FoxO mediates the timing of pupation through regulating ecdysteroid biosynthesis in the red flour beetle, Tribolium castaneum

The steroid hormone 20-hydroxyecdysone (20E), the major developmental hormone in insects, controls all the developmental transitions including ecdysis and metamorphosis. In our study with last larval stages of the red flour beetle, Tribolium castaneum, dsRNA-mediated gene silencing of Forkhead box protein O (FoxO) resulted in reduced food intake and larval mass and this agreed with a reduction in the expression of insulin signaling-related genes (insulin-like peptides 2, 3, 4, and chico). Interestingly, we also observed a significant delay in the moment of the pupation and these FoxO-silenced larvae then turned brown at the middle pupal stage followed by death. The observed delay of pupation concurred with a significant delay in 20E titer in dsFoxO-injected larvae and this in turn agreed with a significant delay in expression of prothoracicotropic hormone (ptth) that is a gene stimulating ecdysteroid biosynthesis, and of spook (spo) that is one of the early Halloween genes involved in ecdysteroid biosynthesis. In addition, there was also a delayed expression of the ecdysteroid response gene hormone receptor 3 (HR3). In an attempt to rescue the effects by dsFoxO, injection of 20E into T. castaneum larvae stimulated the expression of HR3 and induced one extra larval-larval molt, confirming the responsiveness for ecdysteroid signaling in dsFoxO-injected larvae. The observations of this project suggest that FoxO is a player in the timing of pupation via the regulating of ecdysteroid biosynthesis, together with the regulation of both insulin signaling and nutrition.