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 a remove any food. Since the larval eyes, or Bolwig organs, are located di the upper front of the larva, place the larva di the center of an agar plate with the light source above the animal. Turn off the room light and allow the larva a acclimate a the dark.

Since the larva is barely detectable di a light spot made by visible light, turn on infrared LED lights a illuminate the animal for the camera. Wait until the animal begins crawling straight a 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 a test the ability of different genetic strains of Drosophila larvae a escape light.

– Before introducing the larva a the imaging setup, acquire a short video of the light position, and nome 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 a be clearly observed. Use a spoon a remove some larvae from the culture medium, and gently select a third instar larva.

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

Gently brush the larva a the center of the plate. When the larva begins a crawl straight, rotate the plate a make the larva head towards the light spot. Click Capture, Setup, and Video Capture a select the save path. And click Start Recording a record. Allow the lava a 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 nome the post as a unfiltered video, “lightarea2”, and compare this video with the “lightarea1” video a ensure that the light spot position is not changed.