Name: large-scale gravitaxis assay of caenorhabditis dauer larvae
Uploaded: 2022-05-31T15:00:00
Description: Watch this Scientific Journal Video about Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae at JoVE.com

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 t...

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#956;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 &#956;m filter). Keep at 4 &#176;C</td>
		</tr>
		<tr>
			<td>AF16 C. briggsae 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&#34; L x 6&#34; W x 26&#34; 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 KH2PO4, 42 mM Na2HPO4, 86 mM NaCl</td>
		</tr>
		<tr>
			<td>N2 C. elegans 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>
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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...

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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 ...

This high throughput assay can be used to observe robust gravitactic behavior in caenorhabditis dauer larvae. Worm behavior is observed over large distances compared with assays performed on a petri dish. This enhances the sensitivity of the assay and allows for detailed data analysis.Studying gravitaxis behavior can provide insight into how gravity sensation occurs in caenorhabditis, which has relevance for understanding the mammalian vestibular system. Creating the gravitaxis assay chambers, isolating dauers, and injecting the worms into the chambers takes practice. You can save time by isolating the dauers and creating the chambers a day in advance.Begin by setting up a Bunsen burner, one to two razor blades, pliers, tweezers, and a plastic cutting surface, and a fume hood. To make a chamber, gather two 5 milliliter serological pipettes, and with tweezers, remove the cotton plug from one pipette. Hold a razor blade using pliers over the Bunsen burner until hot.Use the heated blade to slice off the tapered end of the second pipette so that the entire pipette has a uniform diameter. Now, quickly bring the two modified pipette ends close to the flame and melt them slightly. Join these ends by pressing them together firmly, ensuring that the walls of both pipettes are continuous.If any gaps are visible after joining, break them apart and repeat this step. Prepare NGM with 4%agar according to standard worm procedures and fill each chamber by attaching it to a serological pipetter while the agar is still molten. To minimize any variations in the consistency of the agar due to uneven cooling, hold the pipetter parallel to the bench top while drawing up the agar.Then slowly draw the solution and seal the tip with parafilm before removing the chamber from the pipetter. Lay the chamber flat to cool and allow the agar to harden before moving. Once cooled, heat a three-millimeter hex key over a Bunsen burner and create a small plastic opening by firmly pressing it into the wall of each chamber about five millimeters to one side of the midline.Next, use a heated blade to remove the cotton and tapered ends of the chamber and seal the ends with paraffin film. 10 to 15 days before the experiment, chunk each strain onto two to three large NGM plates with OP50 bacteria and wrap the paraffin film. Collect worms by rinsing the lids and plates with M9 Buffer and pipetting the solution into 15-milliliter centrifuge tubes.Pellet the worms by spinning at 1600 x G for 30 to 60 seconds at room temperature and aspirate most of the M9 Buffer with a pipette or vacuum aspirator. Add seven milliliters of 1%sodium dodecyl sulfate solution to each worm pellet and leave the worms in it for 30 minutes. To allow aeration, keep rotating the tubes continuously during this time.Then, remove the detergent by rinsing it three to five times with M9.After adding five milliliters M9, add five milliliters of cold filtered 60%sucrose solution. Mix thoroughly and create a separation gradient by centrifuging at 1600 x G for five minutes at room temperature. Fill new 15-milliliter centrifuge tubes with two milliliters of M9 Buffer.Now crush the ends of glass Pasteur pipettes to widen the bore. And use these pipettes to transfer the top layer of the solution from the sucrose gradient to the new tubes. Rinse the dauers three to five times with M9 Buffer.Centrifuge dauers at 1600 x G for five minutes at room temperature and aspirate most of the M9 solution with a pipette or vacuum aspirator. Next, estimate the worm density by manually counting the number of worms in three separate 1-microliter droplets under a dissecting microscope, and use the average of these counts to approximate the number of worms per microliter. Now widen the tip bore by cutting the end of 10, 20, or 200-microliter pipette tips.Set the micropipette to a slightly larger volume than the intended aspiration volume. Aspirate approximately 1 to 2 microliters of the concentrated worm solution and allow a small amount of air to enter the tip. Gently push the pipette tip into the agar while depressing the micro pipette.This will create an injection site without clogging the tip. Release the worms into the agar and use paraffin film to seal the opening. To eliminate as many variables as possible once the chambers are placed within a dark Faraday cage at room temperature, label each chamber and hang it vertically within the Faraday cage.Test horizontal controls concurrently by laying some chambers flat within the same environmental conditions. Then use the vertically-oriented N2 worms as a positive control and horizontally-oriented chambers containing N2 dauers as an additional negative control against the experimental strains. After a few hours, the dauer worms begin to disperse from the start site and take several hours to reach either end of the chamber.Leave the chambers undisturbed during this time and score within 12 to 24 hours following injection. Next, remove the chambers one at a time and score gravitaxis by looking for live dauers under a dissecting microscope and marking their locations with ink. Do not score worms within 2.5 centimeters to either side of the injection site as these worms are not likely to be demonstrating a directional preference.Also, avoid scoring worms that appear dead or are trapped in the liquid and discard any chambers containing more than 50%dead or swimming worms. To quantify the results, use a marker to divide each half of the chamber into seven 3.5 centimeters, starting 2.5 centimeters away from the injection site. Use a manual tally counter to tally the number of worms observed in each section.In caenorhabditis briggsae, a large percentage of dauer worms showed migration toward the top of the chambers. However, the horizontal controls showed distribution around the center of the chambers, indicating negative gravitactic behavior. Also, the vertical distributions of caenorhabditis briggsae and caenorhabditis elegans did not show any difference suggesting that caenorhabditis briggsae dauers show a negative gravitaxis behavior similar to caenorhabditis elegans dauers.Paralytic agents such as sodium azide are not used in this protocol. Therefore, it is important to score the gravitaxis chambers as soon as they're removed from the Faraday cage. This assay led to the discovery of negative gravitaxis behavior in c.elegans.Through testing several mutant strains, it also revealed genetic and neural requirements for gravitaxis in worms.

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