No abstract

Double stranded RNAs (dsRNA) degrading nuclease is responsible for the rapid degradation of dsRNA molecules, and thus accounts for variations in RNA interference (RNAi) efficacy among insect species. Here, the biochemical properties and tissue-specific activities of dsRNA degrading nucleases in four insects (Spodoptera litura, Locusta migratoria, Periplaneta americana, and Zophobas atratus) from different orders were characterized using a modified assay method. The results revealed that all insect dsRNA degrading nucleases tested showed high activity in alkaline environments at optimal Mg2+ concentrations and elevated temperatures. We also found that enzymes from different insects varied in terms of their optimal reaction conditions and kinetic parameters. Whole body enzyme activity differed dramatically between insect species, although enzymes with higher substrate affinities (lower Km) were usually balanced by a smaller Vmax to maintain a proper level of degradative capacity. Furthermore, enzyme activities varied significantly between the four tested tissues (whole body, gut, hemolymph, and carcass) of the insect species. All the insects tested showed several hundred-fold higher dsRNA degrading activity in their gut than in other tissues. Reaction environment analysis demonstrated that physiological conditions in the prepared gut fluid and serum of different insects were not necessarily optimal for dsRNA degrading nuclease activity. Our data describe the biochemical characteristics and tissue distributions of dsRNA degrading activities in various insects, not only explaining why oral delivery of dsRNA often produces lower RNAi effects than injection of dsRNA, but also suggesting that dsRNA-degrading activities are regulated by physiological conditions. These results allow for a better understanding of the properties of dsRNA degrading nucleases, and will aid in the development of successful RNAi strategies in insects.

Double‐stranded RNA (dsRNA) length may affect RNA interference (RNAi) efficacy. Herein, variation in RNAi efficacy associated with dsRNA molecular length was confirmed via comparison of knockdown results following dsRNA injection into Tribolium castaneum. Through in vitro experiments with T. castaneum midgut, dsRNA accumulation in the midgut, degradation by midgut homogenates and persistence in haemolymph after injection were tested to determine the causes of RNAi efficacy variation. The comparative efficacies of dsRNAs were 480 bp ≈ 240 bp > 120 bp > 60 bp >> 21 bp. The combined midgut dsRNA accumulation and midgut homogenate‐induced degradation analyses suggested cellular uptake to be the key barrier for 21 bp dsRNA functioning, but was likely not the main determinant of the variation in longer dsRNAs’ (≥60 bp) bioactivity. In vitro RNAi experiment with T. castaneum midgut showed that long dsRNAs all significantly depleted the expression of corresponding genes, suggesting little variation in intracellular RNAi machinery’s affinity for different dsRNA lengths. In vivo haemolymph content dynamics of different dsRNAs following injection indicated higher persistence of longer dsRNAs. In addition, comparison of the in vivo and in vitro RNAi efficacy also indicated the importance of haemolymph degradation. Thus, the varied efficacy of long dsRNAs resulted from their degradation by nucleases, which varied with dsRNA length.