RNA interference (RNAi) to control Philaenus spumarius, vector of Xyella fastidiosa

The xylem-limited bacterium Xylella fastidiosa poses a significant phytosanitary risk to economically important crops, such as olive trees. In Europe, it is responsible for Olive Quick Decline Syndrome (OQDS), which was first observed in the Salento region in 2013. In Italy, the primary vector is the meadow spittlebug, Philaenus spumarius, a highly polyphagous and widespread insect. As there are currently no effective cures for X. fastidiosa, management strategies mainly rely on controlling the vector and removing infected plants, which often involves agricultural practices and the use of chemical insecticides. However, these approaches are often insufficient and raise environmental concerns, highlighting the need for more sustainable alternatives. RNA interference (RNAi) has emerged as a promising biocontrol strategy due to its high target specificity and rapid environmental degradation. This thesis is the first to comprehensively investigate RNAi in P. spumarius, with the aim of assessing its potential for controlling the vector and reducing the spread of X. fastidiosa. Chapter 1 demonstrated the presence and functionality of the RNA interference (RNAi) machinery in P. spumarius. Delivering double-stranded RNA (dsRNA) via microinjection triggered gene silencing in adults, and via plant-mediated feeding in nymphs. The small RNA profiles and key components of the core RNAi machinery involved in dsRNA systemic spreading were also characterised, which confirmed the activation of RNAi mechanisms in this species. Chapter 2 presents a high-throughput screening of candidate genes to identify effective RNAi targets that induce mortality. Silencing the V-ATPase gene resulted in the highest mortality. Furthermore, downregulating this gene significantly reduced the bacterial load of Xylella fastidiosa in infected insects, suggesting a potential role in limiting vector survival and pathogen transmission. Chapter 3 investigated the effects of silencing the ATP synthase beta gene on reproduction. Treated females exhibited a drastic reduction in egg laying, as well as a reduction of almost 50% in the presence of eggs in the ovaries. This was accompanied by downregulation of vitellogenin, indicating strong impairment of reproductive capacity. Finally, Chapter 4 explored innovative dsRNA delivery systems based on the polymeric nanocarrier, PAEMA, which were applied through hydroponic solutions. It was expected that this approach would enhance the stability of dsRNA and its uptake by plants, facilitating ingestion by xylem-feeding insects and improving RNAi efficacy. However, visible gene downregulation assessed at 8 dpi showed a decrease in the corresponding transcript only when naked dsRNA was used. Polyplex solutions (PAEMA-dsRNA) did not improve delivery or RNAi efficacy in the target organism. Further efforts should be made to test different polyplex compositions in terms of the presence of polymers and dsRNA, as well as exploring the effect of polyplexes on insect and plant material separately. Overall, the results demonstrate that RNAi is functional and effective in Philaenus spumarius, identifying V-ATPase and ATP synthase beta as promising targets for pest control. Although challenges remain for large-scale application, particularly with regard to delivery, this work provides a solid foundation for developing sustainable, targeted strategies to control Philaenus spumarius vector populations and limit the spread of Xylella fastidiosa