Foraging Path-length Protocol for Drosophila melanogaster Larvae

1. Prepare Grape Plates and Holding Bottles for Collection of Larvae

1. To make holding bottles, cut holes in one side of 6 oz fly culture bottles, large enough to fit a fly vial plug for air supply (Fig. 1D).
2. To make grape plates, prepare 250 ml of grape juice medium (1.8% agar, 45% grape juice, 2.5% acetic acid, 2.5% ethanol) by boiling the agar, grape juice and most of the water, cool down to 70 ºC (stir while cooling), then add acetic acid and ethanol and bring up to volume with water.
3. Use a syringe (without needle) to fill 35 x 10 mm Petri dish lids until the surface of the grape juice medium makes a dome above the lip of the lid (Fig. 1A).
4. Store grape plates in a humid and airtight container at 4 ºC until use (up to a maximum of 2 weeks).

2. Preparation of Food Plates

1. Prepare standard fly food recipe.
   Note: To test rearing food quality effects on path-length we used low nutrient fly food: 10% sucrose, 1.3% agar, 0.8% potassium sodium tartrate, 0.1% monopotassium phosphate, 0.05% sodium chloride, 0.05% calcium chloride, 0.05% magnesium chloride, 0.05% ferric sulfate, 5% yeast and 0.5% propionic acid in water; and high nutrient fly food: 1.5% sucrose, 1.4% agar, 3% glucose, 1.5% cornmeal, 1% wheat germ, 1% soy flour, 3% molasses, 3.5% yeast, 0.5% propionic acid, 0.2% Tegosept and 1% ethanol in water. For both foods autoclave the solution and cool to 50 ^oC before adding the propionic acid, Tegosept and ethanol.
2. Pour 40 ml of fly food into 100 x 15mm Petri dishes.
3. Store food plates in a humid and airtight container at 4 ºC until use (up to a maximum of 2 weeks).

3. Prepare Test Plates

1. Prepare 58 x 25 cm, 6 mm thick black Plexiglas plates with 10, 1 mm deep circular wells, 10 cm in diameter and arranged in a 2 x 5 design (Fig. 1C) to test larvae on.
   Note: Up to 30 larvae can be tested at a time if 3 or more of the above Plexiglas plates are available. Alternatively, larvae can be tested in Petri dishes, 10 cm in diameter. This option is much more laborious and allows for lower numbers of larvae to be tested.
2. Obtain a black 39 x 8 cm, 6 mm thick Plexiglas spreader for spreading the yeast paste on the Plexiglas test plates.
3. Prepare 100 x 15 mm Petri dish lids for recording larval path-lengths by separating lids from bottoms in advance.

4. Setting up Parental Populations

1. Keep rearing conditions constant throughout the entire assay (e.g., 25 ºC, 60% humidity and a 12:12 light:dark cycle on standard fly food).
2. Raise parental population in same rearing conditions.
3. Collect 100 – 150 females and 50 – 75 males and age them for 5 ± 1 days for optimal fecundity.
4. Allow females and males to mate overnight in a 6 oz fly culture bottle with standard fly food.
5. Transfer flies into a holding bottle and secure a grape plate (a small amount of fresh yeast paste on the grape plate will promote egg laying; Fig. 1B) over the top with masking tape (Fig. 1E).
6. Leave bottles standing upside down (Fig. 1F), with the grape plate at the bottom to allow adults to lay eggs on the grape plate.
7. Discard the first grape plate after 24 hr and place a new grape plate in the holding bottle. This will be the grape plate from which larvae are collected the next day.
   Note: Grape plates will have to be cleared of larvae 22 hr after being set up, set up plates so that they can be cleared early next day (e.g., set up plate at 12 pm and clear at 10 am the next day). It is advisable to discard the first grape plate to avoid any eggs that females might have been withholding and that might be advanced in development.

5. Collect L1 (First Instar) Larvae from Grape Plates

1. Remove grape plates from bottles 22 hr after they were set up and place in a 60 x 15 mm Petri dish.
2. Place a new grape plate with fresh yeast paste on the holding bottle.
3. Clear and discard all larvae from the seeded grape plate using a dissecting probe.
4. Incubate cleared grape plates for 4 hr in standard conditions.
5. After 4 hr, pick 100 L1 larvae per strain from the grape plates and place on food plates.
   1. Place larvae on the food gently (without perforating the food and burying the larvae) and avoid crowding by distributing the larvae across the food plate.
6. Incubate food plates until test animals are approximately 10 hr before the start of wandering.
   Note: At 25 ºC, 10 hr before wandering generally corresponds to 72 – 96 hr after hatching. The exact amount of incubation time should be determined before starting the experiment by collecting an initial set of larvae for each strain and recording the number of hours until the start of wandering (wandering larvae will move out of the food onto the sides and lid of the food plates).
   Note: If needed, the same parents can be used to collect larvae for 3 – 4 days of testing by repeating steps 4.7 to 5.5. Change the order in which strains are set up and tested across days to avoid a test order effect on path-length. Test the same "control" strain/strains each day of testing to control for day effects.
7. If many strains are tested, stagger the set up and collection times of the larvae to allow for the testing of each strain at the appropriate time point later on.

6. Larval Path-length Test

1. Dissolve dry-active baker's yeast in water at a 1:2 ratio (weight to volume). The finished yeast paste should be thinner than pancake batter, in a way that when lifting a spoon out of the paste, it drips and leaves behind ribbons on the surface of the paste that quickly melt away.
   Note: The consistency of the yeast paste is important and should be constant throughout the experiment. The exact yeast:water ratio might have to be adjusted depending on the brand and batch of yeast.
2. Gently pick larvae of the first strain to be tested from the food plate using a paint brush.
3. Collect larvae in a Petri dish and gently wash with water to remove all food.
   1. Quickly wash larvae with 1 – 2 ml of water and gently dab larvae dry with a laboratory wipe to avoid transferring any water when placing the larvae on the test plate. To avoid stress, keep larvae humid but not submersed in water.
4. Arrange three test plates side by side and set a 5 min timer for each test plate.
5. Apply yeast paste to the top of the test plates and using the spreader, spread a thin layer of yeast paste on each test plate so that all the wells have an even layer of yeast.
6. Gently place one larva in the center of each well, starting the 5 min timer after placing the first larvae on each plate. Note: Three plates can be done at a time, yielding a sample size of 30/strain/test day, up to a total of 500 larvae.
7. Place a Petri dish lid on top of each well.
8. When the timer runs out, without removing the larvae from the plate, start tracing the path-lengths left behind in the yeast using a permanent marker. Trace path-lengths in the same order as larvae were placed on the test plates.
   1. Perform tracing at the same speed as larvae were placed on the plates. For consistency in later data analysis, use the same marker for all path-lengths. Permanent marker is recommended.
9. Repeat steps 6.2 to 6.8.1 for each strain to be tested. Pick each strain from the food immediately before testing to avoid starvation effects on path-length.

7. Food Deprivation

1. If larvae are to be food deprived prior to testing, collect larvae as described in steps 6.2 to 6.3, but the desired amount of food deprivation time earlier (e.g., if larvae are to be food deprived for 4 hr, collect larvae 4 hr before test time).
2. Divide washed larvae into two groups and place the food deprived group in a 60 x 15 mm Petri dish with 5 ml 1% agar and the control group in a Petri dish with 5 ml standard food.
3. Place a weight on top of the inverted lid of the Petri dishes (otherwise larvae are able to crawl out when searching for food).
4. Incubate for the previously established food deprivation period and proceed with testing as described in steps 6.2 to 6.9.
5. If many strains are to be tested, stagger the food deprivation start times to allow for testing of each strain at the appropriate time point.

8. Data Analysis

Note: This section describes a method for data analysis using ImageJ⁷ or Fiji⁸ for processing of path-lengths, although other software can be used.

1. Use a light table to transfer all traced path-lengths from the Petri dish lids to white paper (lids can be washed with ethanol and reused for further path-length recording).
2. Trace a 50 mm straight reference line on one of the sheets of paper.
3. Scan the path-lengths at a resolution of 300 dpi and save as bitmap or tiff image files.
4. Open the image file, select the "Process" tab > "Binary" > "Make binary". Then under the "Process" tab, select "Binary" > "Skeletonize". This produces a one pixel width line, regardless of thickness of trace.
5. Make sure each path-length appears as a continuous single line with no crossovers.
6. Separate any intersections using the "Straight line tool" and coloring the line white (the continuity of the path can be checked with the "Wand" (tracing) tool, the entire path should be highlighted yellow (Fig. 2).
7. Select the "Analyse" tab > Set measurements and select "Perimeter" and "Display label".
8. Select the "Analyse" tab > "Set scale" and select "Remove scale".
9. On the image, select the reference line only, select the "Analyse" tab > "Analyse particles" and select "Show: Outlines", "Display results" and "Clear results table". Note: The output will show the number of pixels corresponding to the 50 mm scale line.
10. Select "Analyse" tab > "Set scale" and fill in the pixel number in "Distance in pixels" and the corresponding distance (50 mm) in "Known distance".
11. Finally, select all the path-length drawings of one genotype at a time, select the "Analyse" tab > "Analyse particles" and select "Show: outlines", "Display results" and "Clear results table".
    1. Remove any written labels or obvious extraneous markings (they will be recognized as path-lengths) within the selected area by selecting the area and filling it white.
12. Visually inspect the "Outlines" output file to ensure the perimeters were correctly ascertained. The output will show a path-length in mm for each path-length drawing in the selection. This can easily be exported to a spreadsheet and analyzed with any statistical software.