. melpomene males and females indicates that 67 of the predicted 73 Gr genes and 67 of the 70 predicted Or genes are expressed in these three tissues. Intriguingly, we find that one-third of all Grs show female-biased gene expression (n = 26) and nearly all of these (n = 21) are Heliconius-specific Grs. In fact, a significant excess of Grs that are expressed in female legs but not male legs are the result of recent gene duplication. This difference in Gr gene expression diversity between the sexes is accompanied by a striking sexual dimorphism in the abundance of gustatory sensilla on the forelegs of H. melpomene, suggesting that female oviposition behaviour drives the evolution of new gustatory receptors in butterfly genomes.

Recombinant expression, detergent solubilisation and purification of insect odorant receptor subunits

Insect odorant receptors (ORs) are seven transmembrane domain proteins that comprise a novel family of ligand-gated non-selective cation channels. The functional channel is made up of an odour activated ligand-binding OR and the OR co-receptor, Orco. However, the structure, stoichiometry and mechanism of activation of the receptor complex are not well understood. Here we demonstrate that baculovirus-mediated Sf9 cell expression and wheat germ cell-free expression, but not Escherichia coli cell-based or cell-free expression, can be used successfully to over-express a selection of insect ORs. From a panel of 19 detergents, 1%w/v Zwittergent 3–16 was able to solubilise five Drosophila melanogaster ORs produced from both eukaryotic expression systems. A large-scale purification protocol was then developed for DmOrco and the ligand-binding receptor, DmOr22a. The proteins were nickel-affinity purified using a deca-histidine tag in a buffer containing 0.2mM Zwittergent 3–16, followed by size exclusion chromatography. These purified ORs appear to form similarly sized protein-detergent complexes when isolated from both expression systems. Circular dichroism analysis of both purified proteins suggests they are folded correctly. We also provide evidence that when DmOrco is expressed in Sf9 cells it undergoes post translational modification, probably glycosylation. Finally we show that the recombinant ORs can be incorporated into pre-formed liposomes. The ability to recombinantly express and purify insect ORs to homogeneity on a preparative scale, as well as insert them into liposomes, is a major step forward in enabling future structural and functional studies, as well as their use in OR based biosensors.

Orco mutant mosquitoes lose strong preference for humans and are not repelled by volatile DEET

Female mosquitoes of some species are generalists and will blood-feed on a variety of vertebrate hosts, whereas others display marked host preference. Anopheles gambiae and Aedes aegypti have evolved a strong preference for humans, making them dangerously efficient vectors of malaria and Dengue haemorrhagic fever1. Specific host odours likely drive this strong preference since other attractive cues, including body heat and exhaled carbon dioxide (CO2) are common to all warm-blooded hosts2, 3. Insects sense odours via several chemosensory receptor families, including the odorant receptors (ORs). ORs are membrane proteins that form heteromeric odour-gated ion channels4, 5 comprised of a variable ligand-selective subunit and an obligate co-receptor called Orco6. Here we use zinc-finger nucleases to generate targeted mutations in the Ae. aegypti orco gene to examine the contribution of Orco and the OR pathway to mosquito host selection and sensitivity to the insect repellent DEET. orco mutant olfactory sensory neurons have greatly reduced spontaneous activity and lack odour-evoked responses. Behaviourally, orco mutant mosquitoes have severely reduced attraction to honey, an odour cue related to floral nectar, and do not respond to human scent in the absence of CO2. However, in the presence of CO2, female orco mutant mosquitoes retain strong attraction to both human and animal hosts, but no longer strongly prefer humans. orco mutant females are attracted to human hosts even in the presence of DEET, but are repelled upon contact, indicating that olfactory- and contact-mediated effects of DEET are mechanistically distinct. We conclude that the OR pathway is crucial for an anthropophilic vector mosquito to discriminate human from non-human hosts and to be effectively repelled by volatile DEET.

Probing insect odorant receptors with their cognate ligands: Insights into structural features

Odorant receptors (ORs) are essential for insect survival in the environment and thus are ideal molecular targets for the design of insect-inspired modern green chemicals to control populations of agricultural pests and insects of medical importance. Although insect ORs are known for more than a decade, their structural biology is still in its infancy. Here, we unravel the first structural features of ORs from the malaria mosquito, the Southern house mosquito and the silkworm moth. The second extracellular loops (ECL-2s) of their predicted structures are much longer than ECL-1s and ECL-3s. The 27 amino-acid-residue-long of the ECL-2s in mosquito and the 43 amino-acid-residue-long ECL2s in moth ORs are well-conserved. About one-third of the residues are identical, including 3–4 Pro residues. Thorough examination of well-conserved residues in these structures, by point mutation and functional assay with the Xenopus oocyte recording system, strongly suggest that these “loops” include three β-turns and some degree of folding. In the Southern house mosquito three Pro residues in ECL-2 are essential for full activation of the receptor, which is finely tuned to the oviposition attractant 3-methylindole. Additionally, the “corner residues” of prolines, including Gly, Tyr, and Leu are functionally important thus suggesting that turns are stabilized not only by backbone hydrogen bonds, but also by side–chain interactions. Examination of ECL-2s from a distant taxonomical group suggests these ECL-2 loops might be functionally important in all insect ORs. Two of the four Pro residues in the predicted ECL-2 of the bombykol receptor in the silkworm moth, BmorOR1, are essential for function. Experimental evidence indicates that these loops may not be specificity determinants, but they may form a cover to the yet-to-be-identified membrane embedded binding cavities of insect ORs.

In situ Tip-Recordings Found No Evidence for an Orco-Based Ionotropic Mechanism of Pheromone-Transduction in Manduca sexta

The mechanisms of insect odor transduction are still controversial. Insect odorant receptors (ORs) are 7TM receptors with inverted membrane topology. They colocalize with a conserved coreceptor (Orco) with chaperone and ion channel function. Some studies suggest that insects employ exclusively ionotropic odor transduction via OR-Orco heteromers. Other studies provide evidence for different metabotropic odor transduction cascades, which employ second messenger-gated ion channel families for odor transduction. The hawkmoth Manduca sexta is an established model organism for studies of insect olfaction, also due to the availability of the hawkmoth-specific pheromone blend with its main component bombykal. Previous patch-clamp studies on primary cell cultures of M. sexta olfactory receptor neurons provided evidence for a pheromone-dependent activation of a phospholipase Cβ. Pheromone application elicited a sequence of one rapid, apparently IP3-dependent, transient and two slower Ca2+-dependent inward currents. It remains unknown whether additionally an ionotropic pheromone-transduction mechanism is employed. If indeed an OR-Orco ion channel complex underlies an ionotropic mechanism, then Orco agonist-dependent opening of the OR-Orco channel pore should add up to pheromone-dependent opening of the pore. Here, in tip-recordings from intact pheromone-sensitive sensilla, perfusion with the Orco agonist VUAA1 did not increase pheromone-responses within the first 1000 ms. However, VUAA1 increased spontaneous activity of olfactory receptor neurons Zeitgebertime- and dose-dependently. We conclude that we find no evidence for an Orco-dependent ionotropic pheromone transduction cascade in M. sexta. Instead, in M. sexta Orco appears to be a slower, second messenger-dependent pacemaker channel which affects kinetics and threshold of pheromone-detection via changes of intracellular Ca2+ baseline concentrations.

Differential expression of two novel odorant receptors in the locust (Locusta migratoria)

BackgroundOlfaction in animals is important for host localization, mating and reproduction in heterogeneous chemical environments. Studying the molecular basis of olfactory receptor neurons (ORNs) systems can elucidate the evolution of olfaction and associated behaviours. Odorant receptors (ORs) in insects have been identified, particularly in the holometabolous model Drosophila, and some of them have been functionally studied. However, ORs in the locust—a hemimetabolous model insect and the most important insect crop pest—have not yet been identified, hindering our understanding of locust olfaction. Here, we report for the first time four putative ORs in Locusta migratoria: LmigOR1, LmigOR2, LmigOR3 and LmigOR4.ResultsThese four putative OR genes encoded proteins with amino acids of 478, 436, 413 and 403 respectively. Sequence identity among them ranged from 19.7% to 35.4%. All ORs were tissue-specifically expressed in olfactory organs, without sex-biased characteristics. However, LmigOR1, LmigOR3 and LmigOR4 were only expressed in the antenna, while LmigOR2 could also be detected in mouthparts. In situ hybridization demonstrated that the LmigOR1antisense probe labelled olfactory receptor neurons (ORNs) in almost all segments of the antenna, but only a few segments housed ORNs expressing LmigOR2. The number of neurons labelled by LmigOR1 antisense probes in each antennal segment was much greater (>10 neurons/segment) than that labelled by LmigOR2 probes (generally 1–3 neurons/segment). Furthermore, some of the labelled neurons could be attributed to the basiconic sensilla, but LmigOR1 and LmigOR2 were expressed in different subtypes.ConclusionsOur results strongly suggested that these newly discovered genes encode locust ORs and the differential expression patterns of LmigOR1 and LmigOR2 implied distinct functions. These results may offer insights into locust olfaction and contribute to the understanding of the evolution of insect chemoreception.

Insect Odorant Response Sensitivity Is Tuned by Metabotropically Autoregulated Olfactory Receptors

Insects possess one of the most exquisitely sensitive olfactory systems in the animal kingdom, consisting of three different types of chemosensory receptors: ionotropic glutamate-like receptors (IRs), gustatory receptors (GRs) and odorant receptors (ORs). Both insect ORs and IRs are ligand-gated ion channels, but ORs possess a unique configuration composed of an odorant-specific protein OrX and a ubiquitous coreceptor (Orco). In addition, these two ionotropic receptors confer different tuning properties for the neurons in which they are expressed. Unlike IRs, neurons expressing ORs are more sensitive and can also be sensitized by sub-threshold concentrations of stimuli. What is the mechanistic basis for these differences in tuning? We show that intrinsic regulation of Orco enhances neuronal response to odorants and sensitizes the ORs. We also demonstrate that inhibition of metabotropic regulation prevents receptor sensitization. Our results indicate that Orco-mediated regulation of OR sensitivity provides tunable ionotropic receptors capable of detecting odors over a wider range of concentrations, providing broadened sensitivity over IRs themselves.

Amiloride Derivatives Are Effective Blockers of Insect Odorant Receptors

Heteromeric insect odorant receptors (ORs) form ligand-activated nonselective cation channels in recombinant expression systems. We performed a pharmacological characterization of Drosophila melanogaster and Bombyx mori ORs expressed in the Xenopus laevis oocyte expression system and characterized them using the 2-electrode voltage clamp. We identified amiloride derivatives as high-affinity blockers, which inhibit the ion current through the channel in a low micromolar range. For the heteromeric Drosophila Or47a + DmelOrco receptor, the potency sequence (IC(50)) is HMA [5-(N,N-hexamethylene)amiloride] (3.9 µM), MIA [5-(N-methyl-N-isobutyl)amiloride] (11.0 µM), and DMA [5-(N,N-dimethyl)amiloride] (113.3 µM). Amiloride itself is nearly ineffective. Other tested insect ORs (Drosophila Or49b + DmelOrco, B. mori BmorOr1 + BmorOrco) were blocked in a similar fashion suggesting that the amiloride derivatives were potential general blockers of all receptor combinations. Our results suggest that pyrazine derivatives of amiloride are useful probes to study the mechanism of chemosensory transduction in insects in more detail.

Blockade of Insect Odorant Receptor Currents by Amiloride Derivatives

Insect odorant receptors (ORs) function as heteromeric odorant-gated ion channels consisting of a conserved coreceptor, Orco, and an odorant-sensitive tuning subunit. Although some OR modulators have been identified, an extended library of pharmacological tools is currently lacking and would aid in furthering our understanding of insect OR complexes. We now demonstrate that amiloride and several derivatives, which have been extensively used as blockers for various ion channels and transporters, also block odorant-gated currents from 2 OR complexes from the malaria vector mosquito Anopheles gambiae. In addition, both heteromeric and homomeric ORs were susceptible to amiloride blockade when activated by VUAA1, an agonist that targets the Orco channel subunit. Amiloride derivatives therefore represent a valuable class of channel blockers that can be used to investigate the pharmacological and biophysical properties of insect OR function.

Sensory receptors—design principles revisited

Sensory receptors—design principles revisited

Bestrophin-Encoded Ca2+-Activated Cl− Channels Underlie a Current with Properties Similar to the Native Current in the Moth Spodoptera littoralis Olfactory Receptor Neurons

Responses of insect olfactory receptor neurons (ORNs) involve an entry of Ca2+ through olfactory heterodimeric receptor complexes. In moths, the termination of ORN responses was found to strongly depend on the external Ca2+ concentration through the activation of unknown Ca2+-dependent Cl− channels. We thus investigated the molecular identity of these Cl− channels. There is compelling evidence that bestrophins form Cl− channels when expressed in heterologous systems. Here we provide evidence that antennae of the moth Spodoptera littoralis express three transcripts encoding proteins with hallmarks of bestrophins. One of these transcripts, SlitBest1b, is expressed in ORNs. The heterologous expression of SlitBest1b protein in CHO-K1 cells yielded a Ca2+-activated Cl− current that shares electrophysiological properties with the native Ca2+-activated Cl− current of ORNs. Both currents are anionic, present similar dependence on the intracellular Ca2+ concentration, partly inactivate over time, have the same anion permeability sequence, the same sequence of inhibitory efficiency of blockers, the same almost linear I–V relationships and finally both currents do not depend on the cell volume. Therefore, our data suggest that SlitBest1b is a good candidate for being a molecular component of the olfactory Ca2+-activated Cl− channel and is likely to constitute part of the insect olfactory transduction pathway. A different function (e.g. regulation of other proteins, maintenance of the anionic homeostasis in the sensillar lymph) and a different role (e.g. involvement in the olfactory system development) cannot be excluded however.

A Conserved Dedicated Olfactory Circuit for Detecting Harmful Microbes in Drosophila

Flies, like all animals, need to find suitable and safe food. Because the principal food source for Drosophila melanogaster is yeast growing on fermenting fruit, flies need to distinguish fruit with safe yeast from yeast covered with toxic microbes. We identify a functionally segregated olfactory circuit in flies that is activated exclusively by geosmin. This microbial odorant constitutes an ecologically relevant stimulus that alerts flies to the presence of harmful microbes. Geosmin activates only a single class of sensory neurons expressing the olfactory receptor Or56a. These neurons target the DA2 glomerulus and connect to projection neurons that respond exclusively to geosmin. Activation of DA2 is sufficient and necessary for aversion, overrides input from other olfactory pathways, and inhibits positive chemotaxis, oviposition, and feeding. The geosmin detection system is a conserved feature in the genus Drosophila that provides flies with a sensitive, specific means of identifying unsuitable feeding and breeding sites.

Insights into subunit interactions within the insect olfactory receptor complex using FRET

Insect olfactory receptors (ORs) are a novel family of ligand-gated cation channels that can respond to volatile organic compounds at low concentrations. They are involved in the detection of odorants associated with mate recognition, food localisation and predator avoidance. These receptors form a complex that is currently thought to contain at least two subunit members: the non-canonical Orco ion channel subunit and a ligand-binding receptor subunit. The integral membrane proteins SNMP1 and 2 are also associated with olfactory function, with SNMP1 required for cis-vaccinyl acetate reception in Drosophila melanogaster. In order to investigate protein–protein interactions among these membrane proteins we measured intermolecular Förster/Fluorescence Resonance Energy Transfer (FRET) in live insect cells by acceptor photobleaching. Fusion proteins containing Cyan Fluorescent Protein or Yellow Fluorescent Protein were produced using baculovirus-mediated expression in High Five™ cells. The majority of the recombinant products were of the expected size for the fusion proteins and located within intracellular membranes. We were able to show FRET efficiencies providing evidence for homomeric and heteromeric interactions of the ligand-binding OR, Or22a, and Orco (Or22a–Or22a, Or22a–Orco, Orco–Orco). There was no evidence for an interaction between SNMP1 and Orco or between SNMP2 and Orco or Or22a. However, fusion proteins of SNMP1 and Or22a did show an interaction by FRET, suggesting SNMP1 may interact with at least some insect olfactory receptor complexes. In summary, this study supports previously observed homomeric and heteromeric interactions between Orco and the ligand-binding OR, Or22a, and identifies a novel interaction between Or22a and SNMP1.

Molecular cloning and functional identification of an insect odorant receptor gene in Spodoptera litura (F.) for the botanical insecticide rhodojaponin III

Specific behaviors of insects are stimulated by the recognition of chemicals in the external environment by sensilla on chemoreceptive organs. Rhodojaponin III is a nonvolatile natural botanical insecticide, which has an intense effect of deterring oviposition activity against many insects. This study aimed to determine whether the Or83b protein is involved in the recognition of rhodojaponin III. The SlOr83b protein has a conserved seven-transmembrane domain and a putative receptor/receptor interaction loop. The Or83b mRNA is first transcribed in 1st larvae, and expressed in later larval stages. A high level of Or83b mRNA is expressed in pupal and adult stages. Knockdown of SlOr83b expression was achieved through RNAi by injecting double-stranded RNA (dsRNA) into the 8th day pupae, and oviposition significantly increased on rhodojaponin III treated cabbage leaves. These results suggest that Or83b is critical for insects to identify the nonvolatile the insecticide rhodojaponin III.