extraction and storage of volatile sex pheromones from c. elegans and c. remanei females

Extraction and Quantification of Volatile Sex Pheromone Responses in &lt;i&gt;C. elegans&lt;/i&gt;

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.

Author Spotlight: Examining Volatile Sex Pheromone Influence on Male &lt;i&gt;C. elegans&lt;/i&gt; Behavior

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Our research examines how the volatile sex pheromone affects male C.elegans behavior using chemotaxis assay. It aims to identify the molecular mechanism of pheromone production and the neuronal mechanism of it is perception. Individual worm to worm variability in response to 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 straight forward and highly reproducible.It is designed to optimize pheromone yet 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.

Extraction and Storage of Volatile Sex Pheromones from C. elegans and C. remanei Females

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.

extraction-storage-volatile-sex-pheromones-from-c-elegans-c-remanei

Research

JoVE Journal

Behavior

60 Views. 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 ...