Key factors determining competitions between double-stranded RNAs in Tribolium castaneum

Combinatorial delivery of different double-stranded RNAs (dsRNAs) can result in competitive inhibition in insect pests and remains one of the obstacles in the way of future applications of the RNA interference (RNAi)-based pest control. In this study, we attempted to discover the basic competition characteristics between dsRNAs and provided insight into the solutions of competitive inhibition. RNAi sensitive insect species Tribolium castaneum were treated, and competitions between dsRNA fragments influencing the effectiveness of RNAi response could be measured. A chimeric dsRNA strategy for conjugating different dsRNA fragments into a single molecule and a nanoparticle carbon quantum dots-mediated dsRNA delivery were confirmed as efficient methods to knock down multiple target genes simultaneously. Furthermore, in vitro assays were conducted for determining the accumulation speed of serially diluted and incubated dsRNA in the midgut tissues. Our data showed that the accumulation of dsRNAs of different treated amounts was 0.25 μg ≈ 0.5 μg > 1 μg ≥ 2 μg > 4 μg, indicating that accumulation speed would be affected by treated dsRNA. Overall, our results strongly suggest that endocytic components influencing cellular uptake might be oversaturated when an excess amount of dsRNAs were treated, thereby causing competitive inhibition of target genes.