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. © 2016 Society of Chemical Industry.

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 prosα2, 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 µg 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.

Transcriptome analysis and systemic RNAi response in the African sweetpotato weevil (Cylas puncticollis, Coleoptera, Brentidae).

The African sweetpotato weevil (SPW) Cylas puncticollis Boheman is one of the most important constraints of sweetpotato production in Sub-Saharan Africa and yet is largely an uncharacterized insect pest. Here, we report on the transcriptome analysis of SPW generated using an Illumina platform. More than 213 million sequencing reads were obtained and assembled into 89,599 contigs. This assembly was followed by a gene ontology annotation. Subsequently, a transcriptome search showed that the necessary RNAi components relevant to the three major RNAi pathways, were found to be expressed in SPW. To address the functionality of the RNAi mechanism in this species, dsRNA was injected into second instar larvae targeting laccase2, a gene which encodes an enzyme involved in the sclerotization of insect exoskeleton. The body of treated insects showed inhibition of sclerotization, leading eventually to death. Quantitative Real Time PCR (qPCR) confirmed this phenotype to be the result of gene silencing. Together, our results provide valuable sequence data on this important insect pest and demonstrate that a functional RNAi pathway with a strong and systemic effect is present in SPW and can further be explored as a new strategy for controlling this important pest.

In vitro antifungal activity of synthetic dsRNA molecules against two pathogens of banana, Fusarium oxysporum f. sp. cubense and Mycosphaerella fijiensis.

BACKGROUND: A key challenge for designing RNAi-based crop protection strategies is the identification of effective target genes in the pathogenic organism. In this study, in vitro antifungal activities of a set of synthetic double-stranded RNA molecules on spore germination of two major pathogenic fungi of banana, Fusarium oxysporum Schlecht f. sp. cubense WC Snyder & HN Hans (Foc) and Mycosphaerella fijiensis Morelet (Mf) were evaluated. RESULTS: All the tested synthetic dsRNAs successfully triggered the silencing of target genes and displayed varying degrees of potential to inhibit spore germination of both tested banana pathogens. When Foc dsRNAs were applied to Foc spores, inhibition ranged from 79.8 to 93.0%, and from 19.9 to 57.8% when Foc dsRNAs were applied to Mf spores. However, when Mf dsRNAs were applied on Mf spores, inhibition ranged from 34.4 to 72.3%, and from 89.7 to 95.9% when Mf dsRNAs were applied to Foc spores. CONCLUSION: The dsRNAs for adenylate cyclase, DNA polymerase alpha subunit and DNA polymerase delta subunit showed high levels of spore germination inhibition during both self- and cross-species tests, making them the most promising targets for RNA-mediated resistance in banana against these fungal pathogens. © 2013 Society of Chemical Industry.