Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

Transcript

By directly applying insecticide on the insect rather than through surface contact, there's minimal variation in insecticide exposure, and this allows us to measure the susceptibility profile of mosquito populations more accurately. The main advantage of this technique is that it allows us to calculate mass-relativized lethal doses of an insecticide rather than lethal concentrations, which greatly improves resistance ratio comparisons. Be careful to store the insecticides properly, avoid contaminating your tools, and ensure the syringe parts are in good shape.

This will help you to obtain consistent results. To begin, remove the needed insecticide solutions from the freezer. Immediately vortex the solution or invert five times, and place it in a light-resistant container at room temperature to let the insecticides warm to room temperature before using.

Set out five microcentrifuge tubes with 0.5 milliliters of acetone each. Fill the syringe barrel completely with acetone from the first tube. Then expel the acetone into a waste container by swiftly pushing down on the plunger, and repeat four more times to complete a total of five acetone washes from the same acetone aliquot.

Then, fill the syringe barrel completely with air, and expel the air and potential acetone remnants into the waste container. Repeat two more times to complete three washes with air. Create an air pocket within the barrel between the syringe plunger and the top of the needle by pulling up the plunger approximately five millimeters into the barrel.

Label the plastic holding cups with the random ID for blind mortality assessment. Using an aspirator powered by suction from inhalation, aspirate the desired number of three-to five-day old adult mosquitoes. Place the tip of the aspirator into the tube with cotton wrapped around the tip.

Gently exhale, and tap the aspirator to transfer the mosquitoes into a conical tube. Use the cotton to plug the tube when the aspirator tip is removed, and then cap with the lid. Briefly knock down the mosquitoes in the tubes by placing them for a minimum of 10 minutes at four degrees Celsius or burying them under the ice in an ice tray.

Transfer the knocked down mosquitoes to the insect handling tent. Carefully tip the mosquitoes out onto a plastic tray placed on the ice, and pour around 50 mosquitoes at a time to ensure each touches the cool tray beneath it and stays knocked down. Sort the mosquitoes by sex by gently picking them up by the legs or wings with forceps, followed by placing each sex into a separate holding cup, and stop when the desired number is reached.

While sorting, remove any mosquitoes that are injured or are extra large or small. To record the weight of the mosquito cup, place an empty cup with a Petri dish as a lid on the scale, and tare the scale. Pour the mosquitoes into the container, and place the lid on top.

Place the container on the scale, and record the final weight, along with the number of mosquitoes. Immediately place the cup of specimens back on the ice to keep them immobilized until all cups of the specimen are weighed. Divide the prepared mosquitoes into five to 10 separate cups placed on ice, labeled with random IDs.

Use tweezers and a handheld counter to achieve 20 to 25 mosquitoes per cup. To sort the fruit flies, anesthetize the flies using carbon dioxide for seven seconds. Pour the flies onto an ice pack wrapped in bench paper.

Using a fine-tipped paintbrush, separate and count the males and females. Use the paintbrush to gently pick up the chosen flies, and place them into a clean, empty stock bottle. After choosing equal number of male and female fruit flies, label the stock bottles with the strain name and fruit fly total.

Anesthetize the bottle of fruit flies using carbon dioxide for seven seconds. Pour the fruit flies onto weighing paper, and use the paper as a funnel to introduce the flies into a tared vial labeled with a random ID.After placing the Petri dish lid on top of the vial of fruit flies, place it on the scale. Record the combined weight and number of specimens on the score sheet.

Immediately place the vial of fruit flies in a tray of ice with the lid still on top to prevent the flies from escaping. Load the syringe with the proper insecticide concentration, starting with the least concentrated dose and work towards the most concentrated dose with each group of organisms. Tip the specimens onto weighing paper placed atop a tray on the ice.

Separate the specimens that are close together using a clean insecticide-free paintbrush or cotton swab to allow easy access to each specimen for dosing. Using the syringe, apply one droplet of insecticide solution to the ventral thorax and abdomen area for mosquitoes and the dorsum for fruit flies. Immediately pour the specimens back into the labeled plastic cup.

Cover the cup with netting and a rubber band. Note an updated specimen count on the cup if any specimens were killed, damaged, or escaped in this process. Place the completed cup into a holding tray.

For the first cup, record the time when dosing is completed. Repeat dosing for each cup until all specimens have been dosed with the proper insecticide concentrations, and record the ending time when all specimens have been dosed. Provide 10%sucrose solution to each cup via a wet cotton ball, and store the mosquitoes and fruit flies at the desired temperature and humidity.

Record specimen mortality at 24 hours after the start of insecticide exposure. Classify mosquitoes as alive if they can fly and hold themselves upright and dead if they are immobile or ataxic, as described by the WHO. Follow the same mortality assessment for fruit flies.

After performing the bioassay, dose response data were obtained featuring two strains that are Rockefeller and IICC genotype for the male and female Aedes aegypti. The mass-relativized median lethal dose for Rockefeller and IICC was 0.008 and 0.336 nanograms per milligram, respectively. The results revealed no differences between the dose response curves of female and male mosquitoes within each strain.

Therefore, data from both sexes were combined to calculate the median lethal dose values. Since the 95%confidence intervals for the median lethal dose of these two strains did not overlap, these values were considered significantly different. Additionally, when calculating the resistant ratios, the IICC strain was over 40 times more resistant than the susceptible Rockefeller strain.

Dose response data for fruit fly, Drosophila melanogaster, Canton-S strain, was also obtained, and the mass-relativized median lethal dose was found to be 0.213 nanograms per milligram. Consistency is critical, especially when storing and applying the insecticide and assessing mortality. Also, take care when handling the specimen to avoid harm or death not induced by the insecticide.

With this method, we can now accurately determine the technical resistance of a mosquito population and how environmental conditions impact resistance or how technical resistance predicts practical resistance.

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Journal (Biology)

Journal (Biology)

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

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