Blood-fed and gravid Anophelesfemales resting indoors were collected in human dwellings between 8:00 and 12:00 during two sampling campaigns conducted in January 2023 and April 2024 in four villages located in central Senegal. The study was carried out in the villages of Camara (13˚38'N, 15˚37'W) and Keur Mamadou (13˚41'N, 16˚02'W) in the Nioro health district, Tawa Mboudaye (13˚58'N, 16˚12'W) in the Ndoffane health district, and Dielmo village (13˚43'N, 16˚24'W) in the Sokone health district (Figure 1).

The Nioro and Ndoffane health districts are located in the Kaolack administrative region, whereas Sokone is situated in the Fatick region. The study area is characterized by a Sudano-Sahelian climate, with annual rainfall ranging from 700 to 1300 mm. Temperatures typically vary between 18˚C and 40˚C, with peak temperatures recorded in May and the lowest in January [14]. The region experiences two distinct seasons: a rainy season from June to October and a long dry season from November to May. The hydrographic network of the study sites includes permanent water bodies bordered by dense aquatic vegetation, predominantly Typha australis, creating favourable breeding habitats for An. funestus larvae. Indoor-resting, blood-fed, and gravid female mosquitoes were collected from human dwellings using mouth aspirators and torches and transferred into holding cups covered with untreated netting. Mosquitoes were transported alive to the insectary, where they were maintained on a 10% sugar solution for 3 - 4 days until they became fully gravid. Individual oviposition was then induced in Eppendorf tubes, after which the egg batches from individual females were pooled for larval rearing and transferred into plastic trays for larval rearing [15]. After oviposition, all F0 females were individually preserved in labelled 1.5 ml Eppendorf tubes containing silica gel and stored at −20˚C for subsequent molecular analysis. Larvae from hatched eggs were reared under standard insectary conditions (25˚C ± 2˚C and 70% ± 10% relative humidity), and emerging F1 adults were pooled and randomly maintained in rearing cages for subsequent WHO susceptibility tests.

Figure 1. Geographic location of the study sites in central Senegal.

Wild-caught female mosquitoes were initially morphologically identified as members of the Anopheles funestusgroup using standard mosquito identification keys [16][17]. Following oviposition, species identification was further confirmed using a multiplexed polymerase chain reaction (PCR) assay specific to the An. funestusgroup as described by Koekemoer et al. [18]. Genomic DNA was first extracted from individual mosquitoes following the method previously described by Collins et al. [19]. The amplified PCR products were visualized under UV illumination following gel electrophoresis to determine species-specific banding patterns.

Insecticide susceptibility assays were conducted on 3 - 5 day-old unfed F₁ females of An. funestuss.s. in accordance with standard WHO [20]. Mosquitoes were exposed to the WHO diagnostic concentration of insecticides from the four major classes used in public health, including pyrethroids (permethrin 0.75%, cyfluthrin 0.15%, deltamethrin 0.05%, lambda-cyhalothrin 0.05% and alpha-cypermethrin 0.05%), organochlorine (dichlorodiphenyltrichloroethane or DDT 4%), the carbamate (bendiocarb 0.1%), and the organophosphates (pirimiphos-methyl 0.25% and fenitrothion 1%). For each insecticide, approximately 100 mosquitoes were tested, divided into four replicates of 25 individuals each. A control group exposed to untreated papers was included for each experiment. Following exposure, mosquitoes were maintained under standard insectary conditions, and mortality was recorded 24 hours later. Resistance status was interpreted according to WHO criteria [20]. All bioassays were carried out under controlled laboratory conditions at 25˚C ± 2˚C and 70% ± 10% relative humidity.

The potential involvement of cytochrome P450s monooxygenases in insecticide resistance was investigated using PBO synergist assays following WHO guidelines [20]. Unfed F₁Anopheles funestus s.s. adult females aged (3 - 5 days) were first exposed to PBO 4% impregnated papers for 1 hour and then immediately transferred to test tubes containing insecticide-treated papers for an additional 1-hour exposure. The insecticides tested included deltamethrin (0.05%), alpha-cypermethrin (0.05%), lambda-cyhalothrin (0.05%), permethrin (0.75%), cyfluthrin (0.15%), DDT (4%), and bendiocarb (0.1%). Mosquito mortality was recorded 24 hours post-exposure. Two controls were included in each experiment: one exposed only to PBO papers and the other exposed to untreated papers. The contribution of cytochrome P450-mediated metabolic resistance was inferred by comparing mortality rates between mosquitoes exposed to insecticide alone and those pre-exposed to PBO prior to insecticide exposure [20].

The intensity of resistance to pyrethroids and carbamate was assessed using WHO resistance intensity assays performed at 5x and 10x the diagnostic concentrations [20]. The insecticides tested included permethrin (3.75% and 7.5%), deltamethrin (0.25% and 0.5%), alpha-cypermethrin (0.25% and 0.5%), lambda-cyhalothrin (0.25% and 0.5%), and bendiocarb (0.5% and 1%). Results were interpreted according to WHO criteria for resistance intensity [20]. Following the assays, dead mosquitoes were preserved individually in silica gel, while surviving mosquitoes were stored in RNALater^® to maintain RNA integrity for subsequent molecular and genomic analyses. All samples were kept at −80˚C until processing.

Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, California USA) with significance set at 5% and confidence intervals (CI) calculated at 95%. Mortality results were interpreted according to WHO criteria (≥98%: susceptible; 90% - 97%: possible resistance; <90%: confirmed resistance). No additional statistical tests were applied, as WHO thresholds alone were considered sufficient for classification.

Among mosquitoes collected indoors, a total of 600 females (F₀) were morphologically identified as members of the Anopheles funestus group. Subsequent molecular characterization confirmed that all identified specimens belonged to Anopheles funestussensu stricto, which will hereafter be referred to as An. funestus.

No mortality was recorded in either control group exposed to PBO-only papers or untreated papers, confirming the validity and reliability of the bioassays.

3.2.1. Phenotypic Resistance of Anopheles funestus to Standard Insecticide Concentrations

A total of 10,834 F₁Anopheles funestus adults aged 3 - 5 days from the study sites were tested against different classes of insecticides at WHO diagnostic concentrations.

WHO susceptibility bioassays revealed varying levels of resistance across insecticide classes and study sites. Overall, An. funestus populations from Nioro and Ndoffane were resistant to pyrethroids, with mortality rates ranging from 46.7% to 88.3% (Figure 2).

Anopheles funestus populations were susceptible to DDT (100%) in Camara (Nioro) and Dielmo (Sokone), but resistance (88.3%, n = 103) to that organochlorine was found in Keur Mamadou (Nioro), whereas in Tawa Mboudaye (Ndoffane), suspected resistance (91.4%, n = 116) was observed.

Regarding organophosphates, An. funestus populations were fully susceptible to pirimiphos-methyl. However, resistance to fenitrothion was detected in Tawa Mboudaye and Keur Mamadou, with mortality rates of 86.4% (n = 103) and 76.1% (n = 109), respectively.

For carbamates, full susceptibility to bendiocarb was observed in Tawa Mboudaye and Dielmo, with mortality rates of 99.1% (n = 107) and 100% (n = 110), respectively. In contrast, populations from Nioro were resistant, with mortality rates of 83.0% (n = 105) in Camara and 68.2% (n = 107) in Keur Mamadou (Figure 2).

Overall, An. funestus populations from central Senegal showed high levels of resistance to pyrethroids and carbamates, along with reduced susceptibility to organochlorines, while remaining fully susceptible to pirimiphos-methyl. These resistance patterns may be associated with local selection pressures, including agricultural insecticide use.

Figure 2. Susceptibility profile of F₁ mosquitoes across the four studied sites, showing mortality rates recorded 24 h post-exposure in An. funestus s.s. The red dotted line indicates resistance (90%) and the green dotted line indicates the susceptibility 98% threshold.

3.2.2. Resistance Intensity to Insecticides

Resistance intensity bioassays conducted using increased concentrations (5× and 10×) of alpha-cypermethrin, deltamethrin, lambda-cyhalothrin, permethrin, and bendiocarb revealed spatial variation in resistance levels across study sites, likely reflecting differences in local environmental conditions and insecticide use practices, particularly in agriculture. Overall, resistance intensity varied across study sites and insecticides, ranging from low to high levels. High intensity to pyrethroids was observed in some locations, while low to moderate intensity predominated for other compounds (Figure 3).

In Keur Mamadou, An. funestus populations exhibited low resistance intensity to alpha-cypermethrin and permethrin, moderate resistance intensity to deltamethrin, and high resistance intensity to lambda-cyhalothrin. In Camara, low resistance intensity was observed for bendiocarb, deltamethrin, and permethrin, whereas moderate to high resistance intensity was recorded for alpha-cypermethrin (5×) and lambda-cyhalothrin (5×), with mortality rates of 95.2% and 92.2%, respectively.

In the Ndoffane health district, high resistance intensity was detected in Tawa Mboudaye, with approximately 85% mortality at 10× for alpha-cypermethrin. In the same locality, moderate resistance intensity was observed for deltamethrin, lambda-cyhalothrin, and permethrin, with complete mortality (100%) recorded at 5×. This resistance profile may reflect selective pressure associated with the intensive use of agricultural insecticides in this horticultural area. In Dielmo, resistance intensity was low for lambda-cyhalothrin and moderate for alpha-cypermethrin (Figure 3). Overall, resistance intensity varied according to study sites and insecticides tested, ranging from low to high levels. High-intensity resistance to pyrethroids was observed in some locations, whereas low to moderate resistance intensity predominated for other insecticides (Figure 3).

Figure 3. Resistance intensity profiles of resistance Anopheles funestus populations to pyrethroids and bendiocarb. Red dotted line indicates the threshold of 98% mortality.

3.2.3. Bioassays, Synergist PBO (Piperonyl Butoxide)

Synergist assays using piperonyl butoxide (PBO) 4% were conducted to investigate the potential involvement of cytochrome P450 monooxygenases in the observed resistance to pyrethroids, bendiocarb, and DDT across the study sites.

Pre-exposure of An. funestus s.s. to PBO resulted in a complete restoration of susceptibility to type I pyrethroids (permethrin and cyfluthrin), bendiocarb, and DDT (p < 0.001), indicating a predominant role of cytochrome P450 monooxygenases in metabolic resistance in these populations.

In contrast, a partial recovery of susceptibility was observed for type II pyrethroids (alpha-cypermethrin, deltamethrin, and lambda-cyhalothrin) following PBO pre-exposure (p < 0.001) (Table 1). This partial restoration suggests that, in addition to cytochrome P450 monooxygenases, other resistance mechanisms may contribute to the observed phenotypic resistance.

No mortality was recorded in the control groups (PBO-only exposure and untreated papers), confirming the validity of the bioassays.

Table 1.Effect of PBO pre-exposure on the susceptibility profile of mosquitoes to pyrethroids, bendiocarb, and DDT.

N: number of mosquitoes exposed, CI: confidence interval, and PBO: piperonyl butoxide.