A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae

The overall goal of this assay is to identify drosophila larvae suffering from tissue hypoxia despite being in normal oxygen levels. In drosophila there are many mutations that produce slow, poor, growth of the larvae and some larvae sluggishness. This assay identifys within that class of larvae, those that are suffering from tissue hypoxia.

Ultimately, this assay will help identify more mutations that produce oxygen deficiency in the tissues through various physiological processes. The idea for this assay arose from the observation that larvae with damaged airways or trachea showed two strange behaviors. These abnormal behaviors are the failure to burrow into food during early larva life and the failure to tunnel into a softer substratum during the wandering phase.

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 and 700 milliliters of deionized water by heating.

Prepare a 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 board 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 a 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 a 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 has died or pupated. Here are examples of plates for a wild type strain for 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 enclose. Take note of pupa formation and evaluate pupae for abnormalities.

To image the tunnels within the assay plate, first carefully remove and discard all the the yeast food from the central well of each plate. Then gently flood each plate with tap water and use a soft paintbrush to carefully rub off any debris that has been tracked onto the agar surface. Some pieces of agar may crumble where tunneling is most intense.

This can be corrected later. Continue to replace the water several times and gently brush away the debris until the plate is completely clean. Then give the plate a final rinse with deionized water, cover it, and let it dry at room temperature overnight.

It is important to use a gentle flow of water and a gentle brushstrokes to clean the agar plates so that you do not break off or damage parts of the agar gel. The next day, prepare a tiff file image of the plate using a black background to show the tunnels as bright white lines. Gel documentation systems work well for this step.

Then, quantitate the tunneling in the image using Image J with the Oval tool, define the agar gel up to, but not including, the plastic edge of the petri plate. Then apply an automatic threshold to produce a reverse image of the plate so the tunnels appear as black lines. To correct dark areas that contain no tunnels, use the Color picker and Paintbrush tools to white out those regions.

Next, under the Analyze pull-down menu, select Set scale and click to remove scale. Then go to the Set measurements window under Analyze and check Area and Limit to threshold. Now press the Control key with the M key to get a measurement of the black area in the image in the unit of pixels which represents the tunneled area.

To quantitate tunneling lost through agar erosion around the central hole, uncheck Limit to threshold. Use the Wand tool to define the central hole and press Control plus M to measure its value in pixels. Subtract from this value the pixel value of a 1.5 centimeter diameter hole.

Add the difference to the pixel value previously obtained to give the total tunneling pixel value. For plates in which the missing agar extends beyond the central ring of tunneling, an additional step is necessary to prevent quantitation from including areas outside the central region. Use the Color picker and Paintbrush tools to add a black bar as shown to limit the area quantified.

The described assay was used to investigate potential hypoxia in larvae with impaired function of the gene uninflatable in the trachae. The gene was suppressed RNAi via Gal4 UAS. Two Gal4 lines were used.

Breathless Gal4, which expresses through all of development in the entire tracheal system. Or cut(ue)Gal4, which expresses strongly in a small tracheal section during larva life. Control larvae contained one of the Gal4 lines, but no uninflatable RNAi.

Larvae with cut(ue)Gal4 driving uninflatable RNAi were indistinguishable from controls throughout development in terms of both growth and behavior. However, strong suppression of uninflatable RNAi throughout the trachae with blt-Gal4 resulted in failure to burrow in the feeding phase and produced a high death rate. These larvae also showed a complete absence of tunneling during the wandering phase.

Furthermore, the breathless Gal4 experimental larvae were smaller, thinner, and more sluggish than controls and remained as larvae long after all other groups pupated. After watching this video, you should have a good understanding of how to test drosophila larvae for tissue hypoxia by monitoring the extent to which they burrow into their food mounds or tunnel into a soft substratum. A complete absence of tunneling is a strong indicator of tissue hypoxia.