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-galactosidasedsRNA 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.
Since the discovery in the early 1980s that 1-octen-3-ol, isolated from oxen breath, attracts tsetse fly, there has been growing interest in exploring the use of this semiochemical as a possible generic lure for trapping host-seeking mosquitoes. Intriguingly, traps baited with 1-octen-3-ol captured significantly more females of the malaria mosquito, Anopheles gambiae, and the yellow fever mosquito, Aedes aegypti, than control traps, but failed to attract the southern house mosquito, Culex quinquefasciatus. Additionally, it has been demonstrated that this attractant is detected with enantioselective odorant receptors (ORs) expressed only in maxillary palps. On the basis of indoor behavioral assays it has even been suggested that 1-octen-3-ol might be a repellent to the southern house mosquito. Our approach was two-prong, i.e., to isolate 1-octen-3-ol-sensitive ORs expressed in maxillary palps and antennae of southern house female mosquito, and test the hypothesis that this semiochemical is a repellent. An OR with high transcript levels in maxillary palps, CquiOR118b, showed remarkable selectivity towards ( R)-1-octen-3-ol, whereas an OR expressed in antennae, CquiOR114b, showed higher preference for ( S)-1-octen-3-ol than its antipode. Repellency by a surface landing and feeding assay showed that not only racemic, but enantiopure ( R)- and ( S)-1-octen-3-ol are repellents at 1% dose thus suggesting the occurrence of other ( S)-1-octen-3-ol-sensitive OR(s). Female mosquitoes with ablated maxillary palps were repelled by 1-octen-3-ol, which implies that in addition to OR(s) in the maxillary palps, antennal OR(s) are essential for repellency activity.
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 occuring 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.
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