我们要感谢亚历克斯Hitchen梅格展位提供的帮助与配合试验。这项工作是由NERC资助NE /支持H015604 / 1 TP。

1.准备飞食品与食品着色<ol><li>取标准果蝇小瓶大约20毫升食物中的底部（ 图1）。使用下面的方法，使用1升沸水混合食物为10g琼脂，85克葡萄糖，60克玉米粉，40克酵母中，并搅拌5分钟憋着的。加25毫升10％的nipagen一旦混合物已经冷却至75℃。 </li><li>后的食品已冷却和固化加两滴-蓝色食用色素（约0.5 1毫升）中的食物的顶部，并散布在所述小瓶的整个表面（ 图1）。使用不同的颜色的染料，如果首选。 </li><li>离开食品两天在冰箱使染料是由食物的顶层吸收;这样就避免了过多的水分在成熟期损坏苍蝇。添加一小片卫生纸，如果过多的水分仍是一个问题，吸干多余的水分，随后将其删除。 </li><li>转印蝇到食品单独或组。 注意：蝇将获得1天之内被放置在食物肠染色。另外，完全成熟的苍蝇在食物染色实验（在成熟期间没有接触到食用色素观察死亡率增加）之前。 </li><li>检查染色的苍蝇可从非染色苍蝇容易辨别。使用一个更高浓度的染料，或者不同的染料1.4 - 如果他们不能区分，重复步骤1.1。 </li></ol> 2.两个男交配试验用食用色素<ol><li>为了生产后代，建立了包含对男性和女性的苍蝇多瓶（小团体的男性和女性都同样适用，但要小心避免幼虫拥挤）。让女性产卵和移动苍蝇新瓶每5 - 7天。在合适的温度为感兴趣的物种商店小瓶（22°C 2 F或D. pseudoobscura和D. subobscura和25℃D.果蝇 ）。 </li><li>之前收集实验苍蝇除去所有的苍蝇从收集小瓶在设定的时间收集男性和女性之前，以确保它们将是处女（ 果蝇 - 6小时，在25°C，D pseudoobscura - 18小时在22℃，并D. subobscura - 24小时，在22°C）。 注意：如果过得不处女这将偏向自己的交配试验15的行为。 <ol><li>存储和单独的标准的75×20毫米的塑料瓶成熟男性（含约20毫升食物）。这样就避免了对男性的交配行为和健身看到一些物种的负面影响时，男性被保存在组16。 </li><li>露出一半的男性到所需的治疗（CO 2麻醉在这种情况下）。使用CO 2垫或点击揭露苍蝇所需的时间。在发作店一半ES在每次治疗的各色美食，直到交配发生。这会让他们在交配试验在视觉上​​区分。 </li><li>对于转移苍蝇使用抽吸17。标记每个小瓶，以确定两者的治疗和阳的颜色状态。这里，可使用四个处理（麻醉，非有色= G-NC，麻醉，有色=气相色谱，无麻醉，无着色，NG-NC和没有麻醉的，有色的，NG-C）。 </li></ol></li><li>转让新出现的女性到新鲜食品小瓶成熟为10组。 </li><li>允许苍蝇成熟于配合年龄（ 果蝇 - 3天，D。pseudoobscura - 5天，D。subobscura - 7天18商店拥有在合适的温度为物种正在研究（ 例如，22℃，程序 D。 。pseudoobscura和D subobscura和25℃ 果蝇 ）。 </li><li>移动雌性个体小瓶（续癌宁约20毫升食物）1天交配试验驯化到配合小瓶之前。标注这些小瓶使小瓶可以分化。小心使用中性标签盲目的实验（ 即 1 - 150），所以它是不可能的猜测苍蝇的身份在任何小瓶。 注意：人谁放置苍蝇到每个小瓶必须知道苍蝇放入每个小瓶，因为它们会注意到其治疗染色的身份。然而，谁手表交配观察者不应该知道自己的身份。要做到这一点的至少两个实验者将需要，一个用于建立和一个观察。 </li><li>开始交配试验间10 - 上午12点，或在一个时间，与在光/暗循环的苍蝇暴露（“黎明”为蝇）的光来上重合。添加两个雄蝇每个交配瓶（含单身女飞）采用吸气。确保这两个男子是从不同的治疗（麻醉或控制）和一个具有肠染色以使其能够彼此区分它们，并注意其男性被染色。 </li><li>如果发生交配，记录下队友男性的地位（无论是颜色还是非 - 彩色）。如果试验持续2小时，假设女性不会交配。 注：2小时是适合这些物种，但其他的果蝇 ，可能需要更多或更少的时间。 </li></ol> 3.单雄性交配试验<ol><li>对于单身男性的试验，重复协议2有两个变化： <ol><li>在步骤2.3并没有以有色食物的男性。 </li><li>在步骤2.7，只添加一个单身男性每小瓶。 </li></ol></li><li>在步骤2.8中，记录飞被添加到小瓶中，配合启动和时间的配合完成后应记录时间的时间。从这些值，计算交配成功率，延迟和持续时间。 </li></ol> 4.数据分析<ol><li>使用适当的统计数据包ANA裂解。如果数据是正常的，只有两种处理中，使用t检验或等效广义线性模型（GLM）。两个男的实验中，使用二项测试或二项式GLM可用在任何基本的统计数据包。 注意：对于示例数据，所有的分析进行了在研发版本3.0.3 19。 </li><li>检查交配延迟和交配持续时间数据为正常，通过绘制每处理20的延迟和持续时间的频率直方图，并使用一个测试为常态如夏皮罗-威尔克。如果它是不正常的，改造它，或者使用非参数统计数据相当于20。 注意：对于从单雄性实验的示例性数据记录变换会见正常和方差相等的要求。 </li><li>如果数据可以是归一化，使用t检验来使用两种处理21时检查在单雄性交配试验交配延迟和持续时间之间的差异。如果多次治疗时，尝试使用方差分析（ANOVA）20。如果该数据不能被标准化，尝试等效非参数检验21。 </li><li>使用二项式测试来测试的任食品着色或CO 2麻醉上竞争的男性20的交配成功的效果。如果多次治疗中使用，为的是与实施例的数据的情况下，使用具有二项式误差结构21一 ​​个GLM。 </li><li>对于本例中的数据的两名男性试验中，使用GLMS与二项式误差结构。一个GLM检查颜色作为响应变量（有色= 0和非着色= 1）与物种，气体状态和气体处理装作为解释variables.One GLM审查的CO 2作为响应变量（脱气= 0和不脱气= 1）种，色状态和气体处理装。在每一种情况下，产生的最大的模式，并执行基于AIC 20模型简化。 </li></ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+cuticular+hydrocarbons+and+their+effect+on+courtship+in+Drosophila+virilis+and+D.+lummei.">Genetic analysis of cuticular hydrocarbons and their effect on courtship in Drosophila virilis and D. lummei.</a> Behaviour Genetics. 37, 713-725 (2007).</li></ol>

作者宣称，他们有没有竞争的财务权益。

这些数据表明，CO 2麻醉的影响是物种之间不一致，用两个3种示出的影响不大。我们的研究结果表明食物染料标记有比二氧化碳对雄性交配成功率较低的影响，2麻醉D.果蝇 。这些实验表明，食用染料能够容易且便宜地被用于标签苍蝇涉及多个雄性交配测定。 这三个模型果蝇的研究中，只有D.果蝇表现出的CO 2麻醉对交配的表现在竞争激烈的情况下的效果。相比之下，没有一个品种表现出交配潜伏期，违背了先前的结果D.方面收集的气体在单交配试验效果果蝇 5。竞争的效果，因此可突出二氧化碳麻醉更微妙的健身效果，这是唯一的检测在情况下，有男男性竞争。曝光在早期收集和前一天的试对D的男性的能力产生不利影响果蝇获得配合。暴露试验前两天但是没有显示出任何效果。 D.两者pseudoobscura和D. subobscura没有表现出暴露于气体中的任何一种试验中没有任何影响。一种解释是D.果蝇易受早期接触的CO 2，因为它必须被前面的寿命收集（0 - 6小时龄）比其他物种，以确保男性是处女。因此，男性D.这个年龄段的果蝇可能更敏感的苍蝇的角质层仍然硬化，相比已不再有他们的角质层硬化的其他物种。在一般情况下，这支持了的CO 2麻醉的效果是特定物种和调查人员应适当地测试在他们的目标物种的效果。 Currently，则多数工作的对CO 2麻醉的效果已经进行了对果蝇 5,11,22，因此可能不适合应用到其他相关的物种。 呈现给区分苍蝇的可选择的非侵入性方法是食品染料。结果表明这种治疗方法已经在所有物种检查没有影响。然而，虽然它的用途是成功的在个人之间进行区分，提供一种便宜和容易看见标记应当注意的是，该染料是更容易辨别在D. pseudoobscura和D. subobscura比D.果蝇。以前的作者已经使用了多种颜色（红，绿和蓝）4,6。我们发现蓝色着色是最容易区分所有物种，特别是D. pseudoobscura和D. subobscura。用几种颜色将有可能让更多的复杂的实验，有许多单独标记flie秒。然而，不同的染料的初步测试是必不可少的，因为有些食物染料未能颜色苍蝇，食用时有可能被消化。其它染料可具有毒性作用，降低了苍蝇生存，并且应避免14）。使用更昂贵的污渍的替代食品着色方法也被用于为D.检查肠道完整性23 果蝇，这些可以提供另一种选择，虽然价格较贵，染色法23。 染料的方法是一样快的CO 2翼剪取苍蝇可存储在染色食品从收集。食物的摄取是快速（〜3小时），所以存储O / N上的各色美食也足以标记苍蝇，在其他研究中使用6。然而，着色的持续时间相对较短（〜4 - 5小时）相比，翼削波（永久）或荧光标记的灰尘（10 - 19天）24。由于果蝇而变化的外观，不同的染料会或多或少有效针对不同的物种，并作为一些菌株（ 例如 ，敲除突变体）可以是易受改变饮食，任何使用的染料要求其有效性尤其是当较长的初步试验长期接触染料可能是有毒的14。与此相反的研究卡劳等人 14，我们发现存储多天之后没有显著死亡率有色食品D.果蝇 （3天），D。 pseudoobscura（5天）或D. subobscura（7天），可能是由于在染料的差使用。 为成功地使用的染料技术的关键步骤是步骤1.5，验证所选择的染料可以很好地被使用的物种和菌株。另一种技术涉及将彩色尘埃向在田间试验24在使用前即时的外侧。这种方法已被用于在该领域，由于吨追踪个体Ø标记的持续时间和质量的易用性标志苍蝇24。虽然我们还没有明确地测试此方法在交配试验中，它以检查任何影响的灰尘有可能对在交配重要，特别是在果蝇 25,26感官将是重要的，在物种，但是，其中肠染色是不可能的，这些方法可能是合适的。 总之，我们发现，在2所测试的3种（D. pseudoobscura和D subobscura）的没有发现任何的CO 2麻醉或雄性的交配能力食用色素的效果。对于D.果蝇的二氧化碳麻醉的负面影响被检测到，但食用色素并没有影响交配成功在这一物种。总体而言，该染料的方法提供了一种鉴定个体果蝇 ，它等效或比需要的CO 2麻醉方法更好的简单和廉价的非侵入性的方法。它很可能是这种方法会在一系列物种的工作。

在过去的几十年中有越来越多的兴趣在男性的选择和竞争性如何影响之间的进化1,2。在交配行为的实验已经起到了开发和测试性选择3,4理论具有重要作用。特别是，使用研究属果蝇的物种，一直对性选择和行为的理解作出了贡献。然而，调查是否常用的技术可能会人为偏差标准的交配实验5,6的结果是很重要的。 麻醉通常用于处理和鉴定在实验7。例如，苍蝇通常收集交配前，或分类成使用二氧化碳（CO 2）的麻醉剂的基因型或实验性治疗。在实验中，其中两个或两个以上的个体需要加以区分，通常的做法是，以anaesthetise的FLIES和翼关闭的夹子部分，以确定每个单独的或治疗组5,8。它是非常重要的，但是，要理解的CO 2处理将如何影响行为。 二氧化碳麻醉的效果已经研究了果蝇中男性接触CO 2显著需要较长的时间进行交配和总体拥有较低的交配成功率比非麻醉男性或男性用挨冻5麻醉。观察到这种效果既当麻醉施用在实验当天以及当苍蝇给予一天恢复。然而，这项研究在一个单一的男性被提交给每个女5只检查试验是有限的。更现实的情况是女性遇到多个雄性9,10，让男性，这可能让男性健身因麻醉更微妙的损失检测之间的竞争。使用二氧化碳麻醉，也可以恩发现，以减少成虫产卵量D.与长寿果蝇当它们不久羽化后露出，如当收集的处女蝇11是常见的。 另一种为CO 2麻醉喂养它们的食物有色染料4,6,10,12,13标记苍蝇。该染料进入飞的肠和可见通过腹部，使有色苍蝇加以区别从无色蝇，或从苍蝇标有不同的颜色。方法的不同之处是如何可以适用：被直接添加到食物12中，通过染色的补充酵母膏6，或经由暴露于一种新型染食品衬底13。这些标记技术出现，以显示对交配性能4,6没有影响。然而，一纸直接检查一样食用色素的成人D.影响果蝇发现了一个强烈的减少寿命14。以前的工作也集中ALMOST完全D.果蝇，既涉及到的CO 2麻醉5,11和食用色素14的方法的效果。目前，很少有关于二氧化碳麻醉或使用肠道着色是如何影响其他果蝇的交配行为。 下面的研究评估二氧化碳麻醉对3种果蝇（黑腹果蝇，D. pseudoobscura和D. subobscura）的交配行为的影响。收集苍蝇对CO 2的效果进行了检查在单和两名男交配试验。 的 CO 2的效果也已发现，以改变在D.果蝇 5和暴露于CO 2和交配之间，以便不同的延迟时间进行了试验。另一种标记方法麻醉和机翼剪裁：使用食品染料染色的苍蝇也评估。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Plastic tubing</td>
			<td>Fisher Scientific</td>
			<td>TWT-200-061G</td>
			<td>Other tubing may be suitable however it must fit within a 1ml pipette tip.</td>
		</tr>
		<tr>
			<td>Fine mesh</td>
			<td>Any haberdashery</td>
			<td>-</td>
			<td>Fine curtain mesh is suitable</td>
		</tr>
		<tr>
			<td>1ml pipette tips</td>
			<td>VWR</td>
			<td>83007-376</td>
			<td>Various brands of pipette tips would be suitable</td>
		</tr>
		<tr>
			<td>Plastic Vials</td>
			<td>Sarstedt</td>
			<td>58.49</td>
			<td>Larger vials or bottles could also be used.</td>
		</tr>
		<tr>
			<td>Cotton Balls</td>
			<td>Lewis Medical Solutions</td>
			<td>28170</td>
			<td>The size of cotton will vary depending on the size of vials used</td>
		</tr>
		<tr>
			<td>Blue Food colouring</td>
			<td>Thesugarcraftcompany</td>
			<td>-</td>
			<td>Other dye colours and brands can have variable results.</td>
		</tr>
		<tr>
			<td>CO2 Tank</td>
			<td>BOC</td>
			<td>BOC 40VK</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharpie Markers</td>
			<td>Steadtler</td>
			<td>Lumocolor 313 S</td>
			<td>Various colours can be used, but Lumocolor S give an excellent combination of durability and fineness</td>
		</tr>
		<tr>
			<td>Stopwatch</td>
			<td>Salter</td>
			<td>SL3920</td>
			<td>Any stopwatch with a good digital display would be fine</td>
		</tr>
	</tbody>
</table>

dyeing insects for behavioral assays: the mating behavior of anesthetized drosophila

利用果蝇交配实验，极大地促进了性选择和行为的理解。实验往往需要操作简单，方便，廉价的方法试验个体进行区分。一个标准的技术，这是二氧化碳麻醉，然后标注或机翼裁剪每个飞行。然而，这是侵入性的，并已被证明影响行为。其它技术已经使用着色识别苍蝇。本文提出了一种标签果蝇一个简单的和非侵入性的方法，使他们能够在实验中单独确定，使用食用色素。这种方法是用在临床试验，其中两个雄性竞争交配雌性。印染允许快速和容易识别。有，但是，在整个测试的3种颜色的强度差一些。数据呈现表示染料具有在果蝇黑色素上交配行为的低级影响比CO 2的法莫替丁。 二氧化碳麻醉的影响表现取决于果蝇物种，与D. pseudoobscura和D. subobscura显示没有影响，而D.果蝇雄性减少了交配成功。提出的染料方法适用于广泛的实验设计。

两名男性交配试验- CO 2麻醉对交配行为的影响发现解释中的CO 2麻醉效果的变化的最佳模式包含物种作为因子（与四pseudoobscura和D subobscura稠，因为它们显示出彼此之间没有差异）。在D. pseudoobscura和D. subobscura有二氧化碳麻醉对交配成功的两位男试验（Z = 1,589 0.087，P = 0.931）无显著影响。对于D.果蝇 ，暴露在CO 2麻醉男性有显著降低交配成功率（Z1,589 = 2.467，P = 0.014）。还有种和治疗（χ2 = 1,589 6.83，P = 0.009）具有更大的影响之间的显著相互作用被看到时D.果蝇暴露于气体收集或前1天的试验（ 表1）。 Howev呃，D.果蝇雄性暴露到CO实验试验前2两天没表现出的 CO 2的效果。 两名男性交配试验 - 肠道色素对交配行为的影响模型简化显示被发现的任何三个品种（P&gt; 0.1）的食用色素没有显著影响。当治疗或气体状态被包括在分析中，这些也没有显著性（P&gt; 0.1）。成功的交配对有色蝇横跨治疗的比例示于表2中 。在果蝇之间的着色的差保持在D中着色和未着色的食品可以在图1中可以看出。肠道食用色素的强度更大pseudoobscura和D. subobscura比D.果蝇 。 单交配试验-用CO 2麻醉对米的效果阿婷行为有交配的延迟对于任何三个品种没有区别，当二氧化碳麻醉用最近收集羽化成虫。效果被发现的交配持续时间D. subobscura当它被暴露于CO 2之前2天交配试验（ 图2和3;表2）。 <img alt="图1" src="/files/ftp_upload/52645/52645fig1.jpg" /> 图1.照片显示彩色和非彩色飞食品（A）的样品瓶和肠道着色的男性D.强度Subobscura（B）中。 <img alt="图2" src="/files/ftp_upload/52645/52645fig2.jpg" /> 图2.均值和95％置信区间交配潜伏期为三物种调查了单身男性试验中，当男性被麻醉D（光棒）或不麻醉的（黑条），当收集到性成熟前处女。 <img alt="图3" src="/files/ftp_upload/52645/52645fig3.jpg" /> 图3.均值和95％置信区间为三个物种调查了单身男性试验交配持续时间，当男性麻醉（光棒）或不麻醉的（黑条），当为性成熟前处女收集。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>治疗</td><td>种类</td><td>号试验</td><td>第苍蝇彩色的交配</td><td> p值</td><td>第苍蝇毒气的交配</td><td> p值</td></tr><tr><td rowspan="3">收集对CO 2 </td><td> D. MEL </td><td> 73 </td><td> 36 </td><td> 1 </td><td> 27 </td><td> 0.0344 </td></tr><tr><td> D. PSE </td><td>79 </td><td> 41 </td><td> 0.8221 </td><td> 44 </td><td> 0.3682 </td></tr><tr><td> D.子</td><td> 71 </td><td> 40 </td><td> 0.3425 </td><td> 33 </td><td> 0.6353 </td></tr><tr><td rowspan="3">暴露在CO 2 18小时前</td><td> D. MEL </td><td> 57 </td><td> 28 </td><td> 1 </td><td> 19 </td><td> 0.0163 </td></tr><tr><td> D. PSE </td><td> 65 </td><td> 32 </td><td> 1 </td><td> 31 </td><td> 0.8043 </td></tr><tr><td> D.子</td><td> 68 </td><td> 38 </td><td> 0.3961 </td><td> 35 </td><td> 0.9036 </td></tr><tr><td rowspan="3">暴露在CO 22天。 </td><td> D. MEL </td><td> 56 </td><td> 19 </td><td> 0.0222 </td><td> 32 </td><td> 0.3497 </td></tr><tr><td> D. PSE </td><td> 70 </td><td> 32 </td><td> 0.5504 </td><td> 33 </td><td> 0.7202 </td></tr><tr><td> D.子</td><td> 56 </td><td> 29 </td><td> 0.8939 </td><td> 26 </td><td> 0.6889 &lt;/ TD&gt; </tr></tbody></table> 从表二男选择实验在所有物种中和处理，审议1.结果。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>种类</td><td>特点</td><td> DF </td><td> t值</td><td> p值</td></tr><tr><td rowspan="2"> 黑腹果蝇 </td><td>潜伏</td><td> 58 </td><td> 1.379 </td><td> 0.174 </td></tr><tr><td>长短</td><td> 58 </td><td> 1.243 </td><td> 0.221 </td></tr><tr><td rowspan="2"> D. pseudoobscura </td><td>潜伏</td><td> 109 </td><td> 0.419 </td><td> 0.676 </td></tr><tr><td>长短</td><td> 109 </td><td> 0.436 </td><td> 0.664 </td></tr><tr><td rowspan="2"> D. subobscura </td><td>潜伏</td><td> 83 </td><td> 0.098 </td><td> 0.922 </td></tr><tr><td>长短</td><td> 83 </td><td> 1.767 </td><td> 0.081 </td></tr></tbody></table> 表2.单交配实验检查收集的对CO 2麻醉效果的交配潜伏期和持续时间的结果。试验共进行了三种果蝇 （ 黑腹果蝇，D. pseudoobscura和D. subobscura）。

Watch this Scientific Journal Video about Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila at JoVE.com

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

染色昆虫行为检测：麻醉的交配行为<em&gt;果蝇</em

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

dyeing-insects-for-behavioral-assays-mating-behavior-anesthetized

Research

JoVE Journal

Neuroscience

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

9.7K 次观看.  University of Liverpool. This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

视频: Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

在昆虫单感器记录果蝇按蚊</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

科研

神经科学

Studying the Neural Basis of Adaptive Locomotor Behavior in Insects

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those leg movements that result from passive forces and interactions with other legs through the common contact with the substrate. If we want to understand, which part of a given movement is produced by nervous system motor output, kinematic analysis of stepping movements, therefore, needs to be complemented with electrophysiological recordings of motor activity. The recording of neuronal or muscular activity in a behaving animal is often limited by the electrophysiological equipment which can constrain the animal in its ability to move with as many degrees of freedom as possible. This can either be avoided by using implantable electrodes and then having the animal move on a long tether (i.e. Clarac et al., 1987; Duch &amp; Pfl&uuml;ger, 1995; B&ouml;hm et al., 1997; Gruhn &amp; Rathmayer, 2002) or by transmitting the data using telemetric devices (Kutsch et al, 1993; Fischer et al., 1996; Tsuchida et al. 2004; Hama et al., 2007; Wang et al., 2008). Both of these elegant methods, which are successfully used in larger arthropods, often prove difficult to apply in smaller walking insects which either easily get entangled in the long tether or are hindered by the weight of the telemetric device and its batteries. In addition, in all these cases, it is still impossible to distinguish between the purely neuronal basis of locomotion and the effects exerted by mechanical coupling between the walking legs through the substrate. One solution for this problem is to conduct the experiments in a tethered animal that is free to walk in place and that is locally suspended, for example over a slippery surface, which effectively removes most ground contact mechanics. This has been used to study escape responses (Camhi and Nolen, 1981; Camhi and Levy, 1988), turning (Tryba and Ritzman, 2000a,b; Gruhn et al., 2009a), backward walking (Graham and Epstein, 1985) or changes in velocity (Gruhn et al., 2009b) and it allows the experimenter easily to combine intra- and extracellular physiology with kinematic analyses (Gruhn et al., 2006).
We use a slippery surface setup to investigate the timing of leg muscles in the behaving stick insect with respect to touch-down and lift-off under different behavioral paradigms such as straight forward and curved walking in intact and reduced preparations.

We describe a method to record motor activity, timed to the electrically recorded tarsal contact signal in a tethered insect, walking on a slippery surface. This is used to study the neural basis of adaptive behavior under reduced influence of mechanical interaction between legs through the substrate.

研究昆虫的自适应运动能力行为的神经基础

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

对检测方法果蝇行为

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

单苍蝇检测方法的觅食行为的<em&gt;果蝇</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

在气味引导行为的高分辨率定量<em&gt;果蝇</em&gt;使用<em&gt; Flywalk</em&gt;范式

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

一个行为分析对机械感觉MARCM为基础的克隆<em&gt;果蝇</em

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their worlds when trying to understand their behavior and ecology. Sensitivity to the ultraviolet spectrum (UV; 300 - 400 nm) is found across multiple genera of birds, fish, reptiles, amphibians, and even mammals. This protocol describes a technique for examining organisms for the presence of UV-reflecting structures and a method for testing whether these cues are used as social signals in the context of mate choice. A spectrophotometer is used to detect the presence of UV reflectance and variation in reflective intensity between individuals and sexes. An example of this technique is presented in which a dichotomous mate choice test exposes sexually receptive individuals to opposite sex individuals whose visual appearance can be manipulated by filters that either transmit full spectrum or block UV wavelengths. This system allowed for the determination that female, but not male, sailfin mollies (Poecilia latipinna) were using UV markings as part of their mating decisions. These types of studies serve to expand our knowledge of the range of organisms that utilize UV and provide insight into how UV plays a role in their lives.

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna

This protocol outlines the use of spectrophotometry to detect ultraviolet-reflecting structures on organisms (in this example, the sailfin molly Poecilia latipinna) and describes dichotomous choice tests for fish that allow inferences to be made on the role of ultraviolet cues during mate selection.

试验以检测紫外线反射结构，并使用茉莉花鳉确定其重要性伴侣偏好<em&gt;孔雀鱼latipinna</em

Many organisms use cues and signals beyond human sensitivity during social interactions. It is important to take into account how organisms perceive their ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

果蝇美容行为的量化

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...