Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Summary

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.

Abstract

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.

Introduction

The interplay between chemical communication and reproductive success is a fundamental principle across the animal kingdom^{1,2,3,4,5,6,7,8,9,10}. 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 t....

Protocol

1. Crude sex pheromone extraction from females and hermaphrodites

1. Protocol for C. elegans Synchronization
   1. Preparation of adult females or hermaphrodites
      1. 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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
10 cm Petri dishes	Falcon	25373-100	Falcon bacteriological Petri dish 100 x 15 mm
6 cm Petri dishes	Falcon	25373-085	Falcon bacteriological Petri dish 60 x 15 mm
C. remanei (EM464)	CGC
Centrifuge	Eppendorf	centrifuge 5418	Any brand should work.
Chemoattraction assay plates	Homemade solution	N/A	1.5% agar, 25 mM NaCl, 1.5 mM Tris-base, and 3.5 mM Tris-Cl
Cholesterol	Alfa Aesar	57-88-5
Dissecting Microscope	Leica	LeicaMZ75	Any brand should work.
E. Coli OP50	CGC
Ethanol	Koptec	64-17-5
fog-2(q71) (JK574)	CGC
him-5(e1490)(CB4088)	CGC
Household bleach	Clorox Germicidal bleach concentrated		Bleach
M9 buffer	Homemade solution	N/A	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.
Magnesium Sulfate, Anhydrous, Powder	Macron	M1063-500GM-EA
Microwave	TOSHIBA	N/A	Any brand should work.
N2	CGC
NaOH	Sigma-aldrich	S318-3	1 M
NGM plates solution	Homemade solution	N/A	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 °C. Put on a hotplate at 65 °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).
Parafilm	Bemis	13-374-10	Bemis Parafilm M Laboratory Wrapping Film
Peptone	VWR	97063-324
Pipet- aid	Drummond Scientific	4-000-100	Any brand should work.
Plastic paper	Octago	Waterproof Screen Printing Inkjet Transparency Film	https://www.amazon.com/Octago-Waterproof-Transparency-Printing-Printers/dp/B08HJQWFGD
Potassium chloride	Sigma-aldrich	SLBP2366V
Potassium phosphate	Spectrum	7778-77-0
Pipette	Eppendorf	SKU: EPPR4331; MFG#: 2231300006	20 - 200 µL, 100 - 1000 µL, any brand should work.
Rotator	Labnet	SKU: LI-H5500	Labnet H5500 Mini LabRoller with Dual Direction Rotator. Any brand should work.
Sodium chloride	VWR	7647-14-5
sodium phosphate dibasic	Sigma-aldrich	SLCG3888
Tris-base	Sigma-aldrich	77-86-1
Tris-Cl	Roche	1185-53-1
Tryptone	VWR	97063-390
Vortex	Scientific industries	Vortex-Genie 2	Any brand should work.
WormLab system	MBF Bioscience	N/A	https://www.mbfbioscience.com/help/WormLab/Content/home.htm; https://www.mbfbioscience.com/products/wormlab/
Wormpicker	Homemade	N/A	made with platinum and glass pipet tips