Species diversity and human biting rates of malaria and dengue vectors in central and northern Benin

Abstract

Background

Malaria and dengue fever are transmitted by female mosquitoes of the Anopheles and Aedes genera, respectively. These diseases are among the most prevalent in sub-Saharan Africa, with dengue fever posing an increasingly significant threat. In this context, studying the species diversity of vectors, as well as their bionomics, including their ecological and behavioral traits, is essential for a better understanding of transmission dynamics, and for guiding appropriate vector control strategies. This study aims to characterize the species diversity and human bite rates of malaria and dengue vectors in central and northern Benin simultaneously, using a multimethod sampling approach combining larval surveys, captures on human volunteers, oviposition trap use, and pyrethrum spraying captures.

Results

Combined diurnal and nocturnal collections yielded a total of 2,337 adult mosquitoes across the five study communes. Species composition was dominated by the genera Aedes and Anopheles, with marked spatial heterogeneity among localities. The statistically significant intercommunal differences (p < 0.001) indicated a heterogeneous spatial distribution of the mosquito populations, which was further supported by the diversity indices. During larval surveys, 993 potential breeding sites were inspected, of which 51.0% were positive. In the study area, household containers such as buckets, jars, and jerry cans are the most abundant and productive breeding sites for Aedes. After emergence, 1,988 adult mosquitoes were identified, representing seven species: Aedes aegypti, Ae. albopictus, Ae. vittatus, Anopheles gambiae, Culex nebulosus, Cx. quinquefasciatus, and Cx. tigripes. Pyrethrum spray collection revealed five species and confirmed the presence of endophilic vectors, notably An. gambiae and Cx. quinquefasciatus. Analysis of biting activity revealed a pronounced circadian pattern: Aedes aegypti exhibited predominantly diurnal biting activity, whereas Anopheles gambiae showed a marked increase in nocturnal activity. The concurrent detection of Anopheles gambiae s.l. and Aedes aegypti during both daytime and nighttime, even at relatively low abundance levels, represents a noteworthy epidemiological signal suggesting a potential combined risk of malaria and dengue transmission.

Conclusion

The multimethod approach highlights the importance of integrated entomological surveillance to anticipate emerging epidemiological dynamics and adapt vector control strategies to local ecological conditions.

Background

Vector-borne diseases represent a major public health challenge in tropical and subtropical regions [1]. Among these diseases, those transmitted by mosquitoes are the most widespread [2]. Malaria and dengue are among the most prevalent infections and are responsible for substantial morbidity and mortality, particularly in sub-Saharan Africa [3–5]. Malaria is transmitted by mosquitoes of the genus Anopheles and remains endemic in many African countries. Furthermore, dengue is transmitted primarily by Aedes aegypti and is promoted by urbanization, climate variability, and environmental change [6–8]. In this global context, Benin is a particularly telling example of these epidemiological trends. Malaria is the leading cause of medical consultations and hospitalizations [9], and dengue outbreaks have been reported in recent years, suggesting that the virus is actively circulating in the country [10, 11]. This situation is closely linked to the diversity and spatial distribution of mosquito vectors, whose dynamics largely depend on the ecological conditions and physicochemical characteristics of their environments [12]. Benin exhibits a marked ecological gradient from south to north, ranging from humid and urbanized coastal zones to drier Sudanian-Sahelian regions, which may influence both the species composition of mosquito populations and their biting behavior [13, 14]. Understanding vector species diversity and human bite rates is therefore essential for assessing transmission risk and guiding vector control strategies [15]. Species diversity provides information on mosquito community composition, the relative abundance of species, and the dominance of major vector species, whereas the human biting rate constitutes a key entomological indicator for estimating the intensity of human–vector contact [16, 17]. However, entomological studies often rely on a single sampling technique, which may lead to underestimation of the true diversity of vector populations or provide only a partial understanding of their ecology [18]. The use of a multimethod approach that combines human landing catches, larval habitat surveys, ovitrap deployment, and pyrethrum spray collection allows for a more comprehensive characterization of mosquito populations. Indeed, the integration of these sampling techniques makes it possible to account for immature stages, oviposition behavior, resting preferences, and biting activity [19]. Understanding biodiversity is important, so this study examined the species diversity of malaria and dengue vectors, as well as the rate at which these vectors bite humans, in central and northern Benin, where such data are scarce. The lack of up-to-date entomological data in Benin is a significant obstacle to developing effective, targeted vector control strategies. A lack of information on vector population composition and feeding behavior, particularly human bite rates, limits our understanding of spatiotemporal variations in transmission risk. This study aims to address this knowledge gap by simultaneously characterizing the species diversity and human biting rates of malaria and dengue vectors in central and northern Benin to improve our understanding of spatial variations in transmission risk.

Methods

Study area

This study was conducted in five communes in Benin: Savalou and Bantè, located in the Central Region, and Bassila, Djougou, and Parakou, located in the North (Fig. 1). These communes are located within the Sudanian-Guinean climatic zone. This zone is characterized by two main seasons: a rainy season and a dry season. The landscape consists of forest vegetation and wooded savanna, with trees and shrubs growing on clay-sandy and lateritic soils. According to the 2013 national population census (RGPH-4), Savalou had a population of 144,549 inhabitants, Bantè 107,181, Bassila 130,091, Djougou 267,812, and Parakou 255,478. In these regions, households commonly store rainwater in cisterns and jars, as well as in containers such as jerry cans, buckets, and barrels for domestic use. These municipalities were selected because of their strategic geographic position in central and northern Benin, areas characterized by intense cross-border mobility with Nigeria and Burkina Faso, countries where dengue outbreaks have been reported. At the national level, confirmed dengue cases were recorded in 2019 in southern Benin, and the recent expansion of Aedes albopictus reflects the ongoing geographic spread of arbovirus vectors [13, 20]. Furthermore, Benin remains endemic for malaria, and the increasing adaptation of certain Anopheles species to artificial breeding sites similar to those exploited by Aedes further highlights the importance of investigating these anthropogenic environments. The selection of these municipalities therefore aimed to expand entomological surveillance to areas that remain poorly documented and to assess the potential co-occurrence of malaria and dengue vectors in ecological contexts undergoing environmental change. Vector sampling was conducted from January 2024 to September 2025, corresponding to a cumulative duration of ten months of effective field investigation.

Fig. 1.

Study area

Sampling of mosquitoes

Mosquito larval surveys

Prior to the inspection of breeding sites in the selected households, consent was obtained from village leaders and household heads. With the assistance of a community guide, the team from the Centre de Recherche Entomologique de Cotonou explained the objectives of the study and highlighted the public health risks associated with mosquito breeding sites located within and around human dwellings. Households were selected through random sampling within each neighborhood to minimize sampling bias. All domestic and peridomestic containers capable of holding water were systematically inspected. These included water storage containers, small discarded containers, and artificial structures exposed to rainwater, such as used tires and abandoned vehicles. At each positive breeding site, immature stages (larvae and pupae) were collected via a dipper and stored separately according to the sampling site and habitat type. The samples were carefully labeled before being transported to the insectary of the Centre de Recherche Entomologique de Cotonou for rearing. In the laboratory, larvae are maintained under controlled conditions, with temperatures ranging from 26 to 30 °C and relative humidity between 70 and 80%, conditions favorable for optimal development [21, 22]. The adult mosquitoes obtained were maintained on a 10% sugar solution to ensure survival until identification. Species identification was performed on the emerged adults on the basis of diagnostic morphological characteristics via standard taxonomic keys, including those of by Edwards [23], Huang [24] for mosquitoes of the genus Aedes, and Coetzee [25] for Anopheles.

Human landing catch (HLC)

In each study commune, two neighborhoods were selected, and within each site, collections were conducted in two households. In each household, one collector was stationed indoors and another outdoors, resulting in a total of four collectors per site. To minimize bias related to individual attractiveness or collector skill, a regular rotation of collectors between households was implemented. Collected mosquitoes were sorted by hourly interval and preserved in RNAlater (Sigma brand), then labeled and pooled according to time interval, locality, date, and collection site for subsequent species identification and estimation of the human biting rate. Daytime collections were conducted from 07:00 to 19:00. Two teams of four collectors were deployed per site, with the first team operating from 07:00 to 13:00 and the second from 13:00 to 19:00. Nighttime collections were carried out from 19:00 to 06:00 using a similar design. Two teams of four collectors worked in shifts, the first from 19:00 to 01:00 and the second from 01:00 to 06:00.

Setting up egg-laying traps

For the selection of houses, random sampling was performed within each neighborhood. Two villages were selected in each study municipality. A total of 12 ovitraps were installed per village. These ovitraps attract gravid female mosquitoes of the genus Aedes to search for suitable oviposition sites. Each ovitrap consisted of a plastic bottle cut at approximately one-third of its upper portion. A rectangular hardboard strip was placed inside the container to provide a substrate for oviposition. The traps were suspended in vegetated areas that were shaded and located near human dwellings. Four to five days after deployment, ovitraps containing larvae resulting from egg hatching were collected. The traps without visible larvae were carefully emptied into separate labeled containers indicating the date, ovitrap number, and locality until egg hatching occurred. All the larvae obtained were reared to the adult stage. Emerged mosquitoes were examined under a stereomicroscope and identified to establish an inventory of the species encountered.

Pyrethrum spray catch

Two villages were selected in each municipality. Prior to the application, the household occupants were informed about the procedure, and their consent was obtained. They were then asked to leave the room during spraying and to cover or remove food items, kitchen utensils, and children's toys. Adult mosquito samples were collected indoors via non-residual insecticide aerosol sprays between 06:00 and 08:00. The equipment used included white cotton sheets measuring 2 m × 3 m, Rambo aerosol insecticide sprays containing pyrethroids (0.25% transfluthrin and 0.20% permethrin), Eppendorf tubes for storing collected mosquitoes, forceps, and flashlights. Eight to ten households were inspected for the collection of indoor resting mosquitoes via the pyrethrum spray catch method.

Statistical analysis

Entomological data were entered and preprocessed via Microsoft Excel to verify the consistency of counts, manage missing values, and structure the datasets (raw counts by species and by municipality). Row percentages corresponding to the distribution of species within each municipality were calculated in Excel to provide an initial description of species composition patterns. Statistical analyses and graphical outputs were produced via R software (version 4.5.2) with scripts designed to ensure the reproducibility of the results. On the basis of the raw counts, the relative proportions of each species were calculated for each municipality and subsequently used to estimate the following ecological diversity indices: the Shannon diversity index (H′), Simpson diversity index (1 − D), and Pielou’s evenness index (J′), according to the classical formulas described below. The Shannon diversity index (H′) was used to quantify species diversity by simultaneously incorporating species richness and the distribution of individuals among species. Its value ranges from 0, corresponding to a community composed of a single species, to a theoretical maximum value (H′max = log S), reached when all recorded species (S) have equal abundances. The index is calculated as follows:

$${\text{H}}^{\prime }=-\mathrm{\Sigma }\left(\left(ni/N\right)\times \text{log}\left(ni/N\right)\right),$$

ni = number of individuals of species i; N = total number of individuals across all species; S = species richness (total number of species).

$${\text{H}}^{\prime }\text{max}=\text{log}\left(\text{S}\right).$$

Species dominance was estimated via Simpson’s diversity index expressed as (1 − D), which represents the probability that two individuals randomly selected from the sample belong to different species. The values approach 0 when the community is dominated by a small number of species and approach 1 when the abundances are more evenly distributed. The applied formula is as follows:

$$1-\text{D}=1-\mathrm{\Sigma }\left[ni\left(ni-1\right)/N\left(N-1\right)\right],$$

ni = number of individuals of species i; N = total number of individuals.

Pielou’s evenness index (J′) was used to assess the degree of uniformity in the distribution of individuals among species. This index, derived from Shannon’s diversity index, expresses the ratio between the observed diversity (H′) and the theoretical maximum diversity (H′max). It ranges from 0, indicating strong dominance by one or a few species, to 1, indicating a perfectly even distribution of abundances. It is defined as follows: J′ = H′/H′max with H′max = log(S).

Comparisons of species distributions among municipalities were performed via contingency tables, with calculations of row percentages and the application of chi-square tests (χ²) or Fisher’s exact test when at least one expected frequency in the contingency table was less than 5.

Hourly bite rate per person (HBR) by Anopheles gambiae s.l. and Aedes aegypti

The HBR is calculated by dividing the number of mosquitoes captured by the number of trappers.

Number of mosquitoes/person/1 night.

Number of mosquitoes/person/1 day.

Results

Species diversity assessed via the four mosquito sampling technique

Culicidae diversity based on larval surveys

Main mosquito larval habitats

A total of 993 potential breeding sites were inspected during the study, of which 506 (51.0%) were positive for mosquito larvae. Among the recorded habitats, jars represented the most frequent category (393 sites), with 195 positive sites, corresponding to a positivity rate of 49.6% (Fig. 2). Tires accounted for 135 sites, of which 89 (65.9%) contained larvae, making them among the most productive breeding habitats. Buckets also presented a high level of infestation, with 72 positive sites out of 120 inspected. Barrels and jerry cans accounted for 154 and 115 sites, respectively, with positivity rates of 46.8% (72/154) and 44.3% (51/115). In contrast, cisterns, animal watering troughs, and metal cans, which were less frequently encountered (40, 12, and 24 sites, respectively), presented more variable positivity rates, estimated at 22.5% (9/40), 41.66% (5/12), and 54.16% (13/24), respectively (Fig. 2). Overall, these findings indicate that domestic containers such as buckets, jars, and jerry cans constitute the most abundant and among the most productive breeding sites for Aedes in the study area.

Fig. 2.

Mosquito breeding sites (potential and positive)

Species diversity based on larval surveys

Larval habitat surveys yielded a total of 1,988 adult mosquitoes, belonging to seven species: Aedes aegypti, Ae. albopictus, Ae. vittatus, Anopheles gambiae, Culex nebulosus, Cx. quinquefasciatus, and Cx. tigripes, originating from various types of breeding sites (Table 1). The analysis of species composition revealed that Aedes aegypti was the most abundant species, accounting for 70.7% of all adults, and occurred in all types of habitats, with particularly high frequencies in buckets (86.6%), jerry cans (83.9%), and barrels (91.4%) (Table 1). Aedes albopictus was minimally represented (0.3%), whereas Ae. vittatus and Anopheles gambiae s.l. accounted for 1.1% and 1.5% of the total, respectively.

Table 1.

Species diversity from larval surveys

Types of breeding sites	Ae. Aegypti %(n/N)	Ae. albopictus %(n/N)	Ae. vittatus %(n/N)	An. gambiae %(n/N)	Cx. nebulosus %(n/N)	Cx. tigripes %(n/N)	Cx. quinquefasciatus %(n/N)	Total
Water troughs	100 (2/2)	–	–	–	–	–	–	100 (2/2)
Can	83.97 (241/287)	1.05 (3/287)	2.79 (8/287)	0.70 (2/287)	–	1.05 (3/287)	10.45 (30/287)	100 (287/287)
Tank	72.22 (91/126)	–	–	2.38 (3/126)	–	–	25.40 (32/126)	100 (126/126)
Tin can	50 (64/128)	–	–	0.78 (1/128)	–	0.78 (1/128)	48.44 (62/128)	100 (128/128)
Jar	71.33 (311/436)	–	0.69 (3/436)	3.21 (14/436)	0.46 (2/436)	1.83 (8/436)	22.48 (98/436)	100 (436/436)
Tires	41.12 (162/394)	0.25 (1/394)	2.79 (11/394)	1.02 (4/394)	1.02 (4/394)	0.51 (2/394)	53.30 (210/394)	100 (394/394)
Buckets	86.55 (296/342)	0.58 (2/342)	–	0.58 (2/342)	–	–	12.28 (42/342)	100 (342/342)
Barrel	91.36 (148/162)	–	–	1.85 (3/162)	–	–	6.79 (11/162)	100 (162/162)
Total	70.67 (1405/1988)	0.30(6/1988)	1.11 (22/1988)	1.46 (29/1988)	0.50 (10/1988)	0.70 (14/1988)	25.25 (502/1988)	1988

%(n/N): percentage (number of species/total number of species in the breeding sites)

Diversity of Culicidae collected via oviposition traps

The species diversity recorded from the oviposition traps included three genera, Aedes, Anopheles, and Culex, comprising eight species (Table 2). These were Aedes aegypti, Ae. albopictus, Ae. palpalis, Ae. vittatus, Anopheles gambiae s.l., Culex nebulosus, Cx. quinquefasciatus, and Cx. tigripes, with Aedes aegypti showing the highest frequency. The genus Aedes overwhelmingly dominated. Aedes aegypti accounted for nearly all the individuals collected across all the communities, with proportions ranging from 85.4% in Bassila to 97.5% in Savalou. Differences between localities were statistically significant (p < 0.001). Aedes albopictus was present at low frequencies, reaching maximum proportions in Djougou (6.5%) and Bassila (6.2%), with significant spatial variation (p < 0.001). Aedes palpalis and Ae. vittatus were marginal across sites (< 1.5%). No significant differences were detected for Ae. palpalis (p = 0.25477), whereas Ae. vittatus exhibited moderate but significant variation (p = 0.02656). Within the genus Anopheles, only Anopheles gambiae s.l. was detected, with very low proportions (< 1%) in the three communities and no significant spatial differences (p = 0.22546) (Table 2).

Table 2.

Composition of mosquitoes emerging from “egg-laying traps.”

Commune species	Bantè %(n/N)	Bassila %(n/N)	Djougou %(n/N)	Parakou %(n/N)	Savalou %(n/N)	p-value
Ae. aegypti	94.2 (211/224)	85.4 (345/404)	88.3 (462/523)	91.6 (630/688)	97.5 (426/437)	0.00001
Ae. albopictus	0.4 (1/224)	6.2 (25/404)	6.5 (34/523)	1.2 (8/688)	0.2 (1/437)	0.00001
Ae. palpalis	–	0.2 (01/404)	0.4 (2/523)	–	–	0.25477
Ae. vittatus	–	1 (04/404)	0.2 (1/523)	–	1.1 (5/437)	0.02656
An. gambiae	–	–	0.2 (1/523)	0.3 (2/688)	0.7 (3/437)	0.22546
Cx. nebulosus	–	–	–	1.6 (11/688)	–	0.00001
Cx. quinquefasciatus	5.4 (12/224)	7.2 (29/404)	4.2 (22/523)	4.9 (34/688)	0.5 (2/437)	0.00001
Cx. tigripes	–	–	0.2 (1/523)	0.4 (3/688)	–	0.07489
Total	100 (224/224)	100 (405/405)	100 (523/523)	100 (688/688)	100(437/437)

%(n/N): percentage (number of species/total number of species in the breeding sites

Mosquito collection by pyrethrum spray

During pyrethrum spray collection, five species were recorded: Ae. aegypti, Anopheles gambiae, An. pharoensis, Culex quinquefasciatus, and Culex nebulosus (Fig. 3). Aedes aegypti was moderately represented, accounting for 11% of the total catch, whereas Anopheles gambiae accounted for 7%, highlighting its relevance for monitoring dengue and malaria vectors. Notably, Ae. aegypti and Anopheles gambiae were observed in engorged states, indicating recent blood feeding and suggesting a potential risk of malaria and dengue transmission to residents (Fig. 3). Semigravid and gravid females were also detected, representing individuals capable of oviposition in the following days, thereby ensuring the persistence of their populations.

Fig. 3.

Graph showing the physiological state of mosquitoes

Mosquito collection via human landing catches

Combined diurnal and nocturnal collections yielded a total of 2,337 adult mosquitoes across the five study communes. Species composition was dominated by the genera Aedes and Anopheles, with marked spatial heterogeneity among localities (Table 3). The relative abundance of Aedes aegypti varied substantially between communes, with higher proportions in Bantè (29.8%) and Savalou (17.2%), whereas lower frequencies were recorded in Djougou (5.7%) and Parakou (3.8%). Aedes vittatus was detected at low proportions across all sites, indicating a marginal contribution to community structure. Within the genus Anopheles, Anopheles gambiae s.l. was widely distributed and accounted for a substantial proportion of captures, particularly in Djougou where it represented 38.5% of individuals, suggesting favorable ecological conditions for its development. In contrast, lower proportions were observed in Parakou (4.9%) and Savalou (7.3%). Anopheles pharoensis exhibited a more localized distribution, with a notable presence in Djougou (10.7%) and a more moderate contribution in Parakou (2.1%), while being absent or rare in the other communes. Anopheles funestus was recorded at very low frequencies. Intercommunal differences in species composition were statistically significant (p < 0.001 for the main species), indicating pronounced spatial heterogeneity in vector populations.

Table 3.

Species diversity based on combined day and night catches

Commune species	Bantè %(n/N)	Bassila %(n/N)	Djougou %(n/N)	Parakou %(n/N)	Savalou %(n/N)	p-value
Ae. aegypti	29.8 (79/265)	8.1 (36/442)	5.7 (41/720)	3.8 (26/677)	17.2 (40/234)	< 0.0001
Ae. vittatus	0.4 (1/265)	–	0.6 (4/720)	0.4 (3/677)	0.9 (2/234)	0.1990
An. funestus	–	0.5 (2/442)	0.1 (1/720)	–	–	0.2548
An. gambiae	18.9 (50/265)	8.8 (39/442)	38.5 (277/720)	4.9 (33/677)	7.3 (17/234)	< 0.0001
An. pharoensis	–	0.9 (4/442)	10.7 (77/720)	2.1 (14/677)	–	< 0.0001
Cx. nebulosus	–	–	–	0.7 (5/677)	0.4 (1/234)	0.0035
Cx. quinquefasciatus	50.9 (135/265)	81.7 (361/442)	44.4 (320/720)	87.6 (593/677)	74.7 (174/234)	< 0.0001
Mansonia uniformis	–	–	–	0.4 (3/677)	–	0.0173
Total	100 (265/265)	100 (442/442)	100 (720/720)	100 (677/677)	100 (234/234)

%(n/N): percentage (number of species/total number of species in the breeding sites)

Diversity indices supported these patterns. Species richness (Taxa S) ranged from four species in Bantè and Savalou to seven species in Parakou, reflecting variability in community complexity (Table 4). The Shannon diversity index (H′) was highest in Djougou (1.17 [1.12–1.21]), indicating a relatively balanced distribution of individuals among species, whereas lower values were observed in Parakou (0.55 [0.47–0.63]) and Bassila (0.65 [0.56–0.73]), suggesting stronger dominance by certain species. Consistently, the Simpson diversity index (1 − D) was higher in Djougou (0.64 [0.62–0.66]) and Bantè (0.62 [0.58–0.65]), indicating greater diversity, while lower values in Parakou (0.23 [0.19–0.27]) and Bassila (0.32 [0.26–0.37]) reflected reduced heterogeneity. Pielou’s evenness index (J′) further highlighted differences in community structure, with higher values in Bantè (0.75 [0.72–0.96]) and Djougou (0.65 [0.63–0.74]), indicating a more even distribution of individuals among species. Conversely, lower values in Parakou (0.28 [0.24–0.33]) and Bassila (0.40 [0.35–0.48]) indicated increased dominance by a limited number of species (Table 4).

Table 4.

Species diversity index

Index	Bantè	Bassila	Djougou	Parakou	Savalou
Individual	265	442	720	677	233
Taxa_S	4	5	6	7	4
Shannon_H (IC_95%)	1.04 [0.97–1.09]	0.65 [0.56–0.73]	1.17 [1.12–1.21]	0.55 [0.47–0.63]	0.75 [0.64–0.85]
Simpson_1-D(IC_95%)	0.62 [0.58–0.65]	0.32 [0.26–0.37]	0.64 [0.62–0.66]	0.23 [0.19–0.27]	0.41 [0.34–0.47]
Pielou_J (IC_95%)	0.75 [0.72–0.96]	0.40 [0.35–0.48]	0.65 [0.63–0.74]	0.28 [0.24–0.33]	0.54 [0.47–0.67]

IC_95%: 95% confidence interval

Hourly human biting rates (HBRs) of Anopheles gambiae s.l. and Aedes aegypti

Figures 4 and 8 illustrate the hourly biting cycles of the two primary vectors of malaria and dengue across the five study communities. During the daytime collection in Bantè, Ae. aegypti exhibited three peaks of biting activity indoors (Fig. 4A). The first peak occurred between 08:00 and 10:00, reaching 3 bites per person per hour, followed by a second peak of 2.5 bites per person per hour between 11:00 and 13:00. A third peak exceeded 6 bites per person per hour from 16:00 to 18:00. In addition to these periods of high activity, biting rates ranged from 0 to 1 bite per person per hour. In contrast, the hourly biting rates of An. gambiae remained low, averaging approximately 0.5 bites per person per hour. Outdoors (Fig. 4B), Ae. aegypti also displayed three periods of high biting activity: the first between 08:00 and 11:00 at 3 bites per person per hour, the second between 13:00 and 14:00 above 2.5 bites per person per hour, and the third from 15:00 to 17:00, reaching up to 4 bites per person per hour. For An. gambiae, only a single peak was observed between 17:00 and 18:00, with biting activity remaining low, ranging from 0 to 0.5 bites per person per hour throughout the day. During the nighttime collection, the pattern was reversed, with An. gambiae exhibited greater biting activity than Ae. aegypti did. For the indices (Fig. 4C), three peaks for An. gambiae activity was recorded as follows: 2.5 bites per person per hour between 21:00 and 23:00, 1.5 bites per person per hour from 00:00 to 01:00, and 1 bite per person per hour between 03:00 and 05:00. Outside of these periods, biting rates varied from 0 to 0.5 bites per person per hour. Conversely, Ae. aegypti showed no activity throughout the night. Outdoors, An. gambiae exhibited three activity peaks (Fig. 4D): the first peak from 20:00 to 22:00 at 1.5 bites per person per hour, the second peak from 22:00 to 00:00 at 1 bite per person per hour, and the third peak from 05:00 to 06:00 at 1 bite per person per hour. Ae. aegypti showed a single, minor peak outdoors from 01:00 to 02:00 at 0.5 bites per person per hour, remaining below the biting activity of An. gambiae; the remainder of the night was characterized by nearly zero activity.

Fig. 4.

HBR of mosquitoes captured during the day indoors (A) and outdoors (B) and at night indoors (C) and outdoors (D)

Fig. 8.

HBRs of mosquitoes captured during the day indoors (A) and outdoors (B) and at night indoors (C) and outdoors (D)

During daytime collection in Bassila, Ae. aegypti exhibited two peaks of biting activity indoors (Fig. 5A). The first peak occurred between 08:00 and 10:00, when 1 bite per person per hour was reached, and the second peak occurred from 15:00 to 17:00, when 1 bite per person per hour was also recorded. In addition to these periods of high activity, biting rates ranged from 0–0.5 bites per person per hour. In contrast, An. gambiae maintained low activity, varying between 0 and 0.5 bites per person per hour. Outdoors (Fig. 5B), Ae. aegypti also displayed two periods of elevated activity: the first between 09:00 and 10:00 at 1 bite per person per hour and the second between 15:00 and 17:00 at 2.5 bites per person per hour. An. gambiae showed a single peak outdoors from 17:00 to 19:00 at 1 bite per person per hour, with the remainder of the day at low levels ranging from 0 to 0.5 bites per person per hour. During nighttime collection, the pattern reversed, with An. Gambiae exhibited higher biting rates than Ae. aegypti did. Indoors (Fig. 5C), An. gambiae showed two peaks: 1 bite per person per hour between 23:00 and 01:00 and 2 bites per person per hour from 03:00 to 05:00. Between these peaks, biting activity remained near zero. Ae. aegypti exhibited a single indoor nocturnal peak of 1.5 bites per person per hour between 21:00 and 22:00, whereas other periods were characterized by low activity, ranging from 0 to 0.5 bites per person per hour. Outdoors (Fig. 5D), An. gambiae displayed three peaks: the first peak from 21:00 to 22:00 at 1 bite per person per hour, the second peak from 00:00 to 02:00 at 2 bites per person per hour, and the third peak from 03:00 to 06:00, reaching up to 2.5 bites per person per hour. In contrast, Ae. aegypti showed no nocturnal biting activity outdoors.

Fig. 5.

HBR of mosquitoes captured during the day indoors (A) and outdoors (B) and at night indoors (C) and outdoors (D)

During daytime collection indoors in Djougou (Fig. 6A), Ae. aegypti exhibited a single peak of biting activity between 08:00 and 10:00, reaching 1 bite per person per hour. Outside this peak period, biting rates ranged from 0 to 0.5 bites per person per hour throughout the rest of the day. An. gambiae consistently presented low indoor activity, varying between 0 and 0.5 bites per person per hour. Outdoors (Fig. 6B), Ae. aegypti displayed three periods of elevated biting activity. The first peak occurred from 08:00 to 10:00, the second peak occurred between 14:00 and 16:00, both at 1 bite per person per hour, and the third peak occurred from 17:00 to 19:00, reaching 2 bites per person per hour. An. gambiae exhibited a single outdoor peak between 11:00 and 12:00 at 1.5 bites per person per hour, with low activity (0–0.5 bites per person per hour) during the remainder of the day. During the nighttime collection, An. gambiae presented substantially higher biting rates than did Ae. aegypti. For the Indian people (Fig. 6C), a pronounced peak was observed between 23:00 and 04:00, exceeding 10 bites per person per hour. Outside these peak hours, biting activity remained near zero. Ae. aegypti maintain very low nocturnal activity indoors, ranging from 0 to 0.5 bites per person per hour. Outdoors (Fig. 6D), An. gambiae exhibited a major peak from 22:00 to 04:00, reaching up to 12 bites per person per hour, with a secondary peak from 00:00 to 02:00 at 2 bites per person per hour. In contrast, Ae. aegypti showed almost no nocturnal biting activity outdoors.

Fig. 6.

HBR of mosquitoes captured during the day indoors (A) and outdoors (B) and at night indoors (C) and outdoors (D)

In the commune of Parakou during the daytime, Ae. aegypti exhibited two peaks of biting activity both indoors (Fig. 7A) and outdoors (Fig. 7B). The first peak occurred between 08:00 and 09:00, when 2 bites per person per hour were reached, and the second peak occurred between 10:00 and 11:00, when 1 bite per person per hour was reached. Outside these periods of elevated activity, biting rates ranged from 0 to 0.5 bites per person per hour for the remainder of the day. The hourly biting rates of An. gambiae remained low, between 0 and 0.5 bites per person per hour, with a single peak between 12:00 and 13:00, reaching 1 bite per person per hour. During the nighttime collection indoors (Fig. 7C), biting rates for both An. gambiae and Ae. aegypti were low, ranging from 0 to 0.5 bites per person per hour. Outdoors (Fig. 7D), An. gambiae exhibited three peaks: the first peak occurred between 21:00 and 22:00 at 1 bite per person per hour, the second peak occurred between 01:00 and 03:00, with more than 4 bites per person per hour, and the third peak occurred from 04:00 to 06:00, with 2.5 bites per person per hour. In contrast, Ae. aegypti displayed nearly no nocturnal biting activity outdoors, with rates ranging from 0 to 0.5 bites per person per hour throughout the night.

Fig. 7.

HBR of mosquitoes captured during the day indoors (A) and outdoors (B) and at night indoors (C) and outdoors (D)

In the commune of Savalou, during daytime collection, Ae. aegypti exhibited two peaks of biting activity indoors (Fig. 8A). The first peak occurred between 08:00 and 11:00, reaching 2 bites per person per hour, and the second peak occurred between 12:00 and 15:00. A third peak was observed between 16:00 and 18:00, with a rate of 1 bite per person per hour. Outside these periods of elevated activity, biting rates ranged from 0 to 0.5 bites per person per hour for the remainder of the day. Outdoors (Fig. 8B), An. gambiae was absent. Ae. aegypti displayed three peaks of activity: the first from 08:00 to 10:00 at 1.5 bites per person per hour, the second from 12:00 to 14:00 at 1 bite per person per hour, and the third between 15:00 and 18:00. During the remaining periods, biting rates were low, ranging from 0 to 0.5 bites per person per hour. During the nighttime collection, the trend reversed, with An. gambiae showing greater biting activity than Ae. aegypti does. Indoors (Fig. 8C), An. gambiae exhibited two peaks: the first peak occurred between 01:00 and 03:00 at 1 bite per person per hour, and the second peak occurred between 04:00 and 05:00 at the same rate. Outside these peaks, biting rates ranged from 0 to 0.5 bites per person per hour throughout the night. The biting activity of Ae. aegypti was absent indoors. Outdoors (Fig. 8D), An. gambiae showed a single peak of 1.5 bites per person per hour between 01:00 and 05:00, with rates ranging from 0 to 0.5 bites per person per hour at other times. Ae. aegypti exhibited low activity throughout the night, ranging from 0 to 0.5 bites per person per hour.

Discussion

This study focused on examining the bionomics and species diversity of the main vectors of malaria and dengue fever in certain communities in central and northern Benin. Our findings show that Culicinae, which are composed of Aedes and Culex species, were more abundant than Anophelinae, which consists of An. gambiae s.l., An. pharoensis, and An. funestus, a pattern that is consistent with previous studies in southern Benin [14, 26]. Earlier surveys on mosquito diversity in Benin reported a broader range of species, not all of which were detected in the present study. For example, Djènontin et al. [26] documented 28 species in the Ouidah-Kpomassè and Tori health zones in southern Benin; Adoha et al. [14] reported 25 species in Covè, Zangnanado, and Ouinhi; Salako et al. [27] recorded 14 species in northern Benin; Tokponnon et al. [28] documented 12 species in the Atacora and Donga departments; Padonou et al. [29] recorded 14 species in Oueme; Agbanrin et al. [30] reported 15 species in Cotonou; and Kiam et al. [31] reported 14 species in Cameroon via human landing catches. Agbanrin et al. [30] and Konkon et al. [32] reported 13 and 5 species, respectively, via larval surveys and 4 species via oviposition traps in Lokossa, Bohicon, and Dassa [33]. Differences in species counts likely reflect variations in sampling methods, collection periods, and climate-driven shifts in mosquito dynamics.

The results also revealed distinct circadian patterns in biting activity between An. gambiae and Ae. aegypti. Ae. aegypti exhibited pronounced daytime biting, whereas An. gambiae showed a marked increase in nocturnal biting. These observations are consistent with previous studies describing nocturnal peaks for An. gambiae [34, 35] and a daytime predominance for Ae. aegypti [32, 36]. Across the studied communities, two to three peaks of Ae. aegypti activity were detected both indoors and outdoors. These patterns align with reports from Konkon et al. [32] in southern Benin and Dalpadado et al. [36] in western Sri Lanka. In contrast, the hourly biting rates of An. gambiae remained low during the day (0–0.5 bites/person/hour), further confirming its nocturnal behavior [34]. During nighttime collection, the pattern reversed, with An. gambiae exhibited higher biting rates than Ae. aegypti did, which intensified from 22:00 to 05:00. Similar observations have been reported in Kilombero Valley, Kamuli District in Uganda, southern Mozambique, and Cameroon [31, 35, 37, 38]. However, the biting profiles in this study differ from those reported in some regions of Africa and the Pacific, where peaks in Anopheles activity shift toward early evening or early morning, reflecting behavioral plasticity [39–41]. These variations suggest that mosquito biting dynamics can adapt to local ecological and anthropogenic pressures, influencing human exposure risk. Environmental factors and adaptation to human behavior can modulate mosquito host-seeking rhythms [42, 43]. Mosquitoes may adjust their activity patterns on the basis of human habits and local control strategies, altering exposure risk [44]. The observed spatial and temporal co-occurrence of these two major vectors indicates that human populations in these localities are potentially exposed to malaria and dengue pathogens [38, 45, 46]. This aligns with Howell et al. [46], who showed that vector-borne disease transmission in tropical regions can be facilitated by co-occurring vectors and simultaneous human exposure to temporally and spatially differentiated bites. Co-occurrence is explained by overlapping ecological niches that suit species with distinct biological requirements: An. gambiae s.l., typically associated with relatively clear, oxygenated water bodies and seasonal environmental conditions, and Ae. aegypti, adapted to artificial containers and domestic environments [12, 13]. The simultaneous presence of nutrients is driven by species-specific breeding and blood-feeding preferences and the physicochemical characteristics of aquatic habitats resulting from human activity [12]. Urbanization and human activity create diverse breeding sites, enhancing human-vector interfaces [47–49]. Although the observed biting rates were modest, the persistent coexistence of these vectors in the same environment increases the likelihood of repeated exposures and the theoretical risk of malaria‒dengue coinfection. Several limitations should be considered. The study employed a primarily cross-sectional design, capturing the entomological situation during surveys but not reflecting full seasonal fluctuations. Additionally, the relatively limited number of communities may restrict spatial representativeness and generalizability. Future studies would benefit from longitudinal designs covering multiple seasons to better characterize temporal variability in vector populations and vector–host interactions. Despite these constraints, the data provide valuable insights into the ecology and diversity of mosquito vectors in central and northern Benin, providing a scientific basis for guiding future entomological surveillance and vector control strategies.

Conclusion

The results of this study reveal a high level of species diversity among mosquito vectors, indicating the presence of primary malaria and dengue vectors in the same environments. Analysis of human bite rates reveals species-specific host-seeking activity: Aedes aegypti contributes notably during the day, while Anopheles gambiae s.l. contributes notably at night. This circadian complementarity of biting behaviors indicates continuous human exposure to vectors and increases the risk of concurrent malaria and arbovirus transmission. The multimethod approach adopted enabled better characterization of vector population structure and dynamics by integrating their diversity and interaction with human hosts. These results can guide targeted interventions against malaria and dengue vectors for the National Malaria Control Program (NMCP) and the National Program for the Control of Communicable Diseases (NPCCD), respectively.

Abbreviations

An

Anopheles

Ae

Aedes

CREC

Centre de Recherche Entomologique de Cotonou

Cx

Culex

HBR

Human biting rate

PSC

Pyrethrum spray catch

NMCP

National Malaria Control Program

NPCCD

National Program for the Control of Communicable Diseases

WHO

World Health Organization

Author contributions

The original study was conducted by GGP, IH, AKK, DMZ, and MCA, who also provided the data. The study concept was conceived and developed by the GGP and IH. The data were collected by IH, AKK, MDZ, AS, RVA, ASS, and AF. IH and AKK drafted the manuscript. The statistical analysis of the data was performed by IH and ACK. The maps were produced by IH and CJA. ASS and LB critically reviewed the manuscript. All the authors have read and approved the final version of the submitted manuscript.

Funding

No funding was received for this study.

Data availability

All the data collected and analyzed in this study and the materials used are available from the corresponding author.

Declarations

Ethics approval and consent to participate

The study protocol was reviewed and approved by the Institutional Ethics Committee for Health Research of the Centre de Recherche Entomologique de Cotonou (CIERS-CREC) under approval number 06-22/CREC/CIERS-CREC/SG. Prior to the initiation of field activities, informed consent was obtained from mosquito collectors, as was authorization from household heads and local authorities at all study sites, in accordance with the Declaration of Helsinki. Mosquito collectors were recruited from local communities, taking into account their usual exposure to mosquito bites. They received prior training on mosquito collection techniques and protective measures aimed at minimizing bites during collection activities. All participants were vaccinated against yellow fever and were monitored regularly for health status throughout the study period. In addition, antimalarial chemoprophylaxis was provided free of charge to volunteers before and after the collection campaigns. In the event of febrile symptoms, participants were immediately referred to a physician at the nearest health center for appropriate medical management.

Consent to publication

All the authors have read and agreed to the publication of this article.

Competing interests

The authors declare no competing interests.