20-hydroxyecdysone upregulates apoptotic genes and induces apoptosis in the Bombyx fat body

During insect metamorphosis, obsolete larval tissues are removed by programed cell death (PCD), mainly apoptosis and autophagy, which is directed by the molting hormone, 20-hydroxyecdysone (20E) and the 20E-triggered transcriptional cascade. Here, we investigated how 20E regulates apoptosis at the transcriptional level in the fat body of the silkworm, Bombyx mori. As detected by TdT-mediated dUTP Nick-End Labeling (TUNEL), apoptosis weakly occurred during the fourth larval molting, decreased to undetected levels during the early fifth instar, and gradually increased from day 4 of fifth instar to the wandering stage to the prepupal stage. Meanwhile, as determined by quantitative real-time PCR, eight genes involved in apoptosis, including Apaf-1, Nedd2 like1, Nedd2 like2, ICE1, ICE3, ICE5, Arp, and IAP, were highly expressed during molting and pupation, when the 20E titer is high. Injection of 20E into day 2 of fifth instar larvae significantly induced apoptosis and upregulated apoptotic genes after 6 h of treatment, and in vitro treatment of larval fat body tissues with 20E upregulated all the eight apoptotic genes. Moreover, RNAi knockdown of USP, a component of the 20E receptor complex EcR-USP, at the early-wandering stage reduced apoptosis and downregulated apoptotic genes after 24 h of treatment. Taken together, we infer that 20E upregulates apoptotic genes and thus induces apoptosis in the Bombyx fat body during larval molting and the larval-pupal transition.