Diapause is, evolutionarily, a physiological state of developmental arrest used by many insects to adapt to environmental changes in their habitats (Denlinger, 2002; Bale and Hayward, 2010). As an adaptive strategy, diapause is common at different developmental stages, such as eggs, larvae, pupae, and adults. Each insect enters diapause at a fixed stage. In Bombyx mori, diapause occurs during the embryonic phase; diapause termination of the daughter eggs occurs via incubating the developing mother eggs at 15°C, which considerably enhances the establishment of germline transformation technology (Zhao et al., 2012). In Antheraea pernyi, diapause occurs during the pupal stage. Previous studies have used 20-hydroxyecdysone (20E) to terminate pupal diapause of A. pernyi (Liu Y. et al., 2015; Li et al., 2020). However, the eclosion incidence, a key factor, has never been discussed in these studies, which limits the application of 20E in production and biotechnology development. A high eclosion incidence after pupal diapause termination can easily raise the rearing frequency per year of A. pernyi, which can promote the development of sericulture and basic research, including the establishment of germline transformation technology.

Environmental cues usually trigger diapause in insects. Notably, photoperiod and temperature cues (Bale and Hayward, 2010) received by the brain subsequently affect the secretion of molting hormone from the prothoracic glands (PGs) (Mizoguchi et al., 2013). PGs, mediated by several Halloween genes, first produce ecdysone (E), the precursor of the major molting hormone (Gilbert, 2004; Rewitz et al., 2013). E is released into hemolymph, and then transformed into its active form, 20-hydroxyecdysone (20E), as catalyzed by 20-monooxygenase in peripheral tissues (Petryk et al., 2003). The inactivation of PGs and the resulting decline in ecdysteroid titer during the wandering stage causes pupal diapause (Zdarek and Denlinger, 1987; Mizoguchi et al., 2013). Low ecdysteroid titers in the hemolymph of pupae are closely related to pupal diapause maintenance (Smith et al., 1996; Takeda et al., 1997); injection of exogenous 20E into diapausing pupae can terminate pupal diapause (Liu Y. et al., 2015; Reynolds et al., 2019). Many studies have also invoked 20E to reboot pupal-adult transition by artificially increasing 20E content in the hemolymph (Yang et al., 2010; Niitsu et al., 2014; Chen et al., 2016). However, pupal diapause termination via the injection of 20E has a dose-dependent effect; injection at a large dose causes mortality in pupae (Williams, 1968; Wu et al., 1994; Chen et al., 2016). This situation limits the efficient use of 20E-induced pupal diapause termination in preparing sufficient number of eggs for production in sericulture and biotechnological development in A. pernyi. To date, studies have only determined the effective dosage of 20E to terminate diapause in 18-week-old pupae of A. pernyi (Liu Y. et al., 2015). Whether this dosage is also suitable for diapause termination in pupae at other ages and consequently results in a high eclosion incidence remains unclear.

A single ecdysone pulse in the hemolymph can trigger molting and metamorphosis in a stage- and tissue-specific manner (Lan et al., 1999; Rewitz et al., 2013; Yamanaka et al., 2013). However, studies have not clearly documented the genetic expression trend associated with the pupal-adult transition in A. pernyi pupae, as triggered by 20E. E93 is a 20E primary-response gene; its expression can be suppressed by the juvenile hormone (JH) via the JH primary-response gene Kr-h1 during the larval stage (Baehrecke and Thummel, 1995; Urena et al., 2014). However, during pupal-adult metamorphosis, E93 represses JH signaling (Urena et al., 2014; Liu X. et al., 2015). These studies demonstrated that E93 is a universal adult specifier for insects, promoting larval tissue dissociation and adult tissue formation (Mou et al., 2012; Urena et al., 2014; Liu X. et al., 2015). βFTZ-F1 is another 20E downstream nuclear receptor. In Drosophila, βFTZ-F1 is involved in fat body dissociation by regulating the expression of matrix metalloproteinase 2 (MMP2) (Bond et al., 2011); the knockdown of βFTZ-F1 during the late pupal stage suppresses the normal development of adult organs (Sultan et al., 2014). Fat body remodeling is a premise to form normal adults, which involves the disruption of the extracellular matrix in the fat body and causes polygonal fat cells to dissociate into spherical fat cells (Nelliot et al., 2006; Zheng et al., 2016). Cathepsin genes (Cats) in A. pernyi (Sun et al., 2017; Sun et al., 2018), B. mori (Lee et al., 2009), and Helicoverpa armigera (Zhang et al., 2013) also regulate this process. In Apis mellifera, pupal-adult transition can be divided into two phases: pupae and pharate adult (Soares et al., 2013). The pharate adult develops underneath the pupal cuticle in the duration between pupal apolysis and adult ecdysis. Eye pigmentation begins at the end of pupal apolysis, and can be used as an indicator for the beginning of pharate adult to assist understanding the pupal-adult transition. In this study, we determined the optimal dosage of 20E and its effect on pupal diapause termination via morphological observation and comparative transcriptome analysis to improve the understanding of possible biological processes during pupal-adult transition in A. pernyi.

The univoltine A. pernyi has a long pupal diapause period. Efficient pupal diapause termination would improve the development of production and biotechnology. Earlier studies demonstrated that 20E treatment or photoperiod manipulation can terminate pupal diapause in A. pernyi (Takeda et al., 1997; Liu Y. et al., 2015). The photoperiodic treatment lasts about 20 days, after which the treated pupae enter the post-termination phase (Li et al., 2020). Our results showed that 20E treatment efficiently led to adult ecdysis approximately half a month later (Figure 1). Therefore, 20E treatment remains convenient for pupal diapause termination at different diapause stages. A high dose of 20E can result in death of pupae and emergence failure (Williams, 1968; Wu et al., 1994), which occurs in A. pernyi. Here, we first documented the malformation effect triggered by a high dose of 20E in A. pernyi (Figure 2). Inappropriate dosage can result in slightly or extremely abnormal development. For the purpose of a high eclosion incidence, we determined the optimal dose of 20E (6 μg/g) for pupal diapause termination at the age of 1 month (Figures 1, 2). Based on another optimal dose of 20E (2 μg/g) for the diapause termination in 4-month-old pupae (Supplementary Figure S4), comprehensive consideration showed that how much 20E was needed to efficiently break pupal diapause depended on when 20E was administered during diapause. A number of different stages should be further included to show a dose response curve during diapause, which could optimize the 20E application in the future. Different natural environments affect the pupal diapause intensity in Antheraea insects (Takeda et al., 1997). The slight difference in the curve fitting of the cumulative eclosion incidences between the two replicates (Figure 1A,B) also confirmed this effect, suggesting slightly stronger diapause tendency of pupae in 2021 than that in 2020.

Antheraea pernyi has been extensively used as a classic organism for examining diapause regulation (Williams and Adkisson, 1964; Li et al., 2020). Subsequent studies have indicated that Cathepsins L and O participate in the disruption of the extracellular matrix and regulate fat body dissociation in A. pernyi (Sun et al., 2017; Sun et al., 2018). Our results provided more evidence that three other Cathepsins were also induced in a similar manner between 1 and 3 dpi; they likely modulated the dissociation process together (Supplementary Figure S5, Supplementary Table S9). Time-series transcriptional responses could provide biological information of stage-specifically molecular events. The expression of genes in Profile 0 and 19 was continuously downregulated and upregulated, respectively, which might be related to diapause maintenance or development reboot. However, Profile 2, 5, 9, 16, and 18 showed a distinct expression fluctuation, indicating stage-specific events of pupal-adult transition. Further enrichment analysis revealed the involvement of upregulated DEGs in the FOXO signaling pathway in Profile 16 (Figure 6). 20E-induced nuclear localization of FOXO controls lipolysis and fat body dissociation by upregulating the expression of lipase, carboxypeptidase, and metalloproteinase genes (Hossain et al., 2013; Cai et al., 2016; Jia et al., 2017). The upregulated expression of Forkhead box O (FoxO), triglyceride lipase Brummer (Bmm), molting fluid carboxypeptidase A (CPA), and matix metalloproteinase genes (MMP) supported the same conclusion for A. pernyi (Figure 6). The transcription activity of FOXO depends on its dephosphorylation (Pan et al., 2018), indicating that the upregulated expression of inhibitor of nuclear factor kappa B kinase (IKK) and phosphoinositide-dependent kinase (PDK) fails to induce the cytoplasmic localization of FOXO by phosphorylation early after 20E treatment. 20E might repress the phosphorylation of PDK and IKK at the post-translation level to maintain FOXO nuclear localization, which controls lipolysis and fat body dissociation of A. pernyi. Further in-depth studies are warranted.

Many studies have showed that E is first synthesized in PG by ecdysteriodogenic enzymes encoded by several Halloween genes, followed by conversion into 20E in peripheral tissues (Petryk et al., 2003; Gilbert, 2004; Rewitz et al., 2013). Earlier reports documented that 20E pulse is necessary for pupal-adult transformation in Tenebrio molitor and Apis mellifera (Quennedey et al., 1983; Soares et al., 2013), and a long-day photoperiodic manipulation could trigger two 20E pulses sequentially in the next 35 days in A. pernyi pupae (Li et al., 2020). In the present study, the intermittent expression of the 20E inactivation enzyme and the upregulated expression of four Halloween genes (Figure 7) suggested a similar role as that of 20E pulse. We speculated that exogenous 20E indirectly triggered the expression of Halloween genes by a downstream activator factor, followed by the synthesis of endogenous 20E. PG involution is a foregone destination before adult ecdysis (Quennedey et al., 1983; Dai and Gilbert, 1997). Now that Halloween genes can be indirectly induced at 1 and 3 dpi, the degeneration of A. pernyi PG may commence later (Figure 3A). Further studies should investigate this degeneration process. Eye pigmentation is an important feature that indicates the beginning of the pharate stage (Shimono et al., 2009; Soares et al., 2013). Between 5 dpi and adult ecdysis, we envisioned that A. pernyi is in a pharate state, before which A. pernyi sustains the pupal state. The high expression of βFTZ-F1 during the late pupal stage modulated the normal development of adult organs in Drosophila (Sultan et al., 2014). We also observed the high expression of βFTZ-F1 at 9 dpi (Figure 7A, 8E), indicating the conserved function of βFTZ-F1 between A. pernyi and Drosophila. In B. mori, FOXO regulates JH degradation by directly binding to the promoter regions of the genes involved in JH degradation (Zeng et al., 2017). In the present study, the expression of the juvenile hormone epoxidase hydrolase gene (JHEH) was also upregulated together with FoxO (Figure 6B, 7B), suggesting their regulatory relationship. EcRB1 and USP form a heterodimeric nuclear hormone receptor to mediate ecdysteroid action in insects. During pupal development, high 20E titers could repress the expression of EcRB1 in Apis mellifera (Mello et al., 2014). The downregulated expression of EcRB1 and USP at 1 dpi showed the similar repressive effect early after 20E treatment (Figure 8A,B). Similar results had also been reported in the study of 20-day-old pupae in A. pernyi (Ru et al., 2017). As two key transcription factors in ecdysone signaling during metamorphosis, the expression of E93 and Br-C are induced by the ternary complex of 20E-EcRB1-USP (Liu X. et al., 2015; Qian et al., 2017). The same expression patterns of EcRB1, E93, and BR-C showed the similar regulation of 20E-EcRB1-USP on E93 and Br-C in A. pernyi (Figure 8).

Three trehalase genes were differentially expressed in 1 dpi versus 0 dpi (Supplementary Figure S6, Supplementary Table S10). In some insects, the expression of trehalase genes is modulated by 20E signaling (Yao et al., 2010; Li et al., 2020). Trehalose is an important form of energy during diapause and is also a basic substance for chitin biosynthesis. Acting as a key enzyme of trehalose metabolism, the three trehalases should function within energy metabolism during the early stages of the pupal-adult transition. Chitin is a structural molecule in the insect midgut and exoskeleton. Chitinases located in the midgut and cuticle regulate pathogen invasion and the cyclical renewal of chitin fibers (Noh et al., 2018; Liu et al., 2020). During metamorphosis, chitinase genes act as late response genes of 20E signaling to regulate wing development and the nymph-adult transition (Xu et al., 2020; Wu et al., 2022). The five chitinase genes upregulated at 1, 6, or 9 dpi (Supplementary Figure S6, Supplementary Table S10) may be necessary to promote proper wing development, liquefaction of the old cuticle, and midgut degeneration during adult development. Confirmation of these hypotheses requires further functional analyses.

In summary, the optimal dosage of 20E for diapause termination in the age of 1 month was first determined for a high eclosion incidence. After 20E treatment, genes related to FOXO signaling pathway, molting hormone biosynthesis, energy metabolism, and tissue remodeling were successively reactivated, which sped up pupal-adult metamorphosis in approximately half a month. This study provides a foundation for further mechanistic research on pupal-adult metamorphosis, the rearing of larvae more than once a year, and the establishment of germline transformation technology in A. pernyi.