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Abstract
Insecticide resistance is a worldwide problem with major impact on agriculture and human health. Understanding the underlying molecular mechanisms is crucial for the management of the phenomenon; however, this information often comes late with respect to the implementation of efficient counter-measures, particularly in the case of metabolism-based resistance mechanisms. We employed a genome-wide insertional mutagenesis screen to Drosophila melanogaster, using a Minos-based construct, and retrieved a line (MiT[w−]3R2) resistant to the neonicotinoid insecticide Imidacloprid. Biochemical and bioassay data indicated that resistance was due to increased P450 detoxification. Deep sequencing transcriptomic analysis revealed substantial over- and under-representation of 357 transcripts in the resistant line, including statistically significant changes in mixed function oxidases, peptidases and cuticular proteins. Three P450 genes (Cyp4p2, Cyp6a2 and Cyp6g1) located on the 2R chromosome, are highly up-regulated in mutant flies compared to susceptible Drosophila. One of them (Cyp6g1) has been already described as a major factor for Imidacloprid resistance, which validated the approach. Elevated expression of the Cyp4p2 was not previously documented in Drosophila lines resistant to neonicotinoids. In silico analysis using the Drosophila reference genome failed to detect transcription binding factors or microRNAs associated with the over-expressed Cyp genes. The resistant line did not contain a Minos insertion in its chromosomes, suggesting a hit-and-run event, i.e. an insertion of the transposable element, followed by an excision which caused the mutation. Genetic mapping placed the resistance locus to the right arm of the second chromosome, within a ∼1 Mb region, where the highly up-regulated Cyp6g1 gene is located. The nature of the unknown mutation that causes resistance is discussed on the basis of these results.
Highlights
Insecticide resistance is an increasing problem that compromises the control of insect pests of medical, veterinary and agricultural impact
A single P450, CYP6P3, was over-expressed in pyrethroid resistant Anopheles gambiae mosquitoes, and it was capable of metabolizing pyrethroids [7]
Karunker et al [8] showed that the B. tabaci cytochrome P450 BTCYP6CM1 is capable of metabolizing the neonicotinoid Imidacloprid, one of the most important insecticides worldwide, and to confer neonicotinoid resistance
Summary
Insecticide resistance is an increasing problem that compromises the control of insect pests of medical, veterinary and agricultural impact. An understanding of insecticide resistance mechanisms is essential for the subsequent development of tools and practices that can improve pest control interventions. Extensive biochemical, genetic and molecular studies have been conducted to elucidate insecticide resistance mechanisms [1,2,3]. Knowledge of the mechanisms underlying target site resistance in major pests to some commonly used insecticides has been established to some extent [4,5,6]. The understanding of detoxification/metabolism-based insecticide resistance mechanisms has not kept similar pace, due to the complexity of the involved multi-gene systems and the lack of genome sequence data. In a few cases, the molecular basis of metabolismbased insecticide resistance mechanisms was identified. Karunker et al [8] showed that the B. tabaci cytochrome P450 BTCYP6CM1 is capable of metabolizing the neonicotinoid Imidacloprid, one of the most important insecticides worldwide, and to confer neonicotinoid resistance
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