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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)., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

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.

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.

The Southern house mosquito, Culex quinquefasciatus--a vector of West Nile virus--is equipped with 130 odorant receptors (ORs), which enable young females to locate plants and blood-meal sources and older females to find suitable sites for oviposition. In our attempts to de-orphanize ORs expressed in female antennae, we identified CquiOR37 and CquiOR99, which were narrowly tuned to two phenolic compounds, 4-methylphenol and 4-ethylphenol. When tested in the Xenopus oocyte recording system the observed EC50s for 4-methylphenol and 4-ethylphenol were 6.4 and 18.2 μM for CquiOR37 and 14.4 and 0.74 μM for CquiOR99 (goodness of fit, R² = 0.88-0.99), respectively. Indoor behavioral assays demonstrated that gravid female mosquitoes laid significantly more eggs in water trays spiked with these compounds than in control water trays. Field studies with gravid traps corroborated that 4-ethylphenol is active in a wide range of doses from 0.1 to 10 μg/l, as required for practical applications. A dsRNA construct based on the two genes, CquiOR37/99-dsRNA was stable in pupa hemolymph for up to 3 h. Pupae injected with CquiOR37/99-dsRNA, β-galactosidase-dsRNA or water had more than 40% survival rate at the peak of oviposition (day-9). qPCR analysis showed individual variation, but significant mean reduction in CquiOR37 and CquiOR99 transcript levels in CquiOR37/99-dsRNA-treated mosquitoes. Water-injected females and those treated with the control gene laid significantly more eggs in trays containing 4-ethylphenol than in water trays, whereas CquiOR37/99-dsRNA-treated mosquitoes laid normal number of eggs, but could not discriminate treatment from control. This study linked for the first time specific receptors for 4-ethylphenol with increased oviposition in the important vector Cx. quinquefasciatus., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Southern house mosquito Culex quinquefasciatus has the largest repertoire of odorant receptors (ORs) of all mosquitoes and dipteran species whose genomes have been sequenced to date. Previously, we have identified and de-orphanized two ORs expressed in female antennae, CquiOR2 and CquiOR10, which are sensitive to oviposition attractants. In view of a new nomenclature for the Culex genome (VectorBase) we renamed these ORs as CquiOR21 (formerly CquiOR10) and CquiOR121 (CquiOR2). In addition, we selected ORs from six different phylogenetic groups for deorphanization. We cloned four of them by using cDNA from female antennae as a template. Attempts to clone CquiOR87 and CquiOR110 were unsuccessful either because they are pseudogenes or are not expressed in adult female antennae, the main olfactory tissue. By contrast, CquiOR1, CquiOR44, CquiOR73, and CquiOR161 were highly expressed in female antennae. To de-orphanize these ORs, we employed the Xenopus oocyte recording system. CquiORx-CquiOrco-expressed oocytes were challenged with a panel of 90 compounds, including known oviposition attractants, human and vertebrate host odorants, plant kairomones, and naturally occurring repellents. While CquiOR161 did not respond to any test compound in two different laboratories, CquiOR1 showed the features of a generic OR, with strong responses to 1-octen-3-ol and other ligands. CquiOR44 and CquiOR73 showed preference to plant-derived terpenoids and phenolic compounds, respectively. While fenchone was the best ligand for the former, 3,5-dimethylphenol elicited the strongest responses in the latter. The newly de-orphanized ORs may be involved in reception of plant kairomones and/or natural repellents., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)