This research was supported by research grants from the National Institutes of Health to JHR (#R01 5R01HD081266 and #R01GM141493). Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). We would like to acknowledge Pradeep Joshi (UCSB) for his editorial input. Statistical consultation provided by the UCSB DATALAB.

The strains used in the present study are C. elegans (N2) and C. briggsae (AF16) (see Table of Materials). A mixed-sex population of dauers was used for each assay.
1. Chamber preparation
<ol>
	<li>Work in a fume hood. Set up the workspace with a Bunsen burner, 1-2 razorblades, pliers, tweezers, and a plastic cutting surface (see Table of Materials).</li>
	<li>For each chamber, gather two 5 mL serological pipettes. Remove the cotton plug from one pipette with tweezers. Hold a razorblade over the Bunsen burner until hot using pliers. Use the heated blade to slice off the tapered end of the second pipette so that the entire pipette has a uniform diameter (Figure 1A).</li>
	<li>Working quickly, bring the two modified pipette ends close to the flame, melting them slightly. Join these ends by pressing them firmly together, ensuring that the walls of both pipettes are continuous. If any gaps are visible after joining, break them apart and repeat this step (Figure 1A,B).</li>
</ol>
2. Filling the chambers with agar
<ol>
	<li>Prepare NGM with 4% agar according to standard worm procedures21. While the agar is still molten, fill each chamber by attaching it to a serological pipettor and slowly drawing up the solution. Seal the tip with paraffin film before removing the chamber from the pipettor. Lay the chamber flat (parallel to the benchtop) to cool. 
	&#8203;NOTE: Covering the flask with cling-wrap (see Table of Materials) after autoclaving helps prevent bubbles from forming in the solution18.
	<ol>
		<li>To minimize any variations in the consistency of the agar due to uneven cooling, hold the pipettor (see Table of Materials) parallel to the benchtop while drawing up the agar. Lay the chambers flat on the benchtop and allow the agar to harden before moving. 
		NOTE: The agar percentage and media content can be tailored to the experiment. 4% agar is stiffer than the roughly 2% concentration used for plate pouring and prevents worms from burrowing through the medium in our hands. However, other experimenters have observed burrowing behavior by adult worms in up to 9% agar18,19.</li>
	</ol>
	</li>
	<li>Once cooled, heat a 3 mm hex key (or another metal tool of the same size, see Table of Materials) over a Bunsen burner. Firmly press it into the wall of each chamber about 5 mm to one side of the midline, creating a small opening in the plastic (Figure 1C). Use a heated blade to remove the cotton and tapered ends of the chamber and seal these ends with paraffin film. 
	&#8203;NOTE: Once prepared, each chamber must be used within 1 day or kept at 4 &#176;C for several days. Cover any exposed openings with paraffin film to prevent the agar from drying out.</li>
</ol>
3. Isolating dauer larvae
<ol>
	<li>10-15 days prior to the experiment, chunk each strain onto 2-3 large NGM plates with OP50 bacteria and wrap with paraffin film. After 10-15 days, each plate must be fully starved with thousands of dauer larva present on the agar, walls, and lids. 
	NOTE: Make plates ahead of time using a thick OP50 recipe for maximum crowding (see Table of Materials). Dauer formation can also be induced through other methods, including the addition of pheromones or by growing at higher temperatures22.</li>
	<li>Collect worms by rinsing the lids and plates with M9 buffer and pipetting the solution into 15 mL centrifuge tubes. Spin at 1,600 x g for 30-60 s at room temperature and aspirate most of the M9 buffer with a pipette or vacuum aspirator.</li>
	<li>Isolate dauers by treating with 1% SDS followed by a 30% sucrose flotation gradient following the previously published report20.
	<ol>
		<li>Add 7 mL 1% SDS solution to each worm pellet. Leave the worms in SDS for 30 min; rotate the tubes continuously during this time to allow for aeration. Rinse 3-5x with M9 to remove the detergent.</li>
		<li>Next, add 5 mL M9 followed by 5 mL of cold, filtered 60% sucrose solution. Mix thoroughly, and then centrifuge at 1,600 x g for 5 min at room temperature to create a separation gradient.</li>
	</ol>
	</li>
	<li>Fill new 15 mL centrifuge tubes with 2 mL of M9 buffer. Crush the ends of glass Pasteur pipettes to widen the bore and use these pipettes to transfer the top layer of the solution (containing isolated dauers) from the sucrose gradient to the new tubes. Rinse the dauers 3-5x with M9. 
	&#8203;NOTE: There will be thousands of isolated dauers in solution at this point. They may be used immediately or kept aerated by rotating in 5-7 mL M9 overnight.</li>
</ol>
4. Adding dauers to the chamber
<ol>
	<li>Centrifuge dauers at 1,600 x g for 5 min at room temperature and aspirate most of the M9 solution with a pipette or vacuum aspirator. To estimate worm density, manually count the number of worms in three separate 1 &#181;L droplets under a dissecting microscope. Use the average of these counts to approximate the number of worms per &#181;L. 
	NOTE: Some small volume pipettors may be unable to reach the bottom of a 15 mL centrifuge tube; in this case, a concentrated droplet of worms can be transferred first to a piece of paraffin film.</li>
	<li>Cut the end of a 10, 20, or 200 &#181;L pipette tip to widen the bore. Set the micropipette to a slightly larger volume than the intended aspiration volume. Aspirate approximately 1-2 &#181;L of the concentrated worm solution (ideally between 100-300 worms) and allow a small amount of air to enter the tip. 
	NOTE: Large numbers of worms (1,000+) in one chamber may increase the range of distances traveled due to overcrowding9.</li>
	<li>Gently push the pipette tip into the agar while depressing the micropipette. This will create an injection site in the agar without clogging the tip. Release the worms into the agar. Use paraffin film to seal the opening.</li>
</ol>
5. Running and scoring the assay
NOTE: Gravitaxis can be tested under various conditions that may affect behavior9.
<ol>
	<li>To eliminate as many variables as possible, place the chambers within a dark Faraday cage (see Table of Materials) at room temperature.</li>
	<li>Label each chamber and hang it vertically within the Faraday cage (perform this using labeling tape). Test horizontal controls concurrently by laying some chambers flat within the same environmental conditions. Test the experimental strains against vertically oriented N2 worms as a positive control. Use horizontally oriented chambers containing N2 dauers as an additional negative control. 
	NOTE: Horizontal and vertical assays must be performed in the same setting within the lab to minimize environmental variability between the two conditions. A large incubator may be used to house both Faraday cages.</li>
	<li>Dauers begin to disperse from the start site after a few hours and take several hours to reach either end of the chamber. Leave the chambers undisturbed during this time and score within 12-24 h following injection. 
	NOTE: Do not use a paralytic (such as sodium azide) to immobilize worms that have reached the ends of the chambers. Because the overall distribution of worms is being measured instead of a preference index (as in a two-choice assay), sodium azide is unnecessary and may alter the results.</li>
	<li>Remove and score gravitaxis chambers one at a time. Look for live dauers under a dissecting microscope and mark their locations in ink (Figure 1C,D). Do not score worms within 2.5 cm to either side of the injection site, as these worms are not likely to be demonstrating a directional preference.
	<ol>
		<li>Also, avoid scoring worms that appear dead or are trapped in the liquid. Discard any chambers containing &#62;50% dead or swimming worms. 
		NOTE: Once the chamber has been removed from the testing area, it needs to be scored promptly for accuracy. Dauers must only be visible between the agar&#39;s surface and the pipette&#39;s wall. If burrowing behavior is observed, consider increasing agar concentration in future assays.</li>
	</ol>
	</li>
	<li>To quantify the results, use a marker to divide each half of the chamber into seven 3.5 cm sections starting 2.5 cm away from the injection site (on some pipettes, 3.5 cm = 1 mL volume). Use a manual tally counter (see Table of Materials) to tally the number of worms observed in each section. 
	NOTE: There should be seven sections on each side of the origin, which can be numbered from -7 (bottom) to +7 (top). These numbers must correspond to the same relative locations based on how the chambers were constructed; agar is drawn from the -7 end to the +7 end in step 2.</li>
</ol>

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The authors declare no competing interests.

Comparison with prior methods 
Unlike chemotaxis, gravitaxis in Caenorhabditis cannot be reliably observed using a traditional agar plate experimental design. A standard Petri dish is 150 mm in diameter, resulting in only 75 mm available in either direction for dauers to demonstrate gravitaxis preference. Although C. elegans&#39; orientational preference can be assayed in solution12, this method is low throughput as worms must be analyzed one at a time. Addi...

Sensing Earth&#39;s gravitational pull is crucial for many organisms&#39; orientation, movement, coordination, and balance. However, the molecular mechanisms and neurocircuitry of gravity sensation are poorly understood compared with other senses. In animals, gravity sensation interacts with and can be outcompeted by other stimuli to influence behavior. Visual cues, proprioceptive feedback, and vestibular information can be integrated to generate a sense of body awareness relative to an animal&#39;s surroundings1,2. Conversely, gravitactic preference can be altered in the presence of other stimuli3,4,5. Therefore, gravitactic behavior is ideal for studying gravity sensation and understanding the nervous system&#39;s complex sensory integration and decision-making.
C. elegans is an especially useful model organism for studying gravitaxis because of its polyphenic lifecycle. When exposed to stressors during development, including heat, overcrowding, or a lack of food, C. elegans larvae develop into dauers, which are highly stress-resistant6. As dauers, worms perform characteristic behaviors, such as nictation, in which worms &#34;stand&#34; on their tails and wave their heads, that may facilitate dispersal to better habitats7. Gravitaxis assays of C. elegans and C. japonica suggest that dauer larvae negatively gravitax, and that this behavior is more readily observed in dauers than in adults8,9. Testing gravitaxis in other Caenorhabditis strains may reveal natural variation in gravitactic behavior.
Mechanisms for gravity sensation have been characterized in Euglena, Drosophila, Ciona, and various other species using gravitaxis assays3,10,11. Meanwhile, gravitaxis studies in Caenorhabditis initially provided mixed results. A study of C. elegans orientational preference found that worms orient with their heads down in solution, suggesting positive gravitactic preference12. Meanwhile, although C. japonica dauers were identified early on as being negatively gravitactic8, this behavior has only recently been described in C. elegans9. Several challenges arise in developing a representative gravitaxis assay in worms. Caenorhabditis strains are maintained on agar plates; for this reason, behavioral assays typically use agar plates as part of their experimental design13,14,15. The earliest reported gravitaxis assay in Caenorhabditis was performed by standing a plate on its side at a 90&#176; angle to the horizontal control plate8. However, gravitaxis behavior is not always robust under these conditions. While adult worms can be assayed for orientational preference in solution12, this directional preference may also be context-dependent, leading to different behaviors if the worms are crawling rather than swimming. Additionally, C. elegans is sensitive to other stimuli, including light and electromagnetic fields16,17, which interfere with their responses to gravity9. Therefore, an updated gravitaxis assay that shields against other environmental variables is important for dissecting the mechanisms of this sensory process.
In the present protocol, an assay for observing Caenorhabditis gravitaxis is described. The setup for this study is based in part on a method developed to study neuromuscular integrity18,19. Dauer larvae are cultured and isolated using standard procedures20. They are then injected into chambers made from two 5 mL serological pipettes filled with agar. These chambers can be oriented vertically or horizontally and placed within a dark Faraday cage for 12-24 h to shield against light and electromagnetic fields. The location of each worm in the chambers is recorded and compared with the vertical taxis of a reference strain such as C. elegans N2.

<table><tbody><tr><td>1% Sodium Dodecyl Sulfate solution</td><td></td><td></td><td>From stock 10% (w/v) SDS in DI water</td></tr><tr><td>15 mL Centrifuge tubes</td><td>Falcon</td><td>14-959-53A</td><td></td></tr><tr><td>3 mm Hex key</td><td></td><td></td><td>Other similar sized metal tools may be used</td></tr><tr><td>4% Agar in Normal Growth Medium (NGM) - 1 L</td><td></td><td></td><td>Prior to autoclaving: 3 g NaCl, 40 g Agar, 2.5 g Peptone, 2 g Dextrose, 10 mL Uracil (2 mg/mL), 500 &amp;mu;L Cholesterol (10 mg/mL), 1 mL CaCl2, 962 mL DI water; After autoclaving: 24.5 mL Phosphate Buffer, 1 mL 1 MgSO4 (1 M), 1 mL Streptomycin (200 mg/mL)</td></tr><tr><td>5 mL Serological pipettes</td><td>Fisherbrand</td><td>S68228C</td><td>Polystyrene, not borosilicate glass</td></tr><tr><td>60% Cold sucrose solution</td><td></td><td></td><td>60% sucrose (w/v) in DI water; sterilize by filtration (0.45 &amp;mu;m filter). Keep at 4 &amp;deg;C</td></tr><tr><td>AF16 &lt;em&gt;C. briggsae&lt;/em&gt; or other experimental strain</td><td></td><td></td><td>Available from the CGC (Caenorhabditis Genetics Center)</td></tr><tr><td>Bunsen burner</td><td></td><td></td><td></td></tr><tr><td>Cling-wrap</td><td>Fisherbrand</td><td>22-305654</td><td></td></tr><tr><td>Clinical centrifuge</td><td></td><td></td><td></td></tr><tr><td>Disposable razor blades</td><td>Fisherbrand</td><td>12-640</td><td></td></tr><tr><td>Faraday cage</td><td></td><td></td><td>Can be constructed using cardboard and aluminum foil; 30&quot; L x 6&quot; W x 26&quot; H or larger</td></tr><tr><td>Ink markers</td><td></td><td></td><td>Sharpie or other brand for marking on plastic</td></tr><tr><td>Labeling tape</td><td>Carolina</td><td>215620</td><td></td></tr><tr><td>M9 buffer</td><td></td><td></td><td>22 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 42 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 86 mM NaCl</td></tr><tr><td>N2 &lt;em&gt;C. elegans&lt;/em&gt; strain</td><td></td><td></td><td>Available from the CGC (Caenorhabditis Genetics Center)</td></tr><tr><td>NGM plates with OP50</td><td></td><td></td><td>1.7% (w/v) agar in NGM (see description: 4% agar in NGM). Seed with OP50</td></tr><tr><td>Paraffin film</td><td>Bemis</td><td>13-374-10</td><td></td></tr><tr><td>Plastic cutting board</td><td></td><td></td><td></td></tr><tr><td>Pliers</td><td></td><td></td><td></td></tr><tr><td>Rotating vertical mixer</td><td>BTLab SYSTEMS</td><td>BT913</td><td>With 22 x 15 mL tube bar</td></tr><tr><td>Serological pipettor</td><td>Corning</td><td>357469</td><td></td></tr><tr><td>Stereo Microscope</td><td>Laxco</td><td>S2103LS100</td><td></td></tr><tr><td>Tally counter</td><td>ULINE</td><td>H-7350</td><td></td></tr><tr><td>Thick NGM/agar plate media - 1 L</td><td></td><td></td><td>See 4% Agar in NGM recipe; replace 40 g Agar with 20 g Agar</td></tr><tr><td>Tweezers</td><td></td><td></td><td></td></tr></tbody></table>

large-scale gravitaxis assay of caenorhabditis dauer larvae

Gravity sensation is an important and relatively understudied process. Sensing gravity enables animals to navigate their surroundings and facilitates movement. Additionally, gravity sensation, which occurs in the mammalian inner ear, is closely related to hearing - thus, understanding this process has implications for auditory and vestibular research. Gravitaxis assays exist for some model organisms, including Drosophila. Single worms have previously been assayed for their orientation preference as they settle in solution. However, a reliable and robust assay for Caenorhabditis gravitaxis has not been described. The present protocol outlines a procedure for performing gravitaxis assays that can be used to test hundreds of Caenorhabditis dauers at a time. This large-scale, long-distance assay allows for detailed data collection, revealing phenotypes that may be missed on a standard plate-based assay. Dauer movement along the vertical axis is compared with horizontal controls to ensure that directional bias is due to gravity. Gravitactic preference can then be compared between strains or experimental conditions. This method can determine molecular, cellular, and environmental requirements for gravitaxis in worms.

Comparing gravitaxis across species 
Following the procedure outlined above, C. briggsae dauer gravitaxis can be compared with C. elegans gravitaxis and horizontal controls. The vertical distribution (maroon) of C. briggsae dauers is skewed toward the tops of the chambers, with a large percentage of worms reaching +7 (Figure 2A). Contrasted with horizontal controls (aqua), in which dauers are distributed in a roughly bell-shaped curve around t...

Watch this Scientific Journal Video about Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae at JoVE.com

Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae

The present protocol outlines methods for conducting a large-scale gravitaxis assay with Caenorhabditis dauer larvae. This protocol allows for better detection of gravitaxis behavior compared with a plate-based assay.

Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae

Gravity sensation is an important and relatively understudied process. Sensing gravity enables animals to navigate their surroundings and facilitates ...

large-scale-gravitaxis-assay-of-caenorhabditis-dauer-larvae

Research

JoVE Journal

Behavior

2.2K Views.  University of California. Deze high throughput assay kan worden gebruikt om robuust gravitactisch gedrag te observeren bij caenorhabditis dauer larven. Wormgedrag wordt waargenomen over grote afstanden in vergelijking met testen uitgevoerd op een petrischaaltje. Dit verhoogt de gevoeligheid van de test en maakt gedetailleerde gegevensanalyse mogelijk.Het bestuderen van gravitaxis-gedrag kan inzicht geven in hoe zwaartekrachtsensatie optreedt bij caenorhabditis, wat relevant is voor het begrijpen van het vestibu...

Video: Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Biochemistry

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Bioengineering

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Biology

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

Genetics

Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms

Caenorhabditis elegans (C. elegans) is a well-established model organism used across a range of basic and biomedical research. Within the nematode research community, there is a need for an affordable and effective way to maintain large, age-matched populations of C. elegans. Here, we present a methodology for mechanically sorting and cleaning C. elegans. Our aim is to provide a cost-effective, efficient, fast, and simple process to obtain animals of uniform sizes and life stages for their use in experiments. This tool, the Caenorhabditis Sieve, uses a custom-built lid system that threads onto common conical lab tubes and sorts C. elegans based on body size. We also demonstrate that the Caenorhabditis Sieve effectively transfers animals from one culture plate to another allowing for a rapid sorting, synchronizing, and cleaning without impacting markers of health, including motility and stress-inducible gene reporters. This accessible and innovative tool is a fast, efficient, and non-stressful option for maintaining C. elegans populations.

Caenorhabditis Sieve: A Low-tech Instrument and Methodology for Sorting Small Multicellular Organisms

The current protocol includes a methodology for the sorting and cleaning of age-matched populations of Caenorhabditis elegans. It uses a simple, inexpensive, and efficient custom-made tool to obtain a large experimental population of nematodes for research.

Caenorhabditis elegans (C. elegans) is a well-established model organism used across a range of basic and biomedical research. Within the nematode ...

Developmental Biology

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

Neuroscience

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...

Investigating Collective Behaviors in Nematodes Using Dog Food Agar Medium

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, 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.

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

Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae

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