The authors thank Mike Crickmore, Dragana Rogulja, and Michelle Frank for comments on the manuscript. Pavel Gorelik provided technical support for manufacturing the behavioral arenas. This work was conducted in Mike Crickmore's lab and is also supported by the Whitehall Foundation (Principal Investigator: Dragana Rogulja). S.X.Z. is a Stuart H.Q. and Victoria Quan Fellow at Harvard Medical School.

备注:本协议描述的准备（第1 - 3），执行（第4节），和分析的2-D饱足感检测（第4节）。然后，用多巴胺能刺激作为一个例子，第5显示了如何thermogenetic刺激的2-D饱足测定结合来诱导亢进。第6节说明3种方式来验证的2-D饱足感测定的结果。最后，第7展示了如何衡量雄蝇交配驱动器的恢复。 1.编造8位和32室行为阿里纳斯注意:每个行为竞技场包括在每个四角（ 图1A）的由六角螺钉和拇指螺母保持在一起激光切割的塑料片材的几层。 <ol><li>制作的舞台上层。制造使用激光切割机切割亚克力吸塑给适当的形状床单每一层（ 见图1B，1C的产品）。 注意:许多研究institutioNS在他们的机器店或制造商的空间激光切割机。如果该机构有获得了激光切割机，继续以下步骤。如果不是，则按照步骤1.2代替说明书。 <ol><li>对于每一层，打开激光切割机的软件设计模式。 注:该设计可以在适当命名的PDF文件中找到（ 例如 ，8商会竞技场- Doors.pdf - 2层 ）将用于补充材料1.本文件也包含塑料种类标注（ 如 1 / 8英寸（ 即 3.175毫米）透明丙烯酸）。每层的精确厚度不是关键的。主要要求是，让0.12英寸（3毫米）或更多的可用垂直空间中的腔室。 </li><li>调整的类型和塑料厚度的激光设置（功率，焦点等 ）的使用。将塑料激光切割机的床和运行激光切割机。 注意:这些设置会差异很大，根据激光cutteR型和激光的强度。查找根据以往的经验或对废塑料制作试切适当的设置。深六角形版画都包含在设计休庭六角螺丝头。此功能允许单手收紧拇指坚果，让赛场休息平放在板凳上（不包括凸出的螺丝头）。尽管如此，使得深雕刻通常很难实现的，耗时的，使用激光切割机。这个步骤是可选的，并且可以在不影响行为的结果被跳过。 </li><li>切割门框（第2层， 图1A）的时候，也可以使用适当的激光设置雕刻包括在设计中的室数（如在图1B，1C）。 </li></ol></li><li>将带有在线激光切割制造者的订单。上传所有的8位和/或32腔舞台上的PDF设计文件和指定材料类型（ 如 1/8英寸（ 即 3.175毫米）CLEAř丙烯酸）的基础上在文件中的注释。结果将显示， 如图1B（8-室舞台）和1C（32室舞台）。 </li><li>装配使用四个六角螺钉和四个螺帽拇指对齐并保持塑料层（ 图1A）一起阿里纳斯。该场馆将出现如图1D，1E（8室）和图1F，1G（32室）。 注:食物壁（第4层中的图1A）仅用于二维饱足测定法和在32室舞台，其在求爱测定中使用被省略。 </li></ol> 2.食品准备2-D饱分析注:由于2-D饱足感检测跨越4.5小时，飞食品在舞台上用来提供营养和水分。该协议使用，但并不限于，常规玉米面琼脂飞食品。 <ol><li>部分组装的2-D饱腹感的舞台（8室）使用图层3 - 6（参见层分配图1A），并用拇指螺母，六角螺丝拧紧。 </li><li>将新鲜飞食品到一个干净，微波安全瓶。添加刚好足够的水以覆盖瓶（ 图2A）的底表面。 </li><li>微波食品高30 - 45秒。检查进度，搅拌每15秒，直到融化（ 图2B）。 警告！飞食品容易沸腾了。 </li><li>使用钝1,000微升枪头（ 图2C），以慢慢转移〜1毫升融化食物进入各腔室（ 图2D）。 </li><li>让食品完全组装舞台（ 图2F）之前在4℃再固化为〜10分钟（ 图2E）。 </li><li>使用实验完成的舞台上（见第4节），或保存在4℃。 </li><li>在4℃和再利用储存吃剩的食物。 </li></ol> 3.准备处女的女性行为测定注:2-D饱足感测定采用大量处女雌蝇（〜120 - 160一个完整的行为体育馆）难以用标准的方法来收集。本节将介绍如何使用Y染色体28，已在求偶化验25,29成功使用在HS-HID转基因的另一种方法。用于生成白眼处女雌股票有基因型w1118（X）/ HS-HID（Y）+ / + + / +（布卢明顿库存编号24638）。 <ol><li>倒装飞入一个新瓶一次20 -蛹的50％已eclosed（参见例如图3A）和有足够的男性（5 - 10）传播的库存。 </li><li>热休克原瓶在37℃的热水浴1小时，充分激活HS-HID杀男性（ 图3B）。热休克后，只有女性会从这些瓶子E关闭，因此将保持处女，直到检测。 注:确保瓶子的四周均匀地加热（见水线图3A）。延长这段时间长达2小时，如果1小时是不够的安乐死所有男性。 </li><li>隔离收集的女性至少3天实验之前，确保他们老得足以当性接受25，29。 注:Y染色体，男性少偶尔此股票产生。这些男性有白色的眼睛，而且不受热冲击，因为它们不包含HS-HID转基因。他们不能产生精子，因此，女性30不能减少感受性，31。 </li></ol> 4.执行并打进2-D饱腹感化验注意:控制男性的年龄蝇与年龄交配驱动变化是重要的。该协议使用了3个男性- 4日龄25 </suP&gt;。 <ol><li>设置培养箱到所需的温度（ 例如 ，23℃的标准，RT实验）和湿度（&gt; 30％）。对于没有孵化器的湿度控制，剩下的一杯水在孵化器就足够了。 </li><li>放置一个2-D饱足测定舞台在培养箱中约30分钟以平衡温度，并防止在实验过程中在所述室凝结。旋转门应在打开位置。 </li><li>使用飞抽吸32抽吸15 - 20处女的女性进入竞技场（ 图4）的各腔室。 注:重要的是，在女性和男性不麻醉上的同一天，在测定。根据我们的经验，这样做可能会影响女性的接受能力和男性的交配行为。 </li><li>吸1男到每个室。 </li><li>放置在孵化器加载的舞台上标准的消费摄像机（ 图5）和电影4.5小时以下。 </li><li>通过记录当每个男性飞行开始和结束每一个交配（ 即交配）进球视频。此步骤可能是耗时的在第一。通过实践，我们可以在得分速度5倍或更高的视频。 注意:由于苍蝇在约23℃（在较高温度下的持续时间短）29相配合为〜20分钟，可以通过每个配合的前15分钟，脱脂。 </li><li>记录所有交配次后，总结交配每个苍蝇的数量（见代表性的结果）。 </li><li>使用所提供的代码生成行为谱。见参考文献2的代码，指令，和实施例的数据。 </li><li>使用二氧化碳或低温环境中删除它们之前，麻醉苍蝇。 </li></ol> 5.使用Thermogenetic操纵反向饱腹感的2-D饱腹感化验注:此协议测试定义的神经元群的thermogenetic刺激是否能够克服饱腹感。实验FLIES表达对热敏感阳离子通道TRPA1神经元（UAS-TRPA1）的特定人群。下面的步骤适用于多巴胺能神经元（TH-Gal4的），它已显示出促进交配驱动25的刺激。这些步骤可以与其它神经元的司机一起使用，以发现，促进交配驱动其他人群。 <ol><li>到thermogenetically诱导交配行为在饱足苍蝇，增加温度在孵化器（ 例如 ，从23℃至28.5℃）在完成一个完整的4.5小时的2-D饱足实验之后。 </li><li>孵化器达到所需的温度后，继续热刺激和得分男性的交配（ 即交配）1小时（见代表性的结果）。 注:刺激器的温度和长度可取决于基因型所以，有必要排除阻止神经元的过度刺激，同时还产生一个robu的最佳条件ST表型。 </li></ol> 6.使用求爱和运动验证饱注意:这部分描述3可用于验证的2-D饱测定结果可选方法。它们不是必需为每一个试验。 <ol><li>使用求爱实验来验证饱腹感。 <ol><li>吸一雄一雌处女到每个在求爱舞台上32室。 </li><li>影片使用标准的消费者摄像机〜20分钟的苍蝇。 </li><li>分数求爱指数（CI）量化求爱25;这是花了表演求偶行为（唱歌，以下等 ）时间超过5分钟的窗口交配（ 即交配）的比例。这个窗口从求偶行为的第一个实例开始。如果雄性苍蝇不会在第15分钟开始求爱，其CI是0朴素WT苍蝇应显示的CI大于0.9，而充分饱足苍蝇应具有的CI below 0.2。其他求爱措施也可考虑23,33。 </li></ol></li><li>在2-D饱试验得分求爱<ol><li>得分超过的2-D饱足测定的时间段（ 例如 ，第一h）说明一个雄性苍蝇花费求爱的时间量。求爱被定义为执行仪式的任何步骤（唱歌，下面等 ）的男性。不像在第6.1.3节，这一步时间并不包括男性花费交配。 </li><li>通过阳未在同一时间段配合（ 即不交尾）的总时间除以上述求偶时间。例如，如果一个男性飞15分钟法院和配合20分钟在第一小时，比为15分钟/（60分钟 - 20分钟）= 0.375。该比率显示为代表性的成果"nonmating求偶时的分数。" </li></ol></li><li>利用运动检测以确定耗尽<ol><li>部分组装32室内竞技场所有部分，但对比表。 </li><li>吸一男飞（无雌蝇）到每一个求爱舞台上32室。 </li><li>影片使用标准的消费者摄像机〜10分钟的苍蝇。 </li><li>跟踪与ImageJ的（http://valelab.ucsf.edu/~nstuurman/ijplugins/MTrack2.html）的MTrack2插件苍蝇。 </li></ol></li></ol> 7.经过2-D饱含量恢复配合驱动器<ol><li>如在第4节中所述进行的2-D饱足测定法，但吸阳在测定结束飞出。 </li><li>隔离雄蝇在23℃的天所需的号码。 </li><li>与恢复男性执行2-D饱足感试验和评分的交配（ 即交配）只需1小时（见代表性的结果）。 </li></ol>

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B., Van Woert, M., Davis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+behavior+during+L-dopa+treatment+for+Parkinsonism.">Sexual behavior during L-dopa treatment for Parkinsonism.</a> The American journal of psychiatry. 127 (12), 1691-1693 (1971).</li><li>Sacks, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Awakenings. , (1999).</li><li>Dietzl, G., Chen, D., 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=A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 448 (7150), 151-156 (2007).</li><li>Crickmore, M. A., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+dopaminergic+and+GABAergic+neurons+control+the+duration+and+persistence+of+copulation+in+Drosophila.">Opposing dopaminergic and GABAergic neurons control the duration and persistence of copulation in Drosophila.</a> Cell. 155 (4), 881-893 (2013).</li><li>Peng, J., Chen, S., Busser, S., Liu, H., Honegger, T., Kubli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradual+Release+of+Sperm+Bound+Sex-Peptide+Controls+Female+Postmating+Behavior+in+Drosophila.">Gradual Release of Sperm Bound Sex-Peptide Controls Female Postmating Behavior in Drosophila.</a> Current biology. 15 (3), 207-213 (2005).</li><li>Yapici, N., Kim, Y. J., Ribeiro, C., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+receptor+that+mediates+the+post-mating+switch+in+Drosophila+reproductive+behaviour.">A receptor that mediates the post-mating switch in Drosophila reproductive behaviour.</a> Nature. 451 (7174), 33-37 (2008).</li><li>Pellegrino, M., Nakagawa, T., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Sensillum+Recordings+in+the+Insects+Drosophila+melanogaster+and+Anopheles+gambiae.">Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae.</a> Journal of Visualized Experiments. 36 (36), 1-5 (2010).</li><li>Cook, R., Cook, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Attractiveness+to+males+of+female+Drosophila+melanogaster:+effects+of+mating,+age+and+diet.">The Attractiveness to males of female Drosophila melanogaster: effects of mating, age and diet.</a> Animal behaviour. 23, 521-526 (1975).</li><li>Toates, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Motivational Systems (Problems in the Behavioural Sciences). , (1986).</li><li>Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mating+of+a+fly.">The mating of a fly.</a> Science. 264 (5166), 1702-1714 (1994).</li><li>Simpson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+manipulating+neural+circuits+in+the+fly+brain.">Mapping and manipulating neural circuits in the fly brain.</a> Advances in genetics. 65 (9), 79-143 (2009).</li><li>Venken, K. J. T., Simpson, J. H., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Manipulation+of+Genes+and+Cells+in+the+Nervous+System+of+the+Fruit.">Genetic Manipulation of Genes and Cells in the Nervous System of the Fruit.</a> Neuron. 72 (2), 202-230 (2011).</li><li>Klapoetke, N. C., Murata, Y., 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=Independent+optical+excitation+of+distinct+neural+populations.">Independent optical excitation of distinct neural populations.</a> Nature methods. 11 (3), 338-346 (2014).</li><li>Bellen, H. J., Levis, R. W., 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+BDGP+gene+disruption+project:+single+transposon+insertions+associated+with+40%+of+Drosophila+genes.">The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes.</a> Genetics. 167 (2), 761-781 (2004).</li><li>Spradling, A. C., Stern, D., 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+Berkeley+Drosophila+Genome+Project+gene+disruption+project:+Single+P-element+insertions+mutating+25%+of+vital+Drosophila+genes.">The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes.</a> Genetics. 153 (1), 135-177 (1999).</li><li>Parks, A. L., Cook, K. R., 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=Systematic+generation+of+high-resolution+deletion+coverage+of+the+Drosophila+melanogaster+genome.">Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome.</a> Nature genetics. 36 (3), 288-292 (2004).</li><li>Matthews, K. A., Kaufman, T. C., Gelbart, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+resources+for+Drosophila:+the+expanding+universe.">Research resources for Drosophila: the expanding universe.</a> Nature reviews. Genetics. 6 (3), 179-193 (2005).</li><li>Ni, J. Q., Liu, L. P., 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=A+Drosophila+resource+of+transgenic+RNAi+lines+for+neurogenetics.">A Drosophila resource of transgenic RNAi lines for neurogenetics.</a> Genetics. 182 (4), 1089-1100 (2009).</li><li>Ni, J. Q., Zhou, R., 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=A+genome-scale+shRNA+resource+for+transgenic+RNAi+in+Drosophila.">A genome-scale shRNA resource for transgenic RNAi in Drosophila.</a> Nature. 8 (5), 405-407 (2011).</li></ol>

The authors have nothing to disclose.

动机状态可以满足，维护，并回收34。我们提出的2-D饱足测定迅速，鲁棒测量所有的在飞配合驱动器的这些方面。该测定中开辟了采用先进的飞基因操作来研究动机的行为的分子和电路元件的可能性。 饱腹感检测依赖于男性的能力，成功地法院和交配，并在适当的时候终止交配。虽然hyposexual苍蝇少法庭，低求爱苍蝇不一定hyposexual;他们可能，例如，具有难以...

在果蝇的工作提供了深刻的开拓洞察许多生物现象，包括基因1的性质，胚胎发育2，昼夜节律3，和神经系统4，5，6的发展和布线的原则。动机仍然因为上迄今已研究了系统的限制可能远不如比这些现象可知，。动机在飞主要研究了饥饿，它提出了许多挑战的背景下，由于每次喂食发作和外骨骼这就排除脂肪沉积明显的迹象了微乎其微的食物摄入量。因此，有必要扩大用于研究动机在飞的系统。 我们描述一个行为框架，配合驱动器的研究果蝇 。该系统以在飞以及辅助7，8，9，10，11，12和其性二态电路8,13的推定连接组优势的神经发生的工具。此外，许多先天的14，15，16，17，18，19，20，21和22的教训，23，24感觉运动电路控制求爱已详细制定出来，提供了一个难得的机遇找到赖以动机撞击的精确电路节点。我们最近报道，在飞，如在人类中，多巴胺水平是中央交配驱动装置25，26，27。我们已经获得了相关产生多巴胺和飞接受神经元，促进详细molecular-和电路级的遗传访问使用试验，我们在这里介绍25这种现象的保守分析。 我们添加到张某等人的行为分析。 25一个新的平行为竞技场，允许视频得分，我们称之为一个2维（2-D）的饱足感测定中，在以前的方法的一个重要的改进。因此，新的检测更可扩展性和可量化的，因此更适合参与积极性的基因和神经元的遗传筛选。我们使用这种新的检测，加上求爱分析和neurogeNETIC操作，演示了如何衡量和改变苍蝇交配的车程。

<table><tbody><tr><td>1/16 inch clear acrylic</td><td>McMaster-Carr</td><td>8589K12</td><td>Used to make arenas; see Supplemental Material 1 for designs.</td></tr><tr><td>1/8 inch clear acrylic</td><td>McMaster-Carr</td><td>8589K42</td><td>Used to make arenas; see Supplemental Material 1 for designs.</td></tr><tr><td>3/16 inch clear acrylic</td><td>McMaster-Carr</td><td>8560K219</td><td>Used to make arenas; see Supplemental Material 1 for designs.</td></tr><tr><td>1/32 inch black delrin</td><td>McMaster-Carr</td><td>8575K132</td><td>Used to make arenas; see Supplemental Material 1 for designs.</td></tr><tr><td>Hex screws, 1 inch long (50x)</td><td>McMaster-Carr</td><td>92314A115&amp;nbsp;</td><td>Used to make arenas. Can be replaced by 3/4 inch screws (92314A113, McMaster-Carr) for 32-chamber arenas.</td></tr><tr><td>Thumb nuts (25x)</td><td>McMaster-Carr</td><td>92741A100</td><td>Used to make arenas. Can be replaced by regular hex nuts (90480A005, McMaster-Carr).</td></tr><tr><td>Camcorder</td><td>Canon</td><td>Vixia HF R700</td><td>Can be replaced by any consumer comcorder.</td></tr></tbody></table>

measuring and altering mating drive in male drosophila melanogaster

尽管几十年的调查，动机状态的神经细胞和分子基础仍然神秘。我们最近开发了一种新型，还原，和可扩展的系统使用雄性果蝇 （果蝇）的交配驱动动机进行了深入调查，采用何种方法在这里，我们的细节。行为模式集中在雄性交配驱动器一起生育过多次交配的过程中减少并恢复了〜3天的发现。在这个系统中，在飞可用的有力神经源性工具收敛与遗传访问性和可用于性行为推定接线图。这种融合允许快速隔离和小神经元群体具有特定功能的动机审讯。在这里，我们详细地是，用于测量和在雄性苍蝇改变求偶动机的饱足感测定的设计和执行。使用此实验中，我们还表明，男性低交配驱动器可通过刺激多巴胺能神经元加以克服。饱腹法简单，价格适中，和强大的遗传背景的影响。我们预计饱腹感检测，以产生许多新的见解激励状态的神经生物学。

为了描述果蝇交配的车程，3日龄，广州WT-S男性被在2-D饱法检测。过测定（4.5小时）的过程中，雄性交配的4.8±0.3的平均（平均均值，SEM±标准误差）次。交配发起大多在最初2小时（78％）（ 图6A，6B）和变得较不频繁的测定法的进展（ 图6A，6B）。这种减少不是由于缺乏配合伙伴的（74％的女性在整个试验保持未配合）或肉体疲劳（ 图...

Watch this Scientific Journal Video about Measuring and Altering Mating Drive in Male Drosophila melanogaster at JoVE.com

Measuring and Altering Mating Drive in Male Drosophila melanogaster

本文介绍了采用果蝇 melanogaste R以学习动机男性交配驱动器中的行为分析。利用这种方法，研究人员可以利用先进的飞神经源性技术来揭开背后这个动机的遗传，分子和细胞机制。

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, ...

measuring-and-altering-mating-drive-in-male-drosophila-melanogaster

Research

JoVE Journal

Neuroscience

Measuring and Altering Mating Drive in Male Drosophila melanogaster

11.0K 次观看.  Boston Children’s Hospital. 本文介绍了采用果蝇 melanogaste R以学习动机男性交配驱动器中的行为分析。利用这种方法，研究人员可以利用先进的飞神经源性技术来揭开背后这个动机的遗传，分子和细胞机制。 

视频: Measuring and Altering Mating Drive in Male Drosophila melanogaster

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are also a useful scientific model to study motion vision and flight dynamics in a controlled laboratory setting. As the larvae develop in organically polluted water, they are useful models for investigating investment in microbial immunity. While large scale commercial breeding for agriculture already occurs, there are no standardized protocols for maintaining captive populations for scientific studies. This is important as commercial captive breeding programs focusing on mass output during peak pollination periods may fail to provide a population that is consistent, stable and robust throughout the year, as is often needed for other research purposes. Therefore, a method to establish, maintain and refresh a captive research population is required. Here, we describe the utilization of an artificial hibernation cycle, in addition to the nutritional and housing requirements, for long term maintenance of Eristalis tenax. Using these methods, we have significantly increased the health and longevity of captive populations of E. tenax compared to previous reports. We furthermore discuss small scale rearing methods and options for optimizing yields and manipulating population demographics.

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

The overall goal of these procedures is to establish, maintain and refresh a captive population of Eristalis tenax in a research setting.

研究性学习的Eristalis 管Hoverflies 的饲养和长期维护

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are ...

科研

环境科学

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

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

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

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

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 ...

行为学

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 ...

神经科学

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 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 ...

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 ...

Assessing Differences in Sperm Competitive Ability in Drosophila

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability 1. Sperm competition represents the competition between males after copulating with the same female 2, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals 3. For example, wild-caught D. melanogaster females usually contain sperm from 2-3 males 4. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males 2,5.
Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory 6,7. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions 7,8. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively) 9, which is assumed to be reflective of different competence attributes.
Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster.

Assessing Differences in Sperm Competitive Ability in Drosophila

Differential sperm competitive ability among Drosophila males with distinct genotypes can be ascertained through double-mating experiments. Each of these experiments involves one of the males of interest and a reference male. Readily identifiable markers in the progeny allow inference of the fraction of individuals fathered by each male.

评估精子竞争能力的差异<em&gt;果蝇</em

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the ...

测量并改变配套驱动马累

摘要

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一个简单的方法来衡量在改建悬赏寻求行为使用

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评估精子竞争能力的差异