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Impact of dual active ingredients long-lasting insecticidal nets on the genetic structure of insecticide resistant populations of Anopheles gambiae in Southern Benin

Malaria Journal volume 24, Article number: 72 (2025) Cite this article

Abstract

Background

Insecticide resistance amongst vector populations is a major challenge, exacerbated by the continued use of the same active ingredients. The present study assessed the impact of long-lasting insecticidal nets (LLINs) bi-treated with chlorfenapyr-alphacypermethrin (PY-CFP LLIN) or pyriproxyfen-alphacypermethrin (PY-PPF LLIN) on the genetic structure of resistant populations of Anopheles gambiae in 60 clusters divided into three arms from three districts in southern Benin.

Methods

The study was conducted between September 2019 and October 2021 in 123 villages grouped in 60 clusters. Mosquitoes were collected indoors and outdoors using human landing catches (HLCs) in 4 households in each cluster every 3 months. After morphological identification, a subsample of An. gambiae sensu lato (s.l.) was analysed by PCR to detect the molecular species and the presence of L1014F vgsc-kdr and G119S-ace-1 mutations.

Results

Anopheles coluzzii (56.9%) and An. gambiae sensu stricto (s.s.) (42.8%), with a few hybrids (0.2%), were identified within 4242 samples of An. gambiae tested. The frequency of L1014F vgsc-kdr decreased in An. coluzzii collected both indoors and outdoors locations in the PY-CFP LLIN and PY-PPF LLIN arms post-intervention compared to baseline. In An. gambiae, the frequency of the L1014F allele decreased in year one but increased above baseline in year 2. In both species, the allelic frequency of G119S-ace-1 was < 10%. For L1014F vgsc-kdr, the fixation index was positive (FIS > 0) in both species. However, it was negative (FIS < 0) for the presence of G119S-ace-1. Weak genetic differentiation, especially in the PY-PPF LLIN and PY-CFP LLIN arms (FST ≤ 0.05), was observed in An. gambiae s.s. populations with L1014F vgsc-kdr, while it was generally higher for both species with G119S-ace-1.

Conclusion

The frequency of the L1014F vgsc-kdr resistance allele was high, while that of the G119S-ace-1 allele was low throughout the study period. Consistent changes in allele frequencies were not observed in any of the treatment arms suggesting that the pyrethroid component of dual AI (active ingredients) nets continues to select for the resistant allele and there is little if any evidence that the non-pyrethroid insecticide selects for the wild-type kdr allele.

Background

Global progress in the fight against malaria has remained stable in recent years due to increased resistance of mosquitoes to insecticides and parasites to treatments, as well as insufficient funding for programmes to combat the disease [1]. Most insecticides used in public health vector control are neurotoxic to mosquitoes [2]. Pyrethroids and organochlorines target receptors in the axons of neurons, while carbamates and organophosphates interfere with acetylcholinesterase, an enzyme involved in synaptic transmission [3, 4]. Studies conducted in Benin have confirmed widespread vector resistance to pyrethroids (alpha-cypermethrin, permethrin and deltamethrin) used to treat long-lasting insecticidal nets (LLINs) [5,6,7]. As a result, the effectiveness of LLINs in areas of high vector resistance has been declining [8].

Resistance by modification of the insecticide target site has been observed in Anopheles gambiae sensu lato (s.l.), mediated by genetic mutations (vgsc-1014F, vgsc-1570Y and vgsc-402L) in voltage-gated sodium channels (vgsc) and ace1-119S mutations in the acetylcholinesterase gene (ace-1) [9, 10]. In addition, resistance can be mediated by metabolic mechanisms, which result in increased activity levels of enzymes involved in insecticide degradation [11]. In Benin, these different resistance mechanisms have been reported in several agro-ecological zones [12,13,14,15].

In view of this situation, new molecules with different modes of action are essential to overcome insecticide resistance mechanisms in mosquitoes [16]. A first attempt to manage insecticide resistance caused by nets was the development of insecticide-treated nets (ITNs) that incorporated a pyrethroid and a synergist, piperonyl butoxide (PBO), which has been shown to increase vector mortality with resistance involving overexpression of mono-oxygenases [17, 18]. However, the effectiveness of these ITNs depends on the extent to which mono-oxygenase enzymes are driving resistance in vector populations [19]. In Benin, the addition of the synergist PBO did not fully restore the sensitivity of vectors to pyrethroids in certain localities [20]. Recent research exploring other insecticide classes has identified bi-treated LLINs as a promising option for vector control [21,22,23]. In addition to pyrethroids, these nets are treated with either pyriproxyfen or chlorfenapyr. Chlorfenapyr, a pyrrole, acts by disrupting ATP (adenosine triphosphate) production in the mitochondria via its oxidizing compound (tralopyril: CL303628). This disruption of oxidative phosphorylation leads to the insect’s death [24]. Pyriproxyfen is a growth regulator, a juvenile hormone analogue known to disrupt female reproduction and egg fertility, as well as larval development in insects [25, 26].

Given the mode of action of the new ingredients (chlorfenapyr and pyriproxyfen) contained in these LLINs, they may exert resistance selection pressure in vectors. For example, in Mali, Norris et al. [27] showed an increase in the frequency of the L1014F vgsc-kdr gene in Anopheles coluzzii after intensive use of LLINs. In certain regions of Benin where the insecticide pirimiphos-methyl was used for indoor residual spraying (IRS), putative resistance was subsequently observed, leading to its replacement by the insecticide clothianidin [28].

For prospective insecticide resistance management strategies to succeed, there needs to be a clear understanding of the specificity of resistance mechanisms to individual insecticides, the likelihood of selecting for cross-resistance mechanisms and the impact of intervention deployment on population gene flow and genetic diversity. In 2020, Interceptor G2 (pyrethroid-chlorfenapyr LLIN) and Royal Guard (pyrethroid- pyriproxyfen LLIN) were distributed in Benin to protect populations in the Cove-Ouinhi-Zangnanado (CoZO) health zone as part of a cluster randomised controlled trial (RCT) in Southern Benin. The present study aimed to evaluate the impact of these nets on the genetic structure of insecticide resistant populations of An. gambiae s.l.

Methods

Study area

The study was nested in a RCT that was carried out in the CoZO health zone, Zou department of Southern Benin [29]. In this region, the malaria prevalence is very high, with a peak of cases between May and October [30]. The main income-generating activities were agriculture, trade, fishing and hunting. LLINs, which are distributed nationwide every 3 years, are the main means of protection against mosquito bites in the region. The CoZO health zone comprises 123 villages with a population of around 220,000. It was grouped into 60 clusters assigned to three study arms: Interceptor LLINs (LLINs treated with pyrethroid only; control arm; PY LLINs), Interceptor G2 (LLINs bi-treated with pyrethroid-chlorfenapyr; PY-CFP LLINs) and Royal Guard (LLINs bi-treated with pyrethroid-pyriproxyfen; PY-PPF LLINs). Each cluster (Fig. 1) comprised an average of 200 households for 1200 residents.

Fig. 1
figure 1

Map of the study area. PY LLIN: standard LLIN, LLIN treated with pyrethroid only; PY-CFP LLIN: LLIN bi-treated with pyrethroid-chlorfenapyr; PY-PPF LLIN: LLIN bi-treated with pyrethroid-pyriproxyfen

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Mosquito collection and morphological identification

Adult mosquitoes were collected in all clusters over three collection rounds [from September to October in 2019 (baseline), September–October 2020 and September–October 2021 (post-LLIN distribution)], using human landing catches (HLCs), i.e. three collections per arm (20 clusters/arm). In each cluster, four houses located approximately 15–20 m apart were selected from the survey census database organized in 2019. In each house, two collectors (1 inside and 1 outside) were used from 7 pm to 1am, and two others from 1 to 7am. A total of 2880 collectors were used in 720 households in this study. Anopheles mosquitoes collected were identified morphologically using the taxonomic identification key of Coetzee [31].

Molecular analyses

A subsample of the An. gambiae complex collected indoors and outdoors was randomly selected. The heads and thoraxes of each of the An. gambiae complex were used to detect infection with Plasmodium falciparum sporozoites by ELISA-CSP [32]. Their abdomens, legs and wings were used for species identification using the PCR protocol of Santolamazza et al. [33]. The genotypes of the L1014F vgsc-kdr and G119S-ace-1 mutations were determined in species of the An. gambiae complex following the protocols of Martinez-Torres et al. [11]; and Weill et al. [34], respectively.

Statistical analysis

Data were entered twice into databases designed with CS Pro 7.2 software and analysed with Stata 15.0 (Stata Corp., College Station, TX). The genetic make-up of the An. gambiae complex was determined by calculating the allelic frequencies of the L1014F vgsc-kdr and G119S-ace-1 mutations. The proportion of each allelic frequency was obtained using the binomial test function in R software version 4.3.2. A Chi-square test was used to assess the difference in the frequencies of infection between resistant and susceptible alleles. The level of significance was set at 0.05.

In the genetic analyses, sub-populations were assigned according to the different types of nets distributed (study arm). These included the PY LLIN sub-population, where standard nets were distributed, the PY-PPF-LLIN sub-population, where pyriproxyfen-incorporated nets were distributed, and the PY-CFP-LLIN sub-population, where chlorfenapyr-incorporated nets were distributed. Panmixia within An. gambiae complex populations in the different study arms was verified using the Hardy–Weinberg equilibrium (HWE) test. Indices of observed heterozygosity (Ho), expected heterozygosity (He), fixation index (FIS) and genetic differentiation (FST) within An. gambiae populations were calculated according to the formulas of Weir and Cockerham [35] and Robertson and Hill [36], integrated into Genepop software version 8.4.2. The fixation index (FIS) was used to quantify divergence from panmixia, where a FIS value < 0 indicates an excess of heterozygosity, while FIS > 0 indicates a deficit of heterozygosity. The variation in FIS ranges from −1, then all loci are heterozygous for the same alleles, to + 1 if all loci are homozygous for different alleles. Similarly, FIS = 0 means that allele frequencies conform to the expectations of HWE. The criteria defined by Hartl et al. [37] were used to assess genetic differentiation within populations, classifying it as weak (FST ≤ 0.05), moderate ([0.05–0.15]), significant ([0.15–0.25]), or highly significant (FST > 0.25). These parameters were compared before and after the nets were deployed in the different study arms.

Results

Anopheles species composition

A total of 29,470 mosquitoes belonging to six different anopheline complexes were collected in the study area. The An. gambiae complex accounted for 88.9% of the total Anopheles collected. There were significantly higher proportions of An. gambiae indoors (55.9%, n = 14,617, 95% CI: 55.2–56.5) versus outside (44.1%, n = 11,549, 95% CI: 43.5–44.7); p < 0.0001. A similar trend was observed for Anopheles funestus [68.2% (n = 396, 95% CI: 64.2–71.9) indoors versus 31.8% (n = 185, 95% CI: 28.1–35.8) outdoors; p < 0.0001]; although this group was collected at comparatively low proportions. Other Anopheles species, namely Anopheles ziemanni, Anopheles pharoensis, Anopheles nili and Anopheles brohieri, were also found in low proportions (≤ 4%) both indoors and outdoors (Fig. 2).

Fig. 2
figure 2

Anopheles species composition

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Allelic frequency of the L1014F vgsc-kdr mutation in An. coluzzii and An. gambiae sensu stricto

Of the 4242 An. gambiae s.l. specimens subjected to molecular analysis, two species, An. coluzzii (56.9%, n = 2423) and An. gambiae sensu stricto (s.s.) (42.8%, n = 1819), and a few hybrids (0.2%, n = 10) (An. gambiae/coluzzii) were identified. Indoors, An. coluzzii predominated (55.9%, n = 1434, CI 95%: 54.0–57.9) over An. gambiae s.s. (44.1%, n = 1128, CI 95%: 42.1–45.9), p < 0.0001). The same trends were observed outdoors.

Of a total of 1,797 specimens analysed during the baseline, 968 were An. coluzzii, distributed as follows: 34% (n = 333) in the PY LLIN arm, 35% (n = 341) in the PY-PPF LLIN arm and 31% (n = 294) in the PY-CFP LLIN arm. The remaining 829 specimens were An. gambiae s.s., distributed as follows: 32% (n = 267) in the PY LLIN arm, 31% (n = 252) in the PY-PPF LLIN arm and 37% (n = 310) in the PY-CFP LLIN arm. In the post-intervention (Post1 and Post2), a total of 2,443 specimens were analysed. Of these, 1,455 were An. coluzzii, distributed as follows: 34% (n = 500) in the PY LLIN arm, 33% (n = 484) in the PY-PPF LLIN arm and 33% (n = 471) in the PY-CFP LLIN arm. The remaining 988 specimens were An. gambiae s.s., with 35% (n = 343) in the PY LLIN arm, 27% (n = 269) in the PY-PPF LLIN arm and 38% (n = 376) in the PY-CFP LLIN arm.

In An. coluzzii, the frequency of the L1014F vgsc-kdr allelic indoors and outdoors after distribution of study LLINs was lower compared with baseline although none of the comparisons were statistically significant (Fig. 3). After distribution of study LLINs, the L1014F vgsc-kdr allelic frequency ranged from 74.6% (95% CI 68.6–79.8) in the PY-CFP LLIN arm to 81.7% (95% CI 77.1–85.6) in PY LLIN arm indoors and 72.4% (95% CI 66.1–77.9) in the PY-CFP LLIN arm to 83.5% (95% CI 77.1–88. 4) in the PY LLIN arm outdoors; at baseline the L1014F vgsc-kdr allelic frequency ranged from 82.2% (95% CI 78.2–85.7) in the PY LLIN arm to 86.9% (95% CI 82.7–90.2) in the PY-CFP LLIN arm indoors; and from 82.1% (95% CI 76.6–86.6) in the PY-PPF LLIN arm to 87.6% (95% CI 82.7–91.3) in the PY LLIN arm outdoors.

Fig. 3
figure 3

Allele frequencies of the L1014F vgsc-kdr mutation in species of the An. gambiae complex. PY LLIN: standard LLIN, LLIN treated with pyrethroid only; PY-CFP LLINs: LLIN bi-treated with pyrethroid-chlorfenapyr; PY-PPF LLIN: LLIN bi-treated with pyrethroid-pyriproxyfen; Post1: 1st year post-intervention; Post2: 2nd year post-intervention

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In An. gambiae s.s., there was a decrease in L1014F vgsc-kdr frequencies compared to baseline (Fig. 3) in the first post-intervention year. In contrast, an increase in L1014F vgsc-kdr frequencies were detected indoors and outdoors during the second post-intervention year in all arms compared with baseline (Fig. 3).

Allele frequency of the G119S-Ace-1 mutation in An. coluzzii and An. gambiae s.s.

G119S-ace-1 allele frequencies were generally low in all three study arms, whether baseline or post-intervention (Fig. 4), ranging from 0.36% (95% CI 0.02–2.57) to 8.33% (95% CI 4.29–15.17). In An. coluzzii, despite the generally low frequency, a decrease in G119S-Ace-1 allele frequency was observed both indoors and outdoors after one year after distribution of LLINs in all study arms compared with baseline. But two years after LLIN distribution, there was a general increase in frequency. These variations were not significantly different (p > 0.05). Similar trends were obtained for An. gambiae s.s with a decrease in the first year then an increase in the second year post-intervention compared to baseline in all study arms (Fig. 4).

Fig. 4
figure 4

Allele frequency of the G119S-ace-1 mutation in species of the An. gambiae complex. PY LLIN: standard LLIN, LLIN treated with pyrethroid only; PY-CFP LLINs: LLIN bi-treated with pyrethroid-chlorfenapyr; PY-PPF LLIN: LLIN bi-treated with pyrethroid-pyriproxyfen; Post1: 1st year post-intervention; Post2: 2nd year post-intervention

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Genotypic and allele frequency of L1014F vgsc-kdr and HWE deviations of An. gambiae s.s. and An. coluzzii populations

Resistant allele frequencies were very high (over 74%) at baseline and post-intervention in the different study arms. Indoors, within the L1014F vgsc-kdr locus, the frequency of homozygous resistant (RR) individuals was predominant in both species, with the highest peak observed at two years post-intervention in An. gambiae s.s. from the PY-CFP LLIN arm (95.6%). Heterozygous resistant (RS) individuals were present at moderate frequencies, especially in An. coluzzii in the different arms. Homozygous susceptible individuals (SS) were found at very low frequencies (Table 1). Significant deviations from HWE (p < 0.05), were observed in An. coluzzii populations (both baseline and post-intervention) except in the PY-PPF LLIN arm at two years post-intervention. However, fewer An. gambiae s.s. exhibited significant deviations from HWE except at one-year post-intervention in all arms and in the PY-CFP LLIN arm at baseline. (Table 1).

Table 1 Genotypic and allelic frequencies of L1014F vgsc-kdr and HWE deviations of indoor collected An. gambiae s.s and An. coluzzii populations
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Similar trends were obtained in outdoor vector populations (An. coluzzii and An. gambiae s.s.) at baseline and post-intervention in the three arms (Supplementary file, Table S1). Indoors and outdoors, the frequency of L1014F vgsc-kdr was similar between the intervention arms (PY-CFP LLIN arm and PY-PPF LLIN arms) and the PY-LLIN arm in both An. coluzzii and An. gambiae s.s. (Supplementary file, Table S2).

Genotypic and allelic frequencies of G119S-ace-1 and HWE deviations of An. gambiae s.s. and An. coluzzii populations

Allelic frequencies of the resistant G119S-Ace-1 mutation were very low in the three different arms study at baseline and post-intervention. Indoor, within the G119S ace-1 locus, the frequencies of homozygous susceptible individuals (SS) were much higher in both species. Resistant heterozygous (RS) individuals were less represented, especially at the first-year post-intervention (ranging from 0.7% to 3.6%). No homozygous resistant individuals were observed at any time point (Table 2). Similar trends were obtained outside (Supplementary file, Table S3). There were no significant deviations from HWE in any population, except in An. coluzzii from the PY-CFP LLIN arm at the first-year post-intervention (Table 2). Indoors, no significant difference was observed between the frequency of G119S-Ace-1 of the intervention arms (PY-CFP LLIN arm and PY-PPF LLIN arms) and that of the PY-LLIN arm in both An. coluzzii and An. gambiae s.s. The same trend was observed outdoors (Supplementary file, Table S4).

Table 2 Genotypic and allelic frequencies of G119S-Ace-1 and HWE test indoor of An. gambiae s.s. and An. coluzzii populations
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Fixation index in An. gambiae s.s. and An. coluzzii

Table 3 shows the fixation index (FIS) in An. gambiae s.s and An. coluzzii populations for L1014F-vgsc-kdr and G119S-ace-1 mutations in different study arms indoors and outside dwellings.

Table 3 Fixation index (FIS) in An. gambiae s.s. and An. coluzzii
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In all An. coluzzii populations possessing the L1014F vgsc-kdr resistance allele, the FIS values obtained were positive (FIS > 0), showing a heterozygosity deficit that was observed before and after distribution of the LLINs to different study arms. Similar results were observed in An. gambiae s.s. populations except in the PY LLIN and PY-CFP LLIN arms post-intervention, where FIS values were negative (FIS < 0) signalling an excess of heterozygosity in these subpopulations. However, Ho was lower within An. coluzzii subpopulations compared with He indoors and outdoors in the different study arms before LLIN distribution (Table S1). In the two years after the intervention, a comparable trend was observed. In An. gambiae s.s., before LLIN distribution, similar trends were obtained where Ho was also lower than He. After the intervention, Ho was slightly higher than He (Supplementary file, Table S5).

However, in all An. coluzzii populations possessing the G119S ace-1 resistance allele, the FIS values obtained were negative (FIS < 0), showing an excess of heterozygosity observed before and after the study LLIN distributions. Similar results were observed in An. gambiae s.s. populations. However, the FIS = 0 obtained mainly in the subpopulation of the PY-CFP LLIN arm suggests a lack of difference between observed and expected heterozygosity and was consistent with HWE (Table 5). Ho and He values before and after intervention in An. coluzzii subpopulations are very close, indicating agreement with panmictic expectations. Similar trends were obtained in An. gambiae s.s. subpopulations (Supplementary file, Table S6).

Genetic differentiation in species of the An. gambiae complex

Differentiating between individuals before and after the distribution of LLINs, we observed a generally low genetic differentiation in indoor populations of An. gambiae s.s. with L1014F vgsc-kdr mutations, especially in the PY-PPF LLIN and PY-CFP LLIN arms (FST ≤ 0.05). By comparison, outside, genetic differentiation (FST) was variable except in the PY-PPF LLIN arm where it was low (Table 4).

Table 4 Genetic differentiation in species of the An. gambiae complex
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With G119S-ace-1, genetic differentiation was predominantly high (either FST = [0.15–0.25], or FST > 0.25) in both species (An. gambiae s.s. and An. coluzzii) except for subpopulations in the PY-CFP LLIN arm where they were low at year one post-intervention indoors. Similarly, in An. gambiae s.s., a low genetic differentiation was observed during the second year in the PY-PPF and PY-CFP LLIN arms (Table 4).

Plasmodium falciparum infection of vectors harbouring the L1014F-vgsc-kdr mutation

Overall, out of 2780 mosquitoes whose head-thoraxes were tested for P. falciparum infection, 93 mosquitoes were found to be positive. In An. gambiae s.l., the infection rate (IR) was 3.7% (95%CI: 2.8–4.7, 61/1659) indoors and 2.9% (95%CI: 1.9–3.9, 32/1138) outdoors (p = 0.25). Indoors, the IR was 4.8% (95%CI: 3.5–6.7, 36/743) in An. gambiae s.s. against 2.7% (95%CI: 1.8–4.1, 25/916) in An. coluzzii (p = 0.03). Outdoors, the IR was 3.9% (95%CI: 2.4–6.2, 18/464) in An. gambiae s.s. versus 2.1% (95%CI: 1.2–3.5, 14/674) in An. coluzzii (p = 0.1).

In An. coluzzii, there was no association between P. falciparum infection and genotype (p > 0.05 for all comparisons) in all study arms, with the exception of the 1st year following the intervention when “SS’’ genotypes were significantly more likely to be infected (Table 5). Similarly, no association (p > 0.05 for all comparisons) was observed in An. gambiae s.s. collected either indoors and outdoors (Table 5).

Table 5  Genotypic infection of L1014F-vgsc-kdr by P. falciparum in Anopheles species
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Discussion

The present study provides information on the genetic diversity of An. gambiae s.l. populations in the communes of Covè-Zagnanado-Ouinhi where two types of bi-treated LLINs were distributed.

Of note, the present study was performed as part of a large randomized controlled trial during which, over the two first years, both PY-CFP LLIN and PY-PPF LLIN reduced significantly the indoor entomological inoculation rate (EIR) by 66% (p = 0.0005) and 58% (p = 0.0028) respectively, while only PY-CFP LLIN significantly reduced the outdoor EIR by 70% (0.0035) [38]. Moreover, both PY-CFP LLIN and PY-PPF LLIN were found to perform similarly on the density of the two primary vectors (An. gambiae s.s. and An. coluzzii) as compared to PY LLIN [39]. In all the three study arms, there was a significant decrease in pyrethroid resistance intensity in An. gambiae s.l. in the first-year compared to the baseline, while a significant increase was observed in the second year compared to the first one [40].

Overall, findings of the present study revealed that the frequency of the L1014F vgsc-kdr resistance allele in the two molecular species remained high (over 70%) in the three study arms, pre- and post-intervention. Also, similar kdr frequencies were observed between the PY-PPF LLIN and PY-CFP- LLIN arms and the PY LLIN arm (control arm) in both molecular species and over years (Supplementary file, Table S2). A decrease in this frequency after the intervention was observed, which is not a common phenomenon as the opposite trend has occurred in several settings and was attributed to either the pressure of insecticide contamination in the soil after agricultural practices [41], or the use of ITNs [42,43,44]. This trend suggests the possibility of interactions between pyrethroids and the new active ingredients (chlorfenapyr and pyriproxyfen), though cross-resistance seems unlikely.

The frequency of the G119S-ace-1 allele was low in both species. This is not unexpected, as this mutation is associated with resistance to carbamates and organophosphates [34, 45], which have not been deployed in the study area. Moreover, none of the new chemicals (chlorfenapyr and pyriproxyfen) are cross-resistant to carbamates and organophosphates at least via the G119S-Ace-1 mutation. This mutation often comes with fitness cost, requiring intense selection pressure prior to high occurrence [46]. Several other studies documented the low frequency of this mutation in different settings in Benin [6, 7, 47, 48].

A deficit of heterozygosity (FIS > 0) was observed in populations of An. coluzzii possessing the L1014F-vgsc-kdr mutation. This observation may reflect the effect of pyrethroid insecticides from study LLINs, or other unknown insecticides deployed on mosquitoes, which would continue to maintain resistance within populations by increasing the number of homozygous resistant individuals. This phenomenon could also lead to the elimination of susceptible individuals in various populations, favouring the survival of resistant ones. During the second year, an excess of heterozygosity was observed within the An. gambiae s.s. populations in the three study arms, which contrasts with findings from the recent work of Fassinou et al. [49]. This may simply be a random variation from one year to the next. Variations in population size, demographic composition, migration and population seasonality [50] can all influence levels of heterozygosity.

Low genetic differentiation was observed indoors post-intervention in the PY-PPF LLIN and PY-CFP LLIN arms in the two species carrying the L1014F-vgsc-kdr mutation. This could account for the continued increased genetic homogeneity in resistant An. gambiae s.l. populations following the widespread use of these new study LLINs. In a cotton-growing area in Benin, Aïkpon et al. [51] showed little genetic differentiation between An. gambiae s.s. populations at the L1014F vgsc-kdr and G119S-ace-1 loci. Selective pressures exerted by insecticides may favour the survival of individuals carrying resistance alleles. However, in the present study, genetic differentiation within the two species with the G119S-ace-1 mutation remained generally high after the distribution of the LLINs in the study. Thus, this results could rule out any selection of the G119S-ace-1 mutation; since no IRS has occurred in the study area to date. Ngufor et al. [52] evaluated in a community trial Fludora® Fusion, (mixture of deltamethrin and clothianidin) and VECTRON™ T500 (TENEBENAL™) in the neigbouring commune of Za-kpota, observing that G119S-ace-1 allele frequencies decreased from 36 to 10% in the VECTRON™ T500 arm and from 15 to 11% in the Fludora® Fusion arm. Moreover, some studies have reported an increase in L1014F vgsc-kdr mutation frequencies in An. gambiae s.s. and An. coluzzii species in response to net distribution in some areas [27, 40, 53]. These results provide some evidence of the effect of the introduction of new insecticides on frequencies of resistance mutations. In the large RCT, the infection rate in An. gambiae s.l. was 1% in both PY-CFP LLIN and PY-PPF LLIN arms over the two first years [38]. Moreover, it has been shown in wild mosquitoes possessing the resistance allele at the L1014F vgsc-kdr locus, that the risks of being infected by oocysts and sporozoites were higher in the “RR” and “RS” genotypes compared to “SS” [54]. However, in the present study, there was no evidence of a difference in the infection rate among the three genotypes, which deserves further investigation.

The study period (two years after the intervention) is short for an in-depth study of the genetic behaviour of the species. Similarly, the restricted geographical area of the study, limited to just three districts and randomized by village, is a potential limitation for the study as the flight of mosquito vectors between habitats could result in significant gene flow, thereby reducing the genetic differentiation between populations. Further analysis, including additional genetic data obtained 4 to 5 years after the deployment of these new-generation LLINs at scale, could provide additional information on the impact of dual AI nets on phenotypic resistance to pyrethroids and their impact on the underlying population genetic structure of malaria vectors.

Conclusion

The L1014F vgsc-kdr resistance allele showed a high allele frequency, while a low frequency was observed for the G119S ace-1 allele. This high frequency of L1014F vgsc-kdr allele indicates that dual AI nets continue to exert selective pressure in favour of this allele that is not counteracted by the non-pyrethroid insecticide.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

RCT:

Randomized controlled trial

AI:

Active-ingredients

Vgsc :

Voltage-gated sodium channel

kdr :

Knock down resistance

ace:

Acetylcholinesterase

IRS:

Indoor residual spraying

LLINs:

Long-lasting insecticidal nets

HWE:

Hardy–Weinberg equilibrium

Ho:

Observed heterozygosity

He:

Expected heterozygosity

F IS :

Fixation index

PY-PPF:

Pyrethroid-pyriproxyfen

PY-CFP:

Pyrethroid-chlorfenapyr

ITNs:

Insecticide-treated nets

PBO:

Piperonyl butoxide

HLC:

Human landing catch

ELISA:

Enzyme-linked immuno sorbent assay

CSP:

Circum-sporozoite protein

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Acknowledgements

We express our gratitude to the Benin NMCP, the communities of the Zou region, and their local leaders for their collaboration during the implementation of this study. We also appreciate the technicians who managed the mosquito processing, and the LSHTM ODK support team for their provision of electronic data solutions through LSHTM Open Research Kits ((http://odk.lshtm.ac.uk/).

Funding

This research was funded by a grant to the London School of Hygiene and Tropical Medicine from UNITAID and the Global Fund through the Innovative Vector Control Consortium (IVCC). This cluster-randomized clinical trial is a component of the larger initiative, “The New Nets Project”. The financial partners had no influence at any stage in the development of the activities, right up to the decision to publish this manuscript.

Author information

Author notes

  1. Louisa A. Messenger and Clément Agbangla contributed equally to this work.

Authors and Affiliations

  1. Faculté des Sciences et Techniques de l’Université d’Abomey-Calavi, Abomey-Calavi, Bénin

    Boulais Yovogan, Armel Djènontin, Constantin J. Adoha, Esdras M. Odjo, Antoine A. Missihoun, Serge Akpodji, Come Zinsou Koukpo, Gil G. Padonou & Clément Agbangla

  2. Centre de Recherche Entomologique de Cotonou, Cotonou, Bénin

    Boulais Yovogan, Armel Djènontin, Martin C. Akogbéto, Arthur Sovi, Constantin J. Adoha, Arsène Fassinou, Albert S. Salako, Esdras M. Odjo, Landry Assongba, Edouard Dangbénon, Idelphonse Ahogni, Serge Akpodji, Fiacre Agossa, Roséric Azondékon, Come Zinsou Koukpo, Gil G. Padonou & Corine Ngufor

  3. Faculty of Infectious and Tropical Diseases, Department of Disease Control, London School of Hygiene and Tropical Medicine, London, UK

    Arthur Sovi, Manfred Accrombessi, Corine Ngufor, Natacha Protopopoff & Louisa A. Messenger

  4. Faculté d’Agronomie, Université de Parakou, Parakou, Bénin

    Arthur Sovi

  5. Disease Control and Elimination Theme (DCE), Medical Research Council Unit, The Gambia at the London School of Hygiene and Tropical Medicine (MRCG-LSHTM), Banjul, The Gambia

    Bénoît S. Assogba

  6. Medical Research Council (MRC) International Statistics and Epidemiology, Epidemiology Group, London School of Hygiene and Tropical Medicine, London, UK

    Jackie Cook

  7. Department of Environmental and Occupational Health, School of Public Health, University of Nevada, Las Vegas, NV, USA

    Louisa A. Messenger

Authors
  1. Boulais Yovogan
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  2. Armel Djènontin
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  3. Martin C. Akogbéto
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  4. Arthur Sovi
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  5. Constantin J. Adoha
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  6. Arsène Fassinou
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  11. Edouard Dangbénon
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  12. Bénoît S. Assogba
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  14. Antoine A. Missihoun
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  24. Clément Agbangla
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Contributions

B.Y., A.S., A.D., C.A., J.C., M.A., C.N., M.C.A, L.A.M. and N.P. contributed to the design of the study. Entomological data were collected by B.Y., C.J.A., A.F., A.S.S., C.Z.K, E.M.O., L.A., I.A., S.A. under the supervision of A.S., G.G.P. and M.C.A.. Laboratory analyses were performed by B.Y. and C.J.A.. Original draft was written by B.Y, A.S. and L.A.M. Data management and statistical analysis were conducted by B.Y., R.A., B.S.A., E.M.O., N.P. and L.A.M. G.G.P., N.P. and M.C.A. provided administrative support to the trial. A.S., A.D., C.J.A., A.S.S., A.F., M.A., F.R.A., C.Z.K., I.A., L.A., A.A.M., C.N., B.S.A., J.C., C.A., M.C.A., N.P. and L.A.M. critically revised the manuscript for intellectual content. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Boulais Yovogan.

Ethics declarations

Ethics approval and consent to participate

The protocol for this study was reviewed and approved by the Benin National Health Research Ethics Committee (N°30/MS/DC/SGM/DRFMT/CNERS/SA, Approval n°6 of 04/03/2019) and the Ethics Committee of the London School of Hygiene and Tropical Medicine (16237-1). Written consent to participate in the study was obtained from the heads of households and adult volunteers carrying out the human bait captures. All field workers were vaccinated against yellow fever. If they tested positive for malaria, they were immediately treated with artemisinin-based combination therapy at the nearest health facility.

Competing interests

The authors declare no competing interests.

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Supplementary Information

12936_2025_5308_MOESM1_ESM.docx

Additional file 1: Table S1. Genotypic and allelic frequenciesof L1014F and HWE test indoor of Anopheles gambiae s.s. and Anopheles coluzzii populations. An.: Anopheles; N: number tested; PY LLIN: standard LLIN, LLIN treated with pyrethroid only; PY-CFP LLIN: LLIN bi-treated with pyrethroid-chlorfenapyr; PY-PPF LLIN: LLIN bi-treated with pyrethroid-pyriproxyfen; RR: homozygous resistant; RS: heterozygous resistant; SS: homozygous susceptible; Frfrequency of resistance allele; p valuep value to the Hardy–Weinberg Equilibrium; Post1: 1st year post-intervention; Post2: 2nd year post-intervention

12936_2025_5308_MOESM2_ESM.docx

Additional file 2: Table S2. Allele frequency of the L1014F vgsc-kdr mutation in An. gambiae s.s. and An. coluzzii in the three study arms. An.: Anopheles; N: number tested; PY LLIN: standard LLIN, LLIN treated with pyrethroid only; PY-CFP LLIN: LLIN bi-treated with pyrethroid-chlorfenapyr; PY-PPF LLIN: LLIN bi-treated with pyrethroid-pyriproxyfen; Post1: 1st year post-intervention; Post2; 2nd year post-intervention, CI: confidence interval

12936_2025_5308_MOESM3_ESM.docx

Additional file 3: Table S3. Genotypic and allelic frequenciesof G119S and HWE test outdoor of Anopheles gambiae s.s. and Anopheles coluzzii populations. An.: Anopheles; N: number tested; PY LLIN: standard LLIN, LLIN treated with pyrethroid only; PY-CFP LLIN: LLIN bi-treated with pyrethroid-chlorfenapyr; PY-PPF LLIN: LLIN bi-treated with pyrethroid-pyriproxyfen; RR: homozygous resistant; RS: heterozygous resistant; SS: homozygous susceptible; p value: p value for Hardy–Weinberg Equilibrium; Post1: 1st year post-intervention; Post2: 2nd year post-intervention

12936_2025_5308_MOESM4_ESM.docx

Additional file 4: Table S4. Allele frequency of the G119S-ace-1 mutation in An. gambiae s.s. and An. coluzzii in the three study arms. An.: Anopheles; N: number tested; PY LLIN: standard LLIN, LLIN treated with pyrethroid only; PY-CFP LLIN: LLIN bi-treated with pyrethroid-chlorfenapyr; PY-PPF LLIN: LLIN bi-treated with pyrethroid-pyriproxyfen; Post1: 1st year post-intervention; Post2: 2nd year post-intervention, CI: confidence interval

12936_2025_5308_MOESM5_ESM.docx

Additional file 5: Table S5. Expected heterozygousand observed heterozygouswithin the locus L1014F-vgsc-kdr in An. gambiae s.s. and An. coluzzii species. An.: Anopheles; N: number tested; PY LLIN: standard LLIN, LLIN treated with pyrethroid only; PY-CFP LLIN: LLIN bi-treated with pyrethroid-chlorfenapyr; PY-PPF LLIN: LLIN bi-treated with pyrethroid-pyriproxyfen; Post1: 1st year post-intervention; Post2: 2nd year post-intervention.

12936_2025_5308_MOESM6_ESM.docx

Additional file 6: Table S6. Expected heterozygousand observed heterozygouswithin the locus Ace-1 in An. gambiae s.s and An. coluzzii species. An.: Anopheles gambiae s.l.; N: number tested; PY LLIN: standard LLIN, LLIN treated with pyrethroid only; PY-CFP LLIN: LLIN bi-treated with pyrethroid-chlorfenapyr; PY-PPF LLIN: LLIN bi-treated with pyrethroid-pyriproxyfen; Post1: 1st year post-intervention; Post2: 2nd year post-intervention.

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Yovogan, B., Djènontin, A., Akogbéto, M.C. et al. Impact of dual active ingredients long-lasting insecticidal nets on the genetic structure of insecticide resistant populations of Anopheles gambiae in Southern Benin. Malar J 24, 72 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12936-025-05308-7

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12936-025-05308-7

Malaria Journal

ISSN: 1475-2875

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