Name: video tracking protocol to screen deterrent chemistries for honey bees
Uploaded: 2017-06-12T14:00:00
Description: Watch this Scientific Journal Video about Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees at JoVE.com

我们要感谢Thomas Kuhar博士使用视觉跟踪软件和设备。我们感谢詹姆斯·威尔逊和斯科特·奥尼尔的技术援助。

1.准备糖琼脂糖立方体<ol><li>称取8克糖并置于50mL锥形瓶中。 </li><li>向锥形瓶中加入20mL去离子水。通过旋转烧瓶来溶解糖。 </li><li>称量170 mg琼脂糖，并加入糖溶液。 </li><li>将糖 - 琼脂糖溶液在微波炉中加热25秒。将琼脂糖溶解在糖溶液中。 </li><li> 让烧瓶和糖琼脂糖溶液冷却。 注意：烧瓶应该是凉爽的接触，但不要让溶液凝固。 <ol><li>为了制备用于对照处理的糖琼脂糖立方体?...

<ol>
	<li>Gallai, N., Salles, J. M., Settele, J., Vaissi&#232;re, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economic+valuation+of+the+vulnerability+of+world+agriculture+confronted+with+pollinator+decline.">Economic valuation of the vulnerability of world agriculture confronted with pollinator decline.</a> Ecol Econ. 68 (3), 810-821 (2009).</li><li>van Engelsdorp, D., Meixner, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+historical+review+of+managed+honey+bee+populations+in+Europe+and+the+United+States+and+the+factors+that+may+affect+them.">A historical review of managed honey bee populations in Europe and the United States and the factors that may affect them.</a> J Invertebr Pathol. 103, S80-S95 (2010).</li><li>Aizen, M. A., Harder, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Global+Stock+of+Domesticated+Honey+Bees+Is+Growing+Slower+Than+Agricultural+Demand+for+Pollination.">The Global Stock of Domesticated Honey Bees Is Growing Slower Than Agricultural Demand for Pollination.</a> Current Biol. 19 (11), 915-918 (2009).</li><li>Smith, K. M., Loh, E. H., Rostal, M. K., Zambrana-torrelio, C. M., Mendiola, L., Daszak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogens,+Pests,+and+Economics+Drivers+of+Honey+Bee+Colony+Declines+and+Losses.">Pathogens, Pests, and Economics Drivers of Honey Bee Colony Declines and Losses.</a> Ecohealth. 10, 434-445 (2014).</li><li>Mullin, C. A., Frazier, M., 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=High+Levels+of+Miticides+and+Agrochemicals+in+North+American+Apiaries:+Implications+for+Honey+Bee+Health.">High Levels of Miticides and Agrochemicals in North American Apiaries: Implications for Honey Bee Health.</a> PLoS ONE. 5 (3), (2010).</li><li>Li, Y., Kelley, R. A., Anderson, T. D., Lydy, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+comparison+of+two+multi-residue+methods+for+the+analysis+of+select+pesticides+in+honey+bees,+pollen,+and+wax+by+gas+chromatography+-+quadrupole+mass+spectrometry.">Development and comparison of two multi-residue methods for the analysis of select pesticides in honey bees, pollen, and wax by gas chromatography - quadrupole mass spectrometry.</a> Talanta. 140, 81-87 (2015).</li><li>Kakumanu, M. L., Reeves, A. M., Anderson, T. D., Rodrigues, R. R., Williams, M. A., Williams, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honey+Bee+Gut+Microbiome+Is+Altered+by+In-Hive+Pesticide+Exposures.">Honey Bee Gut Microbiome Is Altered by In-Hive Pesticide Exposures.</a> Front Microbiol. 7, 1-11 (2016).</li><li>Katz, T. M., Miller, J. H., Hebert, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+repellents:+Historical+perspectives+and+new+developments.">Insect repellents: Historical perspectives and new developments.</a> J Am Acad Dermatol. 58 (5), 865-871 (2008).</li><li>Dickens, J. C., Bohbot, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini+review:+Mode+of+action+of+mosquito+repellents.">Mini review: Mode of action of mosquito repellents.</a> Pestic Biochem Phys. 106 (3), 149-155 (2013).</li><li>Lawrence, K. L., Achee, N. L., Bernier, U. R., Mundal, K. D., Benante, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+Evaluations+of+Topical+Arthropod+Repellents+in+North,+Central,+and+South+America.">Field Evaluations of Topical Arthropod Repellents in North, Central, and South America.</a> J Med Entomol. 51 (5), 980-988 (2014).</li><li>Collins, A. M., Rubink, W. L., Cuadriello Aguilar, ., I, J., Hellmich Ii, ., L, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+insect+repellents+for+dispersing+defending+honey+bees+(Hymenoptera+Apidae).">Use of insect repellents for dispersing defending honey bees (Hymenoptera Apidae).</a> J Econ Entomol. 89 (3), 608-613 (1996).</li><li>Brown, M., Hebert, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+repellents:+An+overview.">Insect repellents: An overview.</a> J Am Acad Dermatol. 36 (2), 243-249 (1997).</li><li>Teeters, B. S., Johnson, R. M., Ellis, M. D., Siegfried, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+video-tracking+to+assess+sublethal+effects+of+pesticides+on+honey+bees+(Apis+mellifera+L.).">Using video-tracking to assess sublethal effects of pesticides on honey bees (Apis mellifera L.).</a> Environ Toxicol Chem. 31 (6), 1349-1354 (2012).</li><li>Vallet, A., Cassier, P., Lensky, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+the+fine+structure+of+the+honeybee+(Apis+mellifera+L.)+workers+and+the+pheromonal+activity+of+2-heptanone.">Ontogeny of the fine structure of the honeybee (Apis mellifera L.) workers and the pheromonal activity of 2-heptanone.</a> J Insect Physiol. 37 (11), 789-804 (1991).</li><li>Free, J. B., Ja Pickett, ., Ferguson, a. W., Simpkins, J. R., Smith, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repelling+foraging+honeybees+with+alarm+pheromones.">Repelling foraging honeybees with alarm pheromones.</a> J Agr Sci. 105 (2), 255 (1985).</li></ol>

这种视觉跟踪协议提供了一种以相对快速和容易的方式筛选蜜蜂的化学阻止剂的简单方法。蜂蜜蜜蜂的化学阻碍实验室测试没有推荐的方案。以前的半场和全场研究已经检查了蜂蜜驱虫剂14,15 ;然而，所描述的协议是耗时的，劳动密集的，并且需要在一般实验室之外的附加设备资源。该方案被设计为对这种化合物与蜂蜜蜜蜜进行半场或全场测试之前对化学威慑进行先决条件的评估...

欧洲蜜蜂， Apis melliferaL。是一种经济和农业上重要的昆虫，提供全球超过2000亿美元的授粉服务1 。在美国和欧洲，蜂蜜的数量一直在减少。美国已经失去了1947-2008年管理的蜂蜜殖民地的60％，而欧洲已经失去了ca。 1961-2007年的27％ 2，3 。有许多因素可能导致殖民地损失数量增加，包括但不限于寄生虫侵袭，病原体感染，养蜂行为和农药使用2-4 。 蜂蜜可能通过两个主要途径暴露于农药。当觅食者接触作物时，可能发生蜂巢外的农药暴露已经喷洒了化学物质以防止害虫。蜂巢中的农药暴露可以发生在养蜂人利用化学物质来控制螨虫，细菌和微孢子虫等害虫和病原体时。在美国和加拿大的5,6个蜂巢中，已经在蜡，花粉和蜂蜜样品中鉴定了农药残留。农药与蜂蜜接触的影响包括急性毒性以及亚致死作用，如麻痹，定向障碍，行为和健康变化1，7 。由于现代农业需要使用农药来维持高产量，这些化学品将来将继续得到依赖2 。为了更好地保护蜂蜜免受农药暴露，需要制定新的议定书和条例5 。一种可能的保护方法是使用驱避剂来减少蜂蜜在杀虫剂中的暴露，同时觅食食物。 驱虫剂（IRs）通常被用作针对节肢动物疾病载体的个人叮咬保护措施8 。超过60年前开发的最广泛使用和成功的IR是DEET 8,9 。被认为是驱虫剂测试的黄金标准，被世界卫生组织和环境保护署用作新型驱虫剂筛选的阳性对照10 。此外，DEET已被发现分散蜂蜜从威胁到他们的殖民地11 。与个人IR相关的当前属性包括：（1）对大量节肢动物的持久影响; （2）用于皮肤或衣服时对使用者无刺激性; （3）无臭或令人愉快的气味（4）对服装无影响; （5）施用于皮肤时不产生油性外观，并能够经受使用者的出汗，洗涤和擦拭; （6）对常用塑料无影响;和（7）化学稳定和价格适中广泛使用12 。用于蜜蜂的驱虫剂只需要一些这些属性，如持久的效果，对施用器无刺激性，无臭或令人愉快的气味，化学稳定且价格实惠，广泛使用，对蜂蜜无毒。然而，在深入探索这些属性之前，需要一种以高通量方式筛选化合物排斥/威慑的方法。在这里，我们描述了实验室测定方法，筛选化合物以抑制蜂蜜蜜蜂，这是确定排斥性的重要步骤。以前的研究修改了以前的研究，描述了一种视觉跟踪方法来评估农药对蜜蜂的亚致死效应13 。豪这个方案的不同之处在于，它旨在测量可能阻止蜂蜜从农药处理的作物中的候选驱虫剂的影响。没有推荐的蜂蜜蜜化学威慑实验室测试方案，因此，该方案提供了一种简单的筛选方法。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mL Erlenmeyer flask</td>
			<td style="width:71px;">Kimax</td>
			<td style="width:119px;">26500-50</td>
			<td style="width:167px;">used for making the sugar/agarose cubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sugar</td>
			<td style="width:71px;">Kroger</td>
			<td style="width:119px;"></td>
			<td>any similar product will sufffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Deionized water</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>acquired in house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:71px;">Apex</td>
			<td style="width:119px;">20-102</td>
			<td>used for making the sugar/agarose cubes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mold for agarose cubes (Weigh Boat)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any mold that will provide the researcher with a 1.5 X 1.5 X 0.3 cm sugar/agarose cube will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EthoVision XT</td>
			<td style="width:71px;">Noldus</td>
			<td style="width:119px;"></td>
			<td>visual tracking software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">633 nm LEDs</td>
			<td style="width:71px;">Cyron</td>
			<td>HTP904E</td>
			<td>These lights were placed into a constructed light box to illuminate the arenas from below.&#160; The box was a simple wooden structure with a frosted plastic/plexi glass cover that allowed the light to disperse upwards without any glare.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laptop or PC</td>
			<td style="width:71px;">Dell</td>
			<td style="width:119px;">Inspiron One 2305</td>
			<td>necessary for video tracking software. Any pc device capable of runnin tbe visual tracking software will suffice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bee Keeping protective clothing</td>
			<td style="width:71px;">Dadant &#38; Sons Inc</td>
			<td style="width:119px;">V0126</td>
			<td>any protective hood and jacket will suffice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hive tool</td>
			<td style="width:71px;">Dadant &#38; Sons Inc</td>
			<td style="width:119px;">M00757</td>
			<td>used to open honey bee hive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Container for honey bees</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>any container suitable for housing and storing honey bees will suffice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Featherweight forceps narrow tip</td>
			<td style="width:71px;">Bioquip</td>
			<td align="right" style="width:119px;">4748</td>
			<td>used to select individual honey bees</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">9 cm (diameter) petri dish</td>
			<td style="width:71px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">S01778</td>
			<td>arena used to contain individual honey bees during video tracking</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Recording Device (Camera)</td>
			<td style="width:71px;">Basler</td>
			<td style="width:119px;">acA-1300-60gm</td>
			<td>any device that can record the subject clearly and transfer the file to a computer will suffice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GraphPad Prism</td>
			<td style="width:71px;">Graphpad</td>
			<td style="width:119px;"></td>
			<td>any statistical software package will suffice</td>
		</tr>
	</tbody>
</table>

video tracking protocol to screen deterrent chemistries for honey bees

欧洲蜜蜂Apis mellifera L.是一种经济上和农业上重要的传粉媒介，每年产生数十亿美元。蜜蜂殖民地数量自1947年以来一直在美国和许多欧洲国家逐渐下降。许多因素在这种衰退中起着重要的作用，包括蜜蜂无意中暴露于农药。制定新的方法和法规有必要减少这些授粉者的农药暴露。一种方法是使用驱避剂化学物质，从最近的农药处理作物中阻止蜂蜜蜜蜂。在这里，我们描述了一个方案，以辨别蜂蜜蜜蜂暴露于选择的驱虫剂化学物质的威慑。蜜蜂饲养员在测试前15小时在培养箱中收集和饿死。将单个蜂蜜蜜蜂放入具有糖琼脂糖立方体（对照处理）或糖琼脂糖复合立方体（驱避剂处理）的培养皿中到盘中间培养皿作为竞技场，放置在灯箱内的照相机下，使用视频跟踪软件记录蜜蜂运动活动。分析总共8个对照和8个驱虫剂处理10分钟，每次处理用新的蜂蜜蜜蜂复制。在这里，我们证明蜂蜜蜜蜂通过复合处理从糖琼脂糖方块中被阻止，而蜂蜜蜂蜜被吸引到糖琼脂糖立方体而没有添加化合物。

开发了一个视觉跟踪方案，用糖琼脂糖（对照处理）或糖 - 琼脂糖复合立方体（威慑处理）记录蜂蜜在目标区域消耗的时间。使用统计软件程序分析记录的时间，并将目标区域的平均消耗时间±标准误差报告为条形图。作为驱虫/威慑试验的黄金标准DEET在本协议中被用作阳性对照。蜂蜜蜜蜂提供糖琼脂糖立方体（阴性对照）在目标区域花费343±26秒，而蜂蜜蜜蜂提供了糖 - ?...

Watch this Scientific Journal Video about Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees at JoVE.com

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

蜜蜂殖民地的丧失对作物授粉服务构成挑战。目前的传粉者保护措施需要采取替代方法，以尽量减少蜂蜜与使用驱虫剂的有害农药的接触。在这里，我们提供了一个可视化跟踪协议的详细方法来筛选蜜蜂的威慑。

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Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...