Foraging Path-length Protocol for Drosophila melanogaster Larvae

The overall goal of this assay is to quantify the locomotory component of Drosophila larval behavior. This method can help answer key questions in the behavior genetics field about genetic and environmental contributions to individual differences in larval behavior. The main advantages of this technique are that it is fast, reproducible, and allows for the collection of large sample sizes.

Generally, people new to this method might struggle because the success of the assay depends on developmental timing and the consistent, careful handling of the test animals. Demonstrating the procedure will be Ina Anreiter and Oscar Vasquez, graduate students in my lab. Prepare the holding bottle by cutting holes into one side of a six ounce, plastic fly culture bottle.

Next, use a syringe to fill Petri dish lids with previously prepared grape juice medium until the surface of the grape juice medium makes a dome above the lip of the lid. Store the grape plates in a humid and airtight container at four degrees Celsius for up to two weeks. Pour 40 milliliters of previously prepared fly food into 100 by 15 millimeter Petri dishes.

Obtain three black Plexiglas plates with ten circular wells ten centimeters in diameter and arranged in a two by five design to test the larvae, and a black Plexiglas spreader used to spread the yeast paste onto the Plexiglas plates. Then, separate the lids from the bottoms of the Petri dishes. Collect 100 to 150 females and 50 to 75 males.

Allow females and males to mate overnight in a six ounce fly culture bottle with standard fly food. Transfer the flies into a holding bottle, and use masking tape to secure a grape plate with a small amount of fresh yeast paste over the top of the bottle. Leave the bottle standing upside down with the grape plate at the bottom to allow adults to lay eggs on the grape plate.

Discard the first grape plate after 24 hours and place a new grape plate in the holding bottle. After 22 hours, remove the grape plates from the bottles and place them into a 60 by 15 millimeter Petri dish. Next, place a new grape plate with fresh yeast paste on the holding bottle.

Clear and discard all larvae from the seeded grape plate using a dissecting probe. Incubate the cleared grape plates for four hours in standard conditions. After four hours, pick 100 L1, or first instar larvae, per strain from the grape plates and place them on the food plates.

Place the larvae on the food gently, without perforating the food and without burying the larvae. Groups of ten larvae can be picked and placed at a time. Avoid crowding by distributing the larvae across the food plate.

Incubate the food plates until the test animals are ten hours before the start of wandering, when larvae leave the food in search of a pupation site. If the larvae have started to wander, and they're moving on the lid of the Petri dish, as you can see here, the animals are too old to test and their developmental time has to be reassessed. Dissolve yeast in water at a one-to-two ratio.

Ensure that the finished paste drips and leaves ribbons on the surface of the paste that quickly melt away. Evaluating the right consistency of the yeast paste is more important than using the exact ratio due to the variability between brands, batches of yeast, and the environment. With a paintbrush, gently pick the larvae out of the food plate from the first strain.

Collect larvae in a Petri dish and gently wash the larvae with water to remove all of the food. Quickly wash the larvae with two milliliters of water and gently dab the larvae dry with a laboratory wipe. Keep the larvae in a humid environment but not submersed in water to avoid stress.

Arrange three test plates side by side, and set a five minute timer for each test plate. Apply yeast paste to the top of the test plates and use the spreader to distribute a thin layer of yeast paste on each test plate so that all of the wells have an even layer of yeast. Gently place one larva into the center of each well.

Start the five minute timer immediately after placing the first larvae on each plate. Then place the rest of the larvae in the remaining wells. Place a Petri dish lid on top of each well.

When the timer runs out, start tracing the path-lengths left behind in the yeast using a permanent marker without removing the larvae from the plate. Trace path-lengths in the same order and speed as the larvae were placed on the plates. Finally, repeat the larval path-length test steps for each strain.

Data collected over three consecutive days of testing revealed a significant strain effect, with rovers traveling farther than sitters. In addition to the strain effect, larvae that were food deprived for four hours prior to testing had significantly lower path-lengths than fed larvae. Overall, path-lengths were longer when both strains were reared on high nutrient food compared to low nutrient food.

If performed properly, this assay allows a single person to test up to 500 larvae with ease in four hours, making it valuable for large scale phenotyping. Following the procedure that we have just shown, other variables such as sex, food deprivation, or food quality can be tested to show the effect of those variables on food related movement patterns. After watching this video, you should have a good understanding of how to measure Drosophila larval food-related locomotion for the assessment of the genetic and environmental contributions to individual differences in behavior.