Cantharidin (CTD),a terpenoid defensive toxin mainly produced by blister beetles,is widely known by its toxicity to both cancer cells and pests.However,little information is known about its biosynthesis in blister beetles.In this study,first we determined the CTD content in various tissues of adult blister beetles on different days after mating,and then detected the temporal and spatial expression pattes of genes related to CTD biosynthesis in Epicauta chinensis.Results revealed that the accessory gland is the source of the highest CTD production.The second highest level was in the fat body in male blister beetles after mating.In females,the highest CTD content was in the reproductive system except the ovary after mating.As revealed by messenger RNA expression level analysis,the highest levels of 3-hydroxy-3-methylglutary-CoA reductase (HMGR) and juvenile hormone epoxide hydrolase (JHEH) transcripts of E.chinensis were observed in the fat body in males after mating.However,the highest transcript level of EcHMGR was in the ovary and EcJHEH was maintained at a nearly similar level in females.The transcript level of methyl-faesoate epoxide was significantly higher in the head and that of CYP4BM1 in the midgut in both male and female E.chinensis.We speculate that the fat body may play a more important role than other tissues on the CTD biosynthesis in male E.chinensis after mating.There may be multiple tissues involved in the process of CTD biosynthesis.These four genes probably play regulatory roles in different tissues in males.