5.1. Classical conditioning

Most studies on learning and memory employ classical olfactory conditioning of the proboscis extension response. Here, the bee learns to associate an odorant with proboscis extension (Fig. 5A, B; Takeda, 1961; Bitterman et al., 1983; Hammer and Menzel, 1995; Sandoz et al., 1995; Scheiner et al., 2003; Matsumoto et al., 2012). A simple olfactory conditioning experiment with one odorant as conditioned stimulus and a 30 % sucrose solution as unconditioned stimulus and reward could be performed like this:

1. Place a harnessed bee (see section 2.4.) in a constant airstream.

2. Add the odorant (for example 2µl citral on a filter paper) to the airstream for a few seconds (1-5 seconds).

3 .Observe whether the bee shows spontaneous proboscis extension.

4. Consider discarding bees with spontaneous response.

5. Test whether the bee shows proboscis extension at stimulation of its antennae with the sucrose solution.

6. Discard bees without PER in response to antennal stimulation with sucrose.

7. Start each conditioning trial by exposing a bee to the odorant for 1-3 seconds.

8. Elicit the proboscis extension response by touching the antennae of the bee with the sucrose solution.

9. Allow the bee to lick from the sucrose solution for up to one second.

Make sure that the odorant is turned off while the bee consumes the reward.

10. Once the bee has completed drinking, proceed with conditioning the next bee.

11. Use an interval of 5 minutes before you train your first bee again.

Most bees learn quickly to associate odorant and reward and will soon extend their probosces when their antennae are only stimulated with the odorant, which demonstrates associative learning (Fig. 5B). Although bees learn better if they are allowed to drink from the sucrose solution, they also learn to associate the odorant with proboscis extension when only their antennae are stimulated with sucrose (Sandoz et al., 2002). Honey bees need few trials for olfactory learning and establish long-lasting memories (Bitterman et al., 1983; Menzel and Müller, 1996; Sandoz et al., 1995; Matsumoto et al., 2012). As three conditioning trials usually suffice to measure the learning ability of a population of bees, this assay allows a comparatively high throughput. Classical olfactory conditioning is also a good example of appetitive learning, where the behaviour of the bee is rewarded. Later on (see section 5.), aversive learning paradigms will be introduced to the reader. In those paradigms, the behaviour of a bee is punished.

Classical olfactory conditioning has not only been used to study the mechanisms underlying different forms of learning and memory processes (Menzel et al., 1993; Hammer and Menzel, 1995; Giurfa, 2007; Haehnel and Menzel, 2012) but also to analyse the effects of diverse pesticides on honey bee behaviour. Taylor et al. (1987) showed that exposure to different pyrethroids prior to conditioning differentially impairs associative learning ability in bees. Short-term treatment with imidacloprid had differential effects on olfactory acquisition and memory (Decourtye et al., 2004). Abramson et al. (1999) tested the effects of a whole set of products on classical olfactory learning and extinction and demonstrated differential effects, depending on the time of application. Ramirez-Romero et al. (2007) demonstrated an effect of genetically modified crops containing the soil bacterium Bacillus thuringiensis on feeding behaviour and proboscis extension learning.

Fig. 5. Classical olfactory conditioning (A, B) and operant tactile conditioning (C, D) of the honey bee. Naïve bees do not show proboscis extension when their antennae are stimulated with a certain odour like citral or geraniol (A) or when their antennae can scan a tactile stimulus (C). After pairing the odour or the tactile stimulus with a sucrose droplet, which is first applied to the antennae and then to the proboscis (not shown), the bees show conditioned proboscis extension to the odour (B) and to the tactile stimulus (D). Note that for tactile conditioning, the eyes are painted over with black colour to improve perception of tactile stimuli.

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