Development The Molecular Technique To Detect The Targeted-resistance Of Imidacloprid In Aphis Gossypii Glover

Cotton aphid, Aphis gossypii Glover(Hemiptera: Aphididae) is one of the most serious pests worldwide, and happens throughout all cotton regions of China. Imidaclorprid, a primary chemical of neonicotinoid, selectively target insect n ACh Rs. And imidacloprid has been widely used to control A. gossypii. However, because of the rampant application, resistance of A. gossypii to imidacloprid has been reported worldwide. Recent study has showed that a single point mutation(R81T) of n ACh R β1 subunit in A. gossypii conferred resistance to imidacloprid. Based on this, the competitive PCR amplification of specific allele(c PASA) protocol and real-time PCR amplification of specific allele(rt PASA) method have been developed to detect the imidacloprid resistance in A. gossypii field populations.1. Bioassay of imidacloprid in A. gossypii field populationsThe susceptibility of Aphis gossypii Glover collected from five regions(Dezhou Sandong, Beijing, Xuchang Henan, Handan Hebei and Langfang) to imidacloprid was assayed by leaf-dipping method. The results indicate that the four field populations of A. gossypii exhibit different susceptibilities to imidacloprid. The Xuchang Henan population(XC) and Beijing strain(BJ) were sensitive to imidacloprid. And the LC50 values were 1.03μg/m L and 0.87μg/m L, respectively. The Handan strain(HD) was also a little susceptible to imidacloprid. Its LC50 value was 3.74μg/m L. The Langfang population(LF) and Dezhou Shandong strain(DZ) were resistant to imidacloprid. And the LC50 values were 20.70μg/m L and 44.90μg/m L, respectively. Compared with BJ strain, the relative resistant ratio of Handan population(HD) was 4.3-fold without reaching the resistant level. And the relative resistant ratios of LF and DZ strains were 23.8-fold and 51.6-fold, respectively, reaching the moderate and high level of resistance.2. PCR amplification of the n ACh R β1 subunit gene fragment of susceptible and resistant A.gossypii strainsThe n ACh R β1 subunit gene fragment was cloned from the susceptible BJ strain and the resistant DZ and LF populations, and the sequences were blasted. The results indicated that a single mutation associated with imidacloprid resistance was observed in n ACh R β1 subunit of the DZ and LF field-strains with an amino acid replacement of arginine(AGA) to threonine(ACA) at amino acid position 81. In contrast, no mutation was presented in the BJ strain.3. c PASA for detection of the imidacloprid resistance of A.gossypii field-resistant strainsAccording to the mutation of the n ACh R gene reported, the c PASA primers were designed to detect the R81 T mutation frequency of LF and DZ A.gossypii field strains. The results showed that the mutation frequencies of LF and DZ field strains were 38% and 48%, respectively.4. Development of rt PASA protocolThere are many methods to detect the single mutation site. A simple, rapid and accurate rt PASA protocol combined the allele specific PCR amplification with the real-time PCR was developed. The homozygous resistant allele(RR) and homozygous susceptible allele(SS) were mixed in various ratios. A standard regression line was generated from the plot of allele frequencies versus Ct values. When the allele frequency was above 1%, the linear regression was y=-3.613x+20.37, R2=0.991; When the allelefrequency was under 1%, the standard curve was y=18.8+2.1/{1+10-(0.7x+0.35)}, R2=0.997. Thecorresponding Ct values of tested sample were obtained by rt PASA protocol to estimate the mutation frequency of A.gossypii field strains using the standard curve.5. Detection of R81 T mutation in imidacloprid-resistant field strains of A. gossypii by rt PASAThe rt PASA protocol was used to estimate the R81 T mutation frequency of A. gossypii field-resistant populations. The R81 T mutation frequencies of LF strain was 34.7%±1.3% with the 95% fiducial limits of 31.4%-38.1%, which agreed well with that determined by c PASA(38%). Similarly, the resistant allele frequency of DZ strain was calculated by rt PASA as 45.2%±5.2%with 95% FL of 32.3%-58.1%, which matched well with that predicted by c PASA(48%). The results showed that the rt PASA format will allow rapid and efficient monitoring of A. gossypii resistance in field populations in a high throughput format.