Classical Conditioning in Honeybees

Free flying honeybees (Apis mellifera) were caught inside a bee house from one single hive. For a detailed description of bee handling, conditioning procedure and odorant stimulation see Szyszka et al. [50] (link). Classical conditioning was performed with either 1-hexanol or 1-nonanol as conditioned odor (CS) (Fig. 1) or only 1-hexanol as CS (Fig. 2,3). The odorants were diluted 10⁻² in mineral oil (Sigma-Aldrich, Deisenhofen, Germany). 100 µl of the odorant solution was applied to a 1cm² piece of cellulose (Sugi pads, Kettenbach, Eschenburg, Germany) and placed in the olfactometer. Four-second long odorant stimuli were given with a custom-made olfactometer. The CS was paired with 1 M sucrose solution as a reward, which was presented 2 seconds after CS onset for 3 seconds. The inter-trial interval was 10 minutes. Bees either experienced one-trial conditioning or three-trial conditioning. Memory retrieval was tested 30 minutes, 1 day or 3 days after training. Every bee was tested only once. During each test, the CS and a new odorant were presented. The sequence of CS and new odorant was balanced in order to exclude sequence effects. 1-hexanol and 1-nonanol were used equally as CS and new odorant (Fig. 1). The different experimental groups were always conditioned in parallel to exclude differences due to daily variation in performance.