Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis

Abstract

RNA interference (RNAi) approaches have been applied as a biotechnological tool for controlling plant insect pests via selective gene down regulation. However, the inefficiency of RNAi mechanism in insects is associated with several barriers, including dsRNA delivery and uptake by the cell, dsRNA interaction with the cellular membrane receptor and dsRNA exposure to insect gut nucleases during feeding. The cotton boll weevil (Anthonomus grandis) is a coleopteran in which RNAi-mediated gene silencing does not function efficiently through dsRNA feeding, and the factors involved in the mechanism remain unknown. Herein, we identified three nucleases in the cotton boll weevil transcriptome denoted AgraNuc1, AgraNuc2, and AgraNuc3, and the influences of these nucleases on the gene silencing of A. grandis chitin synthase II (AgraChSII) were evaluated through oral dsRNA feeding trials. A phylogenetic analysis showed that all three nucleases share high similarity with the DNA/RNA non-specific endonuclease family of other insects. These nucleases were found to be mainly expressed in the posterior midgut region of the insect. Two days after nuclease RNAi-mediated gene silencing, dsRNA degradation by the gut juice was substantially reduced. Notably, after nucleases gene silencing, the orally delivered dsRNA against the AgraChSII gene resulted in improved gene silencing efficiency when compared to the control (non-silenced nucleases). The data presented here demonstrates that A. grandis midgut nucleases are effectively one of the main barriers to dsRNA delivery and emphasize the need to develop novel RNAi delivery strategies focusing on protecting the dsRNA from gut nucleases and enhancing its oral delivery and uptake to crop insect pests.

Enhancing RNAi Efficiency to Decipher the Functional Response of Potential Genes in Bemisia tabaci AsiaII-1 (Gennadius) Through dsRNA Feeding Assays

“…The gut nucleases in hemipteran insects are reported as major bottleneck in efficient RNAi, as these enzymes hamper the concentration of dsRNA reaching the cell (Huvenne and Smagghe, 2010;Joga et al, 2016). Besides hemipteran, the degradation of dsRNA by dsRNases have also been reported from other insect orders (Almeida Garcia et al, 2017;Singh et al, 2017;Prentice et al, 2019;Song et al, 2019). Our studies successfully demonstrated the degradation of dsRNA in hemolymph and gut juices of whitefly.…”

Delivery of gene‐specific dsRNA by microinjection and feeding induces RNAi response in Sri Lanka weevil, Myllocerus undecimpustulatus undatus Marshall

“…These studies demonstrate the importance of evaluating the RNAi sensitivity by microinjection as well as by feeding in the target insect species since it is not possible to predict the RNAi effectiveness based on the RNAi response found in other insects, even in closely related species. The activity of dsRNA degrading enzymes (dsRNase) in the insect gut lumen can impair the RNAi response and has been considered one of the main factors that leads to differences in the level of RNAi sensitivity through dsRNA ingestion among insect species …”

Key factors determining variations in RNA interference efficacy mediated by different double‐stranded RNA lengths in Tribolium castaneum

“…Recent studies have shown that rapid dsRNA degradation by some nucleases can cause differences in dsRNA sensitivity among organisms (Wang et al ., ; Singh et al ., ). The two types of nuclease that are likely to be involved in dsRNA degradation are exonucleases and endonucleases (Kupsco et al ., ; Gabel and Ruvkun, ; Ramachandran and Chen, ; Garbutt et al ., ; Wynant et al ., ; Garcia et al ., ; Song et al ., ; Cao et al ., ; Guan et al ., ). Exonucleases work at the termini of the dsRNAs; so, short molecules that are capable of binding exonucleases for long enough to activate them have more ends and are more easily degraded by the exonucleases than are long molecules of similar weight.…”