Project Description

The Issue and its Relevance

Biodiversity and nature are at risk, threatened by the climate crisis and unsustainable agriculture, to only name two of the most important contributors. Plants provide the foundation of all terrestrial life and most often reproduce through pollination. 90% of wild flowering plants rely on pollinators [1, 2]. The most important animal pollinators are insects, of which bees are the most numerous [3]. They make up half of the world's insect biomass and are responsible and essential for pollinating both wild plants in undisturbed ecosystems and crops in human agriculture [4].

Honey bees are thus considered the third most ecologically valuable domestic animal [2]. This is largely due to their abundance, as Western honey bees are the most commonly managed pollinators, with 81 million hives worldwide [1]. The current decline in honey bee populations has a massive effect on nature worldwide, with significant implications for both ecosystems and food security.

While most honey bee colonies are managed and cared for by humans, there are more than 20,000 species of wild bees [1] that face most of the threats that commercial honey bees face to an even more severe degree, leading to significant declines of wild bees in the US and Europe [1, 5]. Due to their diversity and ubiquity, they have an even greater impact on sustaining our ecosystems. Both wild and honey bees are facing various dangers as the climate crisis and industrialization of agriculture have led to a reduction in diverse flowering areas and an overuse of pesticides [1, 6, 7].

The most substantial threat for both is the spread of the Varroa mite [2, 8-10]. The Varroa mite is an invasive brood parasite, which originally infested Eastern honey bees, Apis cerana. They adapted to the Western honey bee as a host around 1970, spreading over to Western Europe and South America in the 1980s and to the US in the 1990s. In 2022, Australia was the last continent to report Varroa mite infestations [11, 12]. In the US, winter losses in honey bee hives increased from 10% to 30% after the introduction of Varroa mites [13].

Pie chart of the main diseases responsible for colony collapse disorder of bee populations. In decreasing order: Varroosis, American foul brood, Nosemosis, Brood mycosis, Virosis, European foulbrood, other.
Figure 1: Main diseases causing colony mortality reported by beekeepers in 2010, excluding undefined diseases and general problems [10]. AFB: American foulbrood,

Varroa mites weaken entire bee colonies by feeding on the hemolymph of larvae, pupae, and adult bees [2, 9]. Untreated colonies are shown to collapse 2-4 years after the infection [9, 14]. This is mainly due to lack of host-parasite coevolution, as the Western honey bee only recently came into contact with the mites and defensive mechanisms have yet to evolve [9]. These drastic repercussions on individual honey bee colonies also pose existential threats to wild bee populations, as Varroa mites are able to transmit viruses to their hosts [2, 9, 13]. Combined with their suppression of the bee's immune system [2, 9, 13, 15, 16], this leads to highly elevated levels of viruses within a honey bee hive. Although Varroa mites do not infest wild bees, many of these viruses, such as the Deformed Wing Virus (DWV), also infect wild bees [2, 8, 17], rendering Varroa mite infestations a global threat to biodiversity of numerous plants and insects.

As Varroa mites have been a global threat to food security for years, beekeepers adapted and started to take action against the parasite. However, all currently available methods have severe consequences for the honey bees and their products. Due to the lack of suitable treatments of honey bees against Varroa mites, we developed BeeVAX.


BeeVAX - Sustainably Combatting the Varroa Mite

BeeVAX protects Western honey bees (Apis melifera) from entomopathogenic (entomo-: insect-, -pathogenic: -causing disease, entomopathogenic: causing disease to insects) fungi, allowing the fungi to be applied as a natural remedy for Varroa mite infection. These filamentous fungi live parasitically in multiple classes of the phylum Arthropoda and are significantly less costly to develop into a new and commercially accepted pesticide [18]. Studies have shown that numerous species of fungi lead to a substantial reduction in mite populations. The most commonly used species are Metarhizium anisopliae and Beauveria bassiana [14, 18, 19]. B. bassiana is already broadly used as a biopesticide, mostly against insect pests that endanger crop yields [18]. They result in over 90% mite mortality [14]. On a molecular level, they work by having their spores attached to the cuticle of the arthropod host and subsequently form hyphae that penetrate the cuticular layers using hydrolytic enzymes. Once they reach the hemolymph, single-celled structures are formed, which corrupt internal tissues, using up the organism's nutrients and evading the immune system. In this process, various toxic metabolites are secreted that strongly contribute to the fungus's toxicity [18]. Usually, these fungi also gravely affect honey bees, causing exceptionally high mortality rates of 12.5% to 50% [19], and were therefore traditionally not considered a viable option for the treatment of honey bees against Varroa mites.

With BeeVAX we unlock the potential of entomopathogenic fungi as biocontrol agents via trans-generational immune priming (TGIP) against these fungi. This allows their employment for specifically killing just the Varroa mites instead of both the mite and the bee. TGIP is the process through which honey bees produce offspring that carry immune competency against the threats their predecessors encountered [20-22]. As insects lack antibodies, the only way for a queen bee to prime her offspring is by transferring pathogen fragments via certain proteins into the egg yolk. These fragments are then taken up and digested by the developing larvae [20, 21].

The protein that was shown to mediate TGIP in A. mellifera is the egg yolk precursor Vitellogenin [22], which enhances phagocytosis by macrophages, thus preparing the organism for reinfection with the pathogen [23]. Vitellogenin makes up variable portions of the bee's hemolymph throughout their life cycle, with the highest amounts found in bees that perform nursing tasks and in winter bees [13, 24]. If the queen bee is confronted with a high amount of pathogenic cell wall fragments while producing eggs, Vitellogenin takes some of these fragments into the egg yolk, and thus the newly born worker bees begin their lives with an elevated immune response against the pathogens [22].

Vitellogenin binds to pathogen-associated molecular patterns (PAMPs) [20, 22]. These pathogen fragments include cell wall fragments of different types of bacteria and fungi [20]. Honey bee Vitellogenin has been shown to possess the highest binding affinity for peptidoglycan (a signature molecule of Gram-positive bacteria), followed by lipopolysaccharides (signature of Gram-negative bacteria). Zymosan (cell wall signature molecule of fungi) shows the lowest binding affinity to Vitellogenin. This is probably due to adaptation, as the fiercest threats to honey bee health are the Gram-positive bacteria Paenibacillus larvae and Melissococcus plutonius, the causes of American and European foulbrood disease, respectively. Fungi traditionally do not pose a significant threat to social bees, as their hives are too warm for the fungi to effectively grow, and they are not in regular contact with the soil-dwelling fungi hyphae [22].

Western honey bee Vitellogenin consists of four distinct and structurally highly conserved domains. The N-terminal β-barrel domain is a putative transcription factor and is connected to the remaining protein via a cleavable polyserine linker. The α‑helical domain is thought to be the main contributor to PAMP recognition. Nevertheless, studies in fish and coral have shown that the following Domain of Unknown Function (DUF1943) and the von Willebrand factor (vWf) may also contribute to PAMP binding. Therefore, it is possible that Vitellogenin contains more than one PAMP-recognition site. [25]

The binding affinity of honey bee Vitellogenin for zymosan, the main PAMP of fungal cell walls, is very low, which results in low TGIP efficiency against fungi [22]. With BeeVAX, we aimed to develop a modified version of Vitellogenin that has a higher binding affinity to fungi's PAMPs than its natural counterpart. Once the DNA sequence of the optimized protein is determined, modern genetic engineering techniques such as CRISPR/Cas9 could be employed to genetically modify the bee queen, enabling her to produce the optimized Vitellogenin.

Visualization of the engineering prozess of Vitellogenin for a better PAMP binding.
Figure 2: Engineering Vitellogenin for a better PAMP binding.

Methodology

BeeVAX is founded on three key pillars:

  • The mutagenesis of the Vitellogenin binding site
  • Demonstrating improved binding affinity to fungal PAMPs, specifically zymosan
  • Enabling the expression of Vitellogenin for future widespread implementation of our protein engineering system
Figure of three pillars carrying the BeeVAX project. The pillars are expression, mutagenesis and binding affinity.
Figure 3: The three pillars our project is based upon: Expression of Vitellogenin, Mutagenesis and Binding affinity

Directed Evolution

The mutation process involved two complementary approaches. On the one hand, we used bioinformatical tools to predict structural properties of Vitellogenin based on different DNA sequences, as described in greater detail on our Modeling page. Our goal was to predict the structure and binding capacity of chimeric proteins. This investigation led to the development of our side project, CTL14, which aims to create a chimeric protein out of a zymosan-binding protein from a moth fused with the honey bee Vitellogenin. On the other hand, we displayed selected domains of Vitellogenin on yeast and tested their binding capacity experimentally. These domains underwent mutation via error-prone PCR to assess their ability to bind the zymosan in comparison to the wild-type Vitellogenin. From that, we compiled a yeast library with distinctly mutated variants of Vitellogenin.

Overview of the directed evolution approach. A yeast cell is bound to Vitellogenin. The Vitellogenin binds a green-fluorescent labeled PAMP. The Vitellogenin is connected to a red-fluorescent labeled myc-tag. Next to the construct is a fluorescent-activated cell sorting device, sorting out the cells with highest fluorescens.
Figure 4: Our construct to conduct directed evolution via fluorescent-activated cell sorting. PAMP: pathogen-associated molecular pattern, myc: antibody binding site, FACS: fluorescent-activated cell sorting

Binding Assays

We tested the binding affinity of generated Vitellogenin variants by two distinct means: For quick evaluation of the binding properties of wild-type Vitellogenin and our mutated variants, we made use of fluorescent-activated cell sorting (FACS). A FACS instrument analyzes the fluorescence intensity of single cells and separates them based on predefined parameters. We induced the display of our selected Vitellogenin domains on the surface of Saccharomyces cerevisiae. Then, we applied zymosan as our fungal PAMP. Both Vitellogenin and zymosan were tagged with two different fluorescence markers, which could be detected by FACS. Based on the ratio of displayed Vitellogenin to bound zymosan, we calculated the binding affinity of each Vitellogenin variant.

Although the FACS measurement is very precise, it relies on successful yeast surface display of the protein. Moreover, the instrument needed is expensive and not accessible for everyone. To offer an alternative binding assay independent of protein display and accessible to other research groups, we designed a second measurement method.

Fluorescence-labeled zymosan monomers are immobilized on a 96-well plate in known quantities. Subsequently, free Vitellogenin is applied. The Vitellogenin is linked to an antibody-conjugate carrying horseradish peroxidase (HRP-conjugate). When the Vitellogenin successfully binds to the zymosan on the plate, it will not be removed by the following washing procedure. The HRP will cleave a signal substrate, generating a specific luminescence. The fluorescence intensity, relative to the quantity of bound zymosan, indicates the Vitellogenin's binding affinity. You can find a more detailed description of our binding assays on our Measurement page.

Overview of the binding assay approach. Poly-PAMPs are hydrolyzed and marked with a fluorescent marker. The Oligo PAMPs are bound to a plate and bound to a Vitellogenin with a His-Tag and marked with an Anti-His-Tag. This binds a HRP-conjugate, which upon activation shows a fluorescent signal. A well plate with fluorescence in varying intensities is shown.
Figure 5: Mode of operation of our developed binding assay. Pathogen-associated molecular patterns are fluorescence-labeled and fixated on the bottom of a 96-well plate. His-tagged Vitellogenin binds to those PAMPs and the horseradish peroxidase-conjugate alters a signal substrate. The product of this is fluorescent. PAMP: pathogen-associated molecular pattern, Vg: Vitellogenin, His: Histidin-tag, needed to bind Anti-His-tag, HRP: horseradish peroxidase

Expression of Vitellogenin

Lastly, we attempted to obtain free Vitellogenin. We designed and cloned a vector that carried the Vitellogenin DNA sequence in Escherichia coli. Subsequently, the vector would be inserted into the yeast Komagataella phaffii, formerly known as Pichia pastoris. K. phaffii, being an eukaryotic organism, possesses the capacity to transcribe, translate, modify, and fold Vitellogenin into its native conformation. This process would allow us to purify the protein for various applications. Additionally, we constructed a shuttle vector for Komagataella phaffii based on a plasmid provided by the former iGEM team from Münster. This vector is available for use by other iGEM teams.


Outlook

Our project BeeVAX is a helpful contribution to the fight against bee decline worldwide and the resulting threats to nature and biodiversity. Immunization of honey bees by BeeVAX opens two major advantages: First, it allows us to protect domesticated honey bees from the Varroa mite, as described previously. Secondly, this leads to a reduction in the transmission of viruses onto wild bees due to the reduced number of Varroa mites. This fact represents an essential component in the effort to preserve biodiversity.

Moving forward, our measurement protocols provide a reliable and adaptable platform for protein engineering. For instance, it will be possible to immunize the honey bee not only against fungal infestations but also against other pathogenic microbes using PAMPs from other species. Our optimized protein can also be used to combat other entomopathogenic fungi infestations. While B. bassiana and M. anisoplae typically do not pose a significant threat to honey bees, multiple species of the genus Nosema, mainly Nosema ceranae and Nosema apis were determined to be important contributors to Colony Collapse Disorder [8, 16].

This entire process of Vitellogenin optimization can be applied to other organisms as well, since honey bees are not the only insects that possess Vitellogenin. All oviparous animals produce Vitellogenin as an egg yolk protein [26]. Many insect species even have several homologous genes [22], rendering future research on Vitellogenin a vital resource in the fight against the decline of threatened arthropods. It could also be used to develop a new approach to combating insect pests. By editing the Vitellogenin of insect pests to transfer fewer immune elicitors, it could serve as a means to weaken these populations and make them more susceptible to pest control measurements.

In summary, our research on Vitellogenin of honey bees represents a cross-species possibility of immunization or sensitization of species against various pathogens, which will become increasingly relevant in the future. It has the potential to contribute significantly to species conservation and biodiversity in Europe and around the world.


Additional Background Information


Impact of Bees on World Hunger and Economy

Global hunger is on an unstoppable trajectory, claiming more lives each year. If current trends persist, the world will not only fail to reach the UN Sustainable Development Goal of “Zero Hunger” by 2030, but the number of people experiencing hunger will exceed 840 million [4]. Because most of the calories consumed worldwide come from pollinated plants, the Western honey bee, A. mellifera, is arguably one of the most crucial animals for food security in the future.

Globally, plant species dependent on insect pollination range from various fruits to vegetables, seeds, nuts, and oil crops [1]. Together, they account for 35% of global food production [1, 8, 19] and contain the majority of essential vitamins, minerals, and micronutrients in human diets. The absence of bees as pollinators would cause entire industries to collapse in both developing and developed countries, leaving millions without a source of income and food [1]. The global monetary value of pollination was estimated at up to $650 billion annually [19], surpassing the annual revenue of every company on earth [27]. Moreover, bees provide direct monetary value in the form of their production of wax, honey, tallow, bee bread, royal jelly, and propolis [19].


Current Treatment Methods

Current mite combat methods often make use of broad-spectrum, low-specificity chemical pesticides and miticides, or organic acids [14, 19, 28]. Both treatment chemicals pose a significant threat to bees too, as they are also sensitive to the toxic effects of the treatment due to their biological similarity to mites [7, 16, 28]. The plans to combat the mites all contain monitoring to assess the infestation level of the colony. Most commonly used methods kill around 300 bees per sample and colony, up to four times per year [28, 29]. After Varroa infestation levels exceed a certain threshold, mite control is applied [28]. Various studies have shown that mite combatting drastically improves the survival chances of infested hives [9, 13, 16].

Many of the applied chemicals are also lipophilic and have been shown to build up in the wax, exposing the whole hive to their toxic effects even after the treatment has ended [13, 16]. In addition to the negative impact on population health, this poses the risk of chemical residues in bee products [9, 13, 14] which is why effective treatment is limited to seasons in which bees do not produce honey [15, 29]. Leftover residue in bee products poses the risk of having negative impacts on human health as well.

Over the course of the last decades, increasing evidence has been collected stating the development of resistances against widely used pesticides in Varroa destructor [13, 14]. This alarming adaptation is thought to be accelerated by the pesticide residues in the wax and by the oftentimes suboptimal doses of pesticides. The pesticides are applied in limited amounts to spare more bees but therefore sometimes fail to eradicate the mites, which are then able to develop resistance. As a result, larger amounts and more aggressive pesticides need to be applied, which leads to a vicious circle of escalation.


Potential Applications

To generate an elevated immune response against fungi, our improved Vitellogenin needs to be applied correctly. Thus, we naturally engaged in finding potential application systems that allow beekeepers all over the world to use our groundbreaking work to protect their bees and therefore, biodiversity and natural resources.

The best approach to address the application of Vitellogenin was to build upon the usual exchange in the colony: worker bees excrete it into the royal jelly, the sole food source of the queen bee [9]. As the queen bee does not leave the hive under normal circumstances, this royal jelly is the only way she gets in contact with pathogen fragments presented by the Vitellogenin of the worker bee. Our plan involves producing an altered version of the royal jelly that contains zymosan as a PAMP of the entomopathogenic fungi. This altered royal jelly acts as an oral vaccination for the queen bee. For that, we developed three distinct ways to get the improved Vitellogenin into the queen bee's hemolymph.

The first approach would be to encapsulate our Vitellogenin in nanoparticles resistant to the bee's midgut digestion that slowly release the protein into the hemolymph. Nanoparticles can be added to the royal jelly if the production of resistant workers is desirable.

The second approach involves creating genetically modified bees that carry our optimized Vitellogenin in addition to the naturally occurring version. This approach would require less maintenance work; however, the implementation of genetically modified bees into our environment needs to be carefully weighed both ethically and legally.

A third but less desirable solution would be to physically inject the queen bee with a syringe. This is a very invasive treatment and can cause high stress levels to the queen bee, making this alternative the least preferred. During our extensive integrated human practices we came into contact with the iGEM team of the University of British Columbia. They were able to educate us on the possibility of killing the bee during the injection, if not done with the appropriate level of expertise and caution. However, this could be a viable method to do a first test run before adopting a combat plan that can be used on broader scales.

An annual treatment plan could be as follows: monitoring the amount of Varroa infestation is crucial to determine if interventions are needed. Regular monitoring is necessary, as mite population growth is currently impossible to predict reliably [9]. We recommend using monitoring methods with minimal invasive damage to the bees, such as computer vision systems that detect Varroa mites on worker bees [30], as traditionally used methods tend to claim the lives of many worker bees [13, 28], which are essential for the colony's survival.

An annual cycle is shown in four pictures, each representing one of the four seasons. The following is written next to the seasons: Spring: “New, short-lived bees are born into the hive, honey gathering season”; Summer: ”Feeding the queen bee with the altered Vitellogenin”; Fall: “ Introducing fungus spores into the hive”; Winter:” Over the winter, the fungus can eliminate the Varroa mite”
Figure 6: Proposed annual treatment cycle: In summer, PAMPs are fed to the queen bee, so that she produces immune offspring. In autumn, fungal spores can be introduced to the hive to eliminate the Varroa mites over the winter season. In spring, newly born worker bees are born without losses due to Varroa infestations.

Treatment is preferably only implemented after the honey is removed to not have the bees honey production rate diminished. Bee life spans vary enormously over the course of one year, with summer bees living about two to six weeks and winter bees living about 20 weeks [24]. Winter bees have significantly elevated levels of Vitellogenin in their hemolymph, which helps them to stay healthy for a long time [13, 15, 24], facilitating our treatment. Therefore, we suggest beginning the mite extermination by feeding the queen bee our altered Vitellogenin in late July, so that in mid-September the whole hive consists of immune-primed winter worker bees. If genetically modified bees are used, this treatment is facilitated by feeding free PAMPs to the entire hive, which are then passed on to the queen bee, who in turn primes the next generation of worker bees. Then, in mid- to late-September, spores of the entomopathogenic fungus can be introduced to the hive. The cooler temperatures and immune bees will ensure that the fungus can effectively eliminate up to 100% of the Varroa mites [14] while sparing the bees. If the mite infestation rate exceeds the threshold in the honey gathering season, this treatment could also be implemented, as the fungi are non-toxic and non-allergic.

The fungus can be mass-cultured in solid-state fermentation and poses no threat to non-arthropods. Potential undesirable side effects can also be ruled out, as these fungi are omnipresent and have been tested in honey bee applications multiple times. [14, 18]



How it all started

For this year's iGEM team from Münster, committed and curious students from different disciplines joined forces to successfully participate in the competition. Due to our diverse characters and interests, finding a topic was not particularly easy. Our ideas went in various directions, from vegan milk synthesis in algae to bioleaching. Eventually, we came across the topic of bees and the threat posed by the ongoing decline of bee populations. We discovered that this development is largely driven by the ectoparasite V. destructor, the Varroa mite. Given that many of our team members have connections to beekeepers, this subject held significant personal relevance for us. In addition, it presented an opportunity to combine the fields of ecology and synthetic biology. Thus, the BeeVAX project was born as our contribution to stopping the global decline of bees.

Very early in the developing state of our project, we came across the egg yolk protein Vitellogenin, which is responsible for trans-generational immune priming in Western honey bees. We proposed that to improve bee health, we could feed the queen bee an altered version of the protein with a higher binding affinity to fungal PAMPs. But as we conducted further research, we encountered two major obstacles.

Firstly, the Varroa mite itself, as a complex, comparatively large ectoparasite, is hardly vulnerable to internal immune responses by the Western honey bee. Furthermore, the mites down-regulate the immune system of the bees, so that direct control of the mites by the immune system of the bees is not promising. To bypass this problem, we decided to use an entomopathogenic fungus to combat the mite. Entomopathogenic fungi are natural enemies of the Varroa mite. Nevertheless, they also harm honey bees, resulting in mortality rates of 12.5% to 50% [19]. Therefore, we want to use Vitellogenin to prime the bees against said fungus, generating an indirect treatment option against the Varroa mite as well as a direct one against entomopathogenic fungi.

Another challenge is the introduction of our altered Vitellogenin to the Western honey bee. Simply feeding it to the queen would not result in a significant improvement of the worker bee's immune competency, as the intake of pathogen-associated molecular patterns (PAMPs) in the queen bee's digestive tract is not mediated by Vitellogenin. Instead, the worker bees' Vitellogenin is digested in the queen bee's digestive tract, and only the PAMPs pass through the walls of the digestive tract into the hemolymph, where they bind to the queen bee's own Vitellogenin. We tackled this obstacle by adjusting our suggested treatment method to either feed encapsulated modified Vitellogenin produced in microorganisms or by genetically modifying the queen bee to carry the gene for the improved Vitellogenin.

We are proud to hereby present our contribution to the world's path towards truly sustainable living. BeeVAX: Don't let the buzz become silent.


Promotion Video and Presentations

Promotion Video

Presentation Video

Village Presentation

References

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Results
Human Practices
Notebook