volatile sex pheromone chemoattraction assay in c. elegans

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.

Volatile Sex Pheromone Chemoattraction Assay in C. elegans

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%auger, 25 millimolar sodium chloride, 1.5 millimolar Tris base, and 3.5 millimolar Tris hydrochloride. Heat the auger in the chemoattraction solution in a microwave until completely dissolved.After cooling, use a pipette to evenly distribute the chemoattraction auger solution into petri dishes. Leave the lids open in a clean area to allow the surface of the auger 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 worms'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.

volatile-sex-pheromone-chemoattraction-assay-in-c-elegans

Research

JoVE Journal

Behavior

94 Görüntüleme. Başlamak için, yetişkin hermafrodit dişi solucanları içeren kültür plakasını inkübatörden çıkarın. Him-5 solucanlarını senkronize ettikten sonra, solucanları M9 tamponu ile beş kez yıkayın. Bir günlük yetişkin erkekleri tohumlanmış plakadan M9 tamponu içeren bir tüpe aktarın.Kalan bakterileri ortadan kaldırmak ve tahlil sırasında yiyeceklerden kaynaklanan etkileşimi önlemek için solucanları tohumlanmamış bir NGM pl...