This work was funded by the Leverhulme Trust grant (RPG-2012-708) and a BBSRC grant (BB/M00709X/1) to GAW.

1. Capturing Bees from the Colony and the Starvation Period
Note: Experiments described here were performed at Newcastle University, UK with Bombus terrestris audax. Multiple (2-3) commercially purchased colonies were used per treatment. The colonies have been maintained on a bench at laboratory conditions (25 &#177; 2 &#176;C and 28 &#177; 2% RH) in constant darkness and were fed with honey bee collected pollen and sugar solutions ad libitum.
<ol>
	<li>Collect individual worker bumble bees using a plastic vial (7 cm long, 2.8 cm inner diameter) with a perforated plastic stopper, after having opened the gate to the colony just long enough for one bee to exit and be trapped.</li>
	<li>Prior to the experiment, individually starve all bumble bees for 2-4 hr&#160;in the plastic vials and keep at room temperature in complete darkness.</li>
</ol>
2. Transferring Bees into the Holding Tubes and the Habituation Phase
<ol>
	<li>After the starvation period, transfer a bumble bee directly from the plastic vial into a holding tube. The holding tube is a modified 15 ml centrifuge tube (length: 119 mm; diameter 17 mm), with a 4 mm hole drilled at the tip and a piece of steel mesh (base: 8 mm; height 30 mm) fixed inside by melting the plastic of the tube with a heated dissecting steel needle.</li>
	<li>Fix the holding tube containing the bumble bee onto a polystyrene holder with dental wax. Fix two pieces of cardboard on either side of the holding tube. This is to shield the bee from visual stimuli that might interfere with the experiment.</li>
	<li>Position a digital microscopic camera 5 cm above the tip of the holding tube and connect the camera to a compatible laptop.</li>
	<li>Adjust the holding tube so that at least the first 18 mm of the holding tube tip is within the video frame. Prior to the experiment, commence the 3 min habituation period.</li>
</ol>
3. Pre-test Phase: Presenting a Drop of Sucrose
<ol>
	<li>Connect a syringe to a female adapter containing a droplet of sucrose solution (~3.5 &#181;l, 500 mM sucrose dissolved in deionized water). Present the sucrose inside the holding tube tip to motivate the extension of the proboscis.</li>
	<li>Give the bumble bee up to 5 min to consume the sucrose droplet. If the droplet is not consumed, remove the bumble bee from the experiment.</li>
	<li>Begin video recording after the habituation period. In this study, the proboscis activity was recorded at 26.7 frames/sec-1 with a 25X magnification rate.</li>
</ol>
4. Test Phase: Presenting the Test Solution
<ol>
	<li>Fill a 100 &#181;l microcapillary tube with the test solution. Connect it to a piece of silicone tubing (6 cm length, 1 mm inside diameter) and fix it to a micro manipulator.
	<ol>
		<li>Connect the tubing via a male adapter to another silicone tubing (6 cm length, 4 mm inside diameter), which acts as a pipette bulb.</li>
		<li>Position the microcapillary tube 5-10 mm away from the holding tube tip. Gently squeeze the tubing to maintain the feeding solution at the tip of the microcapillary tube.</li>
	</ol>
	</li>
	<li>After the bumble bee consumes the sucrose droplet, immediately remove the syringe containing the 500 mM sucrose solution.</li>
	<li>Begin the 2 min test phase when the bumble bee&#39;s proboscis contacts the solution inside the microcapillary tube.
	<ol>
		<li>To control for possible evaporation, fill two additional microcapillary tubes with sucrose or water and manipulate it exactly as during the test phase.</li>
	</ol>
	</li>
	<li>Before and after each trial scan the liquid levels inside the microcapillary tube using a scanner at 600 dpi to measure the amount of food consumed (Figure 4A).</li>
</ol>
5. Image Analyses
<ol>
	<li>Determine the volume of solution consumption using ImageJ (version 1.48), an image processing software.
	<ol>
		<li>Upload the image file and zoom into the image (~400%). To set the reference scale, select the straight line tool and draw a line between the two ends of the microcapillary tube. Select &#39;Analyze&#39; then &#39;Set Scale&#39;. Input the total length of the tube under &#39;Known distance&#39; and the corresponding unit under &#39;Unit of length&#39;.</li>
		<li>Select the straight line tool again and draw a line between the two ends of the liquid level. Select &#39;Analyze&#39; then &#39;Measure&#39;. In the results window the length of the liquid is given under the &#39;Length&#39; column.</li>
	</ol>
	</li>
	<li>Calculate the volume of solution consumption by using the formula: 
	<img alt="Equation 1" src="/files/ftp_upload/54233/54233eq1.jpg" /> 
	where <img alt="Equation 2" src="/files/ftp_upload/54233/54233eq2.jpg" /> is the length of the microcapillary tube and <img alt="Equation 3" src="/files/ftp_upload/54233/54233eq3.jpg" /> and <img alt="Equation 4" src="/files/ftp_upload/54233/54233eq4.jpg" /> are the measured lengths of the liquid inside the microcapillary tube before and after the test phase, respectively.</li>
</ol>
6. Video Analyses
<ol>
	<li>Score the feeding behaviors during the 2 min testing phase of each video using an event logging software (See Materials Table).
	<ol>
		<li>At first, define the feeding behaviors (i.e. the elements) in the behavioral classes menu of the recording software. The feeding behaviors are as followed: (1) proboscis out/contact: the proboscis extends and is in contact with the solution inside the microcapillary tube (2) proboscis out/no contact: the proboscis extends and is not in contact with the solution inside the microcapillary tube, (3) proboscis stowed: the proboscis is not extended but instead stowed under the head and (4) out of sight: the bumble bee is out of the video frame.</li>
		<li>Set each behavior as a &#39;state&#39; and &#39;mutually exclusive&#39; in the properties menu and make continuous recordings for a 2 min interval. Replay the videos in slow-motion mode (2 times slower) for more precision.</li>
	</ol>
	</li>
	<li>Measure the speed of proboscis retraction from the test solution after the first contact between two consecutive frames (separated by 37.5 msec in the video recordings shown here) using a motion tracking video software (See Materials Table).
	<ol>
		<li>Upload the video file and skip to the frame where the proboscis first contacts the solution.</li>
		<li>To set the reference scale, select the line tool and draw a line on the width of the microcapillary tube in the video frame. Right click on the line and select &#39;Calibrate measure&#39;. Input the width of the capillary tube and the corresponding unit.</li>
		<li>Select &#39;Image&#39; then &#39;Coordinate system origin&#39;. On the new window click on the tip of the proboscis and select &#39;Apply&#39;.</li>
		<li>Select the hand move tool, right click on the tip of the proboscis on the video frame and select &#39;Track Path&#39;. Move to the next frame and readjust the tracking point to the tip of the proboscis.</li>
	</ol>
	</li>
	<li>Right click on the tracking point and select &#39;Configuration&#39;. Select &#39;Complete Path&#39; and select &#39;Speed&#39; under measurement. Select &#39;Apply&#39;. The speed is then displayed.</li>
</ol>

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The authors declare no conflict of interest.

With this novel behavioral assay, quinine is shown to deter feeding of the buff-tailed bumble bee. The reduced proboscis contact time and feeding bout frequency with water or the sucrose solution laced with quinine is interpreted here as a refusal to initiate further feeding on non-nutritive or potentially toxic solutions. When quinine is added to 1 M sucrose solution, bumble bees not only reduce the volume of solution they consume, they also retract the proboscis faster, thus reducing contact time between the mouthparts and the solution containing a toxin. Together, these results suggest that quinine is perceived by the gustatory receptor cells on the mouthparts of the bumble bee, as already previously identified in the honey bee9. Quinine is a toxin for insects that induces malaise-like behavior in the honey bee10 and knockdown in the malaria mosquito (Anopheles gambiae)23. This assay could well lead to the identification of some deterrent and potentially toxic compounds that are perceived by the taste receptor cells on the mouthparts in the bumble bees.
It is crucial for the microcapillary tube to be filled with a sufficient volume of test solution to last throughout the test phase. It is recommended that at least around three quarters of the microcapillary tube (e.g. 70-80 &#181;l) is filled. However, care should be taken to not completely fill the microcapillary tube to reduce the risk of spillage during the process of scanning and attaching the microcapillary tube to the experimental apparatus. Care should also be taken when presenting the 500 mM sucrose droplet to the bumble bee, so that the experimenter avoids leaking the droplet into the holding tube.
The 4 mm hole at the tip of the holding tube is large enough for an adult worker bumble bee to naturally extend its proboscis toward the test solution. However it is possible that bumble bees can taste the solution with their antennae before extending their proboscises. This might affect the probability of proboscis extension as PER could be elicited in bumble bees by stimulating their antennae with a sugar solution15. In fact the antennae of Hymenoptera like the parasitoid wasp (Trissolcus brochymenae)24 or the honey bee13 are equipped with taste sensilla, allowing them to taste sugars and toxins like quinine. Consequently, initial antennal contacts with solutions containing highly deterrent compounds like quinine could also reduce the motivation of a bumble bee to extend its proboscis and therefore affect the experimental success rate. Although antennal contact with the test solution cannot be controlled, in the present study we did not find any significant effect of antennal contact on proboscis extension toward the test solution. In this assay, immediately setting up the microcapillary tube after the pre-test phase when the bumble bees&#39; antennae are still within the holding tube can reduce the opportunity for the bumble bees to taste the test solution with their antennae.
The main limitation of this assay arises when tracking the proboscis retraction away from the test solution after the first proboscis contact using the motion tracking video software. The video footage only displays 2D movement of the proboscis, so the given output of the speed measurement can be under or over estimated. However with some modifications, this aspect of the assay could be improved.
This assay can be used to observe natural feeding responses toward solutions containing different compounds including natural-occurring plant secondary metabolites. Observing the immediate feeding responses with this assay gives detailed information about how bumble bees detect these compounds. This is advantageous over existing &#39;go-no go&#39; methods like PER18,19 and over two-choice assays7 because this method produces several behavioral response measures including food consumption during a discrete feeding bout.
Measuring several parameters simultaneously allows a better evaluation of the palatability of a compound. For example in our assay, bumble bees avoid consuming water or the sucrose solution laced with quinine. Retraction of the proboscis could be caused by a change in the responses of the sugar receptor cells12,13. Our assay shows that bumble bees retract the proboscis faster after contacting the sucrose plus quinine solution than water alone; this could suggest that quinine affects a distinct set of neurons in addition to inhibiting sugar sensing neurons9,12,13,25.
Our assay permits the analysis of the temporal pattern of behavioral responses during feeding. A similar protocol where the consumption time and the number of bouts is measured has already been implemented to evaluate the feeding response of Drosophila to nutritive and non-nutritive sugars26. We predict that bees will exhibit a more reliable response to feeding stimulants in our assay than in other methods such as PER because the bees are free to move in the holding tube21. This technique will permit an exhaustive analysis of the taste thresholds for nutrients and toxins to illuminate the mechanisms of feeding in bumble bees and potentially other bee species.

Plant-pollinator interactions are complex. Pollinators visit flowers to obtain nectar and pollen as food; in turn, pollinators facilitate sexual reproduction in plants. While this relationship is mostly mutualistic, floral nectar and pollen sometimes contain toxins or other plant compounds1-5 which can harm pollinators. The ecological rationale for the presence of such compounds in nectar and pollen is not clear in all settings. One outstanding question in this field is how pollinators such as bees can detect and avoid flowers with nectar containing toxins.
The bumble bee species, Bombus terrestris (Linnaeus, 1758), is a generalist pollinator that visits the flowers of many plant species including those producing nectar containing toxins6. Bumble bees have been shown to avoid consuming solutions containing high concentrations of toxins in a 24 hr two-choice assay7. This assay of food consumption described by Tiedeken et al.7 revealed that bees can detect bitter compounds in solutions. However, this assay was unable to distinguish taste from post-ingestive processes such as malaise that could also affect feeding behavior over this time interval8-10.
Bees possess gustatory sensilla on their antennae, mouthparts and tarsi to detect compounds11-13. The proboscis extension reflex (PER) experiments involve restraining individual bees in a harness and then stimulating the bee&#39;s antennal sensilla to produce the feeding reflex14-17. Bees can be restrained in individual harnesses and then stimulated to produce the feeding reflex as an assay of their ability to taste compounds18,19. Others have modified the PER assay to study the sensitivity of the antennae or mouthparts to toxins9,20. However, bees are subjected to stress during harnessing. This could affect how they respond to compounds21.
Here, a new assay is described to assess the behavioral taste response of freely-moving bumble bees to sucrose and quinine, an alkaloid that has previously been reported to be deterrent9 and toxic10 to honey bees (Apis mellifera) and bumble bees (Bombus terrestris)7,22. Although quinine has not been found in plant nectar, this alkaloid is often used as an aversive stimulus in behavioral and physiological studies in bees7,9,12,13,22. The method involves video recording the bumble bees&#39; mouthparts at great resolution during the initial proboscis contact with the test solutions. Specifically, the fine structure of the feeding response is examined by continuously scoring behavior over a 2 min interval. The volume of solution consumed is measured during the feeding period and so the amount of food eaten can be correlated with the microstructure of the feeding behavior. Also the speed of the proboscis retraction is measured, as an indicator of an active avoidance, and therefore pre-ingestive detection.

<table><tbody><tr><td>Bumblebee colonies</td><td>Koppert Biological Systems</td><td></td><td>NATUPOL Beehive</td></tr><tr><td>Digital microscopic camera&amp;nbsp;</td><td>Dino-lite Europe</td><td>AM4815ZT</td><td>Dino-Lite Edge&amp;nbsp;</td></tr><tr><td>100 &amp;mu;l microcapillary tube&amp;nbsp;</td><td>Blaubrand IntraEND</td><td>709144</td><td></td></tr><tr><td>15 ml polypropylene centifuge tube&amp;nbsp;</td><td>Fisher Scientific</td><td>11849650</td><td></td></tr><tr><td>1 ml disposable plastic luer slip syringe</td><td>BD</td><td>300013</td><td></td></tr><tr><td>Dell Latitude 3550 laptop</td><td>Dell</td><td></td><td>Check for compatibility with video software&amp;nbsp;</td></tr><tr><td>Canon CanoScan LiDE 120</td><td>Canon</td><td></td><td>Check for compatibility with the computer/laptop</td></tr><tr><td>Observer software version 5.0.25</td><td>Noldus</td><td></td><td></td></tr><tr><td>Kinovea software version 0.8.15</td><td>Kinovea&amp;nbsp;</td><td></td><td></td></tr><tr><td>silicone tubing</td><td></td><td></td><td>6&amp;nbsp;cm length, 1&amp;nbsp;mm inside &amp;Oslash; &amp;amp; 6&amp;nbsp;cm length, 4&amp;nbsp;mm inside &amp;Oslash;</td></tr><tr><td>Male luer x1/16&quot; standard hose barbed polypropylene adapter</td><td>Cole-Parmer</td><td>TW-45518-22</td><td></td></tr><tr><td>Female luer x 1/16&quot; standard hose barbed polypropylene adapter</td><td>Cole-Palmer</td><td>TW-45508-12</td><td></td></tr><tr><td>Steel mesh&amp;nbsp;</td><td></td><td></td><td>0.5 mm mesh size</td></tr><tr><td>Sucrose (grade II)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S5391</td><td></td></tr><tr><td>Quinine hydrochloride dihydrate</td><td>Sigma-Aldrich</td><td>Q1125</td><td></td></tr><tr><td>ImageJ software version 1.48</td><td>ImageJ</td><td></td><td></td></tr></tbody></table>

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.

The novel assay is used to test the feeding responses to 1 M sucrose, 1 M sucrose solution plus 1 mM quinine and deionized water alone. The immediate feeding responses to each treatment are determined by quantifying the duration of proboscis contacts with the test solution, the frequency of the feeding bouts and the speed of the proboscis retracting away from the test solution after the first contact during the 2 min test phase. The volume of solution consumed is also measured after the test phase. In this study, we have chosen a bout criterion interval of 5 sec (Figure 1, see Supplemental File) based on previous work by French et al.25 who used a 5 sec threshold to characterize the proboscis retraction behavior by Drosophila in response to deterrent compounds25. Thus, we defined a feeding bout as a contact between the extended proboscis and the solution not interrupted by an absence of contacts of 5 sec or more.
In comparison to sucrose and deionized water alone, adding quinine to sucrose solution evidently deters feeding by bumble bees as they will rapidly move away if they detect an aversive substance (Video Figure 1).
In this experiment, the treatments have a significant effect on the cumulative duration of proboscis contacts during the test phase (ANOVA on the log-transformed data, F2,31 = 41, p &#60;0.001). The cumulative duration of contact time with sucrose containing quinine is significantly reduced in comparison to sucrose alone (p &#60;0.001) but not to deionized water alone (p = 0.219) (Figure&#160;2). Similarly, the treatments have a significant effect on the cumulative duration of feeding bouts (ANOVA on the log-transformed data, F2,31 = 27.95, p &#60;0.001, Figure 3A). The cumulative duration of feeding bouts with sucrose containing quinine is significantly reduced in comparison to sucrose alone (p &#60;0.001) but not compared to deionized water alone (p = 0.41). The treatments have also a significant effect on the frequency of feeding bouts (Poisson GLM with a log link function, change in deviance compared to the c2 distribution: p &#60;0.050), whereby the number of bouts with sucrose containing quinine is significantly higher in comparison to sucrose (p &#60;0.01) but marginally significantly different to the deionized water treatment (p = 0.055, due to one bumblebee displaying seven feeding bouts on water, Figure 3B). Likewise, the speed of proboscis retraction differs significantly between treatments (ANOVA on the log-transformed data, F2,31 = 5.12, p &#60;0.050). Bumble bees retract the proboscis away from the test solution significantly faster after the first contact with sucrose containing quinine than with sucrose or deionized water alone (p &#60;0.050, Figure 3C). These results suggest that quinine triggers an active avoidance behavior in bumble bees. The treatments also have a significant effect on the total volume of solution consumed (ANOVA on the log-transformed data, F2,32 = 62.5, p &#60;0.001), whereby the consumption of sucrose containing quinine is reduced in comparison to sucrose (p &#60;0.001) but not to deionized water (p = 0.457) (Figure 4B). The volume of solution evaporated from the capillary during the test period is negligible. At laboratory conditions (25 &#177;2&#176;C and 28 &#177;2% RH), the evaporation varies between 0.033 to 0.883 &#181;l with an average of 0.276 &#181;l and 0.171 &#181;l for deionized water and 1 M sucrose respectively.
In this assay contacts between the antenna and the test solution cannot be prevented. Nonetheless, the percentage of bumble bees using their antennae to taste the feeding solution during the test phase (sucrose: 46.1%, sucrose plus quinine: 60.0% and deionized water: 33.3%) is not significantly different between the treatments (binomial GLM, change in deviance compared to the c2 distribution: p = 0.450). No effect of the treatments is found on the latency between the first antennal contacts and the test solution and the first contacts of the proboscis (median: 2.67 sec for sucrose; 1.10 sec for sucrose plus quinine; 0.80 sec for deionized water, ANOVA on the log-transformed data, F2,13 = 0.620, p = 0.550). In addition, the percentage of bumble bees extending the proboscis to taste the test solution remains constant across the treatments (sucrose: 66.7%; sucrose plus quinine: 50.0%; deionized water: 52.2%; binomial GLM, change in deviance compared to the c2 distribution: p = 0.840). Together these results suggest that the antennae play a minor role in the detection of the toxins in this assay.
A separate experiment examines whether it is necessary to test bees for a period of time longer than 2 min. The amount of food consumed by bees is tested with the 1 M sucrose or 1 mM quinine in 1 M sucrose solutions in two conditions: a 2 min test period and a 10 min test period. For both treatments, total food consumption does not differ for the test periods and no significant interactions occur between the test period and the treatment (N = 6 - 13, ANOVA on the log transformed data; effect of the treatments: F1,31&#160;= 54.8, p &#60;0.001; effect of the test period: F1,31= 0, p = 0.979; effect of the interaction: F = 0.1, p = 0.457). In summary, a 2 min test period is sufficient to assess the effect of the solution on the total amount of food consumed by bumblebees and the deterrent effects of toxic or repellent substances in this assay. Thus, by measuring food consumption and assaying feeding behavior, it is possible to correlate total food consumption to the fine structure of feeding during the assay.
<img alt="Figure 1" src="/files/ftp_upload/54233/54233fig1.jpg" /> 
Figure 1: Latency Periods between the Proboscis Contacts during the First 2 Min of the Feeding Assay. Density plots of the time latency periods separating each proboscis contact with the 1 M sucrose solution, the 1 M sucrose + 1 mM quinine solution and water. The cumulative data from 13, 10 and 11 bees are represented respectively. <a href="https://www.jove.com/files/ftp_upload/54233/54233fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54233/54233fig2.jpg" /> 
Figure 2: Proboscis Contact Durations during the First 2 Min of the Feeding Assay. Density plots of the proboscis contact durations by bumblebees feeding on 1 M sucrose, 1 M sucrose + 1 mM quinine or water. Sample size as in Figure 1. <a href="https://www.jove.com/files/ftp_upload/54233/54233fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54233/54233fig3.jpg" /> 
Figure 3: Proboscis Activity of Bumblebees Feeding on 1 M Sucrose, 1 M Sucrose + 1 mM Quinine or Water. (A) The cumulative duration of feeding bouts during the test phase (B) the frequency of feeding bouts and (C) the speed of the proboscis retraction after first contact. Lettering indicates a significant difference: treatments with different letters indicate P &#60;0.05. Box plots represent the median (black bars), the lowest and the highest data points still within 1.5 of the interquartile range (whiskers) and the outliers (circles). Sample size as in Figure 1. <a href="https://www.jove.com/files/ftp_upload/54233/54233fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54233/54233fig4.jpg" /> 
Figure 4: Quinine supresses feeding by bumble bees. (A) Scanned images of the microcapillary tubes showing the level of the 1 M sucrose or the 1 M sucrose plus 1 mM quinine solution (indicated by a black line) before and after the test phase respectively. (B) Consumption of 1 M sucrose, 1 M sucrose plus 1 mM quinine or deionized water alone by bumble bees after the test phase. Lettering indicates a significant difference: treatments with different letters indicate P &#60;0.001. Box plots represent the median (black bars), the lowest and the highest data points still within 1.5 of the interquartile range (whiskers) and the outliers (circles). Sample size as in Figure 1. <a href="https://www.jove.com/files/ftp_upload/54233/54233fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video Figure 1: Video Recordings of the Proboscis Activity toward (A) <a href="https://www.jove.com/files/ftp_upload/54233/Video_figure_1A_1M_Sucrose.m4v" target="_blank">1 M Sucrose</a>, (B) <a href="https://www.jove.com/files/ftp_upload/54233/Video_figure_1B_Quinine.m4v" target="_blank">1 M Sucrose Plus 1 mM Quinine</a> and (C) <a href="https://www.jove.com/files/ftp_upload/54233/Video_figure_1C_Water.m4v" target="_blank">Deionized Water during the Test Phase</a>.

Watch this Scientific Journal Video about A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris at JoVE.com

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.

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 ...

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8.7K Views.  Newcastle University. 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. 

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

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 ...

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 ...

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.

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.

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

Vertical T-maze Choice Assay for Arthropod Response to Odorants

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, various technologies are being developed as control tools. A subset of these tools focuses on modifying the behavior of arthropods by attraction or repulsion. Therefore, arthropods are often the focus of behavioral investigations. Various tools have been developed to measure arthropod behavior, including wind tunnels, flight mills, servospheres, and various types of olfactometers. The purpose of these tools is to measure insect or arachnid response to visual or more often olfactory cues. The vertical T-maze oflactometer described here measures choices performed by insects in response to attractants or repellents. It is a high throughput assay device that takes advantage of the positive phototaxis (attraction to light) and negative geotaxis (tendency to walk or fly upward) exhibited by many arthropods. The olfactometer consists of a 30 cm glass tube that is divided in half with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the test subjects to make a choice between two potential odor fields in assays involving attractants. In assays involving repellents, lack of normal response to known attractants can also be measured as a third variable.

A vertical, T-maze olfactometer is described for assaying the behavioral response of arthropods. The olfactometer allows the experimenter to measure choices performed by test subjects when subjected to two potential odor fields. Both attraction to and repulsion from odorants can be measured with this device.

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, ...

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

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). ...

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 ...

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

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis during feeding. The ability to rapidly obtain high-resolution measurements of natural antenna and proboscis movements and assess how they change in response to chemical, developmental, and genetic manipulations can aid the understanding of insect behavior. By extending our previous work on assessing aggregate insect swarm or animal group movements from natural and laboratory videos using the video analysis software SwarmSight, we developed a novel, free, and open-source software module, SwarmSight Appendage Tracking (SwarmSight.org) for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The software processes frames about 120 times faster than humans, performs at better than human accuracy, and, using 30 frames per second (fps) videos, can capture antennal dynamics up to 15 Hz. The software was used to track the antennal response of honey bees to two odors and found significant mean antennal retractions away from the odor source about 1 s after odor presentation. We observed antenna position density heat map cluster formation and cluster and mean angle dependence on odor concentration.

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.

Many scientifically and agriculturally important insects use antennae to detect the presence of volatile chemical compounds and extend their proboscis ...

A 3D Printed Pollen Trap for Bumble Bee (Bombus) Hive Entrances

To verify the plant sources from which bumble bees forage for pollen, individuals must be collected to remove their corbicular pollen loads for analysis. This has traditionally been done by netting foragers at nest entrances or on flowers, chilling the bees on ice, and then removing the pollen loads from the corbiculae with forceps or a brush. This method is time and labor intensive, may alter normal foraging behavior, and can result in stinging incidents for the worker performing the task. Pollen traps, such as those used on honey bee hives, collect pollen by dislodging corbicular pollen loads from the legs of workers as they pass through screens at the nest entrance. Traps can remove a large quantity of pollen from returning forager bees with minimal labor, yet to date no such trap is available for use with bumble bee colonies. Workers within a bumble bee colony can vary in size making size selection of entrances difficult to adapt this mechanism to commercially reared bumble bee hives. Using 3D printing design programs, we created a pollen trap that successfully removes the corbicular pollen loads from the legs of returning bumble bee foragers. This method significantly reduces the amount of time required by researchers to collect pollen from bumble bee foragers returning to the colony. We present the design, results of pollen removal efficiency tests, and suggest areas of modifications for investigators to adapt traps to a variety of bumble bee species or nest box designs.

A 3D Printed Pollen Trap for Bumble Bee Bombus Hive Entrances

We present a non-lethal and automated mechanism to collect pollen from bumble bee (Bombus) workers returning to a hive. Instructions for producing, preparing, installing and using the devices are included. By using 3D-printed objects, modification to the design was timely, efficient and allowed for quick turnaround for testing.

To verify the plant sources from which bumble bees forage for pollen, individuals must be collected to remove their corbicular pollen loads for analysis.  ...

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

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