Potential for gene silencing by piwi-interacting RNA in three lepidopterans

RNA interference (RNAi) has emerged as a promising strategy for insect pest control, yet its application in lepidopterans is hindered by insensitivity to double-stranded RNA (dsRNA). Here, we investigate the piwi-interacting RNA (piRNA) pathway as an alternative, mechanistically distinct gene silencing system in Spodoptera frugiperda, Plutella xylostella, and Cydia pomonella. Small RNA profiling revealed that piRNAs (26-30nt) are the predominant class across all species, marked by strong ping-pong signatures and extensive genomic distribution. Notably, abundant somatic expression, including in gut tissues, indicates that ubiquitous, endogenous piRNA populations may be able to initiate processing of exogenous RNA substrates. To test this possibility, synthetic piRNA-trigger constructs were found to induce gene silencing in Sf9 cells. Further, this approach to gene silencing offers opportunities for nucleic acid engineering, which we demonstrate by incorporating G-quadruplex structures that can enhance RNA stability. Together, these findings establish piRNAs as the dominant small RNA pathway in these lepidopterans and provide a mechanistic and design framework for next-generation RNA-based biopesticides.