<aside> 📚

Adapted from the Povolones lab.

• Original publication in Parasites & Vectors
• Example of the assay in action in Veterinary Parasitology
• Detailed protocol on protocols.io </aside>

Materials

• Warm RPMI-1640 (Sigma R8758)
• dH₂O
• 70% ethanol
• 96-well plates used for ImageXpress (USA Scientific 5665-5180Q)
• Extraction toolbox:
  • Two 2.5 inch strainers (B00428M7OI)
  • Ziploc disposable plastic dishes, 8 oz, top cut off
• Forceps
• Bucket of ice

Equipment

• ImageXpress Nano set to 37°C, for quantifying L3 emergence
• 37°C CO₂ incubator
• Repeating 12-channel p1250 pipette with 1250 μL tips

Protocol

1. This assay should be performed at least 14 days after blood-feeding with microfilaria — see Mosquito Blood Feeding.
2. Fill as many 96-well plates as necessary with 200 μL warm RPMI. Three plates is usually enough for 700 fed mosquitoes.
3. Remove cages and cartons of infected mosquitoes from the incubator and transfer them to the screened room.
4. Use a rubber bulb and glass tube to carefully remove all dead mosquitoes from the cartons. Place dead mosquitoes in the freezer overnight before discarding.
5. Cold anesthetize the mosquitoes by putting the cartons at 4°C for 3–5 minutes.
6. Transfer knocked-down mosquitoes into a glass Petri dish with a piece of filter paper, resting on a bucket of ice. Then transfer the mosquitoes from the filter paper to a tea strainer and place the strainer in a dish filled with 70% ethanol — mosquitoes will sink. Soak for 2 minutes.