HUMAN PRACTICES

Interdisciplinary efforts to achieve sustainability

Understanding and addressing real-world problems using synthetic biology requires a thoughtful and engaging approach. Initially focusing on a localized issue, our project began with the intention to tackle the challenges faced by beekeepers in Estonia. However, through interactions with beekeepers from various regions, it became evident that the Deformed Wing Virus (DWV) is a widespread concern, transcending regional boundaries. Thus, our ambitions grew to encompass a broader scale.

We aim to genetically modify yeast strains to produce specific small interfering RNAs (siRNAs) that can selectively target the messenger RNA (mRNA) of the bee DWV, effectively suppressing viral replication.

Why is this important? Bees are crucial pollinators for many ecosystems and the agricultural sector. Fighting off the viruses that threaten bees is not just an ecological need but an economic one as well. By addressing this prevalent issue, we hope to create a positive impact on both local and global scales.

Throughout our project, we collaborated with both amateur and professional beekeepers, experts in virology, bee biology and RNAi research, as well as specialists from the Agriculture and Food Board of the Republic of Estonia.

We have incorporated the received information, insights and feedback into our project on different stages and parts of the project: from conceptualisation and design of the project to safety, modeling and education. An overview of the human practices is presented in the table below.

Beekeeping Regulations and Legislation

Comprehending the regulatory landscape is as crucial as understanding RNAi mechanisms. Therefore, we got familiar with the relevant legislation in the European Union (EU) and Estonia to ensure that our project is fully compliant with existing policies.

In the EU, national programs for beekeeping are developed every three years. These programs aim to improve the overall conditions for the production and marketing of apiculture products and are partially funded by the EU. The budgets for the national apiculture programs are provisional and need to be approved by the European Commission, and EU countries report annually on the implementation of the national apiculture programs. The EU has also introduced financial provisions to support beekeepers and researchers in adopting and developing innovative and sustainable beekeeping practices (National Apiculture Programmes)

The European Food Safety Authority (EFSA) has an important role in ensuring that healthy bee stocks are maintained in Europe. Bees are covered by the Animal Health Strategy for the EU that regulates animal health standards and sets requirements for the transport of bees between Member States. These requirements are intended to prevent and control a number of bee diseases and pests such as the small hive beetle and the Tropilaelaps mite, which can spread via human-assisted movement of bees and trade in hive products, plants, and fruit. Pesticide residues may be taken up by bees while collecting nectar, pollen and water. A regulation was adopted in 2009. The new regulation (11/07/2009) concerning plant protection products on the market aims to ensure a high level of protection of both human and animal health and the environment (Duan et al., 2022).

The Estonian government supports the integration of modern practices with traditional rural crafts. This dual approach not only preserves the rich heritage associated with beekeeping in Estonia but also ensures that the sector's growth benefits from modern scientific advancements. The Estonian government also operates special grants and training programs, aimed at educating beekeepers about sustainable practices, the importance of biodiversity, and the ecological balance that bees are maintaining (Could Estonia Become the next Buzzword in Digital Beekeeping? - E-Estonia, n.d.).

However, challenges for the bee populations persist. Some farmers still use chemicals that are not safe for bees, threatening the sustainable pollination of crops and wild plants (Khalifa et al., 2021). If our team would be the policy makers, we would create a policy that regulates the use of pesticides that have not been shown to be safe for bees and sets a requirement to spray them only when bees are less active, either early in the morning or late at night (Why Bees Are Essential to People and Planet). This policy would help safeguard bees and their habitats, supporting the sustainability of agriculture and the conservation of biodiversity.

Driven by the importance of bees in ecosystems and the threats they are facing, both the EU and Estonia have demonstrated commitment through their legislative actions. Collaborative initiatives between the two, like research funding, knowledge sharing sessions, and cross-border training programs for beekeeping, are testament to their dedication towards ensuring the health and longevity of bee populations.

Stakeholders

As beekeepers and agriculturalists are most directly impacted by our project, we have prioritized their needs and insights in our project. We had discussions with 10 beekeepers. We also reached out to the governmental authorities to gain an overview of beekeeping regulations in Estonia. The summaries and outcome of these discussions is presented below.

Beekeepers

Viljar Vahur - Estonia
Viljar Vahur's bee colonies, to his knowledge, are free from DWV. However, mite infestation is an issue. He cleans the hives twice a year - in autumn and spring, bracketing the beekeeping season. If mites appear during the active season, he feels constrained as the use of pesticides is banned. Instead of yeast, he provides sugar to the bees during spring. Although skeptical about GMOs, he acknowledges that the rapid pace of technological advancement might make GMOs ubiquitous in a decade. He expressed interest in the proposed project and would consider its adoption once its safety is confirmed. While DWV doesn't impact his modest beekeeping business, he highlighted that mites are a widespread concern in Estonia.

Augusts Liskins - Latvia
Augusts Liskins identifies the Varroa mite as the primary issue in beekeeping. Typically, he treats it using insecticides Bipin and Varostop after honey harvesting at the end of summer. This treatment usually lasts until the next autumn. If mites appear in large numbers during the summer, he places onion, garlic, or bog myrtle inside the hive. Augusts has not noticed signs of DWV in his bee colonies, although he is aware of this disease. He does not give yeast to his bees as they have ample bee bread (perga) in his region. Augusts is skeptical about making significant financial investments in beekeeping. He keeps five hives as for him it is not a business. There is plentiful production of honey in his region, making honey there cheap, priced at 8 euros per kg.

Kandtacienka Genadz - Belarus
Kandtacienka Genadz has noticed signs of wing deformation in his bees, characterized by their inability to fly, but the prevalence remains relatively low, within 3-5%. Currently, he does not take any specific measures against this issue. However, mite infestation is almost universal, with nearly all of the bees affected. To combat this, he uses fumigation treatments, initially with fumizan, followed by Bipin. He does not introduce yeast into the bees' diet. While there's interest in novel solutions to address these challenges, Kandtacienka remains cautious about using genetically modified yeasts due to concerns over potential irreversible negative consequences. Despite these reservations, he is open to investing in new solutions for ensuring the health of his bee colonies.

Mykola Babalyk - Ukraine
Mykola Babalyk regularly confronts mite infestations in his bee colonies, though he has been fortunate not to observe any signs of DWV. To address the mites, he administers treatments twice yearly using either Bipin soaked strips or Apivarol fumigation. Further, he occasionally employs a diluted oxalic acid solution, a more labor-intensive but effective measure. In June, he strategically limits the queen's egg-laying to curb mite proliferation amidst the larvae. While keen on maintaining his bees' health, Mykola refrains from adding yeast or other pollen substitutes to their diet and expresses reservations about using genetically modified yeasts, emphasizing the need for comprehensive research to verify their safety and efficacy. He recognizes the detrimental impact of mites on bee productivity and underscores the importance of continuous research and investment in safeguarding bee colonies' health and longevity.

Taras Bilynskyi - Ukraine
Taras Bilynskyi has not identified DWV within his colonies. Interestingly, he finds that Varroa mite infestations peak once every seven years. To manage these mites, Taras employs several strategies: in May and June, he removes drone broods, and by September, he turns to treatments using plates, specifically "Apivaro" this year. Given that honey harvesting extended later this season, he began these treatments in early September. As colder temperatures approach, he applies a mix of Bipin and kerosene in a 1:20 ratio using a smoke gun. Taras told us that he has a sufficient natural pollen source and sees no need for substitutes. It's worth noting that by his observations the presence of the Varroa mite can diminish the productivity of his hives by up to 20%. Despite being skeptical towards genetically modified yeasts, he is open to investing in solutions that guarantee the long-term health and productivity of his bee colonies.

Sergei and Antonina Kamyshevi - Ukraine
Sergei and Antonina have observed DWV and Varroa mite in their bees. Their strategy for combating the mites involves using anti-mite strips between honeycombs, treatments with an insecticide Bipin as well as smoking the hive. Although they have not used pollen substitutes, they are open to trying genetically modified yeasts for virus protection. As hobbyist beekeepers with six hives, they are not planning for major investments but are willing to experiment with new products on a trial basis.

Nataliia Kuchmii - Ukraine
Nataliia Kuchmii mentions encountering signs of DWV infection primarily when there's excessive mite infestation. However, within her bee colonies, the DWV outbreaks occur sporadically or in particularly weak colonies in spring. She faces the issue of mite infestation annually, especially at the season's end. After honey extraction, she uses "Baiwarol" plates from Bayer with the active ingredient flumethrin. Additionally, when there's no more brood in the colony (around October), she treats them thrice with oxalic acid, each treatment three days apart. She emphasizes the importance of checking for falling mites post-treatment.

She doesn't actively combat the DWV; she believes that if there are no mites and the colonies are strong, things generally run smoothly. She hasn't added anything extra to the bees' diet since they have a good pollen base from early spring. Nataliia recalls facing challenges in the beginning stages of her beekeeping journey due to a lack of knowledge about mite treatments, leading to weakened bee colonies. However, with the current treatment methodology, this problem has been resolved. The primary concern is to ensure timely mite treatment at the season's end. Considering the local prices for bee products in her region, she finds it challenging to invest in new solutions.

Serhii Prylipko - Ukraine
Serhii has observed that 5-10% of his bees show signs of DWV. He acknowledges that the Varroa mite never fully disappears from a hive and without systemic mite control, the mites accumulate until the bee colony dies. He combats mite infections using treatments like flumethrin and amitraz, which cause nerve-paralytic effects for the mite. He believes controlling the mites can also control the DWV, as mites are the primary carriers of the virus. Serhii has previously used yeast as a pollen substitute and is open to genetically modified yeast if proven safe. He is also open to investing in new solutions to ensure the health and longevity of his bee colonies, even if they require substantial initial costs.

Valerii Savchak - Ukraine
Valerii Savchak has noted DWV in his bee colonies, with an estimated infection rate between 1% to 1.5%. He confronts mite infestations annually, resorting to chemical treatments like specialized strips and sprays. To address DWV, his focus is on combating the mites, using chemical agents such as amitraz, tactic, and flumethrin. Although he hasn't supplemented his bees with yeast or other pollen substitutes, Valerii views the potential of genetically modified yeast positively, especially if they can offer protection from viruses and are deemed safe. He noted that mite infestations have weakened his colonies and in some years, reduced honey production by up to 50%. However, he remains hesitant to invest in high-cost innovative solutions.

Serhiy Vereshko - Ukraine
Serhiy Vereshko employs meticulous and nature-oriented practices in beekeeping, specifically focusing on mitigating the DWV and mite infestations, which he particularly notices when relocating bee colonies for foraging purposes. Traditional, herb-based remedies, such as spraying a tincture of greater celandine, are favored, witnessing not only a halt in wing deterioration but also a counteraction to brood diseases. In an ongoing battle against persistent mite infestations, Serhiy prioritizes strengthening the bees' natural immunity by feeding them various herb-infused syrups and employing different tinctures and organic substances, like amitraz and flumethrin, to keep mite populations in check. Despite the clear challenges, he emphasizes preventive care, ensuring bee colonies are robust and healthy by merging weaker ones and utilizing natural remedies, avoiding artificial additives or externally-sourced products due to the risk of transmitting diseases and impacting both bee and human health.

Conclusions from beekeepers

We interviewed 10 beekeepers, and 6 out of them from 4 countries reported that they have encountered bees infected with DWV.

All of the beekeepers recognized the Varroa mite as a key concern in beekeeping. They employ different methods to combat these mites. Some use chemical treatments like Bipin, flumethrin, etc., and some also employ natural remedies and hive smoking for mite control.

Regarding nutrition, we encountered a mix of reliance on natural resources like bee bread and the use of yeast as a pollen substitute. Some of the beekeepers are open to experimenting with genetically modified yeasts for virus protection.

While some beekeepers view it primarily as a hobby, maintaining a few hives and being cautious about large investments, others appear more commercially inclined, willing to invest in the health, longevity and productivity of their colonies.

Governmental authorities

Kärt Jaarma:
As a lead specialist in bee health in the Agriculture and Food Board of the Republic of Estonia, Ms. Jaarma enlightened us on the challenges bees face due to human actions, especially in suburban areas.

• Many suburban families misuse pesticides and herbicides, often without thoroughly reading the instructions, leading to substantial harm to the local insect populations. This harms not only pests, but all insects who get exposed to the harmful chemicals. This leads to a massive decline in honey bees and other wild pollinators. Ms. Jaarma stated that Estonia is banning one of the popular insecticides, Fipronil, sale (from 29.03.2024) and use (from 27.09.2024) to overcome this issue.
• She pointed out the importance of encouraging individuals to plant flowering plants that can offer dual benefits of aesthetic appeal and ecological significance, and provide a source of food for pollinating plants.
• Structured Gardening: Teaching the public about structuring gardens so that plants bloom in different seasons can provide a consistent food source for bees. Our proposed board game will incorporate this idea, offering players the knowledge of bee-friendly gardening. We discussed this idea with Ms. Jaarma as well and she approved it.

Hagbard Räis:
Mr. Räis's expertise in bee diseases in Estonia provided crucial insights into the prevailing conditions affecting bees.

• He confirmed that over 90% of beehives are affected by both Varroa mites and DWV, highlighting the urgency and significance of our project. Our project focuses on innovative methods to tackle DWV while considering the pervasive Varroa mite issue.
• Mr. Räis also described that the prevalent practice of chemically treating beehives against mites at the season's start and end has detrimental impacts on bees. Our synthetic biology approach aims to reduce the dependence on such chemical treatments by providing an alternative, ensuring a healthier bee population.

Conclusions from governmental authorities

Together, the insights from Ms. Jaarma and Mr. Räis have reaffirmed our project's direction and value. Their feedback has provided actionable steps to ensure the health and longevity of our bee populations.

Both Ms. jaarma and Mr. Räis elucidated the issues connected to bee population and health in Estonia. They confirmed that DWV and Varroa mites widespread in Estonia, provided useful insights on how pests and diseases are managed. They pointed out how regular people can help sustain bee populations and improve

Overall conclusion from both beekeepers and governmental authorities.

Both beekeepers and governmental authorities agreed that varroa mites and transmitted by them diseases are quite spread and cause bees’ deaths and colony collapses. Current pest management techniques are only partially effective and may negatively affect the bees themself. Therefore, new, more efficient methods to treat bee infectious diseases could be in high demand. This reassures the importance of the project our team has undertaken this season.

Experts

We contacted experts in the field to get advised regarding our project design, concept, and modeling.

Design and concept

Dr. Eva Zusinaite
a virologist in the University of Tartu, advised us on RNAi and suggested using model systems to avoid working with infectious viruses.

• She advised to design shRNAs that target the 5’ untranslated region of the DWV genome, as these would inhibit both translation and replication of the DWV. She also recommended checking the secondary structure of the viral RNA genome to ensure that our shRNAs target single-stranded RNA sequences and not a double-stranded stem.
• Dr. Zusinaite emphasized the importance of minimizing off-target effects and suggested that our approach adhered to Biosafety Level 1 regulations. She proposed using viral sequences of 21-22 nucleotides and integrating them into a reporter system that would allow testing for RNAi in a safe model organism. This is to ensure that we do not work with the functional viral genome, maintaining project safety. Incorporating the suggestions from Dr. Zusinaite, we developed a method to evaluate siRNA activities in yeast. By introducing the AGO1 and DCR1 genes into S. cerevisiae, we enabled siRNA processing and RNAi. We also created a new RNAi efficiency indicator by introducing siRNA target sequences into a transcript that encodes for GFP, providing real-time visualization and quantification of RNAi. This strategy is both efficient in terms of cost and labor and avoids the ethical issues tied to animal testing and working with viruses. Check the measurements page on our wiki.

While Professor Jeffrey E. Barrick acknowledged that he is not an expert specifically on DWV, his background in controlling the evolution of genetically engineered cells provided valuable insights and references to our project.

• He highlighted that the exact mechanism of dsRNA uptake by bee cells and its propagation to result in a systemic response is not yet understood. However, he noted that the role of SID1 in gut uptake is crucial. Keeping this in mind, we can conduct studies to explore the role of SID1 in the gut uptake of dsRNA, possibly involving experiments that gauge the efficiency of RNAi in the absence or reduced expression of SID1.
• Once inside the bee cells, the dsRNA gets processed into siRNAs. These siRNAs, when bound to RISC, can target and cleave any complementary viral RNA in the cell. RNA-dependent RNA polymerases are believed to play a role in replicating these sequences and this can drive the propagation of siRNAs to other bee cells. Based on this insight, we can optimize our genetic modifications to produce dsRNA that can be efficiently processed into siRNAs.
• He emphasized that while most adult bees are infected by DWV, maintaining low levels of the virus is vital to prevent detrimental infections in larvae. Monitoring viral loads in adult bees should be a part of assessment protocol when testing our siRNA yeast on bees.

As our primary focus is to utilize RNAi to shield bees from the Deformed Wing Virus, Dr. Ines Anna Drinnenberg's research provided key insights to our work.

• Our strategy to employ S. cerevisiae to produce dsRNA precursors for RNAi required deeper insights. Since these yeasts are used as food supplements and lack a native RNAi system, they presented an ideal candidate for our experiments. Dr. Drinnenberg's paper titled "RNAi in Budding Yeast" served as a resource that drove us to test the potency of our designed siRNAs in yeast.
• In response to our inquiry, Dr. Drinnenberg generously shared her research, revealing how she achieved RNAi-mediated silencing of GFP and URA3 in S. cerevisiae. She elaborated on the cloning of hairpin constructs and provided specific lengths of sequences they used. Importantly, she provided more details on the sequences of the plasmids they used.

Design Experts Summary

Our team contacted experts in the field already at the early stages of the project and incorporated the received feedback into project design. We learnt how to create working RNAi in S. cerevisiae , which parts of the viral genome are best to target, how to minimize safety hazards while targeting viral mRNA and keep the lab work on the Biosafety level 1. Without their invaluable advice, our project wouldn’t be the same.

Modeling

Dr. Jay Evans from the United States Department of Agriculture has extensively researched the interaction between bees and resident yeast in their guts, and the implications of this on immunity, microbiota, and Nosema disease.

• He explained that the primary mode of Deformed Wing Virus propagation is through the bite of parasitic mites. This understanding helps in comprehending the initial stages of infection.
• Dr. Evans stressed that there is still much to discover about how dsRNA spreads within bees after it enters their digestive system. He noted that some of it gets absorbed by midgut cells, but the exact mechanism of RNA propagation remains uncertain. Based on this advice, we prioritize further research into the movement and amplification mechanisms of dsRNA within bee tissues.
• The defensive mechanism against the virus using dsRNA in bees appears to be systemic. While the exact amplification methods of these triggers are not clear in bees, Dr. Evans suggested looking into similar mechanisms in the beetle Tribolium. Investigating the siRNA action in Tribolium might provide insights that could be applicable to bees and that could be used to optimize our dsRNA-based defense mechanism.

Dr. Zhanna Khuda, an associate professor at Dnipro State Technical University, provided invaluable expertise into our models on dsRNA consumption by bees. The understanding and modeling of how dsRNA operates within bees is critical to our project.

• Once dsRNA enters the bees' midgut, the final concentration of dsRNA in bee cells that act as a protective measure against the virus is governed by two simultaneous processes: degradation of dsRNA by RNAses and absorption of dsRNA directly into bees' cells mediated by the SID1SID2 complex.
• For a more comprehensive understanding, we listed four mathematical equations representing these two processes. The primary challenge was to integrate these equations to arrive at an equation reflecting the final dsRNA concentration in bee cells that would then interact with the Dicer and RISC complex. Dr. Khuda assisted in combining these equations and determining the optimal ratio between these simultaneous processes within the bee's system.

Modeling experts Summary

Experts pointed out the lack of knowledge about dsRNA transmission in bees and advised studying Tribolium beetles for insights into dsRNA-based defenses. And Dr. Zhanna Khuda kindly offered her expertise and helped integrate mathematical equations to determine the final dsRNA concentration in bee cells. These helped us build the mathematical models to predict the results of our experiments.

Overall experts summary

From the early stages of the project, we consulted with different experts in the fields of Virology, bee biology, mathematics, etc. Their feedback helped drastically improve our project and ensured we achieved better results.

We would like to thank all the experts, beekeepers, government representatives, and everyone who helped us make this project possible! THANK YOU!

Inclusive Wiki design

Recognizing the importance of our project's wiki page as a primary source of information, we have taken proactive steps to ensure that it is user-friendly for everyone, including people with disabilities. Adhering to the globally acknowledged WCAG (Web Content Accessibility Guidelines) set forth by the Web Accessibility Initiative, we tailored our wiki page to meet many of the defined 'A' and 'AA' criteria.

From the 'A' criteria, we've incorporated features like user-controllable animations and consistent page titles. We've ensured our site doesn't rely solely on color for information, avoided rapid flashing elements, and provided subtitles for all spoken content in our videos. From the 'AA' benchmarks, our wiki is adaptable to various screen sizes, employs high text-to-background contrast, offers uniform navigation, and avoids timed access restrictions. We've further enhanced user experience by integrating a dedicated accessibility toolbar, conveniently located at the top right of our page. This toolbar provides:

• Text-to-speech conversion.
• Animation controls for those needing a stable visual environment.
• A dyslexia-friendly reading mode featuring the clear Comic Sans font.
• Font resizing options for better readability.