Resistance Monitoring To Three Insecticides And Resistance Mechanisms To Fenvalerate In Helicoverpa Armigera

The cotton bollworm, Helicoverpa armigera (Hiibner)(Lepidoptera:Noctuidae), is an important cotton pest. Since the1980s this pest has developed resistance to many kinds of insecticides, espicially to pyrethroids. Bt transgenic cotton has been commercialized in China since1997, then the occurrence of H. armigera has been effectively controlled, but mirid bugs and whiteflies gradually become the major pests of cotton field, coupling with the cotton aphid and spider mite. For the limited target insects of transgenic Bt cotton, using of insecticides is still one of the most important methods. In order to understand the resistance status of cotton bollworm after15years planting of Bt cotton, resistance to fenvalerate, phoxim and emamectin benzoate was detemined in13field populations in the present study. Cross-resistsnce, inheritance of resistance and resistance mechanisms were investigated in a field-derived fenvalerate-resistant strain (AY-FenR) of H. armigera. Our results will provide some useful information for the effective and sustainable control of H. armigera. The main results are as follows:1. Resistance monitoring to three insecticides in H. armigeraResistance to fenvalerate and phoxim was measured by the topical application in13field populations of H. armigera collected from Northern China, the Yangtze River region and Xinjiang cotton area in2011. Field populations from Northern China and Qianjiang of Hubei Province had the highest levels of resistance to fenvalerate (RFs:149-to1219-fold). The Yancheng population of Jiangsu Province had high level resistance (44-fold) to this insecticide. Shawan and Shache populations of Xinjiang had low levels of resistance to fenvalerate (RFs:3-and10-fold). Low levels of resistance to phoxim (RFs:1-to9-fold) were detected among all field populations. In13field populations, the LD50values of emamectin benzoate were between0.00063-0.00133μg/larva, even lower than that of the susceptible strain (0.0074μg/larva). The range of variation in these13populations was less than2fold. The correlation analysis and significance test of LD50S of three insecticides among field populations showed that emamectin benzoate, fenvalerate and phoxim have no obvious cross-resistance with each other. The synergism of PBO and DEF at the discriminating doses of fenvalerate (0.08μg/larva) and phoxim (0.4μg/larva) showed that PBO had significant synergism to fenvalerate, and DEF had significant synergism to phoxim. It suggests that enhanced oxidative detoxification is an important mechanism for fenvalerate resistance, and esterase-based detoxification is an important mechanism for phoxim resistance in field populations of H. armigera of China.2. Selection of fenvalerate resistance, cross-resistance pattern and inheritance mode of resistance to fenvalerate in the AY-FenR strainThe AY-FenR strain was selected with fenvalerate from AY strain, which was collected from Anyang, Henan Province of China. After seven generations of selection, the resistance to fenvalerate of AY-FenR was3057and1945-fold, compared with the susceptible strain (SCD) and AY strain, respectively. The AY-FenR strain also showed high cross resistance to cypermethrin(19220and1441-fold) and bifenthrin(58and21-fold), low cross resistance to spinosad(6and5-fold) and chlorfenapyr(6and2-fold). But AY-FenR had no cross resistance to chlorpyrifos, fipronil and emamectin benzoate.The inheritance mode of resistance to fenvalerate was evaluated by using the methods of reciprocal crosses and backcross between the susceptible strain SCD and AY-FenR. The progenies of reciprocal crosses showed469-fold (F1) and628-fold (F1’) resistance to fenvalerate compared with SCD strain, respectively. And no significant difference between the LD50values of F1and F1’ indicated that the inheritance of fenvalerate resistance is autosomal. The degree of dominance (D) of resistance was0～1, suggesting that the inheritance of fenvalerate resistance was incompletely dominant. In the log dosage-probit mortality curve of the backcross progeny to fenvalerate, there was no distinct plateau corresponding to50%mortality. The χ2test of the goodness of fit between the observations and predictions under the assumption of monofactorial inheritance rejected the single gene hypothesis. It suggests that fenvalerate resistance in the AY-FenR strain was controlled by more than one factor.3. Biochemical mechanisms of fenvalerate resistance, cloning and sequence comparison of sodium channel gene in H. armigeraThe biochemical mechanisms of fenvalerate resistance in AY-FenR were investigated by synergism test and metabolic enzyme activity detection. PBO had114-fold synergism to fenvalerate in AY-FenR strain, but the synergism of DEF and DEM were6-fold and2-fold in the resistant strain. The activity of PNOD, MROD and ECOD of cytochrome P450 monooxygenase from fatbody and midgut of AY-FenR strain were compared with the susceptible strain SCD and AY strain. In fatbody, the ECOD activity in AY-FenR increased30-fold compared with the AY strain; the MROD activity was11.5-fold compared with the SCD strain. In AY-FenR strain, there was minor increase in esterase activity (to the substrate a-naphthyl acetate), and no increase in glutathione S-transferase activitiy (to both substrates CDNB and DCNB). The results suggested that monooxygenase plays an important role in fenvalerate resistance in the AY-FenR strain.The cDNA fragment encoding Ⅰ-Ⅳ of sodium channel gene was cloned from AY-FenR strain and SCD strain by using RT-PCR technique. Sequence analysis showed that cDNA fragment was5536bp and encoded1845amino acid residues. The deduced amino acid sequence of sodium channel a subunits of H. armigera showed98%amino acid similarity with Helicoverpa zea. Sequence comparison between SCD and AY-FenR indicated there were24amino acid polymorphism sites and no point mutations of kdr or super-kdr associated with knockdown resistance to fenvalerate. Both AY-FenR and SCD had two alternative splicing variants located in exon17, in which two amino acid differences form two splicing variants. It may have no relationship with fenvalerate resistance.4. Selection for a near-isogenic fenvalerate-resistant strain of H. armigeraThe fenvalerate-resistant strain AY-FenR was repeatedly backcrossed with a laboratory susceptible strain SCD. Progenies from each backcross were selected with a discriminating dose of fenvalerate that killed all susceptible homozygostes and parts of resistant heterozygotes. After seven generations of recurrent backcrosses, the progenies of the last backcross (BC7) were further selected for one generation with fenvalerate to generate a near-isogenic strain (designated as SCD-FenR). To increase the resistance level, the near-isogenic resistant strain was further selected for two generations. Now SCD-FenR has205-fold resistance to fenvalerate compared with the susceptible SCD strain. Besides resistant gene and its closely linked regions, this strain has similar genetic background to the susceptible strain. So it will become a useful material to study the fenvalerate resistance mechanism in H. armigera.