Use of Mutagenesis, Genetic Mapping and Next Generation Transcriptomics to Investigate Insecticide Resistance Mechanisms

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.

Spirotetramat resistance adaption analysis of Aphis gossypii Glover by transcriptomic survey

“…Up-regulated transcription of P450 genes appears to be the general mechanism that increases enzyme levels in resistant individuals [45][46][47][48]. CYP6A2 was known to be involved in neonicotinoid resistance and is highly up-regulated and linked with resistance [49,50]. In this study, CYP6A2 (see sequence in Table S7) was the only P450 gene found to be significantly upregulated in the SR strain (Table 4), suggesting its involvement in the detoxification of spirotetramat.…”

Comparative CYP‐omic analysis between the DDT‐susceptible and ‐resistant Drosophila melanogaster strains 91‐C and 91‐R

“…The observation that the deletion of upstream regulatory regions of Cyp4p1 in 91‐C fails to result in any significant difference in expression compared with 91‐R confirms the RNA‐seq‐based estimates of low basal expression in 91‐C . Regardless, the Cyp4p clade remains functional in 91‐R , and Cyp4p2 is up‐regulated in DDT‐ and imidacloprid‐resistant flies when compared with susceptible counterparts . The exact role of the Cyp4 clade in resistance remains unknown.…”

“…Regardless, the Cyp4p clade remains functional in 91-R, and Cyp4p2 is up-regulated in DDT-and imidacloprid-resistant flies when compared with susceptible counterparts. 84 The exact role of the Cyp4 clade in resistance remains unknown. As the gene family resides outside of genome regions of a selective sweep 47 and shows no significant up-regulation in 91-R compared with the deficient 91-C strain, one might conclude that Cyp4p1/2/3 may not be involved in the DDT resistance trait of 91-R.…”

Effects of neonicotinoids and fipronil on non-target invertebrates

“…Particularly, cytochrome P450 monooxygenases (CYPs) are involved, as is the case in mosquitoes (Riaz et al 2013 ). According to Kalajdzic et al ( 2012 ), three P450 genes (Cyp4p2, Cyp6a2 and Cyp6g1) located on the 2R chromosome were highly up-regulated in imidacloprid-resistant flies. However, the same authors did not find that imidacloprid induced expression of Cyp6g1 and Cyp6a2 (Kalajdzic et al 2013 ).…”