and Motunrayo O. Aina1,2, The rising threat of insecticide resistance in malaria vectors restricts the effectiveness of global malaria control initiatives. The Anopheles gambiae complex is a group of efficient vectors that has demonstrated extensive resistance to copious insecticides, particularly pyrethroids (permethrin). Consequently, alternative insecticides with different mechanisms of action, such as chlorfenapyr, are being applied frequently. Target site mutations, such as knockdown resistance (kdr) mutations (L1014F/S) in the voltage-gated sodium channel gene, along with increased metabolic detoxification mediated by cytochrome P450 monooxygenases, glutathione S-transferases, and esterases, are associated with permethrin resistance in An. gambiae complex. Conversely, chlorfenapyr, a novel insecticide involving metabolic activation, has been introduced as an alternative. Understanding these differential and overlapping resistance mechanisms is vital for strategic deployment of insecticides and designing effective resistance management programs. This review evaluates the susceptibility patterns of An. gambiae complex to both permethrin and chlorfenapyr, an alternative insecticide with a novel mode of action, while emphasizing their molecular resistance mechanisms and implications for malaria vector control. More focus is given to the Interceptor® G2 (IG2) long-lasting insecticidal net, which combines alpha-cypermethrin and chlorfenapyr to enhance the control of resistant mosquito populations. Compared to previous reviews, this paper provides an integrated analysis of the synergetic mechanism of IG2 that circumvents resistance, delay its spread, and revamps the efficacy of malaria interventions. By highlighting recent findings from field trials and molecular studies, this review underscores the need for strategic deployment, resistance surveillance, and policy support to sustain the effectiveness of dual-insecticide tools in endemic regions.
Anopheles gambiae Complex, Insecticide Resistance, Permethrin, Chlorfenapyr, Interceptor G2, Malaria Control, Molecular Mechanisms
Malaria remains a significant health concern in sub-Saharan Africa, where Anopheles mosquitoes serve as key vectors of Plasmodium parasites. It accounts for high rates of morbidity and mortality, contributing to around 94% of global malaria cases and 95% of malaria-associated fatalities worldwide in 2023. Unlike other countries, Nigeria bears approximately 30.9% of the total global malaria mortalities, highlighting the severity of the disease in this region.1 Control of disease-transmitting parasites has improved considerably through the use of vector control and synthetic insecticides such as pyrethroids, coupled with insecticide-treated sprays utilized in IRS and LLINs, owing to their low toxicity to humans and high effectiveness against mosquitoes.2 The main malaria prevention strategy in sub-Saharan Africa involves the use of pyrethroid-treated long-lasting insecticidal nets (LLINs), which have significantly prevented an estimated 1.5 billion cases of malaria and 7.3 million deaths attributed to the disease over the last two decades. However, the decline in the number of cases stagnated after 2015, and certain regions in sub-Saharan Africa experienced increased malaria transmission from 2019 to 2020.3 This resurgence is attributed to sparse coverage of efforts due to the spreading resistance to pyrethroid insecticides among malaria-carrying mosquitoes, coupled with stagnation in malaria control efforts.4 The resurgence that sub-Saharan Africa experienced from the 1980s onward necessitated immediate mitigations.
The Anopheles gambiae complex susceptibility to insecticides affects the control of malaria vectors and determines the effectiveness of LLIN and IRS interventions.5 Susceptibility to pyrethroid permethrin and the newly introduced member of the pyrrole insecticide known as chlorfenapyr can significantly affect malaria transmission, the dynamics of the vector population, and the strategies employed for managing resistance. The effects of these susceptibility patterns extend across multiple domains, including vector control efficacy, resistance evolution, behavioral adaptations, and epidemiological consequences.6
Although pyrethroid resistance in general is a growing concern in malaria vector control, this review specifically focuses on permethrin resistance in the Anopheles gambiae complex. Permethrin, a Type I pyrethroids that act as neurotoxins to mosquitoes has been one of the most exploited insecticide in conventional LLINs across the endemic regions, especially in sub-Saharan Africa. However, commercial use and gross application have led to increased selection pressure and emergence resistance in populations of the Anopheles gambiae complex mosquitoes.7 This focus will facilitate more targeted understanding of these mechanisms, significant for assessing cross-resistance potential with newer tools such as Interceptor® G2 nets and PermaNet® Dual, which incorporate a combination of alpha-cypermethrin and chlorfenapyr as well as chlorfenapyr and deltamethrin. The use of pyrethroids, particularly permethrin insecticides, has been instrumental in the control of malaria vectors because they act as neurotoxins to mosquitoes. However, commercial use and overapplication have led to increased resistance in populations of the Anopheles gambiae complex mosquitoes.7 There is growing interest in promising but unstudied insecticides such as chlorfenapyr due to its different mechanism of action that could address this challenge. Its application in the field, however, also poses potential problems, such as developing resistance if not used judiciously.8
A specific type of pyrethroid, permethrin, induces paralysis and death in mosquitoes through the voltage-gated sodium channel’ (VGSC) in the sodium channel pathway of the nervous system.9 However, target-site mutations and metabolic detoxification mechanisms cause widespread resistance to permethrin, which has been observed in An. gambiae as a result of its extensive use.10 An identified resistance mechanism is the knockdown resistance, which occurs at the VGSC gene. Several single-nucleotide polymorphism mutations, such as L1014F and L1014S decrease the binding affinity of pyrethroids, rendering them ineffective.11
Chlorfenapyr has promise in fighting malaria vectors; a halogenated pyrrole pro-insecticide, it induces energy loss and death in species of concern by inhibiting oxidative phosphorylation through mitochondria.12 As an integrated vector management tool, it is valuable as it has a novel mode of action that minimizes the risk of cross-resistance compared to permethrin. There are also concern of the effect of permethrin resistance mechanisms such as target site mutation or overexpression of detoxifying enzymes in chlorfenapyr.13 Chlorfenapyr was effective against permethrin-resistant mosquitoes when combined with other insecticides.3 Chlorfenapyr-pyrethroid long-lasting insecticidal bed nets (LLINs), including Interceptor® G2 (IG2), were promising in more recent studies, especially in regions where vectors had high resistance to pyrethroids, particularly permethrin.14
These dual-active ingredient nets resulted in a substantial reduction in malaria prevalence compared to traditional pyrethroid-only LLINs.15 The synergistic effect of combining two insecticides with distinct modes of action alpha-cypermethrin (a pyrethroid that targets the mosquito’s nervous system), and chlorfenapyr (a pyrrole compound that inhibits mitochondrial oxidative phosphorylation), enhances efficacy against resistant strains and improves control over insecticide resistance.16 The effectiveness of IG2 bednets is significantly diminished by reducing the potential for spreading resistance to both active components, one of which, similar the other, can develop while simultaneously being used for vector control due to selective pressure from several biological mechanisms. More importantly, these mosquitoes may demonstrate a remarkable capacity to convert chlorfenapyr into its active form, tralopyril, thus inducing resistance, as certain cytochrome P450 enzymes, commonly overexpressed in resistant populations, activate chlorfenapyr.17
Insecticide resistance generally arises from biological mechanisms beyond systematic adaptation, such as metabolic adaptation, behavioural adaptation, or genetic mutation.18 The goal of chlorfenapyr is to enhance its insecticidal effectiveness against resistant populations of Anopheles gambiae. While previous reviews have evaluated insecticide resistance in Anopheles gambiae or studied the individual effects of permethrin and chlorfenapyr, few have integrated their combined implications in the context of dual-treated, long-lasting insecticidal nets including Interceptor® G2. Understanding these resistance pathways is essential not only for evaluating the limitations of current interventions but also for assessing the potential of dual-active IG2 bed nets, which incorporate distinct insecticidal actions to overcome these challenges. This review examines the molecular mechanisms driving the effects of both permethrin and chlorfenapyr against the An. gambiae complex, including the interactions of both insecticides’ metabolites and physiological modulators within the IG2 net, and offers recommendations on optimizing the anti-malaria vector control policy and managing insecticide resistance in Africa.
By addressing these knowledge gaps, this review aims to contribute to creating sustainable, efficient vector control methods and revamps the ongoing reduction of the malaria burden worldwide.
The Anopheles gambiae complex and its role in the spread of malaria
An. gambiae (sensu stricto), An. coluzzii, An. arabiensis, An. merus, An. melas, and An. bwambae are members of the An. gambiae complex. They are physically identical but genetically different.19 Animals from these sibling species exhibit distinctive ecological and behavioural traits, and these differences determine their ability to spread disease and insecticide resistance. An. gambiae s.s., An. coluzzii, and An. arabiensis are the primary malaria vectors due to their anthropophilic and endophilic behaviours.20 An. arabiensis shows remarkable ecological plasticity, feeding on both humans and animals and exhibiting higher exophilic and exophagic tendencies, which reduce its exposure to IRS and LLINs. An. melas and An. merus are saltwater-tolerant species with localized distributions. According to a recent study, Anopheles mosquitoes are expanding their habitats to include contaminated populations, while Anopheles larvae prefer transparent, stagnant water sources that are remote from human regions.21 To implement targeted control strategies, understanding the insecticide resistance of these sibling species is required.
Permethrin resistance in malaria vector control
Permethrin, a type of synthetic pyrethroid insecticide, has historically been among the most effective traditional pyrethroid insecticides due to its long-lasting residual efficacy, low toxicity to mammals, and affordability.22 However, many Anopheles populations have developed resistance because of the extensive and prolonged use of the chemical in large quantities, which threatens the effectiveness of malaria control efforts.23 Permethrin is a commonly used insecticide and repellent that effectively targets numerous arthropod vectors, such as mosquitoes, ticks, flies, and lice (Table 1). It particularly impacts the nervous system by causing sodium ion channels to open, leading to paralysis and death in these insects.24 It demonstrates high efficacy against primary disease vectors, including Anopheles mosquitoes (malaria), Aedes aegypti (dengue, Zika, yellow fever), and Culex mosquitoes (filariasis and West Nile virus).25
Table (1):
Application in Vector Control Programs
Application |
Description |
|---|---|
Insecticide-treated nets (ITNs) |
Permethrin is extensively used to impregnate bed nets to provide dual action: killing mosquitoes upon contact and acting as a physical barrier, significantly reducing mosquito biting rates and disease transmission26 |
Indoor Residual Spraying (IRS) |
It is applied to walls and other indoor surfaces where vector insects rest, effectively controlling endophilic species such as Anopheles gambiae, a primary malaria vector1 |
Clothing and Equipment Treatment |
Permethrin-treated clothing offers protection against ticks, mosquitoes, and other arthropods, which is important for military personnel, travelers, and outdoor workers exposed to vector-borne diseases27 |
Public Health Infrastructures |
Permethrin is employed in aerial and ground-level applications for vector control during disease outbreaks including dengue and Zika24 |
Pattern of permethrin resistance
The pattern of permethrin resistance is a growing concern in the fight against malaria. For decades, traditional insecticide-treated nets (ITNs), especially those impregnated with permethrin, have been a key part of malaria control efforts. Permethrin, a widely used synthetic pyrethroid insecticide, is paramount for global initiatives aimed at controlling mosquitoes causing diseases, including malaria, dengue, Zika, and yellow fever.26 It is a principal component in long-lasting insecticidal nets (LLINs), indoor residual spraying (IRS), and agricultural pest management, helping to significantly reduce malaria rates in many regions. However, the frequent and widespread use of permethrin, without proper resistance management, has inadvertently led to the emergence of resistance in mosquito populations, posing a serious challenge to the effectiveness of these traditional control methods.27
The rise of permethrin resistance is particularly noticeable in areas that have experienced repeated IRS campaigns and long-term distribution of permethrin-treated bed nets.28 Over time, the constant pressure from permethrin exposure has driven evolutionary changes in mosquito populations, increasing the prevalence of resistance alleles and enabling resistant mosquitoes to survive and reproduce. This issue is further exacerbated by the use of agricultural insecticides, particularly in rural areas where crops are treated with insecticides that work similarly to permethrin, as other pyrethroids.29
As these resistance alleles accumulate in mosquito populations, the effectiveness of permethrin-based ITNs begins to decline. The associated mechanisms of this resistance primarily involve mutations in target sites, such as the voltage-gated sodium channels that pyrethroids target, as well as metabolic resistance driven by enzymes, including cytochrome P450 monooxygenases.30 This cross-resistance between permethrin and other pyrethroids, such as deltamethrin and lambda-cyhalothrin, further complicates the situation, limiting the efficacy of these crucial control measures in managing resistant vector populations.31
An additional complication arises from the spread of resistance alleles through gene flow and vector migration. In regions where resistant mutations, such as knockdown resistance, were initially identified in West Africa, these mutations have expanded to East Africa via human and mosquito migrations.32 This geographic spread of resistance complicates efforts to control malaria and other mosquito-borne diseases. The failure of traditional ITNs due to resistance underscores the urgent need for new, innovative solutions to maintain effective malaria control.
Mechanisms of permethrin resistance
Permethrin resistance in malaria vectors arises from various mechanisms. A primary factor is metabolic resistance, where mosquitoes increase the production of detoxifying enzymes such as cytochrome P450 monooxygenases, glutathione S-transferases (GSTs), and carboxylesterases. These enzymes break down or sequester pyrethroids, diminishing their toxicity.33 In resistant populations of Anopheles mosquitoes, cytochrome P450 enzymes, particularly CYP6P3, play a principal role in detoxifying permethrin.34 Another important aspect involves mutations in the voltage-gated sodium channel gene, commonly known as knockdown resistance. These mutations, such as L1014F and L1014S, reduce the binding affinity of permethrin, rendering it less effective.35 The prevalence of knockdown resistance in Africa presents a significant challenge for pyrethroid-based treatments.36 Additionally, there is behavioural resistance, where mosquitoes adapt by avoiding treated areas or resting outdoors to minimize contact with insecticides. Cuticular resistance also contributes, as it involves alterations in the composition of the mosquito cuticle that minimizes the absorption of insecticides. Table 2 consists of various genetic and biochemical mechanisms that contributes to permethrin resistance.
Table (2):
Diverse genetic and biochemical mechanisms contribute to permethrin resistance
Mechanism |
Description |
Key Genes/Proteins |
|---|---|---|
Target Site Insensitivity |
Mutations in voltage-gated sodium channels (VGSC) reduce insecticide binding. |
Knock down resistance (L1014F, L1014S)10 |
Metabolic Resistance |
Overexpression of detoxifying enzymes metabolizes permethrin. |
P450s, GSTs, esterases37 |
Behavioral Resistance |
Altered behaviors minimize contact with insecticides |
Behavioural adaptations10 |
Cuticular Resistance |
A thickened cuticle reduces insecticide penetration |
Cuticle protein genes37 |
Chlorfenapyr: Mode of action and efficacy
Chlorfenapyr is a differential pyrrole insecticide, distinguishing itself from neurotoxic pyrethroids of permethrin. While pyrethroids such as permethrin target the voltage-gated sodium channels in mosquito nerve cells, leading to paralysis and death, chlorfenapyr exhibits a different mechanism as a pro-insecticide.38 It must be activated within the insect, where it is converted into its active form of CL 303268. This active form inhibits mitochondrial respiration by obstructing the cytochrome bc1 complex in the electron transport chain, which halts ATP production, the primary energy source for cells.16 The resulting energy depletion causes paralysis and ultimately leads to the mosquito’s destruction.39
Chlorfenapyr targets cellular respiration rather than neural pathways, making it significantly less vulnerable to the cross-resistance mechanisms that is frequently associated with the application of pyrethroids. For instance, resistance mechanisms, including knockdown resistance, which reduce the effectiveness of pyrethroids by altering sodium channel proteins, along with metabolic resistance that enhances detoxifying enzymes including P450 monooxygenases do not impact chlorfenapyr’s ability to inhibit mosquitoes’ vector capacity.40 Consequently, chlorfenapyr remains highly effective, even against populations of Anopheles gambiae s.l. and Aedes aegypti mosquitoes that have developed resistance to pyrethroids.
One of the standout properties of chlorfenapyr, especially when combined with IG2 nets, is its delayed mortality effect. Unlike permethrin, which kills mosquitoes rapidly upon contact, chlorfenapyr causes delayed mortality, typically occurring within 24 to 72 hours after exposure. This delay enables mosquitoes to continue interacting with their surroundings, including other mosquitoes, for a limited time before they succumb.41 Consequently contributing to a secondary kill effect in which the already exposed mosquitoes can transfer this insecticide to other surfaces or mosquitoes through their interaction.42
This delayed mortality also reduces the chances of behavioural changes avoidance by mosquitoes, an approach with fast-acting insecticides such as permethrin. Mosquitoes are less likely to alter their behaviour in response to exposure to chlorfenapyr, allowing for continued mosquito interactions with treated nets and surfaces before they are destroyed by the insecticide.42
Another prominent benefit of chlorfenapyr is its long-lasting effectiveness. Studies have demonstrated that chlorfenapyr maintains its insecticidal efficacy on treated surfaces for several months, even in the challenging environmental conditions found in malaria-prone regions.41 This extended efficacy ensures that IG2 bed nets can provide ongoing protection against malaria vectors throughout the entire transmission season. The durability of chlorfenapyr not only supports its use in bed nets but also makes it a key player in indoor residual spraying (IRS) programs, where consistent vector control is principal for keeping mosquito populations in check between spray applications.43
Additionally, chlorfenapyr has a low risk of developing resistance, which enhances the long-term effectiveness of IG2 nets. While no insecticide is completely immune to the possibility of resistance development, chlorfenapyr’s potential to target cellular respiration instead of the typical neurotoxic pathways as represented by pyrethroids and other insecticides, makes it significantly less susceptible to the rapid emergence of resistance.44 This low resistance risk positions chlorfenapyr as a component of Integrated Vector Management (IVM) programs, where multiple control methods are integrated to minimize resistance and prolong the effectiveness of interventions.16
Synergistic Potential of Chlorfenapyr in Combination Nets
A promising ally in the battle against insecticide resistance in malaria vector control is chlorfenapyr.45 This compound shows impressive synergistic potential when combined with insecticide-treated nets, increasing the effectiveness of treatments by targeting resistant mosquito populations.46 It is used to address several challenges in malaria vector control. First, it helps fight resistance, particularly since pyrethroid resistance is widespread in Anopheles gambiae and other malaria vectors due to mutations at target sites, such as knockdown resistance, and enhanced detoxification processes.17 Chlorfenapyr’s discrete mode of action sets it apart from pyrethroids, allowing it to work effectively even against resistant mosquito strains.8 Moreover, these synergistic nets are designed to effectively target both resistant and susceptible mosquitoes by combining chlorfenapyr with pyrethroids, enhancing overall vector control. Another advantage is that chlorfenapyr, when used with nets, helps delay the development of resistance. Its distinct mechanisms of action minimizes the selection pressure on pyrethroid resistance alleles, which could slow the emergence of resistance and prolong the effectiveness of pyrethroid-based strategies.14
Furthermore, these combination nets offer extended residual efficacy as chlorfenapyr-treated nets maintain their lethal potency for a longer duration compared to standard pyrethroid-only nets, even after multiple washes. This durability makes them more practical and reliable in malaria-endemic areas, where it is essential to maintain net efficacy for ongoing mosquito control.47
Integration into IG2 net
The Interceptor G2 (IG2) net is specially designed to address the growing problem of insecticide resistance in malaria control.44 Unlike the standard bed nets that are impregnated with only pyrethroids, IG2 nets use a blend of two active ingredients chlorfenapyr and pyrethroid insecticide that disrupts the mosquito’s nervous system, leading to rapid knockdown and death.48 Compared to permethrin another type of pyrethroid, alpha-cypermethrin has shown effectiveness against resistant mosquito populations due to some slight chemical modifications.49 The combination of alpha-cypermethrin and chlorfenapyr in IG2 bednet aims to enhance control over malaria vectors, especially in regions where resistance to pyrethroids, including permethrin, is prevalent.50 Each chemical targets different physiological pathways, creating a powerful synergy. Alpha-cypermethrin focuses on neurotoxic pathways, delivering immediate knockdown and repellent effects against mosquitoes, while chlorfenapyr, acts more slowly by disrupting mitochondrial function, ensures that mosquitoes that survive the initial exposure to alpha-cypermethrin will eventually face mortality, particularly those resistant to pyrethroids.16 This dual-action strategy offers a significant edge in areas with high pyrethroid resistance, providing effective control over both resistant and susceptible mosquito populations.17
In addition to Interceptor® G2, which combines alpha-cypermethrin with chlorfenapyr, studies have shown another dual-active net, PermaNet® Dual, incorporates deltamethrin (a type II pyrethroid) alongside chlorfenapyr.43 This combination similarly targets both the nervous system and mitochondrial pathways of mosquitoes, enhancing efficacy against pyrethroid-resistant Anopheles gambiae populations.
Field studies have demonstrated that IG2 bed nets are highly effective in reducing malaria transmission, even in areas with extensive insecticide resistance. In countries such as Burkina Faso, Cameroon, and Uganda, IG2 nets have been shown to provide long-lasting protection, with research confirming their combined effectiveness in combating resistant mosquito populations.50 The studies highlight that IG2’s dual-action formula is particularly adept at addressing the high levels of resistance found in mosquito populations, especially those that have developed resistance to pyrethroids such as permethrin.44 Remarkably, these bednets remain effective for up to three years, offering extended protection in regions with high malaria transmission rates. These combination approaches significantly reduce mosquito biting rates, which in turn lowers the incidence of malaria.14 The development and deployment of IG2 nets represent a strategic approach to managing resistance, decreasing reliance on single insecticides.44 The dual action of chlorfenapyr and alpha-cypermethrin helps combat this challenge by minimizing dependence on any one class of insecticide.50 This combination not only extends the effectiveness of insecticide interventions but also reduces the risk of resistance developing in malaria vector populations.44
By diversifying the insecticides used, IG2 nets provide resilience against evolving resistance, ensuring that they remain a prominent tool in the fight against malaria. Despite their effectiveness, there are associated challenges that include the costly production of IG2 bednets compared to standard pyrethroid-only nets among others.44 To ensure widespread distribution, especially in resource-limited areas, cost-effective strategies will be essential. Furthermore, while chlorfenapyr has not yet faced significant resistance, its ongoing use could eventually lead to resistance issues.44
Effects of permetehrin and chlorfenapyr susceptibility patterns
Effective vector control and an understanding of insecticide resistance depend on the effects of susceptibility patterns to permethrin and chlorfenapyr in insect populations, particularly in Anopheles gambiae, the primary malaria vector. Table 3 shows the categories and the assocate effects.
Table (3):
Category and the effects of susceptibility patterns to permethrin and chlorfenapyr in insect population
| Category | Effect |
|---|---|
| Impact on Malaria Vector Control Interventions | • Reduced efficacy of LLINs and IRS: Pyrethroid resistance has made LLINs less effective, which raises the risk of malaria transmission and mosquito survival rates. Although introducing nets treated with chlorfenapyr (Interceptor® G2) provides an alternative. However, its long-term effectiveness can be challenged if resistance develops.51 |
| • Dual-insecticide strategies are required: Although vector control strategies have been improved by combining chlorfenapyr with pyrethroids, thus monitoring is necessary to identify early indications of resistance development.52 | |
| Evolution of Resistance Mechanisms | • Genetic adaptations in the Anopheles gambiae complex: kdr mutations and upregulation of metabolic enzymes are well resistance. Although chlorfenapyr resistance mechanisms are still being studied, metabolic alterations and mitochondrial mutations might be involved.53 |
| • Cross-resistance Concerns: Permethrin-resistant mosquitoes may display metabolic pathways that influence their susceptibility to chlorfenapyrs, making vector control initiatives more difficult.54 | |
| Behavioral and Ecological Changes | • Altered feeding and resting behaviour: Resistant mosquitoes avoid treated surfaces by shifting to outdoor resting (exophily) or modifying feeding times, reducing insecticide contact.55 |
| • Shifts in species composition: Selective pressure from insecticide use may prefer species with lower susceptibility, altering vector population structures and potentially increasing malaria transmission.56 |
Molecular interactions between permethrin resistance and chlorfenapyr
Chlorfenapyr exhibit a different mechanism compared to permethrin with regards to circumventing mutations and resistance related to metabolic detoxification. While permethrin is a neurotoxic pyrethroid that targets sodium channels, chlorfenapyr operates as a pro-insecticide that disrupts mitochondrial oxidative phosphorylation. Research has demonstrated its impressive effectiveness against Anopheles populations that show significant resistance to permethrin, particularly in studies using insecticidal nets such as the Interceptor® G2.14 Additionally, in permethrin regions, recent field tests in Tanzania revealed a marked reduction in malaria transmission after the introduction of chlorfenapyr-based nets.57 By targeting the overexpressed P450 enzymes associated with pyrethroid resistance, chlorfenapyr interestingly leverages on the vector’s resistance adaptations for its activation. The molecular interactions between chlorfenapyr and permethrin resistance involve various metabolic and functional pathways. One key factors is cytochrome P450 which is an enzyme that not only contributes to pyrethroid resistance through metabolic detoxification but also activates chlorfenapyr.16 In the presence of P450s, including CYP6M2, chlorfenapyr is converted to its active form more efficiently to effectively target resistance populations thereby turning their resistance mechanisms into vulnerable ones than in susceptible mosquitoes.58 Since chlorfenapyr directly impacts energy metabolism and bypasses the nervous system, it remains effective even in populations with high rates of kdr mutations. This independence ensures that chlorfenapyr maintains its efficacy in these resistant groups.8
Another concern is the risk of adaptive resistance; chlorfenapyr can exploit the overexpression of P450 enzymes, and overuse could lead to resistant strains. Its activation or effectiveness could be compromised by adaptive changes, mutations in mitochondrial target sites, or the downregulation of specific P450 enzymes.16 Due to its differential mechanism of action, chlorfenapyr continues to combat populations that have developed resistance. However, it is crucial to identify and address any emerging resistance to chlorfenapyr.14 Even though chlorfenapyr has proven effective, some mosquito populations are beginning to show reduced susceptibility.8 This could be due to adaptive changes in P450 enzyme activity or mutations in mitochondrial target sites, which might diminish chlorfenapyr’s activation or effectiveness.16 Research indicates that Anopheles gambiae populations across southern Africa exhibit varying levels of susceptibility to chlorfenapyr, highlighting the importance of continuous monitoring.
The interaction between permethrin resistance mechanisms and chlorfenapyr are is crucial for developing effective vector control strategies aimed at reducing malaria transmission.17
Efficacy in permethrin-resistant populations
Chlorfenapyr has proven to be highly effective against malaria vector populations that have developed resistance to pyrethroids, combating one of the biggest hurdles in malaria vector control.59 The mutations that cause target-site and knockdown resistance, which prevent permethrin from binding to voltage-gated sodium channels, do not affect chlorfenapyr, since chlorfenapyr works by disrupting mitochondrial function, a process that operates independently of these resistance mechanisms.57 Additionally, in permethrin-resistant mosquitoes that overproduce cytochrome P450 enzymes which converts the bio-activated chlorfenapyr into its toxic form, making it particularly potent in these resistant populations.60 As a result, chlorfenapyr can effectively manage mosquito populations compared to traditional pyrethroid-based methods.
Potential for combination strategies
The complementary mechanisms of action of permethrin and chlorfenapyr support laudable possibilities of combination strategies consisting of dual-action bed net that combine both permethrin and chlorfenapyr insecticides (Interceptor® G2). This approach effectively targets both permethrin-susceptible and resistant mosquito populations, facilitating a reduction in their survival rates and slowing down the development of resistance.57 Pyrethroids may work synergistically with chlorfenapyr to maximize vector mortality rates in mixed mosquito groups, which could enhance the potential of vector control programs.46 Although chlorfenapyr has shown effectiveness against pyrethroid-resistant mosquitoes, however its effectiveness needs to be managed to sustain its efficacy. Selection pressure could help mosquitoes with P450 variants that can deactivate both pyrethroids and chlorfenapyr. Although there’s limited evidence of this cross-resistance, it highlights the need for resistance surveillance by rotating chlorfenapyr with other insecticides this can minimize the selection pressure and the emergence of new resistance mechanisms.8
Chlorfenapyr’s distinct mode of action presents opportunities and challenges for malaria control efforts. Its efficacy in managing pyrethroid-resistant mosquitoes can restore the effectiveness of long-lasting insecticidal nets (LLINs) and indoor residual sprays (IRS) in areas with significant levels of resistance.14 However, chlorfenapyr formulation, especially those that combine multiple actions, tend to be more expensive than traditional pyrethroid based ones, which can strain budgets in malaria-endemic countries.61
Incorporating chlorfenapyr formulation into the malaria elimination strategies offers several long-term advantages including the significant reduction of mosquito survival, which directly helps limit the spread of Plasmodium parasites.62 By widely using chlorfenapyr combined with existing insecticides, reduces the sole dependence on pyrethroids aiding in the slow the spread of pyrethroid resistance.58
The increasing prevalence of permethrin resistance in malaria vectors, driven by target site mutations and enhanced metabolic detoxification, presents a significant challenge to malaria control and elimination efforts. However, introducing chlorfenapyr as a complementary or alternative insecticide offers a promising solution. Unlike pyrethroids, chlorfenapyr acts through a distinct mechanism involving the disruption of mitochondrial oxidative phosphorylation, making it effective against permethrin-susceptible and resistant mosquito populations.
Notably, the Interceptor® G2 (IG2) bed net, which combines chlorfenapyr and alpha-cypermethrin, has demonstrated substantial efficacy in field studies by addressing both susceptible and resistant vector populations. Its dual-action design leverages the metabolic resistance traits of An. gambiae complex, turning them into vulnerabilities through chlorfenapyr activation.
The molecular interactions between permethrin resistance and chlorfenapyr reveal that the overexpression of P450 enzymes, a hallmark of metabolic resistance, paradoxically enhances chlorfenapyr’s activation and efficacy. This unique relationship allows chlorfenapyr to target resistant populations otherwise unaffected by pyrethroid-based interventions. Furthermore, its lack of dependence on voltage-gated sodium channel function ensures that chlorfenapyr remains effective in mosquitoes harbouring knockdown resistance mutations
The implications for malaria vector control are profound with chlorfenapyr-based interventions, such as dual-action insecticidal nets and indoor residual sprays, which have demonstrated significant potential in reducing malaria transmission in areas burdened by pyrethroid resistance. Nonetheless, the successful integration of chlorfenapyr into vector control strategies requires careful resistance management, routine surveillance, and the adoption of rotational or combination approaches to prevent the emergence of new resistance mechanisms. While chlorfenapyr represents a powerful tool for combating pyrethroid resistance, its long-term efficacy relies on sustainable implementation strategies and continuous investment in developing new insecticides with novel modes of action. Through proactive efforts in resistance monitoring and management, chlorfenapyr could play a pivotal role in maintaining the effectiveness of malaria vector control programs and advancing global malaria elimination goals.
Recommendations
To effectively manage malaria vectors and respond to the growing threat of insecticide resistance in Africa, the African malaria control programs should adopt an Integrated Vector Management (IVM) strategy that combines the use of dual active ingredient long-lasting insecticidal nets (LLINs), such as Interceptor® G2, with indoor residual spraying (IRS) with non-pyrethroid insecticides and larval source management in high-risk breeding locations. This integrated approach reduces the over-reliance on any single intervention and mitigates the emergence of resistance. Additionally, it is important to rotate insecticides and use combinations with different modes of action such as alternations between chlorfenapyr and other insecticide classes (neonicotinoids or organophosphates) across transmission seasons can decrease selection pressure. Deploying combination products of IG2 bed nets that combine chlorfenapyr with alpha-cypermethrin targets multiple resistance mechanisms and enhances intervention efficiency.
ACKNOWLEDGMENTS
The authors would like to thank the Management of Covenant University Centre for Research Innovation and Discovery (CUCRID) for defraying the necessary APC cost and also acknowledge Covenant Applied Informatics and Communication Africa Center of Excellence (CApIC-ACE), Covenant University, Nigeria, for their support.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
AUTHORS’ CONTRIBUTION
Both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.
FUNDING
None.
DATA AVAILABILITY
Not applicable.
ETHICS STATEMENT
Not applicable.
- World Health Organization. WHO Guidelines for Malaria. Geneva: WHO; 2025. Accessed April 28, 2025. https://www.who.int/publications/i/item/guidelines-for-malaria
- Ogunnupebi TA, Oduselu GO, Elebiju OF, Ajani OO, Adebiyi E. In silico studies of benzothiazole derivatives as potential inhibitors of Anopheles funestus and Anopheles gambiae trehalase. Front Bioinform. 2024;4:1428539.
Crossref - Accrombessi M, Cook J, Dangbenon E, et al. Efficacy of pyriproxyfen-pyrethroid long-lasting insecticidal nets (LLINs) and chlorfenapyr-pyrethroid LLINs compared with pyrethroid-only LLINs for malaria control in Benin: a cluster-randomised, superiority trial. Lancet. 2023;401(10375):435-446.
Crossref - Van den Berg H, Velayudhan R, Yadav RS. Management of insecticides for use in disease vector control: Lessons from six countries in Asia and the Middle East. PLoS Negl Trop Dis. 2021;15(4):e0009358.
Crossref - Adedeji EO, Ogunlana OO, Fatumo S, et al. Anopheles metabolic proteins in malaria transmission, prevention and control: a review. Parasitol Vectors. 2020;13(1):465.
Crossref - Shekari A, Enwere M, Adebiyi M, Adebiyi E. Network analysis of the protein-protein interaction of insecticide resistance in Anopheles gambiae. 2024 Proc Int Conf Sci Eng Bus Driving Sustainable Dev Goals (SEB4SDG). 2024:1-13.
Crossref - Kambou SS, Valente A, Agnew P, et al. Non-contact detection of pyrethroids widely used in vector control by Anopheles mosquitoes. PLoS One. 2024;19(7):e0298512.
Crossref - Tchouakui M, Assatse T, Tazokong HR, et al. Detection of a reduced susceptibility to chlorfenapyr in the malaria vector Anopheles gambiae contrasts with full susceptibility in Anopheles funestus across Africa. Sci Rep. 2023;13:2363.
Crossref - Niklas B, Rydzewski J, Lapied B, Nowak W. Toward overcoming pyrethroid resistance in mosquito control: the role of sodium channel blocker insecticides. Int J Mol Sci. 2023;24(12):10334.
Crossref - Saavedra-Rodriguez K, Campbell CL, Lozano S, et al. Permethrin resistance in Aedes aegypti: genomic variants that confer knockdown resistance, recovery, and death. PLoS Genet. 2021;17(6):e1009606.
Crossref - Gong Y, Li T, Li Q, Liu S, Liu N. The central role of multiple P450 genes and their co-factor CPR in the development of permethrin resistance in the mosquito Culex quinquefasciatus. Front Physiol. 2022;12:802584.
Crossref - Jeschke P. Propesticides and their use as agrochemicals. Pest Manag Sci. 2015;72(2)210-225.
Crossref - Huang P, Yan X, Yu B, He X, Lu L, Ren Y. A comprehensive review of the current knowledge of chlorfenapyr: synthesis, mode of action, resistance, and environmental toxicology. Molecules. 2023;28(22):7673.
Crossref - Tchouakui M, Thiomela RF, Nchoutpouen E, et al. High efficacy of chlorfenapyr-based net Interceptor® G2 against pyrethroid-resistant malaria vectors from Cameroon. Infect Dis Poverty. 2023;12(1):81.
Crossref - Barker TH, Stone JC, Hasanoff S, Price C, Kabaghe A, Munn Z. Effectiveness of dual active ingredient insecticide-treated nets in preventing malaria: a systematic review and meta-analysis. PLoS One. 2023;18(8):e0289469.
Crossref - Yunta C, Ooi JMF, Oladepo F, et al. Chlorfenapyr metabolism by mosquito P450s associated with pyrethroid resistance identifies potential activation markers. Sci Rep. 2023;13(1):14124.
Crossref - Gleave K, Guy A, Mechan F, et al. Impacts of dual active-ingredient bed nets on the behavioural responses of pyrethroid resistant Anopheles gambiae determined by room-scale infrared video tracking. Malar J. 2023;22(1):132.
Crossref - Bharadwaj N, Sharma R, Subramanian M, et al. Omics Approaches in Understanding Insecticide Resistance in Mosquito Vectors. Int J Mol Sci. 2025;26(5):1854.
Crossref - Akeju AV, Olusi TA, Simon-Oke IA. Molecular identification and wing variations among malaria vectors in Akure North Local Government Area, Nigeria. Sci Rep. 2022;12(1):7674.
Crossref - Coetzee M, Hunt RH, Wilkerson R, della Torre A, Coulibaly MB, Besansky NJ. Anopheles coluzzii and Anopheles amharicus, new members of the Anopheles gambiae complex. Zootaxa. 2013;3619(3):246-274.
Crossref - Tebamifor ME, Cleanclay WD, Mamudu CO, Ogunlana OO. Surveillance of Wolbachia infection in mosquito species in Ota, Ogun State, Nigeria. Discov Appl Sci. 2025;7(4).
Crossref - Kojima H, Sata F, Takeuchi S, Sueyoshi T, Nagai T. Comparative study of human and mouse pregnane X receptor agonistic activity in 200 pesticides using in vitro reporter gene assays. Toxicology. 2011;280(3):77-87.
Crossref - Machani MG, Nzioki I, Onyango SA, et al. Insecticide resistance and its intensity in urban Anopheles arabiensis in Kisumu City, Western Kenya: Implications for malaria control in urban areas. PLoS One. 2024;19(11):e0303921.
Crossref - Environmental Protection Authority. Classification and Endpoints Science Memo for the Reassessment of Synthetic Pyrethroids – Permethrin (APP203936). Wellington, New Zealand: Environmental Protection Authority; 2022. Accessed April 23, 2025. https://www.epa.govt.nz/assets/Uploads/Documents/Hazardous-Substances/Synthetic-Pyrethroids-consultation/APP203936-Draft-Hazard-classification-and-endpoint-memo-Permethrin-.pdf.
- Danladi C, Goje LJ. Evaluation of insecticides resistance in Culex mosquitoes to DDT, deltamethrin, permethrin and PBO (synergy) in Gombe Metropolis, Nigeria. S Asian J Parasitol. 2024;7(4):401-410.
Crossref - Onen H, Luzala MM, Kigozi S, et al. Mosquito-Borne Diseases and Their Control Strategies: An Overview Focused on Green Synthesized Plant-Based Metallic Nanoparticles. Insects. 2023;14(3):221.
Crossref - Okafor MA, Ekpo ND, Opara KN, et al. Pyrethroid insecticides susceptibility profiles and evaluation of L1014F kdr mutant alleles in Culex quinquefasciatus from lymphatic filariasis endemic communities. Sci Rep. 2023;13(1).
Crossref - Azizi S, Matowo J, Mbewe NJ, et al. Laboratory and semi-field efficacy evaluation of permethrin-piperonyl butoxide treated blankets against pyrethroid-resistant malaria vectors. Sci Rep. 2022;12(1):22166.
Crossref - Urio NH, Pinda PG, Ngonzi AJ, et al. Effects of agricultural pesticides on the susceptibility and fitness of malaria vectors in rural south-eastern Tanzania. Parasit Vectors. 2022;15(1):213.
Crossref - Chen TY, Smartt CT, Shin D. Permethrin resistance in Aedes aegypti affects aspects of vectorial capacity. Insects. 2021;12(1):71.
Crosssref - Moyes CL, Lees RS, Yunta C, et al. Assessing cross-resistance within the pyrethroids in terms of their interactions with key cytochrome P450 enzymes and resistance in vector populations. Parasit Vectors. 2021;14(1):115.
Crossref - Zhang H, Li M, Tan R, et al. Presence of L1014F knockdown-resistance mutation in Anopheles gambiae s.s. from Sao Tome and Principe. Front Cell Infect Microbiol. 2021;11:633905.
Crossref - Muhammad A, Ibrahim SS, Mukhtar MM, et al. High pyrethroid/DDT resistance in major malaria vector Anopheles coluzzii from Niger-Delta of Nigeria is probably driven by metabolic resistance mechanisms. PLoS One. 2021;16(3):e0247944.
Crossref - Kengne-Ouafo JA, Kouamo M, Muhammad A, et al. A single E205D allele of a key P450 CYP6P3 is driving metabolic pyrethroid resistance in the major African malaria vector Anopheles gambiae. bioRxiv. 2024.
Crossref - Samake JN, Yared S, Getachew D, et al. Detection and population genetic analysis of kdr L1014F variant in eastern Ethiopian Anopheles stephensi. Infect Genet Evol. 2022;99:105235.
Crossref - Hemingway J, Ranson H, Magill A, et al. Averting a malaria disaster: Will insecticide resistance derail malaria control? Lancet. 2016;387(10029):1785-1788.
Crossref - Rahman RU, Souza B, Uddin I, et al. Insecticide resistance and underlying target-site and metabolic mechanisms in Aedes aegypti and Aedes albopictus from Lahore, Pakistan. Sci Rep. 2021;11(1):83465.
Crossref - Ngufor C, Fongnikin A, Hobbs N, et al. Indoor spraying with chlorfenapyr (a pyrrole insecticide) provides residual control of pyrethroid-resistant malaria vectors in southern Benin. Malaria Journal. 2020;19:249.
Crossref - Chien S, Chien S, and Su Y. A fatal case of chlorfenapyr poisoning and a review of the literature. J Int Med Res. 2022;50(9):3000605221121965.
Crossref - Boussougou-Sambe ST, Djida Y, Doumba-Ndalembouly AG, et al. Resistance of Anopheles gambiae s.s. against commonly used insecticides and implication of cytochrome P450 monooxygenase in resistance to pyrethroids in Lambarene (Gabon). BMC Infect Dis. 2024;24(1):1221.
Crossref - Che-Mendoza A, Gonzalez-Olvera G, Medina-Barreiro A, et al. Efficacy of targeted indoor residual spraying with the pyrrole insecticide chlorfenapyr against pyrethroid-resistant Aedes aegypti. PLoS Negl Trop Dis. 2021;15(10):e0009822.
Crossref - Kweyamba PA, Hofer LM, Kibondo UA, et al. Sub-lethal exposure to chlorfenapyr reduces the probability of developing Plasmodium falciparum parasites in surviving Anopheles mosquitoes. Parasit Vectors. 2023;16(1):342.
Crossref - Ngufor C, Fongnikin A, Hobbs N, et al. Indoor spraying with chlorfenapyr (a pyrrole insecticide) provides residual control of pyrethroid-resistant malaria vectors in southern Benin. Malar J. 2020;19(1):249.
Crossref - Mbewe NJ, Rowland MW, Snetselaar J, et al. Efficacy of bednets with dual insecticide-treated netting (Interceptor® G2) on side and roof panels against Anopheles arabiensis in north-eastern Tanzania. Parasit Vectors. 2022;15(1):326.
Crossref - Bohmert AL, Logan RAE, Portwood NM, Hartke J, Ingham VA. A descriptive review of next-generation insecticide-treated bed nets for malaria control. Front Malar. 2024;2:1337572.
Crossref - Ngufor C, Critchley J, Fagbohun J, N’Guessan R, Todjinou D, Rowland M. Chlorfenapyr (a pyrrole insecticide) applied alone or as a mixture with alpha-cypermethrin for indoor residual spraying against pyrethroid resistant Anopheles gambiae sl: an experimental hut study in Cove, Benin. PLoS One. 2016;11(9):e0162210.
Crossref - World Health Organization. Vector Control Products Prequalification Programme. WHO; accessed April 22, 2025. https://extranet.who.int/prequal/vector-control-products
- Assenga AA, Ahoua Alou LP, Camara S, et al. PRONet Duo insecticide-treated net incorporated with chlorfenapyr and bifenthrin is superior to Interceptor® G2 nets against pyrethroid-resistant Anopheles gambiae sensu lato: a randomized experimental hut trial in Côte d’Ivoire and Tanzania using non-inferiority design. Front Malar. 2025;3.
Crossref - Menze BD, Tchouakui M, Mugenzi LMJ, et al. Marked aggravation of pyrethroid resistance in major malaria vectors in Malawi between 2014 and 2021 is partly linked with increased expression of P450 alleles. BMC Infect Dis. 2022;22(1):660.
Crossref - Sovegnon PM, Akoton R, Stopard IJ, et al. Efficacy of Interceptor G2, Royal Guard and PermaNet 3.0 against pyrethroid-resistant Anopheles gambiae s.l. from Za-Kpota, southern Benin: an experimental hut trial. Parasit Vectors. 2024;17(1):300.
Crossref - Adoha CJ, Sovi A, Yovogan B, et al. Efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr long-lasting insecticidal nets (LLINs) for the control of non-Anopheles mosquitoes: Secondary analysis from a cluster randomised controlled trial (cRCT). Insects. 2023;14(5):417.
Crossref - Pryce J, Medley N, Choi L. Indoor residual spraying for preventing malaria in communities using insecticide-treated nets. Cochrane Database Syst Rev. 2022;2022(1):CD012688.
Crossref - Keita M, Sogoba N, Kane F, et al. Multiple resistance mechanisms to pyrethroids insecticides in Anopheles gambiae sensu lato population from Mali, West Africa. J Infect Dis. 2021;223(12 Suppl 2):81-90.
Crossref - Sagbohan HW, Kpanou CD, Osse R, et al. Intensity and mechanisms of deltamethrin and permethrin resistance in Anopheles gambiae s.l. populations in southern Benin. Parasit Vectors. 2021;14(1):202.
Crossref - Zhou G, Yu L, Wang X, et al. Behavioral response of insecticide-resistant mosquitoes against spatial repellent: A modified self-propelled particle model simulation. PLoS One. 2020;15(12):e0244447.
Crossref - Sanou A, Nelli L, Guelbeogo WM. Insecticide resistance and behavioral adaptation as a response to long-lasting insecticidal net deployment in malaria vectors in the Cascades region of Burkina Faso. Sci Rep. 2021;11:17569.
Crossref - N’Guessan R, Camara S, Rowland M, et al. Attractive targeted sugar bait: The pyrrole insecticide chlorfenapyr and the anti-malarial pharmaceutical artemether-lumefantrine arrest Plasmodium falciparum development inside wild pyrethroid-resistant Anopheles gambiae s.s. mosquitoes. Malar J. 2023;22(1):344.
Crossref - Martin J. Efficacy and durability (insecticidal and physical) of next generation insecticide-treated nets against pyrethroid resistant malaria vectors in Tanzania: a multi-faceted study [dissertation]. London, UK: London School of Hygiene & Tropical Medicine; 2024.
Crossref - Zoungbedji DM, Padonou GG, Konkon AK, et al. Assessing the susceptibility and efficacy of traditional neurotoxic (pyrethroid) and new-generation insecticides (chlorfenapyr, clothianidin, and pyriproxyfen) on wild pyrethroid-resistant populations of Anopheles gambiae from southern Benin. Malar J. 2023;22(1):245.
Crossref - Oliver SV, Kaiser ML, Wood OR, Coetzee M, Rowland M, Brooke BD. Evaluation of the pyrrole insecticide chlorfenapyr against pyrethroid resistant and susceptible Anopheles funestus (Diptera: Culicidae). Trop Med Int Health. 2010;16(3):289.
Crossref - Mosha JF, Kulkarni MA, Lukole E, et al. Effectiveness and cost-effectiveness against malaria of three types of dual-active-ingredient long-lasting insecticidal nets (LLINs) compared with pyrethroid-only LLINs in Tanzania: a four-arm, cluster-randomised trial. Lancet. 2022;399(10331):1227-1241.
Crossref - Syme T, N’dombidjי B, Todjinou D, Ariori V, Ngufor C. Laboratory evaluation of the regeneration time, efficacy and wash-resistance of PermaNet Dual (a deltamethrin-chlorfenapyr net) against susceptible and pyrethroid-resistant strains of Anopheles gambiae sensu lato. PLoS One. 2024;19(8):e0298513.
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