Non transformative RNA interference protocols for gene silencing in the Japanese beetle Popillia japonica

Strategies based on RNAi emerged in crop protection against fungi, viruses and insect pests. Here, exogenous application of dsRNAs was used to silence essential genes of the Japanese beetle Popillia japonica (Coleoptera: Scarabaeidae), highly polyphagous quarantine invasive species that cause severe damages to crops. The management of P. japonica is mainly based on broad-spectrum chemical insecticides. Innovative and sustainable alternative strategies are urgently needed to cope with this pest. Two target genes in P. japonica were selected to be silenced as potentially responsible for inducing lethal phenotype, based on previous wide-genome screenings on other coleopterans. To avoid off-target effects, genes with identity higher than 80% with pollinator homologs were excluded and P. japonica dsRNAs were designed in the least conserved gene regions, based on alignments with Apis mellifera. Different procedures were used to deliver dsRNAs to larvae (by injection) and to adults (by injection and by plant-mediated feeding assays). Such protocols allowed to select the most effective gene in inducing lethal phenotype. Upon dsRNA injection, all the target transcripts were significantly reduced. In P. japonica adults, either injected or mass-fed on vine leaves treated with dsRNAs, silencing of a gene involved in 26S proteasome complex determined reduction in survival and prolificacy rates. These data pave the way for the use of RNAi-based strategies in the control of the Japanese beetle and proactively awaiting for the EU approval of the exogenous use of dsRNAs.