Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability 1,2 . The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood . Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates 7 . Bees use different floral signals like colors or odors 7,8 , but also tactile cues from the petal surface 9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae 10,11,12,13 . This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees 14 . The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern. Video LinkThe video component of this article can be found at http://www.jove.com/video/50179/ Protocol 1. Preparing the Bees 1. Nectar or Pollen foragers are caught in the field either from a sucrose feeder or directly from the hive entrance while returning from a foraging trip. Each single bee is captured into a glass vial that is closed with a foam plug and taken immediately into the laboratory for further handling. 2. In the laboratory, the captured bees are briefly cooled in the refrigerator at 4 °C until they show first signs of immobility. 3. Each single immobilized bee is mounted in a small metal tube with adhesive tape between head and thorax and over the abdomen. Care should be taken that the proboscis and antennae are freely movable. 4. Paint the compound eyes and ocelli of the fixed bee with white paint (e.g. solvent-free Tipp-Ex) to occlude vision. 5. Add a small drop of melted wax behind the head of the bee to fix it to the tape between head and thorax to prevent head movements during recordings. 6. Mark each single bee with a number on the tape for better identification and place the tube with the fixed bee into a humid atmosphere to prevent dehydration. 7. Feed each single bee for 5 sec with droplets of a 30% sucrose solution presented with a syringe and let all bees recover for 30 min before starting with the tactile conditioning protocol. Tactile Conditioning1. Before conditioning, each single bee has to be tested for the proboscis extension response (PER) to a 30% sucrose stimulus applied to the antennae. Thereby the tip of the proboscis has to cross a virtual line between the opened mandibles. Discard all bees that don't respond with a PER to the sucrose stimulus. 2. For tactile conditioning use a brass cube (e.g. 3 x 5 mm) with a smooth or an engraved pa...

No abstract