SummaryIn this BEEBOOK paper we present a set of established methods for quantifying honey bee behaviour. We start with general methods for preparing bees for behavioural assays. Then we introduce assays for quantifying sensory responsiveness to gustatory, visual and olfactory stimuli. Presentation of more complex behaviours like appetitive and aversive learning under controlled laboratory conditions and learning paradigms under free-flying conditions will allow the reader to investigate a large range of cognitive skills in honey bees. Honey bees are very sensitive to changing temperatures. We therefore present experiments which aim at analysing honey bee locomotion in temperature gradients. The complex flight behaviour of honey bees can be investigated under controlled conditions in the laboratory or with sophisticated technologies like harmonic radar or RFID in the field. These methods will be explained in detail in different sections. Honey bees are model organisms in behavioural biology for their complex yet plastic division of labour. To observe the daily behaviour of individual bees in a colony, classical observation hives are very useful. The setting up and use of typical observation hives will be the focus of another section. The honey bee dance language has important characteristics of a real language and has been the focus of numerous studies. We here discuss the background of the honey bee dance language and describe how it can be studied. Finally, the mating of a honey bee queen with drones is

Laboratory studies in honey bees have shown positive correlations between sucrose responsiveness, division of labour and learning. We tested the relationships between sucrose acceptance and discrimination in the field and responsiveness in the laboratory. Based on acceptance in the field three groups of bees were differentiated: (1) bees that accept sucrose concentrations >10%, (2) bees that accept some but not all of the sucrose concentrations <10% and water, and (3) bees that accept water and all offered sucrose concentrations. Sucrose acceptance can be described in a model in which sucrose- and water-dependent responses interact additively. Responsiveness to sucrose was tested in the same bees in the laboratory by measuring the proboscis extension response (PER). The experiments demonstrated that PER responsiveness is lower than acceptance in the field and that it is not possible to infer from the PER measurements in the laboratory those concentrations the respective bees accepted in the field. Discrimination between sucrose concentrations was tested in three groups of free-flying bees collecting low, intermediate or high concentrations of sucrose. The experiments demonstrated that bees can discriminate between concentrations differences down to 0.2 relative log units. There exist only partial correlations between discrimination, acceptance and PER responsiveness.

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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...

The experiments analyze different forms of learning and 24-h retention in the field and in the laboratory in bees that accept sucrose with either low (≤3%) or high (≥30% or ≥50%) concentrations. In the field we studied color learning at a food site and at the hive entrance. In the laboratory olfactory conditioning of the proboscis extension response (PER) was examined. In the color learning protocol at a feeder, bees with low sucrose acceptance thresholds (≤3%) show significantly faster and better acquisition than bees with high thresholds (≥50%). Retention after 24 h is significantly different between the two groups of bees and the choice reactions converge. Bees with low and high acceptance thresholds in the field show no differences in the sucrose sensitivity PER tests in the laboratory. Acceptance thresholds in the field are thus a more sensitive behavioral measure than PER responsiveness in the laboratory. Bees with low acceptance thresholds show significantly better acquisition and 24-h retention in olfactory learning in the laboratory compared to bees with high thresholds. In the learning protocol at the hive entrance bees learn without sucrose reward that a color cue signals an open entrance. In this experiment, bees with high sucrose acceptance thresholds showed significantly better learning and reversal learning than bees with low thresholds. These results demonstrate that sucrose acceptance thresholds affect only those forms of learning in which sucrose serves as the reward. The results also show that foraging behavior in the field is a good predictor for learning behavior in the field and in the laboratory.