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The present work introduces a form of associative mechanosensory conditioning of the proboscis extension reflex (PER) in honeybees. In our paradigm, harnessed honeybees learn the elemental association between mechanosensory, antennal stimulation and a reward of sucrose solution delivered to the proboscis. Thereafter, bees extend their proboscis to the antennal mechanosensory stimulation alone. We show that bees can learn such an association in a side-specific manner, that is, they learn the association on the antennal side that was rewarded and not on the side that was not rewarded. Responding produced by the paired training does likely contain a substantial Pavlovian component. Responding is only elicited by mechanosensory stimulation and not by spurious cues such as olfactory, visual, and contextual ones. The interstimulus interval (ISI) affects one-trial mechanosensory learning: a bell-shaped curve with a maximum of responding approximately 4 sec ISI was obtained. Mechanosensory memory is still operative 24 h after conditioning. Apart from absolute conditioning in which mechanosensory stimulation of one antenna is paired with sucrose, differential, side-specific, mechanosensory conditioning using two mechanosensory stimulations, one rewarded and the other not, is also possible. This paradigm constitutes, therefore, a new standard procedure for further learning studies in honeybees.

Associative Mechanosensory Conditioning of the Proboscis Extension Reflex in Honeybees