Insecticide susceptibility tests

Knock-down and mortality of An. gambiae (s.l.) females resulting from tarsal contact with insecticide impregnated papers with discriminating doses were assessed using WHO susceptibility test kits [5]. Deltamethrin (0.05%), alphacypermethrin (0.05%), bendiocarb (0.1%), propoxur (0.1%), and pirimiphos-methyl (0.25%) impregnated papers (obtained from Vector Control Research Unit, School of Biological Sciences, Malaysia) and clothianidin (2%) impregnated papers (Sumitomo chemical, Japan; Lot: {"type":"entrez-nucleotide","attrs":{"text":"CL190805","term_id":"40703328","term_text":"CL190805"}}CL190805) were tested for their efficacy against adult An. gambiae s.l. females (later identified to sibling species). In the selection of the test insecticides, current and previous insecticide usage profile of the national malaria control program was considered.

Four replicates of 24–25 non-blood fed, 3–5 days old females mosquitoes were exposed to insecticide impregnated papers for 1hour. A minimum of 99 and a maximum of 100 mosquitoes were exposed and fifty were used as a control for each insecticide tested. The number of knocked down mosquitoes were recorded at 10, 15, 20, 30, 40, 50 and finally after 60 minutes (1hour) exposure time [5]. In parallel, two replicates of mosquitoes (25x2) were exposed to control papers impregnated with silicone oil as control for pyrethroids and olive oil for organophosphate/carbamate insecticides. For clothianidin, water impregnated untreated papers obtained from similar company (Sumitomo chemical, Japan, Lot: UC190815) were used as negative controls.

After one-hour exposure time, mosquitoes were transferred back into holding tubes, provided with 10% sugar solution and the proportion of surviving and dead mosquitoes were recorded 24 hours post exposure. However, for clothianidin tests, 10% sugar solution was changed every 12 hours and mortality was recorded daily until 100% mortality was obtained. All the tests were performed at 25°C ±2°C and 70% ± 10% relative humidity. The quality of each insecticide impregnated paper was checked on a known susceptible laboratory colony of An. arabiensis obtained from Sekoru insectary, Tropical and Infectious Diseases Research Centre, Jimma University (JU TIDRC), Ethiopia.

From each test, randomly selected samples of dead and surviving mosquitoes were preserved individually in Eppendorf tubes over silica-gel and kept in a freezer (-21°C) for subsequent molecular species identification and kdr allele detection [5].