| Summary: | The physiological and biochemical characterization of the “Halloween” genes has fundamental importance in the biosynthesis pathway of ecdysteroids. These genes were found to catalyze the final phases of ecdysteroid biosynthesis from dietary cholesterol to the molting hormone 20-hydroxyecdysone. We report the characterization of the <i>Cs-Phm</i> in a major insect pest in agriculture, the rice striped stem borer, <i>Chilo suppressalis</i> (<i>C. suppressalis</i>). A full-length transcript of <i>Cs-Phm</i> was amplified with an open reading frame (ORF) of 478 amino acids through 5′ and 3′ RACE. <i>Cs-Phm</i> shows five insect-conserved P450 motifs: Helix-C, Helix-I, Helix-K, PERF, and heme-binding motifs. Phylogenetic analysis clearly shows high similarity to Lepidoptera and evolutionary conservation in insects. The relative spatial and temporal transcript profile shows that <i>Cs-Phm</i> is highly expressed in the prothoracic glands and appears throughout the larval development, but with low expression at the start of the larval instar. It seems to peak in 3–4 days and decreases again before the larvae molt. Double-stranded RNA (dsRNA) injection of Cs-Phm at the larval stage efficiently knocked down the target gene and decreased its expression level. The dsRNA-treated group showed significantly decreased ecdysteroid titers, which leads to delayed larval development and higher larval mortality. Negative effects of larval development were rescued by treating 20E in the dsRNA-treated group. Thus, in conclusion, our results suggest that <i>Cs-Phm</i> is functionally conserved in <i>C. suppressalis</i> and encodes functional CYP that contributes to the biogenesis of 20E.
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