Name: Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans (Video) | JoVE Journal
Uploaded: 2024-08-09T14:35:00.000+00:00
Duration: 6 min 49 s
Description: Learn volatile sex pheromone extraction and chemoattraction assays in Caenorhabditis elegans to study chemical communication and male behavior. This reproducible protocol enables quantitative analysis of pheromone responses.

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5 minutes

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.

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

The authors have no conflicts of interest to disclose.

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 populations is essential to control the age and the reproductive state, ensuring the successful extraction of volatile sex pheromones from virgin females or sperm-depleted hermaphrodites29,34. Second, repeated washing steps are necessary to thoroughly remove bacteria, minimizing contamination that could interfere with pheromone signaling in subsequent chemotaxis assays. Next, the choice of assay parameters, such as attractant concentration, plate size, and assay duration, can all influence outcomes and must be tailored to the specific experimental goals.
Accurate drying times and consistent spacing of test spots are critical for reliable chemotaxis assay results. Wet plates significantly affect the chemotaxis index. If a layer of water remains on the agar surface, capillary forces may trap the worms at the starting point. This can hinder their movement, leading to false negative results in the assay. Further, prolonged drying times pose a different challenge for those released later. As the plate dries, volatile pheromone components evaporate and diffuse, creating a less distinct concentration gradient. This makes it difficult for later-released worms to locate the target pheromone, even if they are free to move later. Therefore, ensure that the plates are sufficiently dry to eliminate any surface moisture that could affect the experiment&#39;s outcomes.
The volume of agar solution added to the plates must be adjusted according to the specific requirements of the experiment, ensuring optimal assay conditions. The thickness of the agar layer, adjusted by varying the amount of agar solution poured into the dish. Typically, a shorter distance between the attractant and the agar plate leads to more robust results. Conversely, a longer distance can help eliminate the effects of non-volatile components, providing a clearer assessment of the volatile attractant&#39;s influence. The agar thickness can be tailored to the specific experimental needs, as shown in Figure 2B.
The process of picking 20 worms should not exceed 1-2 min to prevent the early picked worms from drying out and becoming unhealthy, which could affect the results. Ensure the release of worms onto the assay plate simultaneously to prevent the early-released worms from random walking too far from the starting point. Delays in this step can lead to variable starting positions among the samples, resulting in unfair comparisons. The entire process from picking the males to closing the lid should take between 2 and 5 min.
Tailor the spacing between the experimental/control spots and the starting point on the assay plate according to the plate size and the experiment&#39;s specific objectives. Increasing the distance between the spots can make the assay more challenging, which is beneficial for detecting subtle effects. Conversely, reducing the distance creates a more robust assay test that highlights severe defects.
Troubleshooting may be necessary at various points in the protocol. If the extracted pheromone does not induce a chemotaxis response, then carefully verify the age and reproductive status of the source females and hermaphrodites. Only virgin females or sperm-depleted hermaphrodites will produce the target pheromone. Due to the high mating efficiency of C. elegans males, the presence of even a single male on the L4 female isolation plate can drastically affect results. Additionally, ensure control of bacterial contamination throughout the extraction process. While this extraction protocol offers a straightforward way to obtain crude volatile sex pheromones, it does have certain limitations. The crude pheromone extract may contain traces of other signaling molecules, making it difficult to isolate the specific effects of the sex pheromone itself definitively. The functional components of the volatile sex pheromone have not yet been identified. While no functional difference has been reported between the sex pheromones of one-day-old virgin females and six-day-old hermaphrodites, the possibility of a subtle difference cannot be ruled out.
In the chemoattraction assay, a low chemotaxis index could indicate a problem with the health or developmental stage of the tested males. To ensure optimal results, use healthy one-day-old adult males grown at 20 &#176;C on a clean OP50 plate without contamination. Abrupt temperature changes from lower storage temperatures (such as 15 &#176;C) to 20 &#176;C can negatively affect chemoattraction behavior for up to three generations. For best practice, allow strains recently thawed or kept at lower temperatures to adapt to 20 &#176;C for over three generations before use. While isolating test males at the L4 stage on the day before the assay day is recommended, it is not strictly necessary. Pre-exposure to adult hermaphrodites does not significantly impact chemoattraction assay results. One-day-old hermaphrodites do not produce volatile sex pheromones due to the existence of self-sperm. Therefore, the L4 male isolation can be skipped when conducting large-scale screening. However, for studies specifically investigating the effects of previous mating experience or other related studies, isolating males at the L4 stage 1 day before the assay day is necessary.
The him-5 mutation in C. elegans causes a high incidence of males due to an increased rate of X-chromosome nondisjunction during meiosis in the parent of the males. Thus, the males&#39; chemosensory system required for chemotaxis is still intact. This makes it easier to obtain a large number of males for assay. Consequently, him-5 males exhibit robust chemotaxis comparable to WT N2 males, making them a suitable control in sex pheromone chemotaxis assays.
Additional considerations: 
Sodium azide replacement: Sodium azide used to paralyze worms during video recording can be avoided. Instead, use a drop of M9 solution to temporarily immobilize worms though surface tension is enough for video-based assay.
Worm density: Limit the number of worms per plate to avoid overcrowding and maintain optimal conditions. Overcrowding will affect the post-video analysis as the program will have difficulty in identifying worms if they overlap with each other. A maximum of 20 worms is recommended for a 6 cm Petri dish, and 100 worms for a 10 cm dish.
Traditional chemoattraction assays offer a straightforward approach, requiring minimal effort for data acquisition. While these assays provide a quick overview of chemotaxis response, they capture only the endpoint of a complex behavioral response. This limits their ability to reveal the details of pheromone perception and locomotor response. In contrast, the advanced trajectory analysis provides insights into individual and collective movement patterns. This approach uncovers differences missed by endpoint-based measures alone. By allowing the exploration of detailed kinetic patterns and response variability within test populations in detail, this method significantly enhances our understanding of chemosensory mechanisms. This highlights the assay&#39;s applicability to diverse research questions and underscores the importance of considering individual variability in experimental design.
This methodology offers a framework for studying volatile sex pheromones in C. elegans. It can facilitate gene discovery related to volatile sex pheromone synthesis, secretion, and perception, furthering our understanding of chemical communication at the molecular and neural circuit level. Additionally, the chemoattraction assay and trajectory data analysis can be used to investigate other chemoattractants in C. 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.

<table><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>&lt;em&gt;C. remanei &lt;/em&gt;(EM464)</td><td>CGC</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>centrifuge 5418</td><td>&amp;nbsp;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>&amp;nbsp;Any brand should work.</td></tr><tr><td>&lt;em&gt;E. Coli&lt;/em&gt; 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>&lt;em&gt;fog-2(q71)&lt;/em&gt; (JK574)</td><td>CGC</td><td></td><td></td></tr><tr><td>&lt;em&gt;him-5(e1490)&lt;/em&gt;(CB4088)</td><td>CGC</td><td></td><td></td></tr><tr><td>Household bleach</td><td>Clorox Germicidal bleach concentrated</td><td></td><td>&amp;nbsp;Bleach</td></tr><tr><td>M9 buffer</td><td>Homemade solution</td><td>N/A</td><td>3 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 11.3 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O, 5 g NaCl, H&lt;sub&gt;2&lt;/sub&gt;O to 1 L. Sterilize by autoclaving. Add 1 mL 1 M MgSO&lt;sub&gt;4&lt;/sub&gt; 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>&amp;nbsp;Any brand should work.</td></tr><tr><td>N&lt;sub&gt;2&lt;/sub&gt;</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, H&lt;sub&gt;2&lt;/sub&gt;O to 1 L.Sterilize by autoclaving. Once the autoclave is done (2 h), wait until the temperature of the medium drops to 65 &amp;deg;C. Put on a hotplate at 65 &amp;deg;C and stir. Then add the following, waiting 5 min between each to avoid crystallization: 1 mL CaCl&lt;sub&gt;2&lt;/sub&gt; (1 M), 1 mL MgSO&lt;sub&gt;4&lt;/sub&gt; (1 M), 25 mL K&lt;sub&gt;3&lt;/sub&gt;PO4 (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&amp;nbsp;</td><td>4-000-100</td><td>&amp;nbsp;Any brand should work.</td></tr><tr><td>Plastic paper&amp;nbsp;</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 &amp;micro;L, 100 - 1000 &amp;micro;L, any brand should work.</td></tr><tr><td>Rotator</td><td>Labnet</td><td>SKU: LI-H5500</td><td>&amp;nbsp;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&amp;nbsp;</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>&amp;nbsp;Any brand should work.</td></tr><tr><td>WormLab system&amp;nbsp;</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&amp;nbsp;</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

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.

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 a strong preference for the pheromone source. Conversely, experiments with the pheromone receptor mutant srd-1 consistently yield negative results. The C.I. for srd-1 males is typically around zero24. This confirms the assay's ability to detect the absence of a chemotactic response.
In suboptimal experiments, numerous factors can affect the results. For instance, insufficient pheromone concentration may lead to weak responses in him-5 males, while contamination or improper handling could cause unpredictable movement in both strains. These scenarios result in lower C.I. values for him-5 males and potentially misleading non-zero values for srd-1 males. Therefore, the successful implementation of this protocol is marked by a clear distinction in the chemotaxis indices between wild-type and mutant strains, supported by the trajectory analysis visually confirming the behavioral differences (Figure 3). Data analysis should include statistical comparisons of C.I. values to ensure the observed differences are significant.
Demonstration of the video-based chemoattraction assay data analysis and visualization 
Successful assay outcomes: Figure 3 showcases the robust chemotactic response of him-5 males to a volatile sex pheromone. The color-coded trajectories reveal (A) time cost, (B) distance: increasing proximity to the pheromone source over time, confirming the worms' attraction. (C) speed: varying speeds along the trajectories, providing insights into the dynamics of chemotactic responses. (D) straightness: how directly the worms move toward the pheromone source. The straighter the path, the more efficient and targeted their movement is in response to the chemical attractant. (E) Direction correctness: This shows the worms' ability to accurately orient and move themselves toward the source of the pheromone, the chemical attractant. It essentially measures how well the worms can sense the chemical gradient and navigate along it to reach their target.
A drop in speed to zero typically indicates a stop or turn. Combining speed information with straightness data can help identify turning events. Specifically, a turning event is indicated when speed drops to zero and straightness is also very low. In contrast, experiments with sensory defective mutants or those conducted under suboptimal conditions (e.g., low pheromone concentration, inappropriate worm density) may yield different outcomes.
By comparing trajectories and quantifying parameters like directionality, speed, and straightness, researchers can gain insights into the underlying mechanisms of chemotaxis. Successful experiments with clear, directed trajectories toward the pheromone source validate the assay's ability to detect attractive stimuli. Conversely, the absence of such patterns in negative controls or mutant strains confirms the assay's specificity and ability to identify sensory defects. The video-based bulk chemoattraction assay, as illustrated in Figure 3, provides a powerful tool for dissecting the details of worm chemotaxis behavior. By analyzing individual trajectories, researchers can uncover not only the presence or absence of attraction but also the difference in the response, offering a deeper understanding of the underlying genetic and neural mechanisms governing chemotaxis.
<img alt="Flowchart of worm preparation and pheromone extraction; includes QC and storage steps." class="xfigimg" src="/files/ftp_upload/67115/67115fig01v2.jpg" data-altupdatedat="1785211204000" data-alt="Figure 1"> 
Figure 1: Workflow of crude sex pheromone extraction. Crude sex pheromone is extracted from synchronized one-day-old virgin fog-2 mutant females of C. elegans and WT females of C. remanei, where worms are synchronized, washed, isolated, and incubated in M9 buffer for pheromone production. The extracted pheromone-containing supernatant is stored at -80 °C for up to 1 year for use in chemoattraction assays, with quality control tests conducted using N2 or him-5 males to verify attractiveness. For large-scale extraction from 6-day-old virgin C. elegans hermaphrodites, worms are synchronized and washed repeatedly for 6 days, with sex pheromone extracted similarly but modified based on the volume of worm pellets and mixed homogeneously for experimental use. <a href="https://www.jove.com/files/ftp_upload/67115/67115fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Chemotaxis assay diagram with sex pheromone, control buffer; data analysis on chemoattraction index." class="xfigimg" src="/files/ftp_upload/67115/67115fig02.jpg" data-altupdatedat="1785211204000" data-alt="Figure 2"> 
Figure 2: The volatile sex pheromone chemoattraction assay and the chemoattraction Index calculation. (A) This assay involves preparing WT N2 or him-5 males with a standardized bleach synchronization protocol, testing their response to a positive control substance before the volatile sex pheromone chemoattraction assay. The experiment includes marking the plates for control and test spots and using sodium aside to immobilize worms at these spots for accurate scoring. Results are typically scored 30 min after starting the experiment (indicated by closing the lid). (B) Side view of the chemoattraction assay setup with two distinct distances. (C) C.I. calculation and the interpretation. C.I. is a quantitative measure of the attractiveness or repulsiveness of a test stimulus. The C.I. ranges from 1, indicating strong attraction, to -1, indicating strong repulsion. A low C.I. value can arise from two scenarios: either a small number of worms in both the experimental and control spots or an equal distribution of worms between the two spots. (D) Chemoattraction indices for him-5 and srd-1 C. elegans males in response to volatile sex pheromones from C. remanei and C. elegans females. Males of different genetic backgrounds respond differently to the sex pheromone stimulus. him-5 males exhibit a response to both pheromones, with a notably higher index to pheromones from C. remanei females. In contrast, the chemoreceptor mutant strain srd-1 males display no response to either pheromone in this analysis. ***p &lt; 0.01. Abbreviations: WT = wild type; C.I. = chemoattraction index. <a href="https://www.jove.com/files/ftp_upload/67115/67115fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Behavioral analysis charts showing trajectory, distance, speed, and direction correctness over time." class="xfigimg" src="/files/ftp_upload/67115/67115fig03v2.jpg" data-altupdatedat="1785211204000" data-alt="Figure 3"> 
Figure 3: Trajectory analysis of him-5 males in response to volatile sex pheromone. This figure illustrates the movement patterns of him-5 male worms during a chemoattraction assay with a volatile sex pheromone stimulus. Individual trajectories are color-coded to represent (A) Time: Progression of the trajectory over the course of the experiment. The color represents the time elapsed since the beginning of the assay. (B) Distance to pheromone: Proximity to the pheromone source at each time point. (C) Speed: Worm velocity at each point. (D) Straightness: Directness of the path taken by the worm. (E) Direction correctness: Alignment of movement with the direction of the pheromone source. In all three examples, the male worms successfully reached the location of the pheromone. Data were averaged over 20 frames to filter out movement caused by body twisting. Experiments are performed on a 10 cm Petri dish and follow the video-based bulk chemoattraction assay method (see protocol section 7.3.7.2) mentioned in this protocol. <a href="https://www.jove.com/files/ftp_upload/67115/67115fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

Our research examines how the volatile sex pheromone affects male surrogates behavior using chemotaxis assay. It aims to identify the molecular mechanism of pheromone production, and the neuronal mechanism of its perception. Individual worm to worm variability in response to the 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 straightforward and highly reproducible.It is designed to optimize pheromone health 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.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. After synchronizing the healthy adult Caenorhabditis elegans worms, wash them five times with M9 buffer. To synchronize worms at the L1 stage, rotate them in the M9 buffer for 12 to 15 hours to arrest development.Observe the arrested L1 worms onto a 10-centimeter NGM culture plate seeded with OP50 bacteria. To minimize the presence of males on a hermaphrodite plate, check the plate two days after worm release and remove any observed males. After three days of development, observe the appearance of embryos, indicating that the worms have become reproductively mature adults.Wash away the embryos repeatedly with M9 buffer and let them settle until most of the adults are at the bottom of the tube. Pipette the separated adults and transfer them to a new OP50 seeded NGM plate. 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%ager, 25 millimolar sodium chloride, 1.5 millimolar Tris-base, and 3.5 millimolar Tris-hydrochloride. Heat the ager in the chemoattraction solution in a microwave until completely dissolved. After cooling, use a pipette to evenly distribute the chemoattraction ager solution into Petri dishes.Leave the lids open in a clean area to allow the surface of the ager 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 worm's 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.

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Research

JoVE Journal

Behavior

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A sialoglycan microarray assay can be used to evaluate anti-Neu5Gc antibodies in human sera, making it a potential high-throughput diagnostic assay for cancer and other chronic inflammation-mediated human diseases.

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay 

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

This article presents a noninvasive biomonitoring system for the continuous recording and analyses of crayfish cardiac and locomotor activities. This system consists of a near-infrared optical sensor, a video-tracking module, and software for evaluating crayfish heartbeats that reflects its physiological condition and characterizes crayfish behavior during heartbeat fluctuations.

Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

Swimming induced paralysis (SWIP) is a well-established behavioral assay used to study the underlying mechanisms of dopamine signaling in Caenorhabditis elegans (C. elegans). However, a detailed method to perform the assay is lacking. Here, we describe a step-by-step protocol for SWIP.

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

Learn volatile sex pheromone extraction and chemoattraction assays in Caenorhabditis elegans to study chemical communication and male behavior. This reproducible protocol enables quantitative analysis of pheromone responses.

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

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