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.

Abstract:The olon tree, Zanthoxylum heitzii (syn. Fagara heitzii) is commonly found in the central-west African forests. In the Republic of Congo (Congo-Brazzaville) its bark is anecdotally reported to provide human protection against fleas. Here we assess the insecticidal activities of Z. heitzii stem bark, seed and leaf extracts against Anopheles gambiae s.s, the main malaria vector in Africa. Extracts were obtained by Accelerated Solvent Extraction OPEN ACCESSMolecules 2014, 19 21277 (ASE) using solvents of different polarity and by classical Soxhlet extraction using hexane as solvent. The insecticidal effects of the crude extracts were evaluated using topical applications of insecticides on mosquitoes of a susceptible reference strain (Kisumu [Kis]), a strain homozygous for the L1014F kdr mutation (kdrKis), and a strain homozygous for the G119S Ace1R allele (AcerKis). The insecticidal activities were measured using LD50 and LD95 and active extracts were characterized by NMR spectroscopy and HPLC chromatography. Results show that the ASE hexane stem bark extract was the most effective compound against An. gambiae (LD50 = 102 ng/mg female), but was not as effective as common synthetic insecticides. Overall, there was no significant difference between the responses of the three mosquito strains to Z. heitzii extracts, indicating no cross resistance with conventional pesticides.

Additional vector control tools are needed to supplement current strategies to achieve malaria elimination and control of Aedes-borne diseases in many settings in Thailand and the Greater Mekong Sub-region. Within the next decade, the vector control community, Kasetsart University (KU), and the Ministry of Higher Education, Science, Research and Innovation must take full advantage of these tools that combine different active ingredients with different modes of action. Pu Teuy Mosquito Field Research Station (MFRS), Department of Entomology, Faculty of Agriculture, Kasetsart University (KU), Thailand was established in 2001 and has grown into a leading facility for performing high-quality vector biology and control studies and evaluation of public health insecticides that are operationally relevant. Several onsite mosquito research platforms have been established including experimental huts, a 40-m long semi-field screening enclosure, mosquito insectary, field-laboratory, and living quarters for students and researchers. Field research and assessments ranged from ‘basic’ investigations on mosquito biology, taxonomy and genetics to more ‘applied’ studies on responses of mosquitoes to insecticides including repellency, behavioural avoidance and toxicity. In the course of two decades, 51 peer-reviewed articles have been published, and 7 masters and 16 doctoral degrees in Entomology have been awarded to national and international students. Continued support of key national stakeholders will sustain MFRS as a Greater Mekong Subregion centre of excellence and a resource for both insecticide trials and entomological research.

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