Abstract: 【Aim】 To clone and obtain the Halloween family genes involved in ecdysteroid synthesis in the Chinese oak silkworm, Antheraea pernyi, and to characterize their expression patterns during pupal diapause termination under long photoperiod conditions, so as to lay a foundation for elucidating the features of ecdysteroid synthesis throughout diapause termination in A. pernyi pupae. 【Methods】 Five Halloween family genes (ApCyp307a1, ApCyp306a1, ApCyp302a1, ApCyp315a1 and ApCyp314a1) were cloned from A. pernyi using RT-PCR and subjected to bioinformatic analysis. The expression levels of the above five Halloween genes in the brain, prothoracic gland, hemolymph, midgut, fat body, testis/ovary, trachea and cuticle of pupae of A. pernyi at diapause and developmental stages were examined via semi-quantitative RT-PCR. RT-qPCR was employed to analyze the expression levels of the above five Halloween genes, as well as the ecdysone receptor genes ApEcR and ApUSP, in the prothoracic gland and fat body of A. pernyi pupae stored at both room temperature (19-25 ℃) and low temperature (6 ℃) conditions under a long photoperiod of 17L∶7D. RT-qPCR was used to detect the  expression levels of ApCyp314a1, ApEcR and ApUSP in the fat body at 24 h after RNAi of Cyp314a1 in the 4th instar larvae and prepupae of A. pernyi, and the effects of RNAi on the development of larvae and prepupae were further observed and detected. 【Results】Five Halloween family genes of A. pernyi were cloned, and designated as ApCyp307a1, ApCyp306a1, ApCyp302a1, ApCyp315a1 and ApCyp314a1 (GenBank accession numbers were MW677192, MW677193, MW677194, MW677195 and MW677196, respectively), based on homologous sequence alignment. Phylogenetic tree revealed that Halloween genes are highly conserved throughout evolution. Tissue-specific semi-quantitative PCR results indicated high expression levels of ApCyp307a1, ApCyp306a1, ApCyp302a1 and ApCyp315a1 in the prothoracic gland of A. pernyi pupae, with ApCyp302a1 and ApCyp315a1 showing marked upregulation during developmental stage of A. pernyi pupae. ApCyp314a1 was highly expressed in the midgut, fat body and testis/ovary of A. pernyi pupae at the developmental stage. A. pernyi pupae stored at low temperatures and exposed to the photoperiod of 17L∶7D exhibited accelerated emergence compared to those maintained at room temperature. The expression level of ApCyp315a1 in A. pernyi pupae stored under the photoperiod of 17L∶7D at room temperature peaked at 7 d after treatment, those of ApCyp306a1, ApCyp302a1 and ApUSP were increased at 21 d after treatment, whereas those of ApCyp314a1 and ApEcR peaked near eclosion (at 35 d after treatment). After low-temperature storage, the expression level of ApCyp307a1 in A. pernyi pupae was increased substantially during the pre-eclosion phase at 7 d after treatment under the photoperiod of 17L∶7D, while ApCyp302a1 was highly expressed at 21－35 d after treatment under the photoperiod of 17L∶7D, and the expression level of ApCyp315a1 peaked near eclosion, which showed a different expression pattern from that during the termination of pupae diapause at normal temperatures. The expression level of ApCyp314a1 peaked near eclosion, and ApEcR and ApUSP exhibited the expression profiles similar to those in the normal temperature group. Compared to the control group injected with dseGFP, RNAi of ApCyp314a1 significantly decreased the expression levels of ApCyp314a1 in the fat bodies of the 4th instar larvae and prepupae, and significantly suppressed the larval ecdysis and pupation of A. pernyi. 【Conclusion】These results demonstrate that Halloween genes play crucial roles in 20-hydroxyecdysone (20E) synthesis, and growth and development of A. pernyi. During long photoperiod-induced diapause termination, the expression levels of 20E synthesis-related genes increased during 7－21 d, whereas ApCyp314a1 was highly expressed in the pre-eclosion phase. Low-temperature treatment can accelerate diapause termination in A. pernyi pupae. With the exception of ApCyp306a1, which displayed multiple expression level peaks, the other Halloween genes were predominantly upregulated during the middle and late stages of low-temperature treatment.

Key words: Antheraea pernyi, ecdysone, Halloween gene, ecdysone receptor, RNAi