Embryonic microinjections

Culex embryo microinjections were performed as previously described²⁸. A microscopic injection station equipped with FemtoJet 4× microinjector (Eppendorf, Hamburg, DE) was used for injections. Injections were carried out using quartz capillaries (0.7 mm internal diameter and 1.0 mm external diameter) pulled into needles using a Sutter P2000 laser based micro-pipette needle puller (Sutter Instruments, Novato, CA USA) and the following program: HEAT = 729, FIL = 4, VEL = 40, DEL = 128, PUL = 134, Line = 1.

A clear plastic cup containing approximately 100 ml of hay infused water was placed into the adult cages on the 5th day after a blood meal. Cages were placed in the dark to encourage egg laying and allowed to lay for 45–60 min. The egg rafts were disaggregated and aligned horizontally on a piece of moistened chromatography paper and against a nitrocellulose membrane (GE Healthcare, Amersham UK). Lines of embryos were then transferred to a piece of Scotch double-sided tape 665 (3 M, USA) on a plastic coverslip. Prepared eggs were covered with Halocarbon oil 27 (Sigma Aldrich, Gillingham UK) to prevent desiccation and injected. Injected eggs were washed with distilled water to remove as much oil as possible and (still on the coverslip) submerged egg side down into larval rearing trays and allowed to hatch. Surviving larvae were transferred to a new tray with hay infused water and maintained at the standard rearing conditions.