The capacity of mosquitoes to resist insecticides threatens the control of diseases such as dengue and malaria. Until alternative control tools are implemented, characterizing resistance mechanisms is crucial for managing resistance in natural populations. Insecticide biodegradation by detoxification enzymes is a common resistance mechanism; however, the genomic changes underlying this mechanism have rarely been identified, precluding individual resistance genotyping. In particular, the role of copy number variations (CNVs) and polymorphisms of detoxification enzymes have never been investigated at the genome level, although they can represent robust markers of metabolic resistance. In this context, we combined target enrichment with high-throughput sequencing for conducting the first comprehensive screening of gene amplifications and polymorphisms associated with insecticide resistance in mosquitoes. More than 760 candidate genes were captured and deep sequenced in several populations of the dengue mosquito Ae. aegypti displaying distinct genetic backgrounds and contrasted resistance levels to the insecticide deltamethrin. CNV analysis identified 41 gene amplifications associated with resistance, most affecting cytochrome P450s overtranscribed in resistant populations. Polymorphism analysis detected more than 30,000 variants and strong selection footprints in specific genomic regions. Combining Bayesian and allele frequency filtering approaches identified 55 nonsynonymous variants strongly associated with resistance. Both CNVs and polymorphisms were conserved within regions but differed across continents, confirming that genomic changes underlying metabolic resistance to insecticides are not universal. By identifying novel DNA markers of insecticide resistance, this study opens the way for tracking down metabolic changes developed by mosquitoes to resist insecticides within and among populations.

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Several countries continue to experience endemic and reemerging dengue fever (DF) and dengue hemorrhagic fever (DHF). 1) Annually, millions of people are estimated to be infected with dengue virus worldwide. 2,3) In Thailand, outbreaks of DHF were first recognized in 1958 and subsequently the disease has expanded throughout the country. 4) Approximately 40,000 cases of dengue with a mortality rate of 0.09 (per 100,000 people) were reported in 2007. 5) In spite of continued vigilance in control methods, dengue cases remain a major health threat in Thailand. The reason is unclear but is possibly related to the increase of human and economic activities in urban and semi-urban zones. Moreover, traditional water storage practices increase the breeding sites for Aedes aegypti, a primary vector of DF and DHF in Thailand. 6,7) Aedes aegypti, a daytime biting mosquito, is highly anthropophilic, often resides in and near human dwellings and preferentially feeds on humans. 1,8,9) This species has been found to be highly adapted to all man-made and natural environments and is a potential vector of dengue. [10][11][12] The key to preventing dengue transmission is to reduce human-vector contact using synthetic chemicals. [13][14][15][16][17] Several synthetic insecticides, including organochlorines, organophosphates, carbamates and synthetic pyrethroids, have been used in dengue control programs. 18) DDT (organochlorine) was widely used to control Ae. aegypti after the first dengue epidemic in 1958. 19,20) Deltamethrin (synthetic pyrethroids) is currently one of the most commonly used insecticides in public health programs and has been the mainstay for the emergency control of Ae. aegypti adults in Thailand since 1994. 18,21) Temephos (organophosphate) is commonly used in water containers for the control of Ae. aegypti larvae. Ultra-low-volume (ULV) applications of fenitrothion and malathion are used during the peak period of adult Aedes populations, especially during the rainy season. 16 (Received April 24, 2008; Accepted June 26, 2008) In this study, nine strains of Aedes aegypti mosquitoes from different localities in Thailand were subjected to susceptibility tests against commonly used insecticides for vector control in Thailand. Aedes aegypti from different localities were strongly resistant to DDT and permethrin, except for one strain from Chiang Mai (Chiang Mai-P) that was found to be susceptible to permethrin. In contrast, most Ae. aegypti strains are susceptible to deltamethrin and alphacypermethrin, except that Bangkok and Nonthaburi strains showed incipient resistance to deltamethrin and alphacypermethrin, respectively. Various levels of malathion and propoxur susceptibility were also detected in Ae. aegypti strains; however, two strains of Ae. aegypti (Satun and Nonthaburi) were found to be completely susceptible to malathion. The insecticide susceptibility level in three strains of Culex quinquefasciatus was also investigated. The results indicated strong resistance to DDT and moderate resistance to permethri...

BackgroundMalaria is common in hilly, forested areas along national borders in Southeast Asia. Insecticide resistance in malaria vectors has been detected in a few countries in the Greater Mekong sub-region (GMS), representing a threat to malaria control and prevention. This study aims to determine the insecticide resistance status of Anopheles mosquitoes in Ubon Ratchathani province, northeastern Thailand, where increasing number of malaria cases were reported recently.MethodsMosquitoes were collected in 2013–2015 using human landing and cattle bait collections in six sites during both the rainy and dry seasons. Mosquitoes were first morphologically identified to species and their susceptibility status to deltamethrin (0.05%), permethrin (0.75%) and DDT (4%) investigated, according to WHO guidelines. Bioassays with the synergists PBO and DEF were carried out to address the role of detoxifying enzymes in insecticide resistance. DNA sequencing of a fragment of the voltage-gated sodium channel gene was carried out to detect knock-down resistance (kdr) substitutions at position 1014 in resistant species.ResultsDue to low vector abundance, complete bioassays (n ≥ 100 mosquitoes) were only achieved for Anopheles hyrcanus s.l., which was resistant to all insecticides tested (mortality ranged from 45 to 87%). Suspected resistance to DDT was found in Anopheles barbirostris s.l. (mortality 69%), but it was susceptible to deltamethrin (mortality 97–100%) and permethrin (mortality 100%). Although insufficient number of primary vectors were collected, results showed that Anopheles dirus s.l. and Anopheles maculatus s.l. were susceptible to deltamethrin (mortality 100%). Anopheles nivipes and Anopheles philippinensis were susceptible to all three insecticides. PBO significantly increased mortality to deltamethrin and permethrin in pyrethroid-resistant An. hyrcanus s.l. None of the sequenced specimens presented the L1014F or L1014S mutation.DiscussionThis study shows that insecticide resistance is present in potential malaria vectors in northeastern Thailand. The absence of kdr mutations in all Anopheles species tested suggests that metabolic resistance is the main mechanism of pyrethroid resistance. This study provides new findings about insecticide susceptibility status of potential malaria vectors in northeastern Thailand that are deemed important to guide malaria vector control.