Effect of sublethal doses of fenpropathrin on the activities of detoxification enzymes in Cydia pomonella

SHANG Suqin; LIU Yonghua; LIU Ning; ZHANG Caihong; TA Guangchong

doi:10.13925/j.cnki.gsxb.20170280

Abstract：[Objective] The use of insecticides to control coding moths Cydia pomonella in the apple orchards in China is essential and as a result,the development of insecticide resistance in pest populations is of major concern.C.pomonella is one of the main quarantine pests of fruits in apple production and insecticide resistance of this species is little known in a number of countries.The aim of this study is to investigate various detoxification enzymes including multifunctional oxidase (MFO),carboxylesterase (CarE) and glutathione s-transferase (GST) that are involved in C.pomonella resistance to fenpropathrin by testing the effects of fenpropathrin on the activities of detoxifying enzymes in the larvae of C.pomonella,and by also observing the impact of the sublethal effects on the survival of individual insects after using fenpropathrin which can result in the change in ecological behavior,reproduction and development and so on.[Methods] The coding moths were originally collected from abandoned apple orchards in Wuwei city,Gansu province and maintained under standard laboratory conditions of (25 ±1) ℃,a relative humidity of (75±5)％,with a 16∶ 8 light:dark cycle;after which a third of the instar larvae of the coding moths were chosen to be the tested insects.First,prepare the five concentration gradient 2.5,5,10,20 and 40 mg· L-1 by using raw fenpropathrin and 60％ acetone as the solvent,spraying it into the feed block containing 24-holes in which the tested insects were put after being starved for four hours.The LC50 virulence regression equation was established according to the death status of tested insects after being treated for 72 h by using the method of stomach toxicity,of which,different logarithm concentrations of fenpropathrin are set as the abscissa and the mortality probability value of the third instar of C.pomonella is set as the ordinate.Then the sublethal doses of LC10 and LC20 of fenpropathrin on the third instar larvae of C.pomonella were calculated according to the above equation.The enzyme source proteins of MFOs,CarEs and GSTs were derived from the supematant of the crude enzyme solution prepared by mixing the collected larvae with 1.5 mL of pre-cooled 0.04 mol· L-1 (pH=7.0) PBS buffer,and 0.1 mol· L-1 (pH=7.8) PBS,66 mmol· L-1 (pH=7.0) liquid homogenate,centrifuged at 10 000 g for 15 min at 4 ℃.Then the enzyme activity of the MFOs,CarEs and GSTs of the coding moths was tested respectively after being treated with LC10,LC20 concentrations of fenpropathrin for 6,12,24,36,48,60 h.At the same time,the Km and Vmax of these three kinds of detoxification enzymes were calculated by using the Lineweaver-Burk double reciprocal mapping method of Wilkinson's.[Results]The specific activity of CarEs treated with LC20 were raised 1.54,2.19,1.53 and 1.34 times respectively but there were no significant differences at 6 h,60 h,and that of LC10enhanced 1.40,2.27 and 1.42 times at 12 h,24 h,36 h respectively in comparison with the control.The specific activity of GSTs treated with LC20 was higher than that of both the control and LC10 treatment(P ＜ 0.05),and that of LC10 was higher than that of the control at 36 h and 48 h respectively and showed no significant differences at other times.The specific activity of MFOs treated with LC20 was higher than that of the control at 12 h,24 h,36 h and lower at 48 h,and showed no significant differences at other times,and that of LC10 was higher at 12 h,24 h and 36 h but had no significant differences at other times respectively in comparison with the control.The Km value of CarEs treated with LC20 decreased 40.00％,37.23％,32.17％,29.51％,52.94％ and 28.09％ respectively at 6,12,24,36,48 and 60 h in comparison with the control,and the V~ values significantly increased 24.59％,19.36％ and 81.97％ more than that of the control.The change trend of Km and Vmax of the GSTs is contrary to the CarEs.The Km value of the MFOs was significantly lower than the control at 48 h and had no obvious changes at other times.The Vmax of the MFOs had no obvious changes at 12 h and 48 h but was lower than that of the control at all other times.[Conclusion] Insecticide resistance is either based on an increase in levels of detoxification enzymes,or is related to reduced target-site sensitivity.Detoxification enzymes that are associated with insecticide resistance belong to large enzyme families.In this study,MFOs and GSTs all played important roles on detoxification after being treated by LC10,LC20 of fenpropathrin on the third instar larvae of C.pomonella.of which,the greatest degree decline of Km value and the highest degree of increase of Vmax value were shown with CarEs which indicated that CarEs were the main reason for resistance to fenpropathrin of C.pomonella,and also indicated that fenpropathrin played a certain role in the induction of detoxification enzymes of C.pomonella.In this study,we investigated the effects of LC10 and LC20 concentrations on the dynamics and kinetic parameters of detoxification enzymes in the third instar larvae of coding moths respectively.It is necessary to select different chemicals to further investigate the changes of detoxification enzymes in different insecticide resistance levels,and then determine the detoxification enzyme activity,and the ultimate screening out of the useful synergist or proper pesticide mix formula so as to provide essential guidance for integrating the management of the resistance population of C.pomonella in practice.