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?...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Escherichia coli and C. elegans strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli OP50</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>OP50</td>
			<td>Food for C. elegans. Uracil auxotroph. E. coli B.</td>
		</tr>
		<tr>
			<td>lite-1(ce314); ljIs123[mec-4p::ChR2, unc-122p::RFP]</td>
			<td>author</td>
			<td>ZX899</td>
			<td>lite-1(ce314) mutant carrying the genes expressing ChR2 and RFP under the control of the mec-4 and unc-122 promoter, respectively</td>
		</tr>
		<tr>
			<td>N2 Bristrol</td>
			<td>Caenorhabditis Genetics Center</td>
			<td></td>
			<td>Wild-type C. elegans strain</td>
		</tr>
		<tr>
			<td>For worm cultivation</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 E. coli 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>Observation</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&#160;</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>Shutter control</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&#160; light illumination</td>
		</tr>
		<tr>
			<td>Shutter controller</td>
			<td>OptoSigma</td>
			<td>SSH-C2B-A</td>
			<td>For controlling temporal pattern of&#160; light illumination</td>
		</tr>
		<tr>
			<td>Temperature control</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>Data acquisition software</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。

Both nonscientists and scientists are fascinated with animals&#39; collective behaviors, as in flocks of birds and schools of fish. Collective behaviors have been analyzed in a broad range of fields, including physics, biology, mathematics, and robotics. In particular, active matter physics is a growing research field that focuses on systems composed of self-propelled elements, that is, dissipative systems, such as flocks of birds, schools of fish, biofilms of motile bacteria, cytoskeletons composed of active molecules, and groups of self-propelled colloids. The theory of active matter physics maintains that however complex the behaviors of individuals are, the collective motions of enormous numbers of living things are governed by a small number of simple rules. For example, the Vicsek model, a candidate for a unified description of the collective motion of self-propelled particles, predicts that short-range alignment interaction of moving objects is required to form a long-range ordered phase with eccentric fluctuation in 2D, as in herds of animals1. Top-down experimental approaches pertaining to the physics of active matter are developing rapidly. Previous experiments confirmed the formation of a long-range ordered phase in Escherichia coli2. Other recent works&#160;employed cells3,4, bacteria5, motile colloids6, or moving proteins7,8. Simple minimal models such as the Vicsek model successfully described these real phenomena. In contrast with these unicellular experimental systems, collective behaviors by animals are usually observed in the wild, as no one could hope to perform controlled experiments with 10,000 real birds or fish.
Biologists share the same interest as physicists: how individuals interact with each other and functionally behave as a group. One of the traditional research fields for analyzing individual behavior is neuroscience, in which the mechanisms underlying behavior have been examined at the neuronal and molecular levels. Many neuroscientific bottom-up approaches have been developed thus far. Top-down approaches in physics and bottom-up approaches in biology can be facilitated using model animals such as the fruit fly, the worm Caenorhabditis elegans, and the mouse9. However, there have been few findings on the large-scale collective behavior of these model animals in the laboratory10; it is still difficult to prepare genetically tractable model animals on a large scale in the laboratory. Therefore, in current research on collective behaviors in biology and physics, it has been difficult for scientists who usually do research in the laboratory to study animals&#39; collective behaviors.
In this study, we established a method for the large-scale cultivation of nematodes to study their collective behaviors. This system allows us to change environmental conditions and examine the effect of individual-level locomotion on collective behaviors using mutants10. In active matter physics, the parameters of the mathematical model can be controlled in both experiments and simulations, which enables verification of that model for identifying unified descriptions. Genetics is used to understand the neural circuit mechanism underlying collective behavior11.

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

大规模培养线虫以研究其集体行为

We are studying the mechanism underlying collective behavior from the perspective of active metaphysics and neuroscience. In the research field of collective behavior, experimental system has been established using non-living things and at the cellular level. However, it remains still challenging to design the animal-level experimental system to study the collective behavior in the laboratory.We successfully reconstructed the collective behavior with traditionally used model animal, nematode C.elegans. This system make it possible to control the parameter with mathematical model proposed for animals'collective behavior, and studies the neuromechanism underlying collective behavior using molecular genetics.

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?...

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

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。

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

Research

JoVE Journal

Biology

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

427 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

Animals exhibit dynamic collective behaviors, as observed in flocks of birds, schools of fish, and crowds of humans. The collective behaviors of animals have been investigated in the fields of both biology and physics. In the laboratory, researchers have used various model animals such as the fruit fly and zebrafish for approximately a century, but it has remained a major challenge to study large-scale complex collective behavior orchestrated by these genetically tractable model animals. This paper presents a protocol to create an experimental system of collective behaviors in Caenorhabditis elegans. The propagated worms climb on the lid of the Petri plate and show collective swarming behavior. The system also controls worm interactions and behaviors by changing the humidity and light stimulation. This system allows us to examine the mechanisms underlying collective behaviors by changing environmental conditions and examining the effects of individual-level locomotion on collective behaviors using mutants. Thus, the system is useful for future research in both physics and biology.

Here, a system is reported for studying the collective behaviors of nematodes by culturing them in bulk using dog food agar medium. This system allows researchers to propagate large numbers of dauer worms and can be applied to Caenorhabditis elegans and other related species.

科研

生物学

Nematode Worm Culture in Dog Food Agar Plates for Behavioral Analysis

Begin by placing four adult worms on an nematode growth medium or NGM agar plate seeded with E.coli OP50. Grow the first generation worms to starvation on the NGM plate at a temperature of 23 degrees Celsius for seven days. Next, transfer small amounts of dog food agar or DFA medium to the center of a new NGM plate seeded with E.coli OP50.For optogenetic experiments, pour 40 microliters of all-trans-retinal onto the DFA prior to worm inoculation. Next place, 0.5 milligrams of dog food on the DFA medium, about two millimeters away from the plate lid. Avoid contamination by irradiating the NGM plate with ultraviolet light for 15 minutes.Gather the starved worms from the NGM plates using sterilized water. Then inoculate the gathered worms onto the DFA medium located on the NGM. Propagate the worms at 23 degrees Celsius, enabling them to climb towards the plate lid over 10 to 14 days.Increased humidity exhibited larger compartment sizes of the worm network patterns before ultimately collapsing, leaving dormant worm clusters on the inner surface of the lid.

狗粮琼脂平板中的线虫培养用于行为分析

Observation of the Collective Behavior and Optogenetic Experimentation of Nematode Dauer Worms

Begin by placing a new NGM plate devoid of E.coli and DFA onto an aluminum plate On the stage of a macro zoom microscope. Use a Peltier temperature controller unit to maintain the plate bottom at 23 degrees Celsius temperature for at least five minutes. Next, replace the plate with the lid of a plate that the nematode worms have climbed up onto.Increase the temperature of the plate bottom to 26 degrees Celsius to change the interior humidity. Now, capture images of the inner surface of the plate lid at 20 frames per second using the microscope camera, keep the ZX899 worms on DFA, maintaining this condition for five minutes before illumination. Now illuminate the ZX899 worms attached to a Petri plate lid, maintained at 23 degrees Celsius.Capture the images of the inner plate surface at 20 frames per second. Increased humidity exhibited larger compartment sizes of the worm network patterns before ultimately collapsing, leaving dormant worm clusters on the inner surface of the lid.