Name: a novel behavioral assay to investigate gustatory responses of individual, freely-moving bumble bees (bombus terrestris)
Uploaded: 2016-07-21T14:00:00.000+00:00
Duration: 7 min 30 s
Description: 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

这项工作是由利华休姆信托基金赠款（RPG-2012-708）和BBSRC资助（BB / M00709X / 1）GAW资助。

1.捕获从殖民地和饥饿时间蜜蜂注意:这里描述实验在纽卡斯尔大学，英国， 欧洲熊蜂奥达斯执行。多个（2-3）购得菌落每次处理使用。菌落一直保持在实验室条件在不断的黑暗（25±2℃和28±2％RH）的长凳上和饲喂蜜蜂采集花粉和糖溶液自由采食 。 <ol><li>收集使用塑料瓶（7厘米长，2.8厘米内径）用有孔塑料塞个别工人熊蜂，已经打开后，门殖民地刚好够长的一个蜜蜂退出和被困住。 </li><li>在实验前，单独饿死所有熊蜂在塑料小瓶2-4小时，并保持在完全黑暗的房间的温度。 </li></ol> 2.蜜蜂转移到控股管和Habituation阶段<ol><li>饥饿期之后，直接从塑料小瓶转移大黄蜂成保持管。保持管是改性15ml离心管（长119毫米;直径17mm）的，与在前端钻出4毫米的孔和一块钢网（碱:8毫米;高30mm）的通过熔化内部固定管的塑料用加热解剖钢针。 </li><li>修复含有大黄蜂到牙科蜡聚苯乙烯持有人保持管。固定在保持管的任一侧纸板的两件。这是为了从视觉刺激可能与实验干扰屏蔽蜂。 </li><li>数码相机显微位置上的保持管的顶端的上方5厘米，将相机连接到兼容的笔记本电脑。 </li><li>调整保持管，使得至少保持管末端的前18毫米是视频帧范围内。在实验前，开始的3分钟习惯期。 </li></ol> 3。预测试阶段:呈现蔗糖的降<ol><li>注射器连接到含有蔗糖溶液的液滴的阴接头（〜3.5微升，500毫蔗糖溶解在去离子水）。目前保持管内的尖蔗糖激励的喙的扩展。 </li><li>给大黄蜂长达5分钟消耗蔗糖液滴。如果液滴没有被消耗，请从实验中的大黄蜂。 </li><li>习惯性期后开始录像。在这项研究中，长鼻活动被记录在26.7帧/秒-1用25X放大率。 </li></ol> 4.测试阶段:提交测试解决方案<ol><li>填用试验溶液中的100μl的微毛细管。它连接到一个片硅管（6厘米长，内径1毫米）的，并将其固定到一个微操纵器。 <ol><li>通过男性适配器连接管道到另一个硅胶管（6厘米长，内径4毫米），WHI通道充当吸管灯泡。 </li><li>微毛细管5-10毫米从保持管尖端远的地方。轻轻挤压管，以保持在微毛细管的前端馈送溶液。 </li></ol></li><li>大黄蜂消耗液滴的蔗糖后，立即删除含有500毫米蔗糖溶液注射器。 </li><li>开始2分钟测试阶段时，大黄蜂的长鼻接触微细管内的解决方案。 <ol><li>为了控制可能蒸发，填充用蔗糖或水两个附加微细管和准确操纵它作为在测试阶段。 </li></ol></li><li>每次试验之前和之后用扫描仪以600dpi以测量食物消耗（ 图4A）的量扫描微毛细管内的液体水平。 </li></ol> 5.图像分析<ol><li>确定溶液的消费使用ImageJ体积（1.48版本），图象PROC咝声软件。 <ol><li>上传图片文件和放大图像（〜400％）。设置参考尺度，选择直线工具画出微毛细管的两端之间的线路。选择"分析"，然后"设置规模"。输入管的下&#39;的已知距离&#39;和下&#39;长度单位"对应的单位的总长度。 </li><li>再次选择直线工具画出液面的两端之间的线路。选择"分析"然后&#39;对策&#39;。在结果窗口中的液体的长度是在"长度"列下给出。 </li></ol></li><li>通过使用公式计算溶液消耗的体积: <img alt="式（1）" src="/files/ftp_upload/54233/54233eq1.jpg" /> 哪里<img alt="公式（2）" src="/files/ftp_upload/54233/54233eq2.jpg" />是微毛细管的长度和<img alt="公式3" src="/files/ftp_upload/54233/54233eq3.jpg" />和<img alt="式4"SRC ="/文件/ ftp_upload / 54233 / 54233eq4.jpg"/&gt;是微毛细管内的液体的测量长度分别之前和测试阶段之后，。 </li></ol> 6.视频分析<ol><li>期间使用的事件记录软件的每个视频的2分测试阶段得分取食行为（见材料表 ）。 <ol><li>首先，在定义录音软件的行为类别菜单中的摄食行为（ 即元素）。馈送行为如下: （1）长鼻出/接触:长鼻延伸，并与所述微细管内的溶液（2）长鼻出/不接触接触:长鼻延伸，并且不与微细管内的溶液相接触，（3）长鼻存放:长鼻不延长，但头部和（4）淡出人们的视线，而不是下存放:大黄蜂是出了视频帧。 </li><li>设置每个行为作为一个"国家"，并在属性菜单"互斥"，并连续记录了2分钟时间。在回放慢动作模式下的视频（2次更慢）更高精度。 </li></ol></li><li>测量使用运动跟踪视频软件从两个连续帧（37.5毫秒在这里所示的视频记录分开）之间的第一接触后的试验溶液长鼻回缩的速度（见材料表 ）。 <ol><li>上传的视频文件，并跳到帧中长鼻首次接触解决方案。 </li><li>要设置参考比例，选择直线工具画在视频帧的微毛细管的宽度的线。右键单击该行并选择"校准措施"。输入毛细管的宽度和相应的单元。 </li><li>选择"图像"，然后"坐标系原点"。在新的窗口中点击喙的末端，并选择9;应用"。 </li><li>选择手移动工具，右键点击喙的尖端视频帧，然后选择"轨道路径"。移动到下一帧，并重新调整的跟踪点的喙的末端。 </li></ol></li><li>右击跟踪点，并选择"配置"。选择"完整路径"，并在测量中选择"速度"。选择"应用"。然后显示速度。 </li></ol>

<ol>
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作者宣称没有利益冲突。

有了这个新的行为分析，奎宁显示威慑的buff尾大黄蜂的喂养。减小的长鼻接触时间和喂食发作频率与水或与奎宁股价的蔗糖溶液这里解释为一个拒绝启动关于非营养性或潜在有毒的溶液进一步馈送。当奎宁被添加至1M蔗糖溶液，熊蜂不仅降低的溶液所消耗的体积，他们也缩回长鼻快，从而降低了口器和含有毒素的溶液之间的接触时间。总之，这些结果表明，奎宁是通过在大黄蜂的口器的味觉受体细胞感知，如蜜蜂9先前已经识别。奎宁是昆虫毒素诱导的蜜蜂10击倒在疟蚊（ 按蚊 ）23萎靡样行为。此法很可能导致identificat由该味觉受体细胞上的熊蜂口器感知一些威慑和潜在的有毒化合物的离子。 这是至关重要的微毛细管与试验溶液足够体积填充持续整个试验阶段。它建议在微毛细管（ 例如 70-80微升）三至少周围小区被充满。然而，应注意不要完全填充微毛细管，以减少扫描的过程中溢出的风险，并在微毛细管附着在实验装置。呈现的500mM蔗糖液滴时向大黄蜂，使实验者避免泄漏液滴到保持管护理还应该采取。 在保持管的尖端4毫米的孔是为成人工熊蜂足够大，以自然朝向试液延长其长鼻。然而可能的是熊蜂可以延长其proboscises前品尝他们的触角解决方案。这可能会影响如PER可以在熊蜂通过用糖溶液15刺激触角被引出长鼻扩展的概率。事实上膜翅目，如寄生蜂（Trissolcus brochymenae）24或蜜蜂13天线都配有味道感受器，让他们品尝到糖和毒素类似奎宁。因此，提供解决方案含有奎宁一样高度的威慑化合物最初接触触角也能够降低大黄蜂的动机扩大其长鼻，因此影响了实验的成功率。虽然与试验溶液触角接触不能被控制，在本研究中，我们没有发现在长鼻分机朝向测试溶液触角接触的任何显著效果。在该试验中，试验前的W相后，立即建立微毛细管母鸡熊蜂"触角仍保持管内可以减少机会的熊蜂品尝他们的触角供试品溶液。 从使用运动跟踪视频软件第一长鼻接触后的试验溶液跟踪长鼻回缩时远离该测定的主要局限产生。的视频画面仅显示长鼻的二维运动，所以速度测量的给定的输出可以是低于或高于估计。然而有一些修改，测定的此方面还有待改进。 该测定可用于观察朝向包含不同化合物，包括天然存在的植物次生代谢物溶液自然馈送响应。观察用这个方法直接喂养的响应给出了关于熊蜂如何检测这些化合物的详细信息。这是在现有的优势"走出去，没有去"的方法，如PER 18,19 </sup&gt;和200多选择题测定7，因为这种方法会产生一些行为的应对措施，包括离散喂食发作期间食品消费。 测量几个参数允许同时的化合物的适口性的更好的评估。例如，在我们的实验中，熊蜂避免食用水或奎宁里掺蔗糖溶液。长鼻回缩可以通过在糖受体的细胞12,13的响应的变化所导致。我们的分析表明，熊蜂缩回比单独的水​​蔗糖加奎宁溶液接触后长鼻快;这可能表明，奎宁影响一组不同的神经元在除了抑制糖传感神经元9,12,13,25。 我们的分析允许饲养过程中的行为反应的时空格局的分析。其中消费时间和较量的数目测定的类似协议具有人准备实施评估果蝇对营养和非营养性糖26进食反应。我们预测，蜜蜂会表现出在我们的测定法比其他方法如PER进食刺激剂的更可靠的响应，因为蜜蜂自由在保持管21移动。该技术将允许味道阈值的详尽分析营养物质和毒素照亮熊蜂和潜在的其他蜂种饲养的机制。

植物授粉的相互作用是复杂的。传粉者访花获得花蜜和花粉为食;反过来，授粉促进植物有性生殖。虽然这种关系是互惠大部分，花蜜和花粉有时含有毒素或其他植物化合物1-5 这会伤害传粉者。在花蜜和花粉等化合物的存在的理由生态并非所有的设置清晰。在这一领域的一个突出问题是，传粉昆虫如蜜蜂如何检测和避免花果茶含有毒素。 大黄蜂物种， 欧洲熊蜂 （林奈，1758），是一个通才授粉的访问许多植物物种，包括含有毒素6那些生产花蜜的花朵。熊蜂已经示出，以避免含有在24小时的两选择测定7高浓度的毒素消耗的解决方案。此法通过Tiedeken 等 7所描述的食品消费透露，蜜蜂可以检测解决方案的苦味化合物。然而，该测定不能从后摄食工艺如不适也可能影响超过这个时间间隔8-10摄食行为区分的味道。 蜜蜂拥有自己的触角，口器和跗节味觉感受器来检测化合物11-13。长鼻扩展反射（PER）的实验涉及线束抑制个别蜜蜂，然后刺激蜜蜂的触角感受器产生反射喂养14-17。蜜蜂可以在个别线束克制，然后刺激产生的喂养反射作为其味化合物18,19能力的检测。他人已经修改了PER测定来研究天线或口器毒素9,20的灵敏度。然而，蜜蜂是利用过程中受到的压力。这可能会影响如何他们的反应的化合物21。 这里，一个新的测定法被描述为评估的可自由移动的熊蜂蔗糖和奎宁，先前已报道为威慑的生物碱行为味道响应9和有毒10蜜蜂（ 蜜蜂 ）和熊蜂（ 熊蜂蒺藜 ）7，22。虽然奎宁尚未在植物花蜜发现，这种生物碱常被用作在蜂7,9,12,13,22在行为和生理研究厌恶的刺激。该方法包括与测试解决方案最初的长鼻接触时视频录制熊蜂"的口器在大分辨率。具体地，馈送响应的精细结构是通过在2分钟的时间间隔连续地进行为检查。的溶液消耗的体积在馈送期间被测量，因此食用食物的量可与微结构相关联摄食行为。也长鼻回缩的速度被测量，作为主动回避的一个指标，并且因此预摄食检测。

<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)

通才授粉喜欢的buff尾大黄蜂， 欧洲熊蜂 ，遇到既营养物质和毒素，他们从开花植物采集花蜜。只有少数的研究已经描述了朝向食品的毒素蜜蜂的味觉，并且这些实验主要用于抑制蜜蜂的长鼻分机应答。这里，一个新的行为分析提出了测量可自由移动的馈送的响应，每个工人熊蜂到营养物质和毒素。此法测量解决方案由每个大黄蜂摄入量，并确定食品中促味剂如何影响摄食行为的微观结构。 该解决方案在一个微毛细管个别熊蜂先前已饥饿2-4小时出现。该行为是在数字视频捕捉。取食行为的精细结构是由连续得分probo的位置分析SCIS使用的事件记录软件视频录制（口器）。长鼻的位置是由三个不同的行为类别定义如下:（1）长鼻延伸并在与该溶液接触，（2）长鼻延伸但不与溶液及（3）长鼻接触头下收起。此外还估计长鼻从溶液缩回远的速度。 在本测定法的溶液的体积消耗，喂食发作的次数，馈送发作的持续时间和所述第一接触后的长鼻回缩的速度用来评估phagostimulatory或测试的化合物的威慑活性。 这种新口味法将允许研究人员测量花蜜发现的化合物是如何影响蜜蜂的摄食行为，也将是授粉生物学家，毒物学家和研究neuroethologists的大黄蜂的味觉系统是有用的。

新颖的测定用于馈送回复测试至1M蔗糖，1M蔗糖溶液加1mM的奎宁和单独去离子水。直接馈送响应到每个治疗由定量用试验溶液，馈送发作的频率和长鼻期间2分钟试验阶段第一接触后缩回从测试溶液远的速度长鼻接触的持续时间来确定。消耗溶液的体积也在测试阶段后测量。在这项研究中，我们选择了5秒的一个回合标准间隔基于法国等 25谁使用5秒的阈值由果蝇响应表征长鼻回缩行为威慑以前的工作（ 图1，见补充文件 ）化合物25。因此，我们定义一个馈送回合作为扩展长鼻和溶液之间的接触不吨由缺乏5秒以上的接触的中断。 相比于蔗糖和单独的去离子水，加入奎宁蔗糖溶液通过熊蜂显然阻止馈送，因为它们会迅速移开，如果他们检测厌恶物质（ 视频图1）。 在该实验中，处理对长鼻触点的累积持续时间在测试阶段一个显著效果（对数转化数据的ANOVA，女2,31 = 41，P &lt;0.001）。的接触时间与含奎宁蔗糖的累积持续时间比较显著降低到单独蔗糖（P &lt;0.001），但不向去离子水单独（p值= 0.219）（ 图2）。同样的，处理对的馈送发作（ANOVA对数变换的数据，女的累积持续时间的显著效果<sUB&gt; 2,31 = 27.95，P &lt;0.001， 图3A）。喂养含有奎宁蔗糖较量的累积持续时间比较显著降低到单独蔗糖（P &lt;0.001），但不与单独的去离子水（p值= 0.41）。的治疗也有对馈送发作频率的显著效果（泊松GLM具有日志链接函数，在偏差变化相比的C 2配电:P &lt;0.050），由此用含奎宁蔗糖较量的数目是在显著更高比较蔗糖（p &lt;0.01），但略有显著不同的去离子水处理（p值= 0.055，由于一个熊蜂上显示排放在7喂食发作， 图3B）。同样，长鼻回缩的速度处理之间显著不同（ANOVA对数变换的数据，女2,31 = 5.12，P &lt;0.050）。熊蜂收回PRoboscis从测试溶液与含有奎宁比蔗糖或单独的去离子水（P &lt;0.050， 图3C）蔗糖第一接触后显著更快了。这些结果表明，奎宁触发熊蜂一个主动回避行为。的治疗也有溶液的总体积消耗（ANOVA对数变换的数据，女2.32 = 62.5，P &lt;0.001），由此含奎宁蔗糖的消耗相比被减少到蔗糖（ 第一个显著效果&lt;0.001），但不以去离子水（p值= 0.457）（ 图4B）。的溶液在测试期间从毛细管蒸发量是微不足道的。在实验室条件下（25±2℃和28±2％RH）中，在蒸发0.033至0.883微升之间，平均0.276微升和分别0.171微升去离子水和1M蔗糖的不同而不同。 ，C 2分布:p = 0.450）。的治疗没有效果在第一触角接触和测试溶液和长鼻（中位数的第一触头之间的延迟值:2.67秒为蔗糖; 1.10秒为蔗糖加奎宁; 0.80秒为去离子水，方差分析对数转换数据，女2,13 = 0.620，p = 0.550）。此外，延长长鼻品尝测试解决方案熊蜂的比例仍然在整个治疗（蔗糖不变:66.7％;蔗糖加奎宁:50.0％;去离子水:52.2％;二项式GLM，在越轨行为的变化相比，C 2分布:P = 0.840）。一起这些结果表明，该天线在检测在该测定的毒素发挥次要作用。 一个单独的实验检查是否有必要测试蜜蜂一段时间大于2分钟长。一个2分钟的测试周期和10分钟的测试期间:食品的蜜蜂消耗的量与所述的1M蔗糖或1mM的奎宁在1M蔗糖溶液以两种条件下进​​行测试。对于这两种治疗，总食物消耗不为测试周期不同并且在测试期间和治疗之间没有发生显著相互作用（N = 6 - 13，方差分析上的日志变换数据;的治疗效果:F 1,31 = 54.8，p &lt;0.001;在测试期效果:F 1,31 = 0，p为0.979;相互作用的效果:F = 0.1，p值= 0.457）。总之，一2分钟的测试时间足以评估在食物的由大黄蜂和在该试验中的有毒或驱避物质压制作用消耗的总量的溶液的效果。因此，通过测量食物消耗并测定摄食行为，能够在测定期间相关联的总食物消费馈送的精细结构。 <img alt="图1" src="/files/ftp_upload/54233/54233fig1.jpg" /> 图1:摄食测定法的第一2分钟时长鼻联系人之间的潜伏期时间潜伏期与分离的1M的蔗糖溶液各长鼻接触密度图，1蔗糖+ 1毫米奎宁溶液和水。从13，10和11蜜蜂的累积数据分别表示。 <a href="https://www.jove.com/files/ftp_upload/54233/54233fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> &lt;/ P&gt; <img alt="图2" src="/files/ftp_upload/54233/54233fig2.jpg" /> 图2: 长鼻联系工期摄食测定法的第一2分钟期间 ，大黄蜂1蔗糖，1M蔗糖+ 1毫米奎宁或水喂长鼻接触持续时间的密度图。试样尺寸如图1所示。 <a href="https://www.jove.com/files/ftp_upload/54233/54233fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54233/54233fig3.jpg" /> 图3:熊蜂喂养的1M蔗糖，1M蔗糖+ 1mM的奎宁或水的长鼻活性 （A）的进给的较量在测试阶段（B）的喂食发作的频率和（C）的速度的累积持续时间。长鼻重第一次接触后牵引。刻字表示显著区别:用不同的字母表示处理P &lt;0.05。箱形图表示位数（黑网吧），内间距范围的1.5的最低和最高的数据点仍然（威威）和异常值（圆圈）。试样尺寸如图1所示。 <a href="https://www.jove.com/files/ftp_upload/54233/54233fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54233/54233fig4.jpg" /> 图4:试验前和试验阶段之后微细管示出的1M蔗糖的水平或1M的蔗糖加1mM的奎宁溶液（由黑线表示）的奎宁supresses由熊蜂馈送 （A）的扫描图像分别。 （ 二 ）1蔗糖消费，1M蔗糖加1毫米奎宁或去离子水<html>通过测试阶段后熊蜂孤单。刻字表示显著区别:用不同的字母表示处理P &lt;0.001。箱形图表示位数（黑网吧），内间距范围的1.5的最低和最高的数据点仍然（威威）和异常值（圆圈）。试样尺寸如图1所示。 <a href="https://www.jove.com/files/ftp_upload/54233/54233fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 视频图1:长鼻活动对（A）的视频录制<a href="https://www.jove.com/files/ftp_upload/54233/Video_figure_1A_1M_Sucrose.m4v" target="_blank">1蔗糖</a> ，（B） <a href="https://www.jove.com/files/ftp_upload/54233/Video_figure_1B_Quinine.m4v" target="_blank">1蔗糖加1毫米奎宁</a>和（C）D / 54233 / Video_figure_1C_Water.m4v"目标="_空白"&gt;去离子水测试阶段中。

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.

一种新的行为分析调查个人的味觉，可自由移动的熊蜂（<em&gt;欧洲熊蜂</em&gt;）

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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Neuroscience

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

8.6K 次观看.  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. 

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

Proboscis Extension Response (PER) Assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth (labellum), there are hair-like structures called sensilla which houses taste neurons. When an attractive substance makes contact to the labellum, the fly extends its proboscis to consume the material. Proboscis Extension Response (PER) assay measures this taste behavior response, and it is a useful method to learn about food preferences in a single fly. Solutions of various sugars, such as sucrose, glucose and fructose, are very attractive to the fly. The effect of aversive substances can also be tested as reduction of PER when mixed in a sweet solution.Despite the simplicity of the basic procedure, there are many things that can prevent it from working. One of the factors that requires attention is the fly's responsive state. The required starvation time to bring the fly to the proper responsive state varies drastically from 36 to 72 hours. We established a series of controls to evaluate the fly's state and which allows screening out of non-responsive or hyper-responsive individual animals. Another important factor is the impact level and the position of the contact to the labellum, which would be difficult to describe by words. This video presentation demonstrates all these together with several other improvements that would increase the reproducibility of this method.

Proboscis Extension Response PER Assay in Drosophila

Proboscis extension response or PER is a taste behavior assay that has been used in flies as well as in honeybees. When the proboscis makes contact with an attractive substance, the fly extends its proboscis to consume the substance. Solutions of various sugars are very attractive to the fly.

长鼻扩展响应（PER）的含量在果蝇

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth ...

科研

生物学

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.

在蜜蜂的长鼻扩展响应的经典条件反射行为药理学（蜜蜂）

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

神经科学

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.

触觉空调和取样策略在蜜蜂触角的运动分析（<em&gt;蜜蜂</em&gt; 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 ...

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.

的垂直T-迷宫的选择含量，节肢动物响应加味剂

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.

RNAi介导的双基因敲除和味觉感知测量蜜蜂（<em&gt;蜜蜂</em&gt;）

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.

单苍蝇检测方法的觅食行为的<em&gt;果蝇</em

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.

长鼻扩展响应协议为侦查行为可塑性的昆虫：应用基础，生物医学和农业研究

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

Testing Drosophila Olfaction with a Y-maze Assay

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.

Testing Drosophila Olfaction with a Y-maze Assay

We present a choice test to reveal the influence of odorants on Drosophila behavior using a Y-maze assay.

测试<em&gt;果蝇</em&gt;用Y型迷宫测定嗅觉

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, ...

Taste Preference Assay for Adult Drosophila

Olfactory and gustatory perception of the environment is vital for animal survival. The most obvious application of these chemosenses is to be able to distinguish good food sources from potentially dangerous food sources. Gustation requires physical contact with a chemical compound which is able to signal through taste receptors that are expressed on the surface of neurons. In insects, these gustatory neurons can be located across the animal's body allowing taste to play an important role in many different behaviors. Insects typically prefer compounds containing sugars, while compounds that are considered bitter tasting are avoided. Given the basic biological importance of taste, there is intense interest in understanding the molecular mechanisms underlying this sensory modality. We describe an adult Drosophila taste assay which reflects the preference of the animals for a given tastant compound. This assay may be applied to animals of any genetic background to examine the taste preference for a desired soluble compound.

Taste Preference Assay for Adult Drosophila

Taste is an important sensory process which facilitates attraction to beneficial substances and avoidance of toxic substances. This protocol describes a simple ingestion assay for determining Drosophila gustatory preference for a given chemical compound.

口味偏好测定成人<em&gt;果蝇</em

Olfactory and gustatory perception of the environment is vital for animal survival.  The most obvious application of these chemosenses is to be able to ...

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.

SwarmSight: 利用共同的准备和常规硬件实时跟踪昆虫的天线运动和喙的延伸反射

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

一种新的行为分析调查个人的味觉，可自由移动的熊蜂（

摘要

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此视频中的章节

长鼻扩展响应（PER）的含量在果蝇

在蜜蜂的长鼻扩展响应的经典条件反射行为药理学（蜜蜂）

触觉空调和取样策略在蜜蜂触角的运动分析（

的垂直T-迷宫的选择含量，节肢动物响应加味剂

RNAi介导的双基因敲除和味觉感知测量蜜蜂（

单苍蝇检测方法的觅食行为的

长鼻扩展响应协议为侦查行为可塑性的昆虫：应用基础，生物医学和农业研究

测试

口味偏好测定成人

SwarmSight: 利用共同的准备和常规硬件实时跟踪昆虫的天线运动和喙的延伸反射