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PLoS Genet
2023 Jun 29;196:e1010814. doi: 10.1371/journal.pgen.1010814.
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G3'MTMD3 in the insect GABA receptor subunit, RDL, confers resistance to broflanilide and fluralaner.
Zhang Y, Huang Q, Sheng C, Liu G, Zhang K, Jia Z, Tang T, Mao X, Jones AK, Han Z, Zhao C.
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Meta-diamides (e.g. broflanilide) and isoxazolines (e.g. fluralaner) are novel insecticides that target the resistant to dieldrin (RDL) subunit of insect γ-aminobutyric acid receptors (GABARs). In this study, we used in silico analysis to identify residues that are critical for the interaction between RDL and these insecticides. Substitution of glycine at the third position (G3') in the third transmembrane domain (TMD3) with methionine (G3'M TMD3), which is present in vertebrate GABARs, had the strongest effect on fluralaner binding. This was confirmed by expression of RDL from the rice stem borer, Chilo suppressalis (CsRDL) in oocytes of the African clawed frog, Xenopus laevis, where the G3'MTMD3 mutation almost abolished the antagonistic action of fluralaner. Subsequently, G3'MTMD3 was introduced into the Rdl gene of the fruit fly, Drosophila melanogaster, using the CRISPR/Cas9 system. Larvae of heterozygous lines bearing G3'MTMD3 did not show significant resistance to avermectin, fipronil, broflanilide, and fluralaner. However, larvae homozygous for G3'MTMD3 were highly resistant to broflanilide and fluralaner whilst still being sensitive to fipronil and avermectin. Also, homozygous lines showed severely impaired locomotivity and did not survive to the pupal stage, indicating a significant fitness cost associated with G3'MTMD3. Moreover, the M3'GTMD3 mutation in the mouse Mus musculus α1β2 GABAR increased sensitivity to fluralaner. Taken together, these results provide convincing in vitro and in vivo evidence for both broflanilide and fluralaner acting on the same amino acid site, as well as insights into potential mechanisms leading to target-site resistance to these insecticides. In addition, our findings could guide further modification of isoxazolines to achieve higher selectivity for the control of insect pests with minimal effects on mammals.
Fig 1. Responses to GABA and antagonists in Xenopus laevis oocytes expressing wild-type and mutant arthropod or mammalian GABAR subunits.(A) and (B) Concentration-response curves of GABA (A) and inhibition of GABA-induced currents by fluralaner (B) from wild-type or mutant RDL receptors. (C) and (D) Inhibition of GABA-induced currents by fipronil (C) and avermectin (D) in wild-type or G3’MTMD3 RDL. (E) and (F) Effect of the G3’MTMD3 mutation in RDL of different arthropod species on response to GABA (E) or fluralaner (F). (G) and (H) Concentration-response curves of GABA (G) and inhibition of GABA-induced currents by fluralaner (H) from heteromeric Mmα1β2 or mutant Mmα1β2-M3’GTMD3 channels. Error bars indicated the standard error of the mean (SE). Significant difference was determined by Student’s t-test (ns, not significant; **, P < 0.01).
Fig 2. CRISPR/Cas9-mediated point-mutation at G3’TMD3 in the Rdl gene of Drosophila melanogaster.(A) Diagram of the genome editing strategy. Black triangles indicated the gRNA-targeted sites. A 2114 bp homologous region with a modified codon corresponding to the G3’TMD3 residue was cloned into the donor plasmid, and nine synonymous mutations indicated by vertical lines were designed around the G3’TMD3 residue to prevent repeated editing. (B) Genotypes of the heterozygous mutant strains were confirmed by sequencing of genomic DNA. The corresponding codon of the G3’TMD3 residue is boxed, and the synonymous mutations are indicated by black equilateral triangles. (C) Balancer-associated phenotype of heterozygous mutant strains. Additional bristles on the haltere are indicated by a black triangle.
Fig 3. Identification of D. melanogaster embryos bearing homozygous mutations at RDL G3’TMD3.(A) Fluorescence detection of embryos. Heterozygous mutant or TM3 Sb GFP/ TM3 Sb GFP embryos showing a GFP signal. Homozygous mutant embryos labeled with white triangles showed no GFP signal. BF, bright field. (B) The proportion and hatching rate of embryos bearing homozygous mutations. For each strain, genotypes of 100 embryos were identified. 70.83% (17 of 24), 70.37% (19 of 27) and 76.92% (20 of 26) embryos hatched that were homozygous for G3’MTMD3, G3’STMD3 and G3’QTMD3, respectively. (C) Genotypes of the homozygous mutant larvae were confirmed by sequencing of genomic DNA. The codon of the G3’TMD3 residue is boxed and the synonymous mutations are labeled with black equilateral triangles.
Fig 4. Lethality and reduced locomotion in D. melanogaster larvae caused by homozygous mutations at G3’TMD3.(A) Temporal characteristics of lethality in homozygous mutants. Data were analyzed using the Log-rank test for trend and the Mantel-Cox test (ns, not significant; *, P < 0.05; ***, P < 0.001). (B) Crawling speed of homozygous mutants. Error bars indicate the standard error of the mean (SE). Significant difference was determined by Student’s t-test (***, P < 0.001). (C) Representative motion path of homozygous mutants in 2 minutes (S1 Video).
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