Figure 1.
Mutations of Aedes aegypti Vssc investigated in this study.
(A) Schematic illustration of the locations of the Vssc mutations assessed. Positions are numbered according to the amino acid sequence of the most abundant splice variant of housefly Vssc (GenBank accession nos. AAB47604 and AAB47605). (B) Six Vssc types investigated in this study and the positions of primers used for site-directed mutagenesis are shown. Primer sequences are listed in Methods. AaNavS2 is the wild-type Vssc of which the cDNA was isolated from the insecticide-susceptible SMK strain.
Figure 2.
Structural and functional properties of Aedes aegypti Vsscs expressed in Xenopus oocytes.
(A) The cloned AaNavS2 (wild-type) channel was expressed in Xenopus oocytes and a Na+ current trace was recorded. (B) The Na+ current was blocked by the application of 10 nM tetrodotoxin, verifying that this was a Na+ current. (C) Normalized voltage-conductance and inactivation curves of AaNavS2. The peak current was plotted against the depolarizing voltage (solid circles). The peak current amplitude was plotted as a function of the pre-pulse potential (triangles). Error bars indicate standard errors for 7–9 oocytes (D–I). Na+ current-voltage curves for 6 Vssc types. Error bars indicate standard errors for 7–9 oocytes. Reversal potentials were: (D) wild-type (+35 mV); (E) V1016G single mutation (+35 mV); (F) F1534C single mutation (+35 mV); (G) S989P single mutation (+35 mV); (H) S989P+V1016G double mutation (+25 mV); and (I) S989P+V1016G+F1534C triple mutation (+20 mV).
Table 1.
Voltage-dependence of activation and inactivation of Aedes aegypti sodium channels and sensitivity of mutant channels to pyrethroids.
Figure 3.
Pyrethroid-induced tail currents from oocytes injected with various types of Vssc.
(A, G) AaNavS2 (wild-type); (B, H) AaNavR6 (V1016G); (C, I) AaNavR7 (F1534C); (D, J), AaNavR8 (S989P); (E, K), AaNavR9 (S989P+V1016G); (F, L) AaNavR10 (S989P+V1016G+F1534C) in the absence (control) or presence of 100 nM permethrin (A–F) or 100 nM deltamethrin (G–L).
Table 2.
Time constants of the decay (τdecay) of pyrethroid-induced tail currents.
Figure 4.
Sensitivity of Aedes aegypti Vsscs to permethrin and deltamethrin.
The percentages of modified channels were plotted against different concentrations of permethrin (A) and deltamethrin (B), and fitted to a four-parameter logistic equation. Error bars indicate standard errors for 4–8 oocytes. The percentages of modified channels were significantly different between AaNavR6 (V1016G) and AaNavR9 (S989P+V1016G) at deltamethrin concentration of 1.0 µM (P = 0.0091 by Tukey-Kramer test) in Figure 4B.