Drosophila Burrowing and Tunneling Assay: A Method to Assess Tissue Hypoxia in Fly Larvae

- To set up the embryo collection cage, use grape juice agar plates supplemented with fresh yeast paste and place the plates onto cages containing male and female Drosophila flies of the appropriate genotype. The odor from the yeast and grape juice is attractive and promotes egg laying by the females. Allow egg laying for a timed period. Then remove the adults and incubate the embryo plates for 24 hours to obtain first instar larvae. Then gently transfer a few individual larvae from the grape juice plate onto an agar-only test plate where a hole in the agar in the center of the plate was cut out and filled with yeast paste.

Young larvae must feed to gain body weight to develop, so they burrow in the food source, the yeast paste. As they develop, larvae enter a wandering phase and tunnel away from the food to seek out a site for pupation. To assess hypoxia, examine the tunneling patterns larvae form in the substrate. Lack or insufficient tunneling is a sign of oxygen deprivation. In the example protocol, we will see how to set up the burrowing and tunneling assay.

- Set up the control and experimental genetic crosses in egg lay dishes as described in the text protocol. Keep the flies in darkness for at least two days prior to starting timed egg collections. For timed egg collection, transfer the adults to new grape agar plates decorated with a fresh smear of yeast paste early in the day and allow the adults to lay eggs on the plates for four hours. After four hours, transfer the adults to fresh grape agar plates.

Next, incubate the four-hour collection plates overnight at 25 degrees Celsius. By the next afternoon, most of the larvae will have hatched. Plan to have at least 50 for each experimental group.

For a single run of the assay, prepare at least five assay plates per experimental group. Add 16 grams of fly agar in 700 milliliters of deionized water by heating. Prepare 17.5 milliliters Nipagen in 95% ethanol. Heat the agar solution until it is almost completely dissolved. Then add Nipagen solution to the agar solution and heat until agar completely dissolves. Cool the agar solution briefly, then load 10 centimeter plates with 15 milliliters of the agar solution. Once the agar gels, put lids on the plates and let them dry at room temperature for a few hours or overnight.

Cured agar must be firm to the touch and resist crumbling when pieces are cut out of it. Once the agar has cured, use a 1.5 centimeter cork borer to make a central hole in the agar gel in each plate. Then fill the holes neatly with fresh yeast paste.

To transfer the larvae to the assay plate, make a simple tool. Bend a curve into the tip of a plastic microspatula and dip the tip in yeast paste to make it adhesive to larvae. Now pick up the first instar larvae individually and place them on the agar plate close to the food mound. For each experimental group, prepare at least five plates each with 10 larvae.

To ensure reproducibility between assay replicates, it is critical that only 10 larvae are placed on each assay plate. Be sure to check the assay plate carefully to determine that one and only one larva is transferred each time you deliver a larva with the microspatula tip.

Once the plate is loaded, label it, cover it, then place it in darkness with the lid side up at room temperature. Examine each plate daily until all the larvae have died or pupated. Here are examples of plates for a wild type strain from day two through day eight of the assay. On day two, larvae are buried in food and no tunneling occurs. Tunneling begins on day three and reaches a maximum between days five and eight as larvae cease wandering and pupate in their tunnels.

Carefully transfer the pupae with a bent teasing needle to a grape plate and if desired, count how many pupae eclose. Take note of pupae formation and evaluate pupae for abnormalities.