Difference between revisions of "Team:Toulouse/Description"

Bees excel in pollination and thus play an essential role in maintaining ecosystems by participating to the production of seeds. Bees harvest pollen and use proteins to feed their offspring. Moreover, by gathering pollen, they allow the reproduction of 84 % of the plants that grow in Europe. Bees are also responsible for the production of 35 % of edible fruits and vegetables. Among all bees, the domestic bee, Apis mellifera, which builds its swarm in hives provided by man, is directly responsible for the production of honey, wax and propolis.

Figure 1: Examples of products dependent on bee pollination

According to the French INRA (National Institute of Agricultural Research), the bee extinction would represent an economical worldwide loss of 163 billion USD. This huge number reflects the importance of the bees in the global ecosystem.

Varroa destructor is an obligatory ectoparasite of bees. This means it is an external parasite and it cannot survive without its host, the bee. The varroa development cycle takes place in the beehive cells in parallel of the bee development cycle. The key individual of the varroa development cycle is the adult female commonly named “founder”. The founder reproduces exclusively in a brood cell that represents the beehive cell containing a bee larva. The founder reproduction occurs during the phoretic phase where the varroa is moved from one bee colony to the other by an adult bee.

Figure 2: Synchronized life cycle of Honeybees and Varroa destructor. Phoretic phase is the phase were varroa is fixed on an adult bee. This phase is followed by the reproductive phase which takes place in bee broods. Phoretic phase then restarts when the imago (last embryonic stage of the bee) emerges from the brood.

The bee infestation by varroa occurs between the egg laying of the queen bee in beehive cells and the encapsulation of the bee larva inside the beehive cell. After feeding off the larva the founder will start laying varroa eggs. The newborn varroas will develop themselves, go through different stages and will reproduce within the brood cell. When the mature bee finally emerges from the brood cell, female varroas will stick themselves to it therefore becoming “phoretic”. The Varroa destructor itself is of significant danger. However it is the association with the Deformed Wing Virus (DWV) that makes him one of the most virulent threats for honeybees. The study of the interplay between the mite and the DWV shows that an intense viral replication is triggered by the mite feeding off the bee, leading to a devastating viral outbreak.

Figure 3: Effects exerted by Varroa destructor on honeybee and their hypothetical consequences, source [4]. The parasite may weaken the immune system allowing the proliferation of DWV (Deformed Wing Virus) amoung bees. This proliferation may cause disorders responsible of physical and cognitive impairement.

♦ Search of tolerant or resistant bees through selection
♦ Use of “zoo-technic” or “bio-technic” means (Trapping of varroas in empty male broods or installation of fenced entries)
♦ Use of chemical treatments

This last type is the most effective, however it is limited and their use is complicated since bees generate consumable products. The lack of effectiveness of those treatments allows enough varroa to decrease bee population in hives, beyond dangerous thresholds. The use of such treatments (usually yearly) can eliminate up to 90% of the varroa population, which is insufficient to maintain the plague at safe levels.

On top of that, such treatments may have serious disadvantages:

♦ It has been shown that some miticide or degradation metabolites from these molecules accumulate in beewax.
This long term accumulation could explain the emergence of resistant parasites.

♦ Some treatments were shown to have detrimental effects on bees.
Those effects are usually increased if the treatments are misused.

♦ Some treatments can contaminate the production of bees and impact the quality of the products.

Our strategy is based on a genetically modified Escherichia coli strain able to alternate a production of two molecules according to a circadian cycle. During the day, while bees are entering or exiting the beehive, butyrate is biosynthesized by our engineered bacteria, ApiColi, in order to attract the varroa fixed on bees.
By night, ApiColi produces formate, a well-known molecule lethal to the varroa attracted during the day.
Formic acid is currently used to fight varroa, but at very high doses, that also have an impact on bees.

Figure 1: Circadian rhythm switch strategy

In our project, the engineered bacteria ApiColi will be placed at the bottom of a trap, called TrApiColi, positioned at the entrance of the hive. Varroas will be attracted and killed there, leaving the bee colony less exposed to both chemicals, and particularly formic acid.

Furthermore, at the moment, beehives are only treated with formic acid during spring and fall, when no honey is being made. This is due to the fact that formic acid weakens bees, but also that when varroa have entered the brood, they are not affected by the miticide.
The attraction power of ApiColi will enable us to prevent most varroas from entering the beehive and reaching the brood. This way, our treatment would be usable even during summer.

Concerning formic acid, it is currently used by beekeepers as a treatment against varroa infestation but only during spring and fall and at high doses. Thus it has deleterous effects on bees and beekeepers. We also know that bees have a lifecycle based on the sunlight. As we explain above, during the day, bees are active and come in and out of the hive to gather pollen. This is when they bring back the varroas to the hive. During the night on the contrary, bees are less active and stay in the hive.

With all these data, we have been able to design a perfect solution fighting Varroa destructor. A trap containing the engineered bacteria (ApiColi) will be placed at the entrance of the beehive. During the day, Apicoli will produce butyric acid which will diffuse up to where the bees enter the hive. The varroas will be attracted and drop into the trap through a grid. Thus they will never come inside the hive.

To be respectful of the bees lifecycle, our bacteria will only produce formic acid at night. This way, the bees will be much less exposed to it since it will be confined to the trap and at much lower doses. Finally, we do not know if these two molecules effect can interfere, but we do know that they can be harmful to ApiColi if accumulated. That is why alternating production of both acids according to a circadian rhythm will enable us to have a more controlled use of the carbon source, and thus a longer culture length and trap efficiency.
To do so, based on a paper published in Nature by Levskaya et al., Synthetic biology: engineering E. coli (2005), we used a light switch system composed of two membrane proteins: