RNAi‐mediated silencing of Mediterranean fruit fly (Ceratitis capitata) endogenous genes using orally‐supplied double‐stranded RNAs produced in Escherichia coli

BACKGROUND The Mediterranean fruit fly (medfly), Ceratitis capitata Wiedemann, is a major pest affecting fruit and vegetable production worldwide, whose control is mainly based on insecticides. Double‐stranded RNA (dsRNA) able to down‐regulate endogenous genes, thus affecting essential vital functions via RNA interference (RNAi) in pests and pathogens, is envisioned as a more specific and environmentally‐friendly alternative to traditional insecticides. However, this strategy has not been explored in medfly yet. RESULTS Here, we screened seven candidate target genes by injecting in adult medflies gene‐specific dsRNA hairpins transcribed in vitro. Several genes were significantly down‐regulated, resulting in increased insect mortality compared to flies treated with a control dsRNA targeting the green fluorescent protein (GFP) complementary DNA (cDNA). Three of the dsRNAs, homologous to the beta subunit of adenosine triphosphate (ATP) synthase (ATPsynbeta), a vacuolar ATPase (V‐ATPase), and the ribosomal protein S13 (RPS13), were able to halve the probability of survival in only 48 h after injection. We then produced new versions of these three dsRNAs and that of the GFP control as circular molecules in Escherichia coli using a two‐self‐splicing‐intron‐based expression system and tested them as orally‐delivered insecticidal compounds against medfly adults. We observed a significant down‐regulation of V‐ATPase and RPS13 messenger RNAs (mRNAs) (approximately 30% and 90%, respectively) compared with the control medflies after 3 days of treatment. No significant mortality was recorded in medflies, but egg laying and hatching reduction was achieved by silencing V‐ATPase and RPS13. CONCLUSION In sum, we report the potential of dsRNA molecules as oral insecticide in medfly. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. Orally delivered recombinant circular double‐stranded RNAs (dsRNAs) produced in Escherichia coli induce strong silencing of targeted genes in Ceratitis capitata. This result opens the possibility to use RNA interference‐based crop protection strategies against this insect pest.