Description
Features |
Foraging arena: L: 28 cm x W: 28 cm |
Two superposed acrylic plates with 24 holes of 1cm in diameter |
Upper plate: 0.2cm acrylic clear acrylic plate |
Lower plate: 0.7-cm thick grey acrylic plate |
Artificial flower patch: 4cm deep; 480 eppendorf tubes |
Color: diameter of 3.8 cm. 24 colored circles |

Introduction
The Bee Reward Expectations Apparatus (BREA) is employed to investigate reward expectations in honeybees, an area where data is relatively scarce compared to vertebrates, as complex cognitive abilities were traditionally thought to be exclusive to animals with larger brains. Gil et al. developed BREA specifically to observe reward expectations in foraging honeybees under conditions that mimic their natural foraging environments.
Associative learning for rewards hinges on the connection between external cues and the internal representation of past reward experiences. Studying reward learning can enhance our understanding of behavioral changes due to rewards, expectations of rewards, goal-directed behavior, and the cognitive complexity involved in decision-making and planning (Gil, 2010).
BREA consists of an artificial flower patch set within a square foraging arena composed of two overlapping plates. Each plate features 24 holes designed to hold Eppendorf tubes (representing flowers) filled with sucrose solution as a reward for the bees. Surrounding the flowers are colored circles serving as visual stimuli, visible through the overlapping plates.
During training, experimental bees are allowed to inspect and feed on the flowers, which vary in sucrose solution volume across two variable and three constant reward trials. Subsequently, the bees are tested without the presence of sugar rewards. Researchers observe the bees to determine their ability to choose the correct flower based on visual stimuli and measure the total time spent searching for a reward.
Apparatus and Equipment
BREA consists of an artificial flower patch set within a 28 cm × 28 cm foraging arena. This arena is constructed using two square acrylic plates stacked on top of each other, each containing 24 evenly spaced holes, each 1 cm in diameter. The upper plate is crafted from 0.2 cm thick transparent acrylic, while the lower plate is made of 0.7 cm thick opaque acrylic.
The artificial flowers are represented by Eppendorf tubes, each 4 cm deep, placed within the 24 holes of the acrylic plates. These tubes protrude 1.8 cm above the upper transparent acrylic plate. Surrounding each Eppendorf tube are 24 colored circles—12 blue and 12 yellow—with a diameter of 3.8 cm. These circles are visible through the upper transparent plate, centered around the Eppendorf tubes.
Training Protocol
A colony of Apis mellifera carnica bees resides in a two-frame observation hive located indoors. An artificial flower patch containing unscented 50% w/w sucrose solution is positioned 145 meters away from the observation hive. A small cohort of labeled recruiting bees from the colony is allowed to visit and feed on the artificial flower patch. These recruiting bees entice nest-mates to join them in the foraging arena.
New bees arriving at the arena are trapped before they access the sucrose solution and marked with plastic tags. After tagging, these bees are cooled and released as potential experimental subjects. Bees that return to the flower patch undergo a pre-training phase to become experimental bees.
To ensure experimental integrity, the entire apparatus is meticulously cleaned to eliminate any extraneous cues that could influence the bees’ performance. Tracking and recording of trials are facilitated using advanced systems such as the Noldus EthoVision XT tracking and video system.
Data Analysis
The following data can be obtained from the experiment
- Learning Score (LS) – The ratio between the number of inspections of the rewarded flower by color and the total number of inspections of both types of flowers (rewarded/unrewarded color) for each visit.
- Retention score (RS) – The ratio between the number of inspections of the rewarded color and the total number of inspections of both colors.
- Cumulative learning score (CLS) – The sum of individual learning scores.
- Successful Inspection (SI) – The number of times the experimental bee finds sugar reward during its multiple inspections of the flowers.
- Unsuccessful Inspection (UI) – The number of times the experimental bee did not find sugar reward when inspecting a flower signaled by rewarded color.
- Cumulative Inspection Time (CIT) – The amount of time in seconds that the experimental bee spends searching for sugar reward during each test session.
- Visit Time (VT) – the time in minutes the experimental bee spends foraging on the arena during every single visit.
- Total Visit Time (TVT) – the sum of individual VT values for nine successive visits.
- Training Time (TT) – the sum of TVT and the time the experimental bee spends inside the hive in between successive foraging visits to the arena.
- Total volume (Vol) collected during training in microliters (μL).
- Solution Intake Rate throughout the TVT (SIR1) in μL/min – The ratio between Vol and TVT.
- Solution Intake Rate throughout the TT (SIR2) in μL/min – The ratio between Vol and TT.
- Mean Solution Intake Rate (MSIR) in μL/min – Mean ratio between the collected volume and the VT for each of the nine successive visits to the patch.
Summary
- Bee Reward Expectations Apparatus (BREA) is used to study reward expectations in honeybees.
- BREA is an artificial flower patch with a square foraging arena made up of two overlapping plates.
- There are 24 Eppendorf tubes (flowers) placed on the foraging arena with sucrose solution as reward for the bees.
- The experimental bees are trained for two variable and three constant reward trials and then tested in the absence of sugar reward.
- The bees are observed for choosing correct flower based on visual stimuli and the total time spent looking for the reward.
- Studies of reward expectations are essential in increasing understanding of goal-directed behaviors, decision making, and planning.
References
Gil M, De Marco RJ, Menzel R (2007). Learning reward expectations in honeybees. Learning & Memory 14(7), 491-6. DOI: 10.1101/lm.618907
Gil M (2010). Reward expectations in honeybees. Communicative Integrative Biology 3(2), 95-100.