Some strains were provided by the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This work is supported by the Howard Hughes Medical Institute, with which P.W.S. is an investigator.

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans.">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Cell. 74 (3), 515-527 (1993).</li><li>Hilliard, M. A., Bargmann, C. I., Bazzicalupo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+responds+to+chemical+repellents+by+integrating+sensory+inputs+from+the+head+and+the+tail.">C. elegans responds to chemical repellents by integrating sensory inputs from the head and the tail.</a> Curr Biol. 12 (9), 730-734 (2002).</li><li>Hilliard, M. A., Bergamasco, C., Arbucci, S., Plasterk, R. H., Bazzicalupo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worms+taste+bitter:+ASH+neurons,+QUI-1,+GPA-3+and+ODR-3+mediate+quinine+avoidance+in+Caenorhabditis+elegans.">Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans.</a> EMBO J. 23 (5), 1101-1111 (2004).</li><li>Zariwala, H. A., Miller, A. C., Faumont, S., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Step+response+analysis+of+thermotaxis+in+Caenorhabditis+elegans.">Step response analysis of thermotaxis in Caenorhabditis elegans.</a> J Neurosci. 23 (10), 4369-4377 (2003).</li><li>Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+by+the+nematode+Caenorhabditis+elegans:+identification+of+attractants+and+analysis+of+the+response+by+use+of+mutants.">Chemotaxis by the nematode Caenorhabditis elegans: identification of attractants and analysis of the response by use of mutants.</a> Proc Natl Acad Sci U S A. 70 (3), 817-821 (1973).</li><li>Wicks, S. R., de Vries, C. J., van Luenen, H. G., Plasterk, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CHE-3,+a+cytosolic+dynein+heavy+chain,+is+required+for+sensory+cilia+structure+and+function+in+Caenorhabditis+elegans.">CHE-3, a cytosolic dynein heavy chain, is required for sensory cilia structure and function in Caenorhabditis elegans.</a> Dev Biol. 221 (2), 295-307 (2000).</li><li>Jansen, G., Weinkove, D., Plasterk, R. H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+G-protein+subunit+gpc-1+of+the+nematode+C.+elegans+is+involved+in+taste+adaptation.">The G-protein subunit gpc-1 of the nematode C. elegans is involved in taste adaptation.</a> EMBO J. 21 (5), 986-994 (2002).</li><li>Srinivasan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+blend+of+small+molecules+regulates+both+mating+and+development+in+Caenorhabditis+elegans.">A blend of small molecules regulates both mating and development in Caenorhabditis elegans.</a> Nature. 454 (7208), 1115-1118 (2008).</li><li>Choe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascaroside+signaling+is+widely+conserved+among+nematodes.">Ascaroside signaling is widely conserved among nematodes.</a> Curr Biol. 22 (9), 772-780 (2012).</li><li>Ezcurra, M., Tanizawa, Y., Swoboda, P., Schafer, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+sensitizes+C.+elegans+avoidance+behaviours+through+acute+dopamine+signalling.">Food sensitizes C. elegans avoidance behaviours through acute dopamine signalling.</a> EMBO J. 30 (6), 1110-1122 (2011).</li><li>Ward, A., Liu, J., Feng, Z., Xu, X. Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-sensitive+neurons+and+channels+mediate+phototaxis+in+C.+elegans.">Light-sensitive neurons and channels mediate phototaxis in C. elegans.</a> Nat Neurosci. 11 (8), 916-922 (2008).</li><li>Kunitomo, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+memory-dependent+synaptic+plasticity+of+a+taste+circuit+regulates+salt+concentration+chemotaxis+in+Caenorhabditis+elegans.">Concentration memory-dependent synaptic plasticity of a taste circuit regulates salt concentration chemotaxis in Caenorhabditis elegans.</a> Nat Commun. 4, 2210 (2013).</li><li>Snow, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+anterograde+intraflagellar+transport+motors+cooperate+to+build+sensory+cilia+on+C.+elegans+neurons.">Two anterograde intraflagellar transport motors cooperate to build sensory cilia on C. elegans neurons.</a> Nat Cell Biol. 6 (11), 1109-1113 (2004).</li><li>Tobin, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+expression+of+TRPV+channel+proteins+defines+their+sensory+functions+and+subcellular+localization+in+C.+elegans+neurons.">Combinatorial expression of TRPV channel proteins defines their sensory functions and subcellular localization in C. elegans neurons.</a> Neuron. 35 (2), 307-318 (2002).</li><li>Waggoner, L. E., Zhou, G. T., Schafer, R. W., Schafer, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+alternative+behavioral+states+by+serotonin+in+Caenorhabditis+elegans.">Control of alternative behavioral states by serotonin in Caenorhabditis elegans.</a> Neuron. 21 (1), 203-214 (1998).</li><li>Sawin, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genetic and cellular analysis of modulated behaviors in Caenorhabditis elegans. , (1996).</li><li>Ramot, D., Johnson, B. E., Berry, T. L., Carnell, L., Goodman, M. 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The authors have nothing to disclose.

A critical step in the protocol is ensuring that the assay plates have a consistent level of dryness across different experimental days. Different dryness levels will result in different diffusion rates of the solution into the agar and consequently, variations in behavioral outcome. Thus, assay plates should always be made fresh on the afternoon before experiments. The number of worms tested per assay should also be regulated for ease of comparison between treatments. For reference, a wild-type worm lays 4-10 eggs/h on average yielding &#62;360 worms per assay if the worm synchronization protocol above is followed17. If certain mutant strains are egg-laying defective, pick more gravid adult worms for egg-laying to reach the target number of progeny. Another important step in the protocol is to handle worms gently during the washing process and worm placement. Worms are sensitive to mechanical stimuli, which elicit stress responses such as reversals and egg-laying inhibition18. Furthermore, care should be taken to define the ROI accurately and to determine the optimal thresholding constant value for specific lighting conditions before proceeding with the video analysis. It is also recommended that the calibration and thresholding processes be repeated if a lengthy period of time has elapsed since the last experiment was run.
A limitation of this method is that it is not suitable for assaying small worm populations. However, if the appropriate controls to determine the influence of the presence of food on the sensory behavior are performed, then utilizing food to restrict the worms&#39; spatial exploratory location as in the retention assay is also possible with this setup. In addition, this method is not intended for studying stimulus gradient navigation due to the close proximity and small amounts of the control and experimental solution drops used.
In the future, programming software that enables multi-worm tracking and single-worm feature extraction can be integrated into this system19,20. Recording subtle behavior parameters of single worm behavior such as reversal speeds and amplitude of body bends will provide a more detailed picture of an individual worm&#39;s chemosensory behavior in the context of a population-based assay. The assay can also be modified to study habituation by boring holes through the ROI using syringe needles with the appropriate gauge size and filling the holes with agar infused with the experimental compound or control buffer. This will ensure a more consistent surface concentration of the compound over a longer period of recording time as is necessary during habituation studies. Another potential application of this method is to conduct comparative behavior studies across different nematode species. In addition, the behavior chamber can be modified in multiple ways to study behavioral responses to multimodal stimuli. For optogenetic applications, high-intensity LED arrays can be attached next to the camera mount to selectively activate neurons of interest during the assay. Heating elements, cooling systems, and temperature sensors can also be added to the setup to study the effects of temperature on sensory behaviors. Furthermore, odor delivery systems can be installed inside the chamber to investigate interactions between odorsensory and chemosensory modalities.

Foraging animals must integrate inputs from multiple sensory modalities and select appropriate behavioral strategies in order to successfully navigate their environment. Understanding how external sensory inputs are received and transduced into neural information to guide action selection is a central goal in the field of neurobiology. The genetically tractable nematode, C. elegans, is an attractive model organism in which to study the neural mechanisms underlying sensory biology and multimodal integration. Although C. elegans has only 302 neurons, it can detect and discriminate between a wide variety of environmental stimuli including soluble compounds, volatile odorants, and ambient temperature1,2,3,4,5,6,7. The nematode C. elegans relies heavily on its chemosensory apparatus to localize food sources and to alert itself to potential threats. Thus, behavioral assays designed to screen the responses of wild-type and mutant C. elegans to chemical stimuli play a crucial role in dissecting the genetic, cellular, and neural mechanisms underlying C. elegans&#39; remarkable sensory capabilities.
To assay the response to soluble compounds, three types of assays have been described &#8211; the drop test, the chemotaxis assay, and the retention assay. In the drop test, a small drop of the compound is placed at the tail of a moving worm and the worm&#39;s decision to reverse or move forward once the liquid reaches the anterior sensory apparatus is scored4. The drop test requires little experimental preparation and is useful when the sample size of worms is small, as in the case of laser-operated worms. However, as only one worm can be assayed at a time and the experimenter must be present throughout the duration of the assay, the drop test can be time consuming. The drop test is also vulnerable to variations in drop delivery between each worm within a sample, which may influence the overall results of the assay. Another limitation of the drop test is that it can only be used to assay the worm&#39;s response to aversive compounds as it is not possible to discriminate between an attractive or neutral effect of the compound from the worm&#39;s forward movement.
The chemotaxis assay for soluble compounds generally involves dividing an agar plate into four quadrants, with the experimental solution mixed into the agar of two opposing quadrants and the control solution mixed into the other two quadrants8,9. At the start of the assay, a drop of buffer containing worms is placed at the center of the plate and the number of worms in each quadrant is scored at different time points. The chemotaxis assay provides greater statistical power compared to the drop test as large numbers of worms are tested in each assay. However, one limitation of this method is that preparation of the chemotaxis assay plates requires large quantities of the experimental compound. This will make it difficult to conduct large-scale behavior screens if a complicated purification process with limited yields is required to obtain the compound of interest, as in the case of the ascaroside signaling molecules10. In addition, the manual counting of worms throughout the assay is susceptible to errors and the perturbation of the plates during the counting process might affect the results.
Unlike the two aforementioned methods, the retention assay utilizes machine vision, which minimizes error during the scoring process and reduces experimenter&#39;s interference during the assay11. Computerized analysis of video recordings of worm behavior can also potentially reveal subtler behavioral dynamics that will be missed when scoring is only performed at a few discrete time points. In the retention assay, two solutions spots are added on opposite sides of a small circular bacterial food patch followed by the placement of a small number of worms in the middle of the food patch. The worms&#39; behavior is then video recorded, analyzed, and a preference index value is calculated based on the total number of worm pixels in each solution region. Although the presence of an attractive food patch enables smaller populations of worms to be used in each assay, food has previously been shown to sensitize avoidance behaviors to soluble repellants12. Furthermore, worms exhibit a photophobic response to short-wavelength light and the use of microscope light sources that emit white light in the behavior recording setup might affect behavior13.
The purpose of the method discussed in this article is to record and analyze C. elegans&#39; preference for soluble compounds using a population-based assay. To this end, the current method integrates and improves upon aspects from all three of the previously discussed methods. It enables large populations of worms to be tested and requires only small amounts of the experimental solution to be used in each assay. In addition, the assay is conducted within a custom-built enclosed behavior chamber with infrared LED backlighting to minimize the effects of short-wavelength light on behavior. Each chamber can also be outfitted with multiple microscope cameras, which increases experimental throughput without compromising bench space. Finally, video analysis software outputs the preference index value for each video as well as an accompanying worm occupancy plot to visualize population behavior dynamics over time. The chamber setup and assay protocol can be further modified to study multimodal behavior responses such as the effect of odorants or temperature on chemosensory behaviors.
This article describes the construction of the behavior chamber and the assay protocol. It also demonstrates the utility of this method in assaying the response of wild-type worms and chemosensory defective mutants to the known soluble repellant, copper ions4. Finally, the video analysis process using the provided software is detailed.

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automated analysis of a nematode population-based chemosensory preference assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

Figure 3A shows the preference index values obtained for different genotype and treatment pairings. A preference index value of 1 indicates strong attraction to the solution placed in the experimental ROI while a preference index value of -1 indicates strong repulsion. A preference index of 0.02 was obtained when the M13 buffer solution was placed in both the control and experimental ROIs, demonstrating that there is no spatial bias towards either ROI. N2 worms strongly avoided the copper ions resulting in a preference index of -0.67, which corroborates previous findings that copper ions are a strong repellant (Supplementary Movie 1)4. osm-3 mutants, which lacks proper formation of the distal segments of sensory cilia, showed a significantly decreased avoidance of copper ions (PI = -0.19)15. ocr-2 mutants, which are defective in many ASH-mediated nociceptive responses including copper avoidance, also exhibit significantly decreased avoidance and even some mild attraction to copper ions (PI = 0.19) (Supplementary Movie 2)16.
Figure 3B shows representative worm occupancy plots, which indicate the density of worms in the control versus the experimental ROIs over time in each video. The darker the color of the plotting area, the greater the number of worm pixels in the ROI. The occupancy plot for N2 worms treated with the M13 buffer control shows that the number of worms in both ROIs remains similar throughout the assay. However, the occupancy plot for N2 worms treated with copper ions indicates that the worms strongly and consistently avoid the experimental ROI throughout the assay.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55963/55963fig1.jpg" /> 
Figure 1: Photo and Schematic Representation of Behavior Chamber Design. (A) Front view of behavior chamber setup (left) and corresponding schematic representation of chamber frame (right). Opaque polyester sheets enclose the chamber on all faces except the bottom face, which allows light through from the infrared backlight panel. The numbers 1, 2, and 3 correspond to the extrusion layers in (B). (B) Schematic of top view of extrusion layers comprising the behavior chamber frame. This includes the top layer (1), the middle camera mount assembly layer (2), and the bottom stage layer (3). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55963/55963fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55963/55963fig2.jpg" /> 
Figure 2: Solution-placement and Plate-alignment Templates. (A) The solution-placement template has four quadrants and number alignment markers. The control solution is placed in the upper left and bottom right quadrants while the experimental solution is placed in the upper right and bottom left quadrants. (B) The plate-alignment template has only number alignment markers to minimize occlusion of the worms in the field of view during video recording and analysis. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55963/55963fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55963/55963fig3.jpg" /> 
Figure 3: Example Data Collected Using this Assay. (A) Preference Index (PI) values for wild-type N2 and mutant C. elegans in response to M13 buffer and 10 mM CuCl2. N2 worms showed no preference between control and experimental ROIs when M13 control solution was placed in both ROIs but strongly avoided the experimental ROI when copper ions were placed in it (PI = 0.02 and -0.67, respectively). osm-3 (p802) mutants and ocr-2 (ak47) mutants showed significantly decreased avoidance of copper ions compared to N2 (PI = -0.1 and 0.2, respectively). N= 6 assays for each genotype-treatment pairing, &#62;360 worms per assay, error bars indicate &#177; 1 S.E.M., ***p &#60;0.001, one-way ANOVA followed by post-hoc Tukey Honestly Significant Difference (HSD) test. (B) Representative worm occupancy plots for N2 worms with M13 buffer in both ROIs (top) and N2 worms with M13 buffer in the control ROI and copper ions in the experimental ROI (bottom). The color scale below each occupancy plot represents the number of worm pixels. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55963/55963fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Movie 1: Behavioral Response of N2 worms to 10 mM Copper Chloride. Worms are attracted to the control quadrants with M13 buffer (upper left and bottom right) but strongly avoid the quadrants containing copper ions dissolved in M13 buffer (upper right and bottom left). Video was recorded at 1 frame per second and sped up 15x. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55963/Supplementary_Video_1.mp4">Please click here to download this file.</a>
Supplementary Movie 2: Behavioral Response of ocr-2 (ak47) Worms to 10 mM Copper Chloride. Worms roam to a roughly equal extent in both the control quadrants with M13 buffer (upper left and bottom right) and the quadrants containing copper ions dissolved in M13 buffer (upper right and bottom left) throughout the recording duration. The mutants exhibit a slight preference for the quadrants containing copper ions at the start of the recording. Video was recorded at 1 frame per second and sped up 15x. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55963/Supplementary_video_2.mp4">Please click here to download this file.</a>
Supplemental File: Solution-placement and Plate-alignment Templates. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55963/JoVE_template.pptx">Please click here to download this file.</a>

Watch this Scientific Journal Video about Automated Analysis of a Nematode Population-based Chemosensory Preference Assay at JoVE.com

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

automated-analysis-nematode-population-based-chemosensory-preference

Nanyang Technological University

Research

JoVE Journal

Behavior

7.4K Views.  California Institute of Technology. We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage. 

Video: Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

Fat Preference: A Novel Model of Eating Behavior in Rats

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to this multifactorial disorder is the consumption of a high fat diet, a behavior that has been shown to increase both caloric intake and body fat content. However, the elements regulating preference for high fat food over other foods remain understudied.
To overcome this deficit, a model to quickly and easily test changes in the preference for dietary fat was developed. The Fat Preference model presents rats with a series of choices between foods with differing fat content. Like humans, rats have a natural bias toward consuming high fat food, making the rat model ideal for translational studies. Changes in preference can be ascribed to the effect of either genetic differences or pharmacological interventions. This model allows for the exploration of determinates of fat preference and screening pharmacotherapeutic agents that influence acquisition of obesity.

Dietary fat content influences both energy intake and body fat composition in mammals. By examining rats&#8217; preference for high fat food in a series of choice experiments, it is possible to test genetic differences and pharmacological interventions on their preference for high fat food.

Obesity is a growing problem in the United States of America, with more than a third of the population classified as obese. One factor contributing to ...

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays 1. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs 2, with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Behavioral assays provide powerful tools for understanding neuronal function. Here we present several protocols for quantifying predatory feeding behavior found in the model nematode Pristionchus pacificus and its relatives. Additionally, we provide methods for analyzing predatory feeding adaptations including mouth structures and teeth.

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including ...

Environment

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

Neuroscience

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...

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

Author Spotlight: Examining Volatile Sex Pheromone Influence on Male C. elegans Behavior

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 ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Biology

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

C. elegans Chemotaxis Assay: A Method to Test Chemosensation in Worms

Source: Campbell, J. C., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/55939/a-caenorhabditis-elegans-nutritional-status-based-copper-aversion">A Caenorhabditis elegans Nutritional-Status Based Copper Aversion Assay.</a> J. Vis. Exp. (2017).
This video introduces chemotaxis in the nematode C. elegans and shows a sample protocol testing chemotactic aversion to copper sulfate.

Source: Campbell, J. C., et al. A Caenorhabditis elegans Nutritional-Status Based Copper Aversion Assay. J. Vis. Exp. (2017).
This video introduces ...

JoVE Encyclopedia of Experiments

Caenorhabditis elegans (worm)

Examine Chemoavoidance Behavior in Transgenic Worms Using Osmotic Avoidance Assay

Source: Wang, Q. et al., Z. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63081">Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity.</a> J. Vis. Exp. (2021).
This video describes an osmotic avoidance assay to examine chemoavoidance behavior in a transgenic worm model. Avoidance behavior is tested in a plate in the presence of a high-osmotic barrier and chemoattractant. The worms with a functional loss of neurons get attracted by the chemoattractant, cross the high-osmotic barrier, and become paralyzed in the trap zone because of the anesthetic solution.

Source: Wang, Q. et al., Z. Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity. J. ...

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

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Chapters in this video

Fat Preference: A Novel Model of Eating Behavior in Rats

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

C. elegans Chemotaxis Assay

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

C. elegans Chemotaxis Assay: A Method to Test Chemosensation in Worms

Examine Chemoavoidance Behavior in Transgenic Worms Using Osmotic Avoidance Assay