Abstract

RNA interference-based biopesticides are recognized as the third-generation pesticide revolution, offering a sustainable solution to resistance development, toxicological risks, and environmental pollution caused by overuse of conventional chemical pesticide. Phyllotreta striolata , a major global pest of cruciferous crops, serves as an excellent model for developing RNAi-based control strategies minimal dsRNA requirement and high experimental feasibility. However, the molecular mechanisms underlying nuclease-mediated dsRNA degradation in P. striolata remain uncharacterized, posing a critical barrier to achieve a robust RNAi response. Based on bioinformatics analysis and homologous sequence alignment, we identified and characterized a gut-enriched nuclease gene in P. striolata adults. The gel electrophoresis assay demonstrated heterologously expressed and purified recombinant PsdsRNase protein exhibited rapid dsRNA degradation activity. Analysis of biochemical properties revealed that optimal conditions for the dsRNA-degrading activity were alkaline (pH = 9) in low concentration of Mg 2+ (1 mM) at 35 °C. Then we measured the contribution of PsdsRNase to dsRNA-degrading activity in vivo via knockdown of PsdsRNase gene, and found a transient reduction in nuclease efficiency within the gut lumen from silenced adults. Furthermore, pre-silencing of PsdsRNase resulted in a 58.71% greater reduction in PsDre4 mRNA levels and increased mortality by over 20%. Our findings decipher the molecular basis of dsRNA degradation in P. striolata , while providing a theoretical foundation for enhancing RNAi efficiency through nuclease activity modulation.