Experiment 4: learning to avoid an odour
This protocol is extracted from research article:
Hard limits to cognitive flexibility: ants can learn to ignore but not avoid pheromone trails
J Exp Biol, Jun 4, 2021; DOI: 10.1242/jeb.242454

The aim of experiment 4 was to show that it is within the ants’ capacity to learn to avoid a neutral chemical signal (here: lemon odour) after it was associated with a punishment. We tested 12 ants using the same setup as in experiment 2 (Fig. 1) but replaced the pheromone trail on the negative arm of the Y-maze by lemon scent while the positive arm was covered with unscented paper overlays. Lemon-scented paper overlays were produced by placing unscented paper layers in an airtight container with lemon essential oil for at least 24 h; these were taken out of the container just before applying them to the runway. Between trials, all paper overlays (scented and unscented) were replaced to avoid orientation via pheromones left during the previous trial.

Five ants were allowed to walk up a bridge, leading to a Y-maze, one arm of which was covered with an unscented paper overlay, and the other covered with lemon-scented paper. The first ant choosing the lemon-scented arm first was selected to be the subject, while ants choosing the unscented arm of the Y-maze were gently brought back into the nest. This was done in order to ensure that no ants with an innate aversion to lemon odour were tested, thus making the experiment very conservative. The pre-selected subject then carried out two pre-training trials, 12 trials [following a pseudo-randomized order: L–R–L–R–R–L–L–R–L–R–L–R and its reversed sequence, where L (left) and R (right) indicate the arm of the maze containing the sucrose reward] and two unrewarded learning tests after trial 8 and 12, all as described in experiment 2, but with lemon scent instead of a pheromone trail.

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