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

Imagem baseada em smartphone para ensaio de prevenção de gramado 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.

Autor em destaque: um método de imagem baseado em smartphone para ensaio de prevenção de gramado de C. elegans  

Um método de imagem baseado em smartphone para 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.

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

C. elegans Lawn Avoidance Assay  

C. elegans Ensaio de Evitação de Gramado

Usando um palito de fio de platina, transfira 30 worms sincronizados de estágio L4 para a placa PA14. Coloque os vermes no meio do gramado da bactéria PA14. Neste estudo, para cada condição, foram testadas duas placas.Coloque as duas placas no estágio elevado do aparelho de gravação com a tampa voltada para baixo de modo que o lado do ágar esteja voltado para cima em direção à câmera. Na tela do smartphone, toque onde está a placa, para que a câmera possa focar nas placas de ensaio. Inicie a gravação e, em seguida, adicione mais placas ao palco.Recorde de 20 horas a partir do último conjunto de placas colocadas no palco. No vídeo final de lapso de tempo, 20 horas de gravação resultarão em um vídeo de 20 minutos de duração. Vermes selvagens N2 foram comparados com mutantes npr-1.Os vermes selvagens progressivamente deixaram o gramado bacteriano e ficaram do lado de fora. Vermes fora do gramado bacteriano espesso foram claramente vistos no vídeo, mas vermes dentro do gramado bacteriano eram mais difíceis de distinguir.

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 ...