BEHAVIOURAL AND PHYSIOLOGICAL INSECTICIDE RESISTANCE OF THE DENGUE VECTOR AEDES AEGYPTI IN THE KINGDOM OF SAUDI ARABIA

Abstract

ABSTRACT Background Dengue has been endemic in the western region of Saudi Arabia since the 1990s. Insecticide-based control of Aedes aegypti remains the main dengue control option in Saudi Arabia as currently there is no curative medication for dengue, and the recently-approved vaccine is not yet available in the Middle Eastern region. Accumulating evidence suggests that insecticide resistance can reduce the effectiveness of vector control, but very little information is available on the insecticide resistance profile of Ae. aegypti in Saudi Arabia or the Middle Eastern region in general. The present study investigated the prevalence and levels of resistance to commonly used adulticides and larvicides, the mechanisms of physiological resistance and impact of insecticide resistance on host seeking success and behaviours in Ae. aegypti populations from the dengue endemic cities of Makkah and Jeddah in western Saudi Arabia. Methods Insecticide resistance profiles of the mosquito strains were assessed using WHO tube assays (bendiocarb, fenitrothion, deltamethrin and permethrin with or without PBO), cone assays (PermaNet 2.0 and DuraNet) and larval bioassays (Bti and temephos). The impact of mosquito age, duration and frequency of exposure to deltamethrin on mortality was also investigated using WHO tube bioassays. Target site mutations were identified by sequencing and were genotyped using TaqMan quantitative PCR assays, whilst metabolic resistance mechanisms were investigated by qPCR and microarray analysis. The metabolic activity of the leading candidate gene from the microarray study, CYP9J7, was characterised via in vitro insecticide metabolism assays. The effect of insecticide resistance and an insecticide treated barrier on host seeking behaviour was tested using two laboratory assays: a ‘thumb test’ of blood feeding behaviour and a wind tunnel test of barrier penetration. Results Jeddah and Makkah populations exhibited susceptibility to temephos and Bti, suspected resistance to fenitrothion and strong resistance to permethrin, deltamethrin, and bendiocarb. PBO pre-exposure increased pyrethroid mortality significantly in the Jeddah strain, but not in Makkah. Mosquito age and exposure v

duration significantly reduced survival of both strains, but susceptibility decreased after repeated exposures with a proportion of females surviving many successive assays. Three kdr mutations (S989P, V1016G, F1534C) were detected for the first time in Saudi Arabia, two of which were previously only identified in Asia. The S989P and V1016G markers were in perfect linkage disequilibrium (LD) and strongly predicted deltamethrin resistance, but were in negative LD with F1534C, which, probably as a consequence, showed negative association with resistance. Enrichment analysis of microarray data showed significant elevation of cytochrome P450s and zinc finger nucleic acid binding proteins in the overexpressed genes. The leading candidate gene CYP9J7, did not metabolise deltamethrin and permethrin, but did exhibit moderate metabolism of all three of the organophosphates tested. The Jeddah strain and especially the more resistant Makkah strain were much more successful in penetrating holes in PermaNet 2.0 than the New Orleans susceptible strain in the wind tunnel assay. Analysis of behavioural patterns suggested that changes in behaviour were a result of contact irritancy and differential intoxication of the strains rather than changes in amount of flight, contact with the net barrier and resting on the wind tunnel walls in assays with PermaNet 2.0 compared to the untreated assay. Conclusion This study provides the first study on how insecticide resistance mechanisms can impact the behaviour of an Ae. aegypti population from the Middle