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JoVE Encyclopedia of Experiments
Encyclopedia of Experiments: Biology

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Light Spot Assay: A Method to Investigate Drosophila Phototactic Behavior



- Begin by selecting a third instar larva, the largest of the larval forms, from the culture medium, and wash it with water to remove any food. Since the larval eyes, or Bolwig organs, are located in the upper front of the larva, place the larva in the center of an agar plate with the light source above the animal. Turn off the room light and allow the larva to acclimate to the dark.

Since the larva is barely detectable in a light spot made by visible light, turn on infrared LED lights to illuminate the animal for the camera. Wait until the animal begins crawling straight to control for its path. And then rotate the plate so that the larva crawls directly toward the light spot. Record the animal crawling into the light and then away from the light spot. This light-avoidance tactic helps larvae avoid predators by burrowing into food.

In the following example, we will perform the light spot assay to test the ability of different genetic strains of Drosophila larvae to escape light.

- Before introducing the larva to the imaging setup, acquire a short video of the light position, and name the unfiltered control background video "lightarea1". Place the filter back on the camera lens, and turn on a light far from the experimental device at as low a setting as possible while still allowing the larvae to be clearly observed. Use a spoon to remove some larvae from the culture medium, and gently select a third instar larva.

Wash the larva in distilled water, and place the larva in the center of the agar plate. Gently remove any excess water from the larva, and turn off the room light. Allow the larva to acclimate for two minutes in the dark environment before turning on the infrared LED light. The following light-avoidance behavior experiment needs to be carried out in the dark. For the convenience of demonstration, we filmed in the bright light.

Gently brush the larva to the center of the plate. When the larva begins to crawl straight, rotate the plate to make the larva head towards the light spot. Click Capture, Setup, and Video Capture to select the save path. And click Start Recording to record. Allow the lava to crawl toward, enter, and leave the light spot until it is nearly out of the field of view, then click Stop Recording.

Move the filter away from the camera, and acquire a short video of the position of the light spot. Then name the post as a unfiltered video, "lightarea2", and compare this video with the "lightarea1" video to ensure that the light spot position is not changed.

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