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.
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.
Sensing Earth's gravitational pull is crucial for many organisms' 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'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'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 "stand" 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° 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.
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
2. Filling the chambers with agar
3. Isolating dauer larvae
4. Adding dauers to the chamber
5. Running and scoring the assay
NOTE: Gravitaxis can be tested under various conditions that may affect behavior9.
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 the center of the chambers, this trend indicates negative gravitactic behavior. These data can be compared with C. elegans gravitaxis performed on the same experimental days (Figure 2B).
A Kruskal-Wallis test followed by Dunn's test with Bonferroni correction for multiple comparisons determined any significant differences between assays9,23. In this experiment, 1,108 C. briggsae dauers were scored across three vertical chambers from two independent experimental days. These worms migrated significantly upward than horizontal controls (p < 0.001; 1,639 dauers in three horizontal chambers over 2 experimental days). Moreover, the vertical C. briggsae and vertical C. elegans distributions did not differ (p > 0.05; 386 C. elegans dauers in two vertical chambers over 2 experimental days). These results suggest that C. briggsae dauers show a negative gravitaxis behavior similar to that of C. elegans dauers.
Figure 1: Gravitaxis assay chamber setup. Photos depicting steps of gravitaxis assay chamber preparation. (A) Individual 5 mL pipettes were prepared for fusion into a single chamber. Removal of cotton plug indicated with black arrowhead; removal of tip indicated with a white arrowhead. The tubes used in this study have an inner diameter of 6 mm and are each 34.5 cm long (prior to cutting). (B) Completed chamber prior to addition of NGM agar. Pipettes have been fused by first melting over a Bunsen burner. (C) Example chambers filled with NGM agar. The injection site is indicated with an arrow and enlarged in the inset. Worms have been marked in ink, and lines are drawn to distinguish distances along with the chamber as well as to facilitate scoring. (D) View of chambers in their entirety (taken after scoring). Please click here to view a larger version of this figure.
Figure 2: Gravitaxis in C. briggsae vs. C. elegans. Results of gravitaxis in C. briggsae and C. elegans. (A) Histogram of C. briggsae gravitaxis. Movement in vertical chambers (maroon) is compared with horizontal controls (aqua). Distance from the injection point (-7 being the furthest below, +7 furthest above) is plotted as the percent of total worms assayed (x-axis). No data are plotted for the origin (+0) as worms are not counted within 2.5 cm to either side of the injection site. N = 1,639 worms across three tubes in the horizontal condition; vertical N = 1,108 worms across three tubes. (B) Boxplots comparing the distribution of C. briggsae horizontal and vertical worms versus C. elegans vertical worms. C. briggsae sample sizes are given above. C. elegans vertical N = 386 worms across two tubes. Means are indicated with white diamonds; vertical conditions are in maroon and are labeled "V", and horizontal conditions ("H") are in aqua. Comparisons were made using the Kruskal-Wallis test, followed by the Dunn's test with Bonferroni correction for multiple comparisons. No stars = p > 0.05, **** = p < 0.0001. Please click here to view a larger version of this figure.
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' orientational preference can be assayed in solution12, this method is low throughput as worms must be analyzed one at a time. Additionally, gravitactic preferences and behaviors may differ between worms floating in media versus crawling on a surface. For these reasons, we developed a high throughput assay with enhanced sensitivity that can be used to analyze gravitaxis behavior in crawling or climbing worms. Because C. elegans are sensitive to light and electromagnetic fields, both of which interfere with gravitactic preference9, these assays need to be performed without either stimulus. If a homemade Faraday cage is being used, checking the cage's strength and reinforcing it are necessary and recommended.
The assay chambers described in this protocol measure approximately 54 cm in length and are placed within Faraday cages to shield against light and electromagnetic fields. It was found that changing the "arena" for observing gravitaxis behavior has several advantages. First, it allows for detailed quantification and descriptive analysis. The relative distances traveled, and overall distributions of worms can be collected and compared instead of counting the number of negatively versus positively gravitactic worms. Second, the enclosed environment of an agar-filled pipette more closely replicates the conditions that may promote gravitaxis in the wild. As stated above, the dauer stage is a dispersal phase that allows escaping from unfavorable conditions6. Because Caenorhabditis live in compost, where guiding cues such as light may not be available, gravity may enable worms to navigate to the surface6,9. Two-dimensional plate-based assays are unlikely to replicate these conditions even if they are tested without other stimuli. Finally, this larger apparatus tests larger quantities of worms in a single assay.
Limitations and other considerations
While this protocol effectively measures gravitactic preference in large numbers of dauers, it is impractical for small or single worm experiments due to the time and materials required to construct each chamber. By combining counts across trials rather than using a gravitaxis index, statistical power is increased because each worm is treated as an independent event. While this enhanced sensitivity is useful in differentiating gravitactic and non-gravitactic worms, it is important to note that C. elegans dauers exhibit social behaviors7,24 that could affect overall gravitaxis. We found that higher worm densities are correlated with a greater range in the distance traveled over the large-scale assay, though this effect is minimal when the total count is less than approximately 1,000 worms9. Interaction effects resulting from factors such as the total worms in each chamber should be monitored when analyzing the data. As with all behavioral assays, scoring should be blinded when possible.
Finding an average density of worms in the solution can minimize variability in the number of worms injected from chamber to chamber. Worms quickly settle in solution, so mixing the worms before pipetting, either by flicking the tubes or pipetting up and down with a wide-bore tip is important. Even when a consistent density is achieved, the number of worms added may vary because worms are likely to stick to the inside surface of the plastic pipette tips. Coating pipette tips with BSA or other solutions could minimize this effect. Worms must be injected with as little solution as possible; if too much liquid is added to the chamber, worms will become trapped in the solution and may even drift. For this reason, only live, crawling worms may be scored, and any chamber containing more than 50% dead or swimming worms must not be used. Chambers are to be removed from the Faraday cage one at a time and scored within 10-15 min for the greatest accuracy.
Potential applications
This assay may be used to test the environmental, genetic, and cellular requirements for gravitaxis in a variety of Caenorhabditis strains. So far, light and electromagnetic fields have been identified as stimuli that interfere with gravitaxis9. However, C. elegans is sensitive to other stimuli, including temperature, volatile and non-volatile chemicals, texture, humidity, and sound, which influence their behavior25,26,27,28. Understanding how various sensory inputs are integrated within the nervous system is an outstanding question in neuroscience, particularly when integration occurs at the level of individual neurons9,29,30,31,32. Sensory integration is especially important in proprioception, which is itself an integrative modality that draws from multiple sensory cues1.
Understanding gravity sensation in Caenorhabditis has implications for human health. Millions of individuals suffer from vestibular dysfunction in the United States alone33, and many of these disorders have underlying genetic causes34,35. For this reason, the identification of gene candidates and targeted therapies is an active area of research36. Additionally, because vertebrate vestibular and auditory systems are closely linked developmentally and evolutionarily33,36,37, elucidating gravity sensation could also provide insight into hearing and hearing disorders.
The authors have nothing to disclose.
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.
1% Sodium Dodecyl Sulfate solution | From stock 10% (w/v) SDS in DI water | ||
15 mL Centrifuge tubes | Falcon | 14-959-53A | |
3 mm Hex key | Other similar sized metal tools may be used | ||
4% Agar in Normal Growth Medium (NGM) – 1 L | Prior to autoclaving: 3 g NaCl, 40 g Agar, 2.5 g Peptone, 2 g Dextrose, 10 mL Uracil (2 mg/mL), 500 μL 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) | ||
5 mL Serological pipettes | Fisherbrand | S68228C | Polystyrene, not borosilicate glass |
60% Cold sucrose solution | 60% sucrose (w/v) in DI water; sterilize by filtration (0.45 μm filter). Keep at 4 °C | ||
AF16 C. briggsae or other experimental strain | Available from the CGC (Caenorhabditis Genetics Center) | ||
Bunsen burner | |||
Cling-wrap | Fisherbrand | 22-305654 | |
Clinical centrifuge | |||
Disposable razor blades | Fisherbrand | 12-640 | |
Faraday cage | Can be constructed using cardboard and aluminum foil; 30" L x 6" W x 26" H or larger | ||
Ink markers | Sharpie or other brand for marking on plastic | ||
Labeling tape | Carolina | 215620 | |
M9 buffer | 22 mM KH2PO4, 42 mM Na2HPO4, 86 mM NaCl | ||
N2 C. elegans strain | Available from the CGC (Caenorhabditis Genetics Center) | ||
NGM plates with OP50 | 1.7% (w/v) agar in NGM (see description: 4% agar in NGM). Seed with OP50 | ||
Paraffin film | Bemis | 13-374-10 | |
Plastic cutting board | |||
Pliers | |||
Rotating vertical mixer | BTLab SYSTEMS | BT913 | With 22 x 15 mL tube bar |
Serological pipettor | Corning | 357469 | |
Stereo Microscope | Laxco | S2103LS100 | |
Tally counter | ULINE | H-7350 | |
Thick NGM/agar plate media – 1 L | See 4% Agar in NGM recipe; replace 40 g Agar with 20 g Agar | ||
Tweezers |