Abstract

Zona pellucida (ZP)-domain proteins are critical for development and reproduction in insects; however, the underlying mechanisms remain largely unclear. Here we demonstrate that knockdown of papillote ( pot ), which encodes a ZP-domain protein, suppresses larval growth, induces premature metamorphosis, and impairs reproduction in Tribolium castaneum ( T. castaneum ). Pot silencing stimulated juvenile hormone (JH) degradation while reducing phosphorylated levels of insulin receptor β (InR β ) and FOXO, leading to nuclear location of FOXO. Methoprene treatment rescued larval growth and pupation defects as well as phosphorylated InR β (p-InR β ) and phosphorylated FOXO (p-FOXO) levels inhibited by pot knockdown. Similarly, insulin injection reversed pot silencing-mediated reductions in larval growth, pupation, and p-InR β and p-FOXO levels. Silencing pot induced ecdysone biosynthesis, whereas FOXO knockdown suppressed this effect. Interestingly, reducing ecdysone biosynthesis via phm silencing increased p-FOXO levels, repressed FOXO nuclear translocation, and reversed the decline in larval growth and pupation caused by pot RNAi. FOXO or phm knockdown rescued ovarian maturation and egg production decreased by pot silence, which inhibited vitellogenin1 ( Vg1 ) and Vg2 expression via FOXO. These results indicate that Pot orchestrates development and reproduction via a positive hormone feedback axis. This study comprehensively elucidates the mechanism of ZP-domain protein regulating development and reproduction in insects.