WHO cone bioassay with unwashed Royal Guard® nets to determine the fertility of mosquitoes from Ifangni

The efficacy of three types of nets, unwashed PPF-LLIN (Royal Guard^®), unwashed alpha-cypermethrin LLIN (Interceptor^®), and negative control (unwashed untreated net) was evaluated using the WHO cone bioassay. The test was performed on adult female Anopheles gambiae s.l. from the Ifangni district only because of insufficient availability of mosquitoes from the larval sites from Akpro-Missérété, Allada, and Porto-Novo. Six to 12 h after their second blood meal, batches of 5 well-fed mosquitoes were exposed for 3 min to samples with two replicates of each type of net. A total of 80 mosquitoes were used per net. After exposure, mosquitoes were transferred and fed (sugar solution) in veiled cups and labeled with the net sample ID and position (roof or side). Bioassays were performed at 25 ± 2 °C and 70 ± 10% humidity. Mortality was recorded at 24, 48, and 72 h after exposure. Mosquitoes surviving 72 h after each type of net were dissected and data on the egg development status in the ovaries of each mosquito according to Christopher’s stages were reported. Fertile, infertile, and inconclusive mosquitoes were recorded on a data recording sheet and photographs of the ovaries were taken with a digital camera looking through the ocular lens (see the conceptual diagram of the study, Fig. 2).

Conceptual diagram of the fertility study