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 animal11,12. Behavioral genetic studies using C. elegans have contributed to the investigation of individual-level behavioral research. However, in the long history of C. elegans research, although a simple clumping pattern was observed16,17,18,19, no reports have demonstrated dynamic pattern formation via the group-level behavior of C. elegans. A key idea of this study is using DFA medium to facilitate the maintenance of a huge number of worms for a long time in a Petri plate. Using DFA medium, we present the observation of dynamic collective behavior by C. elegans, thus introducing a new behavioral paradigm.
Previously, several mass worm production methods have been reported. In comparison with these methods, the advantage of this method is to enable the investigation of collective behavior on a lid without a process for the isolation of dauer worms. Recently, we published a paper reporting the transfer of nictating dauer worms across a gap between a lid and DFA medium using electrostatic interactions with the lid20. This worm transfer occurs when worms form a nictation column composed of approximately 100 worms. This study shows that only dauer worms can transfer when dauer formations are induced by too much crowding in DFA. The number of worms produced by this method is likely fewer than other methods such as egg yolk-based methods. However, to perform a behavioral assay on a lid, we can use the population of the dauer worms, which hardly include other stage worms such as starved L1 larvae, whereas previous methods require a process for the isolation of dauer worms. Thus, this method allows for a more precise collective behavioral examination using dauer worms. In addition, the experimenter can also control the density of worms in the following procedure. First, autoclaved water was used to collect and wash the worms that moved to the lid. Then, the concentration of worms in water was determined by counting the worms in an aliquot of the worm suspension, and the worm suspension was dropped onto a substrate. Taken together, our system is more controllable in terms of the worm stage and density for behavioral experiments.
Collective behavior has been analyzed from the perspective of active matter physics, which seeks to identify unified descriptions of collective motions of living and nonliving self-propelled particles. Toward this goal, many experimental systems have been developed for nonliving self-propelled particles and cells, but fewer systems have been developed for multi-cellular organisms, which exhibit much more complex behaviors based on the neural circuit. Therefore, our system extends the possibility that the unified description of collective motions exists. Regarding humidity manipulation, our previous numerical simulation based on a model suggested that attraction forces between worms, likely induced by humidity in the experiment, induce pattern changes, which were qualitatively consistent with humidity-induced pattern changes10. However, we think that there is no deterministic experimental evidence showing that pattern changes were induced by humidity rather than temperature. Therefore, the experimenter should exercise caution in whether the collective behavior changes can be attributed solely to the humidity change rather than the temperature change or not.
Understanding the neural mechanism underlying collective behaviors in animals is a new challenge in the field of biology. Collective behaviors lead to the emergence of a new function that does not appear at the individual level. As animals have a nervous system, they have memory and learning abilities, and it is intriguing to examine the differences in these neural functions at the individual and population levels. It has been noted that collective behaviors improve the detection sensitivity for foreign organisms and prey and enhance the ability for correct decision-making21,22,23. C.&#160;elegans also has a nervous system comprised of 302 neurons and thereby memorizes the past cultivation temperature24 and migrates to a location with preferable humidity25. Thus, it would be interesting to investigate the relationship between neural functions and collective behaviors in C. elegans. Furthermore, one can expect to extract mechanical parameters through observation of the behavior of a worm population. For example, observation of the viscoelastic properties in C. elegans crowds would make it possible to estimate the elasticity of a single worm and the surface tension between worms. The size distribution of worm clumps should relate to the surface tension between them. The propulsive force of the C. elegans individual can also be estimated from the frequency that the worm moves out in response to the surface tension. Thus, we can expect to estimate mechanical parameters at the level of individual worms based only on macroscopic information of worm population.
In conclusion, active matter physics aims to identify unified descriptions of collective behaviors, and this field requires more experimental verification of the proposed mathematical models by controlling parameters. Additionally, the functional significance of each animal&#39;s collective pattern formation and its mechanical relevance to neural functions are important open questions. Furthermore, given that one of the objectives of &#39;soft robotics&#39; is the precise control of collectives of robots, we hope that an algorithm can be established through the experiments of worms&#39; collective behaviors for application to controlling the collective motions of soft robots.

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><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

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, 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. Figure 3 shows snapshots of the movie. Worms dynamically remodeled their network patterns during humidity change. As humidity increases, the compartment sizes of the network also become larger. Finally, the networks collapsed, and dormant worm clusters remained on the inner surface of the lid.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65569/65569fig01.jpg" /> 
Figure 1: Photos of DFA medium for cultivating large numbers of worms. (A) Photo of DFA medium prepared in a glass bottle. (B) Photo of NGM plate with DFA medium just after collected worms were inoculated. Abbreviations: DFA = dog food agar; NGM = nematode growth medium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65569/65569fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65569/65569fig02.jpg" /> 
Figure 2: Experimental system for the observation of collective behavior. (A) Microscopy for observation of collective behavior. (B) Mechanical shutter controller and temperature control system using the Peltier system. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65569/65569fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65569/65569fig03.jpg" /> 
Figure 3: Representative data of the dependence of the collective network pattern on humidity. Dependence of the C.&#160;elegans network on ambient humidity. The camera frame rate is 1 fps. Scale bar = 4 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65569/65569fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Video S1: Collective network formations. Wild-type dauer worms were propagated using DFA on NGM in a Petri plate. The worms self-organized inside the lid. The humidity was changed using a Peltier device. Images were taken from above the lid. The movie plays 80 times faster than the real-time recording rate. Abbreviations: DFA = dog food agar; NGM = nematode growth medium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65569/20181213_147crop_80fps.mp4" target="_blank">Please click here to download this File.</a>
Supplementary Video S2: Optogenetic manipulation of worms&#39; collectives. Optogenetics was performed with 1, 2, 4, 8, 32, and 128 s blue light illuminations. This activation initially caused the arborization and collapse of bundles. Finally, a network different from the initial structure was formed. The movie is played 20 times faster than the real-time recording rate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65569/20190111_180_20fps.mp4" target="_blank">Please click here to download this File.</a>

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

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

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

917 Views.  Hiroshima University. We bestuderen het mechanisme dat ten grondslag ligt aan collectief gedrag vanuit het perspectief van actieve metafysica en neurowetenschappen. Op het gebied van collectief gedrag is een experimenteel systeem opgezet met behulp van niet-levende dingen en op cellulair niveau. Het blijft echter nog steeds een uitdaging om het experimentele systeem op dierniveau te ontwerpen om het collectieve gedrag in het laboratorium te bestuderen.We hebben met succes het collectieve gedrag gereconstrue...

Video: Auteur in de schijnwerpers: Collectieve gedragsanalyse van de nematode, Caenorhabditis elegans

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.

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

Genetics

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.

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.

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

Immunology and Infection

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.

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.

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 ...

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.

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.

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.

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.

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.

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

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

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