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

Abstract

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.

Keywords: CquiOR36; EAG; acetaldehyde; odorant receptors; southern house mosquito.

Fig. 1.

Quantitative PCR data. (A) Transcript levels of CquiOR36 in olfactory and nonolfactory tissues from Cx. quinquefasciatus females. For reference, gels obtained before qPCR analysis are displayed. (B) Comparative expression of CquiOR36 in male and female antennae.

Fig. 2.

Responses of oocytes to an Orco agonist and to a series of aldehydes. Oocytes expressing Orco only (top trace) responded to VUAA-1, but not to the aldehydes, whereas oocytes expressing CquiOR36 only (middle trace) did not respond to any tested compound. By contrast, CquiOR36⋅CquiOrco-expressing oocytes (lower trace) yielded robust responses to acetaldehyde and strong responses to propanal. The response to the Orco agonist was somewhat stronger in the oocytes expressing the heteromeric OR complex than the Orco homomer.

Fig. 3.

EAG responses to acetaldehyde recorded from the fruit fly antennae. (A and B) Traces obtained with UAS-CuiOR36/+ and UAS-CquiOR36/DmelOrco-Gal4 flies when challenged with 0 (solvent only), 0.01, 0.1, and 1% acetaldehyde (from top to bottom). (C) Graphic representation of repetitions (n = 3–4) of above experiments. Error bars represent SEM.

Fig. 4.

Behavioral response from gravid females of the southern house mosquitoes to acetaldehyde in a cage oviposition assay. Each pair of bars represents one experiment with two choices: acetaldehyde (2Ald) vs. control (CNTL). Error bars represent SEM. From left to right, n = 15, 15, 16, 11, 16, and 13. Concentrations are indicated inside treatment bars. Data were analyzed with Wilcoxon matched-pair signed-rank tests.

Fig. 5.

Behavioral responses from southern house mosquito females in a dual-choice olfactometer in response to acetaldehyde (0.002%). (A) Response of gravid female mosquitoes, n = 29 releases, P < 0.0001. (B) Responses of blood-seeking (nonblood-fed) mosquitoes, n = 18 releases, P = 0.7207. Data were analyzed using the Wilcoxon matched-pair signed-rank test.