Background

Aedes aegypti is the main indigenous vector of arboviruses in Africa [1]. In Benin, it remains abundant in both the south and the north parts of the country [2, 3]. Recent studies have reported the presence of Aedes albopictus, a vector native to Asia, in Southern Benin [4, 5]. Indeed this mosquito species is known to be invasive, and was involved in several cases of arbovirus epidemics [6, 7]. Dengue which was considered more prevalent in Asia and Latin America, has now spread to several West African countries [8], where all four serotypes of the virus are actively circulating [9]. This worrying situation deserves more attention, as severe forms of the disease are often found in areas where more than two serotypes of the virus coexist [10, 11].

Several cases of dengue fever have been diagnosed in European tourists returning from Benin in recent years [12,13,14]. Indeed, those travelers have tested positive for DENV-1, a virus serotype native to Asia [14]. Moreover, DENV-3 was recently detected in a sample of Aedes aegypti from Porto-Novo, the political capital of Benin [15].

Several factors make Benin a country likely to experience an arbovirus epidemic. These factors include: the high demographic growth, the development of trade of second hand cars and tires, the development of tourism, the presence of two major vectors of arboviruses, the growing urbanization of the country, and its proximity with Nigeria, an endemic country with which it shares a 773 km border. Except for yellow fever for which an effective vaccine is available, the other arboviruses have no curative or preventive treatment [16]. As a result, vector control appears to be the only way to control the disease transmission [17]. This control could be through the use of chemicals for killing immature stages or adult mosquitoes, the destruction of vector breeding sites resulting in the reduction of their density, and the sensitization of the population on good practices for water conservation and sanitation. Even with a strong community engagement, it is quite difficult to identify and destroy all breeding sites in the environment. Therefore, chemical control through the implementation of spatial spraying, and use of long lasting insecticidal nets (LLINs) may be the most effective way to control dengue vectors. Several studies have recently reported resistance of Aedes aegypti to pyrethroids and carbamates in Africa and Asia [18,19,20]. Ae. aegypti is well adapted to urban habitats and, therefore, is generally more susceptible to insecticide exposure and resistance than Ae. Albopictus[21].In West Africa, most of the available data on resistance of Aedes mosquitoes to insecticides, are for Ae. aegypti only, despite the reported introduction of Ae. albopictus in Nigeria since 1971. Recently, 238 and 380 confirmed cases of dengue fever have been recorded in Senegal and Côte d'Ivoire, respectively [22, 23]. To plan an effective and sustainable control strategy against arbovirus vectors in Benin, there is an urgent need to determine the susceptibility level of Ae. albopictus to insecticides commonly used in public health, and update data on the resistance status of Ae. aegypti. The present study aims to assess the resistance profile of these two main vectors of arboviruses, in Benin.

Methods

Study area

The study was carried out in the urban and peri-urban areas of 6 communes belonging to two departments in Southern Benin. These are the communes of Avrankou, Adjara, and Porto-Novo in the department of Ouémé and the communes of Kétou, Ifangni and Pobè in the department of Plateau (Fig. 1). These communes that mostly neighbor Nigeria, were surveyed because of the presence recently reported of Ae. albopictus and Ae. aegypti [6]. The whole study area is characterized by a subequatorial climate with two rainy seasons (March to July, and September to November) and two dry seasons (November to February and July to August) with rainfall ranging from 1200 to 1500 mm/year. There are three groups of ecosystems, namely, coastal plain ecosystems, bar land plateau ecosystems and Lama depression clay ecosystems. Long-lasting insecticidal nets (LLINs) distributed every three years throughout the country, and repellents are the main vector control tools used in the study area.

Fig. 1
figure 1

Map showing the study area

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Mosquito collection

Three sampling techniques were used to collect immature stages and adults of Aedes spp. from July 2021 to October 2022. Irrespective of the collection method, the morphological identification of adult mosquitoes was performed using the taxonomic keys of Edwards [24] and Huang & Rueda [25].

Larvae collection

Immature stages of Aedes spp. were sampled from various breeding sites (abandoned jars, tires, cans, and other containers) using the dipping technique. They were filtered and stored in labeled jars, and then transported to the insectary of the Centre de Recherche Entomologique de Cotonou (CREC) for rearing until adulthood.

Ovitrapping method

The ovitraps were used as part of the present study. They were made of polyethylene bottles which were filled with 50 cl of water. A hardboard plate (5 cm by 20 cm) immersed into the water, served as support for the eggs laid. A total of 12 ovitraps were set per site. These traps were hung on a tree/wall at a height of 1.5 m from the ground, using a nail and a metal string, and left for one week in the domestic or peri-domestic environment. To avoid egg-hatching, the inspection of traps occurred on a daily basis. They were removed after 7 days, and the eggs laid on the hardboard plates were brought back to the insectary and put in water. After the hatching, the larvae were reared until adult stage.

Human landing catches

This method was used to collect adults of Aedes spp. during the day. In all study communes, mosquito collections were conducted both indoors and outdoors, with a first group of collectors that worked from 7:00 am to 1:00 pm and which was replaced by a second group from 1:00 pm to 6:00 pm. At each collection point, a volunteer with bare-legged and barefoot serving as bait, collected mosquitoes using hemolysis tubes and a flashlight. The specimens of Aedes spp. that were collected, were released in cages and transported to the insectary.

Biological materials

Females Ae. aegypti field-collected as larvae, were used for the WHO susceptibility tube testing.

For Ae. albopictus, mosquitoes from the three collection methods were reared at the insectary, in order to have a sufficient number of individuals of F1 generation. Only the females mosquito of this generation, were used for the WHO susceptibility tube testing.

Insecticide susceptibility tests

WHO susceptibility tube tests were performed according to the WHO protocol [26], using non-blood-fed females Ae. aegypti (F0) and Ae. albopictus (F1), aged 2–5 days. These mosquitoes were exposed to the following products:

  • -deltamethrin (0.05%), permethrin (0.75%), and alphacypermethrin (0.05%);

  • -alphacypermethrin (0.05%) + PBO (4%).

Batches of 20–25 mosquitoes were introduced into each tube carpeted with an insecticide-treated paper for a 1-h exposure. The number of mosquitoes knocked down by the insecticide at different time intervals (5, 10, 15, 20, 30, 45, 60 min) was recorded. A batch of 20–25 mosquitoes exposed to an insecticide-free paper was used as a control.

The PBO synergist is an inhibitor of oxidases [27]. It helped assessing the involvement of oxidases in the pyrethroid resistance observed in the populations of Aedes. The PBO-pyrethroid, and pyrethroid-only tests were performed simultaneously on the same mosquito populations. After 60 min of exposure, mosquitoes were transferred into observation tubes and kept at 25 °C and 80% humidity, with free access to a 10% sweetened juice. Mortality after 24 h was determined according to the WHO protocol.

Biochemical analyses

Thirty females Ae. aegypti from each district, aged 2–5 days, and non-previously exposed to any insecticide, were used for biochemical analyses. These were performed to compare the expression level of detoxification enzymes (mixed function oxidases, non-specific esterases and glutathione S-transferases) of different populations of Aedes aegypti to the reference susceptible strain (Rockefeller), following the protocol described by Hemingway et al. [28].

Data analysis

Mortality rates from the susceptibility tube tests were interpreted according to the WHO protocol [26]. When a mosquito population had a mortality rate between 98 and 100%, it was considered susceptible. When mortality was between 90 and 97%, the population was suspected of resistance. Below a mortality rate of 90%, the population was said resistant. The 24-h mortality rates and the 60-min knockdown rates were compared using the Chi-square test for comparison of proportions. A linear regression with analysis of variance was used to assess the variation in enzyme activity for each mosquito population. The Mann–Whitney U test was used to compare the enzyme activity between the field mosquito populations and the susceptible laboratory strain (Rockefeller). Statistical analyses were performed using R 3.3.2 software [29].

Results

Mortality and knockdown rates

In total, 3517 females Aedes (1787 Ae. aegypti, and 1730 Ae. albopictus), were bioassayed.

Table 1 shows the pyrethroid-induced mortalities in the different Aedes mosquito populations. After 60 min of exposure to the different insecticides, the knockdown rates observed in Ae. albopictus was very high in all communes, ranging between 94 and 100% (Table 1). A 100% mortality rate was observed in the different populations of Ae. albopictus exposed to deltamethrin and permethrin, which indicates a full susceptibility of these populations to the two tested pyrethroids (Table 1). Similarly, the population of Ae. albopictus from Kétou showed full susceptibility to alphacypermethrin (100%). However, with the same product, a suspected resistance was observed in Adjarra (94.67% [86.19–98.27]), Ifangni (94.38% [86.78–97.91]) and Porto-Novo (95.7% [88.73–98.61]), and a resistance in Avrankou (83% [73.89–89.50]) (Table 1).

Table 1 Mortality observed 24 h after exposure of Aedes albopictus to permethrin, deltamethrin and alpha-cypermethrin
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In the populations of Ae. aegypti, a different trend was observed, with much more variable 60-min knockdown rates. Indeed, irrespective of the pyrethroid insecticide, these rates ranged from 63.51% [51.45–74.16] in Porto-Novo to 100% [95.29–100] in Ifangni (Table 2). 24-h post-exposure, populations of Ae. aegypti from Avrankou (91% [83.16–95.54]), Adjarra (93.75% [85.38–97.67]), Porto-Novo (93.51% [84.89–97.58]) and Kétou (95.65% [88. 61–98.59]) showed a suspected resistance to alpha-cypermethrin (0.05%), while the one of Ifangni displayed a full susceptibility to the same insecticide (Table 2). With deltamethrin (0.05%), a suspected resistance was observed in Avrankou (95% [88. 17–98.14]), Adjarra (95.29% [87.73–98.48]), Ifangni (90.91% [83.00–95.49]), and Kétou (93.87% [86.62–97.48]), while there was a resistance to the same product in Porto-Novo (63.51% [51.45–74.16]). Furthermore, resistance to permethrin was observed in all tested populations of Ae aegypti, except for the Ifangni strain that displayed a suspected resistance (97% [90.84–99.22]) (Table 2).

Table 2 Mortality observed 24 h after exposure of Aedes aegypti to permethrin, deltamethrin and alpha-cypermethrin
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Figure 2 shows the mortality induced by bendiocarb and pirimiphos methyl. Overall, all tested populations of Ae. aegypti displayed a full susceptibility (mortality rates ≥ 98%) with these two products.

Fig. 2
figure 2

Mortalities observed 24 h after exposure of Ae. aegypti to bendiocarb and pirimiphos methyl

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The PBO-alphacypermethrin tests performed with the four field strains of Ae. albopictus led to a mortality rate = 100% and higher than the one for the insecticide alone, indicating a full involvement of oxidases and esterases in alphacypermethrin resistance (Fig. 3).

Fig. 3
figure 3

Mortality rates of Aedes albopictus with alpha-cypermethrin alone and alpha-cypermethrin + PBO

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Expression of oxidases, esterases and GSTs in Aedes aegypti

Figure 4 shows the mean levels of enzyme activities in the field populations of Ae. aegypti, and in the reference susceptible strain (Rockefeller).

Fig. 4
figure 4

Activities of glutathione-S-transferase (A), mono-oxygenase (B), β-esterases (C), and α-esterases (D) in Ae. aegypti collected at Pobè and Ifangni

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The results show an overexpression of GSTs in the population of Ae. aegypti from Pobè (P < 0.0001) compared to the Rockefeller susceptible strain (Fig. 4A). A significantly elevated oxidase activity was observed in the populations of Ae. aegypti from Pobè and Ifangni, compared with that of the Rockefeller susceptible strain (P > 0.001) (Fig. 4B). In contrast, no overexpression of the α and β esterase activity was seen in all the tested populations compared with the Rockefeller susceptible strain (Fig. 4C, D).

Discussion

Studies on insecticide resistance profile have long been performed in Benin in Anopheles vectors of malaria, but very little is done on Aedes responsible for arboviruses, even though the country is close to countries Nigeria, Ghana, Cote d’Ivoire, Burkina-Faso that in recent years have become dengue endemic. Recently, Ae. albopictus, a highly invasive Asian mosquito, has entered Benin and lives in sympatry with Aedes aegypti in urban and peri-urban areas [4]. Both species are described as the main vectors of arboviruses [3, 4]. The present study provides knowledge on their insecticide resistance profile, which will help to better orient the vector control strategy to implement in case of a dengue epidemic.

Findings of the present study showed full phenotypic susceptibility of the different populations of Ae. albopictus tested to permethrin and deltamethrin, despite the widespread resistance observed of Anopheles vectors and Ae. aegypti to these two pyrethroids in Benin [4, 30]. This could be because permethrin and deltamethrin are among the type II pyrethroids with high toxicity. However, with alpha-cypermethrin, Ae. albopictus was resistant in Avrankou, and suspected of resistance in Adjarra, Porto-Novo and Ifangni. This shows the beginning of the emergence of resistance of Ae. albopictus to alphacypermethrin, a type I pyrethroid with a relatively low toxicity compared to type IIs. Previous studies had already revealed a relative susceptibility of this mosquito species to pyrethroids [21]. This onset of resistance of Ae. albopictus to pyrethroids was reported by Ngo et al. [31] and Yougang et al. [32] in the cities of Douala and Yaoundé in Cameroon, respectively. According to these authors, this rapid expansion of resistance in Ae. albopictus could result from domestic or organic pollutants, as this species is widely distributed in water tanks, spare tires and discarded containers, which are widely distributed in cultivated agricultural sites [33]. It is also possible that the susceptibility of this species has been affected by the increased use of repellent insecticides in peri-urban settings. However, this resistance of Ae. albopictus to alpha-cypermethrin was reversed by the synergist PBO in all communes, confirming the involvement of oxidases in pyrethroid resistance in the localities studied [34]. The deployment of next-generation LLINs combining the PBO synergist with a pyrethroid insecticide could be considered an alternative option for an effective control of populations of Ae albopictus. At the opposite, Ae. aegyptiwas found to be much more resistant or suspected of being resistant to pyrethroids. Similar observations were previously made in Cameroon [32]. This supports the hypothesis of an occurrence of a strong insecticide resistance selection in Ae. aegypti relative to Aedes albopictus. It is likely that these species exhibit different biting and resting behaviors, which could explain variable insecticide exposure. Indeed, Ae. aegypti has been frequently collected indoors in urban environments, while Ae. albopictus is much more sampled outdoors in a peri-urban environment. This particular behavior of Ae. aegypti could expose it more to indoor insecticide-based interventions such as insecticide sprays, aerosols, or LLINs to prevent nuisance in urban settings [35,36,37,38]. This high resistance of Ae. aegypti compared to Ae. albopictus has been reported in different epidemiological settings in Central and West African countries [39,40,41] such as Benin [3]. Reduced susceptibility of Aedes mosquitoes to pyrethroids is widespread and has been reported in several countries [17]. Our results corroborate previous studies from Thailand that reported high resistance to deltamethrin and permethrin in Ae. aegypti [42, 43]. Our biochemical data revealed the overexpression of MFOs and GSTs in populations of Ae. aegypti from Ifangin and Pobè. Oxidases are involved in the detoxification of pyrethroids in mosquitoes [44, 45]. The enormous quantities of insecticides of the same class used in agriculture could be at the origin of this overproduction of oxidases. The glutathione-S-transferase activity observed in our vector populations confirms the strong resistance to DDT observed by Yadouleton et al. [2] in Ae. aegypti in Benin. High GST expression may be due to overexpression of the GST2 gene [45]. In contrast, the low esterase activity observed would certainly be because the vectors from Ifangni and Pobè have not been or are weakly exposed to carbamates and organophosphates. However, a full susceptibility of populations of Ae. aegypti to bendiocarb and pyrimiphos-methyl was observed in all the surveyed communes. This can be explained by the low esterase activity observed in Ifangni and Pobè, where the use of pyrimiphos methyl and carbamates is low. It would be also interesting to characterize the Kdr and Ace-1R resistance mechanisms in populations of Aedes vectors, as they are commonly found in Anopheles mosquitoes. Carbamates and organophosphates could also be considered a good alternative for the control of arbovirus vectors in Benin in case of epidemics. Though our findings suggest that permethrin and deltamethrin could also be used, there is a growing concern that Ae. albopictus develop resistance to these pyrethroid insecticides. Ae. aegypti may develop other resistance mechanisms, such as behavioral or physiological one, to circumvent the modes of action of these insecticides. It would be interesting to investigate all the resistance mechanisms in Ae. aegypti to establish its full resistance profile to make good vector control decisions.

Conclusion

The present study showed full susceptibility of Ae. albopictus to deltamethrin and permethrin but widespread resistance to pyrethroids in Ae. aegypti in the study area. This suggests that these insecticides (deltamethrin and permethrin) can be used in arboviruses control programs in sites where Ae. albopictus is reported as the main vector. Given pyrethroids resistance observed in Ae. aegypti, and the emergence alpha-cypermethrin resistance in Ae. albopictus, rational use of insecticides, especially pyrethroids, should be encouraged to help reduce insecticide pressure. The synergist PBO increased the mortality of Ae. albopictus to alpha-cypermethrin, showing the involvement of oxidases. However, full susceptibility of Ae. aegypti to bendiocarb and pirimiphos methyl was observed in all study communes.

Given the increasing number of dengue cases in neighboring countries, strategies to improve vector control and prevent the spread of the diseases in Benin are urgently needed.