Name: video tracking protocol to screen deterrent chemistries for honey bees
Uploaded: 2017-06-12T14:00:00.000+00:00
Duration: 10 min 19 s
Description: Watch this Scientific Journal Video about Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees at JoVE.com

我们要感谢Thomas Kuhar博士使用视觉跟踪软件和设备。我们感谢詹姆斯·威尔逊和斯科特·奥尼尔的技术援助。

1.准备糖琼脂糖立方体<ol><li>称取8克糖并置于50mL锥形瓶中。 </li><li>向锥形瓶中加入20mL去离子水。通过旋转烧瓶来溶解糖。 </li><li>称量170 mg琼脂糖，并加入糖溶液。 </li><li>将糖 - 琼脂糖溶液在微波炉中加热25秒。将琼脂糖溶解在糖溶液中。 </li><li> 让烧瓶和糖琼脂糖溶液冷却。 注意:烧瓶应该是凉爽的接触，但不要让溶液凝固。 <ol><li>为了制备用于对照处理的糖琼脂糖立方体，将半冷糖 - 琼脂糖溶液倒入称重模具中。 注意:称重模具的尺寸为1.5 x 1.5 x 0.3 cm 3 。 </li><li>为了制备用于驱避剂处理的糖 - 琼脂糖复合立方体，向半冷却溶液中加入所需量的化合物（ 例如 ，1％DEET in sugar-agarose溶液（v / v））。旋转烧瓶以混合化合物，然后将溶液倒入称重模具中。 注意:此时将准备八个控制模具和八个测试模具。控制模具不含驱避剂。 </li></ol></li><li>将糖琼脂糖立方体放入冰箱中的模具中5-10分钟。从称重船模具中取出固化的糖 - 琼脂糖立方体，并将其放入带有湿纸巾的塑料容器中。 </li><li>将容器放入冰箱保存。糖琼脂糖立方体应在制备后7天内使用。 </li></ol> 2.编程视频跟踪软件和实验设置<ol><li> 在实验资源管理器栏中的实验设置栏（在跟踪软件中，请参阅材料表），位于屏幕左侧，确保选择了正确的摄像头，并将视频录制以培养皿为中心。<ol><li>如果视频录制需要居中，请进入摄像机的设置，并在AOI控制下，选择中心X和中央Y选项。 </li></ol></li><li>在实验设置下，将场地数量更改为16.在跟踪功能下，选择中心点检测。 </li><li> 选择竞技场设置来设置测试所需的竞技场。 <ol><li>将16个陪替氏培养皿放置在灯箱顶部的4 x 4图案上，位于相机下方（ 图1 ）。 注意:这些菜肴用于创建录音舞台，并且是空的。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/55603/55603fig1.jpg" /> 图1:灯箱上的培养皿配置。灯箱顶部放置4×4块培养皿。这种安排提供了对v的控制和驱避治疗的简单识别实时跟踪协议。 <a href="http://ecsource.jove.com/files/ftp_upload/55603/55603fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol start="4"><li>在"竞技场设置"中，使用位于右侧工具面板上的抓取背景按钮拍摄16种培养皿。这将用作设置舞台的模板。 </li><li>从"竞技场设置"屏幕顶部的工具栏中选择"创建椭圆"，并创建一个圆圈，该圆圈与抓取图像中的一种培养皿的直径相匹配。将Arena 1标记放入圆圈（ 图2 ）。 </li></ol><img alt="图2" src="/files/ftp_upload/55603/55603fig2.jpg" /> 图2:可视化跟踪软件竞技场设置的屏幕快照。圆内的对角条纹的观察提供了圆圈中检测区域的确认。一个<html>为每个正方形提供1区标记，并定义每个培养皿的目标区域。 <a href="http://ecsource.jove.com/files/ftp_upload/55603/55603fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol start="6"><li>选择位于右侧工具面板"Arena 1"下方的"Zone Group 1"。从屏幕顶部的工具栏中选择"创建矩形"。使用此方法创建一个30 x 30像素的正方形，然后将其放置在上一步中创建的圆的中心。从顶部工具栏中选择"添加区域"，然后单击正方形中间。移动Zone 1标记，使其位于正方形中。 注意:创建的正方形是进料区域，不要求进料区域与所列的尺寸完全相同，只要其略大于糖琼脂糖立方体。 </li></ol>/ etc / ftp_upload / 55603 / 55603fig3.jpg"/&gt; 图3:完成竞技场设置的屏幕截图。完成的竞技场设置应该是这个例子。 <a href="http://ecsource.jove.com/files/ftp_upload/55603/55603fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol start="7"><li> 在右侧工具面板中选择"竞技场1"，然后单击重复的完整竞技场。从下拉菜单中选择"All Other Arenas"，然后单击"确定"。将重复的竞技场设置移到残留的陪替培养皿中（图3）。 <ol><li>选择"Arena 1"，然后在顶部工具栏上选择"Calibrate Scale"。绘制校准线穿过竞技场1培养皿的直径。将直径测量值更改为9厘米（正在使用的培养皿的直径）。在右侧工具面板上选择"验证竞技场设置"。 </li></ol></li><li>选择 "检测设置"在左边的控制栏上。 </li><li>选择"检测设置1"，然后在右侧工具箱的下拉菜单中选择"灰度调整"。在检测下，设置范围为0到83（ 图4A ）。 </li><li>右键单击"检测设置"，并创建一个名为"检测设置2"的新设置。确保"灰度调整"仍然被选中，但不要更改其他参数。 注意:在场景的视频窗口中将会有大的黄色圆圈。这将有助于在记录之前将培养皿排列在正确的位置（ 图4B ）。 </li></ol><img alt="图4" src="/files/ftp_upload/55603/55603fig4.jpg" /> 图4:检测设置1和2的屏幕截图。 （ A ）显示了对a进行灰度比例校正的场景蜜蜂在培养皿中摄取。 （ B ）显示竞技场检测区域，以便在试验之间进行培养皿的定位。 <a href="http://ecsource.jove.com/files/ftp_upload/55603/55603fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol start="11"><li>在"实验资源管理器"中选择"试用列表"，然后单击顶部工具栏中的"添加变量"。将用户定义的变量命名为"处理"。在用户定义的"治疗"列中选择"预定义值"下拉列，并将C（控制）和T（治疗）添加为预定义值。选择顶部工具栏上的"添加试验"按钮。添加两个试验，然后为每个竞技场选择是否是控制竞技场（C）或治疗竞技场（T）（ 图5 ）。 </li></ol><img alt="图5" src="/files/ftp_upload/55603/55603fig5.jpg" /> 网络连接gure 5:试用列表的屏幕截图。因为程序使用这里的信息来分离数据以对其进行统计分析，所以正确标记场地是很重要的。 <a href="http://ecsource.jove.com/files/ftp_upload/55603/55603fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol start="12"><li> 选择"实验资源管理器"面板下的"数据配置文件"。在左侧列中，选择"用户自定义独立变量"标题下的"处理"。这将在流程图的区域中添加一个"过滤器"框。在弹出框中选择"C"，然后将新创建的过滤器放在"开始"框和"结果1"框之间。流程图箭头应该调整，以便它们从起始框指向过滤器，最后到结果1。 <ol><li>重复步骤2.12，但是这次为过滤器选择"T"，然后选择"结果"在"共同元素"部分。将新创建的框移动到流程图区域并用箭头连接框（ 图6 ）。 </li></ol></li></ol><img alt="图6" src="/files/ftp_upload/55603/55603fig6.jpg" /> 图6:数据配置文件的屏幕快照。这显示了如何设置流程图以在统计分析中获得适当的分离。 <a href="http://ecsource.jove.com/files/ftp_upload/55603/55603fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol start="13"><li>保存文件。 </li></ol>收集蜜蜂个人<ol><li>穿上防护服，选择蜂蜜收集蜜蜂（主要是牧民）。 </li><li>拆下蜂巢的外盖和内盖。使用蜂巢工具从顶部的蜂巢体中选择一个不包含巢的框架。轻轻地将框架从蜂巢盒中取出。 </li><li>检查蜂巢蜜蜂的框架。如果她不在那里，将蜜蜂工人从框架上扫出一个容器运输。收集足够的个人能够对每个要测试的化合物进行两次完整的试验（每个试验使用16个对照）。 注意:所收集的个人应主要为牧师;然而，可能还有其他老年人，如收集中包括的护士蜜蜂。 </li><li>记下蜜蜂被去除的时间。将它们转移到装有空气孔的9cm×7cm×9cm塑料盒中，并将它们放入32℃和70％相对湿度的培养箱中。 </li><li>蜜蜂蜜蜂15小时。 注意:如果蜜蜂在被测试之前的晚上被收集，通常效果最佳。 </li></ol> 4.进行视觉跟踪测定<ol><li>启动视觉跟踪程序并打开保存d实验文件是为此测定创建的。 </li><li>选择"检测设置2"。 </li><li>将控制糖琼脂糖立方体放入16种培养皿中8种的中心。在剩下的8道菜中用糖琼脂糖复合物（威慑）重复此过程。 </li><li>使用镊子从塑料盒中取出一只蜂蜜，将其放入控制培养皿中。 </li><li>对剩余的15种培养皿重复步骤4.4。 </li><li>将所有的菜肴放在灯箱上并定位，使得竞技场检测区域（选择检测设置2时在计算机屏幕上显示）适合每个培养皿场地。通过设置为红色光谱的LED灯阵列从下面照亮灯箱。将整个盒子和相机用黑色塑料片包围，以消除外部光线并减少场内阴影。 </li><li>设置培养皿后，确保"检测设置1"为选择设置。 注意:在这一点上，视觉跟踪软件应该只拾取培养皿中蜂蜜。 </li><li>从实验资源管理器中选择"采集"。按右侧的采集控制弹出框中的绿色"开始试用"按钮开始录制。 </li><li>运行测定10分钟，然后单击采集控制弹出框上的红色"停止试验"按钮停止录制。 </li><li>从培养皿中取出个体，将其放入一个独立的容器中，以免两次测试。 </li><li>对同一化合物的第二次试验重复步骤4.4-4.10。 注意:该方法可以修改，以增加用户根据需要处理的蜂蜜数量。 </li><li>在"实验资源管理器"中选择"统计"，然后单击"计算"。 </li><li>将数据导出到数据管理器软件，然后使用one-w分析数据的重要性使用Tukey的后测试进行方差分析，使用优选的统计软件程序进行不配对的t检验。 </li></ol>

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作者没有什么可以披露的。

这种视觉跟踪协议提供了一种以相对快速和容易的方式筛选蜜蜂的化学阻止剂的简单方法。蜂蜜蜜蜂的化学阻碍实验室测试没有推荐的方案。以前的半场和全场研究已经检查了蜂蜜驱虫剂14,15 ;然而，所描述的协议是耗时的，劳动密集的，并且需要在一般实验室之外的附加设备资源。该方案被设计为对这种化合物与蜂蜜蜜蜜进行半场或全场测试之前对化学威慑进行先决条件的评估...

欧洲蜜蜂， Apis melliferaL。是一种经济和农业上重要的昆虫，提供全球超过2000亿美元的授粉服务1 。在美国和欧洲，蜂蜜的数量一直在减少。美国已经失去了1947-2008年管理的蜂蜜殖民地的60％，而欧洲已经失去了ca。 1961-2007年的27％ 2，3 。有许多因素可能导致殖民地损失数量增加，包括但不限于寄生虫侵袭，病原体感染，养蜂行为和农药使用2-4 。 蜂蜜可能通过两个主要途径暴露于农药。当觅食者接触作物时，可能发生蜂巢外的农药暴露已经喷洒了化学物质以防止害虫。蜂巢中的农药暴露可以发生在养蜂人利用化学物质来控制螨虫，细菌和微孢子虫等害虫和病原体时。在美国和加拿大的5,6个蜂巢中，已经在蜡，花粉和蜂蜜样品中鉴定了农药残留。农药与蜂蜜接触的影响包括急性毒性以及亚致死作用，如麻痹，定向障碍，行为和健康变化1，7 。由于现代农业需要使用农药来维持高产量，这些化学品将来将继续得到依赖2 。为了更好地保护蜂蜜免受农药暴露，需要制定新的议定书和条例5 。一种可能的保护方法是使用驱避剂来减少蜂蜜在杀虫剂中的暴露，同时觅食食物。 驱虫剂（IRs）通常被用作针对节肢动物疾病载体的个人叮咬保护措施8 。超过60年前开发的最广泛使用和成功的IR是DEET 8,9 。被认为是驱虫剂测试的黄金标准，被世界卫生组织和环境保护署用作新型驱虫剂筛选的阳性对照10 。此外，DEET已被发现分散蜂蜜从威胁到他们的殖民地11 。与个人IR相关的当前属性包括:（1）对大量节肢动物的持久影响; （2）用于皮肤或衣服时对使用者无刺激性; （3）无臭或令人愉快的气味（4）对服装无影响; （5）施用于皮肤时不产生油性外观，并能够经受使用者的出汗，洗涤和擦拭; （6）对常用塑料无影响;和（7）化学稳定和价格适中广泛使用12 。用于蜜蜂的驱虫剂只需要一些这些属性，如持久的效果，对施用器无刺激性，无臭或令人愉快的气味，化学稳定且价格实惠，广泛使用，对蜂蜜无毒。然而，在深入探索这些属性之前，需要一种以高通量方式筛选化合物排斥/威慑的方法。在这里，我们描述了实验室测定方法，筛选化合物以抑制蜂蜜蜜蜂，这是确定排斥性的重要步骤。以前的研究修改了以前的研究，描述了一种视觉跟踪方法来评估农药对蜜蜂的亚致死效应13 。豪这个方案的不同之处在于，它旨在测量可能阻止蜂蜜从农药处理的作物中的候选驱虫剂的影响。没有推荐的蜂蜜蜜化学威慑实验室测试方案，因此，该方案提供了一种简单的筛选方法。

<table><tbody><tr><td>50 mL Erlenmeyer flask</td><td>Kimax</td><td>26500-50</td><td>used for making the sugar/agarose cubes</td></tr><tr><td>Sugar</td><td>Kroger</td><td></td><td>any similar product will sufffice</td></tr><tr><td>Deionized water</td><td></td><td></td><td>acquired in house</td></tr><tr><td>Agarose</td><td>Apex</td><td>20-102</td><td>used for making the sugar/agarose cubes</td></tr><tr><td>Mold for agarose cubes (Weigh Boat)</td><td></td><td></td><td>any mold that will provide the researcher with a 1.5 cm x 1.5 cm x 0.3 cm sugar/agarose cube will suffice</td></tr><tr><td>EthoVision XT</td><td>Noldus</td><td></td><td>visual tracking software</td></tr><tr><td>633 nm LEDs</td><td>Cyron</td><td>HTP904E</td><td>These lights were placed into a constructed light box to illuminate the arenas from below.&amp;nbsp; The box was a simple wooden structure with a frosted plastic/plexi glass cover that allowed the light to disperse upwards without any glare.</td></tr><tr><td>Laptop or PC</td><td>Dell</td><td>Inspiron One 2305</td><td>necessary for video tracking software. Any pc device capable of runnin tbe visual tracking software will suffice</td></tr><tr><td>Bee Keeping protective clothing</td><td>Dadant &amp;amp; Sons Inc</td><td>V0126</td><td>any protective hood and jacket will suffice</td></tr><tr><td>Hive tool</td><td>Dadant &amp;amp; Sons Inc</td><td>M00757</td><td>used to open honey bee hive</td></tr><tr><td>Container for honey bees</td><td></td><td></td><td>any container suitable for housing and storing honey bees will suffice</td></tr><tr><td>Featherweight forceps narrow tip</td><td>Bioquip</td><td>4748</td><td>used to select individual honey bees</td></tr><tr><td>9 cm (diameter) Petri dish</td><td>Fisher Scientific&amp;nbsp;</td><td>S01778</td><td>arena used to contain individual honey bees during video tracking</td></tr><tr><td>Recording Device (Camera)</td><td>Basler</td><td>acA-1300-60gm</td><td>any device that can record the subject clearly and transfer the file to a computer will suffice</td></tr><tr><td>GraphPad Prism</td><td>Graphpad</td><td></td><td>any statistical software package will suffice</td></tr></tbody></table>

video tracking protocol to screen deterrent chemistries for honey bees

欧洲蜜蜂Apis mellifera L.是一种经济上和农业上重要的传粉媒介，每年产生数十亿美元。蜜蜂殖民地数量自1947年以来一直在美国和许多欧洲国家逐渐下降。许多因素在这种衰退中起着重要的作用，包括蜜蜂无意中暴露于农药。制定新的方法和法规有必要减少这些授粉者的农药暴露。一种方法是使用驱避剂化学物质，从最近的农药处理作物中阻止蜂蜜蜜蜂。在这里，我们描述了一个方案，以辨别蜂蜜蜜蜂暴露于选择的驱虫剂化学物质的威慑。蜜蜂饲养员在测试前15小时在培养箱中收集和饿死。将单个蜂蜜蜜蜂放入具有糖琼脂糖立方体（对照处理）或糖琼脂糖复合立方体（驱避剂处理）的培养皿中到盘中间培养皿作为竞技场，放置在灯箱内的照相机下，使用视频跟踪软件记录蜜蜂运动活动。分析总共8个对照和8个驱虫剂处理10分钟，每次处理用新的蜂蜜蜜蜂复制。在这里，我们证明蜂蜜蜜蜂通过复合处理从糖琼脂糖方块中被阻止，而蜂蜜蜂蜜被吸引到糖琼脂糖立方体而没有添加化合物。

开发了一个视觉跟踪方案，用糖琼脂糖（对照处理）或糖 - 琼脂糖复合立方体（威慑处理）记录蜂蜜在目标区域消耗的时间。使用统计软件程序分析记录的时间，并将目标区域的平均消耗时间±标准误差报告为条形图。作为驱虫/威慑试验的黄金标准DEET在本协议中被用作阳性对照。蜂蜜蜜蜂提供糖琼脂糖立方体（阴性对照）在目标区域花费343±26秒，而蜂蜜蜜蜂提供了糖 - ?...

Watch this Scientific Journal Video about Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees at JoVE.com

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

蜜蜂殖民地的丧失对作物授粉服务构成挑战。目前的传粉者保护措施需要采取替代方法，以尽量减少蜂蜜与使用驱虫剂的有害农药的接触。在这里，我们提供了一个可视化跟踪协议的详细方法来筛选蜜蜂的威慑。

用于蜂蜜蜜蜂筛选化学成分的视频跟踪协议

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

video-tracking-protocol-to-screen-deterrent-chemistries-for-honey-bees

Research

JoVE Journal

Environment

6.6K 次观看.  Virginia Tech. 蜜蜂殖民地的丧失对作物授粉服务构成挑战。目前的传粉者保护措施需要采取替代方法，以尽量减少蜂蜜与使用驱虫剂的有害农药的接触。在这里，我们提供了一个可视化跟踪协议的详细方法来筛选蜜蜂的威慑。 

视频: Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

A Push-pull Protocol to Reduce Colonization of Bird Nest Boxes by Honey Bees

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. These bees select cavities that are suitable nest sites for birds, resulting in competition. The difficulty of removing bees and their defensive behavior makes a prevention protocol necessary. Here, we describe a push-pull integrated pest management protocol to deter bees from inhabiting bird boxes by applying a bird safe insecticide, permethrin, to repel bees from nest boxes, while simultaneously attracting them to pheromone-baited swarm traps. Shown here is an example experiment using Barn Owl nest boxes. This protocol successfully reduced colonization of Barn Owl nest boxes by Africanized honey bees. This protocol is flexible, allowing adjustments to accommodate a wide range of bird species and habitats. This protocol could benefit conservation efforts where AHB are located.

Preventing colonization of bird nest boxes by invasive Africanized honey bees is important for conservation efforts of nest-site limited birds. We provide an integrated pest management approach to &#34;push&#34; bees away from nest boxes with a repellant insecticide, permethrin, and &#34;pull&#34; them toward pheromone baited swarm traps.

推挽议定书蜜蜂会影响家禽的殖民化巢箱

Introduction of the invasive Africanized honey bee (AHB) into the Neotropics is a serious problem for many cavity nesting birds, specifically parrots. ...

科研

环境科学

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

从个体到群体度评价环境化学物质对蜜蜂发展的影响

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

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 ...

神经科学

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 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). ...

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.

无线射频识别和动作感应视频高效地自动Unrewarded选择行为的记录由大黄蜂

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

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

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

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

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

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 ...

Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related division of labor). Young workers are engaged in nursing the brood (nurse bees), while older workers are engaged in foraging for nectar and pollen (foragers). The physiology of the workers changes in association with this role shift. For example, the main function of the hypopharyngeal glands (HPGs) changes from the secretion of major royal jelly proteins (MRJPs) to the secretion of carbohydrate-metabolizing enzymes. Because worker tasks change as the workers age in typical colonies, it is difficult to discriminate the physiological changes that occur with aging from those that occur with the role shift. To study the physiological changes in worker tissues, including the HPGs, in association with the role shift, it would be useful to manipulate the honeybee colony population by preparing single-cohort colonies in which workers of almost the same age perform different tasks. Here we describe a detailed protocol for preparing single-cohort colonies for this analysis. Six to eight days after single-cohort colony preparation, precocious foragers that perform foraging tasks earlier than usual appear in the colony. Representative results indicated role-associated changes in HPG gene expression, suggesting role-associated HPG function. In addition to manipulating the colony population, analysis of the endocrine system is important for investigating role-associated physiology. Here, we also describe a detailed protocol for treating workers with 20-hydroxyecdysone (20E), an active form of ecdysone, and methoprene, a juvenile hormone analogue. The survival rate of treated bees was sufficient to examine gene expression in the HPGs. Gene expression changes were observed in response to 20E- and/or methoprene-treatment, suggesting that hormone treatments induce physiological changes of the HPGs. The protocol for hormone treatment described here is appropriate for examining hormonal effects on worker physiology.

Here we describe our detailed protocol for the preparation of single-cohort honeybee colonies&#160;&#8211; a useful tool for analyzing the role-associated worker physiology. We also describe detailed protocols for treating workers with juvenile hormone and ecdysone to evaluate the involvement of these hormones in the regulation of worker behavior and/or physiology.

单队列殖民地和工蜂的激素治疗的准备来分析生理学与关联作用和/或内分泌系统

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related ...

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

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

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

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

约束和自由飞行，蜜蜂的神经药理学操纵，<em&gt;蜜蜂</em

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

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 ...

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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