Table 1.
Selection of the SP strain of Aedes aegypti in the laboratory by permethrin.
Figure 1.
Log dose-probit mortality lines of Aedes aegypti.
(A) The development of permethrin resistance in a field-collected population of adult A. aegypti under laboratory selection conditions. SPS0 is a field-collected population prior to permethrin selection. SPS10 is the strain established by permethrin selection for 10 generations and is designated as SP. SMK is a susceptible reference strain. (B) Dose–mortality lines of adult F1 (SP♀ × SMK♂ or SMK♀ × SP♂) and BC1 (F1♀ × SMK♂ or SMK♀ × F1♂) showing the inheritance of SP resistance. (C) Synergistic effects of PBO on permethrin toxicity in adults. (D) Susceptibility of larvae to permethrin and the synergistic effects of PBO.
Table 2.
Toxicity of permethrin and pirimiphos methyl by topical application to adult Aedes aegypti and the synergistic effects of PBO.
Table 3.
Toxicity of permethrin, temephos, and pirimiphos methyl to larval Aedes aegypti and the synergistic effects of PBO.
Table 4.
Transition of the frequency of the Vssc genotype during permethrin selection in the SP strain of Aedes aegypti.
Figure 2.
In vivo distribution of [14C]-permethrin topically applied to SP and SMK strains of Aedes aegypti.
(A) Schematic of the procedure of in vivo assay. (B) Percentage of the applied radiolabel recovered in the external rinse. [14C]-Permethrin (600 dpm; 0.88 ng) was applied to the thoracic notum. PBO (5 µg) was also applied to the thoracic notum or thoracic sternum 1 h prior to permethrin application to observe its effect on permethrin penetration. Acetone was also applied 1 h prior to permethrin application to confirm whether the solvent had any side effects. Values are expressed as the means (±SE) for four mosquitoes. (C) Percentage of radioactivity detected in the mosquitoes. (D) Percentage of radioactivity excreted in the vials. (E) Thin-layer chromatogram of the compounds excreted by females of SP strain. Developing solvents: toluene/ethyl acetate (6∶1). The presence of 4′-HO-permethrin was determined by co-chromatography with an authenticated chemical. (F) Percentage of permethrin and its metabolites excreted by females of SP strain. Values represent the average and bars represent the SE of the mean (n = 3). (G) Re-chromatography of the high-polar compounds stuck around origin. Developing solvents: chloroform/methanol/water (65∶25∶4).
Figure 3.
In vitro [14C]-permethrin metabolism studies.
(A) Thin-layer chromatogram of in vitro permethrin metabolism in adult Aedes aegypti with or without NADPH or PBO. Microsomes (100,000 × g pellet) and the soluble fraction (100,000 × g supernatant) were used as the enzyme source. The presence of 4′-HO-permethrin was determined by co-chromatography with an authenticated chemical. (B) Metabolites of permethrin in adult A. aegypti at various incubation times. For detailed data, see Table S2. (C) Thin-layer chromatogram of high-polar compounds (SP) located at the origin of the first HPTLC. Developing solvents: chloroform/methanol/water (65∶25∶4). (D) Inhibition of permethrin metabolism by 4′-HO-permethrin. Microsomes of the SP strain were incubated for 30 min with [14C]-permethrin, NADPH, and ×7-, ×70-, or ×140-fold higher doses of 4′-HO-permethrin than permethrin.
Figure 4.
Microarray screening of cytochrome P450 and b5 genes differentially expressed in SP and SMK strains.
Differential transcription of cytochrome P450 and b5 genes was individually investigated in adult female (A, B), adult male (C, D), and fourth instar larvae (E, F) of Aedes aegypti. Figures 4A–C show volcano plots of the relative changes (log2, x-axis) and statistical significance (–log10(P-value), y-axis). Figures 4D–F show scatter plots of the relative expression level of each P450 and b5 gene. Genes with relative expression >3-fold and a P-value<0.01 are highlighted.
Table 5.
Cytochrome P450 and b5 genes differentially expressed in the SP (R) strain relative to the susceptible SMK (S) strain of Aedes aegypti.
Figure 5.
Real time quantitative PCR analysis of selected genes from the microarray experiments.
Transcription levels of seven P450 genes were individually validated in adult female (A), adult male (B), and fourth instar larvae (C) in SP and SMK strains of Aedes aegypti. Ribosomal protein S3 gene (RPS3) was used to normalize the data. Expression ratios (SP/SMK) are expressed in parentheses. (D) Correlation between the microarray and real time quantitative PCR (R2 = 0.824). (E) Gene copy number of 7 P450s and RPS3 quantified by real time PCR. The ratios of relative copy number (SP/SMK) are expressed in parentheses. (F) Gene clusters of cytochrome P450 and the CYP9M subfamily on the A. aegypti supercontig 1.29 on the 2nd chromosome. The error bars represent standard errors of three (A, B, and C) and eight (E) biological replicates.
Figure 6.
Genotype–gene expression–phenotype associations.
The association between CYP9M6/CYP6BB2 genotypes and mRNA expression and permethrin excretion rate was investigated. The female F2 populations of SP and SMK strains were dosed with [14C]-permethrin, and the amount of permethrin excreted was individually quantified 24 h after treatment. The genotype of each mosquito was identified by the genomic DNA isolated from six legs. mRNA level was also quantified with RNA isolated from the body part of each mosquito. For more information, see Materials and Methods. (A, D) Plot showing the association between the level of mRNA and permethrin excretion in CYP9M6 (A) and CYP6BB2 (D). (B, E) Box plot showing the association between mRNA level and CYP9M6 (B) and CYP6BB2 (E). (C, F) Box plot showing the association between permethrin excretion rate and genotype of CYP9M6 (C) and CYP6BB2 (F). P-values for comparison of the RR and SS genotypes were calculated using Dunnett's test.
Figure 7.
Associations between the gene expression level of six P450s and permethrin excretion rate.
The female F2 populations of SP and SMK strains were dosed with [14C]-permethrin and the amount of permethrin excreted by each insect was quantified 24 h after treatment. The level of mRNA was quantified using RNA isolated from each mosquito. For more information, see Materials and Methods. (A) CYP6BB2, (B) CYP6Z7, (C) CYP6Z8, (D) CYP9M4, (E) CYP9M5, and (F) CYP9M6. Forty-eight individuals were ranked from 1 to 48 according to the level of expression of each P450 gene. The ranks against multiple P450 genes were combined to standardize the level of mRNA and evaluate the association between permethrin excretion rate and expression level of multiple P450 genes. (G) Association between permethrin excretion and standardized level of mRNA for CYP6BB2 and CYP9M6. (H) Association between permethrin excretion rate and standardized level of mRNA for CYP6BB2, CYP9M6 and CYP9M5. (I) Association between permethrin excretion rate and standardized level of mRNA for CYP6BB2, CYP6Z7, CYP6Z8, CYP9M4, CYP9M5, and CYP9M6.
Figure 8.
Thin layer chromatogram of [14C]-permethrin metabolites by heterologously expressed CYP9M6v1, CYP9M6v2, and CYP6BB2.
A-E denote unidentified metabolites. Developing solvents: toluene/ethyl acetate (6∶1). The presence of 4′-HO-permethrin was determined by co-chromatography with an authenticated chemical.
Figure 9.
Proposed model showing the metabolic pathway of permethrin.