Name: C. elegans Lawn Avoidance Assay
Uploaded: 2023-05-19T16:00:00
Description: Watch this Scientific Journal Video about C. elegans Lawn Avoidance Assay at JoVE.com

The video outlines a procedure for recording the behavior of wild-type and npr-1 mutant L4 stage worms on a PA14 bacteria lawn. It involves transferring the worms, focusing the camera, and capturing a 20-hour recording, resulting in a 20-minute time-lapse video of their movement patterns.

c. elegans lawn avoidance assay

用于 秀丽隐杆线 虫草坪回避测定的基于智能手机的成像

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.

作者聚焦：一种基于智能手机的秀丽隐杆线虫草坪回避测定成像方法  

一种基于智能手机的秀 丽隐杆线虫 草坪回避测定成像方法

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.

Watch this Scientific Journal Video about C. elegans Lawn Avoidance Assay at JoVE.com

C. elegans Lawn Avoidance Assay  

秀丽隐杆线虫 草坪回避测定

使用铂线镐将 30 个同步的 L4 级蜗杆转移到 PA14 板上。将蠕虫放在PA14细菌草坪的中间。在这项研究中，对于每种情况，测试了两个板。将两个板放在记录设备的升高台上，盖子朝下，使琼脂侧朝上朝上。在智能手机屏幕上，点击板的位置，以便相机可以聚焦在检测板上。开始录制，然后将更多板添加到舞台。从放置在舞台上的最后一组盘子开始记录 20 小时。在最后的延时视频中，20小时的录制将产生20分钟长的视频。将野生型N2蠕虫与npr-1突变体进行比较。野生型蠕虫逐渐离开细菌草坪并留在外面。视频中可以清楚地看到厚厚的细菌草坪外的蠕虫，但细菌草坪内的蠕虫更难区分。

Research

JoVE Journal

Behavior

C. elegans Lawn Avoidance Assay

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 ...