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Sci Rep
2016 Aug 25;6:32335. doi: 10.1038/srep32335.
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Synergistic and compensatory effects of two point mutations conferring target-site resistance to fipronil in the insect GABA receptor RDL.
Zhang Y
,
Meng X
,
Yang Y
,
Li H
,
Wang X
,
Yang B
,
Zhang J
,
Li C
,
Millar NS
,
Liu Z
.
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Insecticide resistance can arise from a variety of mechanisms, including changes to the target site, but is often associated with substantial fitness costs to insects. Here we describe two resistance-associated target-site mutations that have synergistic and compensatory effects that combine to produce high and persistent levels of resistance to fipronil, an insecticide targeting on γ-aminobytyric acid (GABA) receptors. In Nilaparvata lugens, a major pest of rice crops in many parts of Asia, we have identified a single point mutation (A302S) in the GABA receptor RDL that has been identified previously in other species and which confers low levels of resistance to fipronil (23-fold) in N. lugans. In addition, we have identified a second resistance-associated RDL mutation (R300Q) that, in combination with A302S, is associated with much higher levels of resistance (237-fold). The R300Q mutation has not been detected in the absence of A302S in either laboratory-selected or field populations, presumably due to the high fitness cost associated with this mutation. Significantly, it appears that the A302S mutation is able to compensate for deleterious effects of R300Q mutation on fitness cost. These findings identify a novel resistance mechanism and may have important implications for the spread of insecticide resistance.
Figure 1. Laboratory selection and characterization of fipronil resistant Nilaparvata lugens.(A) The profile of fipronil resistance during laboratory selection of a field population of N. lugens (isolated from Chainat, Thailand) is illustrated. The graph illustrates changes in resistance ratios in subsequent generations during continuous selection with fipronil (diamonds) and after removal of fipronil selection after the 16th generation (triangles). (B) Mutation detection in insects collected during each generation of fipronil selection, illustrating the frequency of A302S and R300Q mutations. In each generation, 100 individuals were examined by nucleotide sequencing of the TM1-TM3 domain of RDL. In each column, the number of heterozygotes for either A302S or R300Q is indicated by a red bar. The frequency of double mutations (individuals containing both the A302S and R300Q mutation) is also illustrated. (C) The effects of synergists on fipronil resistance ratios are illustrated in insect populations isolated during various generations of selection.
Figure 2. Alignment of RDL amino acid sequences from transmembrane domains 1 to 3 (TM1 to TM3) from various insect species.The positions of arginine (R) and alanine (A) amino acids, corresponding to the R300Q and A302S mutations, are indicated by a black background. Three transmembrane regions (TM1, TM2 and TM3) are underlined. Database accession numbers for the sequences are: Aedes aegypti (Aa), AAA68961; Apis mellifera (Am), AAC63381; Blattella germanica (Bg), AAB33733; Ceratitis capitata (Cc), AAD51101; Drosophila melanogaster (Dm), P25123; Drosophila simulans (Ds), AAK00512; Lucilia cuprina (Lc), AAB81966; Musca domestica (Md), AAC23602; Nilaparvata lugens (Nl), AGK30293.
Figure 3. Fipronil resistance monitoring and mutation detections in Nilaparvata lugens field populations from Taizhou (China), Hanoi (Vietnam) and Suphanburi (Thailand) in five consecutive years from 2010 to 2014.(A) Fipronil resistance ratios in three field populations in five years. (B) Mutations detected in field populations. Columns (black, white and grey) indicate the number of individuals with A302S mutation among 300 tested individuals in each population. In each column, the red bar indicates the number of individuals with the double mutation (R300Q/A302S). No individuals with only R300Q mutation were detected.
Figure 4. Effects of fipronil on GABA-activated currents in Xenopus oocytes expressing wild-type and mutant RDL receptors.Representative traces are illustrated from wild-type RDL. (A) A302S (B), R300Q (C) and R300Q/A302S (D). In each column, the top trace is a response to an EC30 concentration of GABA, the middle trace is a response to the same concentration of GABA co-applied with 0.1 μM fipronil and the bottom trace is a response to EC30 of GABA after a 15-min wash. Because the four RDL variants have different sensitivity to activation by GABA, an equipotent concentration of GABA (EC30) was used in each case (25, 10, 206 and 33 μM, respectively).
Figure 5. Characterization of wild-type and mutant RDL receptors expressed in Xenopus oocytes.Data are presented, from wild-type RDL and RDL containing single mutations (A302S or R300Q) or a double mutation (DM; A302S and R300Q), illustrating agonist dose-response curves to a range of concentrations of GABA (A), radioligand binding studies with [3H]-GABA (B) and dose-response curves illustrating the inhibition by fipronil of responses to GABA (C). Antagonist effects to a range of concentrations of fipronil were examined on wild-type and mutated RDL. Because the four RDL variants have different sensitivity to activation by GABA, an equipotent concentration of GABA (EC30) was used (25, 10, 206 and 33 μM for wild-type RDL, A302S mutant, R300Q mutant and A302S/R300Q mutant, respectively). Data are means of at least five independent experiments.
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