Name: high-throughput assays of critical thermal limits in insects
Uploaded: 2020-06-15T14:00:00
Description: Watch this Scientific Journal Video about High-Throughput Assays of Critical Thermal Limits in Insects at JoVE.com

我们感谢艾莉·麦卡比在饲养苍蝇方面提供的援助。这项工作得到了美国农业部国家粮食和农业研究所哈奇项目赠款1010996和国家科学基金会赠款OIA-1826689对N.M.T.的支持。

1. 高通量CT最小 测定
<ol><li>组装套护柱（图1 A，A图2）<ol>
<li>用电锯切割最宽（7 厘米 x 6.35 厘米 x 0.3 厘米）和最窄的（5.7 厘米 x 5.1 厘米 x 0.3 厘米）丙烯酸管，长度相等（31.5 厘米）（图 2A）。这两个管子将是套管柱的外侧和内侧壁。</li>
		<li>切两个环（2厘米宽）从中间（6.35厘米x5.7厘米x 0.3厘...

<ol>
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上面详述的两种方法生成高通量数据，用于最高和较低的热限的生态相关指标。这些协议以以前确立的昆虫热极限研究共同方法为基地（辛克莱等人总结）。6. 两个协议都可以在短时间内完成（每个协议±2小时），生成具有较大样本量的数据集，不牺牲重复性或准确性，并通过消除手动数据记录和输入（CTmin检测 ）或创建每次检测的备份视频记录（热 KD 测定）来最大...

昆虫的热极限 环境条件的变化，包括温度，是影响生物体1，2的性能，健身，生存和地理分布的主要因素2。上部和下热限决定了生物体能够容忍的环境的理论范围，因此，这些限制是植物和动物分布的重要预测因素，特别是在气候变化面前。,4因此，精确测量热极限的协议是生态学家、生理学家、进化生物学家和保护生物学家等的重要工具。
昆虫作为最丰富、最多样化的陆地动物，经常用于测量热极限。临界热最大值（CT最大值）和临界热最小值（CTmin）通常用于评估热公差,5、6、7,6中特异性和特异性变化。虽然CT最大值和CT最小值可以测量多种表型，包括生长，生殖输出和行为，它们最常应用于运动功能5，6，7。,6,7因此，CT最大值（也称为热敲降温度）和CTmin通常被定义为高低温，昆虫失去运动功能，无法保持直立5，6，7，8，9，10，11。6,7,8,9,10,115CTmin与冷昏迷的开始相吻合，冷温6带来的可逆瘫痪。虽然在热极限下瘫痪通常是可逆的，但持续暴露在这些温度下会导致生态死亡5。
测量热极限的常用方法 各种仪器被用来测量热极限（如辛克莱等人）6.简言之，昆虫在12、13、,浸于液体浴池中的容器12、13、11、14、15、16、,14,15,16铝块1310、17,17或带套的容器18中加热或冷却，并监测直至运动停止。在分析过程中监测昆虫，最常见的方法是直接观察，其中对个体进行实时或回顾性监测，并录下6、9、10、11、15、17。,10,11,15,176,虽然直接观测方法对设备要求最低，但都是劳动密集型的，限制了吞吐量。或者，昆虫可以通过收集个体在离散时间，因为他们,从栖息6，19，20，21,19,20或使用活动监测13间接观察。
与直接观测方法相比，测量热极限的间接方法通常比吞吐量高，而且可能不太容易出错。最常见的间接监测方法使用带衣水的温度控制列6、8、19、20、21。6,8,19,20,21昆虫被放置在带栖息的柱内，内室的温度通过从温度控制流体浴中泵送液体通过柱的套管衬里进行控制。达到其热极限的个人从栖息处下降，并在离散的温度或时间间隔内被收集。虽然这种方法适用于CT分钟，但它已被发现不适合CT最大值，因为当温度升高时，苍蝇会自愿走出柱底。此处描述的新方法通过在自动测量过程中单独包含苍蝇来规避此问题。
除了观察方法外，两种类型的温度制度通常用于评估热上限。动态测定包括逐渐升高的温度，直到运动功能丧失;温度是动态CT最大值max7，8，9，13。,8,9,13相比之下，静态测定由恒定的压力温度组成，直到运动功能丧失;时间点是热敲落时间（热KDT），也被称为静态CT最大值（sCT最大值）在最近的论文由Jürgensen等人,7，8，9，16，22。8,16,2279虽然CT最大值和热敲制测定（热KD测定）产生具有不同单位的指标，但两个特性的数学建模表明它们提供了热耐受性方面可比的信息，并且两者都与生态相关8,8，9。动态测定产生的温度可以与环境条件进行比较，当耐热性差异很大时，例如具有广泛不同热散位物种之间的比较，则更可取。然而，由于热损伤积累的Q10值很高，静态测定对于检测小效应尺寸（如热公差9的特异性变化）可能更可取。此外，实际上，静态检测需要的设备不如动态检测。
目的 本文的目的是正式确定CT最小 和热KD测定的方法，可用于未来的研究，以评估动虫的热极限。这些协议根据以前建立的方法进行改编，设计为高吞吐量、自动化且具有成本效益。这两项测定都可以在短时间内完成（±2小时），这意味着可以在一天中进行多个实验，在不牺牲可重复性或准确性的情况下产生大量数据。通过此设置，可以同时测量 96 只苍蝇的耐热性，而 CTmin 的柱 可以容纳 100 多只苍蝇，前提是有足够的表面积进行栖息。
用于观察 CT min 的高通量方法 通过添加红外传感器来自动计数苍蝇，修改了常见的套护柱方法。Shuman等人于1996年23 日首次提议使用红外传感器进行计数，但一直未得到广泛采用。红外传感器的添加允许生成连续数据，而不是以离散的时间间隔收集数据。该协议还通过消除手动数据输入和在离散时间点手动切换千斤列下方的收集管，最大限度地减少了实验者错误。
从之前对昆虫10、12的耐热性两项研究中，对记录热KDT的高通量方法进行了修改。单个苍蝇储存在温度控制培养箱中的 96 井板中，并录制视频。该协议最大限度地减少了实验者在确定热 KDT 时的偏差，因为可以通过播放录制来审查和验证实验。该协议还提供一组自定义 Python 脚本，可用于加快视频分析速度。使用单个井消除了当其他个体移动或摔倒时可能发生的干扰，当在同一竞技场10、17中观察到个体群体时，可能会造成问题。此外，温度控制培养箱在所有96孔中提供稳定的温度，这与有时在温度控制铝块10上观察到的温度梯度不同。另请注意，96 井记录方法可以调整为测量动态 CT最大值和潜在的 CT最小值（请参阅讨论）。
为了证明每个方案，比较了来自 果蝇 黑色素杆菌遗传参考 小组 （DGRP）选择线的成年果蝇黑色素杆菌女性的热极限。之所以选择这些线，是因为初步实验表明热公差存在显著差异。这些测定证明是区分热公差的强健方法。以下两种协议，高通 量CT最小测定（第1节）和高通量热KD测定（第2节），描述了为能够适应 任何可安装于仪器（如成人果蝇）的昆虫生命阶段生成CT最小值和热KDT数据 的必要操作。对于CT 分钟，昆虫能够栖息也是必不可少的。在这里，每个测定都证明在 成人果蝇黑色素。但是，对于其他分类或生命阶段6可能需要修改。细微的变化可能包括使用具有较大开口的栖息材料，以容纳 CTmin 测定 中更大的样本，或使用更高质量的摄像机来识别慢移动昆虫的细微 KDT 或热 KD 测定中的生命阶段。该协议没有描述准备苍蝇的方法，但重要的是要标准化饲养协议，以确保可重复性25（ 见加西亚和蒂茨26和 Teets和Han27）。提供的协议包括有关如何构建和设置设备、如何记录测量以及数据分析的简要说明的信息。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ARCTIC A40 Refrigerated fluid circulator (Programable teperature ramps)</td>
			<td>Thermo Scientific; Waltham, MA</td>
			<td>153-5401</td>
			<td></td>
		</tr>
		<tr>
			<td>C922 Pro Stream Webcam</td>
			<td>Logitech; Newark, CA</td>
			<td>960-001087</td>
			<td></td>
		</tr>
		<tr>
			<td>Circular adjustable steel clamp &#8211; 5.08 cm to 7.62 cm</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear acrylic tubing &#8211; 5.7 cm x 5.1 cm x 0.3 cm</td>
			<td>United States Plastic Corp., OH</td>
			<td>44036</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear acrylic tubing &#8211; 6.35 cm x 5.7 cm x 0.3 cm</td>
			<td>United States Plastic Corp., OH</td>
			<td>440515</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear acrylic tubing &#8211; 7 cm x 6.35 cm x 0.3 cm</td>
			<td>United States Plastic Corp., OH</td>
			<td>44041</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear silicone sealant</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Collection tube (15 ml)</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Cordless Drill</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila Funnel Monitor (DFM)</td>
			<td>TriKinetics; Waltham, MA</td>
			<td>DFM</td>
			<td>Used to count the number of flies that fall through the funnel at a given time point</td>
		</tr>
		<tr>
			<td>DAM data collection software</td>
			<td>TriKinetics; Waltham, MA</td>
			<td></td>
			<td>Records data input from the DFM</td>
		</tr>
		<tr>
			<td>Fly Storage Lid</td>
			<td>FlySorter; Seatle, WA</td>
			<td>FS-96LID-5PK</td>
			<td>Used to load flies into the storage plate for the sCTmax assay</td>
		</tr>
		<tr>
			<td>Fly Storage Plate</td>
			<td>FlySorter; Seatle, WA</td>
			<td>FS-96PLATE-5PK</td>
			<td>Used to hold flies during in the sCTmax assay</td>
		</tr>
		<tr>
			<td>Fly Food Tray</td>
			<td>FlySorter; Seatle, WA</td>
			<td>FS-TRAY-5PK</td>
			<td>Used to keep flies on food after loading into the 96-well plate until the sCTmax assay</td>
		</tr>
		<tr>
			<td>Glass funnel</td>
			<td>Kimax</td>
			<td>28950-75</td>
			<td>75mm</td>
		</tr>
		<tr>
			<td>Gutter guard</td>
			<td>Any</td>
			<td>Any</td>
			<td>~0.5 cm diameter openings</td>
		</tr>
		<tr>
			<td>Hacksaw</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Heratherm Thermo Scientific incubator</td>
			<td>Thermo Scientific; Waltham, MA</td>
			<td>OMS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Hose nylon adapters (2) &#8211; &#188; MNPT x 3/8</td>
			<td>United States Plastic Corp., OH</td>
			<td>61135</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot glue gun and glue</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Source</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnets</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>OMEGA TC-08 Recorder and TC-08 Player Software</td>
			<td>OMEGA; Norwalk, CT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OMEGA thermocouple (Type T)</td>
			<td>OMEGA; Norwalk, CT</td>
			<td>5LRTC-TT-K-20-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic funnel</td>
			<td>Any</td>
			<td>Any</td>
			<td>2&#34; diameter</td>
		</tr>
		<tr>
			<td>Plastic tubing - 0.6 cm diameter</td>
			<td>United States Plastic Corp., OH</td>
			<td>62852</td>
			<td></td>
		</tr>
		<tr>
			<td>Retort ring</td>
			<td>Any</td>
			<td>Any</td>
			<td>2&#34; diameter</td>
		</tr>
		<tr>
			<td>Retort stand</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Retort three-prong clamp</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Rstudio</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serial port connector (PSIU9)</td>
			<td>TriKinetics; Waltham, MA</td>
			<td>PSIU9</td>
			<td>Intermediate connection between the DFM and computer, allows for multiple DFM connections</td>
		</tr>
		<tr>
			<td>Styrofoam (2&#34; thick)</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape</td>
			<td>Any</td>
			<td>Any</td>
			<td></td>
		</tr>
		<tr>
			<td>Uninterrupted Power Supply (PS9-1)</td>
			<td>TriKinetics; Waltham, MA</td>
			<td>PS9-1</td>
			<td>Power supply for the DFM and PSIU9</td>
		</tr>
		<tr>
			<td>Weld-on #4 Acrylic Cement</td>
			<td>United States Plastic Corp., OH</td>
			<td>45737</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput assays of critical thermal limits in insects

植物和动物的上下热限是预测其性能、生存和地理分布的重要指标，对于预测对气候变化的反应至关重要。这项工作描述了测量昆虫热极限的两种高通量协议：一种用于评估临界热最小值（CTmin），另一种用于评估热敲落时间（KDT）以响应静态热能器。在 CTmin 测定中，个体被放置在丙烯酸套柱中，受到温度下降的斜坡的影响，并使用红外传感器从栖息体上下降时进行计数。在热 KDT 测定中，个体被包含在 96 井板中，放置在培养箱中，温度压力大，并录制视频，以确定他们不能再直立和移动的时间。与常用技术相比，这些协议具有优势。这两种检测成本都很低，可以较快完成（+2小时）。CT最小 测定可减少实验者误差，并可同时测量大量个体。热 KDT 协议生成每个检测的视频记录，从而消除实验者偏差和实时持续监控个体的需要。

测量了来自嗜血杆菌黑色素酯遗传参考面板（DGRP）的女性的热极限（即CT min和热KDT），以证明从所述两种协议生成的高通量数据。使用min DGRP 行 714 （n = 37） 和 913 （n = 45） 测定 CT 分钟。对热 KDT 进行了检测，并与 DGRP 行 189（n = 42）和 461（n = 42）进行比较，并手动分析视频文件。实验的总时间（包括观看视频）每个协议都花了 <2 小时。
<p class=...

Watch this Scientific Journal Video about High-Throughput Assays of Critical Thermal Limits in Insects at JoVE.com

High-Throughput Assays of Critical Thermal Limits in Insects

热极限可以预测生物体所容忍的环境，这是面对快速气候变化的宝贵信息。此处描述的高通量协议用于评估昆虫的关键热最小和热敲取时间。两种协议都最大限度地提高了吞吐量，并最大限度地降低测定的成本。

昆虫关键热极限的高通量测定

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are ...

热耐受性是物种分布和应对气候变化的关键组成部分。在这里，我们提供快速评估小昆虫耐热和耐寒性的方法。这些通过临界热最小值和热耐受性测量冷耐受性的方法，通过热敲落时间测量是高吞吐量、半自动化的，并且需要最少的设备。演示这个程序的将是大卫·奥德，一个来自我实验室的博士后。要设置关键的热最小检测，请打开温度控制液浴电源，然后按下播放按钮运行程序，将浴池温度升高并保持在 25 摄氏度。等待 5 到 10 分钟，使流体浴室柱达到 25 摄氏度，然后用直径 5.08 厘米的过滤器更换柱顶的插头。通过漏斗将大约 40 只苍蝇从食物小瓶中插入柱子，然后快速用插头更换漏斗，注意不要让任何苍蝇逃脱。然后让苍蝇安顿下来。偶尔敲打底塞鼓励苍蝇爬。五分钟后，按流体浴增益上的启动按钮，开始关键的热最小斜坡程序。单击以打开计算机上的热情侣记录软件，然后单击记录开始记录列内的温度，在检测持续时间的每一秒。确保每个温度记录都包含特定于第二个温度采样的时间戳，以便以后可以将温度数据与 Drophila 漏斗监视器的数据合并。将五毫升 90%乙醇加入 15 毫升锥形离心管中，并将管放在柱下方的机架中。偶尔点击柱子的底部插头，以吸引底部的任何苍蝇爬。大多数苍蝇将栖息在栖息处或靠近柱顶15摄氏度。在15摄氏度下，将一个75毫米的外径玻璃漏斗放入果蝇漏斗监测器中。拆下底部插头并收集乙醇插头上仍在拔下的任何苍蝇。调整反火环、漏斗监视器和漏斗，使它们在柱子下。确保漏斗的唇完全密封柱底，并将漏斗底部插入 15 毫升收集管中。打开果蝇漏斗监控软件。该软件将立即开始记录苍蝇达到其临界热最小的时间和日期。达到临界热小热量的苍蝇，将失去神经肌肉功能，并通过果蝇漏斗监测器从栖息状态下降。要监控所有苍蝇是否已达到其临界热最小值，当温度降低时，请检查顶部插头和栖息，以查看是否有苍蝇仍然栖息。当所有的苍蝇，已经达到其临界热最小，移动果蝇漏斗监测仪和漏斗远离柱开口。在极少数情况下，苍蝇可能达到其临界热最小值，但会卡在柱子中。打开顶部插头以卸下这些苍蝇。要建立高通量热敲下来测定，使用吸气器和隔膜盖，将个别二氧化碳静种苍蝇装入每个井中，一个经过改造的96井没有底板，用一个清晰的紧固盖密封板。然后将盘子放在食物供应上，让苍蝇适应96井板至少48小时。在测定当天，将孵化器设定为37.5摄氏度，30分钟后将飞培养板放入孵化器，将板的底部与托盘底部的白纸对。请注意托盘上和网络摄像头框架中井柱和行名称的方向，并沿板的两侧和边缘放置彩色胶带，以帮助验证方向。当板就位时，关闭孵化器门，然后单击视频录制软件中的记录。两小时后，检查录音，看看所有的苍蝇，是否已经到达他们最后的休息点，并停止移动。当所有苍蝇停止移动时，单击"停止"并处置苍蝇。打开视频文件，记录每只飞井的击倒时间。在试验和观察之间，最一致的测量热量是苍蝇到达其最后休息点的时间。然后反向跟踪视频，专注于单个井，并注意到苍蝇首先离开其最后休息点的时间。确定每只苍蝇的最后休息时间可能很困难。为了确保准确的分析，重要的是花时间并单独观察每个数据。在这个代表性的分析中，来自果蝇遗传参考面板线的女性比来自DGRP线714的女性具有明显较低的平均热最小温度。此外，热量在37.5摄氏度，与73和461DGRP线的女性差异显著。稍作修改，这种测量热落时间的方法可用于测量其他热耐受性特性，如冷-昏迷恢复时间或临界热最大值。通过显著减少研究人员的动手时间，这些方法使得基因变异和热耐受性特征的探索能够以以前不可能实现的规模进行。

Research

JoVE Journal

Biology

Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

测试拟南芥耐咀嚼昆虫的选择和非选择性检测

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which ...

科研

生物学

High-Throughput Measurement and Classification of Organic P in Environmental Samples

高通量测量和环境样品中的有机磷分类

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

High-throughput Yeast Plasmid Overexpression Screen

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

高通量使用自动染色体的物理图谱<em&gt;原位</em&gt;杂交

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

High Throughput Microinjections of Sea Urchin Zygotes

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

High Throughput Danio Rerio Energy Expenditure Assay

High Throughput Danio Rerio Energy Expenditure Assay

高通量<em&gt;斑马鱼</em&gt;能量消耗分析

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...