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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E., Menzel, R., Fietz, A., Schafer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+conditioning+of+proboscis+extension+in+honeybees+(Apis+mellifera).">Classical conditioning of proboscis extension in honeybees (Apis mellifera).</a> J Comp Psychol. 97, 107-119 (1983).</li><li>Laloi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+conditioning+of+the+proboscis+extension+in+bumble+bees.">Olfactory conditioning of the proboscis extension in bumble bees.</a> Entomol Exp Appl. 90, 123-129 (1999).</li><li>Smith, B. H., Burden, C. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bees+prefer+foods+containing+neonicotinoid+pesticides.">Bees prefer foods containing neonicotinoid pesticides.</a> Nature. 521, 74-76 (2015).</li><li>Mommaerts, V., Wackers, F., Smagghe, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+gustatory+responses+to+different+sugars+in+harnessed+and+free-moving+bumblebee+workers+(Bombus+terrestris).">Assessment of gustatory responses to different sugars in harnessed and free-moving bumblebee workers (Bombus terrestris).</a> Chem Senses. 38, 399-407 (2013).</li><li>Chittka, L., Dyer, A. 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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 border="0" cellpadding="0" cellspacing="0" width="1383">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Bumblebee colonies</td>
			<td style="width:211px;">Koppert Biological Systems</td>
			<td style="width:255px;"></td>
			<td style="width:411px;">NATUPOL Beehive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Digital microscopic camera&#160;</td>
			<td style="width:211px;">Dino-lite Europe</td>
			<td style="width:255px;">AM4815ZT</td>
			<td>Dino-Lite Edge&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">100 &#956;l microcapillary tube&#160;</td>
			<td style="width:211px;">Blaubrand IntraEND</td>
			<td style="width:255px;">709144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">15 ml polypropylene centifuge tube&#160;</td>
			<td style="width:211px;">Fisher Scientific</td>
			<td style="width:255px;">11849650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">1 ml disposable plastic luer slip syringe</td>
			<td style="width:211px;">BD</td>
			<td style="width:255px;">300013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Dell Latitude 3550 laptop</td>
			<td style="width:211px;">Dell</td>
			<td style="width:255px;"></td>
			<td>Check for compatibility with video software&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Canon CanoScan LiDE 120</td>
			<td style="width:211px;">Canon</td>
			<td style="width:255px;"></td>
			<td>Check for compatibility with the computer/laptop</td>
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			<td height="21" style="height:21px;width:507px;">Observer software version 5.0.25</td>
			<td style="width:211px;">Noldus</td>
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			<td height="21" style="height:21px;width:507px;">Kinovea software version 0.8.15</td>
			<td style="width:211px;">Kinovea&#160;</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:507px;">silicone tubing</td>
			<td style="width:211px;"></td>
			<td style="width:255px;"></td>
			<td>6&#160;cm length, 1&#160;mm inside &#216; &#38; 6&#160;cm length, 4&#160;mm inside &#216;</td>
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		<tr height="23">
			<td height="23" style="height:23px;width:507px;">Male luer x 1/16&#34; standard hose barbed polypropylene adapter</td>
			<td style="width:211px;">Cole-Parmer</td>
			<td style="width:255px;">TW-45518-22</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:507px;">Female luer x 1/16&#34; standard hose barbed polypropylene adapter</td>
			<td style="width:211px;">Cole-Palmer</td>
			<td style="width:255px;">TW-45508-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Steel mesh&#160;</td>
			<td style="width:211px;"></td>
			<td style="width:255px;"></td>
			<td>0.5 mm mesh size</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Sucrose (grade II)&#160;</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td>S5391</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:507px;">Quinine hydrochloride dihydrate</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td>Q1125</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:507px;">ImageJ software version 1.48</td>
			<td style="width:211px;">ImageJ</td>
			<td></td>
			<td></td>
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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 ...

a-novel-behavioral-assay-to-investigate-gustatory-responses

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.6K 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

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors. Using animal models to accomplish this goal, many methodological strategies require invasive techniques to manipulate the intensity of the behavior of interest (e.g., lesion methods, pharmacological manipulations, microdialysis techniques, genetically-engineered animal models). The utilization of a comparative species approach allows researchers to take advantage of naturally occurring differences in response strategies existing in closely related species. In our lab, we use two species of the Peromyscus genus that differ in paternal responses. The male California deer mouse (Peromyscus californicus) exhibits the same parental responses as the female whereas its cousin, the common deer mouse (Peromyscus maniculatus) exhibits virtually no nurturing/parental responses in the presence of pups. Of specific interest in this article is an exploration of the neurobiological factors associated with the affiliative social responses exhibited by the paternal California deer mouse. Because the behavioral neuroscience approach is multifaceted, the following key components of the study will be briefly addressed: the identification of appropriate species for this type of research; data collection for behavioral analysis; preparation and sectioning of the brains; basic steps involved in immunocytochemistry for the quantification of vasopressin-immunoreactivity; the use of neuroimaging software to quantify the brain tissue; the use of a microsequencing video analysis to score behavior and, finally, the appropriate statistical analyses to provide the most informed interpretations of the research findings.

The comparative species approach allows behavioral neuroscientists to explore various neurobiological factors associated with specific behaviors viewed as characteristic of a specific animal model. Taking advantage of naturally occurring differences in behavior between closely related species, this technique doesn&#x2019;t require invasive techniques to manipulate the expression of the behavior.

A goal of behavioral neuroscience is to identify underlying neurobiological factors that regulate specific behaviors.  Using animal models to accomplish ...

MPI CyberMotion Simulator: Implementation of a Novel Motion Simulator to Investigate Multisensory Path Integration in Three Dimensions

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. Humans can do path integration based exclusively on visual 2-3, auditory 4, or inertial cues 5. However, with multiple cues present, inertial cues - particularly kinaesthetic - seem to dominate 6-7. In the absence of vision, humans tend to overestimate short distances (&lt;5 m) and turning angles (&lt;30&deg;), but underestimate longer ones 5. Movement through physical space therefore does not seem to be accurately represented by the brain.
Extensive work has been done on evaluating path integration in the horizontal plane, but little is known about vertical movement (see 3 for virtual movement from vision alone). One reason for this is that traditional motion simulators have a small range of motion restricted mainly to the horizontal plane. Here we take advantage of a motion simulator 8-9 with a large range of motion to assess whether path integration is similar between horizontal and vertical planes. The relative contributions of inertial and visual cues for path navigation were also assessed.
16 observers sat upright in a seat mounted to the flange of a modified KUKA anthropomorphic robot arm. Sensory information was manipulated by providing visual (optic flow, limited lifetime star field), vestibular-kinaesthetic (passive self motion with eyes closed), or visual and vestibular-kinaesthetic motion cues. Movement trajectories in the horizontal, sagittal and frontal planes consisted of two segment lengths (1st: 0.4 m, 2nd: 1 m; &plusmn;0.24 m/s2 peak acceleration). The angle of the two segments was either 45&deg; or 90&deg;. Observers pointed back to their origin by moving an arrow that was superimposed on an avatar presented on the screen.
Observers were more likely to underestimate angle size for movement in the horizontal plane compared to the vertical planes. In the frontal plane observers were more likely to overestimate angle size while there was no such bias in the sagittal plane. Finally, observers responded slower when answering based on vestibular-kinaesthetic information alone. Human path integration based on vestibular-kinaesthetic information alone thus takes longer than when visual information is present. That pointing is consistent with underestimating and overestimating the angle one has moved through in the horizontal and vertical planes respectively, suggests that the neural representation of self-motion through space is non-symmetrical which may relate to the fact that humans experience movement mostly within the horizontal plane.

An efficient way to gain insight into how humans navigate themselves in three dimensions is described. The method takes advantage of a motion simulator capable of moving observers in ways unattainable by traditional simulators. Results confirm that movement in the horizontal plane is underestimated, while vertical movement is overestimated.

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

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 ...

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

New Methods to Study Gustatory Coding

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons (GRNs) detect tastant molecules, they encode information about the identity and concentration of the tastant as patterns of electrical activity that then propagate to follower neurons in the brain. These patterns constitute internal representations of the tastant, which then allow the animal to select actions and form memories. The use of relatively simple animal models has been a powerful tool to study basic principles in sensory coding. Here, we propose three new methods to study gustatory coding using the moth Manduca sexta. First, we present a dissection procedure for exposing the maxillary nerves and the subesophageal zone (SEZ), allowing recording of the activity of GRNs from their axons. Second, we describe the use of extracellular electrodes to record the activity of multiple GRNs by placing tetrode wires directly into the maxillary nerve. Third, we present a new system for delivering and monitoring, with high temporal precision, pulses of different tastants. These methods allow the characterization of neuronal responses in vivo directly from GRNs before, during and after tastants are delivered. We provide examples of voltage traces recorded from multiple GRNs, and present an example of how a spike sorting technique can be applied to the data to identify the responses of individual neurons. Finally, to validate our recording approach, we compare extracellular recordings obtained from GRNs with tetrodes to intracellular recordings obtained with sharp glass electrodes.

We present three new methods to study gustatory coding. Using a simple animal, the moth Manduca sexta (Manduca), we describe a dissection protocol, the use of extracellular tetrodes to record the activity of multiple gustatory receptor neurons, and a system for delivering and monitoring precisely timed pulses of tastants.

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...