Mian-Zhi Wu Shu-Ting Fan Yuanchen Zhang Jin-Fang Tan Guan-Heng Zhu ^*

• School of Agriculture, Sun Yat-sen University, Guangdong, China

Juvenile hormone (JH) and 20-hydroxyecdysone (20E) are the central regulating hormones of insect development. The timing of their secretion usually leads to developmental transitions.In the last larval instar, an elevation of the 20E titer in the absence of JH initiates metamorphosis upon reaching the critical weight (CW). This threshold size of Spodoptera frugiperda was determined to be approximately 24 h after the sixth instar larval stage (L6-24 h). Additionally, the expression of the pupal specifier Broad-complex and the potential 'metamorphosis initiation factor' Myoglianin showed a stepwise increase between L4-24 h and L6-24 h. The main endocrine organs, including brain-corpora cardiacum-corpora allatum (BCC) and prothoracic gland (PG), were sampled for RNA-seq analysis. It revealed that both 20E and JH synthesis enzymes were down-regulated in L6-24 h compared to L4-24 h.Besides, strong tendencies in the expression pattern were detected among lists of transcripts, implying the underlying changes of the organs at the CW. The critical rate-limiting enzyme of 20E synthesis, shadow, was knocked down to mimic the down-regulation of 20E synthesis enzymes in the late larval instars. Interestingly, JH acid o-methyltransferase was also downregulated in the RNA interference (RNAi) larvae. These RNAi larvae displayed larval development arrest and subsequently died, indicating that disrupting 20E synthesis before CW is lethal. Thus, the down-regulation of 20E synthesis enzymes at the CW may represent a carefully regulated event, suggesting a slow-down of larval growth and the initiation of some underlying physiological changes to set the stage for the metamorphosis.