Nov 21, 2024
Eradicating Wolbachia: A tetracycline-based approach for Drosophila melanogaster and other fruit flies
- Brandon Cooper1,
- J. Dylan Shropshire2
- 1Division of Biological Sciences, University of Montana;
- 2Department of Biological Sciences, Lehigh University
- Shropshire Lab
Protocol Citation: Brandon Cooper, J. Dylan Shropshire 2024. Eradicating Wolbachia: A tetracycline-based approach for Drosophila melanogaster and other fruit flies. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp9rwpvzp/v1
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 09, 2024
Last Modified: November 21, 2024
Protocol Integer ID: 111837
Keywords: Wolbachia, Drosophila, tetracycline, wolbachia from drosophila melanogaster, valuable tool for the drosophila research community, flies with tetracycline, wolbachia from the host lineage, approach for drosophila melanogaster, including drosophila melanogaster, eliminating wolbachia, drosophila research community, drosophila melanogaster, drosophila, eradicating wolbachia, treating fly, other fruit fly species, wolbachia researcher, using tetracycline antibiotic, tetracycline antibiotic, free insect, wolbachia, arthropod host, host lineage, biology, species, insect
Abstract
Wolbachia, a maternally inherited endosymbiont, can significantly influence the biology of its arthropod hosts, including Drosophila melanogaster. Its presence can confound experimental results. To overcome this challenge, we present a protocol for eliminating Wolbachia from Drosophila melanogaster and other fruit fly species using tetracycline antibiotics. By treating flies with tetracycline during development, we successfully eradicate Wolbachia from the host lineage, enabling precise and controlled experiments. This protocol provides a valuable tool for the Drosophila research community. Wolbachia researchers can also benefit from this protocol to compare Wolbachia-bearing and Wolbachia-free insects.
Materials
Materials
- Centrifuge tubes, safe-lock, Eppendorf quality, 1.5 mLEppendorfCatalog #022363204
- Pipette tips, filtered, PCR clean and sterile, 2 - 100 uLEppendorfCatalog #0030078543
- Tetracycline hydrochlorideFisher ScientificCatalog #BP912
- diH2O
Equipment
- Analytical balance (any)
- Drosophila incubator (Percival, DR-36VL)
- Microwave (any)
- Pipette, 100 μL (Eppendorf, 4861000716)
- Probe thermometer (any)
- Vortex mixer (any)
Protocol materials
Centrifuge tubes, safe-lock, Eppendorf quality, 1.5 mLEppendorfCatalog #022363204
Tetracycline hydrochlorideFisher ScientificCatalog #BP912
Pipette tips, filtered, PCR clean and sterile, 2 - 100 uLEppendorfCatalog #0030078543
Troubleshooting
Safety warnings
This protocol involves the use of various chemicals and reagents that require careful handling and strict adherence to safety guidelines to ensure safe laboratory practices. Please review the Material Safety Data Sheets (MSDS) for each reagent before beginning the protocol and take appropriate precautions. All steps should be performed while wearing gloves, a lab coat, and eye protection.
Before start
Prepare standard fly food. We use the following protocol:
Protocol
CREATED BY
J. Dylan Shropshire
Sterilize the working surface with 10% bleach and 70% ethanol.
Preparing tetracycline-treated fly food.
Using an analytical balance and a pipette, add the following to a Centrifuge tubes, safe-lock, Eppendorf quality, 1.5 mLEppendorfCatalog #022363204 :
- 3 mg Tetracycline hydrochlorideFisher ScientificCatalog #BP912
- 50 µL diH2O
Vortex and invert the tube until the tetracycline to completely dissolve.
Microwave a vial containing 10 mL of fly food until boiling.
Take care not to allow the food to boil up the sides of the vial.
Stir as needed.
Allow food to cool to 55 °C .
Use a probe thermometer to confirm the temperature.
Add the dissolved tetracycline to the food and mix thoroughly.
The final concentration of tetracycline is approximately 0.3 mg/mL .
Allow food to cool to Room temperature .
Use a probe thermometer to confirm the temperature.
Treat flies with tetracycline
8w 5d
Transfer Wolbachia-bearing flies onto the tetracycline-treated media.
After four days, clear vials of adults.
(Optional) Store vials in a Drosophila incubator set to 23 °C or 25 °C .
4d
Monitor vials regularly.
Once adults begin emerging, transfer to a freshly prepared vial of tetracycline-treated food.
1w
Repeat and until flies have been maintained on tetracycline-treated food for three generations.
3w 1d
Test progeny for Wolbachia.
If the flies still have Wolbachia, continue tetracycline treatment for an additional generation and test their offspring for Wolbachia.
Note
We use the following protocol to test if flies have Wolbachia using PCR:
Rear Wolbachia-free flies on regular fly food for 3 generations before using them in experiments.
Note
Tetracycline can interfere with the host's mitochondria. Providing time for the flies to recover is important to avoid confounding effects associated with mitochondrial disfunction.
Ballard JWO, Melvin RG. (2007) Tetracycline treatment influences mitochondrial metabolism and mtDNA density two generations after treatment in Drosophila. Insect Mol Biol. 16(6):799–802. PMID: 18093008
4w