Team:Toulouse/Description

iGEM Toulouse 2015

Project description

Context: Bees and pollination

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

Domestic bees

There are three types of bees living in a beehive: the worker bees, the queen bee and the male bees. In the colony, the work is divided in such a way that each caste is specialized in its activities. The worker bees are infertile female and represent the most numerous caste in the beehive. Their main activities are cleaning the beehive cells, ventilate the beehive, cap the beehive cells containing the bee eggs, feed the larvae, gather pollen and defend the colony.

The queen bee ensures the renewal of the bee colony. The male bees represent the infertilized eggs. Their main activity is the reproductive function with the queen bee outside of the beehive.

The Queen, a Worker and a male (coming soon)

Collapse of Bees

Over the last few decades, a decline in the honeybee population has been observed. In Europe 24% of the domestic bee species are threatened of extinction. The more indicators you consider, the more alarming appears the situation. The bee loss has a multifactorial origin. Among these factors, the Colony Collapse Disorder (CCD) is one of the most important. CCD corresponds to a massive loss of bee colonies. The causes of this syndrome are numerous: pesticides, viruses, parasites… among those, a mite wisely named Varroa destructor is one of the bee’s fiercest enemies.

Varroa destructor

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 menaces 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.[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 cognitiv impairement.

Outbreak of Varroa destructor in the Western World

The mite varroa was originally an ectoparasite of Apis cerana, the Asian honey bee. The first contact between varroa and Apis mellifera occurred in the 1950s in Asia. During the following years, the infestation spread to Europe, Africa and America. The first signs of this infestation were observed in Europe and North America in the early 1980s. The varroa mite is a particularly virulent parasite of Apis mellifera since the European honeybee did not have time to develop an adaptive response to it.

In Asia on the contrary, Apis cerana and Varroa destructor co-evolved over the centuries and reached a certain balance. Interestingly, the Asian honeybee is capable of detecting the parasite’s presence and of getting rid of it.

Means to fight Varroa destructor

There are three types of methods usued to limit against varroas impact:

♦ 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 its effectiveness is limited and their use is complicated since bees generate consumable products. The lack of effectiveness of those treatments induces the presence in hives of enough varroa after treatment to decrease bee population beyond dangerous thresholds. The use of such treatments (usually annual) can eliminate up to 90% of the varroa population, which is insufficient to maintain the epidemic 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 beeswax.
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.

Bees are probably one of the most valuable animal species to mankind through their unique role in pollination. The speed at which they disappear is alarming and considering the emergency of the situation our team decided to use the tools offered by synthetic biology to fight Varroa destructor.

Strategy: An ounce of prevention is worth a pound of cure

Given the influence of the varroa mite in bee population decline, we aimed our project to fit into the fight against varroa. Current chemical treatments used to fight varroa are not satisfying since they are harmful for bees and human health, and beekeepers relate a lack of effectiveness.

Our project, ApiColi, is an alternative solution in the fight against varroa in order to establish balance between Apis mellifera and Varroa destructor and thus contribute to the preservation of ecosystems.

Introducing ApiColi

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 which is 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

It’s a TrapiColi!

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 it.
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 useable even during the summer.

Apicoli circadian rhythm

We want E.coli to produce butyric acid during the day to attract the varroas and formic acid during the night to kill it

To do so we used a light switch composed of two membrane proteins:

PCB (chromophore phycocyanobilin)
Cph8 and hybrid protein

This protein results from the fusion of red light response domain from Cph1 (phytochrome-like protein cph1) and the intracellular histidin kinase EnvZ (osmolarity sensor protein) from E.coli

During the night (no light)

Cph8 activates the OmpR transcription factor which then activates the polycystronic genes from the formate pathway
Along with the genes coding the formate pathway, a negative transcription factor for the butyrate production polycistron is expressed to lock butyrate production

During the day (with light)

PCB prevents OmpR phosphorylation, thus the formate polycistron can’t be transcripted
The negative transcription factor is not produced, the butyrate polycistron is expressed