经费是由美国农业部CRIS 5347-22620-021-00D提供，并且部分，由农业和食品研究计划竞争性赠款没有。 2011-67009-30141来自粮食和农业的美国农业部研究所。我们感谢技术支持约翰娜纳西夫的。我们也感谢保罗·贝克，大卫·霍顿，迭戈涅托和弗朗西斯Sivakoff提供一些在图3中使用的图片。

1.内部标志，保留和检测程序<ol><li>内部标记程序<ol><li>收集感兴趣的昆虫（N≈100人）从实验室群体饲养的人工饲料，或从外地分为两个干净的饲养容器。 </li><li>放置一个20个正规ml食物分组（无标记的阴性对照处理）到容器之一。补充一个每秒20毫升的人工饲料包1.0的1.0毫克/毫升鸡IgG /卵黄抗体溶液，调匀，并将其放置在另一个容器。 注意:如果感兴趣的昆虫没有人工饲料，标记可以放置在或在任何饮食它们通常持续上（例如，绿豆，蛾卵，水，糖，蜂蜜）。 </li><li>让昆虫恢复其取食活动24小时。 </li></ol></li><li>内部标记保留程序<ol><li>取下每个容器中的饮食数据包，并提供昆虫W¯¯第i另一食物来源以维持它们在实验（例如，绿豆）的持续时间。 </li><li>来自每个治疗每天（或在任何期望的时间间隔）收集昆虫的队列（20），并在-20℃下立即冻结。 </li></ol></li><li>夹心ELISA方法<ol><li>加入50微升稀释的1兔抗鸡的IgY初级抗体:500的Tris缓冲盐水（TBS）到一个干净的ELISA培养板的各孔中，并孵育至少1小时的在RT（注:微孔板，也可以培养ö / N，4℃）。 </li><li>丢弃从板的抗体和用300微升30分钟1.0％的无脂干乳溶液以及阻止的每个。 </li><li>在等待前面的孵化步骤，将冻结昆虫单独1.6 ml离心管中与1.0ml TBS的。 </li><li>磨每个昆虫用干净杵直到彻底均化，并添加100微升各样品到ELISA的单个井盘。允许样品在室温下孵育1小时。 </li><li>加入100微升阴性（仅TBS）和正的（蛋清在TBS中）控制到孔中的每个ELISA板的第一列。另外，在各板（图1）的最后一列中添加100μl的阴性对照昆虫（未标记昆虫）到8孔中。允许控制样品在室温下孵育1小时。 注意 ，在图1中提供的模板是，我们使用我们的标准试验的模板。孔中的样品的放置要由研究者决定。 </li><li>洗各孔三次用磷酸缓冲盐水（PBS） - 吐温，然后添加50微升的兔抗鸡IgG /卵黄抗体缀合辣根过氧化物酶稀释1:10,000在1％牛奶溶液孵育1小时，在室温。 </li><li>用PBS-Tween洗各孔三次，并添加50μl的四甲基联苯胺（TMB）底物的每个孔中。 </li><li> 10分钟后，读出井用微孔板分光光度计设定在650nm的波长。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/53693/53693fig1.jpg" /> 图1表示用于一个典型的96标准化以及标号示意图孔ELISA培养板，每块板含有7井专用于Tris-缓冲盐水阴性对照（TBS），一个井专用到正蛋白对照（位置Ctrl键），致力于夺回虫（样品1 ... 80），和8个孔，致力于无人盯防的（负）昆虫控制80个人口井（NEG按Ctrl 1 ... 8）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53693/53693fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.外部标记，保留和检测程序<ol><li>外部标记程序<ol><li>收集感兴趣的昆虫（N≈100个体）从一个实验室群体或从外地和地方成两个1.0升的塑料容器具有直径为2.5厘米的孔冲裁每个容器的侧面。 </li><li>喷在第一容器的昆虫使用的医用喷雾器1.0毫升的dh 2 O（阴性对照处理）。喷在第二容器中的昆虫用1.0毫升鸡蛋清使用医学喷雾器一个5.0％的溶液。 </li><li>连接喷雾器的软管到标准实验室空气出口，然后插入雾化器的口进入容器的2.5厘米的孔。然后，打开出风口逐渐直至雾化器产生蒸汽。 </li><li>继续喷射（标记），直至蛋清溶液已被分配（约2-5分钟）。 </li><li>除去雾化器和放置一个软木入塑料容器的孔。让昆虫站在RT直至溶液完全干燥。 </li></ol></li><li>外部标记保留程序<ol><li>将干燥的昆虫每次治疗到一个单独的干净容器中饲养，以避免进一步暴露的标志。 </li><li>加入食物和水的干净的容器在研究期间维持昆虫。 </li><li>每日收集来自每个处理的昆虫的队列（20），并在-20℃下立即冻结。 </li></ol></li><li>间接ELISA方法<ol><li>将冷冻昆虫分别为1.6毫升离心管中，加入1.0毫升TBS的。 </li><li>浸泡样品1小时在定轨摇动器组在120rpm。 </li><li>加入阴性和阳性对照的步骤1.3.5描述。 </li><li>浸泡后，吸取从每个样品一100μl的等分试样插入一个新的96孔ELISA板的80指定样品孔中的一个，如图1允许样品在室温下孵育1小时（注:在微孔板，也可培养O / N在4°C）。 </li><li>用洗孔三次PBS-吐温，然后添加300微升含有1％牛血清白蛋白（PBS-BSA）到每个孔中的磷酸缓冲盐水。 </li><li>在室温30分钟后，用PBS-Tween洗两次。 </li><li>加入50微升稀释的1兔抗卵清蛋白的一抗的:在PBS-BSA-的Silwet（0.05％）8000至每个孔，并孵育1小时，在室温。 </li><li>用PBS-Tween洗各孔三次，然后加入50微升山羊抗兔IgG缀合的辣根过氧化物酶稀释1:1小时在RT 2000在PBS-BSA-的Silwet（0.05％）。 </li><li>用PBS-Tween洗各孔三次，并加入50微升TMB底物至每孔中。 </li><li> 10分钟后，读出的井微量培养板分光光度计设定在650nm的波长。 </li></ol></li></ol> 3.数据分析<ol><li>计算8阴性对照昆虫的吸光度平均值在每个ELISA板，然后计算基于所述合并阴性对照来回的标准偏差m该给定的研究12的ELISA板。 </li><li>添加六倍汇集标准偏差为每个ELISA板以获得ELISA阳性阈值而得到的平均值。任何昆虫样品得到的吸光度值等于或大于该值被认为是阳性的蛋白质标记的存在。 </li></ol>

本出版物中的商品名称或商业产品的一提的是专为提供特定信息的目的，由农业部美国农业部并不意味着推荐或认可。美国农业部是平等机会提供者和雇主。

节肢动物蛋白immunomarking过程首次被描述一个世纪前9的近四分之一。此后，程序已经适应研究各种各样的使用在内部和外部施用的IgG / IgYs节肢动物的分散型态。这些蛋白质已被证明坚定标记的各种测试迄今的昆虫物种。然而，如上所述，对于使用的IgG / IgYs的主要限制是，它们是非常昂贵的。因此，抗体/ IgYs仅用于MRR和自标记类型的研究，其中的昆虫的大批量可标记在小的空间或标记可以掺入饮食或诱饵有用。在这里，我们提供了怎样的内部标记可以通过喂食目标昆虫的人工饲料富含鸡IgG /卵黄抗体给予一个简单的演示。反过来中，IgG / IgY抗体，使用夹心ELISA检测。该系统可能是特别有利的研究人员已经使用一饮食中含有鸡蛋，因为该法自然会检测到的IgG /卵黄抗体在鸡蛋中。此外，少量的IgG /蛋白的IgY的可局部给药至使用上述（图3A）中描述的医疗喷雾器昆虫。雾化器最早用于质量标志非常微妙和微小的寄生与兔IgG的MRR型研究3。自那时以来，其他方法已被用于施用的IgG / IgY的马克到节肢动物为扩散研究。例如，香料喷雾器已被用于施加于其他寄生物种13,14的IgG / IgY的标记。喂养它们他人内部被标记各种昆虫（即，自标记）兔IgG标记的蜂蜜14,15或糖水16（图3B）。最后，Baker等7馈纸板到已浸渍兔IgG（图3C）的白蚁。这些白蚁很容易自我标记，因为他们摄入的食物诱饵东西。 <img alt="图3" src="/files/ftp_upload/53693/53693fig3.jpg" /> 图 3。 各种方法用于管理的蛋白质标记:（A）精致的寄生蜂被标记鸡卵黄抗体使用医用雾化器，（B）一个寄生于兔IgG富集的蜂蜜水溶液喂，（C）白蚁对兔IgG富集纸板喂养用干牛奶诱饵，（D）的蜜蜂自标记，因为它们经过放置在蜂房的，（E）的入口除尘装置天然昆虫被标以使用常规的拖拉机喷洒钻机，牛奶在棉场 （F ）原生虫被打上鸡蛋白与安装在亚视，（G）在苜蓿条原生虫的繁荣和喷嘴的喷雾装置被打上鸡蛋白一回包喷雾装置，并在紫花苜蓿（H，I）原生昆虫被标记使用空中涂抹鸡蛋清。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53693/53693fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 抗体/卵黄抗体蛋白已经不只是被用于研究昆虫传播。它们也被用于跟踪在节肢动物食物链营养链接。哈格勒和迪朗17首次提出标志着猎物的IgG /卵黄抗体的概念，反过来，进行了抗体/卵黄抗体特异性肠道夹心ELISA内容上消耗的显着猎物的食肉动物。这种技术最近获得了普及之中肠道分析研究，因为它是更少繁琐和昂贵的比导电猎物特异性ELISA或聚合酶链反应（PCR）型分子肠内容分析（见哈格勒18和Fournier等人的 19个的评价的Ë优点和各种肠道测定技术缺点）。在过去的十年中，猎物immunomarking技术已被用于标识兔IgG标害虫18,20,21，交哺和白蚁群饲养的关系用兔IgG处理过的纸的食物来源22，granivory棉花天敌取食活动在标有兔IgG 23侵入蒲公英杂草种子节肢动物组合的，臭的抗体标记的番茄天蛾虫捕食率收集地块受到各种semiochemical治疗24，和IgG抗体/卵黄抗体标记腐肉25捕食者清除活性。 第二代蛋白质检测间接ELISA试剂盒进行了将近十年前开发的。这些ELISA的是专门设计来检测包含在"场外的货架"食品产品，如卵清蛋白蛋白在鸡蛋清，在豆奶大豆胰蛋白酶抑制剂的蛋白质，和酪蛋白的蛋白质在牛乳。第二代蛋白质immunomarkers已经证明了MRR，标记捕获和自我标记类型的研究很有用。例如，欧文等人 26表明，它是可行的，以局部标记细腻寄生有这些现成的，货架蛋白质。此外，哈格勒等人 8,27表明蜜蜂能自我标记用蛋清和牛乳干燥粉末作为通过蛋白质分配器放置在一个蜂巢（图3D）的入口它们退出。也许标记所涉及的最有创意的使用蛋白靶向牛拍使用喷射式喷雾器28蛋清。反过来，面对飞成年人获得了自我的标记，因为他们从自己的蛹期出现，并退出了牛拍。 第二代蛋白immunomarkers的最重要的特点是，它们是大批量地和IgG抗体/ IgY抗体蛋白11的成本的一小部分不详。此功能有转olutionized区域范围内的标记拍摄类型的研究进行的方式。具体而言，研究人员现在有一种可靠的方法能够迅速且均匀地标签节肢动物比使用各种常规的喷雾钻机设备（图3E）的广大地区的领域。例如，害虫已直接使用手提枪式喷雾器29,30，拖拉机驱动的气流式喷雾器31,32，和防滑喷雾器33果园和农田标注。霍顿等人 34使用安装在全地形车要找准蛋清昆虫占据嵌入在梨园（ 图3F）覆盖作物应用的电喷装置。同样，Swezey 等人 5,35采用背负式喷雾器向鸡蛋清精确的应用程序适用于行的苜蓿陷阱作物（优选的寄主植物）嵌入在有机种植的草莓地，纪念害虫及其天敌（ <str翁&gt;图3G）。从该研究的结果表明，害虫和天敌是由苜蓿陷阱作物保留并很少分散到高度重视草莓。也许一个蛋白质immunomarker的最极端的应用是通过使用一个天线直升飞机和飞机作物喷粉的。在这项研究中，Sivakoff 等人 36施加的牛乳和蛋清的广播的应用程序29和16公顷开花苜蓿的，分别为（ 图3H，I）。这项研究的目的是为了纪念驻地节肢动物苜蓿，然后，苜蓿收获靠修剪（引发了分散的事件），跟踪节肢动物到相邻的棉花田的分散模式（作物易受后害虫种类）。 第二代标记捕捉的程序有些用户修改了原来的程序有所以满足其特定的研究需求。身型和T的大小他目标昆虫，连同其行为特征，将决定的体积和需要的标记的浓度和方式标志施用37。此外，用于重新捕获的昆虫在该领域的方法将取决于这些因素。到目前为止，几乎所有的方法， 使用 （例如，粘虫，扫网，真空）收集昆虫已成功地用于收集标志着昆虫3,5,8,11,29,31,32,35,38。然而，由于蛋白检测ELISA试剂盒的灵敏度极限，我们强调，必须采取非常谨慎，以确保无人盯防的标本不会成为在收集或分拣流程38污染。 最初的研究11所描述的第二代蛋白质标记系统（ 即 ，卵白蛋白，乳酪蛋白和大豆胰蛋白酶抑制剂）的报道影响检测灵敏度可中标记的类型而异的因素。具体来说，他们发现，DIF同的水源，添加剂（表面活性剂）和酶标板的连类型中使用受影响（正面或负面）的检测。此外，该研究和随后的研究37,39,40显示这三种蛋白标志检测分析变化的疗效。蛋清标记被保留长于牛奶标记，将其保持长于豆奶标记。这些结果凸显着手实地调研前的测试蛋白标记保留在任何给定的昆虫的重要性。而且，这些第二代间接ELISA中有时显示的某些类型的昆虫（例如，食草动物）背景噪声（个人OBS）低水平。这个背景噪声可以通过在协议加入100微升过氧化氢到ELISA板的各孔30分钟步骤2.3.4和2.3.5。步骤之间上述（unpubl。数据）被减少。 应当指出，所用的间接ELISA，检测第二代蛋白标记是非常有效的对昆虫浸泡在样品缓冲液中检测该蛋白​​。然而，间接ELISA中不一样有效的夹心ELISA在检测对昆虫内部蛋白标志或猎物蛋白。有研究表明，间接ELISA中是不一样有效夹心ELISA中在匀化昆虫样品34,41中检测的蛋白质。 与任何的ELISA要考虑的一个重要因素是在过程往往显示板直接制版（主要归因于运行的测定当天到一天效果）变异42,43。因此，至关重要的是，每一个ELISA板包括:（1）一种或多种的TBS只阴性对照，（2）一种或多种的TBS +纯蛋白阳性对照，和（3）至少八个阴性昆虫控制（即，个人的已知不含蛋白标记）。只有TBS，TBS +蛋白质，和昆虫阴性对照保证该缓冲区不是错误的来源，所用试剂都正常工作，一第二，有没有在昆虫交叉与ELISA试剂反应，分别任何蛋白质。此外，昆虫阴性对照是用于计算的ELISA阈值（见下文）是必不可少的。在此示范所示的板的设计包括7的TBS坯，1蛋白质+ TBS阳性对照（在第一列）和8阴性对照（在最后一列）上的每个ELISA板（ 图1）。 当处理的ELISA数据是确定的阈值的得分的昆虫的蛋白质标记的存在或不存在的另一个重要的考虑因素。最初提出Stimmann 44为计分铷标记的昆虫的方法，被定义为标记在没有标记的昆虫加未标记的个体的三倍的标准偏差的一个池的平均等级。这个方法也被用来作为得分昆虫的蛋白质标记9,17的存在的传统方法。然而，在一些CASES，研究人员已经直观地选择更保守的阈值，以减少获得假阳性的可能性。最简单的方法是只添加四个至6个标准偏差的阴性对照，而不是三个18,34的平均值。提出由Sivakoff 等人 12所述的更复杂的方法，以进一步减少获得假阳性的机会。这种方法需要计算八个阴性对照的昆虫在每个ELISA板的平均吸收值，然后基于从所有的给定研究的ELISA板的汇集阴性对照计算的标准偏差。 总之，一种可靠的方法标记节肢动物是最扩散研究的成功是至关重要的。各种方法已被用来在过去的二十年里，以纪念节肢动物与蛋白质。蛋白质immunomarking和标记的检测是相对容易和便宜，非常适合于第无数案例研究。在immunomarking技术未来的用户应该确保他们的方法是适合他们的研究细节。的浓度和所需的标记的量，以及如何将标记施加将取决于象行为目标种类40，身型和尺寸的因素。

追踪在自然界中节肢动物害虫，天敌（拟寄生物和捕食者），和授粉的运动是必不可少的，更好地了解如何改善生态系统服务。对于大多数类型的扩散研究的关键组件是具有可靠的方法来标记感兴趣的节肢动物（多个）。多种材料（例如，涂料，染料，着色粉尘，标签，稀有元素，蛋白质）已经被用来标记节肢动物评估其种群动态，扩散能力，摄食行为，以及其他生态相互作用1,2。 在用于任何给定的扩散研究的标记物的适当性将取决于正进行研究的类型。有用于标记节肢动物三大分类已:（1）标记释放回捕（MRR），（2）标记捕获，和（3）自标记捕获。对于标记释放回捕研究，研究者通常标志着节肢动物集中在laboratoRy和释放它们在该领域的中心点。节肢动物，然后在不同的空间和时间间隔收复使用不同的采集设备（ 例如 ，扫网，真空，粘纸）3,4,5。在收复标本，然后检查特定的标记，以区分本地个体释放。对于标记捕获的研究中，研究者通常直接应用在使用现场喷涂设备（例如，背负式喷雾器，喷杆和喷嘴喷雾器）的标志。为标记捕获研究的最佳标记是廉价且容易地施加到节肢动物的栖息地。对于自标记捕获的研究中，研究者通常适用痕节肢动物诱饵6,7或巢穴的入口8。反过来，节肢动物通过"刷"了对马克在退出窝吞噬着明显的诱饵或外部内部标记本身。 如上所述，许多类型的标记已经我们编辑标记各种节肢动物物种。然而，很少是所有这三种传播研究类是有用的。蛋白质immunomarking过程的发展是标志着昆虫的一大突破。 Immunomarking把一个蛋白质标签上节肢动物内部或外部，这反过来，是由一种抗蛋白特异性酶联免疫吸附测定（ELISA）进行检测。所使用的第一个这样的蛋白质标记物兔免疫球蛋白（IgG抗体）和鸡IgG /卵黄抗体9,10。它们被证明是非常有效的标记为MRR和自标记捕获的研究（见讨论）。不幸的是，抗体/ IgY的蛋白质是昂贵的，因此不实用的标记捕获的研究和大多数类型的自标记捕获的研究。接着，第二代蛋白质检测的ELISA被用于包含在鸡蛋清（白蛋白），牛奶（酪蛋白）和豆浆（胰蛋白酶抑制剂蛋白）的蛋白质开发的。每个测定是高度灵敏的，特异性的，而且，最重要的是，使用的蛋白质，是比的IgG /蛋白的IgY 11便宜得多。这些蛋白质已被证明有效的MRR，标记捕获和自我标记捕获的研究（见讨论）。 在这篇文章中，我们将介绍并演示了如何进行蛋白质标记实验室保留研究。这种研究是所需的任何类型的场扩散研究的研究的第一阶段。具体而言，至关重要的是，调查人员知道有多长的标记将被踏上字段扩散研究之前保留在靶向节肢动物物种。在这里，我们将介绍并演示了如何在内部和外部标记昆虫MRR，标记捕获和自我标记拍摄类型的实地研究。然后，我们说明如何检测与间接和夹心ELISA中的标记的存在。

<table>
	<tbody>
		<tr>
			<td>Food storage container (for insect rearing)</td>
			<td>Rubbermaid/Tupperware</td>
			<td>Various sizes</td>
			<td>Various manufacturers &#38; vendors. Mesh fabric is glued in a window `</td>
		</tr>
		<tr>
			<td>Small volume nebulizer (Micro Mist)</td>
			<td>Teleflex-Hudson RCI</td>
			<td>1881</td>
			<td>Available online and in medical supply shops. Other brands will work as well.</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 1.6 ml w/cap</td>
			<td>Continental Lab Products</td>
			<td>4445.X</td>
			<td>Any similar type will work. We prefer brands that have easy to open lids.</td>
		</tr>
		<tr>
			<td>Styrofoam storage box for microcentrifuge tubes (5x20 grid)</td>
			<td>RPI Corp.</td>
			<td>145746</td>
			<td>This design is nice because it doesn&#39;t crowd the tubes.</td>
		</tr>
		<tr>
			<td>Dispenser, repeater</td>
			<td>Eppendorf</td>
			<td>4982000322</td>
			<td>Anything similar will help speed up the dispensing.</td>
		</tr>
		<tr>
			<td>Pestles, plastic disposable tissue grinders, 1.5 mL</td>
			<td>Kimble Chase</td>
			<td>749521-1500</td>
			<td>Available with various vendors. Pestles can be cleaned and autoclaved for reuse.</td>
		</tr>
		<tr>
			<td>BD Falcon ELISA plates, 96-well (flat bottom)</td>
			<td>BD</td>
			<td>351172</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovation pipette, manual (20-200 &#181;l)</td>
			<td>VistaLab</td>
			<td>1057-0200 or 1070-0200</td>
			<td>Any similar type will work. This model is ergonomic.</td>
		</tr>
		<tr>
			<td>Reservoirs, sterile reagent</td>
			<td>VistaLab</td>
			<td>4054-1000</td>
			<td>Anything similar will work.</td>
		</tr>
		<tr>
			<td>Electronic pipette, 8-channel (50-1200 &#181;l) (Biohit eLine)</td>
			<td>Sartorius</td>
			<td>730391</td>
			<td>If you had to pick one electronic model, this size is most useful.</td>
		</tr>
		<tr>
			<td>Electronic pipette, 8-channel (10-300 &#181;l) (Biohit eLine)</td>
			<td>Sartorius</td>
			<td>730361</td>
			<td>Anything similar will work.</td>
		</tr>
		<tr>
			<td>Manifold, multiwell plate washer (8-position, straight)</td>
			<td>Sigma-Aldrich</td>
			<td>Z369802&#160;</td>
			<td>Anything similar will work for manual plate washing.</td>
		</tr>
		<tr>
			<td>Microplate reader with absorbance detection</td>
			<td>Molecular Devices</td>
			<td>SpectraMax 250</td>
			<td>Anything similar will work.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken IgG/IgY</td>
			<td>United States Biological</td>
			<td>I1903-15R</td>
			<td>Also available from other sources</td>
		</tr>
		<tr>
			<td>Egg whites</td>
			<td>Grocery store</td>
			<td>&#8220;All Whites&#8221;, &#8220;Egg Beaters&#8221;, etc.</td>
			<td>Mix 5 mL with 95 mL dH2O for 5% solution</td>
		</tr>
		<tr>
			<td>Tris (Trizma Base)</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td>2.42 g/L to make TBS</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>29.22 g/L to make TBS and 8.0 g/L for PBS</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S0876&#160;</td>
			<td>1.14 g/L for PBS</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>0.2 g/L for PBS</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>0.2 g/L for PBS</td>
		</tr>
		<tr>
			<td>Tween 20, EIA grade</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td>Add 0.5 mL per L to PBS after salts are dissolved.</td>
		</tr>
		<tr>
			<td>PBS with 1% BSA</td>
			<td>Sigma-Aldrich</td>
			<td>P3688</td>
			<td>Mix 1 packet in 1 L dH20</td>
		</tr>
		<tr>
			<td>Anti-Chicken IgY (IgG) (whole molecule) antibody produced in rabbit (1:500)</td>
			<td>Sigma-Aldrich</td>
			<td>C6409</td>
			<td>Mix 100 &#181;l in 50 ml TBS</td>
		</tr>
		<tr>
			<td>Dry Milk, Powdered or Fresh Milk (1%)</td>
			<td>Grocery store</td>
			<td></td>
			<td>Mix 10 g with 1 L dH2O for 1% solution</td>
		</tr>
		<tr>
			<td>Anti-Chicken IgY (IgG) (whole molecule)-Peroxidase antibody produced in rabbit (1:10000)</td>
			<td>Sigma-Aldrich</td>
			<td>A9046</td>
			<td>Mix 100 ul in 1 L of 1% milk</td>
		</tr>
		<tr>
			<td>Anti-Chicken Egg Albumin antibody produced in rabbit (1:8000)</td>
			<td>Sigma-Aldrich</td>
			<td>C6534</td>
			<td>Dilute 125 &#181;l in 1 L PBS-BSA</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG Peroxidase Conjugate (1:2000)</td>
			<td>Sigma-Aldrich</td>
			<td>A6154</td>
			<td>Dilute 0.5 ml in 1 L PBS-BSA, then add 1.3 ml Silwet</td>
		</tr>
		<tr>
			<td>Vac-In-Stuff (Silwet L-77)silicon-polyether copolymer</td>
			<td>Lehle Seeds</td>
			<td>VIS-01</td>
			<td>Add as last ingredient</td>
		</tr>
		<tr>
			<td>TMB Microwell One Component Peroxidase Substrate</td>
			<td>SurModics</td>
			<td>TMBW-1000-01</td>
			<td></td>
		</tr>
	</tbody>
</table>

administering and detecting protein marks on arthropods for dispersal research

监测节肢动物运动常常需要更好地理解相关的人口动态，扩散图案，宿主植物偏好，和其它生态相互作用。节肢动物通常跟踪性质通过用一种独特的标记标记它们并重新加以收集在时间和空间，以确定它们的散布能力。除了实际的物理标记，如彩色灰尘或涂料，各种类型的蛋白已被证明非常有效的标记节肢动物生态研究。蛋白质可以在内部和/或外部给药。蛋白质然后可以上夺回节肢动物用蛋白质特异性酶联免疫吸附测定（ELISA）进行检测。在这里，我们描述了外部和内部标记节肢动物与蛋白质协议。两个简单的实验例演示了:（1）通过提供一种富含蛋白质的饮食和（2）的外部的蛋白标记局部一个引入到昆虫的内部蛋白标志pplied使用医用喷雾器昆虫。然后，我们涉及的三明治和用于检测的昆虫蛋白标记间接ELISA方法一步一步的指导。在此示范，采集和检测的蛋白质标志物上的节肢动物对马克释放重捕，标记捕获，和研究自标记捕获类型的各个方面进行了讨论，随着该immunomarking程序，一直以来的各种方式调整，以适应各种各样的研究目标。

内部标记: 内部标记保持试验的结果示于图2A中。计算出的ELISA临界阈值是0.054。总体（所有四个样本数据合并），没有蛋白质治疗的昆虫产生持续偏低ELISA值（x = 0.038±0.002，N = 80）。相反，所有的昆虫喂富含蛋白质的饮食产生持续强劲的ELISA值（x = 0.475±0.221，N = 80）。 外部标记: 外部标记保持试验的结果示于图2B。计算出的ELISA临界阈值为0.082。总体而言，局部打上只有水的昆虫产生持续偏低ELISA值（x = 0.048±0.007，N = 80）。相反，所有的局部标有蛋清液的昆虫产生持续强劲的ELISA值（x = 0.746±0.232，N = 80）。 <img alt="图2" src="/files/ftp_upload/53693/53693fig2.jpg" /> 图 2。 平均（±SD）ELISA检测鸡卵黄抗体并用鸡蛋白（白蛋白）标示外（B）昆虫内部标记（A）昆虫光密度值。黑条的平均值±SD ELISA光密度值产生了由无人盯防的昆虫。每个每日蛋白质的治疗昆虫样本量为20人。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53693/53693fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Administering and Detecting Protein Marks on Arthropods for Dispersal Research at JoVE.com

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Proteins are often used to mark arthropods for dispersal research. Methods for administering internal and external protein marks on arthropods are demonstrated. Protein mark detection techniques, either through indirect or sandwich enzyme-linked immunosorbent assays (ELISAs), are also illustrated.

管理和检测蛋白标记的节肢动物的扩散研究

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

administering-detecting-protein-marks-on-arthropods-for-dispersal

Nanyang Technological University

Research

JoVE Journal

Environment

7.3K 次观看.  Arid-Land Agricultural Research Center. Proteins are often used to mark arthropods for dispersal research. Methods for administering internal and external protein marks on arthropods are demonstrated. Protein mark detection techniques, either through indirect or sandwich enzyme-linked immunosorbent assays (ELISAs), are also illustrated. 

视频: Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

修改和叶鼓风机-VAC的节肢动物领域采样中的应用

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

科研

环境科学

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory by using pumps and automating delivery of sediment and water to simulate suspension of sediment. FLEES data are used to develop exposure-response curves between the effects on aquatic organisms and suspended sediment concentrations at the desired exposure duration. The effects data are used to evaluate management practices used to reduce the interactions between aquatic organisms and anthropogenic causes of suspended sediments. The FLEES is capable of generating total suspended solids (TSS) concentrations as low as 30 to as high as 800 mg/L, making this system an ideal choice for evaluating the effects of TSS resulting from many activities including simulating low ambient levels of TSS to evaluating sources of suspended sediments from dredging operations, vessel traffic, freshets, and storms.

A robust and flexible flow-through exposure system designed to maintain sediment in suspension is presented. The system is used to investigate the effects of suspended sediment on various aquatic species and life stages in the laboratory.

对水生生命值评价悬浮泥沙影响的流通系统曝光

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

用于检测实验方案<em&gt;蓝藻</em&gt;与抗体基因芯片液体和固体样品

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

协议塑料微粒采样的海面和样品分析

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop cultivars against environmental stresses. An experimental protocol is presented for RGB and hyperspectral imaging of root systems. The approach uses rhizoboxes where plants grow in natural soil over a longer time to observe fully developed root systems. Experimental settings are exemplified for assessing rhizobox plants under water stress and studying the role of roots. An RGB imaging setup is described for cheap and quick quantification of root development over time. Hyperspectral imaging improves root segmentation from the soil background compared to RGB color based thresholding. The particular strength of hyperspectral imaging is the acquisition of chemometric information on the root-soil system for functional understanding. This is demonstrated with high resolution water content mapping. Spectral imaging however is more complex in image acquisition, processing and analysis compared to the RGB approach. A combination of both methods can optimize a comprehensive assessment of the root system. Application examples integrating root and aboveground traits are given for the context of plant phenotyping and plant physiological research. Further improvement of root imaging can be obtained by optimizing RGB image quality with better illumination using different light sources and by extension of image analysis methods to infer on root zone properties from spectral data.

An experimental protocol is presented for assessment of soil grown plant root systems with RGB and hyperspectral imaging. Combination of RGB image time series with chemometric information from hyperspectral scans optimizes insights into plant root dynamics.

RGB 和光谱根成像植物表型和生理研究: 实验装置和成像协议

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop ...

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive species known to redox regulate biological processes in health and disease. Pre-clinical and clinical evidence points to beneficial effects of plasma treatment in the healing of chronic ulcer of the skin. Other emerging topics, such as plasma cancer treatment, are receiving increasing attention. Plasma medical research requires interdisciplinary expertise in physics, chemistry, and biomedicine. One goal of plasma research is to characterize plasma-treated cells in a variety of specific applications. This includes, for example, cell count and viability, cellular oxidation, mitochondrial activity, cytotoxicity and mode of cell death, cell cycle analysis, cell surface marker expression, and cytokine release. This study describes the essential equipment and workflows required for such research in plasma biomedicine. It describes the proper operation of an atmospheric pressure argon plasma jet, specifically monitoring its basic emission spectra and feed gas settings to modulate reactive species output. Using a high-precision xyz-table and computer software, the jet is hovered in millisecond-precision over the cavities of 96-well plates in micrometer-precision for maximal reproducibility. Downstream assays for liquid analysis of redox-active molecules are shown, and target cells are plasma-treated. Specifically, melanoma cells are analyzed in an efficient sequence of different consecutive assays but using the same cells: measurement of metabolic activity, total cell area, and surface marker expression of calreticulin, a molecule important for the immunogenic cell death of cancer cells. These assays retrieve content-rich biological information about plasma effects from a single plate. Altogether, this study describes the essential steps and protocols for plasma medical research.

A high-throughput cold physical plasma treatment protocol of liquids and cells is shown. It involves setting up different feed gas compositions for plasma ignition, measuring the plasma&#39;s emission spectra, and the subsequent analysis of liquids and cellular activity after plasma treatment.

等离子体医学基础研究--从液体到细胞的吞吐量方法

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive ...

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic pathway of dimethylarsinic acid (DMAV) thiolation, have been recently found in the environment as well as human organs. DMMTAV and DMDTAV can be quantified to determine the ecological effects of dimethylated thioarsenicals and their stability in environmental media. The synthesis method for these compounds is unstandardized, making replicating previous studies challenging. Furthermore, there is a lack of information about storage techniques, including storage of compounds without species transformation. Moreover, because only limited information about synthesis methods is available, there may be experimental difficulties in synthesizing standard chemicals and performing quantitative analysis. The protocol presented herein provides a practically modified synthesis method for the dimethylated thioarsenicals, DMMTAV and DMDTAV, and will help in the quantification of species separation analysis using high performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The experimental steps of this procedure were modified by focusing on the preparation of chemical reagents, filtration methods, and storage.

Preparation of DMMTAV and DMDTAV Using DMAV for Environmental Applications: Synthesis, Purification, and Confirmation

This article presents modified experimental protocols for dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV) synthesis, inducing dimethylarsinic acid (DMAV) thiolation through mixing of DMAV, Na2S, and H2SO4. The modified protocol provides an experimental guideline, thereby overcoming limitations of the synthesis steps that could have caused experimental failures in quantitative analysis.

使用 DMAv为环境应用 DMMTAv和 DMDTAv的准备: 合成、纯化和确认

Dimethylated thioarsenicals such as dimethylmonothioarsinic acid (DMMTAV) and dimethyldithioarsinic acid (DMDTAV), which are produced by the metabolic ...

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

检测暴露于有机氯农药的肝细胞线粒体功能的实验方案

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

The Hawaii Protocol for Scientific Monitoring of Coffee Berry Borer: a Model for Coffee Agroecosystems Worldwide

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at capturing and quantifying the dynamics and impact of this invasive insect pest as well as the development of its host plant across a heterogeneous landscape. The cornerstone of this comprehensive monitoring system is timely georeferenced data collection on CBB movement, coffee berry infestation, mortality by the fungus Beauveria bassiana, and coffee plant phenology via a mobile electronic data recording application. This electronic data collection system allows field records to be georeferenced through built-in global positioning systems, and is backed by a network of weather stations and records of farm management practices. Comprehensive monitoring of CBB and host plant dynamics is an essential part of an area-wide project in Hawaii to aggregate landscape-level data for research to improve management practices. Coffee agroecosystems in other parts of the world that experience highly variable environmental and socioeconomic factors will also benefit from implementing this protocol, in that it will drive the development of customized integrated pest management (IPM) to manage CBB populations.

Comprehensive monitoring of coffee berry borer and host plant dynamics is essential for aggregating landscape-level data to improve management of this invasive pest. Here, we present a protocol for scientific monitoring of coffee berry borer movement, infestation, mortality, coffee plant phenology, weather, and farm management via a mobile electronic data recording application.

夏威夷咖啡莓科学监测协议: 全球咖啡农田模型

Coffee berry borer (CBB) is the most devastating insect pest for coffee crops worldwide. We developed a scientific monitoring protocol that is aimed at ...

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are also a useful scientific model to study motion vision and flight dynamics in a controlled laboratory setting. As the larvae develop in organically polluted water, they are useful models for investigating investment in microbial immunity. While large scale commercial breeding for agriculture already occurs, there are no standardized protocols for maintaining captive populations for scientific studies. This is important as commercial captive breeding programs focusing on mass output during peak pollination periods may fail to provide a population that is consistent, stable and robust throughout the year, as is often needed for other research purposes. Therefore, a method to establish, maintain and refresh a captive research population is required. Here, we describe the utilization of an artificial hibernation cycle, in addition to the nutritional and housing requirements, for long term maintenance of Eristalis tenax. Using these methods, we have significantly increased the health and longevity of captive populations of E. tenax compared to previous reports. We furthermore discuss small scale rearing methods and options for optimizing yields and manipulating population demographics.

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

The overall goal of these procedures is to establish, maintain and refresh a captive population of Eristalis tenax in a research setting.

研究性学习的Eristalis 管Hoverflies 的饲养和长期维护

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are ...