The main objective of this research hypothesis-driven proposal is to investigate whether latent or chronic non-lethal viral infections interfere with the efficacy of RNAi in insects. Up until now, the impact of such viruses on the RNAi response in insects has not been investigated. The project will make use of multiple biotechnology and insect physiology technologies to realize 5 specific objectives. In brief: deep sequencing technology will be used to identify and assemble viral genomes. Viral particles corresponding to the most abundant genomes will be produced in cell culture and used to infect virus-free populations of bumblebees. Virus-free and infected insects will be tested for efficiency against exogenously applied dsRNAs. Finally, screens will be carried out to identify inhibitors of viral infection, and then these can increase the efficacy of dsRNAs to carry out gene silencing in the insects.

This project will contribute at first to the scientific community with new insights on the presence of viruses in the insect body in relation to the efficiency of RNAi. At present the latter interaction of virus and RNAi is very hot in the scientific community. The observation that many insect cell lines are latently or chronically infected with virus is very recent (2009-2011) and also our lab at UGent confirmed this as one of the pioneers. Recent publications (9,10) demonstrated that viral infection can result in a significant interference with RNAi and it remains untested by any group so far whether exogenous application of dsRNAs can induce a robust silencing response in otherwise refractory insects that are made virus-free. The current project can help to better understand the challenging theme of the relationship between the RNAi core and virus infection in insects under natural conditions. We hope to shed more light on some important questions on the interaction with RNAi: for instance, whether the virus may induce a saturation of the RNAi machinery or whether the virus may prevent the access of siRNAs to Argonaute proteins. This project promotes a unique approach to clarify the mechanisms of RNAi in insects and the interaction with virus and viral regulatory factors. If the hypothesis is (even partly) correct, fundamentally new ways of thinking will be raised regarding the regulation of physiological functions in insects by viral symbiosis/commensalism/parasitism.

In second, an important aspect of this project is the choice to work with the key pollinator Bombus terrestris. Indeed bumblebees are ecologically and agriculturally important insects because they are generalist pollinators, but to date there are major concerns towards a global decline in pollinator diversity, e.g. the problematic ―colony collapse disorder‖ (CCD) in honeybees and also for bumblebees. The project can contribute in the clearance of parasitic infections in these beneficial pollinating insects and in turn to increase their health. Depending on the results obtained, the new RNAi-method can be based on the co-delivery of dsRNAs and inhibitors of viral replication/infection (e.g. chemical compounds, cytokines or antisense oligo’s/siRNAs).