Materials and Methods

The Flies.

Working with flies: We put the drosophila to sleep with CO2 and then manipulated them with fine paintbrushes and tweezers on a box were we could add CO2 at will.

Females / Males: For each experiment we worked exclusively on female flies to have the least variability. We chose to work on females rather than males because the females eat more, as they have to lay eggs, are bigger and have a higher metabolism. We hence assumed that by working with females, we could achieve an higher uptake of bacteria.

Oregon / Relish: We worked on the drosophila WT Oregon. We also conducted some experiments with the drosophila mutant relish, which is immunodeficient, to see if the basic immune system of the WT flies is harmful to asaia.

The Infection

We prepared aliquots of bacteria with OD 100. We then put 50ul of the solution on filters that we placed over the medium which the flies feed on. Therefore the flies take up the bacteria whenever they eat.

Before infecting the flies we starved them for approximatively two hours in order to ensure that they would immediately take up the bacteria, and all at approximately the same time.

The Bacterial Controls

We infected a set of flies with different types of bacteria whose reaction in the drosophila’s gut is are well known, and therefore use these as controls.

Positive control (Bacteria that should persist in the gut of the drosophila). Pseudomonas entomophila (Pe) Erwinia carotovora carotovora 15 (ECC-15)

Negative control (Bacteria that do not persist in the gut of drosophila). Gac: non persistent Pe mutant

Time of measurements

3h: Three hours after the infection we verified that the flies were actually properly infected. We expected to observe the presence of all types of bacteria (asaia, positive and negative control) as the flies would not have had time to produce an immune response against these bacteria.

24h: We should start observing a decline in the negative control whilst the positive control persists. We can also start observing the tendency of asaia.

48h: The positive control should still persist and the negative control should have almost disappeared. We can then compare asaia to the controls and conclude on the persistency of asaia. bove.