This is the protocol we are using to assess the Light/Dark preference in flies.

24h before the experiment, anesthetize 3-6 d old flies under CO2, and clip 2/3 from both wings to half of them. In the same vial, place around 30 flies with clipped wings and another 30 intact flies. Let them recover from anaesthesia until experiment begins. The number of flies you can test at the same time depends on the size of your T-Maze. We have noticed that there is an overcrowding effect on the results.

The light source should be homogenous above the T-Maze.

Experiment begins.

• Place the flies in the entrance tube (see figure) and let them adapt for 10 min.
• Turn on the light source
• Transfer the flies to the elevator by gently tapping the apparatus, and pull the movable part down in order to close it. Be careful not to face the elevator to the transparent and the opaque tubes. Flies are kept in the elevator for 30 sec.
• Pull down the movable part, so that the elevator is placed between the transparent and the opaque tubes. Allow the flies to choose between dark and light for 30 sec.
• Close all tubes by pulling up the movable part, in order to lock the flies in the tube they have selected.
• Count the number of flies with and without wings in each tube.
• Calculate the Choice Index.

The Choice Index is calculated using the formula:

CI=((#FL×1)+(#FD×-1)+(#FE×0))/(#FT )

where #FL, #FD, #FE, and #FT mean the number of flies in the transparent tube, the opaque tube, the number of flies that remained in the elevator, and the total number of flies, respectively. A CI of 1 mean all the flies chose the light, while an index of -1 mean a negative phototaxis and 0 meant no choice. In each experiment a CI is calculated for the wingless flies and other for the intact flies.