Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Samir Mujagić; Simon Michael Würth; Sven Hellbach; Volker Dürr

doi:10.3791/50179

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Summary

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

Abstract

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 brood³. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks⁴, including hive building⁵ and pattern recognition⁶. 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 mates⁷. Bees use different floral signals like colors or odors^{7, 8}, but also tactile cues from the petal surface⁹ 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 bees¹⁴. 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.

Protocol

1. Preparing the Bees

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.
In the laboratory, the captured bees are briefly cooled in the refrigerator at 4 °C until they show first signs of immobility.
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.
Paint the compound eyes and ocelli of the fixed bee with white paint (e.g. solvent-free Tipp-Ex) to occlude vision.
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.
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.
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.

2. Tactile Conditioning

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.
For tactile conditioning use a brass cube (e.g. 3 x 5 mm) with a smooth or an engraved pattern, e.g., horizontal or vertical grooves forming a grating with 150 μm wave length, as the conditioned stimulus (CS). For the unconditioned stimulus (US) use a 30% sucrose solution (household sugar, diluted in water).
The brass cube (CS) is placed into a holder on a micromanipulator (e.g., Märzhäuser MM33) to ensure exact positioning during the conditioning procedure. The US is presented to the bee with a syringe filled with a 30% sucrose solution.
The conditioning procedure consists of five pairings of the tactile stimulus (CS) and sucrose solution (US) with an inter-trial interval (ITI) of 5 min. Place a single bee in front of the micromanipulator with the mounted tactile stimulus (CS). Position the CS slowly, such that the surface of the tactile stimulus is parallel to the head of the bee (Figure 1 A and B). The distance between the animal and the tactile object should be in the range of the antennal working radius of the tested bee, i.e. the bee should be able to scan the tactile stimulus in a comfortable position with both antennae. In the example shown in Figure 1, the distance was 3 mm. Let the bee scan the tactile stimulus (CS) for 5 sec. After the first 3 sec, present a droplet of the 30% sucrose (US) solution with a syringe under the proboscis. Use the tip of the syringe to gently raise the proboscis. Sucrose stimulation under the proboscis will elicit the unconditioned PER¹⁵. Allow the bee to lick the sucrose reward. Use a stopwatch with an alarm signal to maintain the exact time intervals during conditioning.
The conditioned PER is used as a measure for the learning success of a bee. After the first rewarded pairings, bees start to respond to CS presentation by extension of their proboscis, indicating that they expect the forthcoming reward. A fully extended proboscis observed anytime during the 3 sec time window of tactile stimulus presentation and before sucrose presentation is scored as a positive response. No response is counted negative. The occurrence of the PER has to be noted by the experimenter. The percentage of bees showing the PER during CS presentation is plotted for each trial.

3. Kinematic Recordings

Antennal movements of a single harnessed bee are recorded with a digital video camera with a suitable macro lens (e.g. Basler A602f-2 equipped with TechSpec VZM 200, operated at 50 fps via a fire wire connection). The camera is positioned above the animal in a top-down view (Figure 1 A). In Figure 1, the spatial resolution of the recordings is 0.02 mm/pixel.
Calibrate the camera by recording single pictures of a 10 x 10 mm checkerboard with an edge length of 1 mm from different orientations under the camera objective. The calibration can be done with the camera calibration tool box Matlab¹⁶.
Place a single fixed bee below the camera lens. Present the tactile stimulus, fixed to a micromanipulator, to the animal. Proceed in the same way as described for tactile conditioning, and record the antennal movement while the bee is scanning the object. It is important that the full antennal working range and the tactile stimulus are visible. The choices of stimulus patterns tested and trial numbers depend on the experimental design.

4. Data Analysis

The computation of the image background is done in Matlab. First, the median greyscale value over time has to be calculated for each pixel. Static objects, like the fixed head of the bee and the tactile stimulus, will constitute the image background. Moving objects, like the two antennae, will not be part of the background.
Each frame of the recorded video has to be loaded sequentially, and the difference between the current frame and the image background has to be computed. The result of this subtraction emphasizes those parts of the image that are moving from frame to frame. Ideally, the antennae of the bee are the only areas of non-zero values (Figure 1C).
For further processing, the two largest areas with non-zero pixel values are assumed to be the antennae. For both antennae a binary mask is to be generated containing for each pixel a value 1, if the pixel belongs to the antenna and value 0 otherwise. This mask serves as a basis for localizing the antennal tips later. To gain a preliminary mask for the entire image the grey-value of each pixel of the difference image is compared to a pre-defined threshold. Since we take care of noise later this threshold is chosen to be quite low. The preliminary mask then still carries two kinds of errors: Firstly, due to image noise small regions are still part of the mask. Secondly, areas that belong to the antenna might not necessarily be fully connected. The latter mostly occurs if the background has approximately the same luminescence as the antenna. To eliminate these artifacts, standard image processing morphological operation are applied, i.e. a combination of image erosion and dilatation, relying upon the standard Matlab functions imerode and imdilate respectively (see¹⁷ pp. 158-205 for further explanation). After the denoising process the antennae hypotheses are still contained within the same mask. Hence, as a next step the binary mask is clustered for disjoint areas using the standard Matlab function bwlabel (see¹⁷ pp. 40-48 for further details on the image segmentation algorithm).
The number of pixels per cluster is counted and the two largest clusters are selected. The center of gravity is calculated to distinguish between the left and right antenna (Figure 1D).
The antennal tip for each antenna can be defined as the pixel in a cluster with the highest value in the proximal-to-distal direction (see Figure 1D).