We are grateful to Dr. Tingtao Zhou for designing and writing the code for the trajectory visualizations used in our analysis. This work was supported by funding: R01 NS113119 (PWS), Chen senior postdoc fellowship, and the Tianqiao and Chrissy Chen Institute for Neuroscience.

1. Crude sex pheromone extraction from females and hermaphrodites
<ol>
	<li>Protocol for C. elegans Synchronization
	<ol>
		<li>Preparation of adult females or hermaphrodites
		<ol>
			<li>Monitor culture plates daily until a large population of adult female/hermaphrodites exists and the OP50 food source depletes. Using fog-2 C. elegans and WT C. remanei females for crude sex pheromone extraction, prepare synchronized eggs from the mated females. 
			NOTE: In this protoc...

Extraction and Quantification of Volatile Sex Pheromone Responses in C. elegans

This protocol provides a robust methodology for the extraction of volatile sex pheromones from C. elegans, along with establishing a robust chemoattraction assay to measure male chemoattraction responses. Additional information can be found in the WormLab user guide (see the Table of Materials); for a basic code to visualize worm movement trajectory, see protocol section 7.3.8.5. Several crucial steps in the protocol are important for the outcome. First, careful synchronization of worm populatio...

The interplay between chemical communication and reproductive success is a fundamental principle across the animal kingdom1,2,3,4,5,6,7,8,9,10. Sex pheromones trigger a wide array of sexually dimorphic behaviors essential for locating mates, coordinating the steps involved in finding and attracting a partner, and ultimately promoting the propagation of a species11,12,13,14,15,16,17. Significant progress has been made in understanding pheromone signaling, but the molecular mechanisms, neural circuits, and computational processes governing these interactions often remain incompletely defined18,19,20,21,22,23,24,25,26.
The nematode Caenorhabditis elegans provides a powerful model for dissecting these questions. Notably, C. elegans exhibits an unusual reproductive strategy-hermaphrodites can self-fertilize but also outcross with males27,28,29,30,31,32,33. This flexibility requires a robust communication system to signal reproductive status. C. elegans is known for its well-characterized water-soluble pheromones, the ascarosides, which play varied roles in development, behavior, and social interactions. Recent discoveries have unveiled a distinct class of volatile sex pheromones employed by nematodes. These pheromones are specifically produced by sexually mature C. elegans and C. remanei virgin females and sperm-depleted hermaphrodites, serving as an attractant for adult males29,34,35. This attractant exhibits remarkable sexual dimorphism in its production and perception. The female somatic gonad governs the synthesis of this volatile sex pheromone, and production dynamically reflects reproductive status, ceasing upon mating and resuming several hours later29,34.
Understanding nematode sex pheromone communication provides insights into the evolution of chemical communication systems, the interplay between reproductive state and behavior, and the mechanisms underlying sexually dimorphic neural processing24,26,36,37,38,39. Studies implicate the amphid neuron AWA in males as critical for pheromone detection, with the G-protein-coupled receptor SRD-1 playing a key role in pheromone detection in males24. C. elegans is well-suited for studying animal chemical communication, especially sex pheromone signaling, due to its reliance on the olfactory system for mate-searching. While much is known about ascaroside signaling, the volatile sex pheromone system offers unique opportunities for comparison25,26,36,40,41,42,43,44,45,46,47,48,49,50, 
51,52,53,54,55,56,57. Moreover, C. elegans is a powerful genetic model organism due to its fully sequenced genome, clearly defined cellular lineage, and well-characterized olfactory mutants.
However, the complete neural circuitry involved in processing this pheromone, the computations that translate its perception into targeted mate-searching behaviors, and its biosynthesis regulation remain to be fully elucidated. Further investigations into these processes are crucial for understanding the diverse mechanisms governing animal communication and reproductive behaviors. The identification of key genes involved in pheromone synthesis, secretion, and perception promises to unveil novel molecular players in animal communication.&#160;The assays&#160;described here provide a basis to address these&#160;questions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm Petri dishes</td>
			<td>Falcon</td>
			<td>25373-100</td>
			<td>Falcon bacteriological Petri dish 100 x 15 mm</td>
		</tr>
		<tr>
			<td>6 cm Petri dishes</td>
			<td>Falcon</td>
			<td>25373-085</td>
			<td>Falcon bacteriological Petri dish 60 x 15 mm</td>
		</tr>
		<tr>
			<td>C. remanei (EM464)</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>centrifuge 5418</td>
			<td>&#160;Any brand should work.</td>
		</tr>
		<tr>
			<td>Chemoattraction assay plates</td>
			<td>Homemade solution</td>
			<td>N/A</td>
			<td>1.5% agar, 25 mM NaCl, 1.5 mM Tris-base, and 3.5 mM Tris-Cl</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Alfa Aesar</td>
			<td>57-88-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Leica</td>
			<td>LeicaMZ75</td>
			<td>&#160;Any brand should work.</td>
		</tr>
		<tr>
			<td>E. Coli OP50</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Koptec</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>fog-2(q71) (JK574)</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>him-5(e1490)(CB4088)</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Household bleach</td>
			<td>Clorox Germicidal bleach concentrated</td>
			<td></td>
			<td>&#160;Bleach</td>
		</tr>
		<tr>
			<td>M9 buffer</td>
			<td>Homemade solution</td>
			<td>N/A</td>
			<td>3 g KH2PO4, 11.3 g Na2HPO4.7H2O, 5 g NaCl, H2O to 1 L. Sterilize by autoclaving. Add 1 mL 1 M MgSO4 after cool down to room temperature.</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate, Anhydrous, Powder</td>
			<td>Macron</td>
			<td>M1063-500GM-EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>TOSHIBA</td>
			<td>N/A</td>
			<td>&#160;Any brand should work.</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-aldrich</td>
			<td>S318-3</td>
			<td>1 M</td>
		</tr>
		<tr>
			<td>NGM plates solution</td>
			<td>Homemade solution</td>
			<td>N/A</td>
			<td>2.5 g Peptone, 18 g agar, 3 g NaCl, H2O to 1 L.Sterilize by autoclaving. Once the autoclave is done (2 h), wait until the temperature of the medium drops to 65 &#176;C. Put on a hotplate at 65 &#176;C and stir. Then add the following, waiting 5 min between each to avoid crystallization: 1 mL CaCl2 (1 M), 1 mL MgSO4 (1 M), 25 mL K3PO4 (1 M, pH=6), 1 mL Cholesterol ( 5 mg/mL in ethanol).</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>13-374-10</td>
			<td>Bemis Parafilm M Laboratory Wrapping Film</td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>VWR</td>
			<td>97063-324</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet- aid</td>
			<td>Drummond Scientific&#160;</td>
			<td>4-000-100</td>
			<td>&#160;Any brand should work.</td>
		</tr>
		<tr>
			<td>Plastic paper&#160;</td>
			<td>Octago</td>
			<td>Waterproof Screen Printing Inkjet Transparency Film</td>
			<td>https://www.amazon.com/Octago-Waterproof-Transparency-Printing-Printers/dp/B08HJQWFGD</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-aldrich</td>
			<td>SLBP2366V</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate</td>
			<td>Spectrum</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf</td>
			<td>SKU: EPPR4331; MFG#: 2231300006</td>
			<td>20 - 200 &#181;L, 100 - 1000 &#181;L, any brand should work.</td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>Labnet</td>
			<td>SKU: LI-H5500</td>
			<td>&#160;Labnet H5500 Mini LabRoller with Dual Direction Rotator. Any brand should work.</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>VWR</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium phosphate dibasic</td>
			<td>Sigma-aldrich</td>
			<td>SLCG3888</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Sigma-aldrich</td>
			<td>77-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Cl&#160;</td>
			<td>Roche</td>
			<td>1185-53-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>VWR</td>
			<td>97063-390</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Scientific industries</td>
			<td>Vortex-Genie 2</td>
			<td>&#160;Any brand should work.</td>
		</tr>
		<tr>
			<td>WormLab system&#160;</td>
			<td>MBF Bioscience</td>
			<td>N/A</td>
			<td>https://www.mbfbioscience.com/help/WormLab/Content/home.htm; https://www.mbfbioscience.com/products/wormlab/</td>
		</tr>
		<tr>
			<td>Wormpicker</td>
			<td>Homemade&#160;</td>
			<td>N/A</td>
			<td>made with platinum and glass pipet tips</td>
		</tr>
	</tbody>
</table>

volatile sex pheromone extraction and chemoattraction assay in caenorhabditis elegans

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males.
This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.

Author Spotlight: Examining Volatile Sex Pheromone Influence on Male C. elegans Behavior

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Trajectory analysis of volatile sex pheromone perception defective strain in chemoattraction assay 
This chemoattraction assay reliably differentiates between wild-type and mutant strains of C. elegans in their response to volatile sex pheromones. Successful experiments with him-5 males consistently demonstrate robust chemotaxis towards the pheromone source. This is reflected in a high chemotaxis index (C.I.) (Figure 2), often exceeding 0.5, indicating...

This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and ...

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Abstract