Eggs were collected within 30 min after lay and placed on a microscope slide and fixed by glue. Microinjection were performed with final concentrations of 1000 ng/μl Cas9 protein and 800–990 ng/μl sgRNA (Table (Table1)1) using a TransferManNK2 and FemtoJet microinjection system (Eppendorf, Hamburg, Germany). All operations were finished within 2 h after lay. Approximately 245–485 eggs (Table (Table1)1) were injected for each gene and incubated at the condition with 25 °C, 12 h light/12 h darkness, 80% relative humidity and darkness for 4–6 days until hatching. Hatched larvae were transferred to host plant leaves (Zanthoxylum piperitum) for breeding and reared at 27 °C, 16 h light/25 °C, 8 h darkness and keep 80% relative humidity. The morphological changes were observed mainly from the fourth-instar larvae in order to avoiding the disturbance on early young larvae (first to third instar) which have similar morphology to the fourth-instar larvae. Pupae were transferred into plastic baskets before eclosion. Emerged adults were crossed via hand pairing, and then mated females were placed in net rooms with host plants for oviposition.

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