Learning experiments

Associative learning experiments followed established protocols (Gerber and Hendel 2006; Saumweber et al. 2011; Schleyer et al. 2015a,b). For aversive training, one group of larvae was trained such that n-amyl acetate (AM) was presented simultaneously with the quinine substrate for 2.5 min, immediately followed by a blank trial on a Petri dish with empty Teflon containers (EM) and without quinine for another 2.5 min (paired training). In a second experimental group, the larvae were trained such that AM and quinine were presented on separate Petri dishes (unpaired training). This training cycle was performed three times. Across repetitions of the experiment, in half of the cases the odor-containing trial came first (AM + Quinine/EM and AM/EM + Quinine, respectively), in the other half of the cases the sequence was reversed (EM/AM + Quinine and EM + Quinine/AM, respectively). For the subsequent test, the animals were placed in the middle of a 15-cm-diameter Petri dish with an AM-loaded container on one side and an empty container on the other side in order to create a choice situation. Larval behavior was recorded using a camera and analyzed offline as described below.

For appetitive learning experiments, fructose was used instead of quinine. Analogous to the aversive protocol described above, odor and fructose were presented either paired or unpaired, and testing and recording were performed as mentioned.

The test Petri dishes may or may not contain a taste reinforcer as mentioned in the results. For all experiments, experimenters were blind with respect to the test conditions.