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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 stimuli
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
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.......
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. Addi.......
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.
....Name | Company | Catalog Number | Comments |
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 |
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