RNAi-based demonstration of direct link between specific odorant receptors and mosquito oviposition behavior

Abstract

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.

Mosquito odorant receptor for DEET and methyl jasmonate

“…Our RNAi approach has been demonstrated to cause longlasting transcript-level reductions (9). Thus, we injected three groups of female pupae with CquiIR40a-double-strand RNA (dsRNA), water, or β-galactosidase-dsRNA.…”

“…We cloned the other seven putative ORs, CquiOR55, CquiOR64, CquiOR93, CquiOR125, CquiOR132, CquiOR136, and CquiOR151. We screened them for a DEET receptor by using the Xenopus oocyte recording system, as previously done to deorphanize multiple Culex ORs (9,(14)(15)(16)(17). CquiOR136•CquiOrco-expressing oocytes were mainly activated by DEET, PMD, IR3535, picaridin, and two other repellents, γ-octalactone and 3-octanol ( Fig.…”

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

“…Antennae and legs dissected from blood-fed adult females were used for RNA-Seq as well as to generate templates for validation by qPCR and gene cloning. Deorphanization of a unique OR gene was performed with the Xenopus oocyte expression system (9,35). Oocytes expressing CquiOR95b along with CquiOrco were challenged with large panel of physiologically relevant odorants.…”

“…RNA-Seq data explained our failure to clone CquiOR87, CquiOR110 (7), and CquiOR38 (9) given that their transcripts are very low in antennae (0.06, 0.05, and 0.08 FPKM, respectively), thus indicating they are not adult antennal OR genes. More importantly, this transcriptome analysis provides a road map for cloning and deorphanization OR genes enriched in female antennae.…”

“…quinquefasciatus and related Culex pipiens complex mosquitoes have a very high degree of polymorphism and the highest density of single nucleotide polymorphism (SNP) of all mosquito species studied thus far (8). Thus, genetic differences between the Johannesburg strain (the source of genome sequences) and our California strain has retarded progress in our attempts to isolate, clone, and deorphanize ORs on the basis of genomic data (7,9), particularly given the number of potential pseudogenes and genes unlikely to be expressed in female antennae. Here, we used next generation…”