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      The cytochrome P450 CYP6P4 is responsible for the high pyrethroid resistance in knockdown resistance-free Anopheles arabiensis

      a , , a , a , b , a , a , ∗∗

      Insect Biochemistry and Molecular Biology

      Elsevier Science

      Anopheles arabiensis, Pyrethroids resistance, Metabolic, CYP6P4, δ-ALA, δ-aminolevulinic acid, An., Anopheles, cDNA, complementary DNA, CYPED, cytochrome P450 Engineering Database, DDT, dichlorodiphenyltrichloroethane, DDE, dichlorodiphenyldichloroethylene, IPTG, Isopropyl β-d-1-thiogalactopyranoside, NADP, nicotinamide adenine dinucleotide phosphate, ompA, outer membrane protein A, P450cam, P450 camphor hydroxylase, PLANTSPLP, Piece-wise linear potential Protein-Ligand ANT System, qRT-PCR, quantitative reverse transcriptase-polymerase chain reaction, Rdl, resistance to dieldrin, rp1, resistance to pyrethroids 1

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          Pyrethroid insecticides are the front line vector control tools used in bed nets to reduce malaria transmission and its burden. However, resistance in major vectors such as Anopheles arabiensis is posing a serious challenge to the success of malaria control.

          Herein, we elucidated the molecular and biochemical basis of pyrethroid resistance in a knockdown resistance-free Anopheles arabiensis population from Chad, Central Africa. Using heterologous expression of P450s in Escherichia coli coupled with metabolism assays we established that the over-expressed P450 CYP6P4, located in the major pyrethroid resistance ( rp1) quantitative trait locus (QTL), is responsible for resistance to Type I and Type II pyrethroid insecticides, with the exception of deltamethrin, in correlation with field resistance profile. However, CYP6P4 exhibited no metabolic activity towards non-pyrethroid insecticides, including DDT, bendiocarb, propoxur and malathion. Combining fluorescent probes inhibition assays with molecular docking simulation, we established that CYP6P4 can bind deltamethrin but cannot metabolise it. This is possibly due to steric hindrance because of the large vdW radius of bromine atoms of the dihalovinyl group of deltamethrin which docks into the heme catalytic centre.

          The establishment of CYP6P4 as a partial pyrethroid resistance gene explained the observed field resistance to permethrin, and its inability to metabolise deltamethrin probably explained the high mortality from deltamethrin exposure in the field populations of this Sudano-Sahelian An. arabiensis. These findings describe the heterogeneity in resistance towards insecticides, even from the same class, highlighting the need to thoroughly understand the molecular basis of resistance before implementing resistance management/control tools.

          Graphical abstract


          • An. arabiensis is a major malaria vector in Sudano-Sahelian Africa.
          • Functional characterisation established CYP6P4 as a key pyrethroid resistance gene in An. arabiensis.
          • CYP6P4 metabolizes pyrethroids, including permethrin, bifenthrin and λ-cyhalothrin.
          • However, the P450 does not metabolise deltamethrin, though inhibition assay revealed that the P450 binds deltamethrin.
          • In silico analyses revealed that in contrast to other pyrethroids, deltamethrin binds unproductively in CYP6P4.

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          Most cited references 55

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          ZINC--a free database of commercially available compounds for virtual screening.

          A critical barrier to entry into structure-based virtual screening is the lack of a suitable, easy to access database of purchasable compounds. We have therefore prepared a library of 727,842 molecules, each with 3D structure, using catalogs of compounds from vendors (the size of this library continues to grow). The molecules have been assigned biologically relevant protonation states and are annotated with properties such as molecular weight, calculated LogP, and number of rotatable bonds. Each molecule in the library contains vendor and purchasing information and is ready for docking using a number of popular docking programs. Within certain limits, the molecules are prepared in multiple protonation states and multiple tautomeric forms. In one format, multiple conformations are available for the molecules. This database is available for free download ( in several common file formats including SMILES, mol2, 3D SDF, and DOCK flexibase format. A Web-based query tool incorporating a molecular drawing interface enables the database to be searched and browsed and subsets to be created. Users can process their own molecules by uploading them to a server. Our hope is that this database will bring virtual screening libraries to a wide community of structural biologists and medicinal chemists.
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            Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction.

            A ribosomal DNA-polymerase chain reaction (PCR) method has been developed for species identification of individuals of the five most widespread members of the Anopheles gambiae complex, a group of morphologically indistinguishable sibling mosquito species that includes the major vectors of malaria in Africa. The method, which is based on species-specific nucleotide sequences in the ribosomal DNA intergenic spacers, may be used to identify both species and interspecies hybrids, regardless of life stage, using either extracted DNA or fragments of a specimen. Intact portions of a mosquito as small as an egg or the segment of one leg may be placed directly into the PCR mixture for amplification and analysis. The method uses a cocktail of five 20-base oligonucleotides to identify An. gambiae, An. arabiensis, An. quadriannnulatus, and either An. melas in western Africa or An. melas in eastern and southern Africa.
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              Insecticide resistance in insect vectors of human disease.

              Insecticide resistance is an increasing problem in many insect vectors of disease. Our knowledge of the basic mechanisms underlying resistance to commonly used insecticides is well established. Molecular techniques have recently allowed us to start and dissect most of these mechanisms at the DNA level. The next major challenge will be to use this molecular understanding of resistance to develop novel strategies with which we can truly manage resistance. State-of-the-art information on resistance in insect vectors of disease is reviewed in this context.

                Author and article information

                Insect Biochem Mol Biol
                Insect Biochem. Mol. Biol
                Insect Biochemistry and Molecular Biology
                Elsevier Science
                1 January 2016
                January 2016
                : 68
                : 23-32
                [a ]Vector Biology Department, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, United Kingdom
                [b ]Department of Health and Social Sciences, Leeds Beckett University, LS1 3HE, Leeds, United Kingdom
                Author notes
                []Corresponding author. SulaimanSadi.Ibrahim@
                [∗∗ ]Corresponding author. Vector Biology Department, Liverpool School of Tropical Medicine, Pembroke Place, L3 5QA, United Kingdom.Vector Biology DepartmentLiverpool School of Tropical MedicinePembroke PlaceL3 5QAUnited Kingdom charles.wondji@
                © 2015 The Authors

                This is an open access article under the CC BY license (



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