Introduction

We chose to work with Asaia because it could survive in mosquitos. Due to technical issues, we wanted to work in a first phase with a possible substitute to mosquitoes, namely drosophila. We therefore decided to work with two different hosts, first drosophila, which we discuss in the first part of this section, and then mosquitos, which we discuss in the last part of this section.

I) Experiments on Drosophila

The final goal of our project is for our modified Asaia to survive and produce proteins in the mosquito's gut. Working with mosquitoes however, requires special equipment that we do not have at EPFL. We wondered if we could work on another insect which is less demanding, and therefore turned towards Drosophila (commonly known as the fruit fly), which is much easier to work with.

Considering the fact that bacteria that live in the guts of insects are not very common, we assumed that there was a fair chance that Asaia could persist in Drosophila and that we could use the it as an alternative to mosquitoes for our basic experiments.

Using Drosophila melanogaster we aimed to address two questions:
i. Is Asaia toxic for Drosophila?
ii.Is Asaia able to colonize the Drosophila gut and persist?

(See Materials and Methods for details on how the experiments were conducted.)

Our main results

Figure 1Observing Asaia that express GFP in vivo

1) Asaia is not toxic for Drosophila

Figure 2 Asaia is not toxic to Drosophila. Relish (A) and Oregon (B) flies were infected with different bacterial strains and monitored them over time. Deaths were counted and added up over the course of the experiment.

We infected Drosophila with different bacterial strains, a pathogenic control starin (P. entomophila), a non-pathogenic control strain (Ecc 15) and our Asaia bacteria. For these experiments we used two different fly strains (Oregon and Relish).

We found that Asaia does not cause significantly more deaths than the non-pathogenic bacteria or in the uninfected control (Figure 2).

2) Asaia is not persistant in Drosophila

Figure 3 Asaia does not persist in Drosophila.

With this last experiment we wanted to ascertain wether asaia persisted in drosophila and quantize how many asaia were present in the drosophila’s gut after 3h, 24h and 48h.

To be able to “count” the number of asaia at these three different periods of time, we had to retrieve the bacteria inside the flies, plate them and after incubation count the number of colonies present.

To do this we disinfected the exterior of the flies by washing them with ethanol (for less than five seconds) and then rinsing them with water. The flies were then crushed in the medium corresponding to the bacteria we were interested in (i.e: Gly for asaia, LB for Pe, etc). We then did a serial dilution eleven times with a factor of ten with the crushed flies, and plated each dilution with antibiotics to specifically select the bacteria we were interested in.

Conclusion to the drosophila experiments

The survival assay reveals that Asaia is not lethal for Drosophila.

The persistence assay showed that Asaia is unable to establish itself in the fruit fly's gut. This prevents us to use Drosophila as a substitute to mosquitoes.

As mentioned before, bacteria that live in insects’ gut are not common and it is possible that because it cannot survive in Drosophila, it is actually very specific to mosquitoes. With this assumption, we could assume that we can introduce our modified asaia into the wildlife with very little risk of it spreading to other insects.

II) Experiments on mosquitos

The final objective of this project is to determine if the infection of Anopheles stephensi with our engineered Asaia has an effect on the mosquito's vectorial capacities of Plasmodium berguei. To do so we contacted the Institute Pasteur in Paris to collaborate with them and proceed with our experiments. Nevertheless, due to the fact that we were discovering how to work on asaia, we did not finish in time to do our experiments before the iGEM jamboree. The experiments we would like to conduct are the following:

• Infect Anopheles stephensi with our engineered Asaia.
• Infect the Anopheles stephensi strains with Plasmodium berguei.
• Determine of the vectorial capacities of the different Anopheles strains.