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://jove.unicartagenaproxy.elogim.com/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?...

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 &amp;amp; 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&#039;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 &amp;micro;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 (50-1,200 &amp;micro;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 &amp;micro;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&amp;nbsp;</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>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>&amp;ldquo;All Whites&amp;rdquo;, &amp;ldquo;Egg Beaters&amp;rdquo;, &lt;em&gt;etc.&lt;/em&gt;</td><td>Mix 5 ml with 95 ml dH&lt;sub&gt;2&lt;/sub&gt;O 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&amp;nbsp;g&amp;#8288;/&amp;#8288;L for PBS</td></tr><tr><td>Sodium phosphate dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S0876</td><td>1.14 g/L for PBS</td></tr><tr><td>Potassium phosphate monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</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 dH&lt;sub&gt;2&lt;/sub&gt;O</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 &amp;micro;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 dH&lt;sub&gt;2&lt;/sub&gt;O for 1% solution</td></tr><tr><td>Anti-Chicken IgY (IgG) (whole molecule)-Peroxidase antibody produced in rabbit (1:10,000)</td><td>Sigma-Aldrich</td><td>A9046</td><td>Mix 100 &amp;micro;l in 1 L of 1% milk</td></tr><tr><td>Anti-Chicken Egg Albumin antibody produced in rabbit (1:8,000)</td><td>Sigma-Aldrich</td><td>C6534</td><td>Dilute 125 &amp;micro;l in 1 L PBS-BSA</td></tr><tr><td>Goat Anti-Rabbit IgG Peroxidase Conjugate (1:2,000)</td><td>Sigma-Aldrich</td><td>A6154</td><td>Dilute 0.5 ml in 1 L PBS-BSA, then add 1.3&amp;nbsp;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）。 <p class="jove_conten...

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

Universidad de Cartagena UDEC

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

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

科研

免疫与感染

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

The goal of this protocol is to provide a straightforward and inexpensive approach to collecting Drosophila embryos at medium scale (0.5-1 g) and preparing protein extracts that can be used in downstream proteomic applications, such as affinity purification-mass spectrometry (AP-MS).

中等规模的制备<em&gt;果蝇</em&gt;蛋白质组学实验胚胎提取物

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

发育生物学

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells named wing imaginal discs formed during embryonic development. In the larval stages the imaginal discs grow, increasing its number of cells and forming monolayered epithelial&#160;structures. Inside the pupal case, the imaginal discs bud out and fold into bilayers along a line that becomes the future margin of the wing. During this process, the longitudinal primodia veins originate vein cells on the prospective dorsal and ventral surfaces of the wing. During the pupal stage the stripes of vein cells of each surface communicate in order to generate tight tubes; at the same time, the cross-veins begin their formation.
With the help of appropriate molecular markers, it is possible to identify the major elements composing the wing during its development. For this reason, the ability to accurately detect transcripts or proteins in this structure is critical for studying their abundance and localization related to the development process of the wing.
The procedure described here focuses on manipulating pupal wings, providing detailed instructions on how to dissect the wing during the pupal stage. The dissection of pupal tissue is more difficult to perform than their counterparts in third instar larvae. This is why this approach was developed, to obtain rapid and efficient high quality samples. Details of how to immunostain and mount these wing samples, to allow the visualization of proteins or cell components, are provided in the protocol. With little expertise it is possible to collect 8-10 high quality pupal wings in a short amount of time.

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

The ability to accurately detect transcripts or proteins in Drosophila tissues is critical for studying their abundance and localization related to the development process. Here is the description of a straightforward procedure to dissect pupal wings. These wings can be used as samples in several techniques (immunohistochemistry, PCR assay, etc.).

一种快速高效的解剖蛹翅的方法, 适用于 Immunodetections 或 PCR 检测

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

解剖果蝇黑色素组飞行肌肉为奥米克斯方法

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and ...

遗传学

A Method for Quantifying Foliage-Dwelling Arthropods

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimates of density requires a method with high detection probability and a known sampling area. While most described methods provide a qualitative or semi-quantitative estimate adequate for describing species presence, richness, and diversity, few provide an adequately consistent detection probability and known or consistent sampling areas to provide an index or estimate with adequate precision to detect differences in abundance across environmental, spatial, or temporal variables. We describe how to quantify leaf-dwelling arthropods by sealing the leaves and end of branches in a bag, clipping and freezing the bagged material, and rinsing the previously frozen material in water to separate arthropods from the substrate and quantify them. As we demonstrate, this method can be used at a landscape scale to quantify leaf-dwelling arthropods with adequate precision to test for and describe how spatial, temporal, environmental, and ecological variables influence arthropod richness and abundance. This method allowed us to detect differences in density, richness, and diversity of leaf-dwelling arthropods among 5 genera of trees commonly found in southeastern deciduous forests.

We describe how to quantify leaf dwelling arthropods by sealing the leaves and end of branches in a bag, clipping and freezing the bagged material, and rinsing the previously frozen material in water to separate arthropods from the substrate for quantification.

定量叶类节肢动物的方法

Terrestrial arthropods play an important role in our environment. Quantifying arthropods in a way that allows for a precise index or estimates of density ...

环境科学

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

测量的生理反应<em&gt;果蝇</em&gt;感觉神经元脂质费洛蒙使用实时钙成像

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

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 ...

神经科学

Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the mode of action (MOA) of such molecules leverage bulk treatment of experimental specimens with an excess of a specific reactive species. In this approach, the high reactivity of electrophiles renders non-discriminate labeling of the proteome in a time- and context-dependent manner; redox-sensitive proteins and processes can also be indirectly and often irreversibly affected. Against such a backdrop of innumerable potential targets and indirect secondary effects, linking phenotype to specific target engagement remains a complex task. Zebrafish targeting reactive electrophiles and oxidants (Z-REX)-an on-demand reactive-electrophile delivery platform adapted for use in larval zebrafish-is designed to deliver electrophiles to a specific protein of interest (POI) in otherwise unperturbed live fish embryos. Key features of this technique include a low level of invasiveness, along with dosage-, chemotype-, and spatiotemporally-controlled precision electrophile delivery. Thus, in conjunction with a unique suite of controls, this technique sidesteps off-target effects and systemic toxicity, otherwise observed following uncontrolled bulk exposure of animals to reactive electrophiles and pleiotropic electrophilic drugs. Leveraging Z-REX, researchers can establish a foothold in the understanding of how individual stress responses and signaling outputs are altered as a result of specific reactive ligand engagement with a specific POI, under near-physiologic conditions in intact living animals.

Zebrafish targeting reactive electrophiles and oxidants (Z-REX) is a chemical biology-based method for the investigation of reactive small-molecule signaling. This technique can be applied to live fish of different developmental stages. Here, we couple standard assays in zebrafish with Z-REX for signaling pathway analysis.

监测斑马鱼幼虫的靶向信号反应 - Z-REX 揭示亲电药物和代谢物的精确机制

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the ...

生物学

<meta charset="utf8"></head><body>果蝇眼缺陷的高分辨率成像和分析

A Rapid, Simple Workflow for Quantification of External Adult Drosophila Structures

The Drosophila compound eye is a precisely patterned tissue that has revealed molecular mechanisms and biological processes that drive morphogenesis. It is a simple structure of repeating unit eyes, termed ommatidia, that is used to characterize genetic interactions and gene functions. Mutations that affect eye architecture can be easily detected and analyzed; hence, this system is frequently used in under-resourced institutions. Further phenotypic analysis often includes a Scanning Electron Microscope (SEM) to generate high-magnification images suitable for quantitative analysis. However, SEMs are expensive and require costly reagents; sample preparation spans days; and, often, they need full-time staff for sample preparation and instrument maintenance. This limits their utility at under-resourced institutions or during budgetary austerity. In entomology, the use of high-resolution digital imaging technology is a common practice for the identification and characterization of species. This paper describes a method that combines strategies and allows for high-resolution digital imaging of adult Drosophila structures and quantitative analysis using the open software ImageJ. The workflow is a rapid and student-friendly alternative that remedies the limitations of underfunded and under-resourced research facilities with a cost-effective and rapid approach to quantitative phenotypic analysis.

<meta charset="utf8"></head><body>用于定量体外成体 果蝇 结构的快速、简单的工作流程

Here, we present a rapid, low-cost, workflow for high-resolution imaging of adult Drosophila eyes to quantify patterning and growth defects. We describe our protocol for sample preparation by point-mounting, high-resolution image acquisition, and image analysis.

用于定量体外成体 果蝇 结构的快速、简单的工作流程

The Drosophila compound eye is a precisely patterned tissue that has revealed molecular mechanisms and biological processes that drive morphogenesis. It ...

管理和检测蛋白标记的节肢动物的扩散研究

摘要

探索更多视频

此视频中的章节

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

中等规模的制备

一种快速高效的解剖蛹翅的方法, 适用于 Immunodetections 或 PCR 检测

解剖果蝇黑色素组飞行肌肉为奥米克斯方法

定量叶类节肢动物的方法

测量的生理反应

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

监测斑马鱼幼虫的靶向信号反应 - Z-REX 揭示亲电药物和代谢物的精确机制

用于定量体外成体果蝇结构的快速、简单的工作流程

用于定量体外成体果蝇结构的快速、简单的工作流程

管理和检测蛋白标记的节肢动物的扩散研究

摘要

探索更多视频

此视频中的章节

一个钙生物发光检测蚊虫的功能分析（埃及伊蚊）和刻度（镰形扇头microplus）G蛋白偶联受体

中等规模的制备

一种快速高效的解剖蛹翅的方法, 适用于 Immunodetections 或 PCR 检测

解剖果蝇黑色素组飞行肌肉为奥米克斯方法

定量叶类节肢动物的方法

测量的生理反应

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

监测斑马鱼幼虫的靶向信号反应 - Z-REX 揭示亲电药物和代谢物的精确机制

用于定量体外成体 果蝇 结构的快速、简单的工作流程

用于定量体外成体 果蝇 结构的快速、简单的工作流程

用于定量体外成体果蝇结构的快速、简单的工作流程

用于定量体外成体果蝇结构的快速、简单的工作流程