Screening of Positive Controls for Environmental Safety Assessment of RNAi Products

RNA interference (RNAi) represents a promising pest control strategy, applicable to both insect-resistant genetically modified (IRGM) crops and sprayable RNAi insecticides. These products can achieve sequence-specific gene silencing and require rigorous environmental risk assessment (ERA) prior to approval. However, current environmental safety assessments of RNAi products and other RNAi experiments frequently use double-stranded EGFP (dsEGFP) as a negative control, while suitable RNAi-based positive controls are lacking. Sometimes conventional chemical toxins (e.g., chlorpyrifos) or protein inhibitors (e.g., trypsin inhibitors) are used as substitutes, but their distinct mechanisms, persistence, and metabolism make them inappropriate for RNAi-specific evaluations. In this study, we evaluated the suitability of RNAi-based positive controls for assessing non-target effects on Harmonia axyridis, a widely distributed predatory beetle used as a bioindicator in biosafety assessments. Under laboratory conditions, we tested one microRNA (miR-92a) and two double-stranded RNAs (dsHaSnf7 and dsHaDiap1) for their effects on H. axyridis. Injection of miR-92a showed no significant difference in mortality compared to controls, whereas dsHaSnf7 and dsHaDiap1 significantly reduced survival rates and target gene expression, as confirmed by qPCR. These findings suggest that HaSnf7 and HaDiap1 are suitable candidate genes for establishing RNAi-specific positive controls in environmental risk assessments of RNAi-based products.