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Adapted from the Kimber lab.

• Original publication in PLOS Pathogens
• Example of the assay in action in PLOS Biology </aside>

Materials

• Injection needles
• dsRNA
• Ice
• Glass Petri dish with a Whatman filter inside
• Clean small mosquito cartons
• p2.5 pipette
• Forceps

Preparation

Infect Ae. aegypti LVP mosquitoes with filarial nematodes, following the Mosquito Blood Feeding protocol.

• Sort fed females into small cartons of 25 mosquitoes per carton.
• Make sure sucrose pads are re-wet every day.
• Remove the sucrose pads the day before injection to starve the mosquitoes, so they hold more injected material.

Protocol

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This protocol is best performed with two people — one loading needles and the other injecting mosquitoes.

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1. Dilute dsRNA to 1 μg/μL, based on Qubit RNA quantification.
2. Prepare the injection space:
   • Place trays of prefabricated injection needles next to a dissecting microscope.
   • Wrap the glass stage of the dissecting microscope with Parafilm.
   • Place a glass Petri dish with a Whatman filter in an ice box full of ice. Put the ice next to the injection rig.
   • Ensure the mosquito harness is clean and free of debris — attach it to a syringe tube and flush it with 70% ethanol.
3. Once you are ready to inject, take a carton of mosquitoes and place it in the 4°C fridge for 2–3 minutes, until the mosquitoes are all anesthetized.

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Mosquito survival rate depends on how long they are anesthetized, so be careful not to leave them in the fridge or on the ice for too long.

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