Entomological surveillance of malaria vector: species, behaviors, and insecticide resistance in southern and central Benin

Abstract

Background

Malaria transmission in Benin is shaped by ecological heterogeneity, vector biting behavior, and increasing insecticide resistance. However, integrated multi-season data combining vector behavior, infection, and resistance remain limited in southern and central Benin.

Methods

From December 2023 to October 2024, an entomological and molecular survey was conducted in six districts across southern and central Benin over dry and rainy season collection rounds. Adult mosquitoes were collected using human landing catches (HLC) and pyrethrum spray catches (PSC), identified morphologically and molecularly, and tested for Plasmodium falciparum infection by qPCR. WHO insecticide susceptibility tests were performed, complemented by molecular analyses of resistance mutations (kdr L1014F and ace-1R G119S).

Results

Anopheles gambiae s.l. was the predominant vector (> 98%), exhibiting both indoor and outdoor biting with site- and season-specific heterogeneity. Among indoor-resting females collected by PSC, human blood meals predominated, indicating high anthropophagy in this subset. EIRs were highest in Ifangni and Toffo, including during the dry season. Pyrethroid resistance was widespread, with kdr allele frequencies exceeding 65% in all sites; susceptibility to pirimiphos-methyl was generally higher but reduced mortality was observed in some districts.

Conclusion

These findings support stratified, locally tailored vector control in southern and central Benin, including deployment of next-generation nets where pyrethroid resistance is intense, IRS guided by site-specific susceptibility profiles and rotation, and larval source management in areas with stable breeding habitats.

Clinical trial number

Not applicable.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12879-026-12989-1.

Background

Malaria remains a leading public health challenges in sub-Saharan Africa, accounting for more than 90% of global cases and deaths [1]. In Benin, transmission is perennial and highly heterogeneous across ecological zones, particularly in the southern and central regions, where climatic conditions, agricultural practices and the availability of surface water create suitable larval habitats for malaria vectors. The principal vectors are members of the Anopheles gambiae complex and the Anopheles funestus group, and their distribution and abundance vary in response to environmental and anthropogenic factors [2–3]. These ecological heterogeneity shapes vectorial capacity, biting behavior, and overall malaria transmission dynamics.

In recent years, changes in vector behavior have raised concerns about the sustained effectiveness of conventional vector-control tools. Reported shifts include earlier evening biting, increased outdoor biting and resting, and changes in host-seeking behavior, consistent with selective pressure from insecticide-based interventions, particularly insecticide-treated nets (ITNs) [4–5]. Such adaptations can reduce the impact of interventions primarily designed to target indoor, human-biting mosquitoes.

Concurrently, insecticide resistance has expanded substantially among Anopheles populations in Benin. High levels of resistance to pyrethroids, the main insecticide class used in standard ITNs, have been reported and supported by multiple mechanisms including knockdown resistance (kdr) mutations (L1014F/S), the ace-1 mutation (G119S), and the upregulation of detoxification enzymes such as cytochrome P450 monooxygenases, esterases, and glutathione S-transferases (GSTs) [6–7]. Agricultural pesticide use may further intensify selection pressure, particularly in rice-growing and vegetable production areas [8].

In this context, robust entomological surveillance is essential to guide malaria vector-control programs. Surveillance provides critical information into vector species composition, spatiotemporal distribution, feeding and resting behaviors, insecticide resistance profiles, and the field effectiveness of new-generation ITNs or indoor residual spraying (IRS). Southern and central Benin are especially strategic due to high population density, extensive agricultural landscapes, and rapid urbanization, factors that collectively influence vector ecology and human-vector contact [9].

This study therefore aims to generate updated evidence on vector species diversity, behavioral patterns, and insecticide resistance in southern and central Benin, to support more targeted and effective malaria control strategies aligned with national and global elimination goals.

Method

Study area

The study was conducted during both the dry and rainy seasons, with four collections in total: two during the dry season and two during the rainy season. Collections were carried out in six districts located in the southern and central regions of Benin, West Africa (Fig. 1). In the southern region, the districts of Sèmè-Kpodji, Ifangni, Zogbodomey, Abomey-Calavi, and Toffo were selected from the Ouémé, Plateau, Zou, and Atlantique departments, while Bantè, located in the Collines department, represented the central region.

Fig. 1.

Map of Benin showing the study commune

Southern and central Benin have a sub-equatorial climate characterized by two rainy seasons (April to mid-July and September to November) alternating with two dry seasons (December to March and mid-July to August) [10]. These seasonal patterns strongly influence mosquito breeding habitats and vector population dynamics, supporting the rationale for conducting four collection rounds across the year.

Mosquito sampling methods

Mosquito sampling was conducted in six communes during both the dry and rainy seasons using two complementary entomological methods: Human Landing Catches (HLCs) performed by trained adult volunteers, and Pyrethrum Spray Catches (PSCs) using a non-residual insecticide. Sampling was carried out monthly over a ten-month period, with four consecutive sampling nights per district to capture short-term temporal variation in vector abundance and behavior.

For each method, collections were conducted in two villages per commune: one centrally located and one in the peripheral zone. In each village, four households were selected for HLC based on predefined inclusion criteria: (i) permanent occupancy, (ii) presence of household members willing to participate and (iii) structural integrity allowing clear differentiation between indoor and outdoor catching positions. When more eligible households existed than required, simple random sampling was applied to select the four HLC houses.

HLCs were performed from 19:00 to 07:00. Two teams of eight collectors operated each night: the first team from 19:00 to 01:00 and the second from 01:00 to 07:00. Collections were conducted simultaneously indoors and outdoors to quantify variation in endophagic/exophagic biting rates. To reduce collector-related bias, such as differences in mosquito attractiveness or catching skill, collectors were rotated between indoor and outdoor positions every hour according to a predetermined schedule. Mosquitoes were aspirated directly from the exposed legs of collectors using heparinized tubes, then sorted by village, household, sampling position (indoor or outdoor), and hour of capture.

For PSCs, ten dwellings per village were selected using inclusion criteria ensuring comparability: (i) occupancy by residents for at least six months, (ii) no recent chemical spraying or domestic insecticide use in sleeping rooms, and (iii) availability of at least one closed sleeping space suitable for pyrethrum spraying. Eligible households were identified during a pre-survey visit and, where necessary, randomly selected using a numbered household list.

PSC operations were conducted early in the morning. After the occupants vacated the dwelling, a white canvas sheet was spread over the floor and furniture. Rooms were then sprayed with pyrethrum and sealed for 10–15 min. All knocked-down mosquitoes were collected using forceps and transferred into labeled Petri dishes for later morphological identification. For each room, the total number of mosquitoes collected and their physiological status (unfed, blood-fed, half-gravid, or gravid) were recorded to estimate indoor resting density and feeding patterns.

Morphological and molecular identification of vector species

Mosquitoes obtained via HLC and PSC were initially identified to species level based on morphological characteristics using the taxonomic keys of Coetzee [11]. For each specimen, detailed records were maintained, including village type (central or peripheral), collection site (house 1–10), collection environment (indoor or outdoor), and collection time. All Anopheles specimens confirmed as malaria vectors were individually preserved in cryoboxes for subsequent analyses.

Quantification of Plasmodium falciparum in Anopheles vectors

Genomic DNA was isolated from the head and thorax of Anopheles mosquitoes ((n ≥ 40) using DNAzol^® reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) following the manufacturer’s protocol. The extracted DNA was eluted in 20 µL of nuclease-free water and stored at − 20 °C until downstream molecular analyses. Quantitative PCR (qPCR) assays were conducted according to the methodology described by Boissière et al. [13], enabling precise detection and quantification of plasmodium falciparum in Anopheles vectors.

Insecticide resistance

WHO insecticide susceptibility tests

Insecticide susceptibility assays were conducted in accordance with the World Health Organization (WHO) standard protocol [12]. At each collection site, four replicates of 20–25 non-blood-fed female Anopheles gambiae s.l., (3–5 days old) were exposed for one hour to WHO-impregnated papers treated with 0.05% deltamethrin, 0.05% alpha-cypermethrin, or 0.25% pirimiphos-methyl, ensuring a minimum of 100 mosquitoes per insecticide per commune. Two control groups of the same size were exposed to untreated papers. After exposure, mosquitoes were transferred to holding tubes, provided with 10% glucose solution, and maintained under standard conditions for 24 h, after which mortality was recorded.

Species identification within the An. gambiae s.l, and detection of the kdr L1014F and ace-1R mutation

A representative subset of mosquitoes (n ≥ 100) from the WHO bioassays was selected for molecular analyses. Species within the Anopheles gambiae complex were identified following the methodology of Santolamazza et al. [14]. The presence of the West African knockdown resistance mutation (kdr L1014F) was assessed according to Martinez-Torres et al. [15], while detection of the ace-1R resistance allele was conducted following the protocol described by Weill et al. [16]

Data analysis

Data were analyzed using R version 2.8 (R Core Team, 2008) and the stats package [17]. The Chi-square test was applied to compare proportions of Anopheles gambiae s.l. collected indoors and outdoors, blood-feeding rates, sporozoite indices, and allelic frequencies of the kdr (L1014F) and ace-1 (G119S) mutations.

The Poisson test [18] was used to estimate risk ratios (RRs) and compare the confidence intervals of indoor vector densities and An. gambiae s.l. entomological inoculation rates (EIRs) between communes. Differences in Plasmodium falciparum sporozoite densities among members of the An. gambiae s.l. were assessed using the Kruskal–Wallis test.

Bioassay data were recorded on paper forms and entered into Microsoft Excel for analysis. Mortality rates were calculated as the proportion of dead mosquitoes 24 h after exposure relative to the total number tested. Resistance status was determined following WHO criteria [12]:

• Mortality ≥ 98%: population considered susceptible.

• Mortality between 90 and 97%: population suspected of resistance, requiring further verification.

• Mortality < 90%: population confirmed resistant.

Allelic frequency of kdr mutations was calculated using the formula:

[19].

(n = number of mosquito samples with a given genotype).

Results

Vector species composition during the dry season

During the dry season, a total of 6,440 malaria vector specimens were collected, belonging to two species: Anopheles gambiae s.l. and Anopheles funestus s.l. An. gambiae s.l. was predominant across all six surveyed districts, representing 99.82% of the total catch, whereas An. funestus s.l. accounted for only 0.17% (Fig. 2).

Fig. 2.

Composition of malaria vectors during the dry season in 6 districts of Benin

In the rainy season, two vector species were also recorded across the 6 surveyed districts. An. gambiae s.l remained the dominant species (98.29%), followed by An. funestus s.l. (1.65%) (Fig. 2).

Mosquito blood-feeding behaviors

Human biting rate (HBR) of Anopheles gambiae s.l. indoors and outdoors by district

A total of 3,854 Anopheles gambiae s.l. were collected during the dry season (February–March 2024) across all surveyed sites. Table I summarizes the proportions of indoor and outdoor collections throughout the study period. Overall, no significant differences were detected between indoor and outdoor biting proportions, except in Zogbodomey, where the proportion of indoor-biting mosquitoes was significantly higher than outdoor-biting ones (40.47% vs. 11.90%; P = 0.004). This feeding pattern contrasts slightly with observations recorded during December-January.

During the rainy season (August-October 2024), a total of 6,032 An. gambiae s.l. were collected across all study sites. Although biting behavior varied geographically, no significant differences were observed between indoor and outdoor biting rates in any commune (Table 1).

Table 1.

Number and proportion of An. gambiae s.l. caught indoors and outdoors per district in the rainy season and the dry season

Districts	Dec 2023-Jan 2023			Feb-Mar2024			Aug 2024			oct-24
	Indoors	Outdoors		Indoors	Outdoors		Indoors	Outdoors		Indoors	Outdoors
Nb (%)	Nb (%)	P	Nb (%)	Nb (%)	P value	Nb (%)	Nb (%)	P	Nb (%)	Nb (%)	P
		value						value			value
Abomey-Calavi	19 (59.37)	13 (40.63)	0.211	11 (44.00)	14 (56.00)	0.572	No	No	No	No	No	No
Bantè	3 (100.00)	0 (0.00)	0.102	1 (25.00)	3 (75.00)	0.479	No	No	No	No	No	No
Sèmè-Kpodji	93 (50.81)	90 (49.19)	0.834	32 (49.23)	33 (50.76)	1	No	No	No	No	No	No
Toffo	160 (34.93)	298 (65.07)	< 0.001	82 (45.05)	100 (54.94)	0.075	839 (54.58)	698 (45.41)	< 0.001	99 (55.00)	81 (45.00)	0.073
Ifangni	1165 (62.53)	698 (37.47)	< 0.001	1762 (51.04)	1690 (48.95)	0.087	579 (47.73)	634 (52.26)	0.028	972 (50.05)	971 (49.94)	1
Zogbodomey	25 (69.44)	11 (30.56)	0.0021	75 (59.52)	51 (40.47)	0.004	620 (56.67)	474 (43.32)	< 0.001	29 (44.61)	36 (55.38)	0.292

Nb: number of An. gambiae s.l.; %: proportion of An. gambiae s.l.; p-value: p-value of comparison of the proportion of An. gambiae s.l. indoors and outdoors in the same district

(Test used: Chi-square test)

The highest HBR values were recorded in Ifangni, reaching 215.75 bites per person per night during the dry season and 49.31 bites per person per night during the rainy season, highlighting intense vector aggressiveness in this locality regardless of season (Supplementary Files, Table S1).

Hourly human biting rate of malaria vector during the night

The nocturnal biting behavior of malaria vectors exhibited a consistent pattern across both phases of the dry season (early: December 2023-January 2024; late: February-March 2024). Biting activity began increasing around 21:00, reached an initial peak between 00:00 and 01:00, followed by a second peak between 03:00 and 04:00, and then gradually declined toward dawn (Fig. 3).

Fig. 3.

Hourly HBR of malaria vector during the night in the dry season in all districts surveyed

During the rainy season, the biting cycle showed a similar temporal profile across collection periods. Mosquito activity started to rise around 21:00, with a first peak occurring between 02:00 and 03:00, followed by a second peak between 04:00 and 05:00, and a progressive decline thereafter until early morning (approximately 06:00) (Fig. 4).

Fig. 4.

Hourly HBR of malaria vector during the night in the rainy season in all districts surveyed

Blood meal analysis of Anopheles gambiae s.l

During the dry season, approximately 90.2% of An. gambiae s.l. specimens collected indoors using the PSC method contained human blood, yielding an estimated anthropophagic rate of 90.2% (Table 2).

Table 2.

Blood-meal analysis of Anopheles gambiae s.l. per districts

District	Total analyzed	Human only	Bovine only	Goat only	Human + bovine
Ifangni	18	17	1	0	0
Sèmè-Kpodji	11	8	2	1	0
Abomey-Calavi	2	2	0	0	0
Toffo	11	10	0	0	1
Zogbodomey	9	9	0	0	0
Bantè	0	0	0	0	0
Total district	51	46	3	1	1
		(90.2%)	(5.88%)	(1.96%)	(1.96%)

Sporozoïte index (%CS+) of Plasmodium falciparum and entomological inoculation rate (EIR) of An. gambiae s.l. per district in the dry season and rainy season

During the dry season, the highest entomological inoculation rates (EIRs) were recorded in Ifangni (53.57 infective bites per person per month) and Toffo (10 infective bites per person per month). In the rainy season, elevated EIRs were again observed in Ifangni and Toffo, as well as in Zogbodomey, each exceeding 10 infective bites per person per month (Supplementary Files, Table S2).

In Ifangni, the EIR was slightly higher during the dry season than in the rainy season. This pattern likely reflects the abundance of permanent fish-breeding ponds in the district, which provide stable larval habitats for Anopheles mosquitoes throughout the dry period (Supplementary Files, Table S2).

Insecticide resistance profile

Across all study sites, Anopheles gambiae s.l. exhibited resistance to both deltamethrin and alphacypermethrin (Fig. 5). Mortality rates varied substantially among districts and between insecticides. For deltamethrin (0.05%), the lowest mortality was recorded in Zogbodomey (36.52%), whereas the highest occurred in Abomey-Calavi (55.89%). Under exposure to alphacypermethrin (0.05%), mortality ranged from 17.32% in Zogbodomey to 50% in Toffo.

Fig. 5.

Mortality rates of An. gambiae s.l. after exposure to deltamethrine, alphacypermethrin and pirimiphos-methyl using the WHO tube assay

In contrast, An. gambiae s.l. populations remained largely susceptible to pirimiphos-methyl across several districts. Full susceptibility (mortality > 98%) was documented in Abomey-Calavi, Toffo, and Bantè. Suspected resistance (mortality < 98%) was observed in Sèmè-Kpodji, while confirmed resistance (mortality < 90%) was detected in Ifangni and Zogbodomey (Fig. 5).

Multiple insecticide resistance mechanisms in An. gambiae s.l. (kdr, ace-1)

Across the six study districts, An. coluzzii and An. gambiae s.s. were the two species identified within the An. gambiae complex, representing 88.30% (n = 536) and 11.70% (n = 71) of the total analyzed specimens (n = 607), respectively (Table 3).

Table 3.

Distribution of knock-down resistance (kdr) gene frequencies

Districts	Species	Numbers tested	RR	RS	SS	Freq.L1014F (%)	P- value
Abomey-calavi	An.gambiae	3	1	1	1	50.00	0.348
	An.coluzzii	56	33	19	4	75.89
Bantè	An.gambiae	2	2	0	0	100	1
	An.coluzzii	6	4	2	0	83.33
Sèmè-kpodji	An.gambiae	1	1	0	0	100.00	1
	An.coluzzii	157	109	40	8	82.17
Ifangni	An.gambiae	4	4	0	0	100.00	0.125
	An.coluzzii	146	80	39	27	68.15
Toffo	An.gambiae	33	25	6	2	84.84	0.846
	An.coluzzii	87	65	14	8	82.75
Zogbodomey	An.gambiae	28	24	2	2	89.28	0.162
	An.coluzzii	84	62	15	7	82.73

A high frequency of the kdr L1014F mutation was detected in An. gambiae s.l. populations across all districts, exceeding 65% everywhere. In An. coluzzii, kdr frequencies ranged from 68.15% (95% CI: 54–87) in Ifangni to 82.73% (95% CI: 77–100) in Zogbodomey. In An. gambiae s.s., frequencies ranged from 82% (95% CI: 72–90) in Abomey-Calavi to 100% (95% CI: 15–100) in Sèmè-Kpodji, Bantè, and Ifangni (Table 3).

For the Ace-1R mutation, relatively high frequencies were recorded in Bantè among An. gambiae s.s. (25%), whereas frequencies remained below 6% in An. coluzzii across all districts (Table 4).

Table 4.

Distribution of Ace-1R frequencies

Districts	Species	Numbers tested	RR	RS	SS	Freq.	P- value
						G119S (%)
Abomey-calavi	An.gambiae	3	0	0	3	0.00	1
	An.coluzzii	56	0	5	51	4.46
Bantè	An.gambiae	2	0	1	1	25.00	0.551
	An.coluzzii	6	0	0	6	0.00
Sèmè-kpodji	An.gambiae	1	0	0	1	0.00	1
	An.coluzzii	157	0	14	143	4.46
Ifangni	An.gambiae	4	0	1	3	12.5	0.829
	An.coluzzii	146	0	13	133	4.45
Toffo	An.gambiae	33	0	2	31	3.03	0.716
	An.coluzzii	87	0	9	78	5.17
Zogbodomey	An.gambiae	28	0	1	27	1.78	0.555
	An.coluzzii	84	0	8	79	4.76

Discussion

This study provides updated evidence on the species composition, biting behavior, transmission intensity, and insecticide resistance of Anopheles gambiae s.l. populations across six districts in southern and central Benin.

Across both seasons, An. gambiae s.l. overwhelming dominated collections (> 98%) confirming its central role in malaria transmission in these settings and aligning with previous observations from similar ecological zones in Benin [20, 21]. In contrast, An. funestus s.l., represented only a small fraction of the catch, suggesting that local ecological conditions and land-use patterns may currently favor An. gambiae s.l. other taxa in the surveyed district [22–23]. Within the An. gambiae complex, An. coluzzii predominated over An. gambiae s.s. (Table III), consistent with the presence of permanent or semi-permanent aquatic habitats and human-modified environments that can support An. coluzzii populations. This composition has operational relevance because vector control performance may vary by sibling species due to differences in ecology, behavior, and resistance profiles.

The nocturnal biting behavior exhibited two peaks, but their timing differed by season. During the dry-season rounds, biting increased from 21:00 with a first peak between 00:00–01:00, followed by a second peak between 03:00–04:00 (Fig. 3). In the rainy season, the overall profile was similar but shifted later, with peaks between 02:00–03:00 and 04:00–05:00 (Fig. 4). This seasonal shift is epidemiologically important because it affects the degree of overlap between human sleeping/bednet use and vector host-seeking activity, and therefore modifies the expected impact of indoor interventions.

Patterns of indoor and outdoor biting were heterogeneous across districts and collection rounds (Table I), rather than uniformly indicating either dominant endophagy or exophagy. Several site-round strata showed significant indoor-outdoor differences, including Zogbodomey (indoor predominance in multiple rounds) and Toffo (notably higher outdoor biting in early dry season, with indoor predominance during the rainy-season round). Ifangni also showed round-specific variation. Together, these results argue against a single generalized behavioral conclusion (e.g., “no change” or “widespread outdoor biting”) and instead highlight localized behavioral expression that may reflect differences in housing structure, peri-domestic ecology, and microclimate. Given that behavioral shifts (earlier biting and/or outdoor biting) have been reported under sustained LLIN pressure [5, 24], continued longitudinal surveillance remains important to detect trajectories over time.

Blood-meal analysis indicated a high anthropophagic rate (90.2%); importantly, this estimate derives from indoor-resting females collected by PSC (Table II) and therefore most directly reflects host choice among mosquitoes resting indoors after feeding. Nevertheless, combined with the HBR, these findings support intense human-vector contact in key districts. In particular, Ifangni exhibited exceptionally high HBR during the dry season (215.75 bites/person/night) and substantial EIR (53.57 infective bites/person/month), with the dry-season EIR slightly exceeding the rainy-season value (Supplementary Table S2). This atypical seasonal pattern suggests that stable larval habitats can sustain transmission beyond the rains. In Ifangni, the presence of permanent fish-breeding ponds is a plausible driver of perennial larval productivity and dry-season adult emergence (Supplementary Table S2). Toffo also showed elevated EIR values (≥ 10 infective bites/person/month in both seasons), and Zogbodomey exceeded this threshold in the rainy season (Supplementary Table S2). Programmatically, these levels indicate sustained transmission potential and justify intensified, locally tailored interventions.

WHO susceptibility assays demonstrated widespread pyrethroid resistance, with deltamethrin and alpha-cypermethrin mortality frequently < 60% across districts, consistent with severe resistance previously reported in Benin and across West Africa [6, 7]. Genotyping showed high kdr L1014F frequencies (> 65% in all districts, reaching 100% in some sites), indicating strong selection pressure and, in some localities, near fixation of this target-site mutation [8] (Table V). Given the magnitude of phenotypic resistance observed, kdr alone may be insufficient to explain the low mortality rates, and the contribution of metabolic mechanisms (e.g., P450s) is likely, as reported elsewhere in Benin [6, 7]. However, metabolic resistance was not directly measured in the present study.

In contrast to pyrethroids, susceptibility to pirimiphos-methyl remained high in several districts, supporting its continued utility for IRS where local susceptibility is confirmed [25]. Importantly, suspected or confirmed resistance to pirimiphos-methyl was detected in some settings (notably Ifangni and Zogbodomey), underscoring that IRS deployment should be guided by site-specific resistance profiles, with careful insecticide rotation and routine monitoring to preserve effectiveness.

The ace-1R G119S mutation remained rare in An. coluzzii (< 6%) but was detected at higher frequency in An. gambiae s.s. in Bantè (Table V). Because ace-1R can confer resistance to carbamates and organophosphates [16], its detection warrants strengthened surveillance; however, the number of An. gambiae s.s. genotyped in Bantè was small, so these estimates should be interpreted cautiously and confirmed through expanded sampling before inferring a localized “hotspot.”

This study has several limitations. First, molecular species identification and resistance genotyping were conducted on subsets of specimens, which may not capture the full genetic variability across districts and seasons. Second, PSC collections were limited in some areas, reducing the precision of inferences on indoor resting behavior and host choice beyond indoor-resting mosquitoes. Third, resistance characterization relied on standard WHO bioassays; intensity assays and synergist (e.g., PBO) tests were not performed, and metabolic resistance markers were not assessed, limiting mechanistic attribution of the observed pyrethroid resistance.

Despite these limitations, the findings have direct implications for malaria vector control in southern and central Benin. Persistent transmission, often substantial in both seasons in key districts, combined with strong anthropophagy among indoor-resting vectors and widespread pyrethroid resistance, supports the need for district-specific, seasonally adapted strategies. Priority actions include (i) deployment of next-generation LLINs (e.g., PBO or chlorfenapyr nets) in areas with intense pyrethroid resistance, (ii) targeted IRS using pirimiphos-methyl only where susceptibility remains high and is regularly verified, (iii) larval source management in settings with permanent breeding sites (e.g., fish ponds and other stable water bodies), and (iv) strengthened entomological and molecular surveillance to track behavioral heterogeneity and both target-site and metabolic resistance. Together, these measures are essential to sustain intervention effectiveness and support Benin’s malaria control and elimination objectives.

Conclusion

Our study provides robust evidence that An. gambiae s.l., particularly An. coluzzii, is the dominant malaria vector along the south–north transect of Benin, exhibiting high anthropophily, significant indoor and outdoor biting, and widespread pyrethroid resistance. Spatial and temporal heterogeneity underscores the need for district-specific, seasonally adapted vector control strategies. These findings directly inform Benin’s malaria elimination agenda and contribute to a broader understanding of malaria transmission dynamics in West Africa. Malaria transmission in Benin remains persistent, heterogeneous, and seasonal, with An. gambiae s.l. as the primary vector. Strengthening entomological and molecular surveillance, tailoring interventions to local contexts, diversifying vector control approaches, and integrating environmental and climatic data are essential for advancing national, regional, and global malaria control efforts.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

WHO

World health organization

ETI

Entomological transmission indicators

HBR

Human biting rate

SR

Sporozoite rate

EIR

Entomological inoculation rate

HLC

Human landing catch

PSC

Spray catch

NMCP

National malaria control program

Author contributions

The study was designed and its protocol written by SZH, SA, RAO, LBM and MA. Data collection was performed by SZH, SA, SVA, MA, ZA, OO, MSI, EOB, SI, FT, MO, KDK and BA. MJA, and SZH performed Molecular analyses. SZH, AJD, IT, MBMC and RAO wrote the manuscript. SZH and SC performed the statistical analysis of the data. RAO, and MA have revised the manuscript.

Funding

This study received no external funding.

Data availability

1- The raw data analyzed in this study are available upon request. The corresponding author can be contacted to obtain access to the full datasets and materials used. Contact details are as follows: hougbesteve@gmail.com. 2-We confirm that the data availability statement in the submission system and in the article is exactly the same.

Declarations

Ethics approval and consent to participate

The study protocol was reviewed and approved by the Institutional Ethical Committee of the Cotonou Entomological Research Center (IECC) under approval number IORG005698. The Cotonou Entomological Research Center (CREC) operates under the Directorate of Research and Training of the Ministry of Health of Benin, ensuring that all research activities comply with national regulations and ethical standards for health research. Prior to the start of the study, written informed consent was obtained from all local mosquito collectors who voluntarily agreed to participate after being fully informed about the study’s objectives, procedures, potential risks, and benefits. All participants were vaccinated against yellow fever, underwent periodic medical check-ups conducted by the attending physician, and received appropriate treatment in the event of confirmed malaria infection. All ethical procedures adhered to the principles of the Declaration of Helsinki. The ethical framework and consent procedures applied in this study are consistent with those previously implemented and published in other CREC-led investigations (Salako et al. [20])

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.