The experiments were supported by a grant from the Natural Sciences and Engineering Research Council of Canada to CMSP. We thank Koppert Canada for their bumblebee colony donations. Portions of this manuscript, including some figures and tables have been published in Naturwissenschaften11, and reproduced here with permission from Springer.

渥太华大学的动物管理委员会已经批准了我们的实验方案，它描绘的安全程序，人员蜜蜂的工作。 1.测试环境准备<ol><li>准备2米×2米×2μm的空白空间（独立的房间，或金属屏盖飞笼）。 注:如果房间被选为测试空间，确保蜜蜂可以通过Windows无法逃脱，下车门的空间，以及空气交换管道。 </li><li>加小的入口点（ 例如 ，约2厘米直径的孔），以在飞行笼，其中蜜蜂能进入和退出测试空间，而不阻塞。设计一种机制来阻止接入点，以保持蜂出测试空间的过程中保留用于维护和设备的配置周期。 注:我们采用熊蜂凤仙花克勒松工人。 </li><li>连接一个或两个集落箱来使用的连接器的测试空间。确保一个死蜜蜂不能BLOCK上的连接器。 注意:在此，使用两种类型的连接结构:一个木制的"桥"结构，用玻璃顶盖和金属丝网管。他们很容易清洁，它们提供牵引蜜蜂。 </li><li>将两个人造花持有人的测试空间内。 <ol><li>放置花持有者在测试空间的中心，或者将它们连接到所述壁。 </li><li>一个"2K6头"RFID读取器连接到人造花用遮蔽胶带的柱体部分的顶部（参见图1进行定位）。用1.2米高的木架，用来连接人造花。 注:支架的顶部应设有固定机构，其中人造花可以连接。参见图2的示意图</li></ol></li><li>加高频（分钟200赫兹）的荧光发光装置，以充分照亮测试空间。使用的高频电子镇流器，以确保韧带HT闪烁高于大黄蜂"视觉闪光融合阈值6。 注:在这里，用12日光色荧光灯灯泡，产生约1200勒克斯光照强度为2×2×2米的空间。 </li></ol><img alt="图1" src="/files/ftp_upload/52033/52033fig1highres.jpg" /> 通过花的中心安置在开缸的顶部图1的RFID人造花卉设计。在实验1中所述的RFID读取器中使用的RFID功能的人造花的示意图。刺激模式和立场: 一 。周边同心，B。中央同心，C。周缘径向以及 d。中央子午线。这个数字已经被修改欧尔班等。11。 <img alt="图2" src="/files/ftp_upload/52033/52033fig2highres.jpg" /> <s仲&gt;图2.刺激展示架一个示意图显示人造花刺激展台的设计，和固定夹。附接夹粘在人造花和用于快速连接和分离的刺激，并从刺激看台。此配置是在运动敏感的视频用于实验。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52033/52033fig2highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> 2.大黄蜂殖民地准备<ol><li>在商业上有序殖民地的到来，菌落盒连接到飞行笼。 </li><li>提供花粉（由蜜蜂从多种植物中收集的）和糖-水（1:1体积比） 随意直到检测会话的开始。 <ol><li>煮沸1升水中，并混合在糖的同一卷来创建糖水溶液。 </li><li>从养蜂家或玉米花粉购买大黄蜂殖民地的商业性供应商。磨用研钵和研杵的花粉，和与蜂蜜和水（如果需要）混合，使之变成湿的糊状物。 </li></ol></li><li>一旦实验开始，提供15〜40毫升糖水（数量成正比菌落大小）每天。继续提供花粉自由采食 。根据在蜜罐存储电平调整糖水量。 注意:保持粮食储存水平低提供了激励员工离巢寻找更多的食物。 </li><li>让蜜蜂通过连接器结构进入测试环境中，如在步骤1.3中描述。 注:蜜蜂自由移动的窝和整个实验的测试环境之间。 </li></ol> 3.准备观察射频识别<ol><li>立即开始粘合RFID标签的工人的胸部作为殖民地到达，并继续在整个实验中，为新的工作ERS出现。 </li><li>胶合技术<ol><li>放置在单个容器的工人最初的标签中。酷工人倒在冰箱（约700℃）约1小时，慢下来。 注意:冷却下来的工人提前标注有助于使激进的殖民地更易于管理。这最小化了刺的机会。 </li><li>使用该标记装置和无毒的胶（由标签提供者提供的），附上RFID标签，以工人的胸部。胶RFID标签的工人，因为他们的出现，而他们仍然在乳臭未干的阶段（它们能够飞行前）。 </li><li>等待至少10分钟，将所述工回菌落，以确保该RFID标签不能由工人去除之前。 </li></ol></li><li>处置已经失去了他们的RFID标签（所看到的残胶，或在胸部秃顶）工人在他们乳臭未干的阶段，并没有标记的，或工人。 注:这些蜜蜂可能都有这样的经验outsidË殖民地。 </li><li> RFID阅读器软件<ol><li>使用个人计算机（PC）来配置RFID读取器软件。更改操作系统的日期格式YYYY-MM-DD HH:MM:SS，以确保下载的RFID数据被正确编码。调整使用C ++编程语言，允许出口的RFID阅读器作为逗号分隔值（CSV）的数据RFID阅读器提供的程序。 </li><li>使用RS-232到USB连接器连接的RFID读取器到计算机。周期性地将数据下载到RFID读取器，在读出器到达存储容量（高达32000的记录）。使用RFID阅读器来记录日期，时间以及每个标签相关联的13字符的唯一字符串。导入下载的CSV文件导入到关系数据库管理系统（RDBMS）。 </li></ol></li></ol> 4.人造花准备RFID阅读器<ol><li>购买从当地的工艺品商店蓝色和黄色的烤粘土。 </li><li>创建一个BLUE的圆筒，直径为1.5厘米，并切成一个侧面的1.5厘米×3厘米的孔。 注:在气缸的侧面的孔将作为所述容器的RFID阅读器。 </li><li>创建一个蓝色锥形，其直径为8厘米的一端，和1.5厘米的另一端。 </li><li>合并的气缸和锥体。 </li><li>创建一个长20厘米和0.5厘米宽的黄色粘土的链。 </li><li>切黄链规模，并努力变成蓝色圆锥体和圆柱体。 注:径向形状会要求5厘米长的直线，同心形状会要求4-8厘米长圆形线。 </li><li>烘烤的粘土在130°F至完全硬化。 </li></ol> 5.准备观察运动敏感的视频录制<ol><li>开始只要殖民地到达胶胶制号标签工蜂的胸部，并在整个实验中继续为新员工出现。 </li><li>最初Taggi的过程中删除所有工人纳克会议，以确保每个人都得到了一些标记。地方的工人早在殖民地标记之后。 注:与RFID标签，标签数量更难让工人移除。 </li><li>随着新工人的出现，标记他们，而他们仍然乳臭未干。标记频率随菌落循环的状态，但平均约7-10工人每2-3天。处置过程中他们乳臭未干的阶段并没有标记，因为他们可能不再是花，天真的工人。 </li><li>系统标记更多的工人比可用标记号 注:如果实验持续了数月，多殖民地都参与其中，有一个很好的机会，可以标记编号冒了出来。大约有7区分标记的颜色，每一个编号从1到99，允许多达693并发标记的工人。运行3个月用3-4的殖民地将有超过693工人总数，但从来没有同时活着的实验。 &lt;OL&gt; <li>确保数字标记被定位系统（ 例如 ，标记号的顶侧总是与蜂的头对齐），特别是用于以下数字:6，9，66，69，99。 </li><li>去除死皮的工人，并记录他们的标签号为"释放"。保持标记数字和颜色的一个数据库，它是"可用"或"正在使用"，以确保一个独特的标记不同时使用的多个蜜蜂。 注:其他标签组合可以通过添加颜色到已经存在的颜色来制造。例如，添加黄点为蓝色标签与夏普笔可以创建新的组合。 </li></ol></li><li>视频数据处理<ol><li>放置在每个花海前两个Internet协议（IP）摄像机（最低100万像素的图像分辨率），测试环境之外（参见图3）。 <img alt="图3" src="/files/ftp_upload/52033/52033fig3highres.jpg" /> <st荣&gt;图3.测试环境，测试环境显示在前台的两个正面摄录一体机的照片，天花板上的摄像机的灯具，与刺激之间矗立在房间的中间样本的刺激。 注:IP摄像机和人造花之间的玻璃分隔确保人造花都清晰可见。该IP摄像机可达到5米距离的刺激。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52033/52033fig3highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> </li><li>更换股票镜头1.8毫米可变焦距透镜。这些镜头留出足够的变焦和对焦的假花。 </li><li>将一个额外的IP摄像机正上方的人造花，重点区域1-2米的人造花前面。该摄像机捕捉悬停和antennation行为。 </li><li>连接IP摄像机<html> 经由辅助网络接口控制器（NIC），以及一个以太网集线器的PC。 </li><li>配置PC的动态主机配置协议（DHCP）来动态地分配IP地址给IP摄像机。 </li><li>配置PC上的文件传输协议（FTP）服务器。 </li><li>配置FTP客户端程序来自动存入视频片段在PC上。 </li><li>配置IP摄像机记录检测，每次运动10秒的视频剪辑。 </li></ol></li><li>视频剪辑分析<ol><li>打开一个视频剪辑，并查看其中的内容一帧一帧的使用所选择的视频查看器。 </li><li>记录蜜蜂的塑料标签号码，日期和记录在电子表格或RDBMS的时间。这里，定义一个着陆作为蜂的腿进入与人造花接触。 注:放弃那些对社会影响的选择（着陆，而另一蜂出现在刺激）。 </li></ol></li></ol>E"&gt; 6。人造花准备视频观察<ol><li>设计使用图形编辑软件的视觉特性。 注:确保在印刷图案可以被切割并折叠成锥形。利用几何计算以产生所述切口的形状，结果在一个锥体具有一个8厘米直径的。 </li><li>打印，切割，折叠和视觉性成圆锥形。 </li><li>胶水附着剪辑刺激（参见图2）。 </li></ol> 7.统计分析<ol><li>在这两个实验，计算选择的比例为每蜜蜂（ 例如，一个特定的蜜蜂做X选择之一花了总共Y的了）。 </li><li>与体能测试7的复制善分析这些比例。 注:复制的G-试验计算出的异质性值（G 八），指示重复的变异性（例如，每个蜂）的量，和一个共用的值（G p）的表示的整体意义所有的选择比例。对G的值进行比较，以χ在显着性检验的2个值。 </li></ol> 8.刺激呈现顺序<ol><li>附加人造花的测试空间内的花架。改变组合和显示的刺激的位置在规定时间间隔（ 例如，每天），以避免位置的效果。 </li></ol> 9.研究结束<ol><li>放置在深冷冻机的菌落在-10℃下3天，以杀死蜂群。 </li></ol>

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The authors have no competing financial interests.

RFID技术使研究数百个工人轻松，精度高，但记录的行为特点是观察人类和录像不同。记录RFID的选择行为可谓花香探索。这是一个非常严格的喜好标准相比，在其他研究中所使用的标准，如方法8，进入迷宫臂9,10，触角反应8，或登陆图案11,12。为了比较的选择行为的定义的有效性，并验证新的RFID方法unrewarded行为，着陆的视频记录在实验2中观察到的所有的选择的措施是不相等的:如通过花的条目测量的RFID标准，表明图形定位的视觉特性，更重要的蜂的选择，而视频数据表明，图案类型的视觉特性，更重要的蜂的选择。一个学习没有学问选择行为的一般挑战是，它是很难吸引花幼稚，未受过训练的蜜蜂人造花不提供任何花粉或花蜜。事实上，许多以前的实验中采取的对策被认为是无关的行为选择，测试刺激刺激训练蜜蜂在测试环境中。 RFID和运动敏感录像克服这一障碍，允许连续的记录，24小时，一天，而不研究者的恒定监督，通过增加从15-20蜜蜂的样本大小，以几百蜜蜂。而由未经训练的蜜蜂unrewarded的选择仍然是一个罕见的现象，这些新的实验设计参数进行观测成为可能。 通过这两种技术所提供的其它改进包括消除样品偏压的，外部效度的提高，以及跟踪的个体差异。 SAMPL当只有学习十几蜜蜂在殖民地é偏差可能会出台。有即使在同一集落正在可能错过了，因为只有那些工人观察到发生在给定时间"合作"的研究者在整个各个工作者行为特质显著差异。学习15-20蜜蜂中的300或更多的蜜蜂菌落，表示低至5％的总集落，在这种情况下，采样偏压可显著的。标记和观察所有工人的行为完全消除这个问题。同时刺激的选择的数量也可以被操纵。我们在我们的实验中由于技术原因提供了二进制的选择，但单项选择或多项选择的设计也是可行的。 在外部有效性而言，在实验室环境中学习蜜蜂历来高度人工，这阻碍了结果的普遍性。例如，研究人员必须是公关ESENT进行数据收集，蜜蜂有一个觅食，在测试环境中的一个，和检测被限制在一个小的时间窗口。本文介绍的新技术，通过观察无监督和无限制地消除这些人为的限制。最后，行为的个体差异可以被记录，因为我们能确定这些病例是否由一个单一的蜜蜂或蜜蜂的几个重复的选择。 运动敏感，高空间分辨率的摄像机有超过RFID技术的优势在刺激设计的灵活性方面:视觉刺激的外观几乎可以是任何形状或作为主体的身份可以在至少一个被捕获，只要形成几帧。处理视频是一个小更耗时比处理RFID数据，因为鉴定需要由研究人员，这就要求每个视频剪辑的人工检查来读取。如果视觉刺激的设计能满足R的约束FID的读取器（ 即，对蜂的RFID标签必须来至少3-4毫米的RFID读取器的），那么RFID技术拥有自动化大规模数据集合中的边缘。定性研究很可能会继续由视频分析的青睐。如示出在这个实验中，RFID阅读器可以聚敛​​，无需手工编码非常大的数据集。与每个技术相关联的略微不同的优点建议，在将来他们可以以互补的方式使用。 这两种技术的未来可能在于很少发生行为的精确量化。例如，一个明显的可能性，为未来的应用是使用在大棚等更加自然的环境中这些技术。自然主义和实验控制的结合将使解决了以前不可能回答的问题。从广义上讲，这些技术提供了一个严谨的观察行为的两种新方式ND有效的方式。 RFID和运动敏感的视频是一个显著的进步不仅为研究人员在研究传粉昆虫或昆虫，但这些技术也可以向其他行为科学家。

由大黄蜂和蜜蜂学习没有学问选择行为的一个关键问题是，花天真，未经训练的工人不容易进入测试空间，喜好可以衡量的。其结果是，许多研究者依靠一个不太理想的方法:预先训练工人从表面上中性刺激，研究人员认为是从实验刺激不同的测试空间内送入。然而，最近的实验表明，被认为刺激是中性的（ 即刺激不影响在测试会话随后的选择行为）都以意想不到的方式1的影响偏好。自动化系统，包括射频识别（RFID）的2和运动敏感录像可以提供一个机会解决这个问题。该研究的目的是双重的:（1）主要是由大黄蜂有助于文献没有学问花卉的喜好，（2），其次为EValuate 2选择测量系统所记录的两个不同的自动记录设备。 两条自动化系统3是在本研究实施来观察没有学问的选择行为:RFID和运动敏感的视频录制。这两个系统的两个关键元素是选择不能奖励，不同的视觉提示的显示可以被操纵。运动敏感视频（高清晰度，记录在1熔点分辨率）不仅允许在飞行室连续观察自由探索的工人，而且是有效率地观测的相对罕见的事件4的关键。 实验1中的研究问题涉及到一起的时候显示的视觉特性如何交互。本研究的目的是探讨有关图案式图形定位的相对重要性。采用2×2的设计，径向（ 即旭日）和同心（ 即牛市的电子咋）图案类型被置于任中枢或外周上的人造花（参见图1为刺激的例子）。 RFID阅读器中内置了这些专门设计的人造花的刺激，和大黄蜂接收RFID标签的启用，使我们能够记录每个标签的工人进入人造花刺激。 RFID观测工作由读取器机构（内置在人造花）发送的无线电频率信号（13.56MHz的在这种情况下），这是由无源标签的存在下进行调制。读者可以检测并记录这些信号调制，而稍有改变整个标签使标签的唯一标识。 实验2的问题有三个方面。首先，是花港，通过RFID和着陆测量，通过录像相当于选择标准衡量？选择的测量是在不同的点（着陆视频，和对RFID花项），其可以转化为不同的措施偏爱。二，什么是中央VS周边定位的影响？它不知道工人是否会选择一个中央图案是否包括在不同位置的两个径向图案的组合被提出（参见图4b）。三，什么是图案位置VS模式类型的相对重要性？换句话说，将大黄蜂降落在优选的模式的类型，或在优选的图案位置的模式？蜜蜂可能更倾向于中部径向向外围同心图案，但偏爱可能是由于图案类型或它的中心的定位。在该实验中，两个变量被对垒5（参见图4c，d）所示 。 在实验中，我们用在花朵般的刺激动作感应录像。仿真花置于飞行笼子里，和运动感的高清晰度摄像机，指出在这些花从弗朗t和顶部。更具体地，2摄像机被定位成捕获每个在测试空间的两个刺激的的正视图。另外一个摄像机被放置在刺激之间，记录从上面盘旋的行为，并抓获来自人造花行为。确定了使用数字标记，可以读取高清视频短片的大黄蜂。悬停，antennation和着陆的行为进行观察。

<table><tbody><tr><td>Miniaturized mic3 tags</td><td>Microsensys</td><td>mic3 TAG 64 bit RO</td><td>RFID tags to glue to bee</td></tr><tr><td>RFID reader 2k6 head</td><td>Microsensys</td><td>2k6</td><td>RFID readers built into artificial flowers</td></tr><tr><td>IP camcorders</td><td>Vivotek</td><td>IP8161</td><td>Motion-sensitive video recorders</td></tr><tr><td>Opalith Plattchen number tags and non-toxic glue</td><td>Beeworks.com</td><td>n/a</td><td>Number tags to glue to bees</td></tr><tr><td>Bumblebee Colony for Research</td><td>Koppert Canada</td><td></td><td></td></tr><tr><td>Artificial flowers</td><td>N/A</td><td></td><td>Developed by campus biology shop</td></tr><tr><td>Artificial flower stand</td><td>N/A</td><td></td><td>Developed by campus biology shop</td></tr><tr><td>Flight room</td><td>N/A</td><td></td><td>Developed by campus biology shop</td></tr><tr><td>Laptop with Windows</td><td>Generic hardware / Microsoft software</td><td></td><td>Used to download RFID data</td></tr><tr><td>RS 232 to USB converter</td><td>Generic</td><td></td><td>Connect RFID reader to computer</td></tr><tr><td>Desktop</td><td>IBM</td><td></td><td>Used to transmit video data</td></tr><tr><td>Second NIC</td><td>Generic</td><td>10/100M NIC PCI</td><td>Used to transmit video data</td></tr><tr><td>Network hub</td><td>Generic</td><td>4-port</td><td>Used to transmit video data</td></tr><tr><td>High precision tweezer</td><td>SPI</td><td></td><td>Used to glue number and RFID tags to bees</td></tr><tr><td>Sugar</td><td>Generic</td><td></td><td>Used to mix with water to create sugar-water</td></tr><tr><td>Pollen</td><td>Any local apiarist</td><td></td><td>Fed to bumblebees</td></tr><tr><td>Marking cage with plunger</td><td>Beeworks.com</td><td></td><td>Aids tagging process</td></tr><tr><td>Honey</td><td>Generic</td><td></td><td>Used to mix with water ot create pollen paste</td></tr><tr><td>Bake clay</td><td>Sculpey</td><td></td><td>Stimulus for RFID</td></tr><tr><td>Clay shaping tools</td><td>Generic</td><td></td><td>Stimulus for RFID</td></tr><tr><td>White paper</td><td>Generic</td><td></td><td>Stimulus for Video</td></tr><tr><td>Laser printer</td><td>Generic</td><td></td><td>Stimulus for Video</td></tr><tr><td>Wood</td><td>Generic</td><td></td><td>Stimulus for Video -- attachment clip</td></tr></tbody></table>

radio frequency identification and motion-sensitive video efficiently automate recording of unrewarded choice behavior by bumblebees

我们提出两种方法来观察在一个封闭的空间试验熊蜂选择行为。第一种方法是由无线射频识别（RFID）阅读器内置了显示各种视觉线索人造花，并紧盯大黄蜂工人的胸部RFID标签（ 即无源应答器）。在我们的实现的新颖性是，RFID阅读器被直接内置到人造花，其能够显示多个不同视觉属性，例如颜色，图案类型，空间频率（的图案，即 ，"忙碌"），并且对称性（空间频率和的对称性没有在该实验操作）。此外，与自动化系统相结合，这些可视化显示都能够记录unrewarded和未经培训的选择行为。第二种方法包括记录选择行为，在采用运动感的高清晰度摄像机人造花。 Bumblebees有数字标签粘在thoraces的唯一标识。在本实施以上的RFID的优点在于，除了观察着陆的行为，也可观察到的偏好的替代措施，如悬停和antennation。这两种方法的自动化，允许较大规模的研究，考虑到个体差异，提高实验控制和内部效度。外部效度也得到了提高，因为蜜蜂可以自由进入和退出，不限制测试环境，如现场的研究助理的可用性。相比于人观察实时的自动化方法更符合成本效益的和可能更不容易出错。

实验1:RFID数据 所有375工人群体中的RFID标签，而这些工人（85％）318进入飞行笼在一些点在研究过程中。总共有197（蜜蜂留下殖民地的62％）访问至少一个四人造花的刺激。 选择的定义一个选项被定义为进入人造花工人（ 见图1）。我们把这种行为称为"花的探索。"选择的这个定义比文献中使用的那些，其中，根据研究，使用的徘徊，antennation或降落的某种组合更加严格。碎花的探索是选择一个更严格的定义，因为它需要蜜蜂不仅徘徊，antennating，登陆就可以了，而且还通过探索其顾不上刺激。 数据管理<p class="jove_content"&gt; RFID技术具有收集相关数据的大量的其他方法的能力。该实验产生310221条记录。数据库的程序，如MySQL是一个不可缺少的工具来存储这些数据量和用来梳理出答案，研究问题同样重要的是SQL查询。例如，一个问题是有选择的定义。所述RFID阅读器被配置为记录标签的每1毫秒，其在该停留内的人造花为几分钟甚至几小时蜜蜂的情况下，可以转化为几千录音的存在。我们选择的定义是参观持续了至少1毫秒，而是一个新的访问将不能被计数，直到录音的当前字符串是由至少1分钟破断。 实验小结从蜜蜂的"天真会话"来比较选择P在所有的选择都进行了适当的测试，四复制的善良roportions偶然7的理论值。蜜蜂的天真会议指的是蜂"参加"第一试验条件。对G-测试显示偏爱中央定位（ 见表1）和径向图案的类型。 图4b显示了当同心图案的中心位于该图案的偏好反转和径向图案外围定位。但是，如果定位保持恒定， 如图4a中以及d，图案的选择是对放射状图案; 图4示出了针对每个图案的每个组合的第一选择的相对比例是可比于所示的所有选择的比例。 <img alt="图4" src="/files/ftp_upload/52033/52033fig4highres.jpg"/> 图4. RFID实验结果。选择频率在t他四种不同的花卉组合在实验1的深褐色棒图显示，从蜜蜂的天真会议（左侧y轴） 的所有选择 ，而浅棕色的线表示每个工人的第一选择 （右侧y轴）。 "所有的选择"节目相媲美的模式为"第一选择"，但有更好的统计力量。该条形图显示的图案的定位比的图案的类型更重要。位于中心的图案是首选，即使图案类型的显示，否则最好少同心圆图案。星号表示的选择比例是显著不同机会。注。 * P &lt;0.05，** P &lt;0.01，*** P &lt;0.001。这个数字已经被修改欧尔班等。11。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td rowspan="2"> 条件 </td><td colspan="3"> Pooled </td><td colspan="3"> 异质性 </td><td></td></tr><tr><td> GP </td><td> DF </td><td> P </td><td> GH </td><td> DF </td><td> P </td><td></td></tr><tr><td>环径向VS中央同心</td><td> 3.96 </td><td> 1 </td><td> 0.047 </td><td> 197.55 </td><td> 41 </td><td> 0.000 </td><td></td></tr><tr><td>外设径向VS中央同心</td><td> 33.77 </td><td> 1 </td><td> 0.000 </td><td> 210.81 </td><td> 42 </td><td> 0.000 </td><td></td></tr><tr><td>环径向VS外围同心</td><td> 508.31 </td><td> 1 </td><td> 0.000 </td><td> 345.78 </td><td>三十</td><td> 0.000 </td><td></td></tr><tr><td>外设径向VS周边同心</td><td> 7.42 </td><td> 1 </td><td> 0.000 </td><td> 84.06 </td><td> 24 </td><td> 0.000 </td><td></td></tr></tbody></table> 表1中。 </sRFID数据的实验1.本表已经被修改欧尔班等仲&gt; 统计推断。（2013年）11:G P指的是从偶然的一组比例显著的偏差，和G ^ h是指测试个体差异（ 即异质性）。请参阅手稿上的统计检验的全部细节。 实验2:视频数据 共有264选择被记录在四个条件下在三个测试会话。 表2显示了工人和选择从每一集落贡献的数量。 选择的定义视频数据允许的三种选择行为的记录:徘徊，antennation和着陆。虽然所有三种类型的行为，可以观察到，悬停和antennation是difficuLT用一个标签号，由于快速移动的摄像机，分辨率差，或低速无法记录相关联。关键的是要使用高清晰度摄像机（虽然这不是提供给我们，理想的高帧速率摄像机应该被用来最小化模糊），以确保可以在一个小数目的帧只出现的标签号可以被读。该方法也被用来比较的选择模式与RFID技术，该技术检测花香勘探。 运动灵敏度的考虑之一的生产成功实验的关键问题是运动敏感的摄像机的配置。甲摄像机过于敏感会记录太多的数据是不实际的，并且可以成为非常昂贵的加工。例如，最初我们的摄像机通过在建筑物定期振动触发（ 例如，人在走廊，空气调节器等经过），这导致1R11; 2有效数据点，每150-200录制的视频剪辑。另一方面，一个更严重的错误是低灵敏度的配置，它可以错过密钥数据。关键的是要配置所有的摄像机相同的方式，否则，采样误差可影响结果。 实验小结的合适的测试四个复制善发现了三个组的比例，从机会显著偏离，和一个非显著总体比例（ 见表3和图5）。 （1）图案是很重要的:一个显著倾向于中部径向上的中心同心的图案被发现（ 见表3）。 （2）的放射状图案的位置是那么重要:中央径向和周径方向相结合的演示表明，从机会显著差异无。 （3）中央径向和周缘同心组合导致朝向强烈偏好S中的中央放射状。中央同心和外围 - 径向组合引起朝向周缘呈放射状显著偏好。图案莫须有的位置。个体差异在所有四种组合（ 见表3）非显著。 <img alt="图5" src="/files/ftp_upload/52033/52033fig5highres.jpg"/> 图5.运动敏感的视频结果选择频率在四种不同的花卉组合在实验2的结果表明，模式类型的重要性，在模式的定位:是首选径向模式，即使图案定位周边。值表示的显示模式的选择的数目。星号表示的选择比例是显著不同机会。注。 ** P &lt;0.01，*** P &lt;0.001。这个数字已经被修改欧尔班11。 <tablË边界="0"的cellpadding ="0"CELLSPACING ="0"&gt; <tbody><tr><td rowspan="2"> 条件 </td><td> 第一节 </td><td colspan="2"> 会话2 </td><td colspan="2"> 第3节 </td></tr><tr><td>殖民地1 </td><td>菌落2 </td><td>菌落3 </td><td>菌落4 </td><td> 5殖民地</td></tr><tr><td>工号</td><td> 45 </td><td> 7 </td><td> 2 </td><td> 8 </td><td> 23 </td></tr><tr><td>选择的第</td><td> 151 </td><td> 25 </td><td> 2 </td><td> 20 </td><td> 65 </td></tr></tbody></table> 表2.运动敏感的视频数据的描述统计。总数记录在人造花在实验2中为每个菌落的选择，以及工人作出这些选择的数量。这个表已经被修改欧尔班等 11。请参阅手稿完整的细节。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td rowspan="2"> 条件 </td><td colspan="3"> 汇集 </td><td colspan="3"> 异质性 </td></tr><tr><td> GP </td><td> DF </td><td> P </td><td> GH </td><td> DF </td><td> P </td></tr><tr><td>环径向VS中央同心</td><td> 17.98 </td><td> 1 </td><td> 0.000 </td><td> 40.72 </td><td> 29 </td><td> 0.073 </td></tr><tr><td>环径向VS周径方向</td><td> 1.85 </td><td> 1 </td><td> 0.173 </td><td> 53.63 </td><td> 39 </td><td> 0.060 </td></tr><tr><td>周边径向VS中央同心</td><td> 6.57 </td><td> 1 </td><td> 0.010 </td><td> 26.31 </td><td> 27 </td><td> 0.500 </td></tr><tr><td>中央子午线VS外围同心</td><td> 18.18 </td><td> 1 </td><td> 0.000 </td><td> 41.92 </td><td> 37 </td><td>0.256 </td></tr></tbody></table> 表3.推论统计运动敏感的视频数据。实验2该表已经被修改欧尔班等 11 克对指的是从偶然的基团的比例显著偏差，和G ^ h指的是测试个体差异（ 即异质性）。请参阅手稿上的统计检验的全部细节。

Watch this Scientific Journal Video about Radio Frequency Identification and Motion-sensitive Video Efficiently Automate Recording of Unrewarded Choice Behavior by Bumblebees at JoVE.com

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

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 ...

radio-frequency-identification-motion-sensitive-video-efficiently

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.9K 次观看.  University of Ottawa. This video describes Radio-Frequency Identification (RFID) and motion-sensitive video recording methods to monitor choice behavior by bumblebees. 

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

Early Metamorphic Insertion Technology for Insect Flight Behavior Monitoring

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on insects during their metamorphic development. Here, the implants are fused within the structure and function of the neuromuscular system as a result of metamorphic tissue remaking. The implants emerge with the insect where the development of tissue around the electronics during pupal development results in a bioelectrically and biomechanically enhanced tissue interface. This relatively more reliable and stable interface would be beneficial for many researchers exploring the neural basis of the insect locomotion with alleviated traumatic effects caused during adult stage insertions. In this article, we implant our electrodes into the indirect flight muscles of Manduca sexta. Located in the dorsal-thorax, these main flight powering dorsoventral and dorsolongitudinal muscles actuate the wings and supply the mechanical power for up and down strokes. Relative contraction of these two muscle groups has been under investigation to explore how the yaw maneuver is neurophysiologically coordinated. To characterize the flight dynamics, insects are often tethered with wires and their flight is recorded with digital cameras. We also developed a novel way to tether Manduca sexta on a magnetically levitating frame where the insect is connected to a commercially available wireless neural amplifier. This set up can be used to limit the degree of freedom to yawing &#8220;only&#8221; while transmitting the related electromyography signals from dorsoventral and dorsolongitudinal muscle groups.

We present a novel surgical procedure to implant electrodes in Manduca sexta during its early metamorphic stages. This technique allows mechanically stable and electrically reliable coupling with the neuromuscular tissue to study flight neurophysiology dynamics. We also present a novel magnetic levitation platform for tethered studies of insect yaw.

早期变质插入技术昆虫飞行行为监控

Early Metamorphosis Insertion Technology (EMIT) is a novel methodology for integrating microfabricated neuromuscular recording and actuation platforms on ...

科研

行为学

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

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

The loss of honey bee colonies presents a challenge to crop pollination services. Current pollinator protection practices warrant an alternative approach to minimize the contact of honey bees to harmful pesticides using repellent chemistries. Here, we provide detailed methods for a visual tracking protocol to screen deterrents for bees.

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 ...

环境科学

Studying the Neural Basis of Adaptive Locomotor Behavior in Insects

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those leg movements that result from passive forces and interactions with other legs through the common contact with the substrate. If we want to understand, which part of a given movement is produced by nervous system motor output, kinematic analysis of stepping movements, therefore, needs to be complemented with electrophysiological recordings of motor activity. The recording of neuronal or muscular activity in a behaving animal is often limited by the electrophysiological equipment which can constrain the animal in its ability to move with as many degrees of freedom as possible. This can either be avoided by using implantable electrodes and then having the animal move on a long tether (i.e. Clarac et al., 1987; Duch &amp; Pfl&uuml;ger, 1995; B&ouml;hm et al., 1997; Gruhn &amp; Rathmayer, 2002) or by transmitting the data using telemetric devices (Kutsch et al, 1993; Fischer et al., 1996; Tsuchida et al. 2004; Hama et al., 2007; Wang et al., 2008). Both of these elegant methods, which are successfully used in larger arthropods, often prove difficult to apply in smaller walking insects which either easily get entangled in the long tether or are hindered by the weight of the telemetric device and its batteries. In addition, in all these cases, it is still impossible to distinguish between the purely neuronal basis of locomotion and the effects exerted by mechanical coupling between the walking legs through the substrate. One solution for this problem is to conduct the experiments in a tethered animal that is free to walk in place and that is locally suspended, for example over a slippery surface, which effectively removes most ground contact mechanics. This has been used to study escape responses (Camhi and Nolen, 1981; Camhi and Levy, 1988), turning (Tryba and Ritzman, 2000a,b; Gruhn et al., 2009a), backward walking (Graham and Epstein, 1985) or changes in velocity (Gruhn et al., 2009b) and it allows the experimenter easily to combine intra- and extracellular physiology with kinematic analyses (Gruhn et al., 2006).
We use a slippery surface setup to investigate the timing of leg muscles in the behaving stick insect with respect to touch-down and lift-off under different behavioral paradigms such as straight forward and curved walking in intact and reduced preparations.

We describe a method to record motor activity, timed to the electrically recorded tarsal contact signal in a tethered insect, walking on a slippery surface. This is used to study the neural basis of adaptive behavior under reduced influence of mechanical interaction between legs through the substrate.

研究昆虫的自适应运动能力行为的神经基础

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those ...

神经科学

A Precise and Autonomous System for the Detection of Insect Emergence Patterns

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of emerging insects they can detect. In order to obtain precise measurement of insect emergence, it is necessary for systems to be semi-automated and able to measure large numbers of emerging insects. We addressed these issues by designing and building a system that is automated and can measure emergence of up to 1200 insects. We modified the existing &#34;falling-ball&#34; system using Arduino microcontrollers to automate data collection and expand the sample size through multiple data channels. Multiple data channels enable the user to not only increase their sample size, but also allows for multiple treatments to be run simultaneously in a single experiment. Furthermore, we created an R script to automatically visualize the data as a bubble plot, while also calculating the median day and time of emergence. The current system was designed using 3D printing so the user can modify the system to be adjusted for different species of insects. The goal of this protocol is to investigate important questions in chronobiology and stress physiology, using this precise and automated system to measure insect emergence patterns.

Measurement of insect emergence patterns requires precision. Existing systems are only semi-automated and sample size is limited. We addressed these issues by designing a system using microcontrollers to precisely measure the time of emergence of large numbers of emerging insects.

一种精确、自主的昆虫产生模式检测系统

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

生物学

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 ...

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

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

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

在两个神经元处理阶段在做人蜜蜂同时长期录音

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

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

一种测定果蝇运动活性的简单技术

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

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 ...

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle (Mercynorrhina torquata)

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight insect behavior. This technology enables the development of an insect-machine hybrid system using a living insect platform described in this protocol. Moreover, this protocol presents the system configuration and free flight experimental procedures for evaluating the function of the flight muscles in an untethered insect. For demonstration, we targeted the third axillary sclerite (3Ax) muscle to control and achieve left or right turning of a flying beetle. A thin silver wire electrode was implanted on the 3Ax muscle on each side of the beetle. These were connected to the outputs of a wireless backpack (i.e., a neuromuscular electrical stimulator) mounted on the pronotum of the beetle. The muscle was stimulated in free flight by alternating the stimulation side (left or right) or varying the stimulation frequency. The beetle turned to the ipsilateral side when the muscle was stimulated and exhibited a graded response to an increasing frequency. The implantation process and volume calibration of the 3 dimensional motion capture camera system need to be carried out with care to avoid damaging the muscle and losing track of the marker, respectively. This method is highly beneficial to study insect flight, as it helps to reveal the functions of the flight muscle of interest in free flight.

Insect-machine Hybrid System: Remote Radio Control of a Freely Flying Beetle Mercynorrhina torquata

This protocol describes the process of constructing an insect-machine hybrid system and carrying out wireless electrical stimulation of the flight muscles required to control the turning motion of a flying insect.

昆虫机的混合动力系统:一个自由飞翔的甲壳虫无线电远程控制（<em&gt; Mercynorrhina torquata</em&gt;）

The rise of radio-enabled digital electronic devices has prompted the use of small wireless neuromuscular recorders and stimulators for studying in-flight ...

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

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