Early attainment of 20-hydroxyecdysone threshold shapes mosquito sexual dimorphism in developmental timing

In holometabolous insects, critical weight (CW) attainment triggers the onset of pupation and subsequent metamorphosis into the adult stage. However, the mechanism underlying CW assessment and pupation timing has only been explored in few model organisms and remains elusive in other insects. Here, we investigated the role of 20-hydroxyecdysone (20E) in CW assessment and pupation timing in Aedes albopictus and Ae. aegypti, the main vectors of arboviruses including dengue, chikungunya, and Zika. Our results showed that the attainment of CW was contingent upon surpassing a critical 20E threshold, which resulted in entrance into a constant 22-hour interval and the subsequent 20E pulse responsible for larval-pupal ecdysis. The threshold 20E level induced a peak expression of spook, and RNA interference (RNAi)-mediated knockdown of spook led to pupation delays. Furthermore, sexual dimorphism in pupation time was attributed to higher basal 20E levels. which induced earlier attainment of CW in males compared to females, and 20E treatment at 50% of L3/L4 molt, when most of males but not females pass the pulse, resulted in female-specific lethality. These findings indicate that the 20E threshold plays a pivotal role in determining CW, pupation timing, and their sexual dimorphism in mosquitoes. Our results suggest that modulation of 20E levels holds promise for sex separation in mosquito control programs by manipulating pupation timing and inducing a male-skewed population.