Larval Locomotion Assay

Grape Juice Agar Plates 

1. In a 500 ml conical flask, mix 4 g of bacteriological agar with 40 ml of grape juice and 160 ml of dH₂0. 
2. Add a magnetic stir bar and place the flask on a hot plate/stirrer. Bring to a boil to dissolve the agar. Remove the flask from the hot plate when the solution begins to vigorously boil and climb up the sides of the flask (takes about ~5-10 min). 
3. Place the flask on a different magnetic stir plate and let the flask cool to 50°C (or until the flask is tolerable to touch) with continuous mixing at mid-range speed. 
4. Once cooled, add 1 ml glacial acetic acid and 2 ml 95% ethanol to the solution and allow thorough mixing for ~30s.   
5. Dispense the molten grape juice agar solution in 100 × 15-mm petri dishes (about half the volume of the petri dish).
6. Let the grape juice agar plates solidify at room temperature.
7. Store grape juice plates in a humid and air-tight container or a plastic bag at 4 ºC until use (up to a maximum of 2 weeks).
8. Remove the lid of the petri dish and carefully pour the agar medium in. Close the lid and gently rotate the dish to ensure that the medium covers the plate evenly. 

Locomotion Assay

1. Remove a 3^rd instar wandering larva of the desired genotype from the food vial with a paintbrush.
2. Gently wash the larva with a small drop of H₂0 to remove any traces of food. 
3. Place the larva in the center of the 100 × 15-mm petri dish. Start the 3 min timer after the larva’s first stride or wave of contraction.  
4. When the timer runs out, remove the larva from the plate. With a permanent marker, trace the larval path in the agar on the lid. 

Data Analysis

1. Place a ruler alongside the plate lid. Acquire a digital image of the lid with the ruler beside it. The ruler is required to set the scale and calculate the distance traversed by the larvae. Save images as tiff or jpeg files.
2. To set the scale, open the image using ImageJ or Fiji. Select the straight line tool and draw a line along the ruler for a distance of 1 cm. Select: Analyze>Set Scale. In the dialog box, set known distance to 1.00 and unit of length as cm. 
3. Trace the larval tracks with the freehand line tool. 
4. Click Analyze and select Measure. Record the length of the line. 

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