Study On The Toxicity Of Cantharidin-related Compounds To Periplaneta Americana And Detoxification Mechanism Mediated By Glutathione-S-transferases

Cantharidin as animal(insect)derived secondary metabolites and its derivates have toxic effects on many pests and has a variety of modes of action,as well as low toxicity to part of non-target organisms.Therefore,it has a wide range of utilization value and application prospects.The American cockroach is a common sanitary pest which have strong reproductive capacity,widespread distribution,and difficult to control.In this paper,a total of 43cantharidin-related compounds were used to determine their toxicity to American cockroach with bioassay method.At the same time,cantharidin-related compounds with effective toxicity on American cockroaches were used to determine their effects on American cockroaches’ detoxification enzymes.And their effects on the transcriptional level of glutathione-S transferase as well as its activities in vitro were analyzed detailedly.The specific results are as follows:1.A total of 43 species including cantharidin and norcantharidin derivates extracted or synthetized by the Resource Insect Laboratory of Northwest A&F University were used to determine the stomach toxicity on the American cockroach by bioassay(by sample incorporated in the diet).It was found that cantharidin and norcantharidin derivatives I-7 and II-13,which have more effective stomach toxicity to the cockroach,the medium lethal concentration were 50.92 μg/ml,157.307 μg/ml and 306.348 μg/ml,respectively.2.The specific activity of P450,Car E and GSTs enzymes of the American cockroach detoxification enzymes were compared with the control group after 48 hours with treatment of cantharidin,norcantharidin derivatives I-7 and II-13.Results showed that their activity changed after treatment.The specific performance was as follows: the specific activity of Car E enzyme was significantly lower than that of the control group;the specific activity of P450 and GSTs enzymes was significantly higher than that of the control group.3.The complete ORF sequences of GSTs in American cockroaches were cloned,and the predicted molecular weights were 24.98 KDa and 27.75 KDa,respectively.By constructing the phylogenetic trees with sequence analysis and named PaGST1 and PaGST2 as PaGSTd1 and PaGSTo1.After treatment with the sublethal dose of cantharidin,norcantharidin derivatives I-7 and II-13,their effects on PaGSTd1 and PaGSTo1 transcription level were determined by real-time PCR.Results showed the expression level of PaGSTd1 and PaGSTo1 gene has different degrees of increase compared with the control group after 48 h.4.The inhibition rate of cantharidin and its derivatives to recombinant PaGSTd1 and PaGSTo1 activity in vivo was determined.It was found that cantharidin showed inhibitory effects on PaGSTd1 and PaGSTo1,but other norcantharidin derivatives could not inhibit the activity of PaGSTd1 and PaGSTo1.Although cantharidin showed certain inhibitory activity against PaGSTd1 and PaGSTo1,the effect was far less than that of GSTs structural specific inhibitors.