Test Injections of 16Cq DiNV on Male and Female D. innubila Replicate 2, 20 Injections for Kent, and Some D. melanogaster Injections as Well

This is the second replicate of this infection. I also wanted to add day 0 for both male and female just in case. Kent asked me to do 5 of each treatment and sex for him. And Rob suggested doing some melanogaster as well. For this I separated out flies 1 day before hand into vials that had:

5 male innubila to freeze day 0 for control
5 male innubila for Kent for control
10 male innubila for me to monitor for control
5 female innubila to freeze day 0 for control
5 female innubila for Kent for control
10 female innubila for me to monitor for control
20 W1118 melanogaster for me to monitor for control
10 IMD null melanogaster for me to monitor for control

5 male innubila to freeze day 0 for infection
5 male innubila for Kent for infection
20 male innubila for me to monitor for infection
5 female innubila to freeze day 0 for infection
5 female innubila for Kent for infection
30 female innubila for me to monitor for infection
20 W1118 melanogaster for me to monitor for infection
10 IMD null melanogaster for me to monitor for infection

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 5 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 flies to get control medium
  • Separated out sexes
  • Injected each fly with 27.6nl control medium
  • Used toothpick to place flies in new vial
  • Did all other control medium flies in the same way
  • After the 3rd flask, the needle broke, so I went through the process of clipping and filling another needle
  • 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 5 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
  • Did all other virus flies in the same way

Note here: the melanogaster flies were much harder to inject. They are much smaller than innubila, I suspect that the needle needs to be thinner for them, and that the volume to inject with should be smaller. I noticed that it was hard to push the needle through them (they seem to be much more dehydrated flies), and that often a bubble of either the injection fluid or their hemolymph would come out when pressing inject. I am not sure how much fluid actually got into them. Sometimes the bubble would receed into their body, but I don’t think all the time. I decided to not change any parameters during the infection because I had already started it this way.

The time of infection and duration on CO2 was recorded for each fly:

vial species sex mated status days_emerged day_infected age_infected purpose treatment volume time time on CO2 original_N_number
1 D. innubila female mated 9/30-10/2 20231006 5-6 days Kent CCM 27.6nl 2:15 6min 5
2 D. innubila male mated 9/30-10/2 20231006 5-6 days Kent CCM 27.6nl 2:28 5min 5
3 D. innubila female mated 9/30-10/2 20231006 5-6 days me CCM 27.6nl 2:36 5min 10
4 D. innubila male mated 9/30-10/2 20231006 5-6 days me CCM 27.6nl 3:06 8min 10
5 W1118 male mated 10/1 20231006 5-6 days me CCM 27.6nl 3:12 5min 9
6 IMD null male mated 9/30-10/2 20231006 5-6 days me CCM 27.6nl 3:18 6min 10
7 W1118 male mated 10/1 20231006 5-6 days me CCM 27.6nl 3:24 6min 10
8 D. innubila female mated 9/30-10/2 20231006 5-6 days Kent 16Cq DiNV 27.6nl 3:36 7min 5
9 D. innubila female mated 9/30-10/2 20231006 5-6 days me 16Cq DiNV 27.6nl 3:36 7min 10
10 D. innubila male mated 9/30-10/2 20231006 5-6 days Kent 16Cq DiNV 27.6nl 3:43 8min 5
11 D. innubila male mated 9/30-10/2 20231006 5-6 days me 16Cq DiNV 27.6nl 3:43 8min 10
12 D. innubila female mated 9/30-10/2 20231006 5-6 days me 16Cq DiNV 27.6nl 3:52 6min 10
13 D. innubila male mated 9/30-10/2 20231006 5-6 days me 16Cq DiNV 27.6nl 4:00 7min 10
14 D. innubila female mated 9/30-10/2 20231006 5-6 days me 16Cq DiNV 27.6nl 4:09 5min 10
15 W1118 male mated 10/1 20231006 5-6 days me 16Cq DiNV 27.6nl 4:14 5min 10
16 IMD null male mated 9/30-10/2 20231006 5-6 days me 16Cq DiNV 27.6nl 4:19 6min 10
17 W1118 male mated 10/1 20231006 5-6 days me 16Cq DiNV 27.6nl 4:25 5min 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