DESCRIPTION

There are numerous factors that influence the well-being and proliferation of bees. FAO reports that intensive farming practices, mono-cropping, excessive use of agricultural chemicals, and higher temperatures associated with climate change have a detrimental impact on bee populations (FAO - News Article: Declining Bee Populations Pose Threat to Global Food Security and Nutrition, 2022; 'FAO Publications Catalogue 2022', 2022). Moreover, different pathogens, viruses, pests, and parasites pose a significant threat to honey bee populations worldwide (Ellis & Munn, 2005). These beehive diseases follow the trends of globalization and disease previously reported in one part of the world is now being found in other regions and even on other continents (Ellis & Munn, 2005).

Several actions have been taken to combat the decline in bee and other pollinator populations. For instance, the European Union has established a partial ban on three insecticides to protect the bees (EU regulation No 485/2013; ‘Conclusion on the Peer Review of the Pesticide Risk Assessment for Bees for the Active Substance Clothianidin’, 2013; EUR-Lex - 32013R0485 - EN - EUR-Lex, 2013).

The United Nations also declared the 20th of May the World Bee Day and has provided a set of recommendations on how individuals can contribute to bee conservation (United Nations, 2023):

Emerging infectious diseases have been identified as one of the primary factors contributing to the decline in bee population (McMenamin et al., 2016). More than 20 viruses have been documented infecting Western honey bees and other managed and wild bee species (McMenamin & Genersch, 2015).

Among these viruses, one of the most prevalent is the

(Tehel et al., 2016)

Adding another layer of complexity, there is an interplay between viruses and other parasites. Investigating these relationships is of utmost importance, as the cooperation between viruses and parasites can have devastating consequences. For instance, Varroa mites, external parasites that infest honey bee colonies, have been found to interact synergistically with DWV. This synergy occurs when Varroa mites feed on bee pupae, inadvertently transmitting DWV in the process. Furthermore, the mites weaken the bees' immune system, making them more vulnerable to DWV infections. As a result, the combination of Varroa mite infestations and DWV can exert a particularly harmful impact on bee colonies, leading to colony collapse disorder and posing a significant threat to pollinator populations and agricultural ecosystems. Understanding these intricate interactions is crucial for developing effective strategies to protect bee populations and ensure the pollination services they provide for agriculture and the environment (Tehel et al., 2016).

Although pesticides are often a threat for bees, Barbara et al. demonstrated that treatment of beehives with pesticide tau-fluvalinate against Varroa mites has proven to decrease the DWV cases to some extent (Barbara et al., 2012). Even though this finding could be implemented to manage DWV infections, the existing methods do not provide sufficient results to stop DWV-driven decline of bee populations. Moreover, the use of insecticides in beehives negatively impacts not only varroa mites and other pests but also the overall health of the bees. Therefore, there is a need for innovative approaches to safeguard bee populations.

Bees use RNA interference to defend themselves against RNA viruses

RNA interference (RNAi) is the process where small RNA molecules, typically 21-22 nucleotides long, suppress the expression of specific genes by breaking down the target gene mRNA, preventing it from being translated into proteins. There are several RNAi pathways that perform distinct biological functions and involve different proteins. The short interfering RNA (siRNA) pathway triggered by the double-stranded RNA plays an important role in viral defense in invertebrates and, in particular, in honey bees. Although the detailed mechanism of the siRNA pathway in honey bees is not fully characterized, the main components are known.

In case of viral infection, the siRNA pathway is initiated by the cytosolic double-stranded RNA (dsRNA) produced as a result of replication of the viral genome.

Our proposed solution

In our project, we use yeast Saccharomyces cerevisiae to assist bees in

fighting the viral infections

Our strategy involves modifying yeast cells to produce short interfering RNAs (siRNAs), which can block the expression of bee pathogen genes (Fig. 1). S. cerevisiae is commonly used as a food supplement for bees (Pozo et al., 2020). We envision that the antiviral siRNA-producing yeast, in addition to providing nutrients, can equip bees with the tools to defend themselves against viruses. This helps accumulate siRNAs in bee cells, where they are taken up by the bee's native RNAi machinery, and ultimately repress the expression of viral proteins. Consequently, bees would be better prepared to combat viruses, possessing siRNAs for a robust RNAi response even before encountering the virus. As S. cerevisiae lacks native RNAi machinery and does not rely on RNAi to manage its physiology, this yeast is a good host for production of small RNAs.

Figure 1. Production of anti-viral siRNAs in yeast to supplement bees with the optimal tools to fight the Deformed Wing Virus. First, the viral genome is analyzed computationally to find the optimal siRNA sequences (1). These sequences are expressed in yeast as shRNAs (2), where they are processed by a non-native Dicer enzyme to siRNAs (3). The siRNA-containing yeast extract is used as a food supplement for the bees, leading to accumulation of these siRNAs in the bee cell, where they direct the bee’s RNAi machinery to suppress the viral RNAs.

To evaluate the efficacy and potency of our siRNAs, we propose an experimental approach where we can evaluate and optimize the siRNAs in yeast cells before implementing them in bees. Since S. cerevisiae lacks the RNAi machinery, we express the necessary components of the RNAi system (Dicer and Argonaute proteins) from a closely related yeast, Saccharomyces castellii, in S. cerevisiae cells. This engineered yeast strain can enzymatically cleave shRNA into siRNA molecules. To facilitate a quantitative readout of the efficiency of siRNAs against DWV, we introduce the viral target sequences in the same transcript as the green fluorescent protein (GFP) coding sequence. This allows us to evaluate the efficiency of our designed shRNAs by measuring the changes in GFP fluorescence intensity (Drinnenberg et al., 2009). By utilizing this system, we aim to assess the degree of inhibition exhibited by the siRNAs against complementary viral RNA derived from DWV genome sequence. The system can be used for search of the best siRNA candidates against DWV, and, potentially, can be expanded for the screening of efficient target sequences from other viral pathogens.