Summary

管理和检测蛋白标记的节肢动物的扩散研究

Published: January 28, 2016
doi:

Summary

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.

Abstract

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。

Introduction

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。

Protocol

1.内部标志,保留和检测程序内部标记程序收集感兴趣的昆虫(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。 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?…

Representative Results

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_cont…

Discussion

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?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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。

Materials

Food storage container (for insect rearing) Rubbermaid/Tupperware Various sizes Various manufacturers & vendors. Mesh fabric is glued in a window `
Small volume nebulizer (Micro Mist) Teleflex-Hudson RCI 1881 Available online and in medical supply shops. Other brands will work as well.
Microcentrifuge tubes, 1.6 ml w/cap Continental Lab Products 4445.X Any similar type will work. We prefer brands that have easy to open lids.
Styrofoam storage box for microcentrifuge tubes (5×20 grid) RPI Corp. 145746 This design is nice because it doesn't crowd the tubes.
Dispenser, repeater Eppendorf 4982000322 Anything similar will help speed up the dispensing.
Pestles, plastic disposable tissue grinders, 1.5 mL Kimble Chase 749521-1500 Available with various vendors. Pestles can be cleaned and autoclaved for reuse.
BD Falcon ELISA plates, 96-well (flat bottom) BD 351172
Ovation pipette, manual (20-200 µl) VistaLab 1057-0200 or 1070-0200 Any similar type will work. This model is ergonomic.
Reservoirs, sterile reagent VistaLab 4054-1000 Anything similar will work.
Electronic pipette, 8-channel (50-1200 µl) (Biohit eLine) Sartorius 730391 If you had to pick one electronic model, this size is most useful.
Electronic pipette, 8-channel (10-300 µl) (Biohit eLine) Sartorius 730361 Anything similar will work.
Manifold, multiwell plate washer (8-position, straight) Sigma-Aldrich Z369802  Anything similar will work for manual plate washing.
Microplate reader with absorbance detection Molecular Devices SpectraMax 250 Anything similar will work.
Chicken IgG/IgY United States Biological I1903-15R Also available from other sources
Egg whites Grocery store “All Whites”, “Egg Beaters”, etc. Mix 5 mL with 95 mL dH2O for 5% solution
Tris (Trizma Base) Sigma-Aldrich T1503 2.42 g/L to make TBS
Sodium chloride (NaCl) Sigma-Aldrich S9888 29.22 g/L to make TBS and 8.0 g/L for PBS
Sodium phosphate dibasic (Na2HPO4) Sigma-Aldrich S0876  1.14 g/L for PBS
Potassium phosphate monobasic (KH2PO4) Sigma-Aldrich P5655 0.2 g/L for PBS
Potassium chloride (KCl) Sigma-Aldrich P9541 0.2 g/L for PBS
Tween 20, EIA grade Sigma-Aldrich P1379 Add 0.5 mL per L to PBS after salts are dissolved.
PBS with 1% BSA Sigma-Aldrich P3688 Mix 1 packet in 1 L dH20
Anti-Chicken IgY (IgG) (whole molecule) antibody produced in rabbit (1:500) Sigma-Aldrich C6409 Mix 100 µl in 50 ml TBS
Dry Milk, Powdered or Fresh Milk (1%) Grocery store Mix 10 g with 1 L dH2O for 1% solution
Anti-Chicken IgY (IgG) (whole molecule)-Peroxidase antibody produced in rabbit (1:10000) Sigma-Aldrich A9046 Mix 100 ul in 1 L of 1% milk
Anti-Chicken Egg Albumin antibody produced in rabbit (1:8000) Sigma-Aldrich C6534 Dilute 125 µl in 1 L PBS-BSA
Goat Anti-Rabbit IgG Peroxidase Conjugate (1:2000) Sigma-Aldrich A6154 Dilute 0.5 ml in 1 L PBS-BSA, then add 1.3 ml Silwet
Vac-In-Stuff (Silwet L-77)silicon-polyether copolymer Lehle Seeds VIS-01 Add as last ingredient
TMB Microwell One Component Peroxidase Substrate SurModics TMBW-1000-01

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Cite This Article
Hagler, J. R., Machtley, S. A. Administering and Detecting Protein Marks on Arthropods for Dispersal Research. J. Vis. Exp. (107), e53693, doi:10.3791/53693 (2016).

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