Additive effect of knockdown resistance mutations, S989P, V1016G and F1534C, in a heterozygous genotype conferring pyrethroid resistance in Aedes aegypti in Thailand

Background Mutation 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 . Methods We 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. Results The 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 ...