Name: Author Spotlight: A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay
Uploaded: 2023-02-24T13:40:00
Description: Watch this Scientific Journal Video about A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay at JoVE.com

We thank Deok Joong Lee for critical reading of the manuscript and testing the Python code. This research was sponsored by the National Research Foundation of Korea 2017R1A5A2015369 (K.-h.Y.) and 2019R1C1C1008708 (K.-h.Y.).

1. Setting up the imaging apparatus (Figure 1A-E)
<ol>
	<li>Ensure that a smartphone camera with the following minimum requirements is available: 
	12&#160;megapixel (MP) camera 
	1080p resolution video 
	5 GB&#160;of storage space (20 min video is 3-4 Gb) 
	Time-lapse video app from the application store (free applications available)</li>
	<li>Place the LED light box on the bottom rack of the 25 &#176;C incubator where the assay will t...

Smartphone-Based Imaging for C. elegans Lawn Avoidance Assay

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensation+of+bacterial+secondary+metabolites+modulates+neuroendocrine+signaling+and+behavior+of+C.+elegans.">Chemosensation of bacterial secondary metabolites modulates neuroendocrine signaling and behavior of C. elegans.</a> Cell. 159 (2), 267-280 (2014).</li><li>Singh, J., Aballay, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+infection+regulates+behavior+and+learning+via+neuroendocrine+signaling.">Intestinal infection regulates behavior and learning via neuroendocrine signaling.</a> eLife. 8, 50033 (2019).</li><li>Lee, K., Mylonakis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intestine-derived+neuropeptide+controls+avoidance+behavior+in+Caenorhabditis+elegans.">An intestine-derived neuropeptide controls avoidance behavior in Caenorhabditis elegans.</a> Cell Reports. 20 (10), 2501-2512 (2017).</li><li>Singh, J., Aballay, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+lawn+avoidance+and+bacterial+two+choice+preference+assays+in+Caenorhabditis+elegans.">Bacterial lawn avoidance and bacterial two choice preference assays in Caenorhabditis elegans.</a> Bio-Protocol. 10 (10), 3623 (2020).</li><li>Reddy, K. C., Andersen, E. C., Kruglyak, L., Kim, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polymorphism+in+npr-1+is+a+behavioral+determinant+of+pathogen+susceptibility+in+C.+elegans.">A polymorphism in npr-1 is a behavioral determinant of pathogen susceptibility in C. elegans.</a> Science. 323 (5912), 382-384 (2009).</li><li>de Bono, M., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+variation+in+a+neuropeptide+Y+receptor+homolog+modifies+social+behavior+and+food+response+in+C.+elegans.">Natural variation in a neuropeptide Y receptor homolog modifies social behavior and food response in C. elegans.</a> Cell. 94 (5), 679-689 (1998).</li><li>Mathew, M. D., Mathew, N. D., Ebert, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WormScan:+a+technique+for+high-throughput+phenotypic+analysis+of+Caenorhabditis+elegans.">WormScan: a technique for high-throughput phenotypic analysis of Caenorhabditis elegans.</a> PLoS One. 7 (3), 33483 (2012).</li><li>Stroustrup, N., 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=The+Caenorhabditis+elegans+lifespan+machine.">The Caenorhabditis elegans lifespan machine.</a> Nature Methods. 10 (7), 665-670 (2013).</li><li>Churgin, M. 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A., Fang-Yen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+imaging+system+for+monitoring+C.+elegans+behavior+and+aging.">An imaging system for monitoring C. elegans behavior and aging.</a> Methods in Molecular Biology. 2468, 329-338 (2022).</li><li>Barlow, I. L., 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=Megapixel+camera+arrays+enable+high-resolution+animal+tracking+in+multiwell+plates.">Megapixel camera arrays enable high-resolution animal tracking in multiwell plates.</a> Communications Biology. 5 (1), 253 (2022).</li><li>Kawazoe, Y., Yawo, H., Kimura, K. 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Imaging animal behavior, rather than relying on direct observation, is not only convenient but also has the advantage of leaving visual documentation. This allows for blind analysis by an objective third person, or could even be used for automated analysis using image recognition techniques. Despite the advantages, the standard equipment usually offered is high in cost, so one is committed to the setup once purchased.
Using smartphones to collect video recordings of simple C. elegans ...

Among the many advantages of studying C. elegans, its simple nervous system offers the opportunity to study how changes at the genetic and cellular level impact network function and behavioral output. Despite having a limited number of neurons, C. elegans display a wide range of complex behaviors. One of these is lawn avoidance, in which the bacterivorous nematode responds to a harmful food source by leaving the bacterial lawn. C. elegans avoid lawns of pathogenic bacteria1,2,3, lawns of bacteria that produce toxins or are spiked with toxins1,4, and even RNAi-expressing bacteria whose target gene knockdown is detrimental to the health of the worms4,5. Studies have shown that worms respond to external cues such as metabolites produced by the pathogenic bacteria1,6, or internal cues that indicate that the food is making them sick4,7. These cues are processed through conserved signaling pathways, such as the mitogen-activated protein kinase (MAPK) pathway and the transforming growth factor beta (TGF&#946;) pathway, and require communication between the gut and the nervous system4,6,7,8.
Although the assay is simple, the learned behavior develops over many hours, often overnight. While there are mutants that are incapable of leaving, in which case scoring avoidance at just one time point is sufficient to demonstrate the defect, many mutants do leave eventually but are slower to come out. For these, the movement of the worms needs to be tracked every few hours, which can be difficult to do overnight. Counting itself also takes time, creating a lag time between the plates, and thus limits the number of plates that can be tested at the same time. Using an imaging setup to record many plates simultaneously for the whole duration of the assay would be very useful, but the cost of setup can be prohibitive, depending on the funding situation of the research lab.
To address this, we devised a very simple method that uses smartphones to record avoidance assays. Each phone can record time-lapse videos of up to six assay plates. To provide transmitted light, we use a light emitting diode (LED) light box that can be easily purchased online. Assay plates are placed on an elevated platform, supported by hollow rectangular tunnels, that focus the incoming light, creating contrast. We also provide a Python script that converts the videos into audio video interleave (AVI) files showing 10 s clips of each hourly time point. The videos are then cropped to individual plates and saved in separate files to use for manual counting.
The method provides a low-cost procedure that is also extremely easy to use, using items that are readily available to most people. Here, we describe the method using the well-established lawn avoidance assay against the human pathogen Pseudomonas aeruginosa (PA14), whose protocol has been previously described2,9. Finally, we also review the considerations and limitations of the imaging method for those that want to apply it to other C. elegans behavior experiments.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm Petri dish</td>
			<td>SPL</td>
			<td>#10035</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>BD</td>
			<td>#214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>BD</td>
			<td>#211677</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>DAEJUNG</td>
			<td>2507-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>BioBasic</td>
			<td>CD0122</td>
			<td></td>
		</tr>
		<tr>
			<td>Dipotassium hydrogen phosphate (K2HPO4)</td>
			<td>JUNSEI</td>
			<td>84120-0350</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>BioBasic</td>
			<td>GB0232</td>
			<td></td>
		</tr>
		<tr>
			<td>King B Broth</td>
			<td>MB cell</td>
			<td>MB-K0827</td>
			<td></td>
		</tr>
		<tr>
			<td>LED light box multi-pad</td>
			<td>Artmate</td>
			<td>N/A</td>
			<td>This is a USB powered, LED light pad for tracing and drawing purposes. Artmate is a Korean brand, but searching for &#34;LED light box for tracing&#34; in any search engine should yield numerous options from other brands. Overall dimension is around 9&#34; x 12&#34; (A4 size). For example, from amazon US: https://www.amazon.com/LITENERGY-Ultra-Thin-Adjustable-Streaming-Stenciling/dp/B07H7FLJX1/ref=sr_1_5?crid=YMYU0VYY226R&#38;keywords= 
			LED%2Blight%2Bbox&#38;qid=1674183224&#38;sprefix 
			=led%2Blight%2Bbo%2Caps%2C270&#38;sr=8-5&#38;th=1</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>DAEJUNG</td>
			<td>5514-4400</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic paper sleeve (clear)</td>
			<td>Smead</td>
			<td>#85753</td>
			<td>Any clear plastic sheet with a bit of stiffness can be used as stage. For example, from Amazon US: https://www.amazon.com/Smead-Organized-Translucent-Project-85753/dp/B07HJTRCT7/ref=psdc_1069554_t3_B09J48GXQ 
			8</td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate (KH2PO4)</td>
			<td>JUNSEI</td>
			<td>84185-0350</td>
			<td></td>
		</tr>
		<tr>
			<td>Power strip&#160;</td>
			<td></td>
			<td></td>
			<td>To accommodate 3 phones and one LED box, you need at least 4 outlets.</td>
		</tr>
		<tr>
			<td>Smartphone</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Minimum requirement: 12MP wide camera, 1080p HD video recording at 30fps</td>
		</tr>
		<tr>
			<td>Sodium chloride(NaCl)</td>
			<td>DAEJUNG</td>
			<td>#7548-4100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic anhydrous (Na2HPO4)</td>
			<td>YAKURI</td>
			<td>#31727</td>
			<td></td>
		</tr>
	</tbody>
</table>

a smartphone-based imaging method for c. elegans lawn avoidance assay

When exposed to toxic or pathogenic bacteria, the nematode Caenorhabditis elegans displays a learned lawn avoidance behavior, in which the worms gradually leave their food source and prefer to remain outside the bacterial lawn. The assay is an easy way to test the worms' ability to sense external or internal cues to properly respond to harmful conditions. Though a simple assay, counting is time consuming, particularly with multiple samples, and assay durations that span overnight are inconvenient for researchers. An imaging system that can image many plates over a long period is useful but costly. Here, we describe a smartphone-based imaging method to record lawn avoidance in C. elegans. The method requires only a smartphone and a light emitting diode (LED) light box, to serve as a transmitted light source. Using free time-lapse camera applications, each phone can image up to six plates, with sufficient sharpness and contrast to manually count worms outside the lawn. The resulting movies are processed into 10 s audio video interleave (AVI) files for every hourly time point, then cropped to show each single plate to make them more amenable for counting. This method is a cost-effective way for those looking to examine avoidance defects and can potentially be extended to other C. elegans assays.

Author Spotlight: A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay  

A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay

Among the many behaviors displayed by C-elegans, pathogenic lawn avoidance is the worm's defense strategy to minimize its exposure to harmful pathogens. In our lab, we use this behavior as a tool to identify genes involved in the proper functioning of the neurons that mediate this behavior. The lawn avoidance behavior is a simple assay, however it develops over many hours.Hence, it is convenient if the assay can be recorded to observe behavior over long time periods. Also, if multiple assays can be recorded at the same time, that's even better. Although there are many imaging solutions described in the literature, even a few modestly priced cameras may be out of reach for labs with limited resources.Also, some may want to try the assay, but do not plan on doing it long term. For such cases, short-term solutions such as this protocol might help. Our amazing protocol offers an easy, low cost way to record worm behavior.We use smartphones to record the assay, then process the video through a Python script that makes it suitable for counting.

The first video produced by the script is 1 h from the start of the assay. The video for 0 h is not saved, as worms start the assay inside the lawn, so the occupancy rate is always 100%.
Wild-type N2 worms are compared against npr-1 mutants, whose lawn avoidance defect is well-established in the literature6,10 (Figure 3A-E). As can be seen in the wild type, worms ...

Watch this Scientific Journal Video about A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay at JoVE.com

This article describes a simple, low-cost method of recording the lawn avoidance behavior of Caenorhabditis elegans, using readily available items such as a smartphone and a light emitting diode (LED) light box. We also provide a Python script to process the video file into a format more amenable for counting.

When exposed to toxic or pathogenic bacteria, the nematode Caenorhabditis elegans displays a learned lawn avoidance behavior, in which the worms gradually ...