BackgroundResistance to pyrethroid insecticides is widespread among populations of Aedes aegypti, the main vector for the dengue virus. Several different point mutations within the voltage-gated sodium channel (VGSC) gene contribute to such resistance. A mutation at position 1016 in domain II, segment 6 of the VGSC gene in Ae. aegypti leads to a valine to glycine substitution (V1016G) that confers resistance to deltamethrin.MethodsThis study developed and utilized an allele-specific PCR (AS-PCR) assay that could be used to detect the V1016G mutation. The assay was validated against a number of sequenced DNA samples of known genotype and was determined to be in complete agreement. Larvae and pupae were collected from various localities throughout Thailand. Samples were reared to adulthood and their resistance status against deltamethrin was determined by standard WHO susceptibility bioassays. Deltamethrin-resistant and susceptible insects were then genotyped for the V1016G mutation. Additionally, some samples were genotyped for a second mutation at position 1534 in domain III (F1534C) which is also known to confer pyrethroid resistance.ResultsThe bioassay results revealed an overall mortality of 77.6%. Homozygous 1016G individuals survived at higher rates than either heterozygous or wild-type (1016 V) mosquitoes. The 1016G mutation was significantly and positively associated with deltamethrin resistance and was widely distributed throughout Thailand. Interestingly, wild-type 1016 V mosquitoes tested were homozygous for the 1534C mutation, and all heterozygous mosquitoes were also heterozygous for 1534C. Mutant homozygous (G/G) mosquitoes expressed the wild-type (F/F) at position 1534. However, the presence of the 1534C mutation was not associated with deltamethrin resistance.ConclusionsOur bioassay results indicate that all populations sampled display some degree of resistance to deltamethrin. Homozygous 1016G mosquitoes were far likelier to survive such exposure. However, resistance in some populations cannot be explained due to kdr mutations and indicates that other resistance mechanisms are operating. The presence of this mutation alone does not fully explain the resistance phenotype we see among Thai Ae. aegypti populations.

BackgroundMutation in the voltage-gated sodium channel gene that results in knockdown resistance (kdr), is a major mechanism of pyrethroid resistance in several mosquito species. In Aedes aegypti, V1016G (occurring with and without S989P) and F1534C mutations are common and widely distributed throughout Asia. The G1016 allele is known to be associated with resistance to type I and II pyrethroids. The C1534 allele is primarily associated with resistance to type I pyrethroids and is known to be a recessive allele in conferring kdr.MethodsWe performed crossing experiments using a P989 + G1016 homozygous mutant strain (UPK-R), a C1534 homozygous mutant strain (PMD-R) and a pyrethroid susceptible strain (PMD) to determine the insecticide susceptibility of different genotypic hybrids. Allele-specific PCR methods were used to confirm the genotypes. Metabolic resistance caused by oxidative enzymes and esterase enzymes was ruled out by the addition of piperonyl butoxide (PBO) and bis(4-nitrophenyl)-phosphate, BNPP), respectively.ResultsThe median lethal concentration (LC50) of deltamethrin susceptibility of a S/P989 + V/G1016 + F/F1534 double heterozygous hybrid from the UPK-R × PMD cross was 0.57 (95 % CI: 0.51–0.63) μgl-1, which was about 12-fold lower than for UPK-R, 6.98 (6.10–8.04) μgl-1, and only about 4-fold greater than the susceptible PMD, 0.13 (0.12–0.15) μgl-1. This resistance returned to 0.08 (0.07–0.09) μgl-1 on the addition of PBO suggesting that the P989 + G1016 kdr alleles are recessive. The LC50 of the S/P989 + V/G1016 + F/C1534 triple heterozygous hybrid was 3.58 (3.21–3.95) μgl-1, which was intermediate between that of the homozygous mutant genotypes, being 2-fold higher than the C1534 homozygote and 2-fold lower than the P989 + G1016 homozygote. These minor differences and the high LC50 values of the triple mutated heterozygote indicate there is some degree of functional equivalence of the P989 + G1016 and C1534 alleles in the heterozygote. Addition of PBO decreased the LC50 values by 2-fold, from 3.58 (3.21–3.95) to 1.52 (1.35–1.73) μgl-1, suggesting that oxidase enzymes play a partial role in resistance. The results are consistent with the median lethal time (LT50) of the triple mutated heterozygote against 0.05 % deltamethrin paper. An adult susceptibility test also revealed that the triple mutated heterozygote was resistant to deltamethrin and permethrin.ConclusionsThe combination of the three kdr alleles in the triple mutated heterozygote, S/P989 + V/G1016 + F/C1534, confers high resistance to pyrethroids. This heterozygous form is common in Ae. aegypti populations throughout Thailand and may have an adverse effect on the efficacy of a mosquito control program using insecticide-based approaches.

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