Expression profile of several genes on ecdysteroidogenic pathway related to diapause in pupal stage of Bombyx mori bivoltine strain

Highlights

• There was an accumulation of ecdysone in non-diapause eggs producers' group on d-3 pupal stage.

• Inhibition of Shadow upregulated the kynurenine synthesis pathway in B. mori.

• Shadow is involved in the regulation of the silkworm diapause process.

Abstract

Ecdysone is involved in regulation of embryonic diapause in the silkworm, Bombyx mori. However, its mechanism still remains unclear. To explore the role of ecdysteroidogenic pathway (EP) genes in diapause process of bivoltine B. mori, the eggs of “Qiufeng”, a bivoltine strain, were used as the study materials and arranged into diapause eggs producers (DEPs) and non-diapause eggs producers (NDEPs), respectively. The differential expression of EP genes between two groups was analysed during the early pupal stage. The expression of Shadow was significantly increased in the NDEPs in day-3 pupae and reached the peak simultaneously, indicating that Shadow was in coincidence with diapause process. To validate this hypothesis, a repression of Shadow by RNA interference was performed in day-2 pupae of NDEPs. The expression of Shadow was downregulated by RNAi, and βFtz-F1, a downstream gene of EP, was also decreased. Furthermore, the genes encoding the kynurenine-synthetase were upregulated in the ovary, and Brown, AdenoK which link Shadow to the kynurenine-synthase gene were also upregulated in the fat body. The progeny eggs appeared a light purple colour at 48 h after oviposition, revealing a certain tendency to diapause. We speculate that inhibition of Shadow upregulates 3-hydroxy-kynurenine synthesis by increasing the expression of Brown and AdenoK. In addition, Shadow was cloned, and expressed in E. coli for further functional study of Shadow protein. Our study provided insight into the role of EP genes in the process of diapause of B. mori.

Introduction

Diapause is an alternative strategy for insects to survive adverse environmental conditions (Danks, 1987; Hahn and Denlinger, 2011). Many signalling pathways participate in the regulation of diapause such as hormonal control (Denlinger et al., 2004), the circadian clock and photoperiodism (Goto et al., 2010; Koštál and Denlinger, 2011; Saunders, 2010) and energy utilization (Hahn and Denlinger, 2007, Hahn and Denlinger, 2011).

20-Hydroxyecdysone (20E) is one of the important hormones that control insect moulting and metamorphosis (Warren et al., 2002). Ecdysone, as the precursor of 20E, is synthesized in the endocrine organ prothoracic glands (PGs), then secreted into the haemolymph, and finally converted into 20E at peripheral tissues (Gilbert et al., 2002; Rewitz et al., 2013; Uchibori-Asano et al., 2017). The biosynthesis and release of ecdysone are controlled by prothoracicotropic hormone (PTTH) during specific developmental stages to trigger moulting and metamorphosis (Gilbert et al., 2002; Rewitz et al., 2013). In prothoracic cells, PTTH binds its receptor Torso (a tyrosine kinase receptor) and then activates the synthesis of ecdysone through the MAPK/ERK, PI3K/Akt (Gu et al., 2011) pathway and TOR signalling (Shi-Hong et al., 2012), which converts the dietary cholesterol to ecdysone by a group of ecdysteroidogenic enzymes encoded by the Halloween genes including Neverland, Spook(CYP307A1), Phantom(CYP306A1), Disembodied(CYP302A1), Shadow(CYP315A1), Shade(CYP314A1) and CYP18A1 (Gilbert, 2004; Yoshiyama et al., 2006). The Shadow protein is a crucial enzyme because it catalyses the biosynthesis of ecdysone from 2-deoxyecdysone in the PG. When the synthesized ecdysone is released from the PGs into the haemolymph, it is converted to the active form, 20E, in the peripheral tissues by Shade (Petryk et al., 2003). CYP18A1 is a key enzyme for E and 20E inactivation via 26-hydroxylation (Guittard et al., 2011). The Spook is expressed in the PG, converts 7-dehydrocholesterol to Δ⁴-diketol (Gilbert, 2008; Namiki et al., 2005). The Phantom converts 2, 22, 25dE-ketodiol to 2, 22dE-ketotriol (Gilbert, 2008). The Disembodied codes for a cytochrome P₄₅₀ enzyme that adds a hydroxyl group to the carbon-22 position of 2,22, dE-ketotriol to make 2-deoxyecdysone (Gilbert, 2004). In PGs of Bombyx mori, PTTH stimulates the transcription of Spook, Phantom and Disembodied except Shadow (Niwa et al., 2005; Yamanaka et al., 2007), implying that the function of Shadow is different from other Halloween genes.

Bombyx mori is a typical insect that diapause in the egg (embryo) stage. An important indicator of diapause is the biosynthesis of pigment mediated by the 3-hydroxy-kynurenine pathway (Zhang et al., 2017). Ommochromes are major pigments that are widely distributed in eggs, eyes, wings, and epidermis of insects. In ommochrome biosynthesis of Drosophila, tryptophan is converted to 3-hydroxy-kynurenine by three enzymes encoded by vermilion, Kynurenine formamidase (KFase), and cinnabar (Reed and Nagy, 2005; Searles et al., 1990; Searles and Voelker, 1986; Warren et al., 1996); while the homologous genes in B. mori are 2–3-DIO, KFase and Kmo, respectively. Then 3-hydroxy kynurenine is transverted into pigment granules in the serum by the ATP-binding cassette (ABC) transporter encoded by brown, scarlet and white; subsequently, ommochromes are hypothesized to be synthesized through oxidative condensation of pigment granules. High-performance liquid chromatography (HPLC) and full chromatographic scanning analysis shows that 3-hydroxy-kynurenine profiles are significantly different in diapause and non-diapause eggs, suggesting that diapause hormone (DH) might play an important role in the process of metabolizing kynurenine to 3-hydroxy-kynurenine, i.e., the accumulation of 3-hydroxy-kynurenine is controlled by DH (Zhang et al., 2017).

Ecdysteroid has been identified to participate in controlling embryonic diapause in several insects e.g., Bombyx mori (Sonobe and Yamada, 2004), Manduca sexta (Kaplanis et al., 1973), Locusta migratoria (Tawfik et al., 2002) and Schistocerca gregaria (Gande and Morgan, 1979). In B. mori eggs, ecdysone activity is high during development stages while it is low during diapause (Ohnishi et al., 1971). The amount of 20E sharply increased in non-diapause eggs as embryogenesis proceeded, but remained at a low level in diapause eggs (Sonobe, 1997). Therefore, we speculated that the ecdysteroidogenic pathway (EP) may be involved in regulating kynurenine biosynthesis in silkworm diapause process.

Here, we reported that the expression levels of genes encoding ecdysteroidogenic enzymes in ovaries of progeny diapause-destined pupae were significantly different from non-diapause-destined pupae in B. mori, implying that some genes may affect kynurenine biosynthesis pathway. To test this hypothesis, dsRNAi experiment was performed to knockdown of Shadow. The decreased Shadow levels upregulated the expression levels of kynurenine biosynthesis genes, and resulted in a middle colour of progeny eggs between non-diapause- (yellowish) and diapause-colour (brown). Moreover, the Shadow protein was expressed in Escherichia coli (E. coli) successfully. Our results provide an insight into the molecular mechanism of Bombyx diapause.