(A) Schematic diagrams showing training protocols (protocol 1–3) for the examination of the timing of odor presentation. Animals were exposed to an odor pulse of 3s-, 5s- or 10s-duration (a black bar), which is accompanied with single 1.5 s electric shock (a wavy line). An electric shock was presented at the offset of the odor pulse (protocol 1) or before it (protocol 2 or 3). After 30 s of air-exposure to evacuate CS+ odor, flies were exposed to CS- odor for the same length as CS+. (B) Performance index (PI) of flies trained by protocol 1–3. PI was measured 3 min after the training. Protocol 1 induced conditioning aversion to CS+ odor, which was stronger than those of induced by protocol 2 or 3 (n=6, *p < 0.05, **p<0.01, t-test for each pair). Data are presented as mean ±SEM. The same concentrations of odorants were used in the training and test (2% OCT and 2% MCH).