Cacace, Alfonso (2025) dsRNA production and delivery system based on Lactobacillus engineering: a new tool for controlling the honey bee mite Varroa destructor. [Tesi di dottorato]

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Abstract

This thesis presents the development and validation of an innovative RNA interference (RNAi)-based strategy for the control of Varroa destructor, one of the most damaging parasites affecting Apis mellifera colonies worldwide (Noël et al., 2020; Traynor et al., 2020; Roth et al., 2020). Through a multi-phased experimental approach, key biological and technological challenges that have so far limited the practical implementation of RNAi in apicultural settings were addressed. A transcriptomic analysis of Varroa destructor salivary glands provided a detailed overview of the molecular components potentially involved in host manipulation and nutrient acquisition. Among the 54 differentially expressed transcripts, a set of genes encoding secreted proteins with putative hydrolytic, immunomodulatory, and structural functions were identified, along with several long non-coding RNAs that may contribute to the fine regulation of host–parasite interactions. Within this complex transcriptional landscape, three salivary genes, encoding a chitin binding protein (Vd-CHIBIN), a kazal-type serine protease inhibitor, and a papain-like cysteine protease, emerged as promising functional candidates due to their expression profiles and known roles in other parasitic arthropods. RNA interference experiments confirmed the importance of these salivary factors in mite survival. Infact, the direct efficacy of specific double-stranded RNA (dsRNA) molecules was assessed, targeting three different Varroa genes expressed in the salivary glands of Varroa destructor using soaking assays. These experiments confirmed the susceptibility of the parasite to RNAi mediated gene silencing, with significant downregulation observed in key genes involved in parasite survival. Building on this, oral administration experiments where dsRNA was fed to bees prior to infestation with Varroa mites were performed. These assays demonstrated that dsRNA can be indirectly transferred to the mites through the host, resulting in measurable gene silencing effects, albeit with moderate efficiency. To better understand the limitations of this delivery route, the fate of dsRNA molecules within the honey bee host, from ingestion to systemic circulation was investigated. Using quantitative analyses, it was demonstrated that dsRNA is taken up in a dose-dependent manner and reaches the hemolymph, but it undergoes rapid degradation, up to 90% of intact dsRNA is lost within 24 hours, primarily due to nuclease activity. This provided direct evidence that intestinal degradation and limited systemic stability are major bottlenecks to effective RNAi delivery in bees, confirming what has previously been suggested for other insect models. However, a separate experiment demonstrated that RNAi processed products, specifically siRNAs, are present in honey bee hemolymph and retain their biological activity while circulating within the bee’s system. To overcome these biological barriers, a genetically engineered strain of Lactobacillus paracasei capable of expressing a concatenated dsRNA molecule (dsConc) targeting three essential Varroa genes: chitin-binding protein, kazal-type protease inhibitor, and papain-like cysteine protease was developed. The bacteria were heat-inactivated, transforming them into a postbiotic formulation that maintained both the dsRNA cargo and immunomodulatory properties. When administered orally to bees, this system delivered a total of ~11.2 µg of dsRNA over 8 days, leading to strong and specific gene silencing in parasitizing mites: 89% knockdown of chitin-binding, 79% for kazal, and 79% for papain, compared to controls. Importantly, this postbiotic delivery strategy not only facilitated dsRNA-mediated gene silencing, but also induced immune responses in the bee host, as evidenced by increased expression of antimicrobial peptides (AMPs) such as hymenoptaecin, defensin, abaecin, and apidaecin. In addition, a significant reduction in Deformed Wing Virus (DWV) titers was observed in treated mites, suggesting a broader beneficial effect on the bee-parasite-virus triad. Collectively, this work provides a comprehensive demonstration of a dual-action strategy: targeting Varroa at the genetic level via RNAi, while simultaneously enhancing bee immunity through postbiotic stimulation. By quantifying the obstacles to RNAi efficacy, designing a microbial dsRNA expression platform, and confirming both gene silencing and immune activation in vivo, this research fills critical gaps in the development of RNAi-based biocontrol in bees. The use of safe, food-grade probiotic bacteria such as L. paracasei, already recognized for their GRAS (Generally Recognized As Safe) status, adds further feasibility and biosafety for real-world applications. These findings pave the way for the development of integrated, biologically based tools for sustainable varroa management, offering an alternative to chemical miticides and supporting the long-term health and resilience of honey bee colonies.

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