Conditioning, short-term and long-term memory tests of Drosophila melanogaster grown with and without predators

Conditioning (i.e., associative learning) and memory tests were performed following the methods of previous studies. We used samples of 10 adult flies (males only), raised in standard conditions and aged 6-7 days. The conditioning procedure consisted of 5 training sessions separated by 20-minute intervals (i.e., spaced protocol). During associative conditioning, flies were first exposed for 30 seconds (s) to one odorant simultaneously with a mechanical shock of 2000 rpm vibration pulses of 1 s duration, delivered every 5 s by a test tube shaker (Heidolph Instruments, Schwabach, Germany). This period was followed by a 60 s rest period (no odor and no shock). Then, for 30 s, another odorant was delivered without shock. The training session ended with a second rest period of 60 s. 3-octanol and 4-methylcyclohexanol (both 0.6 mL/L of paraffin) were used as odorants. Each fly group was chosen to be conditioned randomly to either 3-octanol or 4-methylcyclohexanol. The results of the final trial (the fifth out of 5 trials, referred to as the “conditioning” group hereafter) of associative conditioning were used to characterize the learning of fruit flies. 

We tested 1 h (short-term memory, STM) and 24 h memory (LTM) retention after associative conditioning. During the memory retention assay, the flies walked to the choice point of a T-maze, in which they were exposed to two converging currents of air, one carrying 3-octanol and the other 4-methylcyclohexanol, and then allowed to choose between the two odors for 60 s. The memory score was calculated as the difference in the proportion of individuals choosing 3-octanol between flies conditioned to avoid 4-methylcyclohexanol and those conditioned to avoid 3-octanol.