Name: measuring crop motility and food passaging in drosophila
Uploaded: 2020-05-09T14:30:00
Description: Watch this Scientific Journal Video about Measuring Crop Motility and Food Passaging in Drosophila at JoVE.com

这项工作得到了国家自然科学基金（第31872287号）、江苏省自然科学基金（NO.BK20181456）和江苏省六大人才高峰项目（第1号SWYY-146）。

1. 维护和准备实验苍蝇
<ol><li>在25°C的孵化器中，在含有10 mL新鲜食物（1%agar，2.4%的啤酒酵母，3%蔗糖，5%玉米粉）的培养箱中保持苍蝇，湿度为60%。将培养箱的光循环设置为 12 小时光：12-h 暗。</li>
	<li>为了确保大量所需的基因型苍蝇同时出现，培养幼蝇（1~3天大）在标准食品中，表面有干酵母，3天。将成人转移到一个新的食物小瓶与标准食品，包括湿酵母，2天，让产卵。将卵子留在孵化器中?...

<ol>
	<li>Kusano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+motility+and+functional+gastrointestinal+diseases.">Gastrointestinal motility and functional gastrointestinal diseases.</a> Current Pharmaceutical Design. 20 (16), 2775-2782 (2014).</li><li>Reiter, L. T., Potocki, L., Chien, S., Gribskov, M., Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+analysis+of+human+disease-associated+gene+sequences+in+Drosophila+melanogaster.">A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster.</a> Genome Research. 11 (6), 1114-1125 (2001).</li><li>Apidianakis, Y., Rahme, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+as+a+model+for+human+intestinal+infection+and+pathology.">Drosophila melanogaster as a model for human intestinal infection and pathology.</a> Disease Models &amp; Mechanisms. 4 (1), 21-30 (2011).</li><li>Lemaitre, B., Miguel-Aliaga, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Digestive+Tract+of+Drosophila+melanogaster.">The Digestive Tract of Drosophila melanogaster.</a> Annual Review of Genetics. 47, 377-404 (2013).</li><li>Miguel-Aliaga, I., Jasper, H., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+Physiology+of+the+Digestive+Tract+of+Drosophila+melanogaster.">Anatomy and Physiology of the Digestive Tract of Drosophila melanogaster.</a> Genetics. 210 (2), 357-396 (2018).</li><li>Strand, M., Micchelli, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quiescent+gastric+stem+cells+maintain+the+adult+Drosophila+stomach.">Quiescent gastric stem cells maintain the adult Drosophila stomach.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (43), 17696-17701 (2011).</li><li>Ren, J., 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=Beadex+affects+gastric+emptying+in+Drosophila.">Beadex affects gastric emptying in Drosophila.</a> Cell Research. 24 (5), 636-639 (2014).</li><li>Xi, J., 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+TORC1+inhibitor+Nprl2+protects+age-related+digestive+function+in+Drosophila.">The TORC1 inhibitor Nprl2 protects age-related digestive function in Drosophila.</a> Aging. 11 (21), 9811-9828 (2019).</li><li>Wei, Y., Reveal, B., Cai, W., Lilly, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GATOR1+Complex+Regulates+Metabolic+Homeostasis+and+the+Response+to+Nutrient+Stress+in+Drosophila+melanogaster.">The GATOR1 Complex Regulates Metabolic Homeostasis and the Response to Nutrient Stress in Drosophila melanogaster.</a> G3. 6 (12), 3859-3867 (2016).</li><li>Ja, W. 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=Prandiology+of+Drosophila+and+the+CAFE+assay.">Prandiology of Drosophila and the CAFE assay.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (20), 8253-8256 (2007).</li><li>Diegelmann, S., 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+CApillary+FEeder+Assay+Measures+Food+Intake+in+Drosophila+melanogaster.">The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster.</a> Journal of Visualized Experiments. (121), e55024 (2017).</li><li>Edgecomb, R. S., Harth, C. E., Schneiderman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+feeding+behavior+in+adult+Drosophila+melanogaster+varies+with+feeding+regime+and+nutritional+state.">Regulation of feeding behavior in adult Drosophila melanogaster varies with feeding regime and nutritional state.</a> Journal of Experimental Biology. 197, 215-235 (1994).</li><li>Peller, C. R., Bacon, E. M., Bucheger, J. A., Blumenthal, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+gut+function+in+drop-dead+mutant+Drosophila.">Defective gut function in drop-dead mutant Drosophila.</a> Journal of Insect Physiology. 55 (9), 834-839 (2009).</li><li>Chtarbanova, S., 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=Drosophila+C+virus+systemic+infection+leads+to+intestinal+obstruction.">Drosophila C virus systemic infection leads to intestinal obstruction.</a> Journal of Virology. 88 (24), 14057-14069 (2014).</li><li>Solari, 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=Opposite+effects+of+5-HT/AKH+and+octopamine+on+the+crop+contractions+in+adult+Drosophila+melanogaster:+Evidence+of+a+double+brain-gut+serotonergic+circuitry.">Opposite effects of 5-HT/AKH and octopamine on the crop contractions in adult Drosophila melanogaster: Evidence of a double brain-gut serotonergic circuitry.</a> PLoS One. 12 (3), 0174172 (2017).</li></ol>

在果蝇中，摄入的食物从作物转移到肠道进行消化。在这个过程中，营养物质被吸收，废物被排出体外作为粪便。因此，将食物摄入与粪便喷射进行比较，可以大致评估食物在体内运动的速度。毛细管喂食器（CAFE）的方法被广泛用于测量食物摄入10，11。10,粪便数计数方法可用于估计粪便的产生量12。然而，果蝇体内的食物运...

大多数动物有一个消化管称为胃肠道（GI），以吸收来自环境的能量和营养。人类胃肠道由四个部分组成：食道、胃、小肠和大肠（结肠）。从胃到肠的食物通道对营养吸收至关重要。一些影响因素，如老化，有毒药物和感染，导致胃肠道运动障碍和胃排空，这与一些疾病及其症状，如消化不良，胃食管反流病，便秘1。
果蝇（果蝇黑色素）是生物医学研究中广泛使用的模型动物，因为它易于基因操作。重要的是，大约77%与人类疾病相关的基因在果蝇2中具有同源性。使用果蝇的研究在了解许多疾病机制方面取得了巨大的进展。作为一种强大的遗传模型生物体，果蝇在胃肠道研究中得到了广泛的应用。果蝇有一个更简单的消化道，分为三个离散域：前脑、中生道和后脑4。作物是前种的一部分，是一个袋式结构，作为摄入食物储存的场地。中肠是一个长管，作为食物消化和营养吸收的站点，通过上皮层，其中包括吸收性肠细胞（CS）和分泌肠分泌细胞5。有趣的是，果蝇的胃功能分为两部分：作物功能作为食物储存和铜细胞区（CCR）是一个高酸性区域与pH <3 6。在果蝇，摄入的食物最初被转移到作物，然后泵入中果7。因此，作物在食物传传中起着至关重要的作用。被内脏肌肉包裹，由一系列复杂的阀门和括约肌组成，作物不断收缩，将食物移动到中游，以作进一步消化。
该协议允许检测食物从作物到中游的果蝇运动。通过计算作物收缩频率来评估作物收缩。此外，通过检测作物和肠道之间的食物分布，对作物对食物传通的影响进行了调查。此外，食物分配可用于反映使用不同喂养期的直接食物运动或基本食物状况。综合起来，该协议提供了快速评估作物运动性和食物传通性的方法。

<table>
	<tbody>
		<tr>
			<td>96-well plate</td>
			<td>Thermo fisher</td>
			<td>269620</td>
			<td></td>
		</tr>
		<tr>
			<td>Brillant Blue FCF</td>
			<td>Solarbio</td>
			<td>E8500</td>
			<td>also called FD&#38;C Blue No. 1</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo fisher</td>
			<td>Heraeus Pico 17</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Spectra Max</td>
			<td>cMax plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Dumont</td>
			<td>11252-30</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring crop motility and food passaging in drosophila

大多数动物使用胃肠道（GI）来消化食物。摄入的食物在胃肠道的运动对营养吸收至关重要。紊乱的GI运动和胃排空导致多种疾病和症状。作为一种强大的基因模型生物体，果蝇可用于GI运动性研究。D罗友病作物是一种器官，它收缩并移动食物进入中腹，以便进一步消化，其功能类似于哺乳动物的胃。介绍是一种使用简单测量工具研究果蝇作物动性的协议。介绍了一种计算作物收缩以评估作物运动性的方法，以及一种利用分光光度计检测作物和肠道之间染蓝色食物分布的方法，以调查作物对食物传传的影响。该方法用于检测控制与nprl2突变苍蝇在作物运动性上的差异。该协议既具有成本效益，又对作物的动性高度敏感。

这些计算作物收缩率和检测染色食物分布的方法可用于评估作物对食物活力的功能。作物收缩反映了将食物推入肠道的频率。短喂养期后，染料在苍蝇中的分布表明食物从作物直接从作物到中游。
拉帕霉素复合物1（TORC1）的目标是调节营养和细胞代谢的主调节器。TORC1抑制可延长许多生物体的寿命，包括果蝇。作为 TORC1 的抑制剂，nprl2 突变苍蝇表现出 TORC1 和 GI 消化缺...

Watch this Scientific Journal Video about Measuring Crop Motility and Food Passaging in Drosophila at JoVE.com

Measuring Crop Motility and Food Passaging in Drosophila

该协议的目标是测量作物收缩和量化食物分布在果蝇肠道。

测量果蝇的作物运动性和食物通度

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. ...

此方法用于通过计算作物收缩和检测消化中的食物分布来评估作物的动能。这种技术的优点是它不断评估作物的动性。成本低，易于执行。这种方法使得使用只是填充物作为模型来研究胃肠道食物通道的消化功能。帮助演示这个过程的将是我实验室的研究生习近平。在 25 摄氏度的培养箱中，保持含有 10 毫升新鲜食物的小瓶中的苍蝇，湿度为 60%，光线为 12 小时，暗周期为 12 小时。确保大量欲望表型同时通过用标准食物培养带干酵母的年轻苍蝇，然后用湿酵母将成年苍蝇转移到小瓶中，并允许两天产卵。将卵子留在孵化器中，以发育和转移成年苍蝇到新瓶收集更多的卵子。每天收集被围住的雄蝇或雌蝇，用标准食物培养它们，达到所需的年龄。用二氧化碳麻醉苍蝇，用200微升PBS将一只苍蝇转移到解剖板井中。用一对钳子抓住苍蝇的胸，用另一对钳子打开胸部，然后向相反的方向拉两端以打开腹部。小心地把庄稼和肠道从身体里拿出来，等待苍蝇醒来。当苍蝇清醒时，在一分钟内计算作物收缩次数。重复收缩计数五次，在每个计数之间等待 30 秒。称重蓝色染料，以20%的浓度溶解在PBS中。然后在食品冷却过程中搅拌至煮沸的液体维护食品，最终浓度为5%。将苍蝇与饥饿的食物一起放在小瓶中四个小时后，用染色的食物将它们转移到新的小瓶中，并培养它们，以度过所需的时间，请记住，在维护条件下，食物在两个小时内通过苍蝇。将麻醉苍蝇转移到含有200微升PBS的解剖板井中。使用钳子抓住胸部的苍蝇，用另一对钳子取出头部，然后用PBS将身体转移到新井中，然后用轻轻的摇动清洗，以去除附着在身上的染料。用两对钳子打开腹部，小心地将整个肠道与身体分开。将作物从整个肠道中切开，放入100微升PBS的管子中。将其余的肠道放在另一个管与100微升PBS，然后使用移液器提示研磨作物和肠道，并溶解在PBS染料。重复解剖，直到收集到足够的作物和内脏。采集样品后，以最高速度离心管一分钟，将90微升上流液转移到96井板的井中。以每毫升1倍到7至10至4克的浓度进行一系列标准蓝色染料稀释，并将其添加到井中。测量井 630 纳米的吸光度，并使用标准测量来绘制吸光度与染料浓度的线图。然后使用曲线计算样品的染料浓度。作物运动性测定用于计算三天老NPRL突变苍蝇的作物收缩和遗传背景控制。发现这种突变可显著降低作物收缩率。为了进一步确认NPRL2对作物的功能，通过给苍蝇喂食染色食物来检测食物运动。所有苍蝇的作物中都有类似的染料量，但NPRL2苍蝇的肠道染料较少，这可能与作物收缩率降低有关。键入此协议时，请确保作物与身体保持接触，并且苍蝇是活的和清醒的。否则，作物将失去收缩能力。在解剖过程中，作物和肠道应接触。如果染料泄漏到解剖介质中，则不能再使用样品。让我们只是填补我们的胃口和食物弹出在短时间内也可以通过检测整个肠道的染料量进行评估。

Research

JoVE Journal

Biology

Measuring Crop Motility and Food Passaging in Drosophila

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

人类胚胎干细胞传代的色调线与胰蛋白酶

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

科研

生物学

Passaging Human Neural Stem Cells

传代人类神经干细胞

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

从14日龄的幼苗组织原生质体的分离拟南芥</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Catheterization of Intestinal Loops in Ruminants

导尿在反刍动物的肠袢

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the ...

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

机器人技术和植物组织的基因表达研究的动态图像分析

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Gastrointestinal Motility Monitor (GIMM)

Gastrointestinal Motility Monitor GIMM

肠胃蠕动监视器（GIMM）

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

使用网关兼容双分子荧光互补（附设）系统在植物蛋白相互作用检测

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Measuring Glutathione-induced Feeding Response in Hydra

测量水润谷胱甘肽诱导饲养响应

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

可视化和跟踪活果蝇黑色素细胞卵室的内源性 mRNA

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...