in squito RNAi

Handling of infected mosquitoes should be performed in defined BSL-2 spaces.

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 (follow 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 sucrose pads the day before injection

  • This will make sure mosquitoes are starved and will allow them to hold more injected material

Protocol

Note: the following protocol is best performed with two people, one loading needles and the other injecting mosquitoes.**

  1. Dilute dsRNA to 1 μg/μL.

  2. Prepare the injection space:

    a. Place trays of prefabricated injection needles next to a dissecting microscope.

    b. Wrap the glass stage of the dissecting microscope with Parafilm.

    c. Place a glass Petri dish with a Whatman filter in an ice box full of ice. Put the ice next to the injection rig.

    d. Ensure the mosquito harness is clean and void of debris (attach it to a syringe tube and flush it with 70% ethanol).

  3. Once you are prepared for injection, take a carton of mosquitoes and place it in the 4° fridge for 3-5 minutes or until the mosquitoes are all anesthetized.

    Note: Mosquito survival rate is dependent upon the amount of time they are anesthetized, so be careful not to leave them in the fridge or on the ice for too long.

  4. Transfer anesthetized mosquitoes to the glass dish, placing the lid on top.

  5. Prepare an injection needle:

    a. Under the dissection microscope, carefully break the injection needle with a pair of forceps. You want to break the needle such that it is small enough to do minimal damage to the mosquito, but large enough so as it doesn't bend when attempting to puncture the mosquito.

    b. Aspirate 0.25 μL dsRNA with a P2.5 pipette.

    c. Fill the injection needle by capillary action by carefully inserting the end of the needle into the pipette tip filled with dsRNA.

    d. Line the needles up to be used by the individual performing the injections.

    Note: Typically needle filling will go much faster than mosquito injections, so be careful not to get too ahead of the person doing injections; dsRNA will evaporate from the needle if left sitting at room temperature for too long.**

  6. Inject a mosquito:

    a. Carefully remove the lid of the glass dish, making sure mosquitoes remain anesthetized.

    b. Hover the mosquito harness above the posterior thorax of a mosquito, trying to line up the harness with the mosquito body (this takes practice).

    c. Press the foot pedal to activate the vacuum and suck up the mosquito.

    d. Place the harness on the injection stage.

    e. Affix the filled needle to the syringe and micromanipulator.

    f. Use the micromanipulator to line up the needle with the mosquito.

    g. Slowly pierce the mosquito cervical membrane between the head and thorax (the head should be bent down in the process). The head should be gently compressed and will rebound once the needle has pierced the membrane.

    h. Slowly depress the syringe plunger to expel the dsRNA, being careful not to inject air.

    i. Remove the needle from the mosquito and dispose the needle in a sharps container.

    j. Place the mosquito in a clean carton.

  7. When finished with a batch of 25, immediately add a sucrose pad and return the carton to the incubator.

  8. Over the next several days, track mosquito mortality and remove dead mosquitoes daily.

  9. Assay worms on day 14 or 15 post-infection.