20‐hydroxyecdysone‐mediated regulation of the ribosomal protein <i>Cs</i>40SRPS4 via <i>CsE74A</i> is essential for vitellogenesis in <i>Chilo suppressalis</i>

Development and reproduction are key processes in the insect life cycle, and vitellogenesis is a critical event in insect reproduction. Vitellogenin (Vg) is generally recognized as a female-specific, reproductive lipoprotein unique to oviparous taxa that plays a crucial role in vitellogenesis. However, its specific functions, and the mechanisms underlying its regulation, remain poorly understood. We here demonstrate how 20-hydroxyecdysone (20E) regulates Vg transcription via the transcription factor CsE74A and the ribosomal protein Cs40SRPS4 in Chilo suppressalis. Disruption of CsVg in G0 generation C. suppressalis via multiplex CRISPR/Cas9 resulted in decreased yolk deposition, ovarian dysplasia, and delayed oocyte maturation. Dual luciferase and DNA pull-down assays identified the CsVg core promoter (-915 to -811 bp) and its binding ribosomal protein Cs40SRPS4. Functional analysis via RNA interference revealed that Cs40SRPS4 is a ribosomal protein capable of regulating vitellogenesis in C. suppressalis. Furthermore, we correctly predicted that the binding site for CsE74A would be on the CsVg core promoter and confirmed its binding capability. Subsequent experiments further confirmed the interaction between CsE74A and Cs40SRPS4. Notably, the expression of CsE74A, Cs40S4, and CsVg significantly increased after 20E treatment. The results revealed that 20E induced the early response factor CsE74A to bind to the CsVg core promoter, thereby regulating Vg transcription and vitellogenesis in conjunction with Cs40SRPS4. These findings not only provide a foundation for gaining deeper understanding of insect reproductive processes, but could also lead to the development of safer, more economical and sustainable, pest control methods.