Cytochrome P450 Monooxygenases In Strain Of Diamondback Moth (Plutella Xylostella L.) Resistance To Avermectin

In this paper, researches were carried out on the level of cytochrome P450, activity of cytochrome P450 monooxygenase, characteristics of P450 spectrum, and induction in strain of diamondback moth (DBM), Plutella xylostella L., resistance to avermectin. Two methods in insecticide methodology were improved and introduced. Finally, cytochrome P450 gene was primary studied in DBM. The main results were as follows. 1 The susceptibility of diamondback moth to several novel insecticides and Development of DBM strain resistant to avermectin. The susceptibility of DBM collected from Yangling to 7 novel insecticides was determined. The results showed that the DBM field population in Yangling had no remarkably resistance to avermectin, fipronil, acetamiprid, chlorfenapry, indoxacarb, spinosad, emamectin benzoate, the resistance ratios were 2.75,1.65,1.10,1.27,1.16,1.14 and 1.32-fold, respectively. Susceptible strain of DBM larvae in fourth instar were topically applied with avermectin in successive generation under laboratory condition (25±1℃, L∶D=16∶8) to develop resistant strain of DBM. After 18 generations, the resistance ratio of DBM to avermectin increased by 20.6-fold. Avermectin was greatly synergized with MFO inhibitor PBO (piperonly butoxide) and carboxylesterase inhibitor TPP (triphenyl phosphate). The resistant mechanism of DBM to avermectin may be related to microsomal oxidation of MFO and carboxylesterases. 2 Different development stages on resistance-related cytochrome P450 level The level of cytochrome P450 was determined along with the growth and development of DBM. The content of cytochrome P450 and b5 gradually increased with the growth of larvae from 2nd instar to 4th instar of susceptible-and resistant strain. Both P450 and b5 were maximal at the 4th instar and prepupa stages, and it decreased at later time. During the different developmental stages, the resistant strain had significantly elevated levels of cytochrome P450 and b5 than that in the susceptible strain. The folds were increased from 1.21-fold to 1.75-fold. The result indicated that resistant performance to avermectin became stronger along with DBM growth and development. The resistant detoxification mechanism may be involved cytochrome P450-mediated insecticide resistance. There was a good correlation between increased contents of b5 and P450, suggestive of some type of coordinated control of the levels of these two components in the development of resistance. 3 Biochemical change in the cytochrome P450 monooxygenase of avermectin-resistant and susceptible strain of DBM Potential metabolized mechanism of resistance to avermectin about cytochrome monooxygenases was examined in susceptible-(SS) and resistant strain (RS) of DBM. The activities of NADPH-cytochrome c (P450) reductase and four indices of monooxygenase (methoxyresorufin O-demethylation (MROD),　ethoxyresorufin O-demethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and p-nitroanisole O-demethylation (PNOD) were compared between the SS and the RS. In comparison to SS, RS had higher activity of P450 reductase with 1.97-fold. Using different substrates, MROD, EROD, ECOD and PNOD were significantly elevated by 9.41,4.15,1.67 and 2.94-fold, respectively, in the resistant strain compared with the susceptible. These results support the idea that the all four-monooxygenase components may play a role in insecticide resistance. Avermectin resistance was correlated with cytochrome P450 monooxygenase in DBM. 4 Induction of microsomal cytochrome P450 by phenobarbital in DBM. The induction of phenobarbitial (PB) on the microsome cytochrome P450s of susceptible and avermectin resistant DBM strain was examined. PB significantly induced the microsome cytochrome P450 and P450 reductase of DBM, but PB treatment had no significant effect on the level of cytochrome b5 above two strains. The induction response was maximal at 48h, and it decreased at later time. There were significantly changes in the CO-difference spectrum of cytochrome P450 (max: 448.52±0.53nm) in the induced groups, in comparison with the control (max: 449.80±0.20nm). PB significantly increased four monooxygenases activity (MROD, EROD, ECOD and PNOD) in susceptible strain, as well as in resistant strain. These results suggested the three-cytochrome P450 components, except b5, can be induced by PB in DBM, as well as four monooxygenases activity. It implies the increase in both quantity and quality of P450 caused the resistance of DBM to avermectin. 5 The characterization of cytochrome P450-CO difference spectrum in DBM The difference spectrum of cytochrome P450 did not show any degradation product at 420nm. The absorption maxima for the CO difference spectra of cytochrome P450 from DBM resistant strain(RS) to avermectin and susceptible strain(SS) at 451.30±0.86nm and 449.80±0.20nm, respectively. In comparison with peak of SS, RS had increased with shift of about 1.5nm. The results suggested that the characterization of cytochome P450 had changed after development resistance to avermectin in DBM. And the avermectin resistance in DBM wasprobably also associated with the cytochrome P450. 6 Primary studies on cytochrome P450 gene in DBM To study the molecular characteristics of avermectin-resistant related P450 in DBM, according to P450 gene CYP6B2 cloned from susceptible-and resistant strain, two primers (named HJ07 and HJ08) were designed. Two distinctive fragments can be obtained using RT-PCR from both strains, respectively. These products were cloned and sequenced. The results of sequence analysis showed that all of them had the conserved α　helix region of P450 gene and the region of terminator of CYP6B2. These sequences may be as a part of structural gene belongs to unknown CYP family. 7 Two improved methods in insecticide methodology One rapid improved CTAB (cetyltrimethyl ammonium bromide) method for macro-extraction genomic DNA in the larvae of Plutella xylostella was reported. The method is a simple and efficient protocol for isolation of high molecular genomic DNA from insect. It showed that the resulting DNA is suitable for PCR and other molecular biology analysis. Another method according to the probit analysis and the least square, in Windows 98/ Me/2000/ XP system, were introduced. It was a computed procedure code that makes use of the Microsoft EXCEL software establishment to calculate quickly in the insecticide toxicological test that concerning toxicological regression line (LD-P), median lethal dosage (LD50), 95% Fiducial Limit of LD50, relation coefficient(r), standard deviation (Sm) and chi square test (?2). After the user input the insecticide concentrations (or dosages), the trial number of overall insect and dead insect, the procedure code will get the result of toxicological test quickly, simple and accurately, and will calculate the chi square test.