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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. ; Author summary: Diflubenzuron is one of the main larvicides used for the control of the West Nile Virus vector Culex pipiens in the Mediterranean. However, the efficiency of control is now under threat due to the selection of insecticide resistance. Two point mutations were previously identified at the Chitin synthase and shown to confer low and high levels of resistance and a diagnostic was developed to monitor the trait. This study reports the identification of a third mutation associated with high levels of diflubenzuron resistance in Italy. This mutation was also detected in France, whereas no resistance mutations were found in Cx. pipiens mosquitoes sampled from Greece, Portugal and Israel. The findings are of major concern for mosquito control programs in S. Europe, which rely on the use of a limited number of larvicides. ; E.A. F. was supported by the General Secretariat for Research and Technology (GSRT) and the Hellenic Foundation for Research and Innovation (HFRI) in the context of the action "1st Proclamation of Scholarships from ELIDEK for PhD Candidates" (Scholarship Code: 532). JV and RB received funds from European Union's Horizon (INFRAVEC) research and innovation programme under grant agreement No 731060 (https://infravec2.eu/). ; info:eu-repo/semantics/publishedVersion

This is the final version. Available from Elsevier / Academic Press via the DOI in this record. ; Insecticides of the tetronic/tetramic acid family (cyclic ketoenols) are widely used to control sucking pests such as whiteflies, aphids and mites. They act as inhibitors of acetyl-CoA carboxylase (ACC), a key enzyme for lipid biosynthesis across taxa. While it is well documented that plant ACCs targeted by herbicides have developed resistance associated with mutations at the carboxyltransferase (CT) domain, resistance to ketoenols in invertebrate pests has been previously associated either with metabolic resistance or with non-validated candidate mutations in different ACC domains. A recent study revealed high levels of spiromesifen and spirotetramat resistance in Spanish field populations of the whitefly Bemisia tabaci that was not thought to be associated with metabolic resistance. We confirm the presence of high resistance levels (up to >640-fold) against ketoenol insecticides in both Spanish and Australian B. tabaci strains of the MED and MEAM1 species, respectively. RNAseq analysis revealed the presence of an ACC variant bearing a mutation that results in an amino acid substitution, A2083V, in a highly conserved region of the CT domain. F1 progeny resulting from reciprocal crosses between susceptible and resistant lines are almost fully resistant, suggesting an autosomal dominant mode of inheritance. In order to functionally investigate the contribution of this mutation and other candidate mutations previously reported in resistance phenotypes, we used CRISPR/Cas9 to generate genome modified Drosophila lines. Toxicity bioassays using multiple transgenic fly lines confirmed that A2083V causes high levels of resistance to commercial ketoenols. We therefore developed a pyrosequencing-based diagnostic assay to map the spread of the resistance alleles in field-collected samples from Spain. Our screening confirmed the presence of target-site resistance in numerous field-populations collected in Sevilla, Murcia and Almeria. This emphasizes the importance of implementing appropriate resistance management strategies to prevent or slow the spread of resistance through global whitefly populations. ; European Union Horizon 2020 ; Australian cotton research and development corporation

This is the final version. Available from the publisher via the DOI in this record. ; There is an on-going need to develop new insecticides that are not compromised by resistance and that have improved environmental profiles. However, the cost of developing novel compounds has increased significantly over the last two decades. This is in part due to increased regulatory requirements, including the need to screen both pest and pollinator insect species to ensure that pre-existing resistance will not hamper the efficacy of a new insecticide via cross-resistance, or adversely affect non-target insect species. To add to this problem the collection and maintenance of toxicologically relevant pest and pollinator species and strains is costly and often difficult. Here we present Fly-Tox, a panel of publicly available transgenic Drosophila melanogaster lines each containing one or more pest or pollinator P450 genes that have been previously shown to metabolise insecticides. We describe the range of ways these tools can be used, including in predictive screens to avoid pre-existing cross-resistance, to identify potential resistance-breaking inhibitors, in the initial assessment of potential insecticide toxicity to bee pollinators, and identifying harmful pesticide-pesticide interactions. ; European Research Council (ERC) ; European Union's Horizon 2020 research and innovation programme ; Biotechnology and Biological Sciences Research Council (BBSRC)

The transmission of the arboviral agents of dengue, yellow fever, Chikungunya, and Zika by Aedes mosquitoes represents expanding threats to global health. At the 69th World Health Assembly [1], the WHO Director-General Margaret Chan declared that the spread of the Zika virus was "the result of the abandon of mosquito control" by governments since the 1970s and urged Member States to mobilize more efforts and resources to prevent further spread of the diseases. The recent rise of microcephaly cases and other neurological disorders reported in Brazil prompted WHO to declare Zika as a Public Health Emergency of International Concern [2]. After limited early outbreaks in the Pacific in 2007 and 2013, the Zika virus has spread to more than 30 countries in the Americas and the Caribbean, affecting over 1.5 million people [3]. With growing evidence supporting the link between microcephaly and Zika [4, 5] and preliminary evidence confirming Aedes aegypti as the primary vector in the Brazilian outbreak [6], the mandate for control is clear and urgent.