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VIRTIGATION Poster presentation during the 15th International Symposium of Plant Virus Epidemiology in Madrid, Spain. The H2020 project VIRTIGATION - Emerging viral diseases in tomatoes and cucurbits: Implementation of mitigation strategies for durable disease management - has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement No. 101000570.

Chemical insecticides are currently the most frequently utilized insect pest management strategy. However, their application causes a number of potentially harmful side-effects that can no longer be ignored. Due to their lack of specificity and limited biodegradation, for example, use of these chemicals can have a large impact on the environment. Moreover, there is a very real chance that their use will disrupt the delicate balance of local ecosystems as not only target insects are affected. In addition, most economically relevant species have started to develop resistance, causing reduced efficacy of these insecticides against their intended target pests. All of these issues result in a pressing need for alternative eradication methods that protect crops in more efficient and environmentally friendly ways. Due to its highly sequence-specific mechanism of post-transcriptional gene silencing, RNA interference (RNAi) has presented itself as an interesting candidate for further application as an alternative crop protection method. Successful application of RNAi technology as an insecticide requires the ability of target insects to induce a systemic RNAi response after feeding with dsRNA. However, not all economically interesting insect species are able to induce a similarly efficient RNAi response after oral administration of double-stranded (ds)RNA. This poses a significant obstacle for the further development of RNAi as a universally applicable insecticide. An intriguing solution is presented in the form of dsRNA delivery systems, carrier techniques designed specifically to facilitate dsRNA uptake and protect it against degradation in aggressive extracellular environments, such as the insect gut. In this thesis we therefore investigate several potential dsRNA delivery system candidates: a selection of 5 distinct cell-penetrating peptides (CPPs), a Galanthus nivalis agglutinin (GNA)-dsRNA binding domain (dsRBD) fusion protein and a genetically modified dsRNA-producing bacteria strain. Application potential of these delivery systems was determined by evaluating: the stability of the carrier in an ex vivo gut environment, its ability to facilitate in vitro cellular uptake in two environmental RNAi-deficient insect cell lines, Sf9 and Cl8, and its capacity to induce RNAi in vivo after feeding/injection in two devastating insect pest species: the beet armyworm Spodoptera exigua, and the desert locust Schistocerca gregaria. Although under the tested conditions none of the selected delivery systems could induce an RNAi response in vivo after feeding, some highly intriguing results were obtained. Two CPPs, EB1 and C6M1, were found to facilitate uptake of both dsRNA and siRNA in Sf9 and Cl8. Most notably, EB1 could also facilitate cellular uptake of siRNA in vivo in S. gregaria, leading to a systemic RNAi response after injection into the hemolymph. Furthermore, complexation with the GNA-dsRBD fusion protein could protect dsRNA in an ex vivo S. exigua midgut environment. Interestingly, while GNA-dsRBD was found to facilitate dsRNA and siRNA uptake in Cl8 cells, no uptake was observed in Sf9. Additionally, whereas feeding with dsRNA-producing bacteria could not lead to a knockdown in S. gregraria, injection of this delivery system into the hemolymph of the desert locust did. Finally, in an attempt to further clarify dsRNA and delivery system uptake mechanisms, a putative S. gregaria scavenger receptor class C sequence was identified and validated through an RNAi-on-RNAi approach. Taken together, this study provides fascinating insights into the application potential of dsRNA delivery systems in insects. status: published