Simultaneous silencing of gut nucleases and a vital target gene by adult dsRNA feeding enhances RNAi efficiency and mortality inCeratitis capitataadults
Authors: Volpe Gennaro , Mazzucchiello Sarah Maria , Rosati Noemi , Lucibelli Francesca , Varone Marianna , Baccaro Dora , Mattei Ilaria , Di Lelio Ilaria , Becchimanzi Andrea , Giordano Ennio , Salvemini Marco , Aceto Serena , Pennacchio Francesco , Saccone Giuseppe 
Published: August 2024

AbstractCeratitis capitata, known as Mediterranean fruit fly (Medfly), is a major dipteran pest significantly impacting fruit and vegetable farming. Currently, its control heavily relies mainly on chemical insecticides, which pose health risks and have effects on pollinators. A friendly and species-specific alternative strategy involves providing double-stranded RNA (dsRNA) through feeding to disrupt essential functions in pest insects, which is poorly explored in dipteran species. Previous reports in Orthoptera and Coleoptera species suggested that dsRNA degradation by two specific nucleases in the intestinal lumen is among the major obstacle to feeding-mediated RNAi in insects. In our study, we experimented with three-day adult feeding using a combination of dsRNA molecules that target the expression of theATPasevital gene and two intestinal dsRNA nucleases. These dsRNA molecules were recently tested separately in two Tephritidae species, showing limited effectiveness [1,2]. In contrast, we observed 79% mortality over seven days, which was associated with a decrease in mRNA levels of the three targeted genes. As expected, we also observed a reduction in dsRNA degradation following RNAi against nucleases. This research illustrates the potential of utilizing molecules as pesticides to achieve mortality rates in Medfly adults by targeting crucial genes and intestinal nucleases. Furthermore, it underscores the importance of exploring RNAi-based approaches for pest managementSimple SummaryThe control of insect pest species, mainly belonging to Orthoptera, Hemiptera, and Coleoptera species, can be based on novel emerging species-specific pesticides. These consist of dsRNA molecules delivered by feeding to insect larvae or adults, which suppress vital gene functions by RNA-RNA sequence complementarity and RNA interference. However, fewer studies of dsRNA feeding have been performed in dipteran pest insects. Two studies in Orthoptera and Coleoptera species have shown that suppressing intestinal enzymes degrading external dsRNA can improve insect mortality rates.Ceratitis capitata(Tephritidae), the Mediterranean fruit fly (Medfly), is a major dipteran pest significantly impacting fruit and vegetable farming. Currently, its control heavily relies mainly on chemical insecticides, which pose health risks and have effects on beneficial pollinators. Previous attempts to induce mortality by adult dsRNA-feeding in this and other Tephritidae species, such asBactrocera tryoniandB. dorsalis, showed some effectiveness, but were often limited. We improved this method by simultaneously silencing two intestinal nucleases and a vital gene. We have found a mix of three dsRNAs able to induce much higher mortality (79%) within one week, following only three days of adult feeding.