Air leakage between the printed components initially made thevacuum pump suction insufficient, rendering the delivered odor-ant concentrations too dilute for effective training. Therefore, weintroduced slots in the printed components to accommodate O-rings sealing the interfaces between the parts, in order to make theentire design airtight (Fig. 1A). The attachment port for the vacuumsuction is located in the center of the RATTS assembled cylinder(Fig. 1B), since symmetric odor evacuation is critical for the sys-tem design. Odorant delivery is controlled by a series of solenoidvalves (Fig. 1C). Sensors are integrated into the system to detectboth vacuum-controlled airflow and odorant concentration. An airpump is used to move Drosophila between training, resting andtesting chambers (Fig. 1C). The frequency and strength of the airmovements can be independently controlled to move the animalseffectively with the minimum required force. Multiple rounds oftesting were required to optimize the dimensions of the 3D printedcomponents, including the rotating cylinder (Fig. S3), filter screen(Fig. S4), non-rotating compartment (Fig. S5) and the assembly cap(Fig. S6). The supplementary figures show the optimal final dimen-sions determined for each printed component; with bottom, frontand left side cross sectional views. A particularly tight fit is requiredbetween rotating cylinder, non-rotating central compartment andthe assembly cap. The 3D printed material (acrylonitrile butadienestyrene; ABS) has to be airtight to maintain vacuum and for effec-tive air movements of animals between each sequential chamber.Drosophila had to be able to move freely without restrictions, andyet also be maintained in each chamber for training, resting andtesting phases.