Malaria remains a globally endemic disease, with control strategies primarily based on the use of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) (1, 2). Four major classes of insecticides are employed in vector control programs: pyrethroids, organophosphates, carbamates, and organochlorines (3, 4). Among these, pyrethroids are currently the only class recommended by the World Health Organization (WHO) for insecticide treatment net (ITN), due to their low irritant effect, rapid action, and low toxicity to humans (5). However, widespread resistance to all four insecticide classes has been reported in Anopheles gambiae s.l. across sub-Saharan Africa, seriously undermining the effectiveness of LLIN- and IRS-based interventions(6–8). These tools, which have been instrumental in significantly reducing Plasmodium falciparum infection prevalence over the past decade, are now facing growing limitations in their efficacy (9, 10). In response to this threat, one of the most promising approaches involves combining two active ingredients with distinct modes of action within a single LLIN. This combined strategy could enhance the sustainability of interventions and support improved management of insecticide resistance in malaria vectors (11, 12). Insecticide resistance among malaria vectors is a dynamic and unstable phenomenon, with intensity that can rapidly increase under multiple selection pressures particularly those induced by public health interventions and intensive agricultural practices using insecticides belonging to the same chemical classes (13, 14). The role of agricultural practices in selection An. gambiae resistant populations to chemicals were shown by several authors. According to Amara and collaborators, a study conducted in the city of Bobo-Dioulasso revealed that the An. gambiae s.l. complex is largely dominated by An. arabiensis, which accounted for most mosquitoes collected across various urban districts (17). This predominance suggests a specific ecological adaptation of An. arabiensis to local urban conditions, with important implications for targeted vector control strategies (17). However, a previous study conducted in Dioulassoba, a city from Bobo-Dioulasso, Burkina Faso, highlighted strong pyrethroid resistance in An. Arabiensis populations. Among the An. gambiae s.l. complex mosquitoes collected, over 31% belonged to the An. arabiensis species (Namountougou et al. 2019). These populations exhibited mortality rates below 30% following exposure to deltamethrin, indicating high phenotypic resistance. Furthermore, a high frequency (0.98) of the kdr 995F mutation known to confer resistance to pyrethroids was detected (6). This study aims to assess and characterize the current level of insecticide resistance in malaria vectors belonging to the Anopheles gambiae s.l. complex in Bobo-Dioulasso, Burkina Faso. It provides an updated data on observed resistance profiles and key entomological determinants of malaria transmission, thereby contributing to a better understanding of local transmission and the optimization of vector control strategies.

During the 2025 rainy season, Anopheles larvae were collected from natural breeding sites using the standard dipping method. At each location, sampling was conducted across multiple distinct larval habitats, including all developmental stages. Specimens were then pooled by locality. The larvae were reared at the IRSS/Centre Muraz insectary in Bobo-Dioulasso under controlled environmental conditions (temperature: 27–30 ± 1°C; relative humidity: 80 ± 10%; photoperiod: 12 hours of light and 12 hours of darkness, including a one-hour transition at dawn and dusk), until adult emergence (F0). They were fed daily with Tetramin® fish food. Upon emergence, female mosquitoes belonging to the Anopheles gambiae complex were maintained on a 10% sugar solution prior for insecticide susceptibility tests. Subsequently, adult specimens were morphologically identified using the taxonomic keys of Gillies and De Meillon, (1968) (19).

Insecticide susceptibility tests were conducted on 2 to 5-day-old, non-blood-fed adult female mosquitoes of the Anopheles gambiae complex using the WHO tube test protocol (20). Test papers impregnated with pyrethroids (alpha-cypermethrin 0.05%, deltamethrin 0.05%, permethrin 0.75%), an organochlorine (DDT 4%), a carbamate (bendiocarb 0.1%), and an organophosphate (pirimiphos-methyl 0.25%) were used. The An. gambiae s.s. Kisumu strain served as the biological control. Four replicates of 20 to 25 mosquitoes were introduced into WHO test tubes and observed for 60 minutes to ensure post-transfer recovery and to exclude any handling-related mortality. Environmental conditions were standardized at a temperature of 25 ± 2°C and relative humidity between 70% and 80%, to ensure optimal testing conditions. Mosquitoes were then exposed to insecticides for a strictly controlled duration of 60 minutes. Following exposure, they were transferred to observation tubes and maintained for 24 hours under the same thermo-hygrometric conditions, with access to a 10% sugar solution provided on cotton pads. Mortality was assessed 24 hours post-exposure. Alive and dead individuals were counted separately and preserved in Eppendorf tubes containing silica gel covered with cotton, according to sampling site and insecticide type. Samples were stored at − 20°C for molecular analyses, including species identification and detection of resistance-associated mutations (West and East kdr).

To investigate the potential role of detoxification enzymes in pyrethroid resistance, complementary bioassays were conducted using the synergist Piperonyl Butoxide (PBO) at 4%, known to inhibit monooxygenases and, to a lesser extent, esterases (21). Non-blood-fed Anopheles gambiae s.l. females aged 2 to 5 days were pre-exposed to PBO for one hour before being transferred onto insecticide-impregnated papers (alpha-cypermethrin 0.05%, deltamethrin 0.05%, permethrin 0.75%). Control mosquitoes underwent identical pre-exposure on untreated papers. Following exposure, individuals were placed in observation tubes containing cotton soaked in a 10% sugar solution and maintained under controlled conditions for 24 hours prior to mortality assessment. The same procedures were applied to the susceptible An. gambiae s.s. Kisumu strain. Each treatment was replicated four times, with batches of 20 to 25 mosquitoes per replicate.

A cross-sectional entomological survey was carried out between March 2023 and September 2024 in the same areas targeted for the bioassays. Human landing collections were performed in accordance with the World Health Organization (WHO) standard protocol (20). This study was conducted with the informed consent of participants, obtained from household heads in agreement with local authorities, including traditional leaders and the presidents of the respective study sites. In each of the six selected neighborhoods, eight trained adult volunteers were mobilized as mosquito collectors, with two individuals (outdoor and indoor) assigned per household. This collection was conducted in four households per neighborhood. Each collection team operated in two shifts: the first from 6:00 p.m. to 1:00 a.m., and the second from 1:00 a.m. to 9:00 a.m. Collectors served as live bait by sitting on chairs with their legs exposed and used flashlights to visually detect and capture mosquitoes landing on their limbs before blood-feeding occurred. Mosquitoes were caught using hemolysis tubes. To reduce potential bias due to differential attractiveness of individuals to mosquitoes, collectors alternated hourly between indoor and outdoor collection positions. Each collection site was supervised by a technician from the Institut de Recherche en Sciences de la Santé (IRSS) / Centre MURAZ and a local field assistant. Mosquitoes captured in hemolysis tubes were grouped by hour of collection into separate bags and transported daily to the entomology laboratory of IRSS/Centre MURAZ. Upon arrival at the laboratory, specimens were immediately sorted. Each mosquito was then individually transferred into a labeled Eppendorf tube containing silica gel to ensure proper desiccation and preservation for subsequent molecular analyses. The captured mosquitoes were first identified at the genus level using a binocular magnifying glass (AmScope; United Scope LLC) and based on the morphological identification key by Gillies et De Meillon (1968).

Mortality rates and 95% confidence intervals for the WHO susceptibility tests were calculated using the exact binomial method with RStudio software (version 4.4.2). The frequency of L995F and L995S mutations was calculated using the following formula: F(kdr) = (2nRR + nRS) / 2N, where nRR represents the number of homozygous resistant individuals, nRS the number of heterozygotes, and N the total number of specimens analyzed. Pearson correlation tests were performed to compare the abundance of mosquito species collected indoors and outdoors. A significance threshold of α = 0.05 was adopted, and results with p-values below 0.05 were considered statistically significant.

Distribution of Anopheles complex Species

A total of 360 individuals An. gambiae s.l were analyzed by PCR for Anopheles gambiae complex species identification. An. arabiensis (96.67%, 348/360) and An. gambiae s.s (3.33%, 12/360) were identified as members of the An. gambiae complex. Our data shown a clear predominance of An. arabiensis across all surveyed localities, with proportions ranging from 91.7% in Dogona to 100% in Kua and Sabaribougou. An. gambiae s.s. was marginally present, with frequencies below 10% in most sites and completely absent in Kua and Sabaribougou. This specific distribution suggests that malaria transmission dynamics in the area are primarily driven by An. arabiensis, a species known for its more exophilic and zoophilic behavior (Fig. 2).

Susceptibility Status of Anopheles gambiae to Pyrethroids

The data revealed heterogeneity in insecticide efficacy across the study sites, with mortality rates frequently below the 90% threshold established by WHO to confirm vector phenotypic resistance status. This observation indicates widespread resistance to pyrethroids, particularly permethrin and deltamethrin, in several surveyed areas. Additionally, DDT although historically used in control programs shows reduced effectiveness, suggesting possible cross-resistance with pyrethroids (Fig. 3). These findings underscore the need for continuous entomological surveillance and a reassessment of insecticides used in vector control strategies.

Susceptibility Status of Anopheles gambiae to PBO + Pyrethroids

Exposure of Anopheles gambiae to insecticide combinations enhanced with the synergist PBO (Piperonyl butoxide) resulted in a notable increase in mortality rates across all surveyed localities. The combinations PBO + permethrin, PBO + deltamethrin, and PBO + alpha-cypermethrin proved more effective than the insecticides used alone, indicating partial inhibition of metabolic resistance mechanisms in most sites. PBO restores either partially or totally the susceptibility to pyrethroids according to investigated localities.

However, in Sabaribougou, the markedly improved efficacy observed with PBO-containing formulations suggests a predominant, if not exclusive, involvement of metabolic resistance mechanisms in the local mosquito population (Fig. 4). These findings confirm the value of PBO as a complementary tool in targeted vector control strategies.

Understanding the current level of insecticide resistance in malaria vectors belonging to the Anopheles gambiae complex in Bobo-Dioulasso, Burkina Faso, is prerequisite for developing efficient vector control strategies. Thus, our data obtained provide a foundation for deeper reflection on the current state of insecticide resistance in malaria vectors in this region. It also extends our knowledge upon local malaria transmission.

The predominance of An. Arabiensis associated with the high prevalence of Kdr mutations, the variability in mortality rates across insecticides and study sites, reflect a complex entomological dynamic shaped by local selective pressures. These updated data are essential for refining vector control strategies, particularly by considering species- and locality-specific resistance profiles. They also highlight the need to integrate complementary tools, such as PBO-based formulations or alternative insecticides, into malaria control programs in order to sustain intervention efficacy and mitigate the evolution of resistance. The predominance of An. arabiensis across our study sites may be explained by its remarkable ecological plasticity and its ability to adapt particularly well to semi-arid environments and temporary larval habitats, which are often abundant during the rainy season. Unlike An. gambiae s.s., An. arabiensis tolerates high temperatures and low humidity levels more effectively, giving it a competitive advantage in dry climates (27).

An. arabiensis is known to exhibit relative zoophily and a marked exophilic tendency, reducing its exposure to indoor interventions such as residual spraying or the use of insecticide-treated nets (28). This behavioral shift has been documented in multiple settings, including Guinea-Bissau and southeastern Tanzania, where An. arabiensis was found to dominate outdoor collections and contribute significantly to residual transmission (29, 30). This contrasts with our findings, which show no significant difference in the proportions of An. arabiensis collected indoors and outdoors, highlighting its ability to adapt its biting behavior. Furthermore, genetic studies have highlighted the strong resilience of these populations to environmental pressures and vector control interventions (31). Finally, the increasing resistance to insecticides, particularly pyrethroids, may also contribute to the persistence and dominance of An. arabiensis in certain areas (32).

In contrast, An. gambiae s.s. and An. coluzzii were more frequently encountered indoors, albeit at lower overall proportions. These species are traditionally considered endophilic and anthropophilic, traits that make them more susceptible to indoor interventions. However, their reduced presence in this study may reflect ecological displacement or behavioral changes in response to sustained vector control pressure, as observed in other West African contexts. The observed species distribution has important implications for malaria control strategies. The occurence of An. arabiensis outdoors underscores the need to complement indoor interventions with approaches targeting outdoor biting, such as larval source management, spatial repellents, or community-based environmental modifications. Moreover, the persistence of An. gambiae s.s. and An. coluzzii indoors suggests that ITNs and IRS remain relevant but may require optimization to address evolving vector behaviors (33).

Molecular resistance observed in An. Arabiensis, although generally less pronounced than in An. gambiae s.s., remains a concern in several regions of Africa, notably in Ethiopia, Sudan, and Zimbabwe, where moderate to high levels of resistance have been documented (34–36). The observation of widespread resistance to pyrethroids particularly permethrin and deltamethrin in our study area confirms a worrying trend already well documented across West and Central Africa. This resistance appears to be primarily driven by metabolic mechanisms and behavioral adaptations such as exophily and zoophily, which reduce exposure to insecticides used in domestic settings. These traits complicate both the detection and management of resistance, especially in areas where pyrethroids were widely deployed through long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) campaigns. Recent studies have confirmed that the efficacy of these insecticides is significantly compromised in regions with high resistance prevalence (32, 37). In response to this situation, it is essential to adopt alternative strategies, such as the use of dual-active nets combining a pyrethroid with a synergist like piperonyl butoxide (PBO), or the deployment of insecticides with distinct modes of action that do not exhibit cross-resistance with conventional compounds (38). Strengthening entomological and molecular surveillance also remains critical for tracking the evolution of resistance markers and adapting interventions in real time. Interestingly, combinations of pyrethroids with the synergist piperonyl butoxide (PBO) specifically PBO + permethrin, PBO + deltamethrin, and PBO + alpha-cypermethrin offer superior efficacy compared to pyrethroid-only formulations expressed by the increased mosquito mortality observed across most sites indicates. PBO functions by blocking the enzymatic activity of detoxification systems, thereby allowing pyrethroids to retain their lethal effect on resistant mosquitoes (39).

At the Sabaribougou site, the markedly improved efficacy of PBO-based formulations suggests a predominant, if not exclusive, role of metabolic resistance mechanisms. This hypothesis is supported by the absence of high-frequency kdr mutations in local populations associated with elevated expression levels of metabolic resistance genes, as documented in similar West African contexts (40). These observations highlight the need for uses of dual-active long-lasting insecticidal nets (LLINs-PBO) in areas where metabolic resistance is dominant. They also reinforce the recommendations of the WHO Global Plan for Insecticide Resistance Management (GPIRM), which advocates for the integration of synergized formulations into vector control programs when metabolic resistance mechanisms are confirmed (21). Finally, these results underscore the need to strengthen molecular and enzymatic surveillance to guide insecticide selection based on local resistance profiles.

Fortunately, tests conducted with organophosphates and carbamates revealed consistently high efficacy for pirimiphos-methyl and bendiocarb, with mortality rates exceeding the 98% threshold recommended by WHO to confirm vector susceptibility (21). This susceptibility suggests that, despite widespread pyrethroid resistance, organophosphates and carbamates remained operationally effective in several entomological settings. Pirimiphos-methyl, in particular, demonstrated strong performance even in areas where kdr and Ace-1 resistance mutations are present at moderate frequencies, highlighting its potential for indoor residual spraying (IRS) campaigns in contexts of multi-resistance (37, 41, 42). Studies conducted in Côte d’Ivoire and Burkina Faso have confirmed that pirimiphos-methyl, when applied to cement or mud walls, maintains residual efficacy for over six months, making it a robust candidate for seasonal interventions (42–45). Similarly, bendiocarb continues to produce high mortality rates in several localities, despite emerging resistance linked to the Ace-1 mutation particularly where its frequency remains below 20% (46, 47). These findings emphasize the importance of diversifying insecticide classes used in vector control programs, prioritizing compounds that remain effective under high selective pressure. Ultimately, a comprehensive understanding of resistance mechanisms and their spatial heterogeneity is critical for optimizing interventions and sustaining the long-term effectiveness of vector control strategies in the face of escalating resistance. As recommended by WHO, plant-derived molecules may also represent alternative tools against resistant mosquito populations (48–51).

As for malaria transmission and implications for vector control, An. arabiensis plays a key role in the local transmission dynamics of malaria. Although less abundant, An. coluzzii exhibits a relatively high infection rate. This level of infectivity suggests an amplified local vectorial capacity, likely driven by strong anthropophilic behavior and ecological conditions favorable to parasite survival (52). Recent studies have shown that An. coluzzii is particularly well adapted to urban and peri-urban environments, with a preference for permanent and polluted larval habitats conditions that may sustain transmission even at low vector densities (54, 55). In contrast, An. gambiae s.s. appears to play a marginal role in this study, with a moderate sporozoite infection rate (SIR) and a limited contribution to the entomological inoculation rate (EIR). This decline may be attributed to ecological replacement dynamics, as observed in the Kou Valley, where An. arabiensis becomes more prevalent at the onset of the dry season. It may also reflect increased selective pressure from the widespread use of insecticide-treated nets and indoor spraying, which differentially affect species within the complex depending on their feeding behavior and chemical susceptibility (56). The spatial heterogeneity of EIRs observed across neighborhoods highlights the importance of local factors in transmission dynamics. Sabaribougou, which shows high infection rates across all three species, may reflect a combination of high vector density, limited coverage of control interventions, and environmental conditions conducive to larval development. Conversely, Farakan and Dogona exhibit lower EIRs despite the presence of infected mosquitoes, possibly due to reduced vector density or better human protection particularly through net usage and reduced access to larval habitats (43). These findings call for a differentiated approach to vector control, one that accounts for species diversity, local ecology, and vectorial capacity. Targeted entomological surveillance and the adaptation of control strategies to the spatial and behavioral specificities of vector populations are essential to enhance intervention effectiveness.

This study highlights a complex entomological situation in the Bobo-Dioulasso region, characterized by high levels of pyrethroid resistance among malaria vectors and heterogeneous transmission dynamics across neighborhoods and vector species. The predominance of Anopheles arabiensis, its ecological and behavioral resilience, and the notable infection of An. coluzzii in certain sites underscore the need for a localized and differentiated approach to vector control. The results confirm the strategic value of PBO-based synergized formulations, which demonstrate enhanced efficacy in contexts of metabolic resistance. Moreover, the continued susceptibility of vectors to organophosphates and carbamates particularly pirimiphos-methyl and bendiocarb offers robust operational alternatives for indoor residual spraying campaigns. In light of rising multi-resistance and persistent transmission in specific areas, it is imperative to strengthen entomological and molecular surveillance, tailor control tools to local resistance profiles, and implement integrated strategies to preserve intervention effectiveness. A nuanced understanding of vector ecology and behavior, combined with rational insecticide management, is essential to contain residual malaria transmission and guide public health policies toward sustainable solutions.