Name: a step-by-step guide to mosquito electroantennography
Uploaded: 2021-03-10T14:30:00
Description: Watch this Scientific Journal Video about A Step-by-Step Guide to Mosquito Electroantennography at JoVE.com

我感谢克莱门特·维诺格博士和杰弗里·里费尔博士的有益讨论。以下试剂通过BEI Resources，NIAID，NIH获得： 按蚊斯蒂芬西，菌株STE2，MRA-128，由Mark Q. Benedict提供; 埃及伊蚊，Strain ROCK，MRA-734，由David W. Severson提供; 五筋膜库蚊，菌株JHB，卵，NR-43025。作者感谢Jake Tu博士，Nisha Duggal博士，James Weger博士和Jeffrey Marano提供 Culex quinquefasciatus 和 Anopheles stephensi （菌株：Liston）蚊卵。 白纹伊蚊 和 弓形虫鹦鹉螺 来自作者在新河谷地区（美国弗吉尼亚州）收集的田间蚊子。这项工作得到了生物化学系和弗拉林生命科学研究所的支持。

1. 盐水溶液制备
<ol><li>提前准备好生理盐水并存放在冰箱中。</li>
	<li>按照Beyenbach和Masia26 准备解决方案。 注意：以mM为单位的盐水配方：150.0 NaCl，25.0 HEPES，5.0葡萄糖，3.4 KCl，1.8 NaHCO3，1.7 CaCl 2和1.0 MgCl2。用1M NaOH将pH调节至7.1。此时不要在制剂中添加葡萄糖或蔗糖，以增加货架储存。在运行EAG之前，将所需量添加到盐水中（每次实验约50 mL）。</li>
</o...

<ol>
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E., Blackwell, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroantennogram+studies+of+potential+oviposition+attractants+for+Toxorhynchites+moctezuma+and+T.+amboinensis+mosquitoes.">Electroantennogram studies of potential oviposition attractants for Toxorhynchites moctezuma and T. amboinensis mosquitoes.</a> Physiological Entomology. 23 (3), 214-219 (1998).</li><li>Seenivasagan, T., Sharma, K. 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J., Sukumaran, D., Ganesan, K., Prakash, S., Sekhar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroantennogram+and+behavioral+responses+of+Culex+quinquefasciatus+(Diptera:+Culicidae)+females+to+chemicals+found+in+human+skin+emanations.">Electroantennogram and behavioral responses of Culex quinquefasciatus (Diptera: Culicidae) females to chemicals found in human skin emanations.</a> Journal of Medical Entomology. 43 (2), 207-213 (2014).</li><li>Cooperband, M. F., McElfresh, J. S., Millar, J. G., Carde, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attraction+of+female+Culex+quinquefasciatus+Say+(Diptera:+Culicidae)+to+odors+from+chicken+feces.">Attraction of female Culex quinquefasciatus Say (Diptera: Culicidae) to odors from chicken feces.</a> Journal of Insect Physiology. 54 (7), 1184-1192 (2008).</li><li>Dekker, T., Ignell, R., Ghebru, M., Glinwood, R., Hopkins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+mosquito+repellent+odours+from+Ocimum+forskolei.">Identification of mosquito repellent odours from Ocimum forskolei.</a> Parasites &amp; Vectors. 4 (1), 183 (2011).</li><li>Choo, Y. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromodulation+and+differential+learning+across+mosquito+species.">Neuromodulation and differential learning across mosquito species.</a> bioRxiv. , 755017 (2019).</li><li>Lahond&#232;re, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+olfactory+basis+of+orchid+pollination+by+mosquitoes.">The olfactory basis of orchid pollination by mosquitoes.</a> Proceedings of the National Academy of Sciences. 117 (1), 708-716 (2020).</li><li>Beyenbach, K., Masia, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+conductances+of+principal+cells+in+Malpighian+tubules+of+Aedes+aegypti.">Membrane conductances of principal cells in Malpighian tubules of Aedes aegypti.</a> Journal of Insect Physiology. 48, 375-386 (2002).</li><li>Oesterle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Pipette Cookbook. , (2018).</li><li>R Core Team. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R:+A+language+and+environment+for+statistical+computing.">R: A language and environment for statistical computing.</a> R Foundation for Statistical Computing. , (2018).</li><li>Afify, A., Betz, J. 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嗅觉介导的行为受许多因素影响，包括生理因素（例如年龄、一天中的时间）和环境（例如温度、相对湿度）30。因此，在进行EAG时，必须使用处于相同生理状态（即监测年龄，饥饿，交配）31 的昆虫，并在制剂周围保持温暖潮湿的环境以避免干燥。25°C左右的温度是理想的，主要航空公司的湿度为60%至80%。这可以通过在主航线上放置一个起泡器来轻松实现。 </...

蚊子是地球上最致命的生物，每年夺走超过100万人的生命，并使世界一半以上的人口面临接触其传播病原体的风险，同时叮咬1。这些昆虫依靠广泛的线索（即热、视觉、机械、嗅觉、听觉）来定位宿主（植物和动物）、交配和产卵，以及在幼虫和成虫阶段避开捕食者2，3。在这些感官中，嗅觉在上述行为中起着关键作用，特别是对于气味分子的中远程检测2，3。宿主或产卵部位发出的气味由位于蚊子触诊长鼻、跗骨和触角上的各种特异性嗅觉受体（例如 GR、OR、IR）检测2，3。
由于嗅觉是它们寻找宿主（植物和动物）、交配和产卵行为的关键组成部分，因此它构成了研究开发蚊子控制新工具的理想目标4。对驱虫剂（例如避蚊胺、IR3535、派卡瑞丁）和诱饵（例如BG哨兵人类诱饵）的研究非常多产5，但由于目前蚊子控制方面的挑战（例如，杀虫剂抗性，入侵物种），必须开发新的有效控制方法，了解蚊子生物学。
许多技术（例如，嗅觉计，着陆测定，电生理学）已被用于评估蚊子中化合物或化合物混合物的生物活性。其中，电图（或电图（EAGs））可用于确定气味是否被蚊子触角检测到。这项技术最初由施耐德6号开发，从那时起已被用于许多不同的昆虫属，包括飞蛾7，8，9，大黄蜂10，11，蜜蜂12，13和果蝇14，15等等。电学也已使用各种协议，包括蚊子16，17，18，19，20，21，22，23，24，25的单触角或多触角。
蚊子是相对较小和脆弱的昆虫，触角相当细。虽然对飞蛾或大黄蜂等较大的昆虫进行EAG相对容易，因为它们的尺寸更大，触角更厚，但在蚊子中进行EAG可能具有挑战性。特别是，保持良好的信噪比和持久的响应式制备是数据再现性和可靠性的两个主要要求。
此处提出的低噪声EAG分步指南直接提供了这些限制的解决方案，并使该协议适用于来自不同属的几种蚊子物种，包括 伊蚊， 按蚊， Culex和 弓形虫，并描述了雌性和雄性的技术。电学提供了一种快速而可靠的方法来筛选和确定生物活性化合物，然后在通过行为测定确定价后，可以利用这些化合物进行诱饵开发。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Air table Clean Bench</td>
			<td>TMC</td>
			<td>https://www.techmfg.com/products/labtables/cleanbench63series/accessoriess</td>
			<td>Noise reducer</td>
		</tr>
		<tr>
			<td>Analog-to-digital board</td>
			<td>National Instruments</td>
			<td>BNC-2090A</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop Flowbuddy Complete</td>
			<td>Genesee Scientific</td>
			<td>59-122BC</td>
			<td>To anesthesize mosquitoes</td>
		</tr>
		<tr>
			<td>Borosillicate glass capillary</td>
			<td>Sutter Instrument</td>
			<td>B100-78-10</td>
			<td>To make the recording and references capillaries</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td>Sigma Aldrich</td>
			<td>Benzaldehyde: 418099-100 mL; Butyric acid: B103500-100mL; 1-Hexanol: 471402-100mL; Mineral oil: M8410-1L</td>
			<td>Chemicals used for the experiments presented here</td>
		</tr>
		<tr>
			<td>CO2</td>
			<td>Airgas or Praxair</td>
			<td>N/A</td>
			<td>To anesthesize mosquitoes</td>
		</tr>
		<tr>
			<td>Cold Light Source</td>
			<td>Volpi</td>
			<td>NCL-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable syringes</td>
			<td>BD</td>
			<td>1 mL (309628)&#160; / 3 mL (309657)</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode cables</td>
			<td>World Precision Instruments</td>
			<td>5371</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode gel salt free</td>
			<td>Parkerlabs</td>
			<td>12-08-Spectra-360</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>TMC</td>
			<td>https://www.techmfg.com/products/electric-and-magnetic-field-cancellation/faradaycages</td>
			<td>Noise reducer</td>
		</tr>
		<tr>
			<td>Flowmeters</td>
			<td>Bel-art</td>
			<td>65 mm (H40406-0010) / 150 mm (H40407-0075)</td>
			<td>One of each</td>
		</tr>
		<tr>
			<td>GCMS vials and caps</td>
			<td>Thermo-fisher scientific</td>
			<td>2-SVWKA8-CPK</td>
			<td>To prepare odorant dilutions</td>
		</tr>
		<tr>
			<td>Glass syringes (Fortuna)</td>
			<td>Sigma Aldrich</td>
			<td>Z314307</td>
			<td>For odor delivery to the EAG prep</td>
		</tr>
		<tr>
			<td>Humbug</td>
			<td>Quest Scientific</td>
			<td>http://www.quest-sci.com/</td>
			<td>Noise reducer</td>
		</tr>
		<tr>
			<td>2 mm Jack Holder, Narrow, 90 deg., With Wire</td>
			<td>A-M Systems</td>
			<td>675748</td>
			<td>Electrode holder</td>
		</tr>
		<tr>
			<td>Magnetic bases</td>
			<td>Kanetec</td>
			<td>MB-FX</td>
			<td>x 2</td>
		</tr>
		<tr>
			<td>MATLAB + Toolboxes</td>
			<td>Mathworks</td>
			<td>https://www.mathworks.com/products/matlab.html</td>
			<td>For delivering the pulses</td>
		</tr>
		<tr>
			<td>Medical air</td>
			<td>Airgas or Praxair</td>
			<td>N/A</td>
			<td>For main airline</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikkon</td>
			<td>SMZ-800N</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulators Three-Axis Coarse/Fine Compact Micromanipulator</td>
			<td>Narishige</td>
			<td>MHW-3</td>
			<td>x 2</td>
		</tr>
		<tr>
			<td>Microelectrode amplifier with headstage</td>
			<td>A-M Systems</td>
			<td>Model 1800</td>
			<td></td>
		</tr>
		<tr>
			<td>Mosquito rearing supplies</td>
			<td>Bioquip</td>
			<td>https://www.bioquip.com/Search/WebCatalog.asp</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>BD</td>
			<td>25G (305127) / 21G (305165)</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-678-6A</td>
			<td>For odor delivery to the EAG prep</td>
		</tr>
		<tr>
			<td>PTFE Tubing of different diameters</td>
			<td>Mc Master Carr</td>
			<td>N/A</td>
			<td>To connect solenoid valve, flowmeter, airline ect.</td>
		</tr>
		<tr>
			<td>30V/5A DC Power Supply</td>
			<td>Dr. Meter</td>
			<td>PS-305DM</td>
			<td></td>
		</tr>
		<tr>
			<td>R version 3.5.1</td>
			<td>R project</td>
			<td>https://www.r-project.org/</td>
			<td>For data analyses</td>
		</tr>
		<tr>
			<td>Relay for solenoid valve</td>
			<td>N/A</td>
			<td>Custom made</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver wire 0.01&#8221;</td>
			<td>A-M Systems</td>
			<td>782500</td>
			<td></td>
		</tr>
		<tr>
			<td>Solenoid valve (3-way)</td>
			<td>The Lee Company</td>
			<td>LHDA0533115H</td>
			<td></td>
		</tr>
		<tr>
			<td>WinEDR software</td>
			<td>Strathclyde Electrophysiology Software</td>
			<td>WinEDR V3.9.1</td>
			<td>For EAG recording</td>
		</tr>
		<tr>
			<td>Whatman paper</td>
			<td>Cole Parmer</td>
			<td>UX-06648-03</td>
			<td>To load chemical in glass syringe / Pasteur pipette</td>
		</tr>
	</tbody>
</table>

a step-by-step guide to mosquito electroantennography

雌性蚊子是地球上最致命的动物，由于它们在获取血粉时传播的病原体，每年夺走超过100万人的生命。为了找到要觅食的宿主，蚊子依靠广泛的感官线索，包括视觉、机械、热和嗅觉。该研究详细介绍了一种技术，即电学（EAG），该技术使研究人员能够评估蚊子是否可以以浓度依赖性的方式检测单个化学物质和化学物质混合物。当与气相色谱（GC-EAG）结合使用时，该技术允许将触角暴露在完整的顶部空间/复杂混合物中，并确定蚊子可以检测到感兴趣的样品中存在的化学物质。这适用于宿主体味以及植物花束或其他与生态相关的气味（例如，产卵部位气味剂）。在这里，我们描述了一种协议，该协议允许长时间的制备反应时间，并且适用于来自多个属的雌性和雄性蚊子，包括 伊蚊， Culex，按 蚊和 弓形虫 蚊子。由于嗅觉在蚊子 - 宿主相互作用和蚊子生物学中起着重要作用，因此EAG和GC-EAG可以揭示感兴趣的化合物，以开发新的疾病媒介控制策略（例如诱饵）。辅以行为测定，可以确定每种化学物质的价态（例如引诱剂、驱虫剂）。

电学是确定昆虫天线是否检测到化学物质或化学物质混合物的有力工具。它还可用于使用浓度的逐渐增加来确定给定化学品的检测阈值（即剂量曲线响应， 图4B）。此外，测试驱虫剂对宿主相关气味的反应的影响是有用的29。
EAG中应始终使用阳性和阴性对照。在这里，苯甲醛被用作阳性对照（图3B，3C...

Watch this Scientific Journal Video about A Step-by-Step Guide to Mosquito Electroantennography at JoVE.com

A Step-by-Step Guide to Mosquito Electroantennography

本文详细介绍了在几个蚊子属（包括雌性和雄性）中成功和低噪声电图的分步方案。

蚊子电图分步指南

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...

对于电图，使用处于特定和一致生理状态的蚊子对于获得可靠的结果至关重要。分步方案允许在蚊子中进行持久的电学记录。该方法可用于几种蚊子种类，以及雄性和雌性。电学技术也可以转移到其他昆虫模型，包括甲虫、苍蝇、接吻虫和蚂蚁。首先，从储存它们的 20 摄氏度冰箱中取出装有气味混合物和溶剂控制的 1.5 毫升琥珀色小瓶，以防止降解。接下来，将10微升的气味溶液移液到一张滤纸上，装入标记的玻璃注射器内。在实验当天隔离蚊子。在显微镜下工作，使用一对镊子用细丝轻轻折断两个硼硅酸盐毛细管的尖端。在运行电图或EAG实验之前，请确保电极支架内部透明，没有硼硅酸盐碎屑。然后通过将电极支架的银线浸泡在纯漂白剂中五分钟来进行氯化化，直到电线变成深色哑光灰色。然后冲洗电线。松开橡胶塞，并使用 20 号针用盐溶液填充毛细管内部。冲洗毛细管并将电线插入两个毛细管中。保持金属丝尖端距离毛细管尖端小于一毫米。将毛细管穿过电极支架内的橡胶圈而不破坏它，并在确认不存在气泡后轻轻拧紧橡胶塞。使用参比电极支架上开口较宽、记录电极支架上开口较小的毛细管。将两个安装的电极支架放在湿清洁湿巾上，以防止吸头变干，直到准备安装头部。确保空气桌已打开。航空公司没有堵塞，空气打开。验证医用空气罐是否已满，以避免在实验过程中更换它，并确认加湿器中的气泡。打开计算机、软件应用程序和阀门电源。验证互联网连接后，发送控制脉冲以验证传输脉冲的阀门是否正常工作。将铝板放在冰上，上面放一块湿清洁纸巾，然后在角落里放一小块电极凝胶。将蚊子杯放在冰上，让蚊子降温。用微型剪刀夹住蚊子每个天线的尖端。使用镊子将蚊子拖到电极凝胶团旁边，然后将每个天线的尖端轻轻浸入凝胶中。用镊子将蚊子触角拉出，同时将它们彼此相邻。用微型剪刀砍掉蚊子的头。轻轻地将参比电极的尖端浸入凝胶中，然后将其与颈部接触，让头部粘在上面。将参比头电极放在微型机械手上。将两个电极支架连接到放大器。使用微型机械手将记录电极尽可能靠近天线尖端放置。然后将天线尖端插入记录电极。将航空管靠近蚊头准备工作一厘米。打开放大器和降噪器，确保基线信号无噪声。一旦噪音水平令人满意，将第一个气味注射器插入空气孔中以进行测试并关闭法拉第笼。然后单击EAG软件上的记录以传递脉冲。以毫伏为单位的振幅测量EAG响应，然后继续下一个气味或浓度。该实验成功地证明，蚊子物种可以对不同的化学物质表现出不同的嗅觉反应。对用作阴性对照的矿物油没有反应。通过使用鳄鱼夹将元件接地到法拉第笼，可以减少由电噪声引起的结果误差。对于不同种类的蚊子，对相同气味剂的反应阈值在幅度上可能有所不同。例如，与埃及伊蚊、斯蒂芬斯按蚊和五筋膜库蚊相比，弓形虫七叶茜蚊会产生非常大的EAG。注意到响应阳性对照苯甲醛的较大偏转。虽然对阴性对照缺乏反应，但矿物油。这些平均触角响应显示在条形图中。此外，通过呈现天线、化学物质浓度的增加和绘制剂量反应曲线来确定检测每种蚊子物种的特定化学物质的阈值。重要的是在头部被切碎和安装后尽快开始录制，以确保准备工作具有出色的响应性。EAG允许确定蚊子触角是否对给定浓度的给定化学物质做出反应。但是，它不允许确定这种化学物质的平衡。例如，无论是引诱还是悔改。

Research

JoVE Journal

Biology

Protocol for Mosquito Rearing (A. gambiae)

Protocol for Mosquito Rearing A. gambiae

蚊子养育的议定书“（冈比亚按蚊）

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

科研

生物学

Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

建设一个更美好的蚊子：确定基因在冈比亚按蚊和埃及斑蚊蚊子启用疟疾和登革热的电阻

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

一个实用的方法遗传诱导的命运映射：一个视觉引导标记和跟踪细胞在体内</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

一个循序渐进的步骤方法的到Nanodisc脂质颗粒的ABC转运蛋白的重组

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

A Practical Guide to Phylogenetics for Nonexperts

实用指南系统发育的非专家

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

生理录音和黄热病蚊子的味觉附属物RNA测序<em&gt;埃及伊蚊</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

指南现代荧光定量免疫印迹与故障排除策略

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Endotracheal Intubation of Rabbits Using a Polypropylene Guide Catheter

用聚丙烯导管引导家兔气管插管

Endotracheal intubation in rabbits can be challenging due to their unusual anatomy. Achieving a patent airway during anesthesia is critical for the ...

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

同步加速器微CT高通量皮质骨样品采购和分析的剖面、科林和图像处理指南

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

用于下一代测序的基于磁珠的蚊子DNA提取协议

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...