| The melon fly,Zeugodacus cucurbitae(Coquillett)is a great quarantine pest in China.The female adult lays eggs in the fruit,and the larvae eat on the internals,destroying the fruit’s value and flavor.Due to the advantages like low toxicity,high efficient,destroy insects a table wide,abamectin has been widely applied in melon fly management.According to field investigation,melon fly has been resistant to several insecticides which contain abamectin and shows an increasing trend year by year.At present,the reports about melon fly insecticides resistance only stay at resistance monitoring and none at molecular mechanism.Therefore,the study of abamectin resistance mechanism will provide a basis theoretical for field insecticide resistance management of melon fly.In this study,we applied two bioassay to determine the toxicity of the adults of susceptible strain(SS)melon fly to thirty insecticides belonging to seventeen pesticide categories and the relative sensitivity baseline of melon fly to different insecticides were established.Meanwhile,the resistance level of an abamectin-resistant strain(RS)was investigated by two bioassay methods.The activities of GSTs and Car E enzymes were assayed by microplate assay method in SS and RS strains and after treatment of molen fly adults for four time periods with abamectin.The enzyme kinetic constant and synergistic effects of TPP and DEM on abamectin were tested and analyzed in the adults of SS and RS strains.The eight candidate reference genes were expressed in different development periods and tissues of SS and RS strains by RT-qPCR.The stability rank of candidate reference genes was established comprehensively by four analysis software.Applied RT-qPCR to express seven GSTs genes,the expression patterns of seven GSTs genes were determined in different development periods and tissues of SS strain,the expression difference of the seven GSTs genes in SS and RS strains at the same developmental periods and tissues was compared,the expression patterns of seven GSTs genes were determined in SS and RS strain adults after induction with LC50 dose of abamectin at 12,24,48 and 72 h.The specific research results are as follows:(1)The LD50 values of the eighteen insecticides to melon fly were 3.422 ng·fly-1~576.842 ng·fly-1 by topical application,LC50 values of the twenty-one insecticides were 0.035 mg·L-1 ~60.542 mg·L-1 by feeding application.Compared with SS strain,the resistance ratio(RR)of RS strain was 18.22 folds based on LD50,while it was17.37 folds based on LC50,and the RR of two applications was similar.(2)The enzymes activities of GSTs and Car E in the RS strain were significantly activated,with their activities 2.92 and 1.46 folds as high as those of the SS strain.After exposed to LC25 and LC50 of abamectin,the enzymes activities of GSTs was induced in SS and RS strainsssssssssssssss,Car E was induced at after LC50 treatments in the SS strain,and which was inhibited in RS strain and at after LC25 treatments in the SS strain.Compared with SS strain,the Km value of GSTs was significantly reduced,and its Vmax significant increase,the Km and Vmax of Car E were no significantly changed.The synergistic ratios of DEM and TPP were 1.03 and 1.04 in the SS strain,3.45 and 2.52 in the RS strain.GSTs and Car E were involved in the detoxification against abamectin in melon fly,the GSTs had the higher affinity with the substrate and the faster reaction rate,and played a leading role in melon fly to abamectin metabolic resistance(3)The results of ge Norm indicated that the expression stability rank of different development periods was RSP13 = Rp L32(0.269)> TUBE(0.623)> TBP(0.702)>β-Tubulin(1.069)> G6PDH(1.366)> Actin3(1.596)> Actin2(1.923),the expression stability rank of different tissues was Rp L32 = RSP13(0.132)> TBP(0.345)> TUBE(0.442)> β-Tubulin(0.552)> G6PDH(0.780)> Actin2(1.101)> Actin3(1.417),and the best combination of reference gene was 2.The results of Norm Finder indicated that the expression stability rank of different development periods was Rp L32(0.399)>RSP13(0.446)> TUBE(0.738)> G6PDH(0.912)> TBP(0.924)> β-Tubulin(1.086)> Actin3(1.092)> Actin2(1.866),the expression stability rank of different tissues was Rp L32(0.090)> RSP13(0.117)> TBP(0.389)> TUBE(0.445)>β-Tubulin(0.705)> G6PDH(0.877)> Actin2(1.009)> Actin3(1.567).The results of Best Keeper indicated that the expression stability rank of different development periods was TUBE > TBP > RSP13 > Rp L32 > β-Tubulin > G6 PDH > Actin3 > Actin2,the expression stability rank of different tissues was β-Tubulin > TBP > RSP13 >Rp L32 > TUBE > G6 PDH > Actin2 > Actin3.The results of Ref Finder indicated that the expression stability rank of different development periods was Rp L32 > RPS13 >TUBE > TBP > G6 PDH > β-Tubulin > Actin3 > Actin2,the expression stability rank of different tissues was RPS13 > Rp L32 > TBP > β-Tubulin > TUBE > G6 PDH >Actin2 > Actin3.The RPS13 and Rp L32 can be combined as the reference gene for different development periods,different tissues,abamectin resistance and physiological molecular mechanism researchs of melon fly.(4)Compared with SS strain,the expression levels of seven GSTs genes were significantly higher at the same development periods and tissues in RS strain,except for few samples;the expression levels of 7 GSTs genes were different at different developmental periods and tissues in SS strain,and the expression patterns of each GSTs genes were different;except GST35,six GSTs genes could be induced to express in SS and RS strains with significant differences compared with the control group after12,24,48 and 72 h treatment with LC50 dose of abamectin,and the expression patterns of same GSTs genes were difference in SS and RS strains after abemctin treatment.The increased resistance of molen fly was closely related to the overexpression of seven GSTs genes in vivo,and the expression of these seven GSTs genes had time and spatial specificity.It was speculated that these seven GSTs genes had detoxification function of abamectin,GST37 and GST38 also had other important physiological functions.The new expression pattern of GSTs genes enhanced the resistance to abamectin by long-term screening of abamectin. |
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