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.)

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 na&iuml;ve 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 conditi...

Watch this Scientific Journal Video about Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L. at JoVE.com

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

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.

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 ...

tactile-conditioning-movement-analysis-antennal-sampling-strategies

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.5K Views.  Bielefeld University. 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.

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

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

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.

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey bee colony numbers have been declining in the United States and many European countries since 1947. A number of factors play a role in this decline, including the unintentional exposure of honey bees to pesticides. The development of new methods and regulations are warranted to reduce pesticide exposures to these pollinators. One approach is the use of repellent chemistries that deter honey bees from a recently pesticide-treated crop. Here, we describe a protocol to discern the deterrence of honey bees exposed to select repellent chemistries. Honey bee foragers are collected and starved overnight in an incubator 15 h prior to testing. Individual honey bees are placed into Petri dishes that have either a sugar-agarose cube (control treatment) or sugar-agarose-compound cube (repellent treatment) placed into the middle of the dish. The Petri dish serves as the arena that is placed under a camera in a light box to record the honey bee locomotor activities using video tracking software. A total of 8 control and 8 repellent treatments were analyzed for a 10 min period with each treatment was duplicated with new honey bees. Here, we demonstrate that honey bees are deterred from the sugar-agarose cubes with a compound treatment whereas honey bees are attracted to the sugar-agarose cubes without an added compound.

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.

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey ...

Environment

Visual Classical Conditioning in Wood Ants

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, studies implementing classical conditioning paradigms with harnessed insects have been important for investigating the exact cues that individuals learn and the neural mechanisms underlying learning and memory formation. Here we present a protocol for evoking visual associative learning in wood ants through classical conditioning. In this paradigm, ants are harnessed and presented with a visual cue (a blue cardboard), the conditional stimulus (CS), paired with an appetitive sugar reward, the unconditional stimulus (US). Ants perform a Maxilla-Labium Extension Reflex (MaLER), the unconditional response (UR), which can be used as a readout for learning. Training consists of 10 trials, separated by a 5-minute intertrial interval (ITI). Ants are also tested for memory retention 10 minutes or 1 hour after training. This protocol has the potential to allow researchers to analyze, in a precise and controlled manner, the details of visual memory formation and the neural basis of learning and memory formation in wood ants.

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.

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, ...

Behavior

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

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

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

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

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.

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

Biology

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

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

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Radio Frequency Identification and Motion-sensitive Video Efficiently Automate Recording of Unrewarded Choice Behavior by Bumblebees

We present two methods for observing bumblebee choice behavior in an enclosed testing space. The first method consists of Radio Frequency Identification (RFID) readers built into artificial flowers that display various visual cues, and RFID tags (i.e., passive transponders) glued to the thorax of bumblebee workers. The novelty in our implementation is that RFID readers are built directly into artificial flowers that are capable of displaying several distinct visual properties such as color, pattern type, spatial frequency (i.e., &#8220;busyness&#8221; of the pattern), and symmetry (spatial frequency and symmetry were not manipulated in this experiment). Additionally, these visual displays in conjunction with the automated systems are capable of recording unrewarded and untrained choice behavior. The second method consists of recording choice behavior at artificial flowers using motion-sensitive high-definition camcorders. Bumblebees have number tags glued to their thoraces for unique identification. The advantage in this implementation over RFID is that in addition to observing landing behavior, alternate measures of preference such as hovering and antennation may also be observed. Both automation methods increase experimental control, and internal validity by allowing larger scale studies that take into account individual differences. External validity is also improved because bees can freely enter and exit the testing environment without constraints such as the availability of a research assistant on-site. Compared to human observation in real time, the automated methods are more cost-effective and possibly less error-prone.

This video describes Radio-Frequency Identification (RFID) and motion-sensitive video recording methods to monitor choice behavior by bumblebees.

We present two methods for observing bumblebee choice behavior in an enclosed testing space. The first method consists of Radio Frequency Identification ...

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

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

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

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

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

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

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

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.

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

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

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Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

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

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

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

Radio Frequency Identification and Motion-sensitive Video Efficiently Automate Recording of Unrewarded Choice Behavior by Bumblebees

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

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