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Abstract In addition to combating vector‐borne diseases, studying the adaptation of mosquitoes to insecticides provides a remarkable example of evolution‐in‐action driving the selection of complex phenotypes. Actually, most resistant mosquito populations show multi‐resistance phenotypes as a consequence of the variety of insecticides employed and of the complexity of selected resistance mechanisms. Such complexity makes the identification of alleles conferring resistance to specific insecticides challenging and prevents the development of molecular assays to track them in the field. Here we showed that combining simple genetic crosses with pool targeted DNA‐seq can enhance the specificity of resistance allele's detection while maintaining experimental work and sequencing effort at reasonable levels. A multi‐resistant population of the mosquito Aedes aegypti was exposed to three distinct insecticides (deltamethrin, bendiocarb and fenitrothion), and survivors to each insecticide were crossed with a susceptible strain to generate three distinct lines. F2 individuals from each line were then segregated based on their survival to two insecticide doses. Hundreds of genes covering all detoxifying enzymes and insecticide targets together with more than 7,000 intergenic regions equally spread over mosquito genome were sequenced from pools of F0 and F2 individuals unexposed or surviving insecticide. Differential coverage analysis identified 39 detoxification enzymes showing an increased gene copy number in association with resistance. Combining an allele frequency filtering approach with a Bayesian FST‐based genome scan identified multiple genomic regions showing strong selection signatures together with 50 nonsynonymous variations associated with resistance. This study provides a simple and cost‐effective approach to improve the specificity of resistance allele's detection in multi‐resistant populations while reducing false positives frequently arising when comparing populations showing divergent genetic backgrounds. The identification of novel DNA resistance markers opens new opportunities for improving the tracking of insecticide resistance in the field.

In addition to combating vector‐borne diseases, studying the adaptation of mosquitoes to insecticides provides a remarkable example of evolution‐in‐action driving the selection of complex phenotypes. Actually, most resistant mosquito populations show multi‐resistance phenotypes as a consequence of the variety of insecticides employed and of the complexity of selected resistance mechanisms. Such complexity makes the identification of alleles conferring resistance to specific insecticides challenging and prevents the development of molecular assays to track them in the field. Here we showed that combining simple genetic crosses with pool targeted DNA‐seq can enhance the specificity of resistance allele's detection while maintaining experimental work and sequencing effort at reasonable levels. A multi‐resistant population of the mosquito Aedes aegypti was exposed to three distinct insecticides (deltamethrin, bendiocarb and fenitrothion), and survivors to each insecticide were crossed with a susceptible strain to generate three distinct lines. F2 individuals from each line were then segregated based on their survival to two insecticide doses. Hundreds of genes covering all detoxifying enzymes and insecticide targets together with more than 7,000 intergenic regions equally spread over mosquito genome were sequenced from pools of F0 and F2 individuals unexposed or surviving insecticide. Differential coverage analysis identified 39 detoxification enzymes showing an increased gene copy number in association with resistance. Combining an allele frequency filtering approach with a Bayesian FST‐based genome scan identified multiple genomic regions showing strong selection signatures together with 50 nonsynonymous variations associated with resistance. This study provides a simple and cost‐effective approach to improve the specificity of resistance allele's detection in multi‐resistant populations while reducing false positives frequently arising when comparing populations showing divergent genetic backgrounds. The identification of novel DNA resistance markers opens new opportunities for improving the tracking of insecticide resistance in the field. [ABSTRACT FROM AUTHOR]

Background and objective: Acenocoumarol (AC) is a coumarin derivative, vitamin K antagonist anticoagulant drug. It has a narrow therapeutic index and shows large pharmacokinetic and pharmacodynamic interindividual variability. Our objective was to investigate the association between AC dose requirements to achieve a target level of anticoagulation and genetic polymorphisms of genes possibly associated with its metabolism ( CYP1A2, CYP2C9, CYP2C19, CYP3A4, CYP3A5) and transport ( ABCB1). Methods: Ninety-six Bulgarian patients treated orally with AC for at least 3 months were included. They were separated into three groups according to their AC dose requirement, i.e. low, medium and high. Results and discussion: CYP2C9* 1/*3 (associated with an intermediate CYP2C9 activity), CYP2C9*2/*2, and CYP2C9* 2/*3 genotypes (associated with a low CYP2C9 activity) were more prevalent in the group with low dose requirement of AC compared with the other two groups ( P = 0·003). The frequency of CYP2C9* 1/*1 genotype, which is associated with an extensive CYP2C9 activity, was higher in the group of patients with high dose requirements (79%), compared with the groups of the medium and low dose requirements (67% and 21% respectively). In addition, the ABCB1 2677GG/3435CC haplotype was associated with use of lower AC dose, whereas the 2677TT/3435TT and 2677GT/3435TT haplotypes were associated with use of higher AC dose ( P = 0·03). The distribution of polymorphisms of other genes did not show significant differences between the three groups. Conclusion: In vivo, cytochromes P450 isoforms other than CYP2C9, and the permeability glycoprotein transporter, which is encoded by the ABCB1 gene, were not significantly associated with dose requirement of AC. In our Bulgarian patients, the presence of CYP2C9*2 or/and CYP2C9*3 alleles, as well as the ABCB1 2677GG/3435CC haplotype were associated with low dose requirement of AC. [ABSTRACT FROM AUTHOR]