Male and Female D. innubila Injections with 0.01, 0.05, and 0.005 FFU DiNV, Males Second Replicate

This is a second replicate of this infection for males and a first replicate for females. I Separated out ~20 males and females for each treatment on the 18th, and they had emerged on the 14th 15th and 16th, making them 5-7 days old for injection. I injected 10 control and 20 undiluted flies for Kent as well. No day 0 flies were made, but flies were frozen after death every 3 days, and the experiment ends in 2 weeks.

Making dilutions:

I planned the dilutions as the exact same as the previous replicate.

  • Stock tube 29 used, thawed on ice
  • Diluted 1:10 in tube “1” 3ul of stock in 27ul of CCM
  • Tube 2 is for 0.05FFU: 3.79ul of tube 1 in 46.21ul of CCM
  • Tube 3 is for 0.01FFU: 10ul of tube 2 in 40ul of CCM
  • Tube 4 is for 0.005FFU: 5ul of tube 2 in 45ul of CCM
  • Tubes were mixed after add additions and before use with pipetting

19 new vials of mushroom food were made fresh for the infection.

The set up of the injection was the same as a previous injections

Of note all the viral dilutions were kept on ice until use, and pipette mixed with 20ul before drawing up the fluid into the needle. The needle did not break so only one needle was used for every fly.

vial species sex mated status days_emerged day_infected age_infected tube treatment volume time time on CO2 original_N_number
1 D. innubila m yes 20240214-20240216 20240221 5-7 days CCM CCM 27.6nl 3:03 8 min 10
2 D. innubila f yes 20240214-20240216 20240221 5-7 days CCM CCM 27.6nl 3:03 8 min 10
3 D. innubila m yes 20240214-20240216 20240221 5-7 days CCM CCM 27.6nl 3:12 10 min 10
4 D. innubila f yes 20240214-20240216 20240221 5-7 days CCM CCM 27.6nl 3:12 10 min 10
5 D. innubila m yes 20240214-20240216 20240221 5-7 days CCM CCM 27.6nl 3:23 6 min 10
6 D. innubila m yes 20240214-20240216 20240221 5-7 days 4 0.005 FFU 27.6nl 3:33 10 min 11
7 D. innubila f yes 20240214-20240216 20240221 5-7 days 4 0.005 FFU 27.6nl 3:33 10 min 10
8 D. innubila m yes 20240214-20240216 20240221 5-7 days 4 0.005 FFU 27.6nl 3:44 9 min 10
9 D. innubila f yes 20240214-20240216 20240221 5-7 days 4 0.005 FFU 27.6nl 3:44 9 min 10
10 D. innubila m yes 20240214-20240216 20240221 5-7 days 3 0.01 FFU 27.6nl 3:57 9 min 9
11 D. innubila f yes 20240214-20240216 20240221 5-7 days 3 0.01 FFU 27.6nl 3:57 9 min 10
12 D. innubila m yes 20240214-20240216 20240221 5-7 days 3 0.01 FFU 27.6nl 4:09 9 min 10
13 D. innubila f yes 20240214-20240216 20240221 5-7 days 3 0.01 FFU 27.6nl 4:09 9 min 10
14 D. innubila m yes 20240214-20240216 20240221 5-7 days 2 0.05 FFU 27.6nl 4:21 10 min 10
15 D. innubila f yes 20240214-20240216 20240221 5-7 days 2 0.05 FFU 27.6nl 4:21 10 min 10
16 D. innubila m yes 20240214-20240216 20240221 5-7 days 2 0.05 FFU 27.6nl 4:32 9 min 10
17 D. innubila f yes 20240214-20240216 20240221 5-7 days 2 0.05 FFU 27.6nl 4:32 9 min 10
18 D. innubila m yes 20240214-20240216 20240221 5-7 days 29 undiluted passage 4 27.6nl 4:44 10 min 10
19 D. innubila m yes 20240214-20240216 20240221 5-7 days 29 undiluted passage 4 27.6nl 4:44 10 min 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
  • The toothpicks will be soaked in ethanol before disposing in the glass disposal box