Characterisation of mechanisms of insecticide resistance in Malaysian populations of the arbovirus vectors Aedes aegypti and Aedes albopictus

Aedes aegypti and Aedes albopictus are vectors of dengue fever in Malaysia. Vector control is the only means for the prevention of this disease. Unfortunately, insecticide resistance in this mosquito is threatening vector control programs. The present study intended to fill important knowledge gaps on the extent, geographical distribution and underlying mechanisms of these resistances in Malaysia. Mosquitoes were collected from four states in Malaysia in 2010 to assess their resistance status. WHO bioassays showed multiple resistance profiles in all populations. Multiple and high levels of resistance was observed for Ae. aegypti particularly in Kuala Lumpur, while resistance was more moderate in Ae. albopictus. PBO synergist assays indicated the presence of multiple resistance mechanisms for Ae. aegypti whereas it suggested a predominance of metabolic resistance through cytochrome P450s for Ae. albopictus populations. Investigation of resistance mechanisms in Ae. aegypti populations detected the F1534C knockdown resistance (kdr) mutation using pyrosequencing in all four populations with frequency ranging from 40% to 80%. However, apart from Penang and Johor Bharu, a correlation was not always found with resistance phenotypes. Furthermore, the sequencing of a cDNA fragment spanning exons 19 to 31 of the voltage gated sodium channel (VGSC),detected the V1016G kdr mutation in the four populations although at a lower frequency than F1534C. No correlation was observed between V1016G and resistance phenotype. Genome-wide transcription analysis using microarray detected the genes associated with metabolic resistance in these populations. Several cytochrome P450 genes (CYP9J27, CYP6CB1, CYP9J26 and CYP9M4) were among the most up-regulated genes in all populations indicating that they may play an important role in the observed resistance. This list also included glutathione-S-transferases, carboxylesterases and other gene families commonly associated with insecticide. Quantitative real-time PCR (qRT-PCR) validated the over-expression of all cytochrome P450s except for CYP6CB1. Analysis of resistance mechanisms in Ae. albopictus revealed significant differences to those observed in Ae. aegypti. Firstly, no kdr mutation was detected in this species across Malaysia. The absence of kdr mutation was further strengthened by the high genetic diversity of a portion of this gene. Secondly, analysis of the genome-wide transcription profile of Ae. albopictus populations revealed that cytochrome P450s from the CYP6 family are playing a predominant role in the observed resistance in contrast to CYP9 family for Ae. aegypti. Microarray analysis using a new 8 x 60k Agilent Ae. albopictus chip detected 40 genes commonly over-expressed including the cytochrome P450 genes CYP6N9, CYP9AE1 and CYP6AG6. A direct comparison of permethrin-resistant against non-exposed Kuala Lumpur mosquitoes revealed that permethrin resistance was most likely conferred by a combination of a reduced cuticle penetration and detoxification through cytochrome P450s. Indeed, several genes from these two gene families were consistently highly over-expressed. P450 transcripts corresponding to CYP6P4 in An. gambiae or CYP6P12 in Ae. aegypti were consistently the most up-regulated P450. Other up-regulated genes included GST, ABC transporters, heat shock protein, oxidaxes, and proteases. qRT-PCR analysis validated these expression patterns with the P450 CYP6N3 the most over-expressed detoxification gene. This study revealed significant differences in the resistance profile and the underlying resistance mechanism between the two dengue vectors across Malaysia. This presents a challenge for the common control of both species as it appears that a single insecticide-based control intervention may not efficiently control both species