RNA interference : a promising biopesticide strategy against the African sweetpotato weevil Cylas brunneus

The African sweetpotato weevil Cylas brunneus is one of the most devastating pests affecting the production of sweetpotatoes, an important staple food in Sub-Saharan Africa. Current available control methods against this coleopteran pest are limited. In this study, we analyzed the potential of RNA interference as a novel crop protection strategy against this insect pest. First, the C. brunneus transcriptome was sequenced and RNAi functionality was confirmed by successfully silencing the laccase2 gene. Next, 24 potential target genes were chosen, based on their critical role in vital biological processes. A first screening via injection of gene-specific dsRNAs showed that the dsRNAs were highly toxic for C. brunneus. Injected doses of 200ng/mg body weight led to mortality rates of 90% or higher for 14 of the 24 tested genes after 14 days. The three best performing dsRNAs, targeting pros alpha 2, rps13 and the homolog of Diabrotica virgifera snf7, were then used in further feeding trials to investigate RNAi by oral delivery. Different concentrations of dsRNAs mixed with artificial diet were tested and concentrations as low as 1 mu g dsRNA/mL diet led to significant mortality rates higher than 50%. These results proved that dsRNAs targeting essential genes show great potential to control C. brunneus

The independent biological activity of Bacillus thuringiensis Cry23Aa protein against Cylas puncticollis

The Cry23Aa/Cry37Aa proteins fromBacillus thuringiensis(Bt) have been described toxic toCylas puncticollislarvae. In general, it is believed that Cry23Aa and Cry37Aa act jointly to exert the insecticidal activity, while there is no evidence of their toxicity individually. Therefore, in the present study, the contribution of each protein in the insecticidal activity towardC. puncticollislarvae has been assessed. The results showed that both proteins were toxic forC. puncticollislarvae when tested individually. Contrary to what was claimed previously, our results suggest that the presence of both proteins is not necessary to exert toxicity againstC. puncticollislarvae. Also, the binding behavior of Cry23Aa protein to midgut receptors ofC. puncticollislarvae has been determined. According to our results, Cry23Aa binds toC. puncticollisbrush border membrane vesicles (BBMV) specifically and independently of Cry37Aa. Due to the lack of common binding sites, Cry23Aa can be pyramided with Cry3Aa protein for better management ofC. puncticollis

Targeting a coatomer protein complex-I gene via RNA interference results in effective lethality in the pollen beetle Brassicogethes aeneus

he pollen beetle Brassicogethes aeneus is a serious pest of oilseed rape (Brassica napus) in Europe. Management of this pest has grown difficult due to B. aeneus's development of resistance to pyrethroid insecticides, as well as the pressure to establish control strategies that minimise the impact on nontarget organisms. RNA interference represents a nucleotide sequence-based, and thus potentially species-specific, approach to agricultural pest control. The present study examined the efficacy of targeting the coatomer gene coatomer subunit alpha (alpha COP), via both microinjection and dietary exposure to exogenous complementary dsRNA, on alpha COP-silencing and subsequent mortality in B. aeneus. Beetles injected with dsRNA targeting alpha COP (at 0.14 mu g/mg) showed 88% and 100% mortality at 6 and 10 days post-injection, respectively; where by the same time after dietary exposure, 43%-89% mortality was observed in the 3 mu g dsRNA/mu L treatment, though the effect was concentration-dependent. Thus, the effect was significant for both delivery routes. In working towards RNA-based management of B. aeneus, future studies should include alpha COP as a target of interest

Nuclease activity decreases the RNAi response in the sweetpotato weevil Cylas puncticollis

RNA interference (RNAi) refers to the process of suppression of gene expression in eukaryotes, which has a great potential for the control of pest and diseases. Unfortunately, the efficacy of this technology is limited or at best variable in some insects. In the African sweet potato weevil (SPW) Cylas puncticollis, a devastating pest that affects the sweet potato production in Sub-Saharan Africa (SSA), the RNAi response was highly efficient when dsRNA was delivered by injection, but it showed a reduced response by oral feeding. In the present study, the role of nucleases in the reduced RNAi efficiency in C. puncticollis is investigated. Several putative dsRNases were first identified in the transcriptome of the SPW through homology search and were subsequently further characterized. Two of these dsRNases were specifically expressed in the gut tissue of the insect and we could demonstrate through RNAi experiments that these affected dsRNA stability in the gut. Furthermore, RNAi-of-RNAi studies, using snf7 as a reporter gene, demonstrated that silencing one of these nucleases, Cp-dsRNase-3, clearly increases RNAi efficacy. After silencing this nuclease, significantly higher mortality was observed in dssnf7-treated insects 14 days post-feeding as compared to control treatments, and the gene downregulation was confirmed at the transcript level via qPCR analysis. Taken together, our results demonstrate that the RNAi efficiency is certainly impaired by nuclease activity in the gut environment of the SPW Cylas puncticollis

RNAi-based gene silencing through dsRNA injection or ingestion against the African sweet potato weevil Cylas puncticollis (Coleoptera: Brentidae)

BACKGROUND. RNA interference (RNAi) technology can potentially serve as a suitable strategy to control the African sweet potato weevil Cylas puncticollis (SPW), which is a critical pest in sub-Saharan Africa. Important prerequisites are required to use RNAi in pest control, such as the presence of an efficient RNAi response and the identification of suitable target genes. RESULTS. Here we evaluated the toxicity of dsRNAs targeting essential genes by injection and oral feeding in SPW. In injection assays, 12 of 24 dsRNAs were as toxic as the one targeting Snf7, a gene used commercially against Diabrotica virgifera virgifera. Three dsRNAs with high insecticidal activity were then chosen for oral feeding experiments. The data confirmed that oral delivery can elicit a significant toxicity, albeit lower compared with injection. Subsequently, ex vivo assays revealed that dsRNA is affected by degradation in the SPW digestive system, possibly explaining the lower RNAi effect by oral ingestion. CONCLUSION. We conclude that the full potential of RNAi in SPW is affected by the presence of nucleases. Therefore, for future application in crop protection, it is necessary constantly to provide new dsRNA and/or protect it against possible degradation in order to obtain a higher RNAi efficacy.Peer Revie