Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Our research examines how the volatile sex pheromone affects male surrogates behavior using chemotaxis assay. It aims to identify the molecular mechanism of pheromone production, and the neuronal mechanism of its perception. Individual worm to worm variability in response to the pheromone, coupled with the influence of environmental factors such as temperature, humidity, and light can introduce significant noise into the data.

Moreover, the complex nature of worm behaviors complicates data interpretation. Addressing those challenges requires careful experimental design. The protocol is straightforward and highly reproducible.

It is designed to optimize pheromone health while minimizing time and resource consumption. The chemotaxis assay provides quantitative data on male attraction, enabling statistical analysis and comparison. This protocol provides a completed picture of pheromone communication.

To begin, separate 200 L4 stage FOG-2 Caenorhabditis elegans virgin females one day prior to pheromone extraction. Distribute the females onto three separate six-centimeter OP50 NGM plates. On the extraction day, transfer 100 virgin one-day old FOG-2 females into a micro centrifuge tube containing one milliliter of M9 buffer.

After the last wash, incubate females in 100 microliters of M9 buffer for six hours at 20 degrees Celsius to allow pheromone production. Similarly, isolate 15 to 20 L4 stage Caenorhabditis remanei females onto three OP50 NGM plates. After washing, incubate five virgin females in 100 microliters of M9 buffer for six hours at 25 degrees Celsius.

Centrifuge the worms at 15, 000 G for 30 to 60 seconds. Carefully transfer the supernatant containing pheromones to a clean tube. Discard the tube containing the worm pellet.

Store the collected volatile sex pheromone extract at minus 80 degrees Celsius. After synchronizing the healthy adult Caenorhabditis elegans worms, wash them five times with M9 buffer. To synchronize worms at the L1 stage, rotate them in the M9 buffer for 12 to 15 hours to arrest development.

Observe the arrested L1 worms onto a 10-centimeter NGM culture plate seeded with OP50 bacteria. To minimize the presence of males on a hermaphrodite plate, check the plate two days after worm release and remove any observed males. After three days of development, observe the appearance of embryos, indicating that the worms have become reproductively mature adults.

Wash away the embryos repeatedly with M9 buffer and let them settle until most of the adults are at the bottom of the tube. Pipette the separated adults and transfer them to a new OP50 seeded NGM plate. To begin, remove the culture plate containing adult hermaphrodite female worms from the incubator.

After synchronizing him-5 worms, wash the worms five times with M9 buffer. Transfer the one-day old adult males from the seeded plate to a tube containing M9 buffer. Place the worms on an unseeded NGM plate to eliminate residual bacteria and prevent interference from food during the assay.

Prepare a chemoattraction assay plate with 1.5%ager, 25 millimolar sodium chloride, 1.5 millimolar Tris-base, and 3.5 millimolar Tris-hydrochloride. Heat the ager in the chemoattraction solution in a microwave until completely dissolved. After cooling, use a pipette to evenly distribute the chemoattraction ager solution into Petri dishes.

Leave the lids open in a clean area to allow the surface of the ager to dry slightly. Once the surface has dried appropriately, close the lids. To conduct the chemoattraction assay, mark three distinct spots on the lid and the underside of the Petri dish.

Apply two microliters of one molar sodium azide to each experimental and control spot on the plate. Pick 20 healthy and freely moving male worms with a worm picker. Under a dissecting microscope, release the worm at the starting point.

Quickly add two microliters of M9 buffer to the control spot, and two microliters of sex pheromone to the experimental spot on the lid. Gently close the lid and place the plate in a quiet temperature-stable area. After 30 minutes, score the assay by counting the number of worms at each spot.

For an enhanced assessment of chemotaxis, record the worm's trajectories using a camera. The chemotaxis index for him-5 males was consistently above 0.5, indicating a strong attraction to the sex pheromone source, while the chemotaxis index for SRD-1 males was around zero. Successful chemoattraction assays showed him-5 males moving closer to the pheromone source over time, with color-coded trajectories indicating time, distance, speed, straightness, and direction correctness.