Identification and expression profile of Halloween genes involved in ecdysteroid biosynthesis in Spodoptera littoralis

20-Hydroxyecdyone (20E), an active form of ecdysteroid, is the key hormone in insect growth and development. The biosynthesis of ecdysteroid is triggered and under the control of the neuropeptide, prothoracicotropic hormone (PTTH). To date, five cytochrome P450 enzymes, namely Spook (Spo), Phantom (Phm), Disembodied (Dib), Shadow (Sad) and Shade (Shd) related to ecdysteroid biosynthesis, are identified and the character of last four enzymes is well studied in Drosophila melanogaster, Bombyx mori and Manduca sexta. These genes are called Halloween genes and mediate the biosynthesis of 20E from cholesterol. In this study, we extended these works to a major pest insect in agriculture, the cotton leafworm Spodoptera littoralis (Lepidoptera: Noctuidae). We identified the sequence of five Halloween genes, and the converted amino acid sequences were compared with those of other insects. The phylogenetic analysis clearly showed separated clusters of each gene and the evolutional conservation in insects with a high similarity in Lepidoptera. Spo, phm, dib and sad were predominantly expressed in prothoracic glands, and shd was expressed in fat body and Malpighian tubules at the last instar larvae. Spo expression was kept high level between day 2 and day 4 after ecdysis. The expression of phm and dib peaked at day 2, and sad and shd expressions peaked at day 2 and day 4 after ecdysis. In addition, the hemolymph ecdysteroid titer showed a small peak at day 2 and a large peak at day 4 after ecdysis. These results suggest the importance of Halloween genes in ecdysone biosynthesis by prothoracic glands and conversion of ecdysone into 20E by fat body in larval–pupal metamorphosis.