This article details a mosquito feeding assay designed to evaluate the effects of active test compounds on mosquito mortality and fecundity. The methodology involves preparing a blood meal, attracting mosquitoes, and assessing their reproductive capabilities post-exposure.
Begin the mosquito feeding assay by preparing a blood meal, adding the desired concentration of an active test compound into defibrinated rabbit blood. The blood meal lacks fibrin, preventing blood clotting.
Load this blood meal into a membrane-feeding unit's delivery port. Attach it to a heating unit, maintaining the blood at an optimum temperature. Coat the membrane with the lactic acid solution.
Place this unit in a cage having adult, pre-starved female mosquitoes to increase the chances of feeding. Incubate in the dark. Lactic acid attracts mosquitoes toward the feeding unit. Dark conditions encourage them to feed.
Transfer the blood-fed mosquitoes into a growth chamber. After digestion of the blood meal, the active test compound enters the mosquito's body, affecting various organs and causing some to die.
Count the number of dead mosquitoes and calculate the mortality rate. Evaluate the effect of the test chemical on the reproductive system of surviving mosquitoes by introducing an egg cup in the chamber.
Female mosquitoes utilize proteins from the blood meal and lay eggs, which get collected inside the cup. Count the number of eggs, estimating the decrease in fecundity - egg-laying capability.
High mortality and low fecundity correspond to the compound's insecticidal capacity.
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.
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