TITLE: Chemical ecology in the classroom: chemotaxis assay using C. elegans
AUTHORS: Nicole Bradon, Katherine Fiocca, Maria D. Sallee, Lauren Cote, Daniel A. Shaykevich, Lauren A O'Connell

[DESCRIPTION]
There are many natural products in the environment that influence other organisms. How nervous systems sense these compounds to initiate a behavioral response remains an active area of chemical ecology research. We developed a Course Undergraduate Research Experience (CURE) utilizing Caenorhabditis elegans chemotaxis assays to test how ant compounds are detected by heterospecific nervous systems. These experiments can be conducted in an undergraduate laboratory course, where new insights into interspecies interactions can be generated through genuine research experiences in a classroom setting.


				

			

		

	

[GUIDELINES]
This experiment can be designed to test multiple worm strains with the same compound, one worm strain with multiple compounds, or multiple strains and compounds. The Caenorhabditis Genetics Center (CGC) and the Caenorhabditis Natural Diversity Resource (CeNDR) are good sources for wild-type and mutant worm strains.

If using this protocol for teaching, we have found the following works well:
- A max of 20-24 students per class
- One round of training with known attractive and repulsive compounds is beneficial prior to exploring unknown compounds or extracts.
- Students typically start out performing 3 assays each during training, but with experience can reliably run up to 9 assays at once.
- During training and early experiments, increased sample size per group is needed to account for data variation and statistics. For example, all students should perform the training assays to maximize the sample size (20 students = 20 samples per compound tested).
- Worm numbers should be scaled with the experiment by calculating the number of worms needed per assay (300) and accounting for some loss (500 worms per plate needed).  For example worms can be grown on an extra large (150 mm) plate and used for steps 2 and 3 by washing with 10 mL of M9 buffer and bleaching in 10mL of bleach solution in a 15 mL falcon.
- Code the compounds or worm strains such that students are unaware of treatment group until they submit their data to the instructor. 
- For ant collections, students construct ant aspirators, collect ants outside, and then sort ants in the laboratory. Instructors perform the extraction steps outside of class time.

[STEPS]
SECTION: Obtaining chemical extracts for use as chemotaxis stimuli
8. Place the collected ants in a freezer for 15 min until immobile.

SECTION: Obtaining chemical extracts for use as chemotaxis stimuli
9. Wearing gloves, sort ants by species into medium weighing boats. 
SECTION: Prepare chemotaxis plates
11. Label chemotaxis plates.
SECTION: Prepare chemotaxis plates
13. Incubate chemotaxis plates for 30 min to establish a chemical gradient. Chemotaxis plates should remain flat, upright, and with lids on.

SECTION: Prepare chemotaxis plates
12. Take the lid off a chemotaxis plate and apply compounds by adding 5 µL of experimental compounds to each of the dots marked "E" and 5 µL of solvent to each of the "X" marks. Place the lid back on the plate. Repeat for other chemotaxis plates.

SECTION: Prepare chemotaxis plates
11.1. Tape a transparent assay label to the bottom plate (the side with the agar). 
 

SECTION: Prepare chemotaxis plates
11.2. With a fine marker, label the side of the bottom plate with the compounds to be tested and the experimenter's initials. If possible, code the compounds such that the experimenter is unaware of treatment.
 

SECTION: Prepare worms for chemotaxis
14. Take a moment to look at the worms under a dissection scope. If applicable, note any phenotypic differences (behavior, movement, body shape, etc) between wild type and other strains such mutant lines or wild isolates. 
SECTION: Set up the chemotaxis assay
19. Add 2 µL of 0.5 M sodium azide to the four compound (E & X) spots on each chemotaxis plate. Take care to not put sodium azide in the center dot. Change pipette tips each time and being careful not to poke the agar. 
 

 


SECTION: Prepare worms for chemotaxis
15. Label a 1.5 mL tube with the worm strain and experimenter's initials. Find the 0.1 mL marking on the tube and use a marker to make the mark more visible. This will let you know how much liquid to take out at the end.
SECTION: Prepare worms for chemotaxis
16. Harvest young adult worms from the NGM worm embryo plate prepared in Step 3.7.  


SECTION: Prepare worms for chemotaxis
17. Wash the worms with chemotaxis buffer. 
 

SECTION: Prepare worms for chemotaxis
16.1. With a P1000 micropipette, add 1 mL of chemotaxis buffer to the worm plate. Gently swirl the plate. 

SECTION: Prepare worms for chemotaxis
16.2. Tilt the plate to allow the buffer and the suspended worms to collect at the lower edge of the plate.
SECTION: Prepare worms for chemotaxis
16.3. Pipette the solution up and over the agar surface and allow the worms and liquid to collect in the bottom. Repeat to get as many worms off the plate as possible (recommended 3 times).
SECTION: Prepare worms for chemotaxis
16.4. Pipette all the buffer and worms off the plate and transfer to the 1.5 mL tube.
SECTION: Prepare worms for chemotaxis
17.2. Carefully remove consecutive batches of 200 µLof the buffer from the tube. This is recommended so as to not to disturb the worm pellet. Discard the liquid in a liquid waster container.

SECTION: Prepare worms for chemotaxis
17.3. Gently add 900 µL of chemotaxis buffer to the tube. Pro tip: do not resuspend worms with a pipette between washes, as worms stick to the plastic tip.

SECTION: Prepare worms for chemotaxis
17.4. Repeat this wash process twice for a total of three washes.
SECTION: Prepare worms for chemotaxis
17.1. Place the tube containing the worms suspended in buffer in a tube rack and incubate 2 min, allowing the worms to fall to the bottom of the tube.

SECTION: Prepare worms for chemotaxis
18. After the final wash, leave roughly 100 µL of buffer on top of the worms by pipetting out the buffer. 

SECTION: Set up the chemotaxis assay
20. Agitate the worms by flicking the tube. Use a micropipette to transfer worms to the center spot of each plate. The goal is to add roughly 100 worms to each plate. Pro tip: use the same pipette tip for adding the worms to each plate.
If four chemotaxis assays are being run, then transfer ~25-30 µl per plate. Scale up or down depending on the number of plates.
SECTION: Set up the chemotaxis assay
21. Incubate chemotaxis plates face up and lid on for 30 min at room temperature.

SECTION: Set up the chemotaxis assay
20.1. Optional: If after 5 m, the worms are still swimming in a puddle in the middle, you may need to remove excess liquid. To do this, touch the edge of a filter paper or kimwipe to the worm drop center. This is best done looking through a dissecting scope. The goal is to remove excess liquid so that the worms can crawl away from the center. This must be done carefully because worms can stick to the Kimwipe. The goal is to have roughly 100 worms per plate. 
SECTION: Worm quantification and data visualization
22. Using a dissecting scope, focus on a chemotaxis plate such that the worms are visible within a single quadrant.
SECTION: Worm quantification and data visualization
23. Using a clicker, quantify the number of worms on the E or X quadrants on each plate. It is recommend that both X or both E quadrants are counted first and then the other two can be counted. 
SECTION: Worm quantification and data visualization
23.1. Counting tips: if the worm is on a line it is counted in the total number of worms, but not assigned to the S or E quadrants. Worms in the center circle or on the plastic rim of the plates are not counted at all.
SECTION: Worm quantification and data visualization
24. Calculate the Chemotaxis Index (CI) for each plate, where positive values suggest attraction and negative values suggest repulsion: CI = (Number of worms in the two experimental quadrants – Number of worms in the two solvent quadrants) / Total number of worms on the plate.
SECTION: Building insect aspirators using household objects
4. Cut a plastic straw in half and tape a small piece of filter around one end of one half of the straw. The filter should cover the open end. Keep the other half of the straw for later. 
 

SECTION: Building insect aspirators using household objects
5. Prepare the plastic cup.
SECTION: Building insect aspirators using household objects
6. Place the lid on top and test the suction by placing your mouth on the open straw end with the filter and sucking in.
 

SECTION: Building insect aspirators using household objects
5.1. Make two holes with the dissection needle on either side of the deli cup. The holes should line up across from one another. Make the holes wide enough for a straw using a pen.
 

SECTION: Building insect aspirators using household objects
5.2. Insert the two halves of the straw into each hole. The filter side of one of the straw halves should be inside of the cup with the open side outside of the cup.
 

SECTION: Building insect aspirators using household objects
5.3. Line the holes on the outside of the cup with clay by rolling a small log of clay and shaping it into a circle around the hole. 
 

SECTION: Obtaining chemical extracts for use as chemotaxis stimuli
10. Prepare chemical extract.
SECTION: Obtaining chemical extracts for use as chemotaxis stimuli
10.1. Label a 12 mL glass vial with ant species name and fill with 8 mL of methanol (>95%).

SECTION: Obtaining chemical extracts for use as chemotaxis stimuli
10.2. Add ant species to their corresponding glass vial until the volume of ants is halfway up the liquid line within the glass vial.
 

SECTION: Obtaining chemical extracts for use as chemotaxis stimuli
10.3. Once ants are added, place glass vials in the -80 °C freezer for 1440 min.

SECTION: Obtaining chemical extracts for use as chemotaxis stimuli
10.4. Pipette the methanol to a clean glass vial, leaving the ants behind.    
 

SECTION: Obtaining chemical extracts for use as chemotaxis stimuli
10.5. Evaporate the methanol-only samples under a constant flow of nitrogen gas using a nitrogen evaporator.
 

SECTION: Obtaining chemical extracts for use as chemotaxis stimuli
10.6. Once dry, add 8 mL of dimethyl sulfoxide (DMSO) to the glass vials to resuspend your chemical extracts.

SECTION: Worm quantification and data visualization
26. Boxplots are useful for highlighting variation between assays without a treatment group. An example dataset (see attached csv file) is shown, where undergraduate students learned how to perform this assay using a known repulsant (carvone), a known attractant (isoamyl alcohol), and solvent (control). 

This boxplot was generated in R using the following code:
library(ggplot2)
library(wesanderson)

data <- read.csv(file="exampledata.csv", row.names=1)

ggplot(data, aes(y=CI, x=compound, fill=compound)) + geom_boxplot() + 
  geom_jitter(position=position_jitterdodge(0.2), shape = 21, size = 4, alpha=0.6) + 
  theme_classic() + theme(legend.position="top", axis.text=element_text(size=12)) + 
  scale_fill_manual(values = wes_palette("FantasticFox1", n = 3)) + 
  geom_hline(yintercept=0)

 

SECTION: Counting and plating C. elegans embryos
3. Plate the embryos
SECTION: Expand C. elegans stock
1. Prepare nematode growth medium (NGM) plates to grow worms.
SECTION: Synchronize C.elegans by bleaching
2. Synchronize worms by bleaching 3 to 4 days before the chemotaxis assay.

 

SECTION: Synchronize C.elegans by bleaching
2.1. Prepare a fresh bleach solution: 9.8 mL MilliQ water, 600 µL 5N KOH, 1.2 mL 12% sodium hypochlorite.

SECTION: Synchronize C.elegans by bleaching
2.2. Place ~60 mL M9 buffer, bleach solution and a new 15 mL conical tube on ice.

SECTION: Synchronize C.elegans by bleaching
2.3. Use a glass Pasteur pipet, transfer the worms from one petri dish to a 15mL conical by washing the adult worms containing many embryos (gravid hermaphrodites) off the NGM plates with approximately 2 mL of M9 buffer.
 


SECTION: Synchronize C.elegans by bleaching
2.4. Repeat with another 2 mL of M9 buffer.

SECTION: Synchronize C.elegans by bleaching
2.5. Centrifuge 800 rcf, 1 min, 4 °C  to pellet worms.  
SECTION: Synchronize C.elegans by bleaching
2.6. Remove the supernatant with a Pasteur pipet (collect as chemical waste).
SECTION: Synchronize C.elegans by bleaching
2.7. Add 4 mLM9 buffer to the 15 mL conical tube and resuspend the worms.

SECTION: Synchronize C.elegans by bleaching
2.8. Centrifuge 800 rcf, 1 min, 4 °C to pellet worms.
SECTION: Synchronize C.elegans by bleaching
2.9. Remove the supernatant with a Pasteur pipet, leaving behind 1mL of M9 buffer and the worm pellet.
 

SECTION: Synchronize C.elegans by bleaching
2.10. Add 5 mL of the bleach solution to the 1 mL worm pellet.

SECTION: Synchronize C.elegans by bleaching
2.11. Vortex for 5 min.
 


SECTION: Synchronize C.elegans by bleaching
2.12. Centrifuge 800 rcf, 1 min, 4 °C to pellet embryos.
SECTION: Synchronize C.elegans by bleaching
2.13. Remove the supernatant leaving 0.5 mL of worm embryo/ bleach solution.  The worm pellet should be slightly yellow. 
 



SECTION: Synchronize C.elegans by bleaching
2.14. Add 12 mL M9 buffer to the 15mL conical tube and resuspend the embryos by shaking.

SECTION: Synchronize C.elegans by bleaching
2.15. Centrifuge 8000 rcf, 1 min, 4 °C  to pellet embryos.

SECTION: Synchronize C.elegans by bleaching
2.16. Remove the supernatant leaving 0.5 mL of embryo mixture.  The embryos will be white or clear in color.

 


SECTION: Synchronize C.elegans by bleaching
2.17. Repeat 3 more times for a total of 4 washes.  
SECTION: Expand C. elegans stock
1.6. Transfer adult C. elegans to NGM plates seeded with OP50 E. coli.  Allow the adult worms to reproduce for 2-4 days at Room temperature .
 


SECTION: Expand C. elegans stock
1.5. Add 500 µL of OP50 E. coli to the center of the NGM plate and gently swirl the plate to allow the bacteria to form a circle. Allow the bacteria to grow on the NGM plate (right side up) for 24-48 hours at room temperature.  The E. coli provides nourishment for the worms. Plates may be stored at 4 °C  upside down.
 



SECTION: Counting and plating C. elegans embryos
3.1. Vortex the 0.5 mL embryo mixture.

SECTION: Counting and plating C. elegans embryos
3.2. Add 2 µL of embryo mixture to 18 µL of M9 buffer in a microcentrifuge tube.

SECTION: Counting and plating C. elegans embryos
3.3. Pipette 1 µL of this dilution into 3 wells of a microscope PFTE coated slide.
 

 


SECTION: Counting and plating C. elegans embryos
3.6. Pipet approximately 500 embryos around the outer edge of a new NGM 60 mm plate seeded with OP50 E. coli.  Prepare 1 plate for every 2 chemotaxis assays.


 



SECTION: Counting and plating C. elegans embryos
3.7. Young adult worms will be present after 2880 min of incubation at 20 °C.
 


 


SECTION: Expand C. elegans stock
1.4. Allow the petri dishes to dry with the lid on and face up at room temperature for 2880 min.  After the petri dishes are dry, they may be inverted and stored at 4 °C or seeded with bacteria.
 


SECTION: Counting and plating C. elegans embryos
3.4. Using a dissecting scope, focus on the slide such that the embryos are visible within a single well. 
SECTION: Counting and plating C. elegans embryos
3.5. Using a clicker, quantify the number of embryos per well and note if there are any worms present.  All of the worms should have dissolved in the bleaching step.  Add the total number of embryos in the 3 wells and take the average.  Multiple the average number of embryos per well by the dilution factor of 10 to get the number of embryos per 1 µL .   Divide 500 by the number of embryos per microliter to determine the volume of the embryo solution needed for the next step.

SECTION: Expand C. elegans stock
1.1. Combine the following materials and stir for 10 min
3 g NaCl
20 g agar
2.5 g peptone
975 mL water
Autoclave and allow to cool to 50 °C




SECTION: Expand C. elegans stock
1.2. Add additional ingredients to the cooled media and mix:
1 mL 5 mg/ml cholesterol in 95% EtOH
1 mL 1M CaCl2
1 mL 1M MgSO4
25 mL 1M potassium phosphate buffer, pH 6



SECTION: Expand C. elegans stock
1.3. Plate nematode growth medium (NGM) using aseptic technique (under a flame) by adding 12.5 mL to each 60 mm petri dish.

SECTION: Obtaining chemical extracts for use as chemotaxis stimuli
7. This protocol is written for extracting compounds from ants, but other arthropods, plants, microbes, or other biological materials can be used.
SECTION: Worm quantification and data visualization
25. Plot the chemotaxis index values for each plate, with the chemotaxis index on the y-axis and compound on the x-axis.