We thank the Caenorhabditis Genetics Center for providing the strains used in this study. This publication was supported by JSPS KAKENHI Grant-in-Aid for Scientific Research (B) (grant number JP21H02532), JSPS KAKENHI Grant-in-Aid on the Innovative Areas &#34;Science of Soft Robot&#34; project (grant number JP18H05474), JSPS KAKENHI Grant-in-Aid for Transformative Research Areas B (grant number JP23H03845), the PRIME from Japan Agency for Medical Research and Development (grant number JP22gm6110022h9904), JST-Mirai Program (grant number JPMJMI22G3), and JST-FOREST Program (grant number JPMJFR214R).

1. Preparation of worms
NOTE: Prepare the wild-type N2 Bristol strain12 and ZX899 strain (lite-1(ce314); ljIs123[mec-4p::ChR2, unc-122p::RFP])13 for the observation of collective behaviors and optogenetic experiments, respectively. Maintain the ZX899 strain under dark conditions.
<ol>
	<li>Deposit four well-fed adult worms on a 60 mm plate containing 14 mL of nematode growth medium (NGM) with agar and seeded with E. coli OP5012.</li>
	<li>Grow F1 worms to starvation in the NGM plate at 23 &#176;C for 7 days. The yield of F1 worms is approximately 100 worms/plate at this point. The stages of worms comprise a mixed population of dauer and starved L1 larvae.</li>
</ol>
2. Preparation of dog food agar (DFA) medium plates
<ol>
	<li>Autoclave a glass bottle containing 2 g of powdered dog food and 5 mL of 1% agar medium and cool it to room temperature (Figure 1A). 
	NOTE: Other dog foods from different manufacturers can be used in this experiment.</li>
</ol>
3. Inoculation of worms to DFA medium plates
<ol>
	<li>Transfer small amounts (approximately 0.5 g) of DFA medium onto the center of an NGM plate seeded with E. coli OP50 (Figure 1B). For optogenetic experiments, pour 40 &#181;L of 50 &#181;M all-trans-retinal, the cofactor of channel rhodopsin 2, onto the DFA before inoculation of worms.</li>
	<li>Collect the starved worms from four NGM plates using autoclaved water.</li>
	<li>Place a small fragment of dog food (approximately 0.5 g) on the DFA medium, approximately 2 mm away from the lid of the plate.</li>
	<li>Illuminate the NGM plate with ultraviolet light for 15 min inside a clean bench to prevent contamination.</li>
	<li>Inoculate the collected worms (approximately 400 worms) onto the DFA medium on NGM plates. Do not seal the plate with parafilm to avoid increasing the humidity inside the Petri plate and generating water droplets that trap worms on a lid.</li>
	<li>Propagate the worms at 23 &#176;C and allow them to climb up to the lid of the plate for approximately 10-14 days. 
	&#8203;NOTE: Because the number of worms on the lids hardly increased after 10-14 days, it was presumed that the worms had likely run out of food.</li>
</ol>
4. Observation of collective behavior
<ol>
	<li>On the day of the experiment, place a new NGM plate that did not include E. coli and dog food agar medium onto an aluminum plate on the stage of a macro zoom microscope (Figure 2A). Keep the bottom of this new NGM plate at 23 &#176;C using a Peltier temperature controller unit for at least 5 min (Figure 2B). Then, replace the lid of this new NGM plate with the lid of the plate that the worms climbed up onto. Use the objective lens (x2, NA = 0.5) as a low-magnification objective (Figure 2A).</li>
	<li>Increase the temperature of the bottom of the Petri plate from 23 &#176;C to 26 &#176;C to alter the humidity inside that plate (Figure 2).</li>
	<li>Acquire images of the inner surface of the plate lid with the camera at 20 frame s&#8722;1 (Supplementary Video S1).</li>
	<li>Save the acquired images in the Tagged Image File format.</li>
</ol>
5. Optogenetic experiment
<ol>
	<li>Use a 100 W mercury lamp to deliver blue light, filtered with a filter set. Control the illumination time precisely using an electromagnetic shutter system (Figure 2B).</li>
	<li>Maintain ZX899 on DFA under these conditions for 5 min before blue light illumination.</li>
	<li>Illuminate ZX899 worms attached to the lid of a Petri plate on the microscope stage maintained at 23 &#176;C.</li>
	<li>Acquire images of the inner surface of the plate lid with a camera at 20 frame s&#8722;1 (Supplementary Video S2).</li>
</ol>

Investigating Collective Behaviors in Nematodes Using Dog Food Agar Medium

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The authors have no conflicts of interest to declare.

In this study, we show a protocol for preparing a system for the large-scale collective behavior of C. elegans in the laboratory. The DFA-based method was originally established with Caenorhabditis japonica14 and Neoaplectana carpocapsae Weiser15, both of which are non-model animals. However, this method was not applied to investigate collective behaviors. The C. elegans is a genetically tractable model animal11<su...

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.

<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

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.

Author Spotlight: Collective Behavioral Analysis of the Nematode, &lt;em&gt;Caenorhabditis elegans&lt;/em&gt;

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

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.

Here, wild-type dauer worms were used for collective behavior observations. Worms were cultivated at 23 &#176;C for approximately 10-14 days and climbed up to the inner surface of the lid of a DFA medium plate. On the experimental day, only the lid was transferred to a new NGM plate without E. coli and DFA medium. The bottom of this Petri plate was initially kept at 23 &#176;C using the Peltier system, and then its temperature was increased to 26 &#176;C. A movie was taken under the microscope. <strong class="xf...

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

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.

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

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Research

JoVE Journal

Biology

571 Views.  Hiroshima University. 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, ne...

Video: Author Spotlight: Collective Behavioral Analysis of the Nematode, Caenorhabditis elegans

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.

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.

Methods Article

Author Spotlight: Collective Behavioral Analysis of the Nematode, <em>Caenorhabditis elegans</em>