TITLE: A simple phototaxis assay for aquatic larvae
AUTHORS: Julie M Butler, Lauren A O'Connell

[DESCRIPTION]
Phototaxis assays are utilized throughout neuroscience research to measure exploratory behaviors and visual capabilities. Here we detail a simple and low cost phototaxis assay for aquatic larvae. The assay chamber is constructed out of concentric petri dishes, where the animal is in the central chamber while the outer chamber can be rotated. The ability rotate the arena allows observers to distinguish between an animal settling on one side of the arena versus a true preference for one environment over the other. This assay is useful for behavior assays in laboratory settings, including undergraduate teaching laboratories. Student can gather data in real time in a relatively-high throughput manner. 


[BEFORE_START]
Experiments involving animals must have prior approval from an Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.

[GUIDELINES]
The size of the behavior arena should be adjusted based on the size of the animal. This protocol serves as a starting point, but chamber size may need to be adjusted for larger animals.

[STEPS]
SECTION: Arena construction
1. Obtain a large (15 cm diameter) petri dish and burn a hole in the middle of the petri dish just large enough to fit a screw through. This is the chamber base. 
SECTION: Arena construction
2. Paint half the base black on the inside of the dish. Repeat with two more paint layers until opaque.
SECTION: Arena construction
3. Spray the inside of the base with a clear sealant to seal the paint. This also creates a "frost" to diffuse light through the unpainted side.  

SECTION: Arena construction
4. Glue a nut to the middle of the inside of a 15 cm petri dish lid using aquarium sealant.  

SECTION: Arena construction
5. Obtain a medium (10 cm diameter) petri dish and glue a bolt to the bottom using aquarium sealant.   

SECTION: Arena construction
6. The bolt attached to the 10 cm base goes through the hole in the painted 15 cm petri dish base and screws into the nut attached to the 15 cm petri dish lid. This way, the painted base can be rotated without adjusting the dish (10 cm base) containing the tadpole.  

SECTION: Arena construction
7. Place the arena in the center of a light pad. 
SECTION: Arena construction
8. Set up a camera above the arena.   

SECTION: Arena construction
8.1. Set up a stack of two pipette tip boxes on each end of the arena with a camera holder spanning them.
SECTION: Arena construction
8.2. Place your phone a the holder, with your camera pointing through the hole. Any board with a hole in the middle big enough for the camera to film through and strong enough to cold the phone/camera is sufficient.
SECTION: Arena construction
7.1. Check to make sure the 10 cm dish is level. If not, adjust the arena or light pad until level by placing a piece of paper towel under one side.
SECTION: Arena construction
7.2. It helps to have the arena positioned such that the border between the light and dark sides is completely vertical or horizontal. This will make flipping it easier.
SECTION: Running the phototaxis trial
9. Fill the 10 cm dish with 0.1x MMR (~40 ml). Make sure there’s no debris in the water.
SECTION: Running the phototaxis trial
13. Transfer a single tadpole from its holding container to the arena. 
SECTION: Running the phototaxis trial
14. Record behavior for 3 m
SECTION: Running the phototaxis trial
13.1. Animal transfer can be done with a transfer pipette with the tip cut off or a spoon, depending on the species and size.
SECTION: Running the phototaxis trial
13.2. Gently place the tadpole in the center in the 10 cm dish.
SECTION: Running the phototaxis trial
14.1. If recording behavior in real time, use a clicker to record the number of movements the tadpole makes and use the timer to record the time spent on the black side. In a classroom setting, this can be done by a pair of students (one measuring time spent and the other the number of movements).
SECTION: Running the phototaxis trial
14.2. Alternatively, behavior can be scored later using software.
SECTION: Running the phototaxis trial
15. After 3m, carefully rotate the 15 cm painted base 180 degrees so that the black and clear sides are flipped.
SECTION: Running the phototaxis trial
16. Start the timer for 3 min after completion of the arena flip and record behavior.
SECTION: Running the phototaxis trial
17. After 3 m, stop the video recording.
SECTION: Running the phototaxis trial
18. Take the tadpole out of the arena and return the tadpole to its container.
SECTION: Running the phototaxis trial
19. Remove the water from the 10cm dish. If running another animal, fill with new 0.1x MMR.
SECTION: Data analysis
20. Calculate behavior scores and compare across experimental groups. This could include total time spent on the dark side and total number of movements.   

SECTION: Running the phototaxis trial
12. Start the recording on the camera.
SECTION: Running the phototaxis trial
11. Turn on the light pad. Make sure the light is at full strength by holding down the power button.
SECTION: Running the phototaxis trial
10. Place a label on the light pad with the trial or animal number so that it is visible in your recording.