Relationship Between PxGABARα1 Mutations And Fipronil Resistance In Plutella Xylostella

Diamondback moth (Plutella xylostella) is a serious pest of cruciferous vegetables. Control of the diamondback moth has been relied heavily on chemical insecticides, and this pest has developed strong resistance to insecticides and thus one of the most difficult pests to control. Fipronil is a phenyl pyrazole insecticide, which acts as a potent blocker of the insect GABA-gated chloride channel (GABA receptor). In P. xylostella, two rdl-homologous GABA receptor genes(PxGABARα1 and PxGABARα2) were cloned and mapped on the Z sex chromosome. In the present study, based on 3 laboratory-selected and field-collected resistant strains, relationship between PxGABARα1 mutations and fipronil resistance was studied through genetic linkage analysis and gene mutation genotyping.1. Genetic linkage analysis between PxGABARα1 A282S mutation and resistance to fipronil and dieldrin.SZ-FSer and SZ-DSer strains were selected from a field-derived SZ strain with fipronil and dieldrin respectively. Both strains are homologous for the A282S mutation of PxGABARα1.Male moths from SZ-DSer (ZSZS) were cross to female moths from ROTH (ZAW), and male F1 progeny (ZSZA) were backcrossed to female moths from ROTH (ZAW). Backcross progeny were treated with dieldrin, fipronil and endosulfan respectively, and survivors were genotyped at A282S of PxGABARα1. The results showed that A282S mutation was tightly linked with dieldrin resistance in the SZ-DSer strain, and this mutation was also related partially to resistance to fipronil and endosulfan.Similarly, male moths from SZ-FSer (ZSZS) were cross to female moths from ROTH (ZAW), and male F1 progeny (ZSZA) were backcrossed to female moths from ROTH (ZAW). Backcross progeny were treated with 3 ppm and 15 ppm fipronil respectively. Percentage of survivors with homologous A282S mutation (37.7%) from 15 ppm of fipronil treatment was higher than that (10.2%) from 3 ppm of fipronil treatment. It also indicated that fipronil resistance in SZ-FSer was related to A282S mutation.2. Inheritance mode of fipronil resistance in a field population.HF population collected in 2009 from Hefei, Anhui Province had 420-fold resistance to fipronil,2 to 27-fold resistance to cyclodiene insecticides. The HF population was selected with fipronil for further 25 generations to generate a highly resistant HF-R strain (2,200-fold to fipronil). LCP lines of fipronil toxicity were determined with HF-R, ROTH, reciprocal crosses between HF-R and ROTH, and their backcross progeny. Genetic analysis results showed that fipronil resistance in HF-R strain was controlled by a single gene with incomplete dominance, and fipronil resistance levels were affected by sex to some extent.3. Diverse mutations of PxGABARα1 in HF-R strain highly resistant to fipronil.A DNA fragment franking 282A of PxGABARα1 was amplified from the HF-R strain of P. xylostella with degenerate primers. Sequencing results indicated that there were two novel mutations (V285M and T286M), besides A282S mutation in the HF-R strain. Full length cDNA was amplified from the HF-R strain and ROTH strain. Three alleles with mutations (282S285V286T;282A285V286M;282G285M286T) were found in the HF-R strain, but only the wild type allele (282A285V286T) in the ROTH strain. The mutated alleles of PxGABARα1 could be different alleles at a single locus, or different locus other than PxGABARα1 (either autochromosome or sex chromsome). It deserves further study on contribution of these mutated alleles to fipronil resistance in P. xylostella.