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

Imagerie sur smartphone pour le test d’évitement de la pelouse de 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.

Pleins feux sur l’auteur : Une méthode d’imagerie basée sur un smartphone pour le test d’évitement de la pelouse de C. elegans  

Une méthode d’imagerie basée sur un smartphone pour le test d’évitement de pelouse de C. elegans

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  

C. elegans Essai d’évitement de pelouse

À l’aide d’un pic en fil de platine, transférer 30 vers synchronisés du stade L4 sur la plaque PA14. Placez les vers au milieu de la pelouse des bactéries PA14. Dans cette étude, pour chaque condition, deux plaques ont été testées.Placez les deux plaques sur la platine surélevée de l’appareil d’enregistrement, le couvercle étant orienté vers le bas, de sorte que le côté gélose soit orienté vers le haut vers la caméra. Sur l’écran du smartphone, appuyez sur l’emplacement de la plaque pour que l’appareil photo puisse effectuer la mise au point sur les plaques d’essai. Démarrez l’enregistrement, puis ajoutez d’autres plaques à la scène.Enregistrez pendant 20 heures à partir du dernier jeu d’assiettes placées sur la scène. Dans la vidéo time-lapse finale, 20 heures d’enregistrement donneront une vidéo de 20 minutes. Les vers N2 de type sauvage ont été comparés aux mutants npr-1.Les vers de type sauvage ont progressivement quitté la pelouse bactérienne et sont restés à l’extérieur. Les vers à l’extérieur de l’épaisse pelouse bactérienne ont été clairement vus dans la vidéo, mais les vers à l’intérieur de la pelouse bactérienne étaient plus difficiles à distinguer.

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 ...