Project Description:

Introduction

It is October, the leaves are turning yellow, and temperatures are dropping. As autumn begins, bees prepare for the winter, but not all will survive to see spring. These tiny, yet mighty, creatures play a pivotal role in our ecosystems by pollinating plants and ensuring our crops bear fruit. However, these unsung heroes face challenges that endanger their survival.

Back in February this year, a few of our iGEMers were excited to visit an apiary in the Okanagan Valley. Planet Bee, was the sign that sat above the front door of this peaceful little shop.

Inside was an array of honey and wax products. After the iGEMers spent some time browsing around they eventually made their way over to the educational side of their establishment. It was on this side that they saw a curious, yet, disturbing image of a mite with the text “KNOW YOUR ENEMY.”

One of the workers noticed the iGEMers and informed them of their honey bee losses by this mite. It seemed that this strange little mite was causing havoc not to just this apiary, but to beekeepers all across the world.

What Happened Next?

Upon further research, the iGEMers learned that varroa infestations cause devastating effects on bee populations, much worse than they originally thought. ^{generating...} This, in turn, reduces beekeepers’ income and can end businesses in the span of a single season. In tandem, these sudden losses in pollinators can negatively affect surrounding ecosystems.

Unfortunately, the continued rise of globalization demands more agriculture, and thus, more pollinators to support them. There is an increase of bees being sold for industrial agriculture all across the globe, and this distribution of bees becomes an ample opportunity for the spread of disease.

A prominent disease spread by varroa mite infestations is the deformed wing virus (DWV). DWV is a single-stranded RNA virus characterized by its effect on bees; shrivelled wings, a shortened abdomen, cognitive impairment, and paralysis of the legs. ^{generating...}

It is particularly dangerous as bees prepare for the winter. Bees begin to forage less and their populations are no longer in flux, those remaining bees become the hope for survival through the cold and harsh winter months. If an outbreak occurs in this remaining population, they may not have enough that survive to replenish the hive in spring. ^{generating...}

Last year in Canada, the Canadian Association of Professional Apiculturists released a statement on the wintering losses.

It was a stagnating 45.5%, nearly half of the hives across the country!

When beekeepers were surveyed, most provinces associated varroa mite infestations as the cause of these losses.

So, why is varroa mite so deadly?

Varroa mites like to take bloodmeals from honey bees. ^{generating...} They stick their long proboscis through the bee’s exoskeleton and pierce directly into their hemolymph. ^{generating...} As they take a drink from the bees, they simultaneously inject deformed wing virus directly into their hemolymph. From there it wreaks havoc on each bee and consequently, the hive dwindling their populations, reducing their income and affecting the surrounding ecosystem. ^{generating...} On the other side, globalization, honey bee distribution, and their ubiquitous nature has made bee diseases spread rapidly and broadly.

One major contributor to why deformed wing virus affects bees so readily is simply due to the fact that their immune systems were not built to protect them against single-stranded viruses. ^{generating...} Specifically, bees’ immune systems are only able to detect and defend against double-stranded viruses, allowing single-stranded viruses - like DWV - to slip under their radar. ^{generating...}

Our Mission

Honey bees are direct competitors of other important native pollinators. In fact, they pose a danger to native bees by decreasing food availability and spreading diseases but decreasing their numbers is an unlikely solution. They fuel businesses and if used correctly can prove beneficial for the environment and agriculture.

We want bees to survive these deadly infections. We considered our options and found that there may be hope in the creation of a vaccine for bees. Orally distributed, easily used by beekeepers, and safe for the hive. Our plan for a vaccine not only saves each individual bee but saves every bee born from a vaccinated queen.

We want to help beekeepers worldwide safeguard their colonies while creating a safer environment for native bee populations.

This is where the ImmuniBee project was born.

The Importance of Bees

Bees are more than just buzzing insects. They are essential pollinators that enable the growth of countless plant species. From fruits and vegetables to wildflowers, they are the diligent workers who make it all possible.

An Ecological Symphony

There are thousands of bee species, each with its own specialization, serving crucial ecological niches. They pollinate specific crops and native plants, playing a unique part in the delicate balance of nature.

The Honey Bee’s Contribution

The Western honey bee (Apis mellifera) stands out as an invaluable contributor to both agriculture and ecosystems. It is a domesticated species ubiquitous around the world. These industrious insects offer a myriad of economic and ecological benefits that enrich our lives.

Economical Value:

1. Honey Production: Honey bees are renowned for their honey production. Honey products, including honey itself and derivatives like beeswax, have been used by humans for centuries. These products have diverse applications, from sweetening our foods to producing beeswax candles, cosmetics, and medicinal salves.

2. Crop Pollination: Perhaps the most well-known contribution of honey bees is their role as pollinators. They facilitate the reproduction of numerous crops, ensuring bountiful harvests of fruits, vegetables, nuts, and even oilseeds. The agricultural industry heavily relies on honey bee pollination, which has been estimated to be worth billions of dollars annually. Bee-pollinated crops include apples, almonds, cherries, and blueberries, among many others. Their diligence in pollinating these crops significantly boosts agricultural productivity, leading to a more abundant and diverse food supply.

3. Biodiversity Support: Honey bees not only assist in agricultural pollination but also play a pivotal role in supporting biodiversity in natural ecosystems. By pollinating native plants, they contribute to the reproduction of a wide variety of wildflowers, shrubs, and trees. This, in turn, sustains other wildlife that depends on these plants for food and habitat. Hence, honey bees are instrumental in maintaining the overall ecological balance of our natural landscapes.

The Bee Predicament

Bees, including the Western honey bee, are grappling with a slew of biological threats, some of which are inadvertently caused by human activities. Climate change affects their behaviour and also the flowers availability during the year; insecticides leach into the environment and poison colonies; monocultures and habitat destruction reduce and alter food availability for bees.

These threats jeopardize not only the bees themselves but also the delicate balance of the ecosystems they sustain.

A recent threat among beekeepers is an increase in pathogens. Most colonies around the world are infected or in high risk of infection by the Varroa mite (Varroa destructor).

This small red ectoparasite feeds on bees’ hemolymph while simultaneously bringing down their immune systems. They hitch a ride on the back of foraging bees and bring the mites back to the hive where the mites reproduce in the cells of honeycomb.

And what is worse, they are vectors for other pathogens like viruses. One being typically associated with the mite called Deformed Wing Virus (DWV).

In tandem, an infestation of Varroa mite and DWV is the major reason for the high mortality. If protected against DWV, bees will have a much greater chance of survival.

Current Solutions

In the case of A. mellifera, traditional interventions against bee diseases and pests include the use of toxic chemicals and straightforward burning the hive to avoid further infections.

These methods are neither practical nor effective. Some can be quite devastating to the environment if is not under control!

One big obstacle is that bees are very small and numerous making them difficult to treath individually. As well as not possessing an immune system like ours to build antibodies to defend against these infections.

We want to create a tool to make this possible!

Fortifying Bee Immunity

Our goal is to defend bees against the DWV which is currently impossible using natural means. Our idea is to use the bee’s innate immune system to our advantage.

Bee Immunity

Bees have evolved natural defences against the mite and virus.

Behavioural defences

Honey bees are able to use hygienic behaviour to physically remove deceased bees and varroa mite from the hive. Hygienic behaviour can help with varroa control in low amounts, however, once an infestation is too high hygienic behaviour is limited.

Molecular defences

Bees have no inherent immune defense against single-stranded RNA viruses. Instead, they are only able to recognize and defend against double-stranded RNA viruses. To defend against double-stranded viruses, bees use siRNAs to label dsRNA viruses and then degrade them. ^{generating...}

To defend against bacteria, bees produce antimicrobial peptides which are activated by the recognition of PAMPs, peptidoglycan in the cell walls of bacteria, and beta-glucans in the cell walls of fungi. ^{generating...} Additionally, the composition of bacterial and fungal cell walls is important for the activation of trans-generational immune priming, a key defense mechanism bees use to pass immunity to their offspring.

Trans-Generational Immune Priming (TGIP)

Even tough bees do not posses an adaptive immune response, a vaccine is available for these organisms. A team of researchers at the university of Helsinki managed to demonstrate adaptable immunity on A. melifera after feeding them deactivated Panelobacilus larvae cells mixed in sugar water. Further studies showed that this immunity got passed from worker to queen and from queen to her offspring!

This was partially explained thanks to Trans-Generational Immune Priming (TGIP). This natural phenomenon occurs when a queen bee’s innate immune response influences the immunity of her entire colony, without direct genetic alteration.

It has been shown that vitellogenin (Vg), a highly conserved multipurpose protein found in bees, has a crucial role in this system.

• A pathogen like bacteria or fungi is ingested

• It finds its way to the haemolymph through the GI tract

• Vg attaches to the pathogen’s cell wall

• The attached bacteria travels around the bee body

• Vg interacts with the worker’s hypopharyngeal glands and gets incorporated into royal jelly

• The bacteria fragments are then consumed by the queen

• The same process repeats but Vg binds to the queen’s ovaries

• The pathogen fragments are then incorporated into the eggs of the next bee generation with increased immunity

This process’ mechanism is not entirely understood passed this point but specific immunity is indeed improved.

What about viruses?

Attempts to replicate this with deactivated viruses were done but there was no effect. This seems to be due to viruses like DWV not having a cell wall containing peptidoglycan.

This will prevent Vg to bind to the virus and eventually create a reservoir in the queen’s ovaries.

Naturally there is no way for bees to evolve this system to work against viruses as it would take too many steps and they already have molecular defences against them but synthetic biology can help us make them more sturdy.

An early hypothesis was that this process primes bee larvae with an immunoelecitors early on heightening their innate immune responses. But specific immunity was observed instead of an overal improved defense showing an specificity mechanism.

Let’s Make a Bee Vaccine!

Our vision is to engineer a bee vaccine against the deformed wing virus by combining TGIP with Synbio. We will use bacteria as a shuttle for viral particles that the bee can recognize.

By genetically modifying Bacillus subtilis, a common generally regarded as safe (GRAS) bacterium, to express a portion of the deformed wing virus capsid, we hope to stimulate an innate immune response in bees. When the queen is exposed to this modified bacterium, she will pass this immunity to all her eggs, ensuring that future generations of bees are protected.

What We Know and What We Do

We’re committed to understanding the bee immune system better and using this knowledge to create a sustainable solution to bee health problems. This research will allow us to understand more about the complexities of bee immunity, other eusocial insects, and perhaps other arthropods which are of incredible importance for our society.

Why synthetic biology?

There are many questions unanswered:

• Does the bacteria travel intact through the hive?
• Can Vg bring with it other membrane associated proteins?
• Does Vg get degraded when delivering its cargo?
• How does immunity specificity get developed?

We do not know this process very well and we believe that synthetic biology is a unique opportunity to answer these questions.

By creating a modular system that allows us to bring any viral protein to the exterior of a vegetative cell we can create a tool to tackle many viruses and perhaps even improve defences against several pathogens. By equipping our modified bacteria with several pathogen particles we can prime bees against many pathogens and reduce the disease reservoirs we created.

Furthermore, this method uses the bees natural immune system without using harsh and costly chemicals. And once this method is developed, it will be a cheap and safe product as growing bacteria is a quick and effortless process compared to organic synthesis of mitesides and other previous methods.

What’s more, by using TGIP we can treat several colonies in an easy delivery system by just feeding workers which will deliver the treatment to their respective colonies. A highly appreciated aspect by commercial honey producers

This will pave the way for helping endangered invertebrate species, develop new epidemiological controls, possibly create an unique and cheap alternative for beekeepers to safeguard their colonies.

Environmental Benefits

Our project extends beyond protecting bee colonies; it benefits the environment at large. By securing bee populations and maintaining the delicate pollination balance, we’re helping ensure the reproduction of countless plant species and preserving biodiversity.

What We Could Achieve

Our ultimate goal is to safeguard the heroes of our ecosystem. By fortifying their defenses against the deformed wing virus, we aim to secure not only the future of beekeeping but also the ecological balance that relies on these tiny, vital pollinators.

Join us in this journey to make our world a safer place for the keepers of nature’s nectar - the honey bees.