5.4.1. Pupae

1. Lay the frame horizontally at a slight angle, bottom-to-top, under good light.

2. Using needle-forceps, remove the wax capping from 10-50 cells containing white-eyed pupae, by cutting along the inside of the cell.

It is easiest to work from the bottom of the frame upwards, clearing room underneath for opening up the cells higher up the frame and picking up the pupae from behind.

3. With blunt, curved forceps remove the top part of each cell, exposing the head of the pupa.

4. Place the curved forceps underneath the head of the pupa, from the back, and carefully lift the pupa out of its cell.

It is critical to remove the white-eye pupae very carefully from the comb, to avoid damage.

5. Collect the pupae in a plastic Petri dish containing a circular filter paper dampened with sterile water.

6. For the purpose of propagation, you need:

  • a 10~50 µl syringe,
  • a thin needle (around 28G~30G)
  • a semi-automatic volume dispenser control unit that can dispense 1~5 µl volumes.

The Hamilton Company produces both dispenser units and 10~50 µl syringes with Luer locks that fit disposable needles. The pupae can be injected by hand, which is faster (but less precise) than doing so under microscope, with moveable trays.

7. Attach the syringe and control unit horizontally to a stand.

8. Close the four fingers of your hand and carefully lay a white-eye pupa on its back in the groove between index and middle finger.

The abdomen of the pupa points towards the tip of the fingers and the head is supported by the tip of the thumb.

9. Move the pupa towards the needle, inserting the needle at the narrowest angle possible under the skin of the pupa, on the lateral side, between the 2nd and 3rd integuments of the abdomen.

10. Inject 1~5 µl of virus suspension using the control unit.

11. Move the pupa backwards off the needle.

12. Move the pupa carefully from the hand to a plastic, disposable tissue culture plate, using forceps to support the pupa underneath.

Use plates with matching lids, both to prevent cross-contamination between wells and to control the humidity.

13. Incubate the plate at 30oC in a humidity-controlled incubator.

If this is not available, place the plates in a closed plastic box containing moistened paper towels.

14. Check the progress of the infection by monitoring the change in eye colour of the pupae.

Those pupae injected with virus that remain alive will change eye colour, but more slowly than those pupae injected with only buffer.

15. Include a control series with buffer-only inoculations, and a control series without inoculation, just incubation of the pupae.