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Research Project: Biological Control of Invasive Arthropod Pests from the Eastern Hemisphere

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Title: Identification and detection of a novel point mutation in the Chitin Synthase gene of Culex pipiens associated with diflubenzuron resistance

item FOTAKIS, EMMANOULI - Agricultural University Of Athens
item MASTANTONIO, VALENTINA - University Of Rome Sapienza
item GRIGORIKA, LINDA - Liverpool School Of Tropical Medicine
item PORRETTA, DANIELE - University Of Rome Sapienza
item PUGGIOLI, ARIANNA - Caa G Nicoli
item CHASKOPOULOU, ALEXANDRA - European Biological Control Laboratory (EBCL)
item OSORIO, HUGO - National Institute Of Health (INSA)
item WEILL, MYLENE - University Of Montpellier
item BELLINI, ROMEO - Caa G Nicoli
item URBANELLI, SANDRA - University Of Rome Sapienza

Submitted to: PLOS Neglected Tropical Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/9/2020
Publication Date: 5/1/2020
Citation: Fotakis, E., Mastantonio, V., Grigorika, L., Porretta, D., Puggioli, A., Chaskopoulou, A., Osorio, H., Weill, M., Bellini, R., Urbanelli, S. 2020. Identification and detection of a novel point mutation in the Chitin Synthase gene of Culex pipiens associated with diflubenzuron resistance. PLOS Neglected Tropical Diseases. 14(5): e0008284.

Interpretive Summary: Insecticide resistance is a major threat for vector control and prevention of mosquito borne diseases. In the Culex pipiens mosquitoes, major vectors of West Nile virus world wide, high levels of resistance against diflubenzuron insecticide were recently detected in Italy in 2015. The resistant mosquitoes were associated with two mutations within an important gene encoding the enzyme chitin synthase, with both mutations located in the same codon for amino acid 1043. Here we report, for the first time in mosquitoes, the identification of a third mutation at the same codon position of the chitin synthase gene in mosquitoes collected from Italy and France. This mutation was shown to confer striking levels (>15,000 fold) of diflubenzuron resistance in Drosophila flies. The selection of resistance to diflubenzuron in Italy seems to be associated with the extensive use of its active ingredient over the past twenty to thirty years against agricultural pests, also potentially driven by the use of diflubenzuron against mosquitoes over the last ten to fifteen years. The findings are of major concern for public health workers and researchers in Europe and worldwide, as diflubenzuron remains a very important insecticide used for controlling arbovirus mosquito vectors, where alternative larvicides are extremely limited.

Technical Abstract: Diflubenzuron (DFB) is one of the most used insecticides in mosquito larval control including that of Culex pipiens, the proven vector of the recent West Nile virus epidemics in Europe. Two mutations (I1043L and I1043M) in the chitin synthase (CHS) putative binding site of DFB have been previously reported in Cx. pipiens from Italy and associated with high levels of resistance against this larvicide. Here we report the identification of a third mutation at the same I1043 position of the CHS gene resulting in the substitution of Isoleucine to Phenylalanine (I1043F). This mutation has also been found in agricultural pests and has been functionally validated with genome editing in Drosophila, showing to confer striking levels (>15,000 fold) of DFB resistance. The frequency of the I1043F mutation was found to be substantially higher in Cx. pipiens mosquitoes surviving DFB doses largely exceeding the recommended field dose, raising concerns about the future efficient use of this insecticide. We monitored the presence and frequency of DFB mutations in Cx. pipiens mosquitoes from several Mediterranean countries, including Italy, France, Greece, Portugal and Israel. All but one of the Cx. pipiens populations collected in Northern Italy had at least one of the three DFB mutations at frequencies reaching 93.3% for the I1043M, 64.8% for the I1043L and 10% for the I1043F. The newly reported I1043F mutation was also identified in two heterozygote individuals from France (4.2% allelic frequency). In contrast to Italy and France, no DFB resistant mutations were identified in the Cx. pipiens mosquitoes sampled from Greece, Portugal and Israel. The findings of our study are of major concern for mosquito control programs in Europe, that rely on the use of a limited number of available larvicides, and highlight the necessity for the development of appropriate Insecticide Resistance Management (IRM) programs, to ensure the sustainable use of DFB.