Mosquito Feeding Assay: A Method to Evaluate Test Compound Effects on Mortality and Fecundity of Adult Female Mosquitoes

Collect approximately 150 four- to five-day-old adult female mosquitoes with an aspirator, and transfer them to a separate cage. Remove the source of sugar 1 to 24 hours prior to the feeding assay.

Prepare an 80-millimolar stock solution of the testing compound in water. Then, serially dilute it with water to obtain working stock solutions of the desired concentrations.

To obtain desired testing concentrations, add 40 microliters of each dilution to 960 microliters of defibrinated rabbit blood in a 1.5-milliliter tube, and mix by pipetting. Place a membrane filter on a feeding unit, and seal it with a rubber ring.

Next, use a pipette to transfer 1 milliliter of the blood with testing solution through the delivery port located on the reverse side of the feeding unit. Attach the feeding unit to a heating unit. Then, swab the membrane surface gently with freshly prepared 10% lactic acid solution.

Place the feeding unit in the cage. Cover the cage with a dark cloth, and allow the mosquitoes to feed for one hour. After the mosquitoes feed, place the cage in a refrigerator at 4 degrees Celsius for five minutes to anesthetize the mosquitoes. Then, count and record the total number of mosquitoes in the cage.

By examining the abdomen, count and record the total number of fully- and partially-fed female mosquitoes. A minimum of 50 blood-fed mosquitoes should be obtained per dose. Remove any mosquitoes that have not fed. Next, transfer the cage to a growth chamber and use a score sheet to record the number of dead mosquitoes at the appropriate time points.

On day 3 post-blood feeding, place an egg cup in the cage for 72 hours. Use a dissecting microscope to count the total number of eggs produced per treatment.