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In addition to its primary function as an insect repellent, DEET has many "off-label" properties, including a deterrent effect on the attraction of gravid female mosquitoes. DEET negatively affects oviposition sites. While deorphanizing odorant receptors (ORs) using the Xenopus oocyte recording system, we have previously observed that DEET generated outward (inhibitory) currents on ORs sensitive to oviposition attractants. Here, we systematically investigated these inhibitory currents. We recorded dose-dependent outward currents elicited by DEET and other repellents on ORs from Culex quinquefasciatus, Aedes aegypti, and Anopheles gambiae. Similar responses were observed with other plant-derived and plant-inspired compounds, including methyl jasmonate and methyl dihydrojasmolate. Inward (regular) currents elicited by skatole upon activation of CquiOR21 were modulated when this oviposition attractant was coapplied with a repellent. Compounds that generate outward currents in ORs sensitive to oviposition attractants elicited inward currents in a DEET-sensitive receptor, CquiOR136. The best ligand for this receptor, methyl dihydrojasmolate, showed repellency activity but was not as strong as DEET in our test protocol.

Reception of odorants by two main head appendages, antennae and maxillary palps, is essential for insects' survival and reproduction. There is growing evidence in the literature suggesting that the proboscis is also an olfactory appendage and its function as an additional "antenna" has been previously proposed. We surmised that movements of the labrum toward a blood vessel might be chemically oriented and, if so, there should be odorant receptors expressed in the labrum. To test this hypothesis, we first compared by quantitative PCR expression of odorant receptors (OR) from the Southern house mosquito, Culex quinquefasciatus in antennae and proboscis and, subsequently compared OR expression in various proboscis parts. Our data suggested that a receptor for the oviposition attractant, skatole, CquiOR21, was not expressed in proboscis, whereas a receptor for another oviposition attractant, 4EP (4-ethylphenol), CquiOR99, and a receptorf for the insect repellent DEET, CquiOR136, were expressed in the stylet of the proboscis, particularly in the tip of the labrum. In a dual-choice olfactometer, mosquitoes having the stylet coated with nail polish were attracted to 4EP in the same manner as the untreated mosquitoes. By contrast, in an oviposition assay, the stylet-treated mosquitoes did not discriminate 4EP from control oviposition cups, whereas the untreated mosquitoes (as well as mosquitoes having the labella coated) laid significantly more egg rafts in cups treated with 4EP. Ablation experiments confirmed that 4EP was sensed by the labrum where CquiOR99 is highly expressed. Stylet-coated, labella-coated, and untreated mosquitoes laid significantly more egg rafts in skatole-treated cups than in control cups. Likewise, coating of proboscis structures with nail polish had no effect on DEET-mediated oviposition deterrence. In a behavioral arena designed to mimic a human arm, mosquitoes showed significantly reduced probing time when blood was impregnated with 4EP, i.e., they engaged more rapidly in continuous blood feeding as compared to untreated blood. The time of engagement for feeding in skatole-containing blood vs. untreated blood did not differ significantly. Taken together, these data suggest that 4EP reception by the labrum is important not only for oviposition decisions, but also for reducing probing and initiation of blood feeding.

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.

Reception of odorants by two main head appendages, antennae and maxillary palps, is essential for insects’ survival and reproduction. There is growing evidence in the literature suggesting that the proboscis is also an olfactory appendage and its function as an additional antenna has been previously proposed. We surmised that movements of the labrum towards blood vessel might be chemically oriented and, if so, there should be odorant receptors expressed in the labrum. To test this hypothesis, we first compared by quantitative PCR expression of odorant receptors (OR) from the Southern house mosquito, Culex quinquefasciatus in antennae and proboscis and, subsequently compared OR expression in various proboscis parts. Our data suggested that a receptor for the oviposition attractant, skatole, CquiOR21, was not expressed in proboscis, whereas a receptor for another oviposition attractant, 4EP (4-ethylphenol), CquiOR99, and a receptor for the insect repellent DEET, CquiOR136, were expressed in the stylet of the proboscis, particularly in the tip of the labrum. In a dual-choice olfactometer, mosquitoes having the stylet coated with nail polish were attracted to 4EP in the same manner as the untreated mosquitoes. By contrast, in an oviposition assay, the stylet-treated mosquitoes did not discriminate 4EP from control oviposition cups, whereas the untreated mosquitoes (as well as mosquitoes having the labella coated) laid significantly more egg rafts in cups treated with 4EP. Ablation experiments confirmed that 4EP was sensed by the labrum where CquiOR99 is highly expressed. Stylet-coated, labella-coated, and untreated mosquitoes laid significantly more egg rafts in skatole-treated cups than in control cups. Likewise, coating of proboscis structures with nail polish had no effect on DEET-mediated oviposition deterrence. In a behavioral arena designed to mimic a human arm, mosquitoes showed significantly reduced probing time when blood was impregnated with 4EP, i.e., they engaged more rapidly in continuous blood feeding as compared to untreated blood. The time of engagement for feeding in skatole-containing blood vs. untreated blood did not differ significantly. Taken together, these data suggest that 4EP reception by the labrum is important not only for oviposition decisions, but also for reducing probing and initiation of blood feeding.