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      Genome-Wide Transcription and Functional Analyses Reveal Heterogeneous Molecular Mechanisms Driving Pyrethroids Resistance in the Major Malaria Vector Anopheles funestus Across Africa

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          Abstract

          Pyrethroid resistance in malaria vector, An. funestus is increasingly reported across Africa, threatening the sustainability of pyrethroid-based control interventions, including long lasting insecticidal nets (LLINs). Managing this problem requires understanding of the molecular basis of the resistance from different regions of the continent, to establish whether it is being driven by a single or independent selective events. Here, using a genome-wide transcription profiling of pyrethroid resistant populations from southern (Malawi), East (Uganda), and West Africa (Benin), we investigated the molecular basis of resistance, revealing strong differences between the different African regions. The duplicated cytochrome P450 genes ( CYP6P9a and CYP6P9b) which were highly overexpressed in southern Africa are not the most upregulated in other regions, where other genes are more overexpressed, including GSTe2 in West (Benin) and CYP9K1 in East (Uganda). The lack of directional selection on both CYP6P9a and CYP6P9b in Uganda in contrast to southern Africa further supports the limited role of these genes outside southern Africa. However, other genes such as the P450 CYP9J11 are commonly overexpressed in all countries across Africa. Here, CYP9J11 is functionally characterized and shown to confer resistance to pyrethroids and moderate cross-resistance to carbamates (bendiocarb). The consistent overexpression of GSTe2 in Benin is coupled with a role of allelic variation at this gene as GAL4-UAS transgenic expression in Drosophila flies showed that the resistant 119F allele is highly efficient in conferring both DDT and permethrin resistance than the L119. The heterogeneity in the molecular basis of resistance and cross-resistance to insecticides in An. funestus populations throughout sub-Saharan African should be taken into account in designing resistance management strategies.

          Most cited references64

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          DnaSP, DNA polymorphism analyses by the coalescent and other methods.

          DnaSP is a software package for the analysis of DNA polymorphism data. Present version introduces several new modules and features which, among other options allow: (1) handling big data sets (approximately 5 Mb per sequence); (2) conducting a large number of coalescent-based tests by Monte Carlo computer simulations; (3) extensive analyses of the genetic differentiation and gene flow among populations; (4) analysing the evolutionary pattern of preferred and unpreferred codons; (5) generating graphical outputs for an easy visualization of results. The software package, including complete documentation and examples, is freely available to academic users from: http://www.ub.es/dnasp
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            A cocktail polymerase chain reaction assay to identify members of the Anopheles funestus (Diptera: Culicidae) group.

            Anopheles funestus Giles is a major malaria vector in Africa belonging to a group of species with morphologically similar characteristics. Morphological identification of members of the A. funestus group is difficult because of overlap of distinguishing characteristics in adult or immature stages as well as the necessity to rear isofemale lines to examine larval and egg characters. A rapid rDNA polymerase chain reaction (PCR) method has been developed to accurately identify five members of the A. funestus group. This PCR is based on species-specific primers in the ITS2 region on the rDNA to identify A. funestus (approximately 505bp), Anopheles vaneedeni Gillies and Coetzee (approximately 587bp), Anopheles rivulorum Leeson (approximately 411bp), Anopheles leesoni Evans (approximately 146bp), and Anopheles parensis Gillies (approximately 252bp).
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              A single mutation in the GSTe2 gene allows tracking of metabolically based insecticide resistance in a major malaria vector

              Background Metabolic resistance to insecticides is the biggest threat to the continued effectiveness of malaria vector control. However, its underlying molecular basis, crucial for successful resistance management, remains poorly characterized. Results Here, we demonstrate that the single amino acid change L119F in an upregulated glutathione S-transferase gene, GSTe2, confers high levels of metabolic resistance to DDT in the malaria vector Anopheles funestus. Genome-wide transcription analysis revealed that GSTe2 was the most over-expressed detoxification gene in DDT and permethrin-resistant mosquitoes from Benin. Transgenic expression of GSTe2 in Drosophila melanogaster demonstrated that over-transcription of this gene alone confers DDT resistance and cross-resistance to pyrethroids. Analysis of GSTe2 polymorphism established that the point mutation is tightly associated with metabolic resistance to DDT and its geographical distribution strongly correlates with DDT resistance patterns across Africa. Functional characterization of recombinant GSTe2 further supports the role of the L119F mutation, with the resistant allele being more efficient at metabolizing DDT than the susceptible one. Importantly, we also show that GSTe2 directly metabolizes the pyrethroid permethrin. Structural analysis reveals that the mutation confers resistance by enlarging the GSTe2 DDT-binding cavity, leading to increased DDT access and metabolism. Furthermore, we show that GSTe2 is under strong directional selection in resistant populations, and a restriction of gene flow is observed between African regions, enabling the prediction of the future spread of this resistance. Conclusions This first DNA-based metabolic resistance marker in mosquitoes provides an essential tool to track the evolution of resistance and to design suitable resistance management strategies.
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                Author and article information

                Journal
                G3 (Bethesda)
                Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes|Genomes|Genetics
                Genetics Society of America
                2160-1836
                19 April 2017
                June 2017
                : 7
                : 6
                : 1819-1832
                Affiliations
                [* ]Liverpool School of Tropical Medicine, L3 5QA, United Kingdom
                []Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale (OCEAC), P.O. Box 288 Yaoundé, Cameroon
                []Department of Biochemistry, Bayero University, PMB 3011 Kano, Nigeria
                [§ ]Uganda Virus Research Institute, P.O. Box 49 Entebbe, Uganda
                [** ]International Institute of Tropical Agriculture, Cotonou 08 BP 0932, Benin
                [†† ]School of Biological Sciences, Universiti Sains Malaysia, Gelugor, 11800 Penang, Malaysia
                Author notes
                [1 ]Corresponding author: Vector Biology Department, Liverpool School of Tropical Medicine, Pembroke Place, L3 5QA, UK. E-mail: Charles.Wondji@ 123456lstmed.ac.uk
                Article
                GGG_040147
                10.1534/g3.117.040147
                5473761
                28428243
                3cf1f536-844a-4d97-bacd-c8f5f50ed372
                Copyright © 2017 Riveron et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 01 February 2017
                : 04 April 2017
                Page count
                Figures: 5, Tables: 3, Equations: 0, References: 76, Pages: 14
                Categories
                Investigations

                Genetics
                malaria,anopheles funestus,pyrethroids,insecticide resistance,cytochrome p450
                Genetics
                malaria, anopheles funestus, pyrethroids, insecticide resistance, cytochrome p450

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