Evolution of circadian genes
Five circadian genes were annotated in the PFM genome and the other reference insect species. The PER gene was not found in currently assembled genomes of Cydia pomonella and Anoplophora glabripennis . Two types of CRY gene were annotated in 16 species, mammalian-type cryptochrome (CRY-m) and Drosophila type cryptochrome (CRY-d). For most of the 16 insects, two types of CRY gene were found, while only CRY-m was found in two Coleoptera species and only CRY-d was found in Drosophila melanogaster . In the PFM, FPKM values of CRY-m were higher than CRY-d in each stage, indicating that CRY-m may be a major element in the circadian clock of PFM. Domains of circadian genes were conserved among the 16 species (Fig. 4). PAS domains were common in CLK, CYC and PER genes. The phylogenetic tree of PAS domains revealed six clades, corresponding to two domains of three genes (Fig. 5).
Gene expression in diapause and non-diapause PFM
Compared with larvae that developed under a normal day photoperiod, 11 genes were significantly up-regulated and 9 genes were down-regulated in larvae that developed under long-day or short-day photoperiods (Table S1, Fig. S1 ). Genes highly expressed in pre-diapause larvae (SD and LD photoperiod) included genes encoding CUSOD2 (CS_07203), an enzyme that destroys radicals, and that plays an important role in diapause and cold tolerance of insect (Bi, Yang, Yu, Shu, & Zhang, 2014; He, Meng, Yang, & Hua, 2013; Isobe et al., 2006; Y. I. Kim et al., 2010; Sim & Denlinger, 2011; Zhao & Shi, 2009). We identified a cytochrome P450 gene (CS_20496) showing weak expression in pre-diapause larvae and high expression in the other stages, which was also found in diapausing larvae of the wild silk moth, Antheraea yamamai (Yang, Tanaka, Kuwano, & Suzuki, 2008).
We identified 44 genes specifically up-regulated under a long-day photoperiod, and 14 genes specifically up-regulated under a short-day photoperiod (Table S1, Fig. S1 ). Four genes (CS_04235, CS_05017, CS_15183, CS_01854) related to digestion of proteins were up-regulated in larvae developing under a long-day photoperiod. This is congruent with previous reports suggesting that photoperiod had significant effects on digestive enzyme activity (Espinosa-Chaurand, Vega-Villasante, Carrillo-Farnes, & Nolasco-Soria, 2017; Ramzanzadeh, Yeganeh, JaniKhalili, & Babaei, 2016; Shan, Xiao, Huang, & Dou, 2008; Subala & Shivakumar, 2017). The functional link of many of these genes to diapause is not really clear. The circadian genes, which are important in diapause in another moth (Kozak et al., 2019), did not show significant changes for larvae under different photoperiods.