This work was supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grant RGPIN36481-08 to William G. Bendena.

1. Preparation of Experimental Organisms
<ol>
	<li>Pick 10 L4 staged nematodes per strain 24 h prior to commencing the assay to ensure that organisms are young adults when tested. For each mutant or control nematode tested, pick 10 L4s (10 for the control and 10 for the assay).
	<ol>
		<li>Maintain L4 organisms using standard methods26,27 for 24 h on standard agar plates seeded with OP50 Escherichia coli. If organisms are lost during the subsequent wash steps, compensate by increasing the starting sample size (i.e. pick 20 worms rather than 10). 
		Note: Behavior is an innately variable phenotype. Perform the method in triplicate for each strain on three separate days. Include additional control strains and conditions for novel strains, as highlighted in the discussion section.</li>
	</ol>
	</li>
	<li>Immediately prior to assay, transfer experimental organisms to an agar plate with no bacteria and allow nematodes to move freely for 1 min to remove excess bacteria.
	<ol>
		<li>If experimental organisms experienced contamination conditions during the 24 h prior to the assay, discard them.</li>
	</ol>
	</li>
	<li>Pipette 1 mL of M9 onto the bacteria-free plate to wash worms into a microcentrifuge tube.</li>
	<li>Centrifuge at 3,000 x g for 1 min. Worms should form a pellet at the bottom of the tube. Aspirate M9 solution without disrupting the worm pellet. Add 1 mL of M9 to the worm pellet, invert tube to mix worms with the solution.</li>
	<li>Repeat steps 1.4 three more times.
	<ol>
		<li>If excess bacteria was initially transferred with the worms, repeat for a total of 5 times. No bacteria should be transferred to the copper food race plates. If food is transferred to the assay plate, it will interfere with accurate data collection.</li>
	</ol>
	</li>
	<li>After the final wash, aspirate supernatant until 100 &#181;L of M9 and the worm pellet remains. 
	Caution: Starvation related behaviors become evident after 30 min. Consequently worms should be immediately transferred from solution once the wash steps have been completed.</li>
</ol>
2. Preparation of Assay Plates
<ol>
	<li>Prepare standard NGM agar plates two days prior to the assay.
	<ol>
		<li>If plates are kept in a humid environment, make agar plates 3 days prior to assay. Alternatively, remove the lid of the plate for 3 - 6 h to ensure proper dryness (if in a sterile environment).</li>
	</ol>
	</li>
	<li>With a thick permanent marker, make a line on the underside of the plate along the outer edge and another to form a mid-line barrier (Figure 1). The mid-line barrier should be equidistant from each edge of the plate. Use a ruler to ensure precise measurements. These lines will serve as a guide when transferring bacteria and the copper solution. Provided that E. coli is transferred before the copper solution, these lines will serve as indicators.</li>
	<li>Seed plate with 50 &#181;L of OP50 E. coli on only one side of the copper barrier to create a uniform lawn (Figure 2). Bacterial concentration should remain consistent across assays; however, little variability has been observed in response to mild differences in concentration.
	<ol>
		<li>Use the marked lines on the underside of the plate to ensure the bacteria do not come into contact with the copper solution. Provided that the copper solution lines the edge of the plate and forms a mid-line barrier, transfer the bacteria so that the copper solution will not come into contact with the food source.</li>
	</ol>
	</li>
	<li>Mark a second set of plates and transfer no OP50 E. coli to them (Figure 2). These plates will serve to evaluate the negative control. These plates should also have a marking on one half of the plate to denote the initial transfer origin.</li>
	<li>Allow bacteria to dry then incubate plates at 37 &#176;C overnight. Ensure that bacterial patches are not disturbed when transferring to a 37 &#176;C incubator or room. Excessive disturbance could alter the location or shape of the food patch.</li>
</ol>
3. Chemotaxis Assay
<ol>
	<li>Freshly prepare a 0.5 M copper (II) sulfate solution prior to assay start time. Provided that 125 &#181;L of the solution is used per plate, scale up this volume dependent on the number of assay plates used (e.g. 5 assay plates, 625 &#181;L).</li>
	<li>Pipette 100 &#181;L of the copper (II) sulfate solution on the edge of the agar to create an outer copper barrier. The marked underside of the plate should serve as a guide.</li>
	<li>Pipette 25 &#181;L of the copper (II) sulfate solution to create a midline barrier.
	<ol>
		<li>Ensure that the copper (II) sulfate solution does not come into contact with the bacterial patch. Use a spotted technique as streaking may affect locomotion due to indentations/scratches on the agar.</li>
	</ol>
	</li>
	<li>Allow the copper solution to dry onto plate. Time period can vary depending on plate and lab conditions. Visually check for dryness every 5 min after transfer. 
	Note: The copper solution displays a blueish tint and is easily identifiable. Use a laboratory tissue to lightly dab the solution near the edge of the plate to discern dryness.</li>
	<li>Pipette 20 &#181;L of the worm pellet from the bottom of the tube onto the bacteria-free half of the assay plate.
	<ol>
		<li>Ensure that 10 worms are transferred to the assay plate. If extra worms were accidentally included, remove them by picking with halocarbon oil to ensure that no bacteria are added to the plate. Each assay should have a consistent number of nematodes during the assay. 
		NOTE: If too few worms are transferred during assays, increasing the initial sample size will mitigate potential losses during washes and transfers.</li>
	</ol>
	</li>
	<li>Remove excess M9 from the plate with a laboratory tissue. M9 should not come into contact with the copper (II) sulfate solution. 
	Caution: Ensure that worms and the agar surface remain unaffected while performing this step. If used too harshly, the laboratory tissue can create indentations to the agar surface of the plate and can remove worms. Worms accidentally removed via KimWipe should be discarded.</li>
	<li>Once the M9 solution has been removed and all worms have commenced non-liquid locomotory patterns, start the assay stopwatch.
	<ol>
		<li>Optimally, remove the M9 solution within a min. The essential parameter is the identification of sinusoidal locomotion. Worms locomote differently, e.g. thrashing rather than sinusoidal, when in liquid. Start the assay stop watch once each of the experimental organisms stops thrashing.</li>
	</ol>
	</li>
	<li>Check assay plates every 30 min.
	<ol>
		<li>For the assay plates with bacterial patches, positively score organisms if they reach the food patch over a 4 h period. For the negative control plates, positively score organisms if they have crossed the barrier.</li>
	</ol>
	</li>
</ol>

<ol><li>White, J. G., Southgate, E., Thomson, J. N., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+structure+of+the+nervous+system+of+the+nematode+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Philos.+Trans.+R.+Soc.+Lond.+B.+Biol.+Sci%22%5BJournal%5D)">The structure of the nervous system of the nematode Caenorhabditis elegans.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 314 (1165), 1-340 (1986).</li>
<li>Bargmann, C. I. Chemosensation in C. elegans (October 25, 2006). <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBargmann%2C+CI+%22The+C.+elegans+Research+Community%2C+WormBook%22">The C. elegans Research Community, WormBook</a>. , wormbook.1.123.1, http://www.wormbook.org (2006).</li>
<li>Bargmann, C. I., Hartwieg, E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Cell. 74 (3), 515-527 (1993).</li>
<li>Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemotaxis+by+the+nematode+Caenorhabditis+elegans%3A+identification+of+attractants+and+analysis+of+the+response+by+use+of+mutants%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U+S+A%22%5BJournal%5D)">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>Ramot, D., MacInnis, B. L., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bidirectional+temperature-sensing+by+a+single+thermosensory+neuron+in+C.+elegans%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">Bidirectional temperature-sensing by a single thermosensory neuron in C. elegans.</a> Nat. Neurosci. 11 (8), 908-915 (2008).</li>
<li>Russell, J., Vidal-Gadea, A. G., Makay, A., Lanam, C., Pierce-Shimomura, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Humidity+sensation+requires+both+mechanosensory+and+thermosensory+pathways+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci%22%5BJournal%5D)">Humidity sensation requires both mechanosensory and thermosensory pathways in Caenorhabditis elegans.</a> Proc. Natl. Acad. Sci. 111 (22), 8269-8274 (2014).</li>
<li>van Campen, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensory+modulation+disorders+in+childhood+epilepsy%5BTitle%5D)+AND+%22J.+Neurodev.+Disord%22%5BJournal%5D)">Sensory modulation disorders in childhood epilepsy.</a> J. Neurodev. Disord. 7 (34), (2015).</li>
<li>Festa, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neocortical+disconnectivity+disrupts+sensory+integration+in+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Neuropsych%22%5BJournal%5D)">Neocortical disconnectivity disrupts sensory integration in Alzheimer's disease.</a> Neuropsych. 19 (6), 728-738 (2005).</li>
<li>Boecker, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensory+processing+in+Parkinson%27s+and+Huntington%27s+disease%3A+investigations+with+3D+H%282%29%2815%29O-PET%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">Sensory processing in Parkinson's and Huntington's disease: investigations with 3D H(2)(15)O-PET.</a> Brain. 122 (9), 1651-1665 (1999).</li>
<li>Markaki, M., Tavernarakis, N. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/21154667">Modeling human disease in Caenorhabditis elegans.</a> Biotechnol. J. 5 (12), 1261-1276 (2010).</li>
<li>O'Reilly, L. P., Luke, C. J., Perlmutter, D. H., Silverman, G. A., Pak, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((C.+elegans+in+high-throughput+drug+discovery%5BTitle%5D)+AND+%22Adv.+Drug+Deliv.+Rev%22%5BJournal%5D)">C. elegans in high-throughput drug discovery.</a> Adv. Drug Deliv. Rev. 0, 247-253 (2014).</li>
<li>Thompson, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+million+mutation+project%3A+a+new+approach+to+genetics+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">The million mutation project: a new approach to genetics in Caenorhabditis elegans.</a> Genome Res. 23 (10), 1749-1762 (2013).</li>
<li>Chao, M. Y., Komatsu, H., Fukuto, H. S., Dionne, H. M., Hart, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Feeding+status+and+serotonin+rapidly+and+reversibly+modulate+a+Caenorhabditis+elegans+chemosensory+circuit%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U+S+A%22%5BJournal%5D)">Feeding status and serotonin rapidly and reversibly modulate a Caenorhabditis elegans chemosensory circuit.</a> Proc. Natl. Acad. Sci. U S A. 101 (43), 15512-15517 (2004).</li>
<li>Maricq, A. V., Peckol, E., Driscoll, M., Bargmann, C. I. <a target="_blank" href="http://www.nature.com/nature/journal/v378/n6552/abs/378078a0.html">Mechanosensory signaling in C. elegans mediated by the GLR-1 glutamate receptor.</a> Nat. 378 (6552), 78-81 (1995).</li>
<li>Chalasani, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissecting+a+circuit+for+olfactory+behaviour+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nat%22%5BJournal%5D)">Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans.</a> Nat. 450 (7166), 63-70 (2007).</li>
<li>Hilliard, M. A., Bargmann, C. I., Bazzicalupo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((C.+elegans+responds+to+chemical+repellents+by+integrating+sensory+inputs+from+the+head+and+the+tail%5BTitle%5D)+AND+%22Curr.+Biol%22%5BJournal%5D)">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>Hart, A. C., Kass, J., Shapiro, J. E., Kaplan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+signaling+pathways+mediate+touch+and+osmosensory+responses+in+a+polymodal+sensory+neuron%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Distinct signaling pathways mediate touch and osmosensory responses in a polymodal sensory neuron.</a> J. Neurosci. 19 (6), 1952-1958 (1999).</li>
<li>Ishihara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HEN-1%2C+a+secretory+protein+with+an+LDL+receptor+motif%2C+regulates+sensory+integration+and+learning+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">HEN-1, a secretory protein with an LDL receptor motif, regulates sensory integration and learning in Caenorhabditis elegans.</a> Cell. 109 (5), 639-649 (2002).</li>
<li>Saeki, S., Yamamoto, M., Iino, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasticity+of+chemotaxis+revealed+by+paired+presentation+of+a+chemoattractant+and+starvation+in+the+nematode+Caenorhabditis+elegans%5BTitle%5D)+AND+%22J.+Exp.+Biol%22%5BJournal%5D)">Plasticity of chemotaxis revealed by paired presentation of a chemoattractant and starvation in the nematode Caenorhabditis elegans.</a> J. Exp. Biol. 204 (10), 1757-1764 (2001).</li>
<li>Chao, M. Y., Komatsu, H., Fukuto, H. S., Dionne, H. M., Hart, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Feeding+status+and+serotonin+rapidly+and+reversibly+modulate+a+Caenorhabditis+elegans+chemosensory+circuit%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Feeding status and serotonin rapidly and reversibly modulate a Caenorhabditis elegans chemosensory circuit.</a> Proc. Natl. Acad. Sci. U.S.A. 101 (43), 15512-15517 (2004).</li>
<li>Campbell, J. C., Polan-Couillard, L. F., Chin-Sang, I. D., Bendena, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NPR-9%2C+a+Galanin-Like+G-Protein+Coupled+Receptor%2C+and+GLR-1+Regulate+Interneuronal+Circuitry+Underlying+Multisensory+Integration+of+Environmental+Cues+in+Caenorhabdities+elegans%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">NPR-9, a Galanin-Like G-Protein Coupled Receptor, and GLR-1 Regulate Interneuronal Circuitry Underlying Multisensory Integration of Environmental Cues in Caenorhabdities elegans.</a> PLoS Genet. 12 (5), (2016).</li>
<li>Harris, G. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three+distinct+amine+receptors+operating+at+different+levels+within+the+locomotory+circuit+are+each+essential+for+the+serotonergic+modulation+of+chemosensation+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Three distinct amine receptors operating at different levels within the locomotory circuit are each essential for the serotonergic modulation of chemosensation in Caenorhabditis elegans.</a> J. Neurosci. 29 (5), 1446-1456 (2009).</li>
<li>Harris, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissecting+the+serotonergic+food+signal+stimulating+sensory-mediated+aversive+behavior+in+C.+elegans%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Dissecting the serotonergic food signal stimulating sensory-mediated aversive behavior in C. elegans.</a> PLoS One. 6 (7), (2011).</li>
<li>Mitchell, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+differential+role+for+neuropeptides+in+acute+and+chronic+adaptive+responses+to+alcohol%3A+behavioural+and+genetic+analysis+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A differential role for neuropeptides in acute and chronic adaptive responses to alcohol: behavioural and genetic analysis in Caenorhabditis elegans.</a> PLoS One. 5 (5), (2010).</li>
<li>Colbert, H. A., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+signals+modulate+olfactory+acuity%2C+discrimination%2C+and+memory+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Learn+Mem%22%5BJournal%5D)">Environmental signals modulate olfactory acuity, discrimination, and memory in Caenorhabditis elegans.</a> Learn Mem. 4 (2), 179-191 (1997).</li>
<li>Brenner, S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/4366476">The genetics of Caenorhabditis elegans.</a> Genet. 77 (1), 71-71 (1974).</li>
<li>Behavior (July 3, 2006). <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22The+C.+elegans+Research+Community%2C+WormBook%22">The C. elegans Research Community, WormBook</a>. Hart, A. C. , wormbook.1.87.1, http://www.wormbook.org (2006).</li>
<li>Sambongi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensing+of+cadmium+and+copper+ions+by+externally+exposed+ADL%2C+ASE%2C+ASH+neurons+elicits+avoidance+response+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22NeuroReport%22%5BJournal%5D)">Sensing of cadmium and copper ions by externally exposed ADL, ASE, ASH neurons elicits avoidance response in Caenorhabditis elegans.</a> NeuroReport. 10 (4), 753-757 (1999).</li>
<li>Gray, J. M., Hill, J. J., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+circuit+for+navigation+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U+S+A%22%5BJournal%5D)">A circuit for navigation in Caenorhabditis elegans.</a> Proc. Natl. Acad. Sci. U S A. 102 (9), 3184-3191 (2004).</li>
<li>Rechavi, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Starvation-Induced+Transgenerational+Inheritance+of+Small+RNAs+in+C.+elegans%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Starvation-Induced Transgenerational Inheritance of Small RNAs in C. elegans.</a> Cell. 158 (2), 277-287 (2014).</li>
<li>Gloria-Soria, A., Azevedo, R. B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((npr-1+Regulates+Foraging+and+Dispersal+Strategies+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">npr-1 Regulates Foraging and Dispersal Strategies in Caenorhabditis elegans.</a> Cell. 18 (21), 1694-1699 (2008).</li>
<li>Beron, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+burrowing+behavior+of+the+nematode+Caenorhabditis+elegans%3A+A+new+assay+for+the+study+of+neuromuscular+disorders%5BTitle%5D)+AND+%22Genes+Brain+Behav%22%5BJournal%5D)">The burrowing behavior of the nematode Caenorhabditis elegans: A new assay for the study of neuromuscular disorders.</a> Genes Brain Behav. 14 (4), 357-368 (2015).</li>
<li>Wang, S. J., Wang, Z. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Track-A-Worm%2C+An+Open-Source+System+for+Quantitative+Assessment+of+C.+elegans+Locomotory+and+Bending+Behavior%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Track-A-Worm, An Open-Source System for Quantitative Assessment of C. elegans Locomotory and Bending Behavior.</a> PLoS One. 8 (7), (2013).</li>
</ol>

We have nothing to disclose.

This assay design modifies the food race assay24 to include a copper solution to create an aversive midline barrier and around the edge of the plate to prevent a loss of nematodes. Organisms are tested for their ability to cross the aversive barrier and reach a food patch over a 4 h period. In the context of npr-9(GF), we have utilized this assay to evaluate how starvation conditions could affect aversive responses and the detection of food. Provided that we had previously characterized npr-9(GF) as defective for responsiveness to food and aversive cues, we combined multiples environmental cues with nutritional status to evaluate if starvation could modulate the defective npr-9(GF) behaviors. This assay relies on a worm&#39;s ability to detect and respond to chemical stimuli and food cues3,4. Given the diverse variables analyzed, the conditions of the experimental organisms must be meticulously controlled to eliminate confounding factors. For uncharacterized mutants, i.e. those with unknown aversive or food responsiveness, additional control strains would need to be utilized. Aversive defective strains, e.g. che-2 or odr-328, and mutants that do not alter locomotory patterns off food, e.g. tph-129, should also be evaluated in parallel to ensure that the copper and starvation conditions are appropriately controlled. Moreover, to more directly highlight the contribution of starvation to the evaluated behavior, separate assay conditions can also be developed for additional controls. For example, the nematodes can be starved immediately prior to the assay and transferred to the assay plate (with food) to identify how quickly an organism in a starved condition will respond to copper and reach the food patch. Our current assay highlights aversive responses as the experimental organisms progress from a well-fed to starved state.
Our assay is a modification of the commonly used C. elegans chemotaxis assay4 in which a stronger stimulus (i.e. high concentration) has been incorporated in order to evaluate behaviors over a more extended time period. Rather than evaluating instantaneous responses, this assay can be utilized to measure changes in aversive responses as organisms progress towards a starved state (if the necessary controls are included). Similar evaluations have been used in the absence of copper in order to evaluate ethanol withdrawal responses, i.e. nematodes locating food, over time24.
As is the case with any behavioral assay in C. elegans, controlling for environmental variables is essential to ensuring consistently reproducible results. Organisms must all be of the same age, given that the behavioral responses can vary with age. Moreover, variability in the nutritional status of the worms can alter acclimation to starvation conditions. Therefore, the experimental worms should not undergo starvation at any point prior to this protocol. Starvation experience of the parental generation can also influence progeny behavior30; hence, it is advisable to ensure that the experimental organisms are well-fed for at least two generations. Moreover, the number of organisms transferred to the assay plates should remain consistent given that the local population density can influence dispersal rates31. Once worms have been transferred to the plates, it is critical to remove the excess M9 solution with a laboratory tissue without damaging the agar surface. Alteration to the surface may interfere with spontaneous locomotory patterns. Excess solution needs to be appropriately removed before commencing the official start of the assay to ensure that thrashing behavior, i.e. a nematode locomotory pattern observed in liquid, is not present. A clear indicator is to look for crawling behavior.
Assay plates must be controlled so that plate and bacterial dryness are consistent. Overly fresh plates can cause locomotory defects and can also interfere with the detection of environmental stimuli6. The application of the copper solutions need to be as consistent as possible so that aversive barriers are appropriately thick and they do not come into contact with the food source. Applying too little copper solution could lessen the wild type aversive response time, while too much could prove lethal to the nematodes. Aiming for a uniform thickness to the copper solution yields the best results. If indentations are created in the agar during copper solution application, the assay plate should be discarded. Worms may burrow through such holes32, especially when faced with an aversive substance or starvation. To ensure plate uniformity amongst samples, mixed populations of worms (i.e. a mutant and wild type) could be measured on the same plate if one were distinctly labeled (e.g. via GFP).
Although this assay was performed on standard-sized agar plates, larger plates can be used (e.g. 100 mm diameter). In this scenario, the assay duration should be extended to 6 h to provide sufficient time for worm movement. Larger plates could allow for the testing of larger populations and could be used to investigate how population density affects aversive responses over extended periods of time. More technically advanced procedures could also be incorporated with the use of worm tracking equipment coupled to a movable stage33. Worm tracking would allow for more precise measurements and allow for the collection of additional variables (e.g. turning frequency, reversal frequency). Moreover, data collection would be continuously recorded over the 4 h period to give a more precise timeline of starvation-induced behaviors.
The assay can be easily adapted to allow for the measurement of alternative phenotypes. The age of the worm can be varied to allow for measurements on age-related starvation-induced aversive changes. Moreover, variations to the nutritional status of the experimental worms could also allow for analysis of habituation to starvation in the context of food and chemical aversion. So long as protocols are consistent amongst comparable data sets, almost any pre-conditioning of experimental organisms could be evaluated via the chemotaxis assay.

Caenorhabditis elegans has been used as a model for the study of neurobiology for decades due to the relative ease in analyzing the circuitry of a nervous system composed of only 302 neurons1. Provided that the organism is reliant on responding to environmental cues, much of the nervous system is dedicated to regulating the integration of environmental signals2. Despite the simplicity of its nervous system, C. elegans can detect and respond to diverse environmental signals including repellents3, attractants4, temperature5, and even humidity6. A failure to properly integrate environmental signals has been linked to a number of behavioral disorders and neurodegenerative conditions in mammalian model systems7,8,9. With a range of available neural disease models10 in C. elegans and the development of nematode pharmaceutical screens11, this organism has proven to be a useful system for the study of neurobiology. Given the availability of a mapped nematode connectome1 and mutations to almost every gene in the nematode genome12, our understanding of the nematode nervous system, and by extension our own, is partially limited by the design of creatively appropriate assays.
A number of chemotaxis assays have been developed over the past 40 years to evaluate nematode responsiveness to diverse aversive stimuli3,4,13,14,15. Initial experimentation involved the introduction of an acute environmental stimulus while a single worm roamed on an agar plate3,14,16. Immediate changes to locomotory responses were recorded. For example, the volatile odorant octanol can be applied to a hair and wafted in front of a nematode&#39;s nose to stimulate the initiation of backwards locomotion in wild type worms17. More complex assays have also been developed to incorporate multiple variables as a means of assessing behavioral choice18. A variation of this assay entails the use of a copper solution to create an aversive midline barrier4. An attractant, namely diacetyl, was placed on one side of the chemical barrier with worms transferred away from the diacetyl source. Worms defective for copper aversive responses immediately crossed the barrier to reach the diacetyl, while wild type worms were initially repulsed by the barrier. Responses were scored when worms first approached the copper barrier without long term observations.
When worms are evaluated after undergoing starvation conditions, their sensitivity to environmental stimuli is decreased19. When the aversive chemical octanol is wafted in front of the nematode nose, wild type organisms stimulate backwards movement within 3 - 5 s when on food. After these organisms have been removed from food for 10 min, they exhibit a delayed response of 8 - 10 s20. Thus, with increased starvation, nematodes display a decreased aversive response to harmful environmental signals as the search for food becomes more essential to survival. Conversely, nematodes that over-express neuropeptide receptor 9 (npr-9), do not respond to octanol on or off food and exhibit an inability to respond to a number of aversive stimuli21. These npr-9(GF) organisms also do not modulate their reversal frequency in the presence of food, but can reverse in response to harsh touch stimulations indicating that they are capable of backwards locomotion21. We have also evaluated npr-9(LF) mutants given that they exhibit an abnormally decreased reversal frequency off food yet can modulate their behavior in the presence of food21. Coupling the nutritional state of the worm with the introduction of acute external stimuli has aided in elucidating the mechanisms by which a food-related pathway can broadly modulate sensory signaling pathways22,23. The presence of food in the nematode environment has also been used to evaluate ethanol withdrawal responses24. In this experiment, worms were incubated in varying concentrations of ethanol and then were placed on an agar plate with a patch of food known as a &#34;food-race assay&#34;. The food patch was placed on one edge of the plate while the nematodes were placed away from the food source. Ethanol withdrawal was evaluated by measuring the duration of time required for worms to reach the patch of food.
This nutritional-based copper aversion assay builds upon the food-race assay to integrate additional environmental variables, namely food and copper, while assessing behavioral changes over time. This is an adaptation of a commonly use protocol throughout the C. elegans community4. This protocol has been used to evaluate aversive responses and the detection of food over a four-hour period21. Since worm&#39;s exhibit starvation behaviors after 30 min of food deprivation25, we are also able to evaluate how changes to nutritional status can influence environmental responses. The conditions of this assay measure how experimental organisms change responsiveness to aversive stimuli over time, hence this evaluates behavioral changes as organisms progress towards a starved state (and continued measurements of prolonged starvation). Since the npr-9(GF) animals do not alter their behavior in response to food or many aversive cues, we sought to identify if these behavioral deficits would persist in the context of starvation. Ultimately, this assay design has been formulated to specifically evaluate the npr-9(GF) mutants but can be further adapted to also characterize novel strains.

<table><tbody><tr><td>M9 Solution [3 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 6 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 5 g NaCl, 1 ml 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;, H&lt;sub&gt;2&lt;/sub&gt;O to 1 litre. Autoclave to sterilize before use.]</td><td></td><td></td><td>Produced in lab</td></tr><tr><td>Cupric Sulfate</td><td>Sigma</td><td>C-1297</td><td>Use water to appropriately suspend to a concentration of 0.5M</td></tr></tbody></table>

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.

We utilized wild type (N2), npr-9(tm1652), and an npr-9 overexpression strain, i.e. npr-9(GF) (IC836 -npr-9::npr-9;sur-5::gfp;odr-1::rfp), to evaluate responses to starvation and copper aversion. Wild type organisms are capable of detecting and responding to the aversive copper barrier, while npr-9(GF) mutants do not initiate an aversive response to the copper over the 4 h assay21. After 30 min of starvation, roughly 50 - 60% of wild type (N2) organisms cross the copper barrier and reach the food patch. At the 2 h mark, 75% of wild type nematodes reach the food source. By the end of the assay, 100% of N2 organisms have relocated to the food source. In contrast, the majority of the npr-9(GF) organisms do not modulate locomotory patterns in response to food and will continue to cross the aversive barrier even after coming into contact with a food source. The npr-9(GF) worms continuously fail to alter their locomotion in response to food over the 4-h assay and only 30% of the test organisms are found on the food at any given time. The npr-9(LF) animals do not perform as well as N2 organisms, but do modulate their locomotory patterns as starvation increases to reach the food patch (Figure 3). When N2 or npr-9(LF) organisms are evaluated via this assay without food, they rarely cross the copper barrier. The npr-9(GF) mutants repeatedly cross the barrier back and forth (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55939/55939fig1.jpg" /> 
Figure 1: A Visual Representation of Markings to Indicate Copper Solution Placement on the Underside of a 5 cm Petri Dish. These indications are used to ensure that the sections of the plate have been appropriately measured and serve as a guideline when transferring the bacterial patch and then copper solution onto the agar surface. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55939/55939fig1large.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/55939/55939fig2.jpg" /> 
Figure 2: The 5 cm Petri Dish is Divided into Two Sections for on and off Food Assay Plates and a Bacterial Lawn is Formed in the Center of One of These Sections for the On-food Plate. The food patch should not come into contact with the test compound which lines the edges of the plate and forms a midline barrier. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55939/55939fig2large.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/55939/55939fig3.jpg" /> 
Figure 3: Percentage of N2, npr-9(tm1652), and npr-9(GF) Worms that Reach the Food Source Over a Four-hour Period in Response to the Nutritional-status Based Copper Aversion Assay. Data points are averages of at least three experiments (n &#62; 30 worms) performed on separate days for both strains. Data are presented as mean &#177; standard error and analyzed with a two-way repeated measures ANOVA. ** p &#60;0.01, significantly different from N2 animals under identical conditions. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55939/55939fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55939/55939fig4.jpg" /> 
Figure 4: Percentage of N2, npr-9(tm1652) and npr-9(GF) Worms that Cross the Copper Barrier with no Food Source Over a Four-hour Assay. The aforementioned strains were evaluated for copper aversion in the absence of food. Positive responses are scored as crossing the copper barrier and remaining on the non-origin half of the plate. Data points are averages of at least three experiments (n &#62; 30 worms) performed on separate days for both strains. Data are presented as mean &#177; SE and at least 10 animals were assayed in three independent experiments analyzed with a two-way repeated measures ANOVA. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55939/55939fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay at JoVE.com

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.

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 ...

a-caenorhabditis-elegans-nutritional-status-based-copper-aversion

Nanyang Technological University

Research

JoVE Journal

Neuroscience

6.5K Views.  Queen's University.  Hier presenteren we een Caenorhabditis elegans -specifieke test die is ontworpen om veranderingen in het koperen aversiegedrag te evalueren en het vermogen om een ​​gemeenschappelijke voedingsbron te lokaliseren, als het organisme zich voortbrengt van een goed gevoed aan hongerige voedingswaarde. 

Video: A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

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.

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 ...

Behavior

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 ...

Environment

High-Throughput Assay to Measure Caenorhabditis elegans Food Intake and Lifespan

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans&#160;(C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol describes an assay to quantify Caenorhabditis elegans feeding rate based on measuring the clearance of bacteria in liquid culture.

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Biology

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Evaluating Metabolism and Aging Pattern in Caenorhabditis elegans

Using Low-Cost Dyes to Visualize Glycogen Accumulation and Gut Integrity in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences teaching through the staining of the cells or their molecular content. Lugol dye (iodine-potassium iodide solution) has been widely used in biochemistry to stain glycogen stores. In this context, it is possible to observe differences between fed and starved animals, besides the effects of different conditions, such as different diets and oxygen levels. Erioglaucine is a blue dye that indicates the loss of the intestinal barrier. When the intestinal barrier is intact, the blue dye stains inside the lumen; however, when this integrity is disrupted, the dye leaks into the body cavity. Using a stereomicroscope or a microscope, teachers can demonstrate physiological and biochemical alterations, or they can instigate students to ask a scientific question and hypothesize and test their hypothesis using these assays. The present protocol describes two staining techniques in C. elegans that can be easily carried out by students.

Author Spotlight: Exploring Metabolic and Aging Processes in C. elegans Using Low-Cost, High-Impact Assays

Teaching biological sciences can be made more stimulating for students through the use of experimentation. This manuscript presents two different yet complementary protocols that can be utilized in the classroom to encourage students to formulate and test hypotheses related to high-calorie diets, starvation, and aging.

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences ...

C. elegans Survival Assay: A Short-Term Toxicity Test

Source: Possik, E. and Pause, A. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/2496/measuring-caenorhabditis-elegans-life-span-96-well-microtiter">Measuring Oxidative Stress Resistance of&#160;Caenorhabditis elegans&#160;in 96-well Microtiter Plates.</a>&#160;J. Vis. Exp. (2015).
This video introduces a short-term survival assay that tests a treatment for acute toxicity or tests a genetic strain for resistance to a toxic condition.&#160; The example protocol shows an example survival assay in which worms are tested for their ability to&#160; resist oxidative stress induced by paraquat (PQ).

Source: Possik, E. and Pause, A. Measuring Oxidative Stress Resistance of&#160;Caenorhabditis elegans&#160;in 96-well Microtiter Plates.&#160;J. Vis. Exp. ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Samenvatting

Meer video's verkennen