Metabolic resistance to insecticides threatens malaria control. However, little is known about its fitness cost in field populations of malaria vectors, thus limiting the design of suitable resistance management strategies. Here, we assessed the association between the glutathione S-transferase GSTe2-mediated metabolic resistance and life-traits of natural populations of Anopheles funestus. A total of 1200 indoor resting blood-fed female An. funestus (F0) were collected in Mibellon, Cameroon (2016/2017), and allowed to lay eggs individually. Genotyping of F1 mosquitoes for the L119F-GSTE2 mutation revealed that L/L119-homozygote susceptible (SS) mosquitoes significantly laid more eggs than heterozygotes L119F-RS (odds ratio (OR) = 2.06; p < 0.0001) and homozygote resistant 119F/F-RR (OR = 2.93; p < 0.0001). L/L119-SS susceptible mosquitoes also showed the higher ability for oviposition than 119F/F-RR resistant (OR = 2.68; p = 0.0002) indicating a reduced fecundity in resistant mosquitoes. Furthermore, L119F-RS larvae developed faster (nine days) than L119F-RR and L119F-SS (11 days) (X2 = 11.052; degree of freedom (df) = 4; p = 0.02) suggesting a heterozygote advantage effect for larval development. Interestingly, L/L119-SS developed faster than 119F/F-RR (OR = 5.3; p < 0.0001) revealing an increased developmental time in resistant mosquitoes. However, genotyping and sequencing revealed that L119F-RR mosquitoes exhibited a higher adult longevity compared to RS (OR > 2.2; p < 0.05) and SS (OR > 2.1; p < 0.05) with an increased frequency of GSTe2-resistant haplotypes in mosquitoes of D30 after adult emergence. Additionally, comparison of the expression of GSTe2 revealed a significantly increased expression from D1-D30 after emergence of adults (Anova test (F) = 8; df= 3; p = 0.008). The negative association between GSTe2 and some life traits of An. funestus could facilitate new resistance management strategies. However, the increased longevity of GSTe2-resistant mosquitoes suggests that an increase in resistance could exacerbate malaria transmission.

Growing resistance is reported to carbamate insecticides in malaria vectors in Cameroon. However, the contribution of acetylcholinesterase (Ace-1) to this resistance remains uncharacterised. Here, we established that the G119S mutation is driving resistance to carbamates in Anopheles gambiae populations from Cameroon. Insecticide bioassay on field-collected mosquitoes from Bankeng, a locality in southern Cameroon, showed high resistance to the carbamates bendiocarb (64.8% ± 3.5% mortality) and propoxur (55.71% ± 2.9%) but a full susceptibility to the organophosphate fenitrothion. The TaqMan genotyping of the G119S mutation in field-collected adults revealed the presence of this resistance allele (39%). A significant correlation was observed between the Ace-1R and carbamate resistance at allelic ((bendiocarb; odds ratio (OR) = 75.9; p < 0.0001) and (propoxur; OR = 1514; p < 0.0001)) and genotypic (homozygote resistant vs. homozygote susceptible (bendiocarb; OR = 120.8; p < 0.0001) and (propoxur; OR = 3277; p < 0.0001)) levels. Furthermore, the presence of the mutation was confirmed by sequencing an Ace-1 portion flanking codon 119. The cloning of this fragment revealed a likely duplication of Ace-1 in Cameroon as mosquitoes exhibited at least three distinct haplotypes. Phylogenetic analyses showed that the predominant Ace-1R allele is identical to that from West Africa suggesting a recent introduction of this allele in Central Africa from the West. The spread of this Ace-1R represents a serious challenge to future implementation of indoor residual spraying (IRS)-based interventions using carbamates or organophosphates in Cameroon.

Metabolic resistance threatens the sustainability of pyrethroid-based malaria control interventions. Elucidating the fitness cost and potential reversal of metabolic resistance is crucial to design suitable resistance management strategies. Here, we deciphered the fitness cost associated with the CYP6P9a (P450-mediated metabolic resistance) in the major African malaria vector Anopheles funestus. Reciprocal crosses were performed between a pyrethroid susceptible (FANG) and resistant (FUMOZ-R) laboratory strains and the hybrid strains showed intermediate resistance. Genotyping the CYP6P9a-R resistance allele in oviposited females revealed that CYP6P9a negatively impacts the fecundity as homozygote susceptible mosquitoes (CYP6P9a-SS) lay more eggs than heterozygote (OR = 2.04: P = 0.01) and homozygote resistant mosquitoes. CYP6P9a also imposes a significant fitness cost on the larval development as homozygote resistant larvae (CYP6P9a-RR) developed significantly slower than heterozygote and homozygote susceptible mosquitoes (χ2 = 11.2; P = 0.0008). This fitness cost was further supported by the late pupation of homozygote resistant than susceptible mosquitoes (OR = 2.50; P < 0.01). However, CYP6P9a does not impact the longevity as no difference was observed in the life span of mosquitoes with different genotypes (χ2 = 1.6; P = 0.9). In this hybrid strain, a significant decrease of the resistant CYP6P9a-RR genotype was observed after ten generations (χ2 = 6.6; P = 0.01) suggesting a reversal of P450-based resistance in the absence of selection. This study shows that the P450-mediated metabolic resistance imposes a high fitness cost in malaria vectors supporting that a resistance management strategy based on rotation could help mitigate the impact of such resistance.