Odorant inhibition in mosquito olfaction mediated by inverse agonists.

The insect repellent methyl salicylate elicits excitatory responses upon interaction with CquiOR32, an odorant receptor (OR) from the southern house mosquito, Culex quinquefasciatus. By contrast, eucalyptol binds to CquiOR32 to generate electrophysiological and behavioral inhibitory responses. In an attempt to identify CquiOR32 variants displaying more robust inhibitory responses for more accurate current-voltage analysis, we sequenced 31 CquiOR32 clones. In the Xenopus oocyte recording system, CquiOR32V2/CquiOrco-expressing oocytes yielded eucalyptol-elicited outward (inhibitory) currents relatively larger than methyl salicylate-generated inward (excitatory) currents. Rescuing experiments showed that two of the amino acid substitutions in CquiOR32V2 located in a predicted transmembrane helix of the receptor are determinants of the outward/inward ratios. These findings, along with co-stimulus assays, suggest that odorant and inhibitor may bind to the same binding pocket. Current-voltage relationships obtained with standard perfusion buffer and those devoid of Na+ or Cl- indicated that both excitatory and inhibitory currents are mediated, at least in part, by cation. We then concluded that eucalyptol is an inverse agonist, which shifts the open ⇔ closed equilibrium of the receptor toward the closed conformation, thus reducing the spontaneous activity. By contrast, the binding of methyl salicylate shifts the equilibrium towards the open conformation and, consequently, leads to an increase in cation influx.

Reverse chemical ecology approach for the identification of an oviposition attractant for Culex quinquefasciatus.

Pheromones and other semiochemicals play a crucial role in today's integrated pest and vector management strategies. These semiochemicals are typically discovered by bioassay-guided approaches. Here, we applied a reverse chemical ecology approach; that is, we used olfactory proteins to lead us to putative semiochemicals. Specifically, we used 7 of the top 10 odorant receptors (ORs) most expressed in the antennae of the southern house mosquito, Culex quinquefasciatus, and which are yet to be deorphanized. We expressed these receptors in the Xenopus oocyte recording system and challenged them with a panel of 230 odorants, including physiologically and behaviorally active compounds. Six of the ORs were silent either because they are not functional or a key odorant was missing. CquiOR36, which showed the highest transcript levels of all OR genes in female antennae, was also silent to all odorants in the tested panel, but yielded robust responses when it was accidentally challenged with an old sample of nonanal in ethanol. After confirming that fresh samples were inactive and through a careful investigation of all possible "contaminants" in the old nonanal samples, we identified the active ligand as acetaldehyde. That acetaldehyde is activating CquiOR36 was further confirmed by electroantennogram recordings from antennae of fruit flies engineered to carry CquiOR36. Antennae of female mosquitoes also responded to acetaldehyde. Cage oviposition and dual-choice assays demonstrated that acetaldehyde is an oviposition attractant in a wide range of concentrations and thus of potential practical applications.

Functional and Nonfunctional Forms of CquiOR91, an Odorant Selectivity Subunit of Culex quinquefasciatus.

In Culex quinquefasciatus, CquiOR91 is the ortholog of 2 larvae-specific odorant receptors (ORs) from Anopheles gambiae (Agam\Or40, previously shown to respond to several odorant ligands including the broad-spectrum repellent N,N-diethyl-3-methylbenzamide, DEET) and Aedes aegypti (Aaeg\Or40). When we cloned full-length CquiOR91 from a Culex quinquefasciatus larval head RNA sample, we found 2 alleles of this OR, differing at 9 residues. Functional analysis using the Xenopus oocyte expression system and 2-electrode voltage clamp electrophysiology revealed one allele (CquiOR91.1) to be nonfunctional, whereas the other allele (CquiOR91.2) was functional. Receptors formed by CquiOR91.2 and Cqui\Orco responded to (-)-fenchone, (+)-fenchone, and DEET, similar to what has been reported for Agam\Or40. We also identified 5 novel odorant ligands for the CquiOR91.2 + Cqui\Orco receptor: 2-isobutylthiazole, veratrole, eucalyptol, d-camphor, and safranal, with safranal being the most potent. To explore possible reasons for the lack of function for CquiOR91.1, we generated a series of mutant CquiOR91.2 subunits, in which the residue at each of the 9 polymorphic residue positions was changed from what occurs in CquiOR91.2 to what occurs in CquiOR91.1. Eight of the 9 mutant versions of CquiOR91.2 formed functional receptors, responding to (-)-fenchone. Only the CquiOR91.2 Y183C mutant was nonfunctional. The reverse mutation (C183Y) conferred function on CquiOR91.1 , which became responsive to (-)-fenchone and safranal. These results indicate that the "defect" in CquiOR91.1 that prevents function is the cysteine at position 183.

Mosquito odorant receptor for DEET and methyl jasmonate.

Insect repellents are important prophylactic tools for travelers and populations living in endemic areas of malaria, dengue, encephalitis, and other vector-borne diseases. DEET (N,N-diethyl-3-methylbenzamide) is a 6-decade-old synthetic repellent, which is still considered the gold standard of mosquito repellents. Mosquitoes use their sense of smell to detect DEET, but there are currently two hypotheses regarding its mode of action: activation of ionotropic receptor IR40a vs. odorant receptor(s). Here, we demonstrate that DEET, picaridin, insect repellent 3535, and p-menthan-3,8-diol activate the odorant receptor CquiOR136 of the southern house mosquito, Culex quinquefasciatus. Electrophysiological and behavioral assays showed that CquiIR40a knockdown had no significant effect on DEET detection and repellency. By contrast, reduction of CquiOR136 transcript levels led to a significant decrease in electroantennographic responses to DEET and a complete lack of repellency. Thus, direct activation of an odorant receptor, not an ionotropic receptor, is necessary for DEET reception and repellency in Culex mosquitoes. Interestingly, methyl jasmonate, a repellent derived from the nonvolatile jasmonic acid in the signaling pathway of plant defenses, elicited robust responses in CquiOR136•CquiOrco-expressing Xenopus oocytes, thus suggesting a possible link between natural products with long insect-plant evolutionary history and synthetic repellents.

Pheromone discrimination by a pH-tuned polymorphism of the Bombyx mori pheromone-binding protein.

The Bombyx mori pheromone-binding protein (BmorPBP) is known to adopt two different conformations. These are BmorPBP(A), where a regular helix formed by the C-terminal dodecapeptide segment, α7, occupies the ligand-binding cavity, and BmorPBP(B), where the binding site is free to accept ligands. NMR spectra of delipidated BmorPBP solutions at the physiological pH of the bulk sensillum lymph near pH 6.5 show only BmorPBP(A), and in mixtures, the two species are in slow exchange on the chemical shift frequency scale. This equilibrium has been monitored at variable pH and ligand concentrations, demonstrating that it is an intrinsic property of BmorPBP that is strongly affected by pH variation and ligand binding. This polymorphism tunes BmorPBP for optimal selective pheromone transport: Competition between α7 and lipophilic ligands for its binding cavity enables selective uptake of bombykol at the pore endings in the sensillum wall, whereas compounds with lower binding affinity can only be bound in the bulk sensillum lymph. After transport across the bulk sensillum lymph into the lower pH area near the dendritic membrane surface, bombykol is ejected near the receptor, whereas compounds with lower binding affinity are ejected before reaching the olfactory receptor, rendering them susceptible to degradation by enzymes present in the sensillum lymph.

Differential expression of olfactory genes in the southern house mosquito and insights into unique odorant receptor gene isoforms.

The southern house mosquito, Culex quinquefasciatus, has one of the most acute and eclectic olfactory systems of all mosquito species hitherto studied. Here, we used Illumina sequencing to identify olfactory genes expressed predominantly in antenna, mosquito's main olfactory organ. Less than 50% of the trimmed reads generated by high-quality libraries aligned to a transcript, but approximately 70% of them aligned to the genome. Differential expression analysis, which was validated by quantitative real-time PCR on a subset of genes, showed that approximately half of the 48 odorant-binding protein genes were enriched in antennae, with the other half being predominantly expressed in legs. Similar patterns were observed with chemosensory proteins, "plus-C" odorant-binding proteins, and sensory neuron membrane proteins. Transcripts for as many as 43 ionotropic receptors were enriched in female antennae, thus making the ionotropic receptor family the largest of antennae-rich olfactory genes, second only to odorant receptor (OR) genes. As many as 177 OR genes have been identified, including 36 unique transcripts. The unique OR genes differed from previously annotated ORs in internal sequences, splice variants, and extended N or C terminus. One of the previously unknown transcripts was validated by cloning and functional expression. When challenged with a large panel of physiologically relevant compounds, CquiOR95b responded in a dose-dependent manner to ethyl 2-phenylacteate, which was demonstrated to repel Culex mosquitoes, and secondarily to citronellal, a known insect repellent. This transcriptome study led to identification of key molecular components and a repellent for the southern house mosquito.

Facile functional analysis of insect odorant receptors expressed in the fruit fly: validation with receptors from taxonomically distant and closely related species.

With the advent of genomic sequences and next-generation sequencing technologies (RNA-Seq), multiple repertoires of olfactory proteins in various insect species are being unraveled. However, functional analyses are lagging behind due in part to the lack of simple and reliable methods for heterologous expression of odorant receptors (ORs). While the Xenopus oocyte recording system fulfills some of this lacuna, this system is devoid of other olfactory proteins, thus testing only the "naked" ORs. Recently, a moth OR was expressed in the majority of neurons in the antennae of the fruit fly using Orco-GAL4 to drive expression of the moth OR. Electroantennogram (EAG) was used to de-orphanize the moth OR, but generic application of this approach was brought to question. Here, we describe that this system works with ORs not only from taxonomically distant insect species (moth), but also closely related species (mosquito), even when the fruit fly has highly sensitive innate ORs for the odorant being tested. We demonstrate that Orco-GAL4 flies expressing the silkworm pheromone receptor, BmorOR1, showed significantly higher responses to the sex pheromone bombykol than the control lines used to drive expression. Additionally, we show that flies expressing an OR from the Southern house mosquito, CquiOR2, gave significantly stronger responses to the cognate odorants indole and 2-methylphenol than the "background noise" recorder from control lines. In summary, we validate the use of Orco-GAL4 driven UAS-OR lines along with EAG analysis as a simple alternative for de-orphanization and functional studies of insect ORs in an intact olfactory system.

Unusual macrocyclic lactone sex pheromone of Parcoblatta lata, a primary food source of the endangered red-cockaded woodpecker.

Wood cockroaches in the genus Parcoblatta, comprising 12 species endemic to North America, are highly abundant in southeastern pine forests and represent an important prey of the endangered red-cockaded woodpecker, Picoides borealis. The broad wood cockroach, Parcoblatta lata, is among the largest and most abundant of the wood cockroaches, constituting >50% of the biomass of the woodpecker's diet. Because reproduction in red-cockaded woodpeckers is affected dramatically by seasonal and spatial changes in arthropod availability, monitoring P. lata populations could serve as a useful index of habitat suitability for woodpecker conservation and forest management efforts. Female P. lata emit a volatile, long-distance sex pheromone, which, once identified and synthesized, could be deployed for monitoring cockroach populations. We describe here the identification, synthesis, and confirmation of the chemical structure of this pheromone as (4Z,11Z)-oxacyclotrideca-4,11-dien-2-one [= (3Z,10Z)-dodecadienolide; herein referred to as "parcoblattalactone"]. This macrocyclic lactone is a previously unidentified natural product and a previously unknown pheromonal structure for cockroaches, highlighting the great chemical diversity that characterizes olfactory communication in cockroaches: Each long-range sex pheromone identified to date from different genera belongs to a different chemical class. Parcoblattalactone was biologically active in electrophysiological assays and attracted not only P. lata but also several other Parcoblatta species in pine forests, underscoring its utility in monitoring several endemic wood cockroach species in red-cockaded woodpecker habitats.

Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes.

Our knowledge of the molecular basis of odorant reception in insects has grown exponentially over the past decade. Odorant receptors (ORs) from moths, fruit flies, mosquitoes, and the honey bees have been deorphanized, odorant-degrading enzymes (ODEs) have been isolated, and the functions of odorant-binding proteins (OBPs) have been unveiled. OBPs contribute to the sensitivity of the olfactory system by transporting odorants through the sensillar lymph, but there are competing hypotheses on how they act at the end of the journey. A few ODEs that have been demonstrated to degrade odorants rapidly may act in signal inactivation alone or in combination with other molecular traps. Although ORs in Drosophila melanogaster respond to multiple odorants and seem to work in combinatorial code involving both periphery and antennal lobes, reception of sex pheromones by moth ORs suggests that their labeled lines rely heavily on selectivity at the periphery.

Circabidian rhythm of sex pheromone reception in a scarab beetle.

Animals have endogenous clocks that regulate their behavior and physiology. These clocks rely on environmental cues (time givers) that appear approximately every 24 h due to the Earth's rotation; thus, most insects exhibit a circadian rhythm. One notable exception is the scarab beetle, Holotrichia parallela, a severe agricultural pest in China, Japan, South Korea, and India. Females emerge from the soil every other night, reach the canopy of host plants, evert an abdominal gland, and release a pheromone bouquet comprising l-isoleucine methyl ester (LIME) and l-linalool. To determine whether this circa'bi'dian rhythm affects the olfactory system, we aimed to identify H. parallela sex pheromone receptor(s) and study their expression patterns. We cloned 14 odorant receptors (ORs) and attempted de-orphanizing them in the Xenopus oocyte recording system. HparOR14 gave robust responses to LIME and smaller responses to l-linalool. Structural modeling, tissue expression profile, and RNAi treatment followed by physiological and behavioral studies support that HparOR14 is a sex pheromone receptor-the first of its kind discovered in Coleoptera. Examination of the HparOR14 transcript levels throughout the adult's life showed that on sexually active days, gene expression was significantly higher in the scotophase than in the photophase. Additionally, the HparOR14 expression profile showed a circabidian rhythm synchronized with the previously identified pattern of sex pheromone emission. 48 h of electroantennogram recordings showed that responses to LIME were abolished on non-calling nights. In contrast, responses to the green leaf volatile (Z)-3-henexyl acetate remained almost constant throughout the recording period.

The greening-causing agent alters the behavioral and electrophysiological responses of the Asian citrus psyllid to a putative sex pheromone.

The Asian Citrus Psyllid (ACP), Diaphorina citri, is a vector of the pathological bacterium Candidatus Liberibacter asiaticus (CLas), which causes the most devastating disease to the citrus industry worldwide, known as greening or huanglongbing (HLB). Earlier field tests with an acetic acid-based lure in greening-free, 'Valencia' citrus orange groves in California showed promising results. The same type of lures tested in São Paulo, Brazil, showed unsettling results. During the unsuccessful trials, we noticed a relatively large proportion of females in the field, ultimately leading us to test field-collected males and females for Wolbachia and CLas. The results showed high rates of Wolbachia and CLas infection in field populations. We then compared the olfactory responses of laboratory-raised, CLas-free, and CLas-infected males to acetic acid. As previously reported, CLas-uninfected males responded to acetic acid at 1 µg. Surprisingly, CLas-infected males required 50 × higher doses of the putative sex pheromone, thus explaining the failure to capture CLas-infected males in the field. CLas infection was also manifested in electrophysiological responses. Electroantennogram responses from CLas-infected ACP males were significantly higher than those obtained with uninfected males. To the best of our knowledge, this is the first report of a pathogen infection affecting a vector's response to a sex attractant.

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 ß-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.

Bombykol receptors in the silkworm moth and the fruit fly.

Male moths are endowed with odorant receptors (ORs) to detect species-specific sex pheromones with remarkable sensitivity and selectivity. We serendipitously discovered that an endogenous OR in the fruit fly, Drosophila melanogaster, is highly sensitive to the sex pheromone of the silkworm moth, bombykol. Intriguingly, the fruit fly detectors are more sensitive than the receptors of the silkworm moth, although its ecological significance is unknown. By expression in the "empty neuron" system, we identified the fruit fly bombykol-sensitive OR as DmelOR7a (= DmOR7a). The profiles of this receptor in response to bombykol in the native sensilla (ab4) or expressed in the empty neuron system (ab3 sensilla) are indistinguishable. Both WT and transgenic flies responded with high sensitivity, in a dose-dependent manner, and with rapid signal termination. In contrast, the same empty neuron expressing the moth bombykol receptor, BmorOR1, demonstrated low sensitivity and slow signal inactivation. When expressed in the trichoid sensilla T1 of the fruit fly, the neuron housing BmorOR1 responded with sensitivity comparable to that of the native trichoid sensilla in the silkworm moth. By challenging the native bombykol receptor in the fruit fly with high doses of another odorant to which the receptor responds with the highest sensitivity, we demonstrate that slow signal termination is induced by overdose of a stimulus. As opposed to the empty neuron system in the basiconic sensilla, the structural, biochemical, and/or biophysical features of the sensilla make the T1 trichoid system of the fly a better surrogate for the moth receptor.

Crystal and solution structures of an odorant-binding protein from the southern house mosquito complexed with an oviposition pheromone.

Culex mosquitoes introduce the pathogens responsible for filariasis, West Nile virus, St. Louis encephalitis, and other diseases into humans. Currently, traps baited with oviposition semiochemicals play an important role in detection efforts and could provide an environmentally friendly approach to controlling their populations. The odorant binding proteins (OBPs) in the female's antenna play a crucial, if yet imperfectly understood, role in sensing oviposition cues. Here, we report the X-ray crystallography and NMR 3D structures of OBP1 for Culex quinquefasciatus (CquiOBP1) bound to an oviposition pheromone (5R,6S)-6-acetoxy-5-hexadecanolide (MOP). In both studies, CquiOBP1 had the same overall six-helix structure seen in other insect OBPs, but a detailed analysis revealed an important previously undescribed feature. There are two models for OBP-mediated signal transduction: (i) direct release of the pheromone from an internal binding pocket in a pH-dependent fashion and (ii) detection of a pheromone-induced conformational change in the OBP·pheromone complex. Although CquiOBP1 binds MOP in a pH-dependent fashion, it lacks the C terminus required for the pH-dependent release model. This study shows that CquiOBP binds MOP in an unprecedented fashion using both a small central cavity for the lactone head group and a long hydrophobic channel for its tail.

Specificity of the receptor for the major sex pheromone component in Heliothis virescens.

In a previous study, the Drosophila melanogaster OR67d(GAL4);UAS system was used to functionally characterize the receptor for the major component of the sex pheromone in the tobacco budworm, Heliothis virescens Fabricius (Lepidoptera: Noctuidae), HvOR13. Electrophysiological and behavioral assays showed that transgenic flies expressing HvOR13 responded to (Z)-11-hexadecenal (Z11-16:Ald). However, tests were not performed to determine whether these flies would also respond to secondary components of the H. virescens sex pheromone. Thus, in this study the response spectrum of HvOR13 expressed in this system was examined by performing single cell recordings from odor receptor neuron in trichoid T1 sensilla on antennae of two Or67d(GAL4 [1]); UAS-HvOR13 lines stimulated with Z11-16:Ald and six H. virescens secondary pheromone components. Fly courtship assays were also performed to examine the behavioral response of the Or67d(GAL4[1]); UAS-HvOR13 flies to Z11-16:Ald and the secondary component Z9-14:Ald. Our combined electrophysiological and behavioral studies indicated high specificity and sensitivity of HvOR13 to Z11-16:Ald. Interestingly, a mutation leading to truncation in the HvOR13 C-terminal region affected but did not abolish pheromone receptor response to Z11-16:Ald. The findings are assessed in relationship to other HvOR13 heterologous expression studies, and the role of the C-terminal domain in receptor function is discussed. A third line expressing HvOR15 was also tested but did not respond to any of the seven pheromone components.

Acute olfactory response of Culex mosquitoes to a human- and bird-derived attractant.

West Nile virus, which is transmitted by Culex mosquitoes while feeding on birds and humans, has emerged as the dominant vector borne disease in North America. We have identified natural compounds from humans and birds, which are detected with extreme sensitivity by olfactory receptor neurons (ORNs) on the antennae of Culex pipiens quinquefasciatus (Cx. quinquefasciatus). One of these semiochemicals, nonanal, dominates the odorant spectrum of pigeons, chickens, and humans from various ethnic backgrounds. We determined the specificity and sensitivity of all ORN types housed in different sensilla types on Cx. quinquefasciatus antennae. Here, we present a comprehensive map of all antennal ORNs coding natural ligands and their dose-response functions. Nonanal is detected by a large array of sensilla and is by far the most potent stimulus; thus, supporting the assumption that Cx. quinquefasciatus can smell humans and birds. Nonanal and CO(2) synergize, thus, leading to significantly higher catches of Culex mosquitoes in traps baited with binary than in those with individual lures.

Mosquito odorant receptor sensitive to natural spatial repellents and inhibitory compounds.

Previously, we have identified an odorant receptor (OR) from the southern house mosquito Culex quinquefasciatus, CquiOR32, which responded to both odorants (agonists) and inhibitory compounds (antagonists). CquiOR32/CquiOrco-expressing oocytes responded to methyl salicylate and other odorants with inward (regular) currents but gave currents in the reverse direction when challenged with eucalyptol and other inhibitors. To determine whether hitherto unknown ORs show this intrareceptor inhibition, we have now examined two other receptors in the same cluster, CquiOR27 and CquiOR28. We cloned and tested four variants of CquiOR28, but none of the 250 compounds in our panel of odorants, including an Orco ligand candidate (OLC12), elicited inward or upward deflections of the current traces. By contrast, CquiOR27/CquiOrco-expressing oocytes gave robust, dose-dependent inward currents when challenged with γ-octalactone and other odorants. On the other hand, octylamine and other phenolic compounds elicited dose-dependent currents in the reverse direction. When stimulatory and inhibitory compounds were presented in binary mixtures, γ-octalactone-elicited inward currents were attenuated in a dose-dependent manner according to the concentration of octylamine. As part of our chemical ecology approach, we tested the repellency activity of the most potent ligands in the surface landing and feeding assay and a newly reported hand-in cage assay. Protection elicited by γ-octalactone did not differ significantly from that of DEET at the same dose. In the hand-in cage assay, a cream formulation of γ-octalactone showed 97.0 ± 1.3% protection, with 47.6 ± 8.3% and 1.4 ± 0.7% landings per trial in the hands covered with a control and γ-octalactone cream, respectively (N = 8, p = 0.0078, Wilcoxon matched-pairs signed-rank test).

Single amino acid residue mediates reciprocal specificity in two mosquito odorant receptors.

The southern house mosquito, Culex quinquefasciatus, utilizes two odorant receptors, CquiOR10 and CquiOR2, narrowly tuned to oviposition attractants and well conserved among mosquito species. They detect skatole and indole, respectively, with reciprocal specificity. We swapped the transmembrane (TM) domains of CquiOR10 and CquiOR2 and identified TM2 as a specificity determinant. With additional mutations, we showed that CquiOR10A73L behaved like CquiOR2. Conversely, CquiOR2L74A recapitulated CquiOR10 specificity. Next, we generated structural models of CquiOR10 and CquiOR10A73L using RoseTTAFold and AlphaFold and docked skatole and indole using RosettaLigand. These modeling studies suggested space-filling constraints around A73. Consistent with this hypothesis, CquiOR10 mutants with a bulkier residue (Ile, Val) were insensitive to skatole and indole, whereas CquiOR10A73G retained the specificity to skatole and showed a more robust response than the wildtype receptor CquiOR10. On the other hand, Leu to Gly mutation of the indole receptor CquiOR2 reverted the specificity to skatole. Lastly, CquiOR10A73L, CquiOR2, and CquiOR2L74I were insensitive to 3-ethylindole, whereas CquiOR2L74A and CquiOR2L74G gained activity. Additionally, CquiOR10A73G gave more robust responses to 3-ethylindole than CquiOR10. Thus, we suggest the specificity of these receptors is mediated by a single amino acid substitution, leading to finely tuned volumetric space to accommodate specific oviposition attractants.