我们感谢布卢明顿果蝇库存中心的苍蝇股票; C. Gahr，JT Alkema和S. van Hasselt早期尝试开发加压空气测定;碧玉博士为培养酵母提供建议;和Rezza Azanchi和Joel Levine最初开发了果蝇交配行为的延时监测。 JA Gorter得到了神经科学研究学校BCN / NWO研究生课程资助的支持。这项工作得到了荷兰科学研究组织（NWO）（参考文献:821.02.020）对JC Billeter的支持。

环保接头盒<ol><li> 为了确保受控和易于清洁的测试区域，请设置一个120厘米x 64厘米x 85厘米的不锈钢厨柜，如图1A所示。 <ol><li>在天花板正下方的机柜背面钻一个孔，在侧面钻四个四孔，每个孔的直径为2厘米。在盒子的每一侧，距离盒子底部7厘米的高度和12.5厘米的孔之间钻第一组四个孔。在距离底部35厘米的高度处，在盒子的每一侧钻另外两套。 注意:四组四个孔用于摄像机电源线和气泵管进出柜。背面的孔用于灯板的电力电缆。 </li><li>建立一个18行40个交替的白色和红色发光二极管（LED）的灯板，每个灯之间的间距为2.5厘米。将白色和红色LED放在一个circui电源供应。将每个LED与560Ω，0.25 W和5％容差的电阻串联连接。 注意:红色光的发射波长从590-661 nm跨越，峰值为627 nm。实验区域的结果光强度约为900勒克斯，两个灯亮，90勒克斯，只有红灯亮;这是使用智能手机光度计应用程序测量的。 </li><li>将灯板连接到不锈钢柜顶部，并将电源线穿过后面的孔。 </li><li>将白色LED的适配器连接到电源控制定时器，以便在实验的黑暗阶段关闭白色LED。将红色光，这苍蝇是盲目到25的适配器，在正常供电，让他们对实验的整个过程。 </li><li>将一个110厘米和两个54厘米长的金属支架（宽度为0.5厘米）固定在盒子的内侧，高度为50厘米从盒子的底部。在这些支架上放置磨砂玻璃扩散板（尺寸为119.0厘米x 54.5厘米x 0.5厘米）。 </li><li>在光板和玻璃板之间添加三层滤纸（120厘米×50厘米），以扩散光线并限制在配合场地表面上的眩光（在第4节中描述）。使用磁铁（侧面）将长度为120厘米长的木杆的两边的两张滤纸贴在一起，将其粘贴在金属机柜的内部;执行这个动作3次。 </li><li>在箱子的两侧安装四个风扇，以创建一个连续通风的配对箱的空气流。在灯板和玻璃板之间安装第一组8厘米风扇，左侧空气入口和箱体右侧排气，以最大限度地减少由光板产生的热量。 </li><li>将第二组12厘米的风扇安装在机柜底部25厘米以上，以产生通向我的外部气流在柜子的正面，将其冷却至稳定的26°C。将排气侧的风扇连接到抽吸软管，并将气流引导出室外，以防止机柜内的空气重新循环。 </li></ol></li><li>设置两个立柱（约48厘米高），安装有两个夹具，一个在28厘米，一个距离支架底座30厘米。将网络摄像头固定到每个夹具上。将4台摄像机连接到运行监视软件的计算机。 </li><li>将A4纸放在每个网络摄像头的下面。使用未打印的白色纸张或纸张，其预编号格栅为7×5平方厘米，4厘米的轴线以适应配合场地（在第4节中描述）。 注意:具有78°广视角和500万像素分辨率的高清摄像头摄像头可以覆盖对应于A4纸张的21厘米×30厘米的面积，并在20到35个交配场地之间进行监视。 </li></ol>飞行收集和收集<ol><li> 放置20个男性和20个女性野生型Canton-S飞入含有45毫升富含飞蝇食物介质的飞行饲养瓶（见第3部分），持续三至四天。首先将相同的成年人三次转移到新鲜的瓶子里。 <ol><li>将瓶子放置在25℃的培养箱中，12小时:12小时的暗夜循环，灯亮时间为09:00（Zeitgeber time（ZT）0））。大约10天后，新一代将会出现。 </li></ol></li><li> 在二氧化碳垫上麻醉所产生的新近生成的苍蝇不超过5分钟，并使用油漆刷将其收集到飞行食品小瓶中。 <ol><li>将来自野生型Canton-S储藏瓶的处女（新近生长的）雌性和未处理的男性收集到具有6.5mL富含飞行食物介质的2.5cm×9.5cm飞行饲养小瓶中。 </li></ol></li><li>在25℃和12小时:12小时:12小时的暗夜循环和09:00（ZT 0）的照明下，将飞行饲养小瓶中的每只20只苍蝇的同一性别群体的苍蝇期龄为5至8天。 </li><li> TRANSF在实验前一天将苍蝇飞到新鲜的饲养小瓶。 </li></ol>食物中准备<ol><li> 准备1升富飞培养基如下。 <ol><li>将1升自来水用磁力搅拌棒倒入2​​升玻璃烧杯中，并将烧杯放在磁性热板上。保持搅拌，加热至300°C，直到达到沸点温度。 注意:在以下步骤的长沸腾时间内，一定比例的水将蒸发，但是与添加的成分一起，当在约22℃的室温下制备时，该方案产生1升富飞培养基。 </li><li>开启搅拌至500转/分钟（rpm），并将以下成分加到沸水中:10g琼脂，30g葡萄糖，15g蔗糖，15g玉米粉，10g小麦胚芽，10g大豆粉，30g糖蜜，35克活性干酵母。等待酵母膨胀，然后调低热板温度侵蚀到120°C。 </li><li> 10分钟后，将热板转到30℃，搅拌混合物直到冷却至48℃。通过将温度计直接插入食物来监测温度。 </li><li>将2g对羟基苯甲酸甲酯（tegosept，100％）溶解在10mL的96％乙醇中。将此和5 mL 1 M丙酸加入到混合物中。搅拌3分钟</li><li>将飞行食物介质倒入舞台（在第4节中描述），在舞台底部创建一个0.3厘米厚的层。 <ol><li>使用200 mL玻璃烧杯浇注。当精确量很重要时，请使用10 mL血清移液管。 </li></ol></li></ol></li><li>准备如第3.1.1节所述的完全按照3.1.1中描述的飞粉培养基减酵母，但在步骤3.1.2中省去酵母。 </li><li>通过将10g琼脂和35g蛋白胨混合在1L沸水中来制备具有或不具有胨的琼脂的培养基，并进行步骤3.1.4至3.1.5。 </li></ol>马丁g竞技场准备<ol><li>使用加热的准备针（在本生灯加热至发红），在3.5cm×1.0cm塑料培养皿的上侧上穿一个直径大约0.3cm的孔。或者，使用烙铁。 </li><li>当准备含有恶臭化合物的食物介质时，首先将所需化合物30μL（最终食物介质的1％，例如醋酸冰醋酸100％）移液到一半的实验皿中。将另一半的菜肴放空，以作比较。 注意:通过这里描述的设置，最多可以一次测试140个竞技场。 </li><li>使用10mL血清移液管，将3 mL食物介质倒入培养皿底部的所需化合物上。用干酪布覆盖以防止污染，并使培养基在室温下固化约1小时。 </li><li>将餐具和胶带上的盖子放在两边封闭。准备小石蜡膜塞以覆盖孔通过将石蜡膜片轧制成0.2cm厚的辊，然后将其切成0.5cm的片段。 </li></ol>酵母菌气味<ol><li> 在14.0厘米x 2.06厘米培养皿中的酵母提取物胨葡萄糖（YPD）琼脂上生长干活性酵母。戴上手套以防止在此步骤中受到污染。 <ol><li>通过向1L沸腾的超纯水中加入10g酵母提取物，20g蛋白胨，22g葡萄糖（0（+） - 葡萄糖一水合物）和15g琼脂（纯））来制备YPD琼脂平板。一旦所有物质溶解并将其倒置在4°C的冰箱中，将培养皿的底部分层，最长可达2个月。 </li><li>在YPD培养板上撒上几粒干酵母，使其溶解。然后使用无菌回路连续培养基板。将板放在30℃的培养箱中过夜。之后，将文化存放在冰箱中不超过1周。 </li></ol></li><li> 准备YPD液体介质通过加入10克酵母提取物，20克蛋白胨，22克葡萄糖（0（+） - 葡萄糖一水合物）和1升超纯水将搅拌棒加入1升瓶中。 <ol><li>在120℃和1巴压力下高压灭菌25分钟。然后，将瓶子在4℃下储存长达2个月，直到使用。 </li></ol></li><li> 将开口的瓶盖（4.5厘米）与0.32厘米厚的硅胶隔垫合上。 <ol><li>在隔垫上切下两个小孔，以紧密配合带倒钩的隔板配件。将小型聚氯乙烯（PVC）管（直径:外部0.8厘米和内部0.5厘米）连接到出口的两个出口和只有一个入口进入瓶子。参见图1B说明。 </li><li>将合适的盖子和管子在铝箔和高压釜中包装在120℃和1巴压力下25分钟。 </li></ol></li><li> 在这一步中戴上手套以防止污染。将无菌的100μL移液管吸头从YPD琼脂平板（d5.1），并将其放入高压灭菌的YPD液体培养基瓶中。 <ol><li>盖上这种酵母接种的YPD液体培养基瓶以及安装有入口和出口的高压灭菌瓶盖的YPD培养基对照瓶（不添加酵母）（在步骤5.3中描述）。将两个瓶子放在分开的磁性板上，并在开始实验之前在室温下以100rpm搅拌24小时，以使酵母培养物生长。 </li><li>将两个瓶子的入口连接起来，分开水族馆的水泵，向酵母菌提供空气。确保将实验酵母瓶的出口连接到将酵母气味从实验室中排出的管中，以防止干扰实验。 </li></ol></li></ol> 6.气泵装置<ol><li>将大型PVC管（直径:外部1.2厘米，内部0.9厘米）连接到加压空气源，并将其通过两个装有活性炭的1升玻璃锥形瓶装入800 mL线路净化空气。使用实验室通常提供的加压空气作为供气，或将管连接到空气泵（此处使用加压空气）。 注意:管道材料应根据挥发物的化学性质进行选择，并进行测试，以防止挥发物粘到管道衬里（如聚四氟乙烯，尼龙或不锈钢）上。 </li><li> 从15 mL管中分别制造两个分流器，每个分别有三个1,000μL移液管。 <ol><li>制作直径约1厘米的三个孔。首先烧两个孔，使用加热的准备针（本生灯加热红色），紧邻15 mL管的盖子下方。然后通过移除管的底部来制作第三个孔。 </li><li>将1,000μL移液器吸头胶入到狭窄端向外的孔中。切割吸头的末端以允许更大的气流。 </li></ol></li><li>从c的出口连接大型PVC管将装满哈龙的锥形瓶放入15mL管底部的移液管尖端。向两个水平移液器吸头添加小PVC管出口，并将其引导到控制台和实验瓶。 </li><li>为了防止用微生物污染YPD培养基，请将小管连接到无菌注射器过滤器（0.45μm孔径）上，并朝向过滤器的隔板管连接器上的塑料推动器，以及离开过滤器的塑料隔板连接器上的螺钉。然后，将管道连接到YPD培养瓶的入口（ 见图1B ）。 </li><li> 为了防止空气中的酵母从培养瓶行进到实验场，使用小管将每个YPD培养瓶的出口附上一个玻璃管（6.5cm长，外径= 0.5cm，内径= 0.3cm）。将PVC管连接到玻璃管的另一侧，并将其引导到实验箱的下部孔（每侧钻孔）。<ol><li>用玻璃纤维填充管并在使用前对其进行高压灭菌。 </li></ol></li><li>在实验箱的每一侧，将另外的15 mL管式分流器（见6.2节）介绍给小型PVC管，以便在实验侧（最接近风扇空气流的排气口，右侧）运行两个管，和控制侧（最靠近风扇空气流的入口，左）。 </li><li>准备8x 25 mL血清移液管，每个10个出口同时测试80匹配对， 即每个空气条件下为40个。 <ol><li>烧10孔直径约0.8厘米，距离移液管2厘米。 </li><li>将1 mL注射器的外部部分切割成一个小（2.5 cm）和一个大（5 cm）出口。 </li><li>将这些出口用热胶粘在孔中。 </li><li>将一小段塑料石蜡包裹在出口的末端，并附上1,000μL移液器吸头。尖端开口的直径为0.1厘米。为每个实验使用干净的提示。 </li></OL&gt; </li><li>使用两个血清移液器，使用外径≥0.5厘米的T型分流器和短的小PVC管，两侧两个出口的每个。将移液器平放在白纸上（钢箱内的相机下）。 </li><li>使用气流监测器，设置空气流量，使1000μL移液管尖端出口的空气流速为0.5 m / s。这对应于每个尖端的气流为0.0017L / s。 </li></ol> 7.配合行为的监控<ol><li>使用吸液管（如参考文献26所述 ）将一名实验女性在15:00（ZT 6）放置在小培养皿（第4部分描述）中，并给予1小时适应交配场所。 </li><li> 设置实验箱（如第1节所述）如下: <ol><li>打开灯， 即在12h:12h亮暗周期的白光LED连接到打开的定时器在09:00（ZT0）和连续的红色LED指示灯，以允许在实验的黑暗阶段监测苍蝇。打开风扇以限制由光源升温的柜子，并确保在测试区域外排出多余的气味。 </li><li>将网络摄像头摄像机连接到计算机，并使用监控软件启动图像监控。 </li><li>对于每个摄像机，设置监控软件中的对焦，亮度和变焦。 <ol><li>右键单击相机屏幕，打开"相机属性"，然后单击"自动对焦"。调整"焦点"以澄清网格或纸张上的任何书面单词。如有必要，请更改"亮度"和"缩放"。 </li></ol></li><li>设置监控软件的程序，每2分钟拍摄1张照片。右键单击每个相机屏幕，然后选择"编辑相机"，然后选择"操作"选项。点击开始动作"在常规int删除"并将时间更改为"2分钟"。选择"拍照"进行选择操作，最后点击"确定"。 </li><li>右键单击每个摄像机屏幕，然后选择"开始监视"。 </li></ol></li><li> 1小时后（在ZT 7），使用吸移管将野生型雄性转移到培养皿中，将盘放在网络摄像机的A4纸上，然后点击"开始监测"24小时。对于气泵实验，将盘放置在移液管出口连接到配合竞技场的入口处。 </li><li> 要分析这对夫妇的交配行为，请执行以下操作。 <ol><li>按照时间顺序选择并打开图像查看软件和页面中的所有图片。 </li><li>将日期，实验号，盘数和开始时间记录在同一行中的电子表格中。从放置在网箱下面的时刻开始每个竞技场的开始时间m相机。从图片记录时间戳。 </li><li>将同一行中每个交配的开始时间标记到电子表格中。计算一个交配作为一个事件，当男性已经安装女性和夫妇保持适度的静止和相同的姿势至少五个连续的帧（10分钟）。 注意:此标准是基于交配的报告的长度，范围从12到在黑腹果蝇 27分钟，并观察其的10分钟，过交配肥沃27,28。 </li><li>计算电子表格中每行的交配数以确定交配频率。或者，从每行的第一次交配时间中减去实验的开始时间作为交配等待时间的度量，或者从第二次交配时间中减去第一次配对的时间作为重建等待时间的度量。 <ol><li>要计算提醒延迟，请确保在电子表格软件中将第一次和第二次交配的日期定义为连续的天数。 </li></ol></li><li>使用混合效应模型分析数据，假设数据的正态分布，并将实验日期作为随机因素，使用统计软件（参见材料表）来确定独立变量 - 食品介质的统计学意义，空气类型和相互作用 - 如前所述5 。 </li><li>通过使用对数似然比测试和相关的Akaike信息执行非重要独立变量的反向消除来选择最佳解释模型。运行模型后，目视检查数据残差以确定正常。使用Levene测试确认差异的均匀性。在不均匀的情况下，平方根转换数据。 </li></ol></li></ol>

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作者没有争议的利益披露。

该协议描述了在24小时内测试交配行为的测定，同时连续控制耦合对被假设用于确定交配频率的环境线索。当培养基含有酵母时，可以响应于通过加压空气系统输送的酵母空气来增加交配频率（ 图2B ）。另外，用介质中仅含有琼脂，蛋白胨和乙酸气味的简化食品介质可以观察到交配频率中的类似反应（ 图3B ） 通过这里展示的实验，只能对夫妻的一般...

生殖行为通常具有高能量成本，特别是对于产生比男性更大的配子的女性，并且必须仔细选择条件来提高其发育后代。由于能源成本，繁殖与营养条件相关并不奇怪。这是真正的大多数，如果不是所有的动物包括哺乳动物，其可以青春期营养不良，且其性欲可通过食物，限制1的负面影响被推迟。 遗传模式生物体黑腹果蝇的繁殖也受营养条件的影响。男性法院在食物挥发2的存在较高的水平，并且女性更性酵母，鸡蛋的生产和后代存活3，4，5的主要营养素的存在接受。这个对食物的进化保守的生殖反应提供了机会，研究将环境食物可利用性与有遗传易感性和时效性生物体中的有性繁殖联系起来的机制。事实上，在果蝇的工作有牵连胰岛素通路作为食物和交配行为6之间连接的重要调节器。它也表明，交配行为本身改变女性的食品的偏好以及相关联的神经元化学感应7，8，9。 很明显，食物线索影响黑腹果蝇的生殖行为。这些影响似乎主要影响女性，特别是那些已经交配过的人5 。然而，为了测试环境条件的这些急性效应，经典地用于雌性交配行为的测定法可能会由于交配事件之间的长时间中断，不太适合。在经典的重建测定中，一个处女女性首先与男性交配，并立即分离出来，并在24到48小时后呈现了一个新的男性。此经典测定法已被用于取得了巨大成功，以确定修改的女性行为和阴响应12，13，14，15，16，17，18阳射精的组件。因此，这里证实的连续交配测定是经典交配测定法的补充，可用于研究环境条件对生殖行为的急性影响。 使用这里说明的交配行为的连续测定，我们以前表明，一对暴露于酵母的苍蝇会恢复在24小时观察期5，19，20，21 everal倍，而苍蝇不暴露于食品只会remate一次5。这一发现可在指示女性不初始交配后remate数天的黑腹果蝇文献的大部分的光被令人费解的（参考文献10综述，11）。然而，这种差异可以通过测定条件容易地解释，其中在提供新的交配机会之前女性被隔离一至几天。如果在这个小时长的观察期间该对不配合，那么女性的特征就是不接受。此外，由于来自野生捕蝇的数据显示，女性在其存储器官中含有4至6名男性的精子，所以交配频率高不应该是令人惊讶的。因此在dicating，女性自然remate几次22,23。 在这里，我们展示了使用这种连续交配测定来揭示苍蝇如何收集和组合关于环境条件的信息来调节交配频率。该测定允许测试相对大量的交配对进行遗传研究并测试挥发性和非挥发性环境线索的影响。该测定通常运行24小时，但可以延长至48小时，允许测试循环环境线索，例如光暗（LD）循环。我们通过测试来自加压空气系统中的酵母培养物的挥发性线索的影响与食品基质中非挥发性酵母营养物的可用性的结合来证明该测定。 加压空气系统将挥发性线索连续地泵入包含的配合场地sa食物底物和一对测试夫妇（其交配行为被监控）。为了进一步确定酵母影响交配的细节，我们测试酵母的主要挥发性化合物，即乙酸24 ，与蛋白胨（氨基酸）形式的与食品底物中酵母相当的氨基酸含量衍生自动物蛋白酶消化的酸）。这些实验一起证明了可以通过该测定来测试环境线索对黑腹果蝇交配行为的影响。

<table><tbody><tr><td>&lt;strong&gt;Cabinet&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Stainless steel kitchen cabinet</td><td>Horecaworld</td><td>7412.0105</td><td></td></tr><tr><td>White LEDs</td><td>Lucky Light</td><td>ll-583wc2c-001</td><td>Cold white, 20 mAmp and 2 V</td></tr><tr><td>Red LEDs</td><td>Lucky Ligt</td><td>ll-583vc2c-v1-4da</td><td>Wavelength between 625 nm, 20 mAmp and 6 V</td></tr><tr><td>Resistor</td><td>Royal Ohm</td><td>CFR0W4J0561A50</td><td>560 ohm, 0.25 W, 250 V and 5 % tolerance</td></tr><tr><td>Smartphone light meter app</td><td>Patrick Giudicelli</td><td></td><td>Light/Lux Meter FREE, version 1.1.1</td></tr><tr><td>Power timer</td><td>Alecto</td><td>TS-121</td><td></td></tr><tr><td>Metal brackets</td><td></td><td></td><td>Sharp angle 5 by 5 mm,&amp;nbsp; 2 x 5450 and 1 x 1100 mm long</td></tr><tr><td>Frosted glass plate</td><td></td><td></td><td>1190 x 545 x 5 mm</td></tr><tr><td>Filter paper sheets</td><td>LEE filters</td><td>220</td><td>White frost</td></tr><tr><td>Small fan</td><td>Nanoxia Deep silence</td><td>4260285292828</td><td>80 mm Ultra-Quiet PC Fan, 1200 RPM</td></tr><tr><td>Big fan</td><td>Nanoxia Deep silence</td><td>4260285292910</td><td>120 mm Ultra-Quiet PC Fan, 650-1500 RPM</td></tr><tr><td>Webcam camera</td><td>Logitech</td><td>950270</td><td>B910 HD WEBCAM OEM, Angle: 78-degree, resolution: 5-million-pixel&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Camera software</td><td>DeskShare</td><td></td><td>Security monitor pro</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Fly rearing&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fly rearing bottles</td><td>Flystuff</td><td>32-130</td><td>6oz Drosophila stock bottle</td></tr><tr><td>Flypad</td><td>Flystuff</td><td>59-114</td><td></td></tr><tr><td>Wild-type flies</td><td></td><td></td><td>Canton-S</td></tr><tr><td>Fly rearing vials</td><td>Dominique Dutscher</td><td>789008</td><td>Drosophila tubes narrow 25x95 mm</td></tr><tr><td>Incubator</td><td>Sanyo</td><td>MIR-154</td><td></td></tr><tr><td>Magnetic hot plate</td><td>Heidolph</td><td>505-20000-00</td><td>MR Hei-Standard</td></tr><tr><td>Agar</td><td>Caldic Ingredients B.V.</td><td>010001.26.0</td><td></td></tr><tr><td>Glucose</td><td>Gezond&amp;amp;wel</td><td>1019155</td><td>Dextrose/Druivensuiker</td></tr><tr><td>Sucrose</td><td>Van Gilse</td><td></td><td>Granulated sugar</td></tr><tr><td>Cornmeal</td><td>Flystuff</td><td>62-100</td><td></td></tr><tr><td>Wheat germ</td><td>Gezond&amp;amp;wel</td><td>1017683</td><td></td></tr><tr><td>Soy flour</td><td>Flystuff</td><td>62-115</td><td></td></tr><tr><td>Molasses</td><td>Flystuff</td><td>62-117</td><td></td></tr><tr><td>Active dry yeast</td><td>Red Star</td><td></td><td></td></tr><tr><td>Tegosept</td><td>Flystuff</td><td>20-258</td><td>100%</td></tr><tr><td>Peptone (bacto)</td><td>BD</td><td>211677</td><td></td></tr><tr><td>Acetic Acid</td><td>Merck</td><td>1000631000</td><td>Glacial, 100%</td></tr><tr><td>Small petridish</td><td>Greiner bio-one</td><td>627102</td><td>35 x 10 mm with vents</td></tr><tr><td>Paraffin film</td><td>Bemis NA</td><td></td><td>Parafilm</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Yeast and pressurised air set-up&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Big petridish</td><td>Gosselin</td><td>BP140-01</td><td>140 x 20.6 mm</td></tr><tr><td>Ultrapure water</td><td>Millipore corporation</td><td></td><td>MiliQ</td></tr><tr><td>Yeast extract</td><td>BD</td><td>212750</td><td></td></tr><tr><td>Agar (pure)</td><td>BD</td><td>214530</td><td>bacto</td></tr><tr><td>Glucose (0(+)-glucose monohydrate)&amp;nbsp;</td><td>Merck</td><td>18270000004</td><td></td></tr><tr><td>Open caps</td><td>Schott</td><td>29 240 28&amp;nbsp;</td><td>GL45</td></tr><tr><td>Silicone septum</td><td>VWR</td><td>548-0662</td><td></td></tr><tr><td>Barbed bulkhead fittings</td><td>Nalgene</td><td>6149-0002</td><td></td></tr><tr><td>Large PVC tubing</td><td></td><td></td><td>diameter: outer 1.2 cm and inner 0.9 cm</td></tr><tr><td>Small PVC tubing</td><td></td><td></td><td>diameters: outer 0.8 cm and inner 0.5 cm</td></tr><tr><td>15 ml tube</td><td>Falcon</td><td></td><td></td></tr><tr><td>Aquarium pump</td><td>Sera precision</td><td></td><td>Sera air 110 plus, AC 220-240 V, 50/60 Hz, 3 W and pressure &gt;100 mbar</td></tr><tr><td>Activated charcoal</td><td>Superfish</td><td>A8040400</td><td>Norit activated carbon</td></tr><tr><td>Disposible filter unit</td><td>Whatman</td><td>10462100</td><td></td></tr><tr><td>Serological pipettes</td><td>VWR</td><td>612-1600</td><td></td></tr><tr><td>Syringe</td><td>BD Plastipak</td><td>300013</td><td></td></tr><tr><td>Hot glue</td><td>Pattex</td><td></td><td></td></tr><tr><td>Syringe filter</td><td>Whatman</td><td></td><td>FP 30/pore size 0.45 mm CA-S</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Statistics software</td><td>R</td><td></td><td>lme4 package</td></tr></tbody></table>

a method to test the effect of environmental cues on mating behavior in drosophila melanogaster

个人的性行为受基因型，经验和环境条件的影响。这些因素如何相互作用来调节性行为仍然很少了解。在黑腹果蝇中 ，环境线索（如食物供应）影响交配活动，提供易处理系统来调查调控性行为的机制。在黑腹果蝇中 ，环境线索通常通过化学感觉味觉和嗅觉系统感觉到。在这里，我们提出了一种测试环境化学物质对交配行为的影响的方法。该测定由含有食物介质和交配对的小交配竞技场组成。连续监测每对夫妇的交配频率24小时。在这里，我们介绍了该测定法通过加压空气系统从外部来源测试环境化合物的适用性，以及直接在交配领域操纵环境成分。你加压空气系统对于测试非常易挥发的化合物的影响是特别有用的，而直接在配合领域操作组分可以有助于确定化合物的存在。该测定可以适应于关于遗传和环境线索对交配行为和繁殖力以及其他男性和女性生殖行为的影响的问题。

使用这种连续测定，可以在实验环境条件下确定具体的交配行为和交配频率。为了控制环境条件，我们将不锈钢厨柜改造成具有自己的光源和扩散的测试区域，确保了高度丰富的光线和从配套场地顶部的最少量的眩光（ 图1A ） 。内部测试区域完全由不锈钢和玻璃包裹，可以用有机溶剂如己烷或乙醇清洗。此外，机柜配备有用作管道入口的孔，从加压空气系统...

Watch this Scientific Journal Video about A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster at JoVE.com

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

我们展示了一种分析影响果蝇黑腹果蝇交配行为的环境和遗传线索的测定方法。

测试环境线索对交配行为的影响的方法<em&gt;黑腹果蝇</em

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

a-method-to-test-effect-environmental-cues-on-mating-behavior

Universidad San Sebastian

Research

JoVE Journal

Behavior

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

9.0K 次观看.  University of Groningen. 我们展示了一种分析影响果蝇黑腹果蝇交配行为的环境和遗传线索的测定方法。 

视频: A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

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 ...

科研

神经科学

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 ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

简单的检测方法的社交回避的量化<em&gt;果蝇</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

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

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

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 ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

在条件影响社会空间<em&gt;果蝇</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This complex behavior is influenced by genetic, hormonal and environmental factors. As in many organisms, previous fighting experience influences the fighting strategy of flies and the outcome of later contests: losing a fight increases the probability of losing later contests, revealing &#34;loser&#34; effects that likely involve learning and memory 2-4. The learning and memory that accompanies expression of complex social behaviors like aggression, is sensitive to pre-test handling of animals 5,6. Many experimental procedures are used in different laboratories to study aggression 7-9, however, no routinely used protocol that excludes handling of flies is currently available. Here, we report a new behavioral apparatus that eliminates handling of flies, using instead their innate negative geotactic responses to move animals into or out of fighting chambers. In this protocol, small circular fight arenas containing a food cup are divided into two equal halves by a removable plastic slider prior to introduction of flies. Flies enter chambers from their home isolation vials via sliding chamber doors and geotaxis. Upon removal of plastic sliders, flies are free to interact. After specified time periods, flies are separated again by sliders for subsequent experimentation. All of this is done easily without handling of individual flies. This apparatus offers a novel approach to study aggression and the associated learning and memory, including the formation of &#34;loser&#34; effects in fly fights. In addition, this new general-purpose behavioral apparatus can be employed to study other social behaviors of flies and should, in general, be of interest for investigating experience-related changes in fundamental behavioral processes.

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

During fruit fly fights, the behavioral patterns observed, fight dynamics, and associated learning and memory are influenced by experimental conditions. The protocol presented here describes a novel procedure that entirely eliminates handling of flies during experiments. This improves fight dynamics and allows formation of strong &#34;loser&#34; effects.

一种新的方法，省去了处理的研究侵略和"失败者"的影响<em&gt;果蝇</em

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This ...

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 ...

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 ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila&#160;melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

测量并改变配套驱动马累<em&gt;果蝇</em

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

果蝇调理作为学习和记忆的措施

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

测试环境线索对交配行为的影响的方法

摘要

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此视频中的章节

对检测方法果蝇行为

单苍蝇检测方法的觅食行为的

简单的检测方法的社交回避的量化

染色昆虫行为检测:麻醉的交配行为

在条件影响社会空间

一种新的方法，省去了处理的研究侵略和"失败者"的影响

一个行为分析对机械感觉MARCM为基础的克隆

一个简单的方法来衡量在改建悬赏寻求行为使用

测量并改变配套驱动马累

果蝇调理作为学习和记忆的措施