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Reversing insecticide resistance with allelic-drive in Drosophila melanogaster
Monday, 2022/01/24 | 08:14:59

Bhagyashree KaduskarRaja Babu Singh KushwahAnkush AuradkarAnnabel GuichardMenglin LiJared B. BennettAlison Henrique Ferreira JulioJohn M. MarshallCraig Montell & Ethan Bier

Nature Communications volume 13, Article number: 291 (2022). Publishe 12 January 2022

Abstract

A recurring target-site mutation identified in various pests and disease vectors alters the voltage gated sodium channel (vgsc) gene (often referred to as knockdown resistance or kdr) to confer resistance to commonly used insecticides, pyrethroids and DDT. The ubiquity of kdr mutations poses a major global threat to the continued use of insecticides as a means for vector control. In this study, we generate common kdr mutations in isogenic laboratory Drosophila strains using CRISPR/Cas9 editing. We identify differential sensitivities to permethrin and DDT versus deltamethrin among these mutants as well as contrasting physiological consequences of two different kdr mutations. Importantly, we apply a CRISPR-based allelic-drive to replace a resistant kdr mutation with a susceptible wild-type counterpart in population cages. This successful proof-of-principle opens-up numerous possibilities including targeted reversion of insecticide-resistant populations to a native susceptible state or replacement of malaria transmitting mosquitoes with those bearing naturally occurring parasite resistant alleles.

 

See https://www.nature.com/articles/s41467-021-27654-1

Figure 1: kdr mutations paraL1014F and paraI1011M confer insecticide resistance in Drosophila melanogaster.

A Generation of kdr mutations. First, a GFP transgene was inserted at the L1014 site using Cas9-mediated editing. Next, the GFP insertion was deleted with a pair of gRNAs cleaving adjacent sequences and replaced with a donor template containing one of the desired mutations (L1014F, I1011M or I1011V). Successful transformants were screened as GFP negative and later confirmed by genotyping. B DDT resistance: Flies (total number of flies n = 200; number of independent replicates N = 8) were exposed to different concentrations of DDT. Survival, a proxy for DDT resistance, was determined 24 h post exposure. At the reference dose of 50 ppm DTT, the mean resistance values ± s.e.m were: WT = 8.4 ± 4.3; L1014F = 100 ± 0; I1011M = 65.6 ± 6.15; I1011V = 8.75 ± 3.6. C Survival plot for paraWT (Median lifespan = 52), paraL1014F (Median lifespan = 33), paraI1011M (Median lifespan = 33), paraI1011V (Median lifespan = 32) males in absence of any insecticide. All three mutants displayed shortened lifespans compared to the control. Data were analyzed using the Log-rank test for trend and the Mantel-Cox test. D Comparison of embryonic survival. No significant differences in hatching rate were observed among WT, paraL1014F and paraI1011M genotypes. N = 4; n = 400. E paraL1014F are resistant to oxidative stress. paraL1014F flies had significantly higher survival rates at 48 h compared to WT or to paraI1011M mutants. N = 5; n = 125. F paraL1014F are paralytic at high temperature. Male flies were exposed to 37 °C for 4 h and their ability to climb above 5 cm was recorded. N = 4; n = 100. GH para mutants manifested abnormal sleep patterns in both females and males. Significant day-time sleep latency for paraI1011M mutants is indicated by the arrowhead. Control = Black; para1014F = Orange; para1011M = Blue; para1011V = purple. N: independent biological replicates, n: number of animals tested; Means ± s.e.m are plotted; B two-way ANOVA with Sidak’s multiple comparison, DEF one-way ANOVA with Sidak’s multiple comparison. *p < 0.033, **p < 0.0021, ***p < 0.0002, ****p < 0.0001, and ns not significant.

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