Inducing blunt force trauma in third-instar Drosophila melanogaster larvae using the High-Impact Trauma (HIT) device

This protocol details the method used in Lika et al. (2021) to induce trauma to Drosophila melanogaster third-instar larvae using the High-Impact Trauma (HIT) device.

Culturing third-instar larvae
1. Anesthetize 20 adult flies (10 males and 10 females) using CO₂ and place them on molasses-agar egg-laying plates with yeast paste (Bai et al. 2009).
2. Keep adults on the agar-molasses plates for 2 hours at 25°C.
3. After 2 hours, plates should have many eggs spread throughout.
4. Incubate plates for 4 days at 25°C for the larvae to develop to the third-instar stage. 

Collecting third-instar larvae
1. Collect third-instar larvae using Dumont #5 forceps, and place 60 larvae on the bottom of an empty plastic fly vial (FisherBrand, catalog # AS-510, K-resin wide diameter vial).
    a. Carefully handle larvae. Avoid damaging them with forceps as this can induce melanization. If a larva melanizes or is killed from handling, it should be removed from the experiment.
2. Plug the vial using a cotton ball wrapped in plastic wrap (to prevent larvae sticking to the cotton ball, which makes them difficult to collect after strikes from the HIT device).
3. Prepare an equal number of control and treatment vials. 

Inducing blunt trauma using the HIT device
1. Place a vial onto the HIT device spring. Pull the spring back to 90° and release the spring, as described in Katzenberger et al. 2013 and Katzenberger et al. 2015.

    a. Control larvae are not subjected to strikes from the HIT device.
2. Take the vial off the HIT device and let it sit between strikes.
    a. Remove plastic wrapped cotton ball between strikes.
    b. If there are multiple treatment groups, perform strikes in succession.
3. Repeat this process three more times for a total of 4 strikes with 5 min between strikes.

Scoring phenotypes
1. Using forceps, carefully collect larvae and transfer them to a vial with cornmealmolasses-yeast food. Place a maximum of 20 larvae per vial.
2. Incubate vials at 25°C, and each day, visually inspect for dead larvae, melanized larvae, dead pupa, dead adults, and live adults.
    a. Larvae are considered dead if they do not show obvious locomotor activity
    b. Larvae are considered melanized if they have any black areas on their bodies
    c. Pupa are considered dead if they do not eclose as adults.
    d. Adults that survived were monitored daily, the number of surviving flies is counted until all flies have died, and cornmeal molasses food is changed every three days.
 

Materials
• Forceps (Dumont #5, Fine Scientific Instruments)
• Plastic fly vials (FisherBrand, catalog # AS-510, K-resin wide diameter vial)
• Plastic dishes and egg laying media (Bai et al. 2009)
• HIT device (Katzenberger et al. 2013, Katzenberger et al. 2015)

References
Bai J, Sepp KJ, Perrimon N. 2009. Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening. Nat Protoc 4: 1502-12. PMID: 19798083.
Katzenberger, R. J., Loewen, C. A., Wassarman, D. R., Petersen, A. J., Ganetzky, B., and Wassarman, D. A. (2013) A Drosophila model of closed head traumatic brain injury. Proc. Natl. Acad. Sci. USA 110, E4152-E4159. PMCID: PMC3816429
Katzenberger, R. J., Loewen, C. A., Bockstruck, R. T., Woods, M. A., Ganetzky, B., and Wassarman, D. A. (2015) A method to inflict closed head traumatic brain injury in Drosophila. J. Vis. Exp. 100, e52905. PMCID: PMC4544997
Lika, J., Katzenberger, R. J., Ganetzky, B., and Wassarman, D. A. (2021) Effects of blunt force injuries in third-instar Drosophila larvae persist through metamorphosis and reduce adult lifespan. MicroPubl. Biol. 2021, 10.17912/micropub.biology.000423. PMCID: PMC8278228