Nov 21, 2024

Public workspaceEradicating Wolbachia: A tetracycline-based approach for Drosophila melanogaster and other fruit flies

DOI
dx.doi.org/10.17504/protocols.io.dm6gp9rwpvzp/v1
  • 1Division of Biological Sciences, University of Montana;
  • 2Department of Biological Sciences, Lehigh University
  • Shropshire Lab
J. Dylan Shropshire
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DOI: dx.doi.org/10.17504/protocols.io.dm6gp9rwpvzp/v1
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
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
  • ReagentCentrifuge tubes, safe-lock, Eppendorf quality, 1.5 mLEppendorfCatalog #022363204
  • ReagentPipette tips, filtered, PCR clean and sterile, 2 - 100 uLEppendorfCatalog #0030078543
  • ReagentTetracycline 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
ReagentCentrifuge tubes, safe-lock, Eppendorf quality, 1.5 mLEppendorfCatalog #022363204
Materials, Step 1
ReagentTetracycline hydrochlorideFisher ScientificCatalog #BP912
Materials, Step 1
ReagentPipette tips, filtered, PCR clean and sterile, 2 - 100 uLEppendorfCatalog #0030078543
Materials
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
Preparing food for Drosophila melanogaster and its relatives
NAME

Preparing food for Drosophila melanogaster and its relatives

CREATED BY
J. Dylan Shropshire
Sterilize the working surface with 10% bleach and 70% ethanol.
Preparing tetracycline-treated fly food.
Preparing tetracycline-treated fly food.
Using an analytical balance and a pipette, add the following to a ReagentCentrifuge tubes, safe-lock, Eppendorf quality, 1.5 mLEppendorfCatalog #022363204 :
  • Amount3 mg ReagentTetracycline hydrochlorideFisher ScientificCatalog #BP912
  • Amount50 µL diH2O

Vortex and invert the tube until the tetracycline to completely dissolve.
Mix
Microwave a vial containing Amount10 mL of fly food until boiling.
Take care not to allow the food to boil up the sides of the vial.
Stir as needed.
Temperature
Allow food to cool to Temperature55 °C .
Use a probe thermometer to confirm the temperature.
Critical
Temperature
Add the dissolved tetracycline to the food and mix thoroughly.
The final concentration of tetracycline is approximately Concentration0.3 mg/mL .
Mix
Allow food to cool to TemperatureRoom temperature .
Use a probe thermometer to confirm the temperature.
Temperature
Treat flies with tetracycline
Treat flies with tetracycline
8w 5d
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 Temperature23 °C or Temperature25 °C .
4d
Monitor vials regularly.
Once adults begin emerging, transfer to a freshly prepared vial of tetracycline-treated food.
1w
Repeat Go togo to step #7 and Go togo to step #8 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:
Protocol
PCR-based cytotyping to detect Wolbachia in insect tissues
NAME

PCR-based cytotyping to detect Wolbachia in insect tissues

CREATED BY
J. Dylan Shropshire


PCR
Analyze
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
Critical