This article describes a topical testing assay used to evaluate the insecticidal properties of a dopamine receptor antagonist on adult female mosquitoes. The method involves anesthetizing the mosquitoes and applying the test compound directly to their dorsal thorax.
Topical testing assay in mosquitoes depends on the direct deposition of an active test compound on the dorsal thorax of adult mosquitoes to test its insecticidal property.
To begin, anesthetize adult female mosquitoes by exposing them to the critical thermal minimum - a low temperature which ceases their neuromuscular activity.
Place individual anesthetized female mosquitoes under a dissecting microscope. Take a micro-applicator-connected glass syringe filled with the test compound - a dopamine receptor antagonist - in a non-polar solvent. Position the needle near the thorax and use the micro-applicator to carefully deposit a drop of the test compound on the dorsal surface.
Post application, transfer the treated mosquitoes to a growth chamber with controlled environmental conditions for a determined period.
The non-polar solvent in the droplet solubilizes lipids in the cuticle - an outer covering - allowing the compound to penetrate inside. The compound diffuses through the epidermal layer underneath and reaches the thoracic ganglia - a dense group of nerve cells connected to the nervous system.
Upon reaching the nerve cells, the compound binds to an allosteric site - an additional binding site for regulatory molecules - on the dopamine receptor. The allosteric binding hinders the downstream signaling cascade upon binding to dopamine - a neurotransmitter controlling critical biological processes - leading to death.
Following the experimental duration, record the number of dead mosquitoes - treated individuals lacking any movement - to assess the test compound's insecticidal potential.
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.
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