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

This protocol is an excerpt from Qiang et al., A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae, J. Vis. Exp. (2018).

1. Preparation of larvae

1. Two or more days before beginning the assay, set up crosses of the desired experimental and control combinations (minimum 20 females and 10 males each) in egg-lay dishes as described by Wieschaus and Nusslein-Volhard but using 50 mL polypropylene beakers and 6.0 cm disposable Petri dishes containing grape agar and smeared with yeast paste just before use. Keep flies in egg-lay dishes in darkness at room temperature until needed.
2. Grape agar recipe – combine 100 mL frozen 100% grape juice concentrate, 350 mL deioinized water, 5 mL glacial acetic and 13 g D. melanogaster agar in a 1L glass beaker. Microwave in 1-2 min bursts, stirring between bursts, until agar is all dissolved. Cool the solution for a few minutes then add 10 mL of 10% (w/v) of Nipagen (methyl-p-hydroxybenzoate) in 95% ethanol. Mix, then pipette 7 mL per plate into disposable 6.0 cm Petri dishes, allow to gel and dry at room temperature for 3-4 h. Store at 4 °C in plastic bags.
3. Yeast paste recipe – 7 g dried yeast, 10 mL deionized water, mix with a spatula until uniform consistency.
4. Timed egg collections to generate experimental and control larvae. Using new, yeast-smeared, grape plates, collect eggs at room temperature for 4 h in darkness during the morning hours. Remove these 4 h collection plates and replace with fresh plates. Incubate the 4 h collection plates overnight at 25 °C until the afternoon of the next day, when most of the larvae will have hatched. Collect these first instar larvae and use them to set up the experiment.

2. Setting up assay plates

1. Preparation of assay plates. For a single run of the assay, prepare five assay plates each of 10 experimental larvae and five plates of 10 control larvae. Use a 1.5 cm cork borer to remove a central core of agar from each plate creating a hole for the food. Put yeast paste (0.8 – 0.9 g) into the hole and pat gently with a spatula to make a mound filling the hole.
2. Agar plate preparation – combine 700 mL deionized water with 16 g D. melanogaster agar in a 2L glass beaker and microwave for 5 min. Remove from microwave and stir with a glass rod to bring undissolved agar into the solution. Add 17.5 mL of 10% (w/v) Nipagen in 95% ethanol, mix, then continue microwave heating in 30 s bursts followed by stirring, until all agar is completely dissolved. Cool for a minute or two, then pipette 15 mL aliquots into 10 cm plastic Petri dishes. Allow agar to gel, cover plates with lids and let them dry at room temperature for a few hours or overnight. Store plates in sealed plastic bags at 18 °C.
   NOTE 1: To avoid formation of Nipagen crystals on the surface of the plates due to excess dessiccation, use plates within a few days of preparation. If necessary, crystals can be re-dissolved by dropping a few microliters of 95% alcohol onto them.
   NOTE 2: batches of agar can have different gelling properties. The dry gel plates should be firm to the touch and sufficiently resilient that a clean, intact circle of agar can be removed when using the cork borer to create the food hole (see above).
3. Setting up larvae in assay plates. Use mechanical pressure to curve the tip of a plastic microspatula and dip the tip in yeast paste to provide "adhesive" for picking up larvae. Pick up first instar larvae individually and place them on the agar plate close to the food mound. Prepare at least five replicate plates of 10 larvae for both experimental and control genotypes. Once completed, cap and label each plate and set the plates (lid side up) in a dark space at room temperature (in our lab this is 22 °C).

3. Monitoring assay plates

1. Examine plates daily under a dissecting microscope and record the number of larvae on top of the food or out on the agar surface. Note any visible dead or dying larvae. Note when, and if, tunneling into the agar substratum is seen as larvae move into third instar. Note dates on which pupae begin to form and note any pupal abnormalities. Continue daily observations until all larvae have died or pupated.
2. Remove pupae carefully from the plate with a bent teasing needle and transfer to a grape plate. If desired, continue monitoring pupae to determine how many eclose as adults.