Pyrethroid Resistance in Malaysian Populations of Dengue Vector Aedes aegypti Is Mediated by CYP9 Family of Cytochrome P450 Genes

Abstract

Background: Dengue control and prevention rely heavily on insecticide-based interventions. However, insecticide resistance in the dengue vector Aedes aegypti, threatens the continued effectiveness of these tools. The molecular basis of the resistance remains uncharacterised in many endemic countries including Malaysia, preventing the design of evidence-based resistance management. Here, we investigated the underlying molecular basis of multiple insecticide resistance in Ae. aegypti populations across Malaysia detecting the major genes driving the metabolic resistance.

Methodology/principal findings: Genome-wide microarray-based transcription analysis was carried out to detect the genes associated with metabolic resistance in these populations. Comparisons of the susceptible New Orleans strain to three non-exposed multiple insecticide resistant field strains; Penang, Kuala Lumpur and Kota Bharu detected 2605, 1480 and 425 differentially expressed transcripts respectively (fold-change>2 and p-value ≤ 0.05). 204 genes were commonly over-expressed with monooxygenase P450 genes (CYP9J27, CYP6CB1, CYP9J26 and CYP9M4) consistently the most up-regulated detoxification genes in all populations, indicating that they possibly play an important role in the resistance. In addition, glutathione S-transferases, carboxylesterases and other gene families commonly associated with insecticide resistance were also over-expressed. Gene Ontology (GO) enrichment analysis indicated an over-representation of GO terms linked to resistance such as monooxygenases, carboxylesterases, glutathione S-transferases and heme-binding. Polymorphism analysis of CYP9J27 sequences revealed a high level of polymorphism (except in Joho Bharu), suggesting a limited directional selection on this gene. In silico analysis of CYP9J27 activity through modelling and docking simulations suggested that this gene is involved in the multiple resistance in Malaysian populations as it is predicted to metabolise pyrethroids, DDT and bendiocarb.

Conclusion/significance: The predominant over-expression of cytochrome P450s suggests that synergist-based (PBO) control tools could be utilised to improve control of this major dengue vector across Malaysia.

Fig 1. Transcription profiling of Ae. aegypti in Malaysia.

A) Map of the Malaysian peninsular showing the four collection sites of Penang (PG), Kota Bharu (KB), Kuala Lumpur (KL) and Johor Bharu (JB). B) Venn diagram of differentially transcribed genes from microarray data (p = 0.01); C) Relative fold-change of candidate genes from qRT-PCR analysis; D) Relative fold-change of candidate genes from qRT-PCR analysis for resistant samples from Penang. Perm stands for Permethrin, Delta for deltamethrin, Bend for bendiocarb.

Fig 2. Genetic diversity of CYP9J27 across Malaysian populations of Aedes aegypti.

A) Schematic representation of CYP9J27 haplotypes from across Malaysia and in the NO susceptible strain. The polymorphic positions for CYP9J27 amino acid sequences are highlighted. A number is given to each haplotype preceded by location origin (KL, KB, JB, PG and NO, for Kuala Lumpur, Kota Bharu, Johor Bharu, Penang and New Orleans, respectively). The column Nb stands for the number of individual mosquitoes sharing a haplotype. B) Neighbor joining tree of CYP9J27 haplotypes. C) Genetic distance tree between the different samples based on the Nst genetic distances.

Fig 3

Binding conformation of permethrin in (A) KL, (B) JB, (C) PG and (D) NO CYP9J27 models. Distances between the probable sites of attack on permethrin and the heme iron are annotated.