| 1. Enhanced cytochrome P450 monooxygenase activity associated with fenvalerate resistance in Helicoverpa armigeraA field strain collected from Yanggu County, Shandong Province in 2001 was divided into three sub-populations. The YG strain was originated from one sub-population which was maintained in the laboratory without exposure to insecticides. The YGF and YGFP strains were derived from the other two sub-populations by laboratory selection with fenvalerate and fenvalerate+PBO respectively for 13 generations. Compared with the susceptible SCD strain, the YG strain exhibited 7-, 14- ,14-and 21-fold resistance to fenvalerate, cypermethrin, deltamethrin and cyhalothrin respectively. In the fenvalerate-selected YGF strain resistance increased to 1630-, 535- and 72- and 160-fold respectively. In the fenvalerate+PBO-selected YGFP strain this increased to 2420-, 2898-, 282 and 729-fold respectively.The synergistic ratios of PBO to fenvalerate, cypermethrin, deltamethrin and cyhaolothrin in the YGF strain were 458-, 53-, 4-and 5-fold respectively, whereas DEF only gave 4-,3-,0.8- and 0.8-fold synergism. In the YGFP strain, the synergistic ratios of PBO were 12-,46-,16- and 22-fold and the synergistic ratios of DEF were 11-, 2- ,3- and 9-fold. These results suggested that cytochrome P450s were possibly involved in fenvalerate-resistance in H. armigera.In order to confirm the role of cytochrome P450 monooxygenases in fenvalerate resistance, metabolic enzyme activities against model substrates were assayed in the YGF,YGFP and SCD strains. Compared with the susceptible SCD strain, cytochrome P450 mediated PNOD (using p-nitroanisole) of fourth-instar larvae in the YGF and YGFP strains was 23.1- and 9.3-fold respectively. Esterase activity (using substrate a-naphthyl acetate) of third-instar larvae was 2.4- and 1.1-fold. Glutathione S-transferases activity (using substrate l-chloro-2,4-dinitrobenzene,CDNB) was 1.3- and 1.8-fold respectively. These results further confirmed that the enhanced cytochrome P450 monooxygenase was the major metabolic mechanism of fenvalerate resistance in H. armigera.2. Correlation between fenvalerate resistance and cytochrome P450 mediated 0-demethylation activity in H. armigeraCytochrome P450 activities of H. arimgera by resolution with six detergents Chaps (Polyoxyethylene ether w-1, Cholic acid, Emulgen 911, Emulgen 913 and Triton-X100) were assayed using microtier plate reader. The results indicate that Polyoxyethylene ether w-1 is a useful detergent for enhancing the cytochrome P450 enzyme activity of H. arimgera. With addition of 0.1% polyoxyethylene ether w-1 in enzyme, PNOD activity was 34% higher than that of control (without any cosolvent).Cytochrome P450 monooxygenases were a major metabolic mechanism responsible for pyrethroid resistance in H. armigera from Asia. Cytochrome P450 mediated O-demethyla tion activity towards p-nitroanisole (PNOD) of individual fourth-instar larva was determined in five strains of Helicoverpa armigera using a microplate reader. The four resistant strains of YS, HD, YGF and YG59 had 6-, 71-, 2540- and 11800-fold resistance respectively to fenvalerate in comparison to the susceptible BK77 strain. Their mean PNOD activity was 4-, 10-, 24- and 60- fold respectively compared with the BK77 strain. A strong positive correlation (correlation coefficient r= 0.98) between PNOD activity and fenvalerate resistance was found. Of 48 larvae from each strain, only 4% larvae of the susceptible BK77 strain had detectable PNOD activity, whereas 25%, 33%, 79% and 96% larvae from the resistant strains YS, HD, YGF and YG59 exhibited PNOD activity respectively. There was a clear discrimination of patterns of PNOD frequency distribution between H. armigera strains and their magnitudes of fenvalerate resistance. The PNOD activity can be used as a biochemical marker for monooxygenase-mediated pyrethroid resistance in field populations of H. armigera.3. Cloning and mRNA expression levels of cytochrome P450 genes in H. armigeraThree novel cytochrome P450 genes from CYP9 subfam... |
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