Insect repellents: modulators of mosquito odorant receptor activity

Abstract

Background: DEET, 2-undecanone (2-U), IR3535 and Picaridin are widely used as insect repellents to prevent interactions between humans and many arthropods including mosquitoes. Their molecular action has only recently been studied, yielding seemingly contradictory theories including odorant-dependent inhibitory and odorant-independent excitatory activities on insect olfactory sensory neurons (OSNs) and odorant receptor proteins (ORs).

Methodology/principal findings: Here we characterize the action of these repellents on two Aedes aegypti ORs, AaOR2 and AaOR8, individually co-expressed with the common co-receptor AaOR7 in Xenopus oocytes; these ORs are respectively activated by the odors indole (AaOR2) and (R)-(-)-1-octen3-ol (AaOR8), odorants used to locate oviposition sites and host animals. In the absence of odorants, DEET activates AaOR2 but not AaOR8, while 2-U activates AaOR8 but not AaOR2; IR3535 and Picaridin do not activate these ORs. In the presence of odors, DEET strongly inhibits AaOR8 but not AaOR2, while 2-U strongly inhibits AaOR2 but not AaOR8; IR3535 and Picaridin strongly inhibit both ORs.

Conclusions/significance: These data demonstrate that repellents can act as olfactory agonists or antagonists, thus modulating OR activity, bringing concordance to conflicting models.

Figure 1. Compounds identification and repellent effectiveness on AaORs.

(A) Structural formulas of odorants and (B) half maximal inhibitory concentrations (IC₅₀) of insect repellents. Asterisks indicate chiral centers.

Figure 2. DEET and 2-undecanone alone, selectively activate AaORs.

Response traces and concentration-response curves of AaOR2+AaOR7 and AaOR8+AaOR7 exposed to DEET (red), 2-U (blue), IR3535 and Picaridin are recorded in nano-ampere (nA). (A) DEET activates AaOR2+AaOR7. (B) (C) and (D) 2-U, IR3535 and Picaridin do not activate AaOR2+AaOR7. (E) The concentration-response plots of AaOR2+AaOR7 to increasing amounts of repellents. (F) (H) and (I) DEET, IR3535 and Picaridin do not activate AaOR8+AaOR7. (G) 2-U activates AaOR8+AaOR7. Horizontal arrows indicate prolonged recovery times. Inward currents are shown as downward deflections. (J) The concentration-response plots of AaOR8+AaOR7 to increasing amounts of repellents. Odorant concentrations were plotted on a logarithmic scale. All concentrations are in molarity. Each point represents the mean current response and vertical error bars are s.e.m. n = 5 oocytes for each treatment.

Figure 3. DEET and 2-undecanone selectively inhibit odorant-induced responses of AaORs.

The concentration-response plots of AaOR2+AaOR7 and AaOR8+AaOR7 to repeated exposures of 10⁻⁷ M indole (open squares) and 10⁻⁷ M octenol [(R)-(−)-1-octen3-ol] (open circles) were duplicated in each panel for comparative purposes. (A) Response traces of AaOR2+AaOR7 (blue) and AaOR8+AaOR7 (red) to 10⁻⁷ M agonist alone and in combination with increasing concentrations of DEET (10⁻⁷ M to 10⁻² M) are recorded in nano-ampere (nA). (B) Concentration-response plots of AaOR2+AaOR7 (solid blue squares) and AaOR8+AaOR7 (solid red circles) to 10⁻⁷ M indole and 10⁻⁷ M octenol in the presence of increasing amounts of DEET. (C) Response traces of AaOR2+AaOR7 (blue) and AaOR8+AaOR7 (red) to 10⁻⁷ M agonist alone and in combination with increasing concentrations of 2-undecanone (2-U) (10⁻⁷ M to 10⁻² M) are recorded in nano-ampere (nA). Horizontal arrow indicates prolonged recovery time. (D) Concentration-response plots of AaOR2+AaOR7 (solid blue squares) and AaOR8+AaOR7 (solid red circles) to 10⁻⁷ M indole and 10⁻⁷ M octenol in the presence of increasing amounts of 2-U. Inward currents are shown as downward deflections. Odorant concentrations were plotted on a logarithmic scale. Each point represents the mean current response; error bars are s.e.m. n = 5–6 oocytes for each treatment. Treatments with high DEET concentrations (10⁻³ M and 10⁻² M) and 2-U (10⁻³ M and 10⁻² M) differed significantly from the no-repellent controls (two-way ANOVA, Bonferroni posttests, **: P<0.01; ***: P<0.001). Vertical and horizontal scale bars represent 100 nA and 1 min, respectively.

Figure 4. Octenol is not modified by DEET in solution.

The presence of DEET (10⁻³ M) does not affect the mean amount of octenol [(R)-(−)-1-octen-3-ol)] (10⁻⁵ M) in physiological solution. Vertical bars represent s.e.m. (n = 5). Note broken x-axis for data representation. Same letters above histograms indicate non significant differences (P>0.05, ANOVA test with Tukey posttest).

Figure 5. IR3535 and Picaridin inhibit odorant-induced responses of AaORs.

The concentration-response plots of AaOR2+AaOR7 and AaOR8+AaOR7 to repeated exposures of 10⁻⁷ M indole (open squares) and 10⁻⁷ M octenol [(R)-1-octen3-ol)] (open circles) were duplicated in each panel for comparative purposes. (A) Response traces of AaOR2+AaOR7 (blue) and AaOR8+AaOR7 (red) to 10⁻⁷ M agonist alone and in combination with increasing concentrations of IR3535 (10⁻⁷ M to 10⁻² M) are recorded in nano-ampere (nA). (B) Concentration-response plots of AaOR2+AaOR7 (solid blue squares) and AaOR8+AaOR7 (solid red circles) to 10⁻⁷ M indole and 10⁻⁷ M octenol in the presence of increasing amounts of IR3535. (C) Response traces of AaOR2+AaOR7 (blue) and AaOR8+AaOR7 (red) to 10⁻⁷ M agonist alone and in combination with increasing concentrations of Picaridin (10⁻⁷ M to 10⁻² M) are recorded in nano-ampere (nA). (D) Concentration-response plots of AaOR2+AaOR7 (solid blue squares) and AaOR8+AaOR7 (solid red circles) to 10⁻⁷ M indole and 10⁻⁷ M octenol in the presence of increasing amounts of Picaridin. Inward currents are shown as downward deflections. Odorant concentrations were plotted on a logarithmic scale. Each point represents the mean current response; error bars are s.e.m. n = 5–6 oocytes for each treatment. Treatments with high IR3535 concentrations (10⁻⁴ M, 10⁻³ M and 10⁻² M) and Picaridin (10⁻³ M and 10⁻² M) differed significantly from the no-repellent controls (two-way ANOVA, Bonferroni posttests, **: P<0.01; ***: P<0.001). Vertical and horizontal scale bars represent 100 nA and 1 min, respectively.