Nov 21, 2024

Public workspaceLong-term maintenance of diverse Drosophila species with and without Wolbachia endosymbionts

DOI
dx.doi.org/10.17504/protocols.io.rm7vzk85rvx1/v1
  • 1Department of Biological Sciences, Lehigh University;
  • 2Division of Biological Sciences, University of Montana
  • Shropshire Lab
J. Dylan Shropshire
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzk85rvx1/v1
Protocol Citation: Helene Hartman, Tim Wheeler, Brandon Cooper, J. Dylan Shropshire 2024. Long-term maintenance of diverse Drosophila species with and without Wolbachia endosymbionts. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzk85rvx1/v1
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 12, 2024
Last Modified: November 21, 2024
Protocol Integer ID: 112002
Keywords: Drosophila, Wolbachia, fly food, propionic acid
Abstract
This protocol provides a step-by-step guide for maintaining diverse Drosophila species in the laboratory. It assumes that the user has prepared food and established fly colonies. The protocol begins by outlining the preparation of food vials. This includes adding Kimwipes soaked in 0.5% propionic acid to the vials to provide a pupation substrate and increase humidity. We have found this addition to be particularly beneficial for more challenging species. We also detail the process of transferring flies between vials, maintaining accurate records, and establishing a fly emergency room for struggling lines. Using these procedures, we have successfully maintained nearly 90 Drosophila species, including those from the Sophophora and Drosophila subgenera, as well as mushroom specialists like Scaptomyza pallida with the addition of mushrooms.
Materials
Materials
  • ReagentDissection ProbeAmazonCatalog #B00M04W6NI OR ReagentPaint Brushes, SmallAmazonCatalog #B092CN8XJ1
  • ReagentDroso-Plugs, narrow vialsGenesee ScientificCatalog #59-200
  • ReagentDrosophila vials, narrow, polystyrene, reload systemGenesee ScientificCatalog #32-109RL with flies.
  • ReagentDrosophila vials, narrow, polystyrene, reload systemGenesee ScientificCatalog #32-109RL with food.
  • ReagentFlystuff Narrow Fly Vial Reload TrayGenesee ScientificCatalog #59-207
  • ReagentKimtech Science™ Kimwipes™ Delicate Task WipesKimberly-ClarkCatalog #34155
  • ReagentLab Tape, Rainbow Pack, 19mm x 13mFisher ScientificCatalog #15-901-5R
  • ReagentMouse PadAmazonCatalog #B08W47P44Y
  • Concentration0.5 % (v/v) propionic acid from ReagentPropionic acid, ACS reagent, ≥99.5%Merck MilliporeSigma (Sigma-Aldrich)Catalog #402907 and diH2O.
  • ReagentRubber Band, 2 x 1/8Genesee ScientificCatalog #64-104
  • ReagentSharpie Permanent MarkerAmazonCatalog #B00G4CJ8GK
Equipment
  • Drosophila incubator (Percival, DR-36VL)
  • Fridge (any)
  • Freezer (any)


Protocol materials
ReagentKimtech Science™ Kimwipes™ Delicate Task WipesKimberly-ClarkCatalog #34155
Materials, Step 4
ReagentDissection ProbeAmazonCatalog #B00M04W6NI
Materials, Step 7
ReagentMouse PadAmazonCatalog #B08W47P44Y
Materials, Step 12
ReagentGlad Pressn SealGenesee ScientificCatalog #88-142
Step 2
ReagentDrosophila vials, narrow, polystyrene, reload systemGenesee ScientificCatalog #32-109RL
In Materials, Materials, Step 1
ReagentFlystuff Narrow Fly Vial Reload TrayGenesee ScientificCatalog #59-207
Materials, Step 1
ReagentLab Tape, Rainbow Pack, 19mm x 13mFisher ScientificCatalog #15-901-5R
Materials
ReagentPaint Brushes, SmallAmazonCatalog #B092CN8XJ1
Materials, Step 7
ReagentRubber Band, 2 x 1/8Genesee ScientificCatalog #64-104
Materials, Step 13
ReagentSharpie Permanent MarkerAmazonCatalog #B00G4CJ8GK
Materials, Step 2
ReagentDroso-Plugs, narrow vialsGenesee ScientificCatalog #59-200
Materials, Step 2
ReagentPropionic acid, ACS reagent, ≥99.5%Merck MilliporeSigma (Sigma-Aldrich)Catalog #402907
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 Concentration0.5 % (v/v) propionic acid solution.
  1. Using Amount10 mL serological pipet, add Amount2.5 mL of propionic acid to a Amount500 mL graduated cylinder.
  2. Add diH2O to the Amount500 mL mark.
  3. Pour solution into appropriately labeled wash bottle.
  4. Label bottle with the date the solution was made and your initials.

Clean the workspace with Concentration10 % (v/v) bleach and Concentration70 % (v/v) ethanol.
Clean a dissecting probe or brush with Concentration10 % (v/v) bleach and Concentration70 % (v/v) ethanol.
Prepare vials with closures
Prepare vials with closures
1h 6m
1h 6m
Remove a ReagentFlystuff Narrow Fly Vial Reload TrayGenesee ScientificCatalog #59-207 with ReagentDrosophila vials, narrow, polystyrene, reload systemGenesee ScientificCatalog #32-109RL containing fly food from the fridge.

Note
We use the following protocol to produce fly food:
Protocol
Preparing food for Drosophila melanogaster and its relatives
NAME

Preparing food for Drosophila melanogaster and its relatives

CREATED BY
J. Dylan Shropshire


1m
If vials were sealed with ReagentGlad Pressn SealGenesee ScientificCatalog #88-142 , peel it back and insert clean ReagentDroso-Plugs, narrow vialsGenesee ScientificCatalog #59-200 or another closure into each vial.


Note
If using previously autoclaved plugs, mark with a ReagentSharpie Permanent MarkerAmazonCatalog #B00G4CJ8GK . In our experience, plugs can only be autoclaved once. A black dot indicates that plugs should be discarded after use and not re-used.

We use the following protocol to clean and sterilize Droso-Plugs:
Protocol
Cleaning and sterilizing Droso-Plugs for Drosophila maintenance
NAME

Cleaning and sterilizing Droso-Plugs for Drosophila maintenance

CREATED BY
J. Dylan Shropshire




5m
Allow food vials to warm to room temperature.
Note
Warming the vials is very important.
Exposure to temperature extremes can impact Wolbachia abundance in flies.
Flipping flies into cold food vials could result in loss of Wolbachia.

Warming the vials can be sped up by moving half of the vials in a tray to a separate tray so there is an empty space next to each vial.
You can also move the vials to a Temperature23 °C to Temperature25 °C incubator to warm them up faster.



1h
Critical
Prepare vials with Kimwipes
Prepare vials with Kimwipes
10m
10m

Note
We add Kimwipes soaked in Concentration0.5 % (v/v) propionic acid to fly vials to provide an attractive pupation substrate and add additional humidity to the vial.

Not all flies require this preparation. It is unlikely this will ever hurt a line, but it may not be necessary for all lines.

As a rule of thumb, use this preparation when an established stock already has it or if a stock does not have it and looks unhealthy.

Remove several ReagentKimtech Science™ Kimwipes™ Delicate Task WipesKimberly-ClarkCatalog #34155 from the box and place on top of one another.


1m
Rip pile of Kimwipes in half and then into quarters.


1m
Fold quarter in half and then into an accordion-style rectangle.


2m
Push the folded Kimwipe into the fly food using the back of aReagentDissection ProbeAmazonCatalog #B00M04W6NI or ReagentPaint Brushes, SmallAmazonCatalog #B092CN8XJ1 .


2m
Add Concentration0.5 % (v/v) propionic acid to wet the Kimwipe.
The Kimwipe should be fully wet but not so wet that there is liquid pooling atop the food.


1m
Push the moist Kimwipe against the side of the vial so that it does not block flies from food.


Note
The Kimwipe must not touch the vial plug.
If the Kimwipe touches the plug, it can pull moisture into the plug and into the air, drying the vial.

2m
Label Kimwipe prepped tray with:
  • The date of prep
  • Your initials
  • The bottle of propionic acid used
The top label displays the date fly food was prepared and the initials of the person who prepared the fly food. The bottom label displays the date and initials for the person who prepared the Kimwipe vials. The propionic acid bottle used to prepare the vials is also indicated on the label. In this case the propionic acid bottle was labeled "Insect 7".

1m
Flip flies
Flip flies
5m 30s
5m 30s
Open the stock maintenance sheet in your lab notebook.
Note
We use digital lab notebooks with the following columns in the sheet:
  • Box name (Each tray of stocks receives a unique name.)
  • Vials needed (The number of vials needed to maintain the stocks.)
  • Week of (The week that stocks are maintained.)
  • Date transferred (The date that stocks are maintained.)
  • Notes on stocks (Any notes on the health of the stocks.)
  • Initials (The initials of the person who performed stock maintenance.)

Every time stocks are maintained, the table is duplicated with the dates, notes, and initials replaced.

1m
Set a tray of fly stocks to your non-dominant side.
Set a tray of fresh vials to your dominant side.
Put a ReagentMouse PadAmazonCatalog #B08W47P44Y between them.


Note
While flipping, the vial will be repeatedly tapped against the work surface to move flies away from the opening. The mouse pad is used to soften the impact. It is optional.

1m
Grab the oldest healthy vial and put it in your non-dominant hand.
Grab a new clean vial in your dominant hand.


Note
Most healthy lines will have 2-3 vials in the stock, at minimum.
Use a ReagentRubber Band, 2 x 1/8Genesee ScientificCatalog #64-104 to keep vials of the same stock together.

5s
In a quick motion:
  1. Tap the fly-containing vial on the mousepad to get the flies to the bottom.
  2. Quickly remove the closures from both vials.
  3. Flip the new vial on top of the old vial so that the openings touch.


5s
Flip both vials over so that the fly-containing vial is on top.
Gently tap on the mousepad to transfer the flies into the new clean vial.



Note
To prevent dislodging food from the fly-containing vial during transfer, avoid using excessive force when tapping the vial. Gentle tapping is sufficient to encourage the flies to move.

5s
After the flies have been transferred, tap down again briefly to transfer them to the bottom.
Quickly replace the closure.




5s
Take the tape label affixed to the old vial and transfer it to the new vial with flies.
Alternatively, label the vial with a sharpie marker.
If expanding a stock, add a new label.
5s
Put the old vial into a discard tray.
5s
Repeat steps Go togo to step #13 to Go togo to step #18 for each fly line.
Between fly lines, inspect the immediate area for stray flies.
30s
When finished with a tray, record the date and appearance of all stock in your lab notebook.
Move the flies to a Drosophila incubator or store at room temperature.
30s
When finished for the day, move the discard tray of vials to Temperature-20 °C DurationOvernight to kill the flies.
Treat as biohazardous waste.
1m
Clean the workspace with Concentration10 % (v/v) bleach and Concentration70 % (v/v) ethanol.
1m
Maintaining a fly emergency room (ER)
Maintaining a fly emergency room (ER)
If a line of flies looks unhealthy or their numbers are diminishing, remove it from the stock tray and place in a new tray designated as a fly ER.
Monitor lines in the ER on a routine basis (i.e., every other day).
Observing the line should reveal why the line is unhealthy.
Take appropriate steps to improve the health of the stock, such as adjusting humidity, quickly transferring emerged flies to new vials after adult emergence, or adjusting the temperature.