Extraction of Brugia or Dirofilaria L3 from Infected Mosquitoes

Note: If working with Brugia infected mosquitoes, all members of the Zamanian and Bartholomay lab need to be notified 24 hours in advance. Screened-in room will need to be reserved and scheduled on the Google calendar Injection Schedule prior to BSL2 work. Before bringing out the mosquitoes from the insectary, place BSL2 signs on all entrances to the lab and to the screened dissection room. Put on PPE (lab coat, goggles and gloves).

A. Bulk Extraction of L3

Materials

  • RPMI-1640 (Sigma-Aldrich R8758)
  • 2.5 inch strainer (B00428M7OI)
  • Porcelain mortar/pestle
  • Ziploc disposable plastic dishes 8oz (top cut off)
  • Gentamicin 100X stock (0.4 mg/µL, Sigma G1272-100ML)
  • Pen-Strep 100X stock (10,000 U/mL)
  • Ice bucket

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Methods

  1. Aliquot 500 mL of RPMI-1640 into two bottles, with equal volume in each (2 x 250 mL). Add 2.5 mL of 100X Pen-Strep stock solution and 2.5 mL of 100X Gentamicin stock. Then separate the RPMI into 2 separate bottles. Place one bottle in the fridge or on ice to cool and the other in a 37°C incubator.

  2. Cold anesthetize the mosquitoes in the cartons (place the cartons in -20°C refrigerator for about 20-30 s., or until knocked down). You can put up to ~600 mosquitoes in the mortar, so combine the cartons as needed (2 cartons into 1 or 3 cartons into 2). When ready, quickly transfer, at once, all mosquitoes in the carton to the mortar.

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  3. Using the pestle, gently tap the mosquitoes to disrupt the cuticle (use a tapping motion, not a grinding motion). Do not over tap; more is not better. Gently tap until only a few intact mosquitoes are remaining.

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  4. Transfer the mosquitoes to the mesh strainer and place the strained into a Ziploc plastic dish containing cold RPMI (dish should be ~ half full of RPMI). Dip the strainer up and down 3-5 times to remove scales, eggs, etc (the RPMI should appear cloudy). Do not dip too much as you can lose the L3s in the strainer.

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  5. Discard the RPMI and add clean cold RPMI to the dish (½ full) and dip up and down 3-5x to further rinse the L3s in the strainer.

  6. Transfer the strainer of L3s to a clean second dish containing warm RPMI (40-50 mL, enough to cover the L3s) and incubate at 37°C for Brugia spp. or 39°C for Dirofilaria for 30 min. The incubation time will allow the L3s to migrate out of the strainer and into the warm RPMI.

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  7. Remove strainer from dish and transfer all L3s into a Petri dish containing a small amount (~20 mL) of fresh, 37°C RPMI.

  8. Count the total number of L3s harvested and from how many mosquitoes crushed, and aliquot L3s as necessary.

  9. Once the extractions are complete, rinse and wash all materials used. Dispose of the used plates in the biohazard bin. Do not throw away the sieve or Ziploc dishes. They can be re-used.

B. Extraction of L3 via Dissections

Materials

  • 1X Aedes saline
  • Slides
  • Dissection Probes
  • Disposable transfer pipettes
  • Warming plate
  • Bucket of Ice
  • Glass Petri plate with filter paper
  • Forceps
  • KimWipes
  • Eppendorf tubes
  • Small Petri dish containing pre-warmed RPMI + 1% Pen-Strep (v/v)

Methods

  1. Clean and ethanol the work area, stage of dissecting scope, and tools prior to starting.

  2. Turn on warm plate to 37°C and allow to warm up.

  3. Aliquot 1X Aedes saline into 1.5 mL eppendorf tubes. Place tubes in warming plate to allow to come to 37°C.

  4. Under sterile conditions, Aliquot ~10 mL of pre-warmed RPMI amended with P/S and Gentamicin into a small Petri dish. Place the Petri dish on top of the warmer to keep RPMI warm.

  5. On ice, place a large glass Petri dish lined with filter paper. Cold anesthetize the mosquitoes in the cartons (place the cartons in 4°C refrigerator until knocked down). Once knocked down, put mosquitoes on top of the filter paper in the Petri dish.

  6. Take a clean slide and in the center add a puddle of 1X Aedes saline using a transfer pipette.

  7. Using a dissecting probe or a forceps, transfer one adult female mosquito to the pool of saline. Using dissecting probes decapitate mosquito. You may notice L3 beginning to migrate out of the back of the head.

  8. Pierce the mosquito head with one probe and use another probe to cut the mosquito proboscis in half. This will allow the L3 to escape the mouth parts.

  9. Allow the dissected head and mouthparts to incubate in the saline for a few minutes, to allow all the L3 to migrate into saline.

  10. Once all L3 are in saline, individually transfer the L3 into the Petri dish containing the RPMI using a small worm hook (dissecting probe that has been curved into a small hook).

  11. Once all L3 have been transferred, place the dish of RPMI and L3 back on the warm plate and wipe off the slide with a Kimwipe. You can reuse the slide.

  12. Repeat the above process until all mosquitoes have been dissected or until enough L3 have been collected.

  13. If there are mosquitoes remaining, dump mosquitoes into an old mosquito carton and place in freezer. Discard spent tubes of media, Kimwipes and slides in the proper biohazard bins. Place Petri dish of L3 in the 37°C incubator until ready to use. Discard ice, take down BSL-2 signage, and ethanol work area and tools.