Name: Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.) (Video) | JoVE Journal
Uploaded: 2012-12-12T11:50:00.000+00:00
Duration: 10 min 14 s
Description: Learn tactile conditioning and 2D motion capture techniques to analyze antennal sampling kinematics in harnessed honey bees. This method enables precise study of associative and non-associative learning in Apis mellifera.

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5 minutes

We thank Joachim Erber for introducing us to the tactile learning paradigm in honey bees. This work was supported by the Cluster of Excellence 277 CITEC, funded in the framework of the German Excellence Initiative.

1. Preparing the Bees
<ol>
	<li>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.</li>
	<li>In the laboratory, the captured bees are briefly cooled in the refrigerator at 4 &deg;C until they show first signs of immobility.</li>
	<li>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.</li>
	<li>Paint the compound eyes and ocelli of the fixed bee with white paint (e.g. solvent-free Tipp-Ex) to occlude vision.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Tactile Conditioning
<ol>
	<li>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.</li>
	<li>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 &mu;m wave length, as the conditioned stimulus (CS). For the unconditioned stimulus (US) use a 30% sucrose solution (household sugar, diluted in water).</li>
	<li>The brass cube (CS) is placed into a holder on a micromanipulator (e.g., M&auml;rzh&auml;user 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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Kinematic Recordings
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Data Analysis
<ol>
	<li>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.</li>
	<li>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).</li>
	<li>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).</li>
	<li>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).</li>
	<li>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).</li>
</ol>

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No conflicts of interest declared.

Preparation of bees: Collecting and fixing the bees should be done quickly, in order to keep the stress level of the animal low. Stress has an effect on the PER-responsiveness and, therefore, could have an indirect effect on the learning performance in bees19, 20. The stress level can be decreased by placing the glass vials with the bees directly on ice immediately after collection to immobilize them quickly. It has to be taken into account that bees need more time to recover the longer the an...

<table><tbody><tr><td>Pollen foragers, &lt;em&gt;Apis mellifera&lt;/em&gt; L.</td><td></td><td></td><td>Private hives</td></tr><tr><td>Glass vials (22 x 45 mm)</td><td>Carl Roth GmbH Co</td><td>X655.1</td><td></td></tr><tr><td>Brass tubes, 10 x 30 mm</td><td></td><td></td><td>Self-produced</td></tr><tr><td>Brass cube, 3 x 5 mm</td><td></td><td></td><td>Self-produced</td></tr><tr><td>Tipp-Ex (solvent-free)</td><td>Tipp-Ex GmbH Co KG</td><td></td><td></td></tr><tr><td>Sucrose solution (30%)</td><td></td><td></td><td>Household suger diluted in water</td></tr><tr><td>Ethanol (70%)</td><td>Carl Roth GmbH Co</td><td>9065.3</td><td></td></tr><tr><td>Syringe (1 ml) and needle (0.6 x 25)</td><td>Carl Roth GmbH Co</td><td>59.1</td><td></td></tr><tr><td>Stop Watch</td><td>Carl Roth GmbH Co</td><td>L423.1</td><td></td></tr><tr><td>Micromanipulator</td><td>M&amp;#228;rzh&amp;#228;user MM33</td><td>00-42-101-0000</td><td></td></tr><tr><td>Digital video camera</td><td>Basler A602f-2</td><td></td><td></td></tr><tr><td>Macro lens for camera</td><td>TechSpec VZM 200</td><td></td><td></td></tr><tr><td>Matlab R2009b</td><td>The MathWorks</td><td></td><td></td></tr></tbody></table>

tactile conditioning and movement analysis of antennal sampling strategies in honey bees (apis mellifera l.)

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

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 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 odors7, 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 antennae10, 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.

In the following experiment we studied how tactile learning affects antennal sampling behavior in honey bees. For this, we monitored the movement of the antennal tip in naïve and conditioned bees before and during presentation of a tactile stimulus.
First, the spontaneous antennal movement of a group of pollen foragers (N = 42) was recorded for 1 min. One half of the bees (N = 21) was then conditioned by pairing five times a tactile stimulus with a 30% sucrose reward. This was the conditioned ...

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.

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

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.

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Research

JoVE Journal

Neuroscience

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

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Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

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A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

The loss of honey bee colonies presents a challenge to crop pollination services. Current pollinator protection practices warrant an alternative approach to minimize the contact of honey bees to harmful pesticides using repellent chemistries. Here, we provide detailed methods for a visual tracking protocol to screen deterrents for bees.

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

This protocol describes steps for using the novel software, SwarmSight, for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The free, open-source software processes frames about 120 times faster than humans and performs at better than human accuracy.

SwarmSight: Real-time Tracking of Insect Antenna Movements and Proboscis Extension Reflex Using a Common Preparation and Conventional Hardware

We present a protocol for the classical conditioning of harnessed ants that permits researchers to study visual learning and memory formation at a level of analysis not possible with freely moving individuals.

Visual Classical Conditioning in Wood Ants

Learn tactile conditioning and 2D motion capture techniques to analyze antennal sampling kinematics in harnessed honey bees. This method enables precise study of associative and non-associative learning in Apis mellifera.

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

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