Topical Application of Insecticidal Active Ingredients

Take a container of all female mosquitoes and gently introduce carbon dioxide gas to anesthetizeindividuals. From a gas cylinder equiped with a regulator and a hose, slowly increase the rate of flowing CO₂ to a gentle stream. Flow CO₂ into container with mosquitoes (cover hand over container to ensure CO₂ concentration builds within container) for 30 seconds

Place ice in another container and place two, 90-mm glass petri dish bottoms on ice. On the bottom surface of the petri dishes, place 2 90-mm filter papers to prevent the build up of excess condensation, as condensation may adversely affect some species of mosquito ( Anopheles spp. are particularly sensitive). Carefully transfer anesthetized mosquitoes into petri dishes on ice. The ice will prevent mosquitoes from reanimating. Make sure to transfer less than 200 mosquitoes to the petri dish at a given time. This will allow you to complete testing on each mosquito and prevent each individual from remaining on the ice for too long. Mosquitoes left on ice for too long may not reanimate. 

Individually pick up each mosquito by the hind legs with featherweight forceps (Bioquip) and carefully apply 0.2 μL of insecticidal solution to the pronotum of each individual mosquito. This corresponds to one pump of the repeating dispenser. It is good to make sure the repeating dispenser is dispensing by pumping and tapping the tip of the syringe to a kimwipe to detect a solvent droplet. For each treatment group (i.e. insecticide concentration), treat a total of 25 mosquitoes and move treated mosquitoes to the next petri dish on ice. Transferring mosquitoes to the final observation container too soon may cause them to reanimate quickly and escape. Also, transferring mosquitoes quickly to a observation container may cause mosquitoes to stick to the cup, if the insecticidal agent is oily. The subsequent transfer to a secondary petri dish will prevent these issues from arising. After all 25 mosquitoes are treated with your insecticide concentration of choice, quickly transfer them to the observation container. The obsevation container should be a small deli cup (8-counce deli cup) with tulle placed over the top to allow adequate ventilation. 

Move mosquitoes (now in observation container) into an incubator that can maintain 28 C with approximately 80% relative humidity. Provide cotton pad moistened with 10% sucrose water to prevent mosquitoes from dessicating. 

Record percentage knockdown at 1 hr and percentage mortality at 24 hr. Toxicity endpoints may be changed to appropriately characterize insecticidal efficacy if necessary (e.g. 2-hr knockdown, 48-hr mortality). A minimum of 3 biological cohorts should be included in the data set (mosquitoes from different rearing groups) before analysis. Mortality should be defined as ataxia (inability to move) after prodding each individual with a camelhair brush. Knockdown is defined as the inability to fly or orient in the upright direction. 

SAS code is featured below (be sure to modify the data to match your dose-response data): 

PROBIT ANALYSIS (for variable amount of insects per replicate)

# “Enter data in Italicized section as concentration _ total insect number _ number dead ”

# 'Underscore (_) corresponds to space'

# 'Be sure to remove italicized data before copying into SAS software, as this font style may adversely affect the software analysis. It is used here to communicate where to enter data.'

# This code may be used to calculate a KD₅₀using knockdown percentages instead of mortality.

data mgk;

input dose number res;

cards;

0.0001 30 29

0.00005 30 24

0.00002 30 5

0.00001 30 2

0.000002 30 0

0 30 0

;

proc probit data=mgk log10 optc plots=(all);

model res/number=dose/inversecl lackfit;

output out=new p=p_hat;

run ;

ods graphics on;

quit ;