<meta charset="utf8"></head><body>Estrazione e quantificazione delle risposte volatili ai feromoni sessuali in C. elegans

<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

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.

<meta charset="utf8"></head><body>Autore in primo piano: Esame dell'influenza volatile dei feromoni sessuali sul comportamento del maschio di C. elegans

Estrazione di feromoni sessuali volatili e test di chemioattrazione in Caenorhabditis elegans

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

volatile-sex-pheromone-extraction-chemoattraction-assay

Research

JoVE Journal

Behavior

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

1.2K Views.  California Institute of Technology. La nostra ricerca esamina come il feromone sessuale volatile influenzi il comportamento maschile di C.elegans utilizzando il test della chemiotassi. Ha lo scopo di identificare il meccanismo molecolare della produzione di feromoni e il meccanismo neuronale di esso è la percezione. La variabilità individuale da verme a verme in risposta ai feromoni, unita all'influenza di fattori ambientali come temperatura, umidità e luce, può introdurre un rumore signifi...

Video: Autore in primo piano: Esame dell'influenza volatile dei feromoni sessuali sul comportamento del maschio di C. 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.

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.