Two odours indicated forced choices, left or right; a third odour indicated free choice.
Immediately after training, learning was measured by allowing flies to choose between the two odours for 120 s.
Absolute odour avoidance was quantified by a T-maze with one of the two odours (octanol [103 (vol/vol)] or methylcyclohexanol [103 (vol/vol)]) coming from one side and air from the other side.
As olfactory stimuli [35], 2 µl of each of two odours (carnation and cineole) were applied to separate pieces of filter paper, which were then placed into two different 10 ml syringes (BD Luer-Lock™ Tip).
The test synthetic chemicals were two odours behaviourally relevant to bumble bees: isoamylacetate (Sigma-Aldrich, Milano, Italy; >99.7% purity), both component of their pheromone blends and a floral compound, and -linalool (Sigma-Aldrich, >98.5% purity), a common floral compound, [22], [23].
The spatial position of the two odours on any given trial was randomly determined except that no more than three identical trials could occur in succession.
Mice demonstrating discrimination learning were compared with mice that were exposed to the same two odours, but reward was not contingent upon discriminative responding (pseudo-training).
The flies are then tested to see if they have learnt these associations by giving them a choice of two odours that they can walk towards after training.
