Name: a simple approach to manipulate dissolved oxygen for animal behavior observations
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The Authors would first like to acknowledge all students from the freshman Biology 121- Ecology Module lab at Juniata College for their help in generating data used in this study. We would also like to thank Dr. Randy Bennett, Chris Walls, Sherry Isenberg, and Taylor Cox for their assistance in acquiring materials necessary to develop this methodology. Additionally, we would like to thank Dr. Norris Muth and Dr. John Unger for their advice on methodological development and Dr. Jill Keeney and the Biology department for their support of this endeavor. We would also like to thank the anonymous reviewers that have helped to shape and focus this manuscript.&#160; Last but not least, I&#39;d like to thank Hudson Grant for his help with the initial stonefly collection for use in development of this technique

注意:这个实验没有使用脊椎动物，因此没有需要由杰尼阿塔学院研究所的动物护理和使用委员会的批准。然而，对于个人适应与脊椎动物使用这种方法，IACUC批准，应寻求。 领域样品采集<ol><li>确定并收集，存储能力评估潜在的野外场地，运输石蝇迅速在1小时过境最大推荐时间减少在途时间。 </li><li>在选定的领域站点执行球网采样下列标准球网的程序足够的时间来收集至少35石蝇12。 </li><li>收集50升流的水和岩石的与流2厘米的最大直径。 </li><li>放置水族馆中设定为数据流部位的温度的冰箱。在分发流现场收集到的岩石和水族馆，每水族馆4升溪水填补。将20每水族馆-30收集石蝇和放置连接到水族馆鼓泡到每个罐中鼓泡石和打开鼓泡向室内空气连续地添加到水中。 </li><li>允许石蝇调整到在水族馆的48小时期间新的环境。 </li></ol><img alt="图1" src="/files/ftp_upload/54430/54430fig1.jpg" /> 图1.为溶解氧操作。（A）1）安装铜管男性软管倒钩2）塞密封的位置，以检查确保密封性好烧瓶中。 （B）1）2 L侧臂烧瓶装满1.9升水2）气体管和分别空气鼓泡器（蓝色），于氮气鼓泡和房间空气鼓泡使用，3）氮罐和衡量值4）2升烧瓶洋溢着0.4升的水与真空管淹没5）溶解氧测定仪。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54430/54430fig1large.jpg" target="_blank">请点击这里</a>查看该图的放大版本。 2.实验设置<ol><li>上的台顶，一个标准的薄壁真空管连接到2L侧臂烧瓶的侧臂如图所示（ 图1B 1）。 </li><li>与3升的塑料容器保持收集流水在冰箱设置为12℃的1.9升流的水的填充烧瓶中。 </li><li>放在一个托盘大到足以容纳周围的侧臂瓶冰浴不遮挡烧瓶内部的看法和填充用冰托盘中的烧瓶和油管。 </li><li>钻两个直径3mm的孔的橡胶塞，以允许1）的铜管的通道，以输送气体到容器和2）的DO计， 如图1B中的探头插入2升侧臂烧瓶（1） 。 </li><li>使从塞子的边缘的横向切口的孔中的一个，以使DO探头的导线的座位到塞子。 </li><li>连接耦合器带有3毫米公软管倒钩（ 图1A 1）一块直径为2毫米铜管。确保该管足够长以10厘米烧瓶底部的内达到通过塞子到达时间。 </li><li>放置管具有耦合器虽然在止动件的第二孔，直到从止动件的底部的长度是足够到10cm烧瓶底部的内达到。 </li><li>连接0.75米长，薄壁聚乙烯燃气管的直径为3mm到管道上的耦合器。 </li><li>两者DO探头和铜管滑入烧瓶中并密封与止动烧瓶。 </li><li>检查塞子和烧瓶，以及管和塞子中的探针导线之间的紧密配合之间的安全密封。 </li><li>补的1L烧瓶加入0.4升的自来水，并放置相邻的冰浴，并在真空烧瓶中的托盘。 </li><li>淹没聚乙烯管从大保温瓶来进1升烧瓶中的水。固定用胶带管，使得其将通过实验保持浸没。 </li><li>从保温瓶3毫米直径的天然气管线连接到一个水族馆室内空气鼓泡。在水族馆起泡器，它引入了室内空气和氧气送到堵水开始泡在2升烧瓶上的水。 </li><li>监控溶解氧浓度及水分的温度与DO仪5分钟，或直至溶解氧的平衡在腔室内建立，使得在溶解氧很少改变正在发生。 </li></ol> 3.测试实验装置的稳定性<ol><li>测试每个设置为之前除了石蝇的DO稳定性。 </li><li>添加三个或四个石头到2升瓶，使石蝇有基材利于俯卧撑。 </li><li>通过从鼓泡断开气体管，并将其附加到氮气行开始的DO的试用操作。 </li><li>开始在立方英尺每小时（CFH）20约40氮气鼓泡秒至1分钟。 </li><li>一旦溶解氧已0.5毫克/升的目标浓度的内下降到时，流程降低到15 CFH和允许的浓度降低到目标。 </li><li>立即停止氮气流下一旦达到目标浓度。 </li><li>使用水族馆室内空气鼓泡至浓度返回到目标浓度如果溶解氧减小低于目标。 </li><li>如果做一套行动的测试过程中是不稳定的再检查水量仍处于1.9 L和没有水已经起泡了，水温稳定，不改变，并在所有接头密封显得紧，密封。 </li><li>一旦三个试验已经完成，并实验者在控制DO的能力有信心，附加气体线起泡和气泡再平衡。 </li><li>泡到平衡通过附加3mm直径的气体管线的水族馆起泡和开始加入室内空气的到水中，直到浓度氧气在水中不增​​加或更改3分钟。 </li><li>一旦处于平衡状态，停止冒泡和启封烧瓶。 </li></ol> 4. StoneFly的俯卧撑实验<ol><li>由观察员人数除以石蝇的总数确定试验来执行的数目。 </li><li>确定2和10毫克/升之间不同DO水平来评价石蝇的行为反应（俯卧撑的数量）。 </li><li>设置每试验一锥形瓶并添加石蝇的数目相等，因为有观察员至烧瓶（该设计内4石蝇），探针和管放回烧瓶，然后重新密封的橡胶塞的烧瓶中。 注:初始DO为10mg / L浓度被选为第一个观察点，因为它是流从其中石蝇进行取样的溶解氧浓度。 </li><li>一旦水是在10毫克/升通过鼓泡以下步骤2.10-2.11，记录开始水温，并允许石蝇附加到烧瓶中的岩石基体。 </li><li>仅指派一名观察员观看一个StoneFly的保证俯卧撑的行为，这是由StoneFly的表现上下肢体动作计数准确。 </li><li>算并记录在3分钟的观察期的过程中观察到的俯卧撑的数目。 </li><li>操纵DO到下一个试验的DO水平，并重复3分钟的观察期，附加的实验水平。 注意:在这个实验设计，三个不同的DO水平进行了评价。 </li></ol> 5.统计分析<ol><li>向在一组为给定的DO试验执行在四个石蝇俯卧撑的统计分析使用平均数。 </li><li>使用免费的R统计计算软件12在俯卧撑和使用每个实验试的顺序DO浓度的数量进行方差分析的分析（DO级）和温度COVariates。分析DO作为单个因素的离散电平。 </li><li>使用残差的安德森-达林正态性检验，检查正常13。 </li><li>通过绘制针对DO浓度俯卧撑的平均数对数据执行线性回归。 </li></ol>

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The authors declare that they have no competing financial interests.

关键步骤 这个程序提供了一个简单而有效的方式来操作DO在实验室环境对水生生物进行行为研究。我们发现有以几个关键步骤/项意识到执行该实验是直接相关的结果时的感觉。在一个试验中，它是保持室压力，以避免在水上方的气体的分压的变化的关键，和随后的DO波动。按照协议的"试实验装置的稳定"小节中所列的步骤是关键的。检查与烧瓶塞子的密封，保证真空管的完全浸没到水的1升烧?...

溶解氧（DO）是在调解一些水生生态系统中的生物和生态过程的重要关键生理参数。暴露于急性和慢性亚致死缺氧减少某些水生昆虫的生长率，降低暴露1昆虫的生存。该协议的开发提供一个可控的方法来操纵溪水DO水平，观察动物行为的影响。由于所有的有氧水生生物的生存，为了生存和繁殖依赖于氧浓度，溶解氧浓度的变化往往反映在生物行为变化。更多的移动水生无脊椎动物和鱼类已观察到寻求语言环境具有较高的DO 2,3低氧浓度（缺氧）响应。对于流动性较差的水生生物，行为适应增加DO的摄入量可能是唯一可行的选择。 PLEC的水生底栖动物秩序的OPTera（StoneFly的）已被指出以执行"上推"运动，以增加水中的氧的流量，和吸收，在其外部鳃4 - 6。这些适应性行为已在自然的环境，并在实验室实验中观察到。 在水中溶解氧的实验室操作开辟了动物行为研究显著的机会，但在方法上部署显著存在差距。例如，一项研究使用的大型水族馆评估以下用氮气脱气大嘴低音的生理响应时间（ 加州鲈 ）缺氧的环境中，但很少详细给出的方法7。使用氮气和多孔石将气体输送到水，减少水分8 DO描述上斑马鱼（ 斑马鱼 ）进行的另一项研究。对于基于化学的应用程序，对于溶剂的脱气方法使用专门的设备9 - 11从溶剂除去氧气，但不会是适合动物行为的研究。虽然这些研究采用的方法来从水中除去氧气，没有描述的方法可以查明将允许对动物行为评价响应于DO变化。 下面描述这种方法是试图通过使用氮气充分描述了的水的DO操作的协议。此外，这种方法对观察StoneFly的行为（如俯卧撑）之间的关系发展，去做一些在大一级别的生物实验室使用。其中一个这种方法的主要好处是，它可以很容易地与常用玻璃器皿和大多数中等和高等教育机构可获得的材料在实验室内进行。该协议也很容易适应，允许个人扩展，以满足提出的研究或教学应用目标的过程。 </p&gt;

<table><tbody><tr><td>Filter flask 2 L</td><td>Pyrex</td><td>5340</td><td></td></tr><tr><td>Rubber Stopper size 6</td><td>Sigma-Aldrich</td><td>Z164534</td><td></td></tr><tr><td>Nalgene 180 Clear Plastic Tubing</td><td>Thermo Scienfitic</td><td>8001-1216</td><td></td></tr><tr><td>Whisper 60 air pump</td><td>Tetra</td><td></td><td></td></tr><tr><td>Standard flexible Air line tubing</td><td>Penn Plax</td><td>ST25</td><td></td></tr><tr><td>0.25 inch Copper tubing</td><td>Lowes Home Improvement</td><td>23050</td><td></td></tr><tr><td>Male hose barb</td><td>Grainger</td><td>5LWH1</td><td></td></tr><tr><td>Female Connector</td><td>Grainger</td><td>20YZ22</td><td></td></tr><tr><td>Heavy Duty Dissolved Oxygen Meter</td><td>Extech</td><td>407510</td><td></td></tr><tr><td>Nitrogen gas</td><td>Matheson TRIGAS</td><td></td><td></td></tr><tr><td>Radnor AF150-580 Regulator</td><td>Airgas</td><td>RAD64003036</td><td></td></tr></tbody></table>

a simple approach to manipulate dissolved oxygen for animal behavior observations

操纵溶解氧（DO）在实验室环境的能力有显著应用调查若干生态和生物体的行为的问题。这里所描述的协议提供了一种简单的，可重复，并且控制方法来操纵DO来研究从低氧和缺氧条件下得到的水生生物的行为反应。在执行的水脱气用氮气在实验室设置常用生态（水生）应用没有明确的方法存在于文献中，并且该协议是第一个描述的协议以degasify水来观察生物体反应。这种技术和协议是为水生无脊椎动物直接应用开发;然而，小鱼类，两栖类，和其它水生脊椎动物可以很容易被取代。它允许从2毫克/升，以11毫克/升与稳定性DO水平的容易操纵长达5分钟的动物观察期。超过5分钟的观察期水温开始上升，并在10分钟DO水平变得太不稳定维护。该协议可扩展到研究有机体，重现性好，可靠，允许快速实施到入门教学实验室和高层次的研究应用。这种技术的预期的结果应与溶解氧改变生物体的行为反应。

<html>所描述的设置六个试验是由24新生本科生教学实验室环境进行量化石蝇响应不同的溶解氧浓度的水进行俯卧撑的数量。俯卧撑的DO水平内，每个试验中进行的平均数为汇集暗算溶解氧水平俯卧撑如图2所示 。方差分析进行最初利用溶解氧浓度，试验的顺序，温度，以及所有的变量之间的相互作用。结果显示，只有做浓度显著影响由石蝇进行俯卧撑的数量（R ...

Watch this Scientific Journal Video about A Simple Approach to Manipulate Dissolved Oxygen for Animal Behavior Observations at JoVE.com

A Simple Approach to Manipulate Dissolved Oxygen for Animal Behavior Observations

本文介绍了一个简单的和可重复的协议来操作溶氧条件在实验室环境对动物行为的研究。该协议可能在教学和研究实验室的设置可用于评估在溶解氧浓度的变化无脊椎动物，鱼类，两栖类或生物体的反应。

一个简单的方法来操作的动物行为观察溶解氧

The ability to manipulate dissolved oxygen (DO) in a laboratory setting has significant application to investigate a number of ecological and organismal ...

a-simple-approach-to-manipulate-dissolved-oxygen-for-animal-behavior

Universidad San Sebastian

Research

JoVE Journal

Environment

9.2K 次观看.  Juniata College. 本文介绍了一个简单的和可重复的协议来操作溶氧条件在实验室环境对动物行为的研究。该协议可能在教学和研究实验室的设置可用于评估在溶解氧浓度的变化无脊椎动物，鱼类，两栖类或生物体的反应。 

视频: A Simple Approach to Manipulate Dissolved Oxygen for Animal Behavior Observations

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

Ø的底栖交易所<sub&gt; 2</sub&gt;，N<sub&gt; 2</sub&gt;和营养盐使用小的核心孵化

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

科研

环境科学

A Strain Gauge Monitor (SGM) for Continuous Valve Gape Measurements in Bivalve Molluscs in Response to Laboratory Induced Diel-cycling Hypoxia and pH

An inexpensive, laboratory-based, strain gauge valve gape monitor (SGM) was developed to monitor the valve gape behavior of bivalve molluscs in response to diel-cycling hypoxia. A Wheatstone bridge was connected to strain gauges that were attached to the shells of oysters (Crassostrea virginica). The recorded signals allowed for the opening and closing of the bivalves to be recorded continuously over two-day periods of experimentally-induced diel-cycling hypoxia and diel-cycling changes in pH. Here, we describe a protocol for developing an inexpensive strain gauge monitor and describe, in an example laboratory experiment, how we used it to measure the valve gape behavior of Eastern oysters (C. virginica), in response to diel-cycling hypoxia and cyclical changes in pH. Valve gape was measured on oysters subjected to cyclical severe hypoxic (0.6 mg/L) dissolved oxygen conditions with and without cyclical changes in pH, cyclical mild hypoxic (1.7 mg/L) conditions and normoxic (7.3 mg/L) conditions. We demonstrate that when oysters encounter repeated diel cycles, they rapidly close their shells in response to severe hypoxia and close with a time lag to mild hypoxia. When normoxia is restored, they rapidly open again. Oysters did not respond to cyclical pH conditions superimposed on diel cycling severe hypoxia. At reduced oxygen conditions, more than one third of the oysters closed simultaneously. We demonstrate that oysters respond to diel-cycling hypoxia, which must be considered when assessing the behavior of bivalves to dissolved oxygen. The valve SGM can be used to assess responses of bivalve molluscs to changes in dissolved oxygen or contaminants. Sealing techniques to better seal the valve gape strain gauges from sea water need further improvement to increase the longevity of the sensors.

A Strain Gauge Monitor SGM for Continuous Valve Gape Measurements in Bivalve Molluscs in Response to Laboratory Induced Diel-cycling Hypoxia and pH

Understanding the behavioral responses of bivalve suspension-feeders to environmental variables, such as dissolved oxygen, can explain some ecosystem processes. We have developed an inexpensive, laboratory-based, strain gauge monitor (SGM) to measure valve gape responses of oysters, Crassostrea virginica, to diel-cycling hypoxia and cyclical pH.

基于实验室诱导的昼夜循环缺氧和 pH 值的双壳软体动物连续气门目瞪口呆测量用应变计监测仪 (通用)

An inexpensive, laboratory-based, strain gauge valve gape monitor (SGM) was developed to monitor the valve gape behavior of bivalve molluscs in response ...

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and KATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

氧微环境中的单个胰岛水平的定量和临时控制

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological ...

生物工程

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

生物化学

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

发育生物学

A Low-Cost Method of Measuring the In Situ Primary Productivity of Periphyton Communities of Lentic Waters

Measuring the in situ primary productivity of periphyton during the growing season gradient can elucidate the quantitative effect of environmental drivers (mainly phosphorus concentration and light intensity) and species composition on primary productivity. Primary productivity is mainly driven by light intensity, temperature, availability of nutrients, and distribution of the ionic species of the carbonate system in the respective depths of the euphotic zone. It is a complex system that is very difficult to simulate in the laboratory. This cheap, transportable, and easy-to-build floating barge allows measuring the primary productivity accurately-directly under the actual natural conditions. The methodology is based on measuring the primary productivity in real time using noninvasive oxygen sensors integrated into tightly sealed glass jars, enabling online oxygen flux monitoring and providing new insights into metabolic activities. Detailed seasonal in situ measurements of gross primary productivity of microbial mats (or other benthic organisms) can improve current knowledge of the processes controlling primary productivity dynamics in lentic waters.

A Low-Cost Method of Measuring the In Situ Primary Productivity of Periphyton Communities of Lentic Waters

Presented here is a cost-effective and transportable method/facility for measuring the primary productivity of microbial mats under actual in situ environmental temperature and light conditions. The experimental setup is based on widely available materials and can be used under various conditions while offering the advantages of laboratory-based models.

一种低成本的透镜水周边植物群落原 位初级生产力 测量方法

Measuring the in situ primary productivity of periphyton during the growing season gradient can elucidate the quantitative effect of environmental drivers ...

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, Drosophila research lacks popularity in developing countries, possibly due to the misinformed idea that establishing a lab and performing relevant experiments with such tiny insects is difficult and requires expensive, specialized apparatuses. Here, we describe how to build an affordable flylab to quantitatively analyze a myriad of behavioral parameters in D. melanogaster, by 3D-printing many of the necessary pieces of equipment. We provide protocols to build in-house vial racks, courtship arenas, apparatuses for locomotor assays, etc., to be used for general fly maintenance and to perform behavioral experiments using adult flies and larvae. We also provide protocols on how to use more sophisticated systems, such as a high resolution oxygraph, to measure mitochondrial oxygen consumption in larval samples, and show its association with behavioral changes in the larvae upon the xenotopic expression of the mitochondrial alternative oxidase (AOX). AOX increases larval activity and mitochondrial leak respiration, and accelerates development at low temperatures, which is consistent with a thermogenic role for the enzyme. We hope these protocols will inspire researchers, especially from developing countries, to use Drosophila to easily combine behavior and mitochondrial metabolism data, which may lead to information on genes and/or environmental conditions that may also regulate human physiology and disease states.

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

We provide protocols for anyone with a "maker culture" mind to start building a flylab for quantitative analysis of a myriad of behavioral parameters in Drosophila melanogaster, by 3D-printing many of the necessary pieces of equipment. We also describe a high resolution respirometry protocol using larvae to combine behavioral and mitochondrial metabolism data.

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, ...

行为学

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

创建定义的气体环境研究缺氧对<em&gt; C。线虫</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

生物学

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

使用时间的推移显微镜可视化缺氧诱导假死<em&gt; C.线虫</em&gt;胚胎

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

适用于 果蝇 的微量呼吸测定法 - 深入了解 CASK 突变体代谢

Measuring O2 Consumption in Drosophila melanogaster Using Coulometric Microrespirometry

Coulometric microrespirometry is a straightforward, inexpensive method for measuring the O2 consumption of small organisms while maintaining a stable environment. A coulometric microrespirometer consists of an airtight chamber in which O2 is consumed and the CO2 produced by the organism is removed by an absorbent medium. The resulting pressure decrease triggers electrolytic O2 production, and the amount of O2 produced is measured by recording the amount of charge used to generate it. In the present study, the method has been adapted to Drosophila melanogaster tested in small groups, with the sensitivity of the apparatus and the environmental conditions optimized for high stability. The amount of O2 consumed by wildtype flies in this apparatus is consistent with that measured by previous studies. Mass-specific O2 consumption by CASK mutants, which are smaller and known to be less active, was not different from congenic controls. However, the small size of CASK mutants resulted in a significant reduction in O2 consumption on a per-fly basis. Therefore, the microrespirometer is capable of measuring O2 consumption in D. melanogaster, can distinguish modest differences between genotypes, and adds a versatile tool for measuring metabolic rates.

作者聚焦:温度对黑腹果蝇和沙漠适应性甲虫的影响 

Coulometric respirometry is ideal for measuring the metabolic rate of small organisms. When adapted for Drosophila melanogaster in the present study, measured O2 consumption was within the range reported for wildtype D. melanogaster by previous studies. Per-fly O2 consumption by CASK mutants, which are smaller and less active, was significantly lower than the wildtype.

使用库仑显微呼吸法测量黑腹果蝇的 O2 消耗量

Coulometric microrespirometry is a straightforward, inexpensive method for measuring the O2 consumption of small organisms while maintaining a stable ...

一个简单的方法来操作的动物行为观察溶解氧

摘要

探索更多视频

此视频中的章节

Ø的底栖交易所

基于实验室诱导的昼夜循环缺氧和 pH 值的双壳软体动物连续气门目瞪口呆测量用应变计监测仪 (通用)

氧微环境中的单个胰岛水平的定量和临时控制

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

诱导生活青蛙和斑马鱼胚胎缺氧

一种低成本的透镜水周边植物群落原位初级生产力测量方法

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

创建定义的气体环境研究缺氧对

使用时间的推移显微镜可视化缺氧诱导假死

使用库仑显微呼吸法测量黑腹果蝇的 O₂ 消耗量

一个简单的方法来操作的动物行为观察溶解氧

摘要

探索更多视频

此视频中的章节

Ø的底栖交易所

基于实验室诱导的昼夜循环缺氧和 pH 值的双壳软体动物连续气门目瞪口呆测量用应变计监测仪 (通用)

氧微环境中的单个胰岛水平的定量和临时控制

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

诱导生活青蛙和斑马鱼胚胎缺氧

一种低成本的透镜水周边植物群落原 位初级生产力 测量方法

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

创建定义的气体环境研究缺氧对

使用时间的推移显微镜可视化缺氧诱导假死

使用库仑显微呼吸法测量黑腹果蝇的 O2 消耗量

一种低成本的透镜水周边植物群落原位初级生产力测量方法

使用库仑显微呼吸法测量黑腹果蝇的 O₂ 消耗量