To observe phenotype, third early-instar larvae (2 days old) was fed with 6 g food mixed with 60 μl dsRNA or dsGFP (1,000 ng/μl) or DEPC for 48 h and transferred to a new artificial diet with the same treatment for another 48 h. After 96 h, larvae were shifted to soil for pupation. Two individuals from each replication of each group were killed every 24 h until the pupal stage to determine RNAi efficiency, while the others continued to feed. Similarly, two individuals were killed at the adult stage (24 h old), to test the RNAi efficiency. The stability of dsRNAs in the artificial diet, 1 g of each diet was collected 24 h post-feeding. The artificial diet was diluted in 50 μl distilled water, and the dsRNAs were observed in 1% agarose gel electrophoresis. Mortality was recorded by counting the flies number in each group after 24 h. The phenotype effects were observed in each developmental stage until 10 days of the adult’s emergence.

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