Larval Contact Assay: A Procedure to Evaluate Mosquito Larval Mortality following Direct Contact with Test Larvicidal Compound

To evaluate the potential of a test larvicidal compound, a dopamine receptor antagonist, using larval contact assay, begin with live mosquito larvae at their desired developmental or instar stage in aqueous media. Transfer equal numbers of the larvae to the wells of a multi-well plate.

Replace the media in the wells with deionized water to prevent any interference from contaminants that may negatively impact the larvae. Add increasing concentrations of the test compound to the respective wells.

Gently swirl the plate to facilitate contact between larvae and test compound. Incubate the plate under appropriate conditions.

Upon contact with the larvae, the test compound enters the body wall or outermost layer and reaches the spiracles, openings of the tracheal system, which are connected to the ganglia - cluster of nerve cells - comprising the central nervous system.

On the nerve cells, the test compound binds to the secondary binding sites of dopamine receptors, which are involved in critical cellular processes. This binding, even with neurotransmitter dopamine bound to the dopamine receptor, prevents receptor activation and downstream signaling, disrupting larval development, ultimately causing death. 

Record the number of non-responsive larvae, lacking movement, in each well at specific time point following test compound exposure across varying compound concentrations.

Increased larval mortality rate with increasing concentration indicates the larvicidal potential of the test compound.

To begin, label the wells of a clear 24-well tissue culture plate. Use an analytical balance to weigh the test compound. Then, dissolve the compound in sterile, double-distilled water in a 1.5-milliliter tube, resulting in an 80-millimolar stock solution. Next, serially dilute the stock solution using double-distilled water to prepare working stock solutions of the desired concentrations.

Note that care must be taken when the larvae are added to the wells, during the removal of excess water, and when the test chemistry is added, to avoid physical damage to the larvae. Injury may increase mortality, and thus, produce false positive results or invalidate the assay.

Use a wide-bore plastic transfer pipette to transfer five third-instar larvae to each well of the plate. Then, use a 1-milliliter pipette to gently remove the water, and replace it with the desired volume of double-distilled water.

Take care not to touch the larvae when removing the water. Work quickly to ensure the larvae do not desiccate, and gently add the test chemistry by pipetting against the opposite side of the plastic well.

Add an appropriate volume of testing solution to each well, and gently rotate the plate to ensure uniform mixing. Place the plate in a growth chamber, maintaining 12-hour light-dark cycle at 25 degrees Celsius with 75% to 85% relative humidity.

To check larval movement, gently tap the plate. If no movement is observed, gently touch the larva with a sterile toothpick. Score the larva as dead if no response is noticed, and use a score sheet to record the total number of dead larvae in each well at the time points described in the text protocol.