The overall goals of this procedure are to show how to condition honeybees to tactile stimuli, and to introduce a 2D motion capture technique for analyzing the kinematics of fine scale and 10 sampling patterns. At the beginning, the procedure requires capturing free flying bees in front of their hive. The second step is to harness bees into metal tubes and to occlude their eyes with white paint.
Next, the bees are trained to tactile stimulus using the classical conditioning procedure. The final step is to record the an antenna movement of the conditioned bee with a digital video camera. Ultimately, 2D motion capturing in MATLAB is used to analyze changes in an antenna movement behavior in honeybees after both associative and non associative tactile learning paradigms.
This method can be used to study tactile discrimination and perception in various alterable species. It can differentiate changes due to non associative tactile learning from changes occurring from associative learning like during conditioning. Generally, individuals new to this method will struggle because the bees have to be handled very carefully and the timing of the tactile conditioning procedure is critical.
The method can help answer key questions in the field of active touch sensing. It uses a natural reflex behavior as a reporting behavior, allowing us to tell whether or not the animal can discriminate between two tactile stimuli. To begin collect bees directly from the hive entrance.
Capture each bee in a glass vial closed with a foam plug, and take it immediately into the laboratory for further handling in the laboratory. Briefly, cool the bees in a four degrees Celsius refrigerator until they begin to show signs of immobility. First, immobilize each bee onto a metal tube.
Place adhesive tape around the head and thorax and over the abdomen. Confirm that the probus and an antennae are freely movable to occlude the bee's vision. Paint the compound eyes and selli with white paint.
Place a small drop of melted wax between the head and the tape to prevent the head from moving. During recordings. For easier identification, mark the tape of each honeybee with a number and place the tube into a humid atmosphere to prevent dehydration.
Lastly, use a syringe filled with 30%sucrose solution to feed each bee for five seconds and let them recover for 30 minutes. Before starting the tactile conditioning protocol, before conditioning, test each B for the psis extension response or PER. To do this, apply a 30%sucrose stimulus to the antennae for a correct response.
The tip of the psis has to cross a virtual line between the opened mandibles. Only bees with a correct PER are used for the study. For tactile conditioning, metal surface structures are used as the conditioned stimulus or cs.
Their surface can be smooth or grooved to create a different texture. The unconditioned stimulus or US is a 30%sucrose solution. Place the brass cube into a holder on a micro manipulator.
This will allow for exact positioning during the conditioning procedure. Once the tactile stimulus has been mounted, place a bee in front of the micro manipulator. Next, slowly position the Cs such that the surface of the tactile stimulus is parallel to the head of the bee.
The bee should be able to scan the tactile stimulus in a comfortable position with both an antenna. To begin the conditioning procedure, allow the bead to scan the tactile stimulus for five seconds. After the first three seconds, use a syringe to present a droplet of the 30%sucrose solution under the probos.
Use the tip of the syringe to gently raise the probus. This stimulation should elicit the unconditioned PER. Allow the bee to lick the sucrose.
Reward for one second. Proceed with the conditioning procedure until the bee receives five pairings. After the first rewarded pairings, bees should start to respond to CS presentation by extension of their psis indicating that they expect the forthcoming reward.
A fully extended psis observed any time during the first three seconds of tactile stimulus presentation before sucrose presentation is scored as a positive response. Finally, a learning curve is generated for a group of bees. For this, the percentage of bees showing the PER during CS presentation is plotted for each trial.
After conditioning, a digital video camera with a suitable macro lens is used to record the antenna movements. Here we use a basler a 602 F camera, equipped with a tech spec VZM 200 lens operated at 50 frames per second via a fire wire connection, adjust the camera such that the harnessed bee and tactile stimulus will be visible. Next, calibrate the camera by taking single pictures of a calibrated checkerboard from different orientations.
Use specialized software with a camera calibration tool to analyze the images. Once calibrated, place a single fixed B below the camera lens and present the tactile stimulus. As shown earlier.
Record the enseal movement while the bee is scanning the object. Once the data have been collected, MATLAB is used to define the image background first, calculate the median gray scale value over time for each pixel. Static objects like the fixed head of the bee and the tactile stimulus will constitute the image background by subtracting the image background from each frame the moving parts in the video.
Ideally, the antennae of the bee are isolated as the only areas of non-zero values for further processing. The two largest areas with non-zero pixel values are assumed to be the an antennae. A mask is created to localize the antenna tips and for the entire image to reduce the amount of error.
A combination of image erosion and dilatation tools are applied once these techniques have reduced the noise level. A binary mask function is used to cluster the disjointed areas. The center of gravity is calculated for the two largest of these clusters in order to distinguish between the left and right antenna.
Here, they're color labeled in red and green. Finally, the antenna tip for each antenna can be defined as the pixel in a cluster with the highest value in the proximal to distal direction, a two dimensional histogram is created that shows the spatial distribution of the an tip shown. Here is the learning curve of tactile PER conditioning in pollen forger.
The percentage of positive responses after the first pairing was significantly higher when compared to spontaneous behavior, and the learning curve shows saturation after the third reward. In this example, the movement of the anal tip in both the conditioned and the naive group was recorded for one minute in the presence or absence of the tactile stimulus seen. Here are spontaneous an tunnel movements before stimulus presentation.
The absa shows the lateral position of the an tunnel tip while the ordinate shows the distal position. The color scale indicates the percentage of time that the anin tip spent at any given location. Yellow indicates very frequent locations red, less frequent, and black locations are out of reach.
This data show that both the and conditioned group changed their antal movement pattern in presence of a tactile stimulus compared to their spontaneous behavior. Note, the rectangular yellow region near the surface of the stimulus indicating that the ant heel tip spent much time sampling The surface. Bees from the conditioned group spent significantly more time with their an antenna tip in the area of the stimulus than the naive bees.
Following this procedure, electrophysiological and pharmacological methods can be found to answer additional questions like, what is the neurophysiological basis for active touch behavior in honeybees After its development? This technique of tactile conditioning paved the way for researchers in the field of active touch sensing to explore various aspects of tactile stimulus discrimination, but also of tactile learning and memory in Alpha Pots. After watching this video, you should have a good understanding of how to analyze active tactile searching and sampling movements and insects.
Note that the movement analysis can be expanded to three dimensional analysis if at least one further camera is added in synchrony to the first one.
