Test Injections of 16Cq DiNV on Male and Female D. innubila Replicate 1
We want to test the difference in infection between male and female innubila because this has not been looked at yet, especially with the very efficient injection method. We have some evidence from the Kallatheia virus paper that female may die less than males. But we don’t know. Flies had emerged between the 19th and the 21st. All females were placed with males so the assumption is that the females are mated in these cases.
For this I separated out flies 1 day before hand into vials that had:
10 males for control
10 females for control
20 males for virus infection
30 females for virus infection
I decided not to take any day 0 samples (I did not have enough flies ready at the time)
Set up area
- Thawed 16Cq virus on ice and changed gloves after touching
- Made 7 new vials of mushroom food
- Wiped down fly room bench with 95% ethanol before using/putting things on it
- Things taken to the fly room
- mineral oil
- sterile Co2 pad
- pulled needles
- sterile forceps (2)
- two sets of gloves
- 95% ethanol
- Nanoject
- autoclaved toothpicks
- Notebook
- Virus and control medium on ice
- Used one of the forceps to clip off the tip of one of the pulled needles
- Used the metal needle to backfill the pulled needle with mineral oil, avoiding any bubbles
- Placed the pulled needle on the machine
- Slide the collet onto the needle
- Pushed the 2nd o-ring onto the needle
- Slid the needle onto the machine
- Tightened the collet down
- (this method is easier than pushing the needle through the ring on the machine)
- Ejected the mineral oil until the beep
- Filled the needle with control medium (10% FBS, 4% mushroom Schneider’s medium with antibiotics)
- Tested needle to make sure fluid ejects when pressing inject
Injecting flies
- Placed 1st vial of 10 male flies on CO2
- Separated out males with toothpick
- Injected each fly with 27.6nl control medium
- Used toothpick to place flies in new vial
- Placed 2nd vial of 10 female flies on CO2
- Injected each fly with 27.6nl control medium
- Used toothpick to place flies in new vial
- I ejected the rest of the medium, and refill the needle with the 16Cq virus fluid. I think some bubbles got into the needle at this time but I proceeded anyways (this might not have been the correct choice)
- For virus infected flies, infections went the same way:
- Placed vial of 10 male flies on CO2
- Separated out males with a new toothpick
- Injected each fly with 27.6nl 16Cq virus
- Used toothpick to place flies in new vial
- Placed next vial of 10 flies on CO2
- Separated out males with the virus toothpick
- Used virus toothpick to place flies in new vial
- Placed next vial of 10 female flies on CO2
- Injected each fly with 27.6nl 16Cq virus
- Used virus toothpick to place flies in new vial
- And repeated those steps for the 2 more female virus receiving sets of flies
The time of infection and duration on CO2 was recorded for each fly:
| vial | species | sex | mated status | days_emerged | day_infected | age_infected | treatment | volume | original_N_number |
|---|---|---|---|---|---|---|---|---|---|
| 1 | D. innubila | male | mated | 9/19-9/21 | 20230927 | 6-8 days | cell culture medium | 27.6nl | 10 |
| 2 | D. innubila | female | mated | 9/19-9/21 | 20230927 | 6-8 days | cell culture medium | 27.6nl | 10 |
| 3 | D. innubila | male | mated | 9/19-9/21 | 20230927 | 6-8 days | 16Cq DiNV | 27.6nl | 10 |
| 4 | D. innubila | male | mated | 9/19-9/21 | 20230927 | 6-8 days | 16Cq DiNV | 27.6nl | 10 |
| 5 | D. innubila | female | mated | 9/19-9/21 | 20230927 | 6-8 days | 16Cq DiNV | 27.6nl | 10 |
| 6 | D. innubila | female | mated | 9/19-9/21 | 20230927 | 6-8 days | 16Cq DiNV | 27.6nl | 10 |
| 7 | D. innubila | female | mated | 9/19-9/21 | 20230927 | 6-8 days | 16Cq DiNV | 27.6nl | 10 |
Mortality is assessed every day, and flies are transferred on CO2 every 3 days. When transferred, dead flies are frozen. Mortality information can be found here and frozen fly information can be found here
For transferring flies and freezing dead ones, this is the process that is followed:
- Make new food vials the day you need them
- Use the sterilized CO2 pad, toothpicks, and forceps for this
- Place flies on CO2 to transfer, count the number when adding the flies to the vial
- Use new 1.5mL tubes from the molec lab only (less likely to be contaminated)
- Use clean forceps to move a dead fly to the 1.5mL tube
- Between each fly move, put the forceps in 10% bleach, then DI water, then 95% ethanol (these are in tubes that are for dipping in)
- The CO2 pad will be sterilized afterwards, along with the forceps