大规模培养线虫以研究其集体行为
Summary
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。
Abstract
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。
Introduction
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.
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| Escherichia coli and C. elegans strains | |||
| E. coli OP50 | Caenorhabditis Genetics Center | OP50 | Food for C. elegans. Uracil auxotroph. E. coli B. |
| lite-1(ce314); ljIs123[mec-4p::ChR2, unc-122p::RFP] | author | ZX899 | lite-1(ce314) mutant carrying the genes expressing ChR2 and RFP under the control of the mec-4 and unc-122 promoter, respectively |
| N2 Bristrol | Caenorhabditis Genetics Center | Wild-type C. elegans strain | |
| For worm cultivation | |||
| Agar purified, powder | Nakarai tesque | 01162-15 | For preparation of NGM plates |
| All-trans retinal | Sigma-Aldrich | R2500 | For optogenetics |
| Bacto pepton | Becton Dickinson | 211677 | For preparation of NGM plates |
| Calcium chloride | Wako | 036-00485 | For preparation of NGM plates |
| Cholesterol | Wako | 034-03002 | For preparation of NGM plates |
| di-Photassium hydrogenphosphate | Nakarai tesque | 28727-95 | For preparation of NGM plates |
| Dog food | Nihon Pet Food | VITA-ONE | For preparation of dog food agar medium |
| LB broth, Lennox | Nakarai tesque | 20066-95 | For culture of E. coli OP50 |
| Magnesium sulfate anhydrous | TGI | M1890 | For preparation of NGM plates |
| Petri dishes (60 mm) | Nunc | 150270 | For preparation of NGM plates |
| Potassium Dihydrogenphosphate | Nakarai tesque | 28720-65 | For preparation of NGM plates |
| Sodium Chloride | Nakarai tesque | 31320-05 | For preparation of NGM plates |
| Observation | |||
| Computer | CT solution | CS6229 | Windows10 Pro with Intel Xeon Gold 6238R CPU and 768 GB of RAM |
| CMOS Camera | Hamamatsu photonics | ORCA-Lightning C14120-20P | For data acquisition |
| CMOS Camera | Olympus | DP74 | For data acquisition |
| Microscope with SZX-MGFP set | Olympus | MVX10 | For data acquisition |
| x2 Objective lens | Olympus | MV PLAPO 2XC | Working distance 20 mm and numerical aperture 0.5 |
| Shutter control | |||
| Shutter | OptoSigma | BSH2-RIX | For controlling temporal pattern of light illumination |
| Shutter controller | OptoSigma | SSH-C2B-A | For controlling temporal pattern of light illumination |
| Temperature control | |||
| Peltier temperature controller unit | VICS | WLVPU-30 | For controlling humidity inside a Petri plate |
| UNI-THEMO CONTROLLER | Ampere | UTC-100 | For controlling humidity inside a Petri plate |
| Data acquisition software | |||
| HCImage | Hamamatsu photonics | For data acquisition |
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