Introduction

Honeybees are incredibly important pollinators responsible for the abundance and diversity of our food. Unfortunately, honeybees are in trouble: the fittingly named parasitic mite Varroa destructor (from here on ‘Varroa’ or ‘Varroa mite’)weakens the bees and spreads diseases. iGEM Wageningen UR 2016 aims to save the bees from Varroa.

Honeybees can pollinate an enormous range of plants through active collection of pollen¹, making them important for pollination of crops. Apples, almonds and numerous other crops rely heavily on bee pollination, tying the diversity of our diet to the well-being of these insects¹. Therefore, it is of key importance to keep bees alive and healthy.

Sadly, bees are not doing well at all. There has been a sustained loss of bee colonies in the western world for at least 10 years. In 2014-2015, US beekeepers lost over 40% of their colonies²! Such numbers are unacceptable!

Varroa destructor

What causes the loss of bee colonies? According to beekeepers we have spoken to and as has been suggested in scientific studies^3,4,5, the most important factor is the mite Varroa destructor. Varroa mites feed on haemolymph (‘bee-blood’) weakening the bees, while spreading serious diseases like the deformed wing virus. Beekeepers and bee researchers stress that the first and most important step to save the bees should be to control Varroa more effectively.

Currently in the Netherlands and other European countries, beehives are treated against Varroa with a combination of organic products consisting of thymol, formic acid and oxalic acid. These compounds can contaminate beeswax and honey⁶. More importantly, beekeepers are wary that such compounds can be harmful to bees and humans when the concentration used is too high^7,8. On the other hand, too low concentrations fail to kill Varroa and facilitate resistance⁹. The hobbyist character of beekeepers further adds to the concerns that accompany Varroa treatment. Beekeepers often care for their bees in their spare time and might not have time, resources or the experience to use existing treatments in the intended manner.

Bee T

We propose a Varroa specific treatment through the use of engineered bacteria that releases toxin in a specified and regulated manner. The released toxin is not harmful to bees or humans. To do so, the bacteria will employ a network of regulation mechanisms to produce the toxin only when mites are present and when enough bacteria are present to effectively kill the mite. Other mechanisms are intended to strictly confine the bacteria to the treated hive, preventing them from spreading and mixing with natural ecosystems.

Specificity

No Varroa specific toxin was available when we started. There were however results¹⁰ suggesting that a crystal protein produced by a Bacillus-like strain is able to specifically kill Varroa mites. It proved impossible to acquire the strains that were tested. Therefore we tried to find and create similar crystal protein toxins.

However, testing crystal toxins on Varroa mites proved very problematic. Their parasitic nature makes the mites incredibly vulnerable for laboratory experiments. To facilitate testing of the toxins, we developed a new in vitro method for testing Varroa toxicity. This method involves the use of vesicles created from Varroa mites and release of fluorophores from these vesicles.

In an attempt to create a Varroa specific toxin, we modified an existing crystal toxin to specifically target the mites. Additionally we tried to actually create a protein domain able to bind Varroa gut cells. This domain could then be used to make existing protein toxins target Varroa mites.

To improve our chances of getting a working Varroa toxin, we tried to identify other bacteria that naturally produces a useful crystal protein. Our efforts yielded one interesting candidate which was sequenced to identify any potential toxins. To be able to identify the protein responsible for killing Varroa, we constructed a toxin scanner.

Regulation

As constant low levels of toxin facilitate resistances¹¹, we wanted to only produce toxic proteins when Varroa mites are near the bacteria. Therefore we created and tested two systems where protein expression is dependent on the presence of mites. These systems are based on guanine and B12, indicators of Varroa mite presence.

Another measure to prevent low concentrations of toxin was to only produce toxin when enough bacteria are present. This minimizes effects of the toxin on bacterial growth when the density of bacteria is low. To further increase the growth and survival of BeeT, we designed a system to sustain a subpopulation of non-producers. Both systems were modelled to assess their functioning and how they would combine.

Bacteria might not be able to grow very well in beehives. Therefore, regulation based on bacterial density may not be the best approach for toxin expression. Bacteria may not even be able to grow to the density required for toxin production. As an alternative we have created a toggle switch that combines the mite sensing with B12 or guanine with one of our safety measures: vulnerability of BeeT to light. This allows functional and safe regulation of toxin production even if BeeT cannot grow inside beehives.

Biocontainment

We wanted to limit BeeT to the treated hive. This prevents unknown effects the bacteria could have on larger ecosystems. The first, most intuitive mechanism here is making BeeT dependent on the shade that beehives provide. Incorporated as part of the toggle-switch we have attempted to create an optogenetic kill switch, killing BeeT when it is exposed to light. To better understand this system a model was constructed.

As a final biocontainment, we envisioned BeeT to depend on the presence of an artificial amino acid. Such a system had already been constructed¹². We tried to improve this system, causing Cas9 to degrade DNA when the bacteria run out of synthetic amino acid.

Real-world Functionality

Lastly, to assess the applicability of BeeT in a real-world scenario, we created a new module for the BEEHAVE model¹³. The BEEHAVE model assesses population dynamics and foraging of honeybees. The newly constructed module includes BeeT in the model, giving information on what real-life factors influence its effectiveness.

References

1. Spivak, M., Mader, E., Vaughan, M., & Euliss Jr, N. H. (2010). The Plight of the Bees†. Environmental science & technology, 45(1), 34-38. ↩

2. Seitz, N., Traynor, K. S., Steinhauer, N., Rennich, K., Wilson, M. E., Ellis, J. D., ... & Delaplane, K. S. (2016). A national survey of managed honey bee 2014–2015 annual colony losses in the USA. Journal of Apicultural Research, 1-12. ↩

3. Van Der Zee, R., Gray, A., Pisa, L., & De Rijk, T. (2015). An observational study of honey bee colony winter losses and their association with Varroa destructor, neonicotinoids and other risk factors. PloS one, 10(7), e0131611. ↩

4. Genersch, E., Von Der Ohe, W., Kaatz, H., Schroeder, A., Otten, C., Büchler, R., ... & Meixner, M. (2010). The German bee monitoring project: a long term study to understand periodically high winter losses of honey bee colonies. Apidologie, 41(3), 332-352. ↩

5. Guzmán-Novoa, E., Eccles, L., Calvete, Y., Mcgowan, J., Kelly, P. G., & Correa-Benítez, A. (2010). Varroa destructor is the main culprit for the death and reduced populations of overwintered honey bee (Apis mellifera) colonies in Ontario, Canada. Apidologie, 41(4), 443-450. ↩

6. Serra Bonvehí, J., Ventura Coll, F., & Ruiz Martínez, J. A. (2016). Residues of essential oils in honey after treatments to control Varroa destructor. Journal of Essential Oil Research, 28(1), 22-28. ↩

7. Bonnafé, E., Alayrangues, J., Hotier, L., Massou, I., Renom, A., Souesme, G., ... & Armengaud, C. (2016). Monoterpenoid‐based preparations in beehives affect learning, memory, and gene expression in the bee brain. Environmental Toxicology and Chemistry. ↩

8. Charpentier, G., Vidau, C., Ferdy, J. B., Tabart, J., & Vetillard, A. (2014). Lethal and sub‐lethal effects of thymol on honeybee (Apis mellifera) larvae reared in vitro. Pest management science, 70(1), 140-147. ↩

9. Goodwin, M., & Van Eaton, C. (2001). Control of Varroa. A guide for New Zealand Beekeepers. New Zealand Ministry of Agriculture and Forestry (MAF). Wellington, New Zealand. ↩

10. Alquisira-Ramírez, E. V., Paredes-Gonzalez, J. R., Hernández-Velázquez, V. M., Ramírez-Trujillo, J. A., & Peña-Chora, G. (2014). In vitro susceptibility of Varroa destructor and Apis mellifera to native strains of Bacillus thuringiensis. Apidologie, 45(6), 707–718. ↩

11. Tabashnik, B. E., Brévault, T., & Carrière, Y. (2013). Insect resistance to Bt crops: lessons from the first billion acres. Nature Biotechnology, 31(6), 510–521. ↩

12.Mandell, D. J., Lajoie, M. J., Mee, M. T., Takeuchi, R., Kuznetsov, G., Norville, J. E., … Church, G. M. (2015). Biocontainment of genetically modified organisms by synthetic protein design. Nature, 518(7537), 55–60. ↩

13.Becher, M. A., Grimm, V., Thorbek, P., Horn, J., Kennedy, P. J., & Osborne, J. L. (2014). BEEHAVE: a systems model of honeybee colony dynamics and foraging to explore multifactorial causes of colony failure. Journal of Applied Ecology, 51(2), 470–482. ↩