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PLoS Negl Trop Dis
2019 Jun 03;136:e0007432. doi: 10.1371/journal.pntd.0007432.
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Molecular evidence of sequential evolution of DDT- and pyrethroid-resistant sodium channel in Aedes aegypti.
Chen M
,
Du Y
,
Wu S
,
Nomura Y
,
Zhu G
,
Zhorov BS
,
Dong K
.
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BACKGROUND: Multiple mutations in the voltage-gated sodium channel have been associated with knockdown resistance (kdr) to DDT and pyrethroid insecticides in a major human disease vector Aedes aegypti. One mutation, V1016G, confers sodium channel resistance to pyrethroids, but a different substitution in the same position V1016I alone had no effect. In pyrethroid-resistant Ae. aegypti populations, V1016I is often linked to another mutation, F1534C, which confers sodium channel resistance only to Type I pyrethroids including permethrin (PMT), but not to Type II pyrethroids including deltamethrin (DMT). Mosquitoes carrying both V1016G and F1534C exhibited a greater level of pyrethroid resistance than those carrying F1534C alone. More recently, a new mutation T1520I co-existing with F1534C was detected in India. However, whether V1016I or T1520I enhances pyrethroid resistance of sodium channels carrying F1534C remains unknown.
METHODOLOGY/PRINCIPAL FINDINGS: V1016I, V1016G, T1520I and F1534C substitutions were introduced alone and in various combinations into AaNav1-1, a sodium channel from Aedes aegypti. The mutant channels were then expressed in Xenopus oocytes and examined for channel properties and sensitivity to pyrethroids using the two-electrode voltage clamping technique. The results showed that V1016I or T1520I alone did not alter the AaNav1-1 sensitivity to PMT or DMT. However, the double mutant T1520I+F1534C was more resistant to PMT than F1534C, but remained sensitive to DMT. In contrast, the double mutant V1016I+F1534C was resistant to DMT and more resistant to PMT than F1534C. Furthermore, V1016I/G and F1534C channels, but not T1520I, were resistant to dichlorodiphenyltrichloroethane (DDT). Cryo-EM structures of sodium channels suggest that T1520I allosterically deforms geometry of the pyrethroid receptor site PyR1 in AaNav1-1. The small deformation does not affect binding of DDT, PMT or DMT, but in combination with F1534C it increases the channel resistance to PMT and DDT.
CONCLUSIONS/SIGNIFICANCE: Our data corroborated the previously proposed sequential selection of kdr mutations in Ae. aegypti. We proposed that mutation F1534C first emerged in response to DDT/pyrethroids providing a platform for subsequent selection of mutations V1016I and T1520I that confer greater and broader spectrum of pyrethroid resistance.
Fig 1. Gating properties of AaNav1-1 and V1016I channels.(A) Membrane topology. (B) Voltage dependence of activation. (C) Voltage dependence of fast inactivation. (D) Voltage dependence of slow inactivation. (E) Recovery from fast inactivation. (F) Recovery from slow inactivation. Voltage step protocols used to generate the curves are shown above each panel.
Fig 2. Gating properties of the AaNav1-1, T1520I and T1520I/F1534C channels.(A) Positions of T1520I and F1534C. (B) Voltage dependence of activation. (C) Voltage dependence of fast inactivation. (D) Voltage dependence of slow inactivation. (E) Recovery from fast inactivation. (F) Recovery from slow inactivation. Voltage step protocols are the same as in Fig 1.
Fig 3. Effects of mutations V1016I and F1534C on the channel sensitivity to permethrin (PMT) and deltamethrin (DMT).(A) Representative tail currents induced by 1.0 μM PMT. (B) Representative tail currents induced by 1.0 μM DMT. (C) Dose-response curves of the channels modification by PMT. (D) Dose-response curves of the channels modification by DMT. The dose-response curve was fitting with Hill equation. Statistical significance was determined by using one-way ANOVA with Scheffé's post hoc analysis, and significant values were set at p < 0.05. The number of oocytes for each mutant construct was more than 6.
Fig 4. Effects of mutations T1520I and F1534C on the channel sensitivity to pyrethroids.(A) Channel modification by 1.0 μM PMT. (B) Channel modification by 1.0 μM DMT. (C) Channel modification by 1.0 μM cypermethrin, cyfluthrin, NRDC 157 and bifenthrin. The number of oocytes for each mutant was > 6. The asterisks indicate significant differences from the AaNav1-1 channel as determined by using the one way ANOVA with Scheffé's post hoc analysis (p < 0.05). The pound sign indicates a significant difference in sensitivity to PMT between mutants as determined using one-way ANOVA with Scheffé's post hoc analysis (p < 0.05).
Fig 5. Effects of mutations on the channel sensitivity to DDT.(A) Representative traces from AaNav1-1, V1016G, V1016I, T1520I, F1534C, T1520I+F1534C and V1016I+F1534C channels after incubation with DDT (100 μM). (B) Percentages of channel inactivation inhibited by DDT (100 μM). The number of oocytes for each mutant was more than 8. Error bars indicate mean ± s.e. The asterisks indicate significant differences in sensitivity of mutants versus wildtype to DDT as determined by using one-way ANOVA with Scheffé's post hoc analysis (p < 0.05). The pound sign indicates a significant difference in sensitivity to DDT between mutants as determined using one-way ANOVA with Scheffé's post hoc analysis (p < 0.05).
Fig 6. (A) Sequence alignment of sodium channel segments involved in the proposed mechanism by which mutation T1520/3i(-1)I allosterically induces small changes in the PyR1 site geometry. Highlighted are residues (except position 2i18), which are shown in panels B and C. Residue numbers in NavPaS and EeNav1.4 are sequential numbers in the PDB files of the cryo-EM structures where some segments are lacking. (B) and (C), Cryo-EM structures of eukaryotic sodium channels NavPaS (B) and EeNav1.4 (C). The pore-module helices in repeats I, II, III, and IV are magenta, yellow, green and gray, respectively. Side chains in positions 2i18 and 3i13, which correspond to AaNav1-1 residues 1016 and 1534, are space-filled. In both channels, these residues are in the II/III repeat interface that harbors the pyrethroid receptor site PyR1. Threonine T3(i-1) in the extracellular loop of NavPaS, which correspond to T1520/3(i-1) in AaNav1-1, is close to the N-terminus of IIIS6 and in AaNav1-1 it cannot directly interact with PyR1-bound pyrethroids.
Fig 7. Sequential selection of kdr mutations for pyrethroid resistance in Ae. aegypti.Darker background colors indicate higher levels of resistance. V1016 and T1520I are selected in mosquitoes carrying F1534C. F1534C (and probably V1016I) emerged under the DDT pressure prior to usage of pyrethroids. T1520I was selected under pressure of Type I pyrethroids.
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