Infecting Mosquitoes with Microfilaremic Blood

Note: Remove the sucrose pad(s) the night before feeding for LVP or 2 hr. before feeding for AaSD.

Preparing blood for blood feeding

  • Appropriate concentrations for blood feeding:

  • B. pahangi: 120 – 160 mf / 20 µL

  • D. immitis: 80 – 160 mf / 20 µL

Starting with microfilaremic blood

  1. Invert the tube of infected blood to mix the microfilariae (mf).

  2. Add 20 µL of infected blood to 50 µL of dH2O on a slide and mix with a pipette tip.

  3. Place the slide on a compound microscope and count the mf. Start at a corner of the cover slip and count the mf in the fluid outside of the coverslip. Once the perimeter has been counted, count the mf using an s-pattern.

  4. Calculate the mf number in the stock solution. Dilute the infected blood, using C1V1=C2V2 to determine how much control blood (Defibrinated Sheep’s Blood—Hemostat Laboratories DSB100) to add to get the desired blood feeding concentration.

    Note: The blood feeders used for feeding hold 10 mL of blood. When calculating final volume use 11 mL for 1 mL overage. Multiply this by number of feeders you will need.

  5. Re-enumerate the mf in the diluted stock blood in duplicate.

Starting with mf in IP fluid

  1. Invert the tube in order for mf to be evenly distributed throughout IP fluid.

  2. Use the concentration printed on the outside of the tube containing the IP fluid. Determine how much volume of IP fluid you will need to get the desired mf concentration for blood feeding by using C1V1=C2V2.

    Note: The blood feeders used for feeding hold 10 mL of blood. When calculating final volume use 11 mL for 1 mL overage. Multiply this by number of feeders you will need.

  3. Add the calculated amount of IP fluid to a 15 mL conical tube. Spin at 900 rpm for 10 min. You should see a pellet of mf at the bottom of the tube. Remove all supernatant without disturbing pellet. Add the final volume of blood you desire (11 mL x the number o feeders).

  4. Re-enumerate the mf in blood in duplicate to verify that it is within target concentration for the designated species (see target concentrations above). If concentration is off, re-calculate dilutions based on the new numbers and add defibrinated sheep's blood as needed.

Assembling blood feeders and feeding mosquitoes

  1. Use the largest glass blood feeders. Cover the bottom of the feeder with parafilm (pull the parafilm once in both directions, so that parafilm is very thin).

  2. Setup water circulator and tubing and begin circulating water thru the tubes and feeders, so that equilibrate to 37°C prior to feeding.  

  3. In these feeders carefully place ~10 mL of infected blood into the top of the blood feeders using a disposable pipet. You need to make sure that the feeders are level so that the blood doesn’t pool in one side of the membrane since you want an even distribution of mf.

  4. Place a carton of mosquitoes under a membrane feeder and slowly lower the feeder so that it rests atop the carton and is flush with the mesh of the carton.

  5. Allow the carton of females to feed for 30 – 45 min. or until a majority of females have fed.

  6. When feeding is complete, remove an aliquot of blood to confirm that the mf are still alive and active. Do not separate blood fed from non-blood fed mosquitoes at conclusion of feeding.

  7. Place blood fed mosquitoes in insect incubator and label with infection date and information. Allow the infection to incubate for 14-15 days before you extract L3 from the mosquitoes.