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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.

SUBMITTER: Kalajdzic P

PROVIDER: S-EPMC3386967 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Use of mutagenesis, genetic mapping and next generation transcriptomics to investigate insecticide resistance mechanisms.

Kalajdzic Predrag P Oehler Stefan S Reczko Martin M Pavlidi Nena N Vontas John J Hatzigeorgiou Artemis G AG Savakis Charalambos C

PloS one 20120629 6

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 retriev ...[more]

PMID: 22768270

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Project description:BackgroundThe emergence and spread of insecticide resistance in the major African malaria vectors Anopheles gambiae (s.s.) and An. arabiensis may compromise the current vector control interventions and threatens the global malaria control and elimination efforts.MethodsInsecticide resistance was monitored in several study sites in Ethiopia from 2013 to 2015 using papers impregnated with discriminating concentrations of DDT, deltamethrin, bendiocarb, propoxur, malathion, fenitrothion and pirimiphos-methyl, following the WHO insecticide susceptibility test procedure. Mosquitoes sampled from different localities for WHO bioassay were morphologically identified as An. gambiae (s.l.) using standard taxonomic keys. Samples were identified to species using species-specific polymerase chain reaction (PCR) and screened for the presence of target site mutations L1014F, L1014S and N1575Y in the voltage gated sodium channel (VGSC) gene and G119S in the acethylcholinesterase (AChE) gene using allele-specific PCR. Biochemical assays were performed to assess elevated levels of acetylcholinesterases, carboxylcholinesterases, glutathione-S-transferases (GSTs) and cytochrome P450s monooxygenases in wild populations of An. arabiensis, compared to the fully susceptible Sekoru An. arabiensis laboratory strain.ResultsPopulations of An. arabiensis were resistant to DDT and deltamethrin but were susceptible to fenitrothion in all the study sites. Reduced susceptibility to malathion, pirimiphos-methyl, propoxur and bendiocarb was observed in some of the study sites. Knockdown resistance (kdr L1014F) was detected in all mosquito populations with allele frequency ranging from 42 to 91%. Elevated levels of glutathione-S-transferases (GSTs) were detected in some of the mosquito populations. However, no elevated levels of monooxygenases and esterases were detected in any of the populations assessed.ConclusionsAnopheles arabiensis populations from all surveyed sites in Ethiopia exhibited resistance against DDT and pyrethroids. Moreover, some mosquito populations exhibited resistance to propoxur and possible resistance to bendiocarb. Target site mutation kdr L1014F was detected in all mosquito populations while elevated levels of glutathione-S-transferases (GSTs) was detected in some mosquito populations. The reduced susceptibility of An. arabiensis to propoxur and bendiocarb, which are currently used for indoor residual spraying (IRS) in Ethiopia, calls for continuous resistance monitoring, in order to plan and implement evidence based insecticide resistance management.

Project description:The southern root-knot nematode (SRKN; Meloidogyne incognita) is a typical parasitic nematode that affects sweetpotato [Ipomoea batatas (L.) Lam.], causing a significant decrease in crop yield and commercial value. In Japan, the SRKN is classified into 10 races: SP1-SP5, SP6-1, SP6-2, and SP7-SP9, with the dominant race differing according to the cultivation area. Soil insecticides have previously been used to reduce the soil density of SRKNs; however, this practice is both costly and labor intensive. Therefore, the development of SRKN-resistant sweetpotato lines and cultivars is necessary. However, due to the complexity of polyploid inheritance and the highly heterogeneous genomic composition of sweetpotato, genetic information and research for this species are significantly lacking compared to those for other major diploid crop species. In this study, we utilized the recently developed genome-wide association approach, which uses multiple-dose markers to assess autopolyploid species. We performed an association analysis to investigate resistance toward SRKN-SP2, which is the major race in areas with high sweetpotato production in Japan. The segregation ratio of resistant and susceptible lines in the F1 mapping population derived from the resistant "J-Red" and susceptible "Choshu" cultivars was fitted to 1: 3, suggesting that resistance to SP2 may be regulated by two loci present in the simplex. By aligning the double digest restriction-site associated DNA sequencing reads to the published Ipomoea trifida reference sequence, 46,982 single nucleotide polymorphisms (SNPs) were identified (sequencing depth > 200). The association study yielded its highest peak on chromosome 7 (Chr07) and second highest peak on chromosome 3 (Chr03), presenting as a single-dose in both loci. Selective DNA markers were developed to screen for resistant plants using the SNPs identified on Chr03 and Chr07. Our results showed that SRKN-SP2-resistant plants were selected with a probability of approximately 70% when combining the two selective DNA markers. This study serves as a model for the identification of genomic regions that control agricultural traits and the elucidation of their effects, and is expected to greatly advance marker-assisted breeding and association studies in polyploid crop species.

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Use of mutagenesis, genetic mapping and next generation transcriptomics to investigate insecticide resistance mechanisms.

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Use of mutagenesis, genetic mapping and next generation transcriptomics to investigate insecticide resistance mechanisms.

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