Materials and Methods
Mosquito samples
This study involved field-collected mosquitoes and an insecticide-susceptible laboratory strain. Mosquito collections were carried out in 4 villages in Cameroon where An. funestus s.s. is the predominant vector: Gounougou (9°03′00″N, 13°43′59″E) in 2014, 2017, 2020, 2021; Elende (3°41’57.27”N, 11°33’28.46”E) in 2020 (Nkemngo et al., 2020); Mibellon (6°46′N, 11°70′E) in 2016, 2018, 2020 (Menze et al., 2018) and Tibati in 2018 and 2021 (6°28’ N, 12°37’ E) (Tchouakui, Fossog, et al., 2019). Indoor resting mosquitoes were collected with Prokopack aspirators between 06:00 am and 9:00 am, following verbal consent from the house owner. The collected blood-fed Anophelesfemales were kept for 5 days until fully gravid before putting them in 1.5ml tubes for forced egg-laying. The F1s were reared at the Centre for Research in Infectious Diseases (CRID) until the emergence of adults. F1 mosquitoes from Gounougou were used for susceptibility testing and evaluating bed net efficacy.
Molecular identification followed the cocktail PCR method of Koekemoer et al. (Koekemoer et al., 2002) to discriminate members of the An. funestus group and confirm the species as An. funestus s.s was done. The An. funestus s.s laboratory strain FANG, maintained at the insectary of CRID, was used for the crossing and qRT-PCR as the reference strains to determine gene expression.