Competitive fitness assays

This protocol allows for fitness comparisons between a C. elegans mutant strain to that of wild type on a single experimental plate. This can be modified to enable the testing of any experimental variable on two different strains simultaneously. 

Strains used: 

• N2 [Control strain] 
• AWR73:  aaim-1 (kea22) [This was our strain of interest. Use the strain you are trying to compare to the below marked strain]
• YY1446:  RFP::Znfx1 (gg634)  [Contains RFP in germ granules of all life stages, enabling rapid identification of wild-type animals]  

Pathogens used: 

• Microsporidia: Nematocida parisii 
• Bacteria: Pseudomonas aeruginosa PA14::DsRed 

Microsporidia competitive fitness assay: 

1. Prepare a microcentrifuge tube containing 1 ml of 10x OP50-1 and desired concentration of N. parisii spores. For uninfected controls, replace the volume of spores with that of M9. 
2. Pipette this mixture up and down, and plate it onto an unseeded 10 cm NGM plate. 
3. Dry the plate in a drying hood. 
4. Transfer 10 bleach synchronized L1’s from each strain onto lawns of spores and E. coli OP50-1 immediately after drying. [E.g. One plate would contain 10 N2 L1’s and 10 YY1446 L1’s] 
5. Incubate plates at 21°C for 8 days. 

Pseudomonas aeruginosa competitive fitness assay: 

1. Streak out PA14::DsRed onto an LB agar plate and incubate overnight at 37°C. 
2. Pick a single colony into 3ml of LB and grow overnight at 37°C 220 RPM for 16-18 hours. 
3. Pipette 20 μl of culture onto the center of a 3.5 cm SK plate and incubate at 37C for 24 hours, followed by incubation at 25C for 24 hours. 
4. Transfer 10 bleach synchronized L1’s from each strain onto PA14::DsRed plates. 
5. Incubate plates at 21°C for 8 days. 

Washing, fixing and imaging. [Same for microsporidia and Pseudomonas aeruginosa PA14::DsRed assays.] 

1. Wash animals off plates with 1 ml of M9 +0.1% Tween-20 and place in a microcentrifuge tube. 
2. Gravity settle worms, and aspirate supernatant. 
3. Repeat step 1-2 twice more, to remove bacteria from supernatant. 
4. Add 500 μl of 4% Paraformaldehyde (PFA). 
   1. Note: RFP germ granules are not visible when acetone is used as a fixative. 
5. Incubate at room temperature for 30 minutes. 
6. Spin down samples at 10,000 rpm for 30 seconds.
7. Aspirate supernatant and transfer PFA to appropriate hazardous waste container.
8. Resuspend worm pellet in 20 μl of EverBrite Mounting Medium (Biotium). 
9. Mount entire sample in 10 μl volumes onto glass slide to quantify the entire population. 
10. Quantify the number of animals from each strain present in the population using a fluorescent microscope. The strain possessing the RFP granules is your marked wild-type strain, whereas that missing the signal is your query strain. This will allow you to assess the fraction of the population composed of each strain. The more abundant strain in the population displays increased fitness under the experimental conditions tested. Note that by the end of the assay (8 days at 21°C), the population will be composed of F1 adults and young L1/L2 animals.