Topical Testing Assay: A Technique to Evaluate the Insecticidal Potential of Dopamine Receptor Antagonists on Adult Female Mosquitoes by Topical Application

Culture 3 to 5-day-old adult female mosquitoes in a 20-liter plastic cage. Label 9-ounce paper cups with the name and concentration of testing compound.

Next, prepare a 10 µg/µL stock solution of the testing compound in acetone in a 20-milliliter glass vial. Then, serially dilute the stock solution to obtain the desired working concentrations.

Clean a 1-milliliter glass syringe with acetone, and then, fill it with the test solution at the appropriate concentration. Secure the syringe in a micro-applicator, adjusted to deliver a volume of 0.25 microliters.

Next, use an aspirator to remove ten 3 to 5-day-old adult female mosquitoes from the cage, and anesthetize them for 5 minutes at 4 degrees Celsius. Then, transfer the mosquitoes to a Petri dish, and place the dish on ice for 10 minutes.

In order to avoid damaging the adult mosquito, which could contribute to mortality, ensure that they are not anesthetized in the fridge for more than 5 minutes, or immobilized on ice for more than 10, and minimize the handling of the mosquitoes. If you have access to a cold plate and a micromanipulator with a movable stage, that may further minimize handling of the mosquitoes.

Use fine tweezers to remove each mosquito from the dish. With the syringe micro-applicator, apply 0.25 microliters of testing solution to the dorsal thorax under a dissecting microscope. Next, transfer the mosquitoes to a labeled paper cup on ice. Seal the cup with a 10 by 10-centimeter mesh square, and a rubber band. Then, transfer it to the growth chamber, and record the number of dead mosquitoes.