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

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

Overview of identified genes associated to RISC complex in <i>C. puncticollis.</i> (FS) frame shift; (RF) reading frame.

<p>Overview of identified genes associated to RISC complex in <i>C. puncticollis.</i> (FS) frame shift; (RF) reading frame.</p

Sequence comparison to other insect genera from the distribution of BLASTX hit (bitscore >50) against the nr protein database of the National Center for Biotechnology Information.

<p>Sequence comparison to other insect genera from the distribution of BLASTX hit (bitscore >50) against the nr protein database of the National Center for Biotechnology Information.</p

Primers used in PCR for cloning and real-time quantitative PCR.

<p>Primers used in PCR for cloning and real-time quantitative PCR.</p

Inhibition of <i>laccase2</i> expression in second-instar larvae of <i>Cylas puncticollis</i> at 1, 3, 5 and 10 days after injection with dsRNA targeting <i>laccase2</i> at 0.2 µg/mg of body weight.

<p>Injection with dsRNA targeting <i>gfp</i> was used as a control. As internal controls, ribosomal protein L32 and Actin were used. Values are based on two repetitions of three biological samples and expressed as mean ± SEM. Each sample contains 5 pooled insects. The p-values were calculated by unpaired t-test.</p

Overview of identified genes related to the RNAi pathways in <i>C. puncticollis</i>.

<p>(FS) frame shift; (RF) reading frame.</p><p>Overview of identified genes related to the RNAi pathways in <i>C. puncticollis</i>.</p