我们感谢 Caenorhabditis 遗传学中心提供本研究中使用的菌株。本出版物得到了 JSPS KAKENHI 科学研究补助金 （B）（资助号 JP21H02532）、JSPS KAKENHI 创新领域“软机器人科学”项目补助金（资助号JP18H05474）、JSPS KAKENHI 变革性研究领域 B 补助金（资助号JP23H03845）、日本医学研究开发机构 PRIME（资助号 JP22gm6110022h9904）、JST-Mirai 计划（资助号 JPMJMI22G3）、 和 JST-FOREST 计划（批准号 JPMJFR214R）。

1.蠕虫的制备
注：制备野生型N2布里斯托尔菌株12 和ZX899菌株（lite-1（ce314）;ljIs123[mec-4p：：ChR2，unc-122p：：RFP]）13 分别用于观察集体行为和光遗传学实验。在黑暗条件下保持 ZX899 菌株。
<ol><li>将四条喂养良好的成虫沉积在含有14mL线虫生长培养基（NGM）的60mm板上，并接种大 肠杆菌 OP5012。</li>
	<li>在23°C的NGM板中将F1蠕虫培养至饥饿7天。此时，F1 蜗杆的产量约为 100 只蜗杆/板。蠕虫的各个阶段包括 dauer 和饥饿的 L1 幼虫的混合种群。</li>
</ol>2.狗粮琼脂（DFA）培养基板的制备
<ol><li>高压灭菌含有2g狗粮粉和5mL 1%琼脂培养基的玻璃瓶，并将其冷却至室温（图1A）。 注意：本实验可以使用来自不同制造商的其他狗粮。</li>
</ol>3. 将蠕虫接种到 DFA 培养基板上
<ol><li>将少量（约0.5g）DFA培养基转移到接种有 大肠杆 菌OP50的NGM板的中心（图1B）。对于光遗传学实验，在接种蠕虫之前，将 40 μL 50 μM 全反式视网膜醛（通道视紫红质 2 的辅助因子）倒入 DFA 上。</li>
	<li>使用高压灭菌水从四个NGM板中收集饥饿的蠕虫。</li>
	<li>将一小块狗粮（约0.5g）放在DFA培养基上，距离板盖约2mm。</li>
	<li>在洁净的工作台内用紫外线照射 NGM 板 15 分钟，以防止污染。</li>
	<li>将收集的蠕虫（约400条蠕虫）接种到NGM板上的DFA培养基上。不要用封口膜密封板，以免增加培养皿内的湿度并产生水滴，将蠕虫困在盖子上。</li>
	<li>在23°C下繁殖蠕虫，并让它们爬到板盖上约10-14天。 注意：由于盖子上的蠕虫数量在 10-14 天后几乎没有增加，因此推测蠕虫可能已经耗尽了食物。</li>
</ol>4. 集体行为的观察
<ol><li>在实验当天，将不包括 大肠杆菌 和狗粮琼脂培养基的新NGM板放在微距变焦显微镜载物台上的铝板上（图2A）。使用Peltier温度控制器将这种新的NGM板的底部保持在23°C至少5分钟（图2B）。然后，将这个新的NGM板的盖子换成蠕虫爬上去的板盖。使用物镜（x2，NA = 0.5）作为低倍率物镜（图2A）。</li>
	<li>将培养皿底部的温度从23°C提高到26°C，以改变该板内的湿度（图2）。</li>
	<li>使用相机以 20 帧 s-1 采集板盖内表面的图像（补充视频 S1）。</li>
	<li>将采集的图像保存为标记图像文件格式。</li>
</ol>5. 光遗传学实验
<ol><li>使用 100 W 汞灯提供蓝光，并用滤光片组过滤。使用电磁快门系统精确控制照明时间（图2B）。</li>
	<li>在这些条件下将 ZX899 在 DFA 上保持 5 分钟，然后再亮起蓝光。</li>
	<li>在保持在23°C的显微镜载物台上照亮附着在培养皿盖上的ZX899蠕虫。</li>
	<li>使用相机以 20 帧 s-1 采集板盖内表面的图像（补充视频 S2）。</li>
</ol>

使用狗粮琼脂培养基研究线虫的集体行为

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作者没有利益冲突需要声明。

在这项研究中，我们展示了一种在实验室中为秀丽隐杆线虫的大规模集体行为准备系统的方案。基于DFA的方法最初是用Caenorhabditis japonica14和Neoaplectana carpocapsae Weiser15建立的，两者都是非模式动物。然而，这种方法并未应用于调查集体行为。秀丽隐杆线虫是一种遗传上可处理的模式动物11,12。使用秀丽隐杆线虫的行为遗传学研究有助于个体水平行为研究的调查。然而，在秀丽隐杆线虫研究的悠久历史中，尽管观察到简单的团块模式16,17,18,19，但没有报告证明通过秀丽隐杆线虫的群体水平行为形成动态模式。本研究的一个关键思想是使用DFA培养基促进在培养皿中长期维持大量蠕虫。使用DFA介质，我们提出了秀丽隐杆线虫对动态集体行为的观察，从而引入了一种新的行为范式。
此前，已经报道了几种大规模蠕虫生产方法。与这些方法相比，该方法的优点是可以在没有分离 dauer 蠕虫的过程的情况下调查盖子上的集体行为。最近，我们发表了一篇论文，报告了使用与盖子 20 的静电相互作用在盖子和 DFA 介质之间的间隙中转移 nictating dauer 蠕虫。当蠕虫形成由大约 100 个蠕虫组成的 nictation 柱时，就会发生这种蠕虫转移。这项研究表明，当 DFA 中过度拥挤诱导 dauer 形成时，只有 dauer 蠕虫才能转移。这种方法产生的蠕虫数量可能少于其他方法，例如基于蛋黄的方法。然而，为了在盖子上进行行为测定，我们可以使用dauer蠕虫的种群，其中几乎不包括其他阶段的蠕虫，如饥饿的L1幼虫，而以前的方法需要分离dauer蠕虫的过程。因此，这种方法允许使用 dauer 蠕虫进行更精确的集体行为检查。此外，实验者还可以在以下程序中控制蠕虫的密度。首先，使用高压灭菌水收集和清洗移动到盖子上的蠕虫。然后，通过对蠕虫悬浮液等分试样中的蠕虫进行计数来测定水中蠕虫的浓度，并将蠕虫悬浮液滴到基质上。综上所述，我们的系统在蠕虫阶段和行为实验密度方面更具可控性。
从活性物质物理学的角度分析了集体行为，该物理学试图确定对有生命和非有生命的自走粒子的集体运动的统一描述。为了实现这一目标，已经为无生命的自走式粒子和细胞开发了许多实验系统，但为多细胞生物开发的系统较少，这些生物表现出基于神经回路的更复杂的行为。因此，我们的系统扩展了集体运动的统一描述存在的可能性。关于湿度操纵，我们之前基于模型的数值模拟表明，蚯蚓之间的吸引力（可能是由实验中的湿度引起的）诱发了模式变化，这与湿度引起的模式变化在质量上是一致的10。然而，我们认为没有确定性的实验证据表明模式变化是由湿度而不是温度引起的。因此，实验者应谨慎对待集体行为变化是否可以仅归因于湿度变化而不是温度变化。
了解动物集体行为背后的神经机制是生物学领域的一项新挑战。集体行为导致了一种不出现在个人层面的新功能的出现。由于动物具有神经系统，因此它们具有记忆和学习能力，因此在个体和群体水平上研究这些神经功能的差异很有趣。已经注意到，集体行为提高了对外来生物和猎物的检测灵敏度，并增强了正确决策的能力21,22,23。秀丽隐杆线虫还具有由302个神经元组成的神经系统，因此可以记住过去的培养温度24，并迁移到湿度较高的位置25。因此，研究秀丽隐杆线虫的神经功能与集体行为之间的关系将是有趣的。此外，人们可以期望通过观察蠕虫种群的行为来提取机械参数。例如，观察秀丽隐杆线虫群体的粘弹性特性可以估计单个蠕虫的弹性和蠕虫之间的表面张力。蜗虫团块的尺寸分布应与它们之间的表面张力有关。秀丽隐杆线虫个体的推进力也可以通过蠕虫响应表面张力而向外移动的频率来估计。因此，我们可以期望仅根据蠕虫种群的宏观信息来估计单个蠕虫水平的机械参数。
总之，主动物质物理学旨在识别集体行为的统一描述，该领域需要更多的实验验证所提出的数学模型通过控制参数。此外，每种动物集体模式形成的功能意义及其与神经功能的机械相关性是重要的开放性问题。此外，鉴于“软机器人”的目标之一是精确控制机器人的集体，我们希望通过蠕虫集体行为的实验建立一种算法，以应用于控制软机器人的集体运动。

非科学家和科学家都对动物的集体行为着迷，比如成群的鸟和鱼群。集体行为已经在广泛的领域进行了分析，包括物理学、生物学、数学和机器人技术。特别是，活性物质物理学是一个不断发展的研究领域，专注于由自走式元素组成的系统，即耗散系统，如鸟群、鱼群、运动细菌的生物膜、活性分子组成的细胞骨架和自走胶体组。活性物质物理学理论认为，无论个体的行为多么复杂，大量生物的集体运动都受到少数简单规则的支配。例如，Vicsek模型是统一描述自走式粒子集体运动的候选者，它预测运动物体的短程对准相互作用需要形成具有二维偏心波动的长程有序相位，就像在动物群中一样1。与活性物质物理学有关的自上而下的实验方法正在迅速发展。先前的实验证实了大肠杆菌2中长程有序相的形成。最近的其他工作使用了细胞3,4，细菌5，运动胶体6或移动蛋白质7,8。简单的最小模型（如Vicsek模型）成功地描述了这些真实现象。与这些单细胞实验系统相比，动物的集体行为通常是在野外观察到的，因为没有人希望对10,000只真正的鸟类或鱼类进行对照实验。
生物学家与物理学家有着相同的兴趣：个体如何相互影响，如何作为一个群体在功能上表现。分析个体行为的传统研究领域之一是神经科学，其中行为背后的机制已经在神经元和分子水平上进行了研究。到目前为止，已经开发了许多神经科学自下而上的方法。物理学中的自上而下方法和生物学中的自下而上方法可以使用模式动物（如果蝇、 蠕虫秀丽隐杆线虫和小鼠）来促进 9.然而，在实验室中，关于这些模式动物的大规模集体行为的发现很少10;在实验室中大规模制备遗传上可处理的模式动物仍然很困难。因此，在目前生物学和物理学中对集体行为的研究中，通常在实验室进行研究的科学家很难研究动物的集体行为。
在这项研究中，我们建立了一种大规模培养线虫的方法，以研究它们的集体行为。该系统使我们能够改变环境条件，并使用突变体10 检查个体水平运动对集体行为的影响。在活性物质物理学中，数学模型的参数可以在实验和模拟中控制，从而可以验证该模型以识别统一描述。遗传学用于理解集体行为背后的神经回路机制11.

<table><tbody><tr><td>&lt;strong&gt;&lt;em&gt;Escherichia coli&lt;/em&gt; and &lt;em&gt;C. elegans&lt;/em&gt; strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli &lt;/em&gt;OP50</td><td>&lt;em&gt;Caenorhabditis &lt;/em&gt;Genetics Center</td><td>OP50</td><td>Food for &lt;em&gt;C. elegans.&lt;/em&gt; Uracil auxotroph. &lt;em&gt;E. coli&lt;/em&gt; B.</td></tr><tr><td>&lt;em&gt;lite-1(ce314); ljIs123[mec-4p::ChR2, unc-122p::RFP]&lt;/em&gt;</td><td>author</td><td>ZX899</td><td>&lt;em&gt;lite-1(ce314) &lt;/em&gt;mutant carrying the genes expressing ChR2 and RFP under the control of the &lt;em&gt;mec-4 &lt;/em&gt;and &lt;em&gt;unc-122 &lt;/em&gt;promoter, respectively</td></tr><tr><td>N2 Bristrol</td><td>&lt;em&gt;Caenorhabditis &lt;/em&gt;Genetics Center</td><td></td><td>Wild-type &lt;em&gt;C. elegans&lt;/em&gt; strain</td></tr><tr><td>&lt;strong&gt;For worm cultivation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agar purified, powder</td><td>Nakarai tesque</td><td>01162-15</td><td>For preparation of NGM plates</td></tr><tr><td>All-trans retinal</td><td>Sigma-Aldrich</td><td>R2500</td><td>For optogenetics</td></tr><tr><td>Bacto pepton</td><td>Becton Dickinson</td><td>211677</td><td>For preparation of NGM plates</td></tr><tr><td>Calcium chloride</td><td>Wako</td><td>036-00485</td><td>For preparation of NGM plates</td></tr><tr><td>Cholesterol</td><td>Wako</td><td>034-03002</td><td>For preparation of NGM plates</td></tr><tr><td>di-Photassium hydrogenphosphate</td><td>Nakarai tesque</td><td>28727-95</td><td>For preparation of NGM plates</td></tr><tr><td>Dog food</td><td>Nihon Pet Food</td><td>VITA-ONE</td><td>For preparation of dog food agar medium</td></tr><tr><td>LB broth, Lennox</td><td>Nakarai tesque</td><td>20066-95</td><td>For culture of &lt;em&gt;E. coli&lt;/em&gt; OP50</td></tr><tr><td>Magnesium sulfate anhydrous</td><td>TGI</td><td>M1890</td><td>For preparation of NGM plates</td></tr><tr><td>Petri dishes (60 mm)</td><td>Nunc</td><td>150270</td><td>For preparation of NGM plates</td></tr><tr><td>Potassium Dihydrogenphosphate</td><td>Nakarai tesque</td><td>28720-65</td><td>For preparation of NGM plates</td></tr><tr><td>Sodium Chloride</td><td>Nakarai tesque</td><td>31320-05</td><td>For preparation of NGM plates</td></tr><tr><td>&lt;strong&gt;Observation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Computer</td><td>CT solution</td><td>CS6229</td><td>Windows10 Pro with Intel Xeon Gold 6238R CPU and 768 GB of RAM</td></tr><tr><td>CMOS Camera</td><td>Hamamatsu photonics&amp;nbsp;</td><td>ORCA-Lightning C14120-20P</td><td>For data acquisition</td></tr><tr><td>CMOS Camera</td><td>Olympus</td><td>DP74</td><td>For data acquisition</td></tr><tr><td>Microscope with SZX-MGFP set</td><td>Olympus</td><td>MVX10</td><td>For data acquisition</td></tr><tr><td>x2 Objective lens</td><td>Olympus</td><td>MV PLAPO 2XC</td><td>Working distance 20 mm and numerical aperture 0.5</td></tr><tr><td>&lt;strong&gt;Shutter control&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Shutter</td><td>OptoSigma</td><td>BSH2-RIX</td><td>For controlling temporal pattern of&amp;nbsp; light illumination</td></tr><tr><td>Shutter controller</td><td>OptoSigma</td><td>SSH-C2B-A</td><td>For controlling temporal pattern of&amp;nbsp; light illumination</td></tr><tr><td>&lt;strong&gt;Temperature control&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Peltier temperature controller unit</td><td>VICS</td><td>WLVPU-30</td><td>For controlling humidity inside a Petri plate</td></tr><tr><td>UNI-THEMO CONTROLLER</td><td>Ampere</td><td>UTC-100</td><td>For controlling humidity inside a Petri plate</td></tr><tr><td>&lt;strong&gt;Data acquisition software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HCImage</td><td>Hamamatsu photonics</td><td></td><td>For data acquisition</td></tr></tbody></table>

the large-scale cultivation of nematodes to study their collective behaviors

动物表现出动态的集体行为，如在成群的鸟类、鱼群和成群的人类中观察到的那样。动物的集体行为已经在生物学和物理学领域进行了研究。在实验室中，研究人员使用各种模式动物（如果蝇和斑马鱼）大约一个世纪，但研究这些基因可处理的模式动物精心策划的大规模复杂集体行为仍然是一个重大挑战。本文提出了一种在 秀丽隐杆线虫中创建集体行为实验系统的协议。繁殖的蠕虫爬上培养皿的盖子，并表现出集体蜂拥行为。该系统还通过改变湿度和光刺激来控制蠕虫的相互作用和行为。该系统使我们能够通过改变环境条件和使用突变体检查个体水平运动对集体行为的影响来检查集体行为背后的机制。因此，该系统对物理学和生物学的未来研究都很有用。

在这里，野生型dauer蠕虫被用于集体行为观察。将蠕虫在23°C下培养约10-14天，并爬到DFA培养基板盖的内表面。在实验当天，仅将盖子转移到没有 大肠杆菌 和DFA培养基的新NGM板上。该培养皿的底部最初使用帕尔贴系统保持在23°C，然后将其温度提高到26°C。 在显微镜下拍摄了一部电影。 图 3 显示了影片的快照。蠕虫在湿度变化期间动态重塑了它们的网络模式。随着湿度的增加，网络的隔间尺寸也变得更大。最后，网络崩溃，休眠的蠕虫簇留在盖子的内表面。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65569/65569fig01.jpg"> 图1：用于培养大量蠕虫的DFA培养基的照片。 （A）在玻璃瓶中制备的DFA培养基的照片。（B）接种收集的蠕虫后，NGM板与DFA培养基的照片。缩写：DFA = 狗粮琼脂;NGM = 线虫生长培养基。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65569/65569fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65569/65569fig02.jpg"> 图2：用于观察集体行为的实验系统。 （A）用于观察集体行为的显微镜。（B） 使用帕尔帖系统的机械快门控制器和温度控制系统。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65569/65569fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65569/65569fig03.jpg"> 图3：集体网络模式对湿度依赖性的代表性数据。秀丽隐杆线虫网络对环境湿度的依赖性。相机帧速率为 1 fps。比例尺 = 4 毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65569/65569fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
补充视频S1：集体网络形成。 在培养皿中使用DFA在NGM上繁殖野生型dauer蠕虫。蠕虫在盖子内自组织。使用帕尔帖设备改变湿度。图像是从盖子上方拍摄的。影片播放速度比实时录制速度快 80 倍。缩写：DFA = 狗粮琼脂;NGM = 线虫生长培养基。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65569/20181213_147crop_80fps.mp4" target="_blank">请点击此处下载此文件。</a>
补充视频S2：蠕虫集体的光遗传学操作。 在 1、2、4、8、32 和 128 s 蓝光照明下进行光遗传学。这种激活最初导致了树束的树化和塌陷。最后，形成了一个不同于初始结构的网络。电影的播放速度比实时录制速度快 20 倍。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65569/20190111_180_20fps.mp4" target="_blank">请点击此处下载此文件。</a>

Watch this Scientific Journal Video about The Large-Scale Cultivation of Nematodes to Study Their Collective Behaviors at JoVE.com

作者聚焦：线虫 Caenorhabditis elegans 的集体行为分析

在这里，报道了一种通过使用狗粮琼脂培养基大量培养线虫来研究线虫集体行为的系统。该系统允许研究人员繁殖大量的dauer蠕虫，并可应用于 秀丽隐杆线虫 和其他相关物种。

大规模培养线虫以研究其集体行为

Animals exhibit dynamic collective behaviors, as observed in flocks of birds, schools of fish, and crowds of humans. The collective behaviors of animals ...

我们正在从活跃的形而上学和神经科学的角度研究集体行为背后的机制。在集体行为的研究领域，已经建立了使用非生物和细胞水平的实验系统。然而，设计动物水平的实验系统来研究实验室中的集体行为仍然具有挑战性。我们成功地用传统使用的模式动物线虫秀丽隐杆线虫重建了集体行为。该系统可以利用动物集体行为的数学模型控制参数，并利用分子遗传学研究集体行为背后的神经机制。

the-large-scale-cultivation-nematodes-to-study-their-collective

Nanyang Technological University

Research

JoVE Journal

Biology

The Large-Scale Cultivation of Nematodes to Study Their Collective Behaviors

620 次观看.  Hiroshima University. 我们正在从活跃的形而上学和神经科学的角度研究集体行为背后的机制。在集体行为的研究领域，已经建立了使用非生物和细胞水平的实验系统。然而，设计动物水平的实验系统来研究实验室中的集体行为仍然具有挑战性。我们成功地用传统使用的模式动物线虫秀丽隐杆线虫重建了集体行为。该系统可以利用动物集体行为的数学模型控制参数，并利用分子遗传学研究集?...

视频: 作者聚焦：线虫 Caenorhabditis elegans 的集体行为分析

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

大规模混合阶段 卡诺哈布迪蒂斯电子 人种群的文化和分析

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...

科研

遗传学

Rapid Isolation of Wild Nematodes by Baermann Funnel

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild nematodes in their natural environments are valuable for understanding many aspects of biology, including the selective regimes in which distinctive genomic and phenotypic characters evolve, the genetic basis for complex trait variation, and the natural genetic diversity fundamental to all animal populations. This manuscript describes a simple and efficient method for extracting nematodes from their natural substrates, including rotting fruits, flowers, fungi, leaf litter, and soil. The Baermann funnel method, a classical nematology technique, selectively isolates active nematodes from their substrates. Because it recovers nearly all active worms from the sample, the Baermann funnel technique allows for the recovery of rare and slow-growing genotypes that co-occur with abundant and fast-growing genotypes, which might be missed in extraction methods that involve multiple generations of reproduction. The technique is also well suited to addressing metagenetic, population-genetic, and ecological questions. It captures the entire population in a sample simultaneously, allowing an unbiased view of the natural distribution of ages, sexes, and genotypes. The protocol allows for deployment at scale in the field, rapidly converting substrates into worm plates, and the authors have validated it through fieldwork on multiple continents.

This protocol outlines a method for efficiently extracting live nematodes from natural substrates in the field.

由Baermann Funnel快速分离野生线虫

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

利用广域跟踪平台对大型线虫种群进行自动行为分析

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

免疫与感染

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the context of the intestines. Recently, ecological sampling of wild Caenorhabditis nematodes has discovered a diverse array of associated microbes, including bacteria, viruses, fungi, and microsporidia. Many of these microbes have interesting colonization or infection phenotypes that warrant further study, but they are often unculturable. This protocol presents a method to enrich the desired intestinal microbes in C. elegans and related nematodes and reduce the presence of the many contaminating microbes adhering to the cuticle. This protocol involves forcing animals into the dauer stage of development and using a series of antibiotic and detergent washes to remove external contamination. As dauer animals have physiological changes that protect nematodes from harsh environmental conditions, any intestinal microbes will be protected from these conditions. But, for enrichment to work, the microbe of interest must be maintained when animals develop into dauers. When the animals leave the dauer stage, they are singly propagated into individual lines. F1 populations are then selected for desired microbes or infection phenotypes and against visible contamination. These methods will allow researchers to enrich unculturable microbes in the intestinal lumen, which make up part of the natural microbiome of C. elegans and intracellular intestinal pathogens. These microbes can then be studied for colonization or infection phenotypes and their effects on the host fitness.

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

选择性清洁野生 卡诺菌线 虫以富集肠道微生物组细菌

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

栽培<em&gt;秀丽隐杆线虫</em&gt;在三个维度实验室

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

生物学

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative sparsity of information about C. elegans in its natural context comes from the challenges involved in the identification of the small nematode in nature. Despite these challenges, an increasing focus on the ecology of C. elegans has caused a wealth of new information regarding its life outside of the laboratory. The intensified search for C. elegans in nature has contributed to the discovery of many new Caenorhabditis species and revealed that congeneric nematodes frequently cohabitate in the wild, where they feed on microbial blooms associated with rotting plant material. The identification of new species has also revealed that the androdioecious mating system of males and self-fertilizing hermaphrodites has evolved three times independently within Caenorhabditis. The other two selfing species, C. briggsae and C. tropicalis, share the experimental advantages of C. elegans and have enabled comparative studies into the mechanistic basis of important traits, including self-fertilization. Despite these advances, much remains to be learned about the ecology and natural diversity of these important species. For example, we still lack functional information for many of their genes, which might only be attained through an understanding of their natural ecology. To facilitate ecological research of selfing Caenorhabditis nematodes, we developed a highly scalable method to collect nematodes from the wild. Our method makes use of mobile data collection platforms, cloud-based databases, and the R software environment to enhance researchers&#39; ability to collect nematodes from the wild, record associated ecological data, and identify wild nematodes using molecular barcodes.

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

This protocol can be used to perform large-scale ecological surveys of selfing Caenorhabditis nematodes. The primary advantage of this method is the efficient organization and analysis of ecological and molecular data associated with the nematodes collected from nature.

一种高度可扩展的方法，用于对自发 性Caenorhabditis 线虫进行生态调查

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their gut microbiomes. While experiments with well-characterized bacteria or defined bacterial communities can facilitate the analysis of molecular mechanisms, studying nematodes in their natural microbial context is essential for exploring the diversity of such mechanisms. At the same time, the isolation of worms from the wild is not always feasible, and, even when possible, sampling from the wild restricts the use of the genetic toolkit otherwise available for C. elegans research. The following protocol describes a method for microbiome studies utilizing compost microcosms for the in-lab&#160;growth in microbially diverse and natural-like environments.
Locally sourced soil can be enriched with produce to diversify the microbial communities in which worms are raised and from which they are harvested, washed, and surface-sterilized for subsequent analyses. Representative experiments demonstrate the ability to modulate the microbial community in a common soil by enriching it with different produce and further demonstrate that worms raised in these distinct environments assemble similar gut microbiomes distinct from their respective environments, supporting the notion of a species-specific core gut microbiome. Overall, compost microcosms provide natural-like in-lab environments for microbiome research as an alternative to synthetic microbial communities or to the isolation of wild nematodes.

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

Compost microcosms bring the microbial diversity found in nature into the laboratory to facilitate microbiome research in Caenorhabditis elegans. Provided here are protocols for setting up microcosm experiments, with the experiments demonstrating the ability to modulate environmental microbial diversity to explore the relationships between environmental microbial diversity and worm gut microbiome composition.

堆肥微观世界作为秀丽隐杆线虫微生物组研究的微生物多样化、类似自然的环境

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

在固体培养基上长期培养单个 秀丽隐杆线虫 ，用于纵向荧光监测和厌恶干预

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

多孔设备固体培养基上分离秀 丽隐杆线虫 的长期培养和监测

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, ...

大规模培养线虫以研究其集体行为

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大规模混合阶段卡诺哈布迪蒂斯电子人种群的文化和分析

由Baermann Funnel快速分离野生线虫

利用广域跟踪平台对大型线虫种群进行自动行为分析

选择性清洁野生卡诺菌线虫以富集肠道微生物组细菌

栽培

一种高度可扩展的方法，用于对自发性Caenorhabditis 线虫进行生态调查

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

堆肥微观世界作为秀丽隐杆线虫微生物组研究的微生物多样化、类似自然的环境

在固体培养基上长期培养单个秀丽隐杆线虫，用于纵向荧光监测和厌恶干预

多孔设备固体培养基上分离秀丽隐杆线虫的长期培养和监测

大规模培养线虫以研究其集体行为

副本

摘要

探索更多视频

此视频中的章节

大规模混合阶段 卡诺哈布迪蒂斯电子 人种群的文化和分析

由Baermann Funnel快速分离野生线虫

利用广域跟踪平台对大型线虫种群进行自动行为分析

选择性清洁野生 卡诺菌线 虫以富集肠道微生物组细菌

栽培

一种高度可扩展的方法，用于对自发 性Caenorhabditis 线虫进行生态调查

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

堆肥微观世界作为秀丽隐杆线虫微生物组研究的微生物多样化、类似自然的环境

在固体培养基上长期培养单个 秀丽隐杆线虫 ，用于纵向荧光监测和厌恶干预

多孔设备固体培养基上分离秀 丽隐杆线虫 的长期培养和监测

大规模混合阶段卡诺哈布迪蒂斯电子人种群的文化和分析

选择性清洁野生卡诺菌线虫以富集肠道微生物组细菌

一种高度可扩展的方法，用于对自发性Caenorhabditis 线虫进行生态调查

在固体培养基上长期培养单个秀丽隐杆线虫，用于纵向荧光监测和厌恶干预

多孔设备固体培养基上分离秀丽隐杆线虫的长期培养和监测