Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

This method can help answer key questions in the insecticide discovery and vector control fields. Such as, how toxic is an unformulated chemistry to a population of mosquito larvae or adults? Does the chemistry have potential for development as a larvicide or adulticide?

And, what might be the most effective delivery route? The main advantages of this technique are that it can be used to evaluate the toxicity of unformulated chemistries The toxicity of a chemistry against multiple species of vector mosquitoes, and it can be scaled up to evalute hundreds of compounds in high throughput capacity. Demonstrating the procedure will be Jasleen Kaur, a scientific technician from my laboratory.

To begin, label the wells of a 24-well tissue culture plate. Use an analytical balance to weigh the test compound. Then, desolve the compound in sterile double distilled water in a 1.5mL tube, resulting in an 80mM 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 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 one mL 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 that 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 percent 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. Culture three to five day old adult female mosquitoes in a 20 liter plastic cage.

Label nine ounce paper cups with the name and concentration of testing compound. Next, prepare a 10mg/mL stock solution of the testing compound in acetone in a 20mL glass vial. Then, serially dilute the stock solution to obtain the desired working concentrations.

Clean the one mL glass syringe with acetone. Then fill it with the test solution at the appropriate concentration. Secure the syringe in a micro-applicator adjusted to deliver a volume of 0.25uL.

Next, use an aspirator to remove 10, three to five day old adult female mosquitoes from the cage. Anesthetize them for five minutes at four degrees celsius. Then transfer the mosquitoes to a petri dish.

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 five 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 0.25uL 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 10cm mesh square and a rubber band Then, transfer it to the growth chamber, and record the number of dead mosquitoes as previously described.

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 one to 24 hours prior to the feeding assay. Prepare an 80mM 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 40uL of each dilution to 960uL of defibrinated rabbit blood in a 1.5mL tube, and mix by pipetting. Place a member filter on a feeding unit and seal it with a rubber ring.

Next, use a pipette to transfer one mL 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 percent 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 four 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 three post-blood feeding, place an eggcup in the cage for 72 hours.

Use a dissecting microscope to count the total number of eggs produced per treatment. A larval contact assay was performed to evaluate the effect of amitriptyline, a dopamine receptor antagonist, on larval mortality over time. The data revealed that LC 50 value decreases over time course of the experiment.

The effect of amitriptyline on mortality of adult female mosquitoes was compared with bifenthrin, and two negative controls. As compared to acetone treated and untreated negative controls, both amitriptyline and bifenthrin induced significant mortality at each experimental time point. A quantitative feeding assay was performed to evaluate the effect of three different doses of amitriptyline on fecundity and percent mortality of adult female mosquitoes.

The data revealed no statistically significant difference in the fecundity of amitriptyline fed mosquitoes at highest dose compared to only blood-fed controls. Once mastered, any of the assays we have described here, can be completed in approximately two hours by a single individual, if properly performed. While attempting this procedure, it's important to remember to gently handle the organism, and there should be consistency during topical applications and during scoring larval or adult assay.

Following this procedure, other methods like bio-assays to evaluate toxicity via absorption, through the mosquito tarsi, the CDC bottle assay, and the WHO tube assay can be performed in order to answer additional questions regarding the efficacy of the unformulated chemistry, or a formulated product, and potential for development as a contact insecticide or space spray.