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Background  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.    Methodology/Principal findings  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. Among the  Cx .  pipiens  populations collected in Northern Italy all but one had at least one of the three DFB mutations at allele 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.    Conclusions/Significance  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.

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