Transcriptome Analysis and Systemic RNAi Response in the African Sweetpotato Weevil (Cylas puncticollis, Coleoptera, Brentidae)

Abstract

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.

RNA interference: a promising biopesticide strategy against the African Sweetpotato Weevil Cylas brunneus

“…Confirming laccase2 silencing at the transcript level is not easy, given the very variable natural expression profile of this gene. This is reflected in the high variation of laccase2 gene expression in the controls, as previously observed and discussed20. Nevertheless, gene expression at 1, 4, 10 and 12 dpi were significantly different from the controls.…”

“…The functionality of RNAi in C. brunneus was confirmed by an experiment where the laccase2 gene expression was knocked down. This gene, encoding a copper-containing enzyme which oxidizes phenolic compounds to their corresponding quinones, was chosen based on its involvement in cuticle tanning and sclerotization and on the expectation that silencing this gene would result in a clear phenotypical effect, as reported earlier in C. puncticollis 20, T. castaneum 22, D. virgifera 23, Nephotettix cincticeps 24, Monochamus alternates 25 and Aedes albopictus 26. Treated C. brunneus weevils clearly showed defects in their cuticle development during and after pupation.…”

“…The same list of RNAi-related genes was used as in Prentice et al . and Swevers et al 2044. and is comprised of 51 genes, covering the RNAi core machinery genes, auxiliary factors, nucleases, genes related to the antiviral RNAi response and to dsRNA uptake (Supplementary Data 1).…”

“…Species belonging to the order of Coleoptera (beetles, weevils) generally seem to be very sensitive to oral RNAi, while insects belonging to hemipteran and lepidopteran orders exhibit a much lower or a very variable RNAi efficiency1617181920. Recently, a first proof-of-concept experiment has shown that RNAi works in the close relative of C. brunneus , namely C. puncticollis , at least by microinjection20. In this research, we investigate the efficacy and efficiency of oral RNAi in C. brunneus and hence investigate whether an RNAi-based approach is a viable option for control of the sweetpotato weevil C. brunneus .…”

Phenotypic screen for RNAi effects in the codling moth Cydia pomonella

“…Despite these successes suggesting the development of novel tools for pest management, the ability to experimentally induce RNAi effects varies greatly among different insect species. Although strong RNAi effects have been induced in some insects (Whyard et al, 2009;Gu and Knipple, 2013;Prentice et al, 2015), in others RNAi caused low rates of visible phenotypic changes, or none at all (Saleh et al, 2006;Iga and Smagghe, 2010;Marcus, 2005). A recent review of successful and unsuccessful attempts to affect RNAi-induced gene silencing in lepidopteran species discussed variable efficiency of RNAi related to species, tissue and gene function (Terenius et al, 2011).…”