Study area
This study was carried out in Benin, more specifically in 23 of the 77 communes in the country (Fig. 1). Ten of the investigated communes were selected due to their closeness to Nigeria, where Ae. albopictus was first detected three decades ago.
The 23 communes were selected according to their representativeness of the 03 major eco-climatic zones in the country:
Area of degraded forests with a subequatorial climate
This area is characterized by two rainy seasons (Mid-March to Mid-July, and September to November), and two dry seasons (December to Mid-March, and Mid-July to August). Its annual rainfall varies from 1300 to 1500 mm/year [28]. The communes surveyed in this area, as part of this study include: Abomey-Calavi, Porto-Novo, Adjara, Avrankou, Ifangni, Sakété, Pobè, Kétou, Lokossa, Klouékanmè, Zagnanado, Bohicon and Grand Popo (Fig. 1). Abomey-Calavi and Porto-Novo are the most urbanized while the others are peri-urban or rural.
Area of savannas with a Sudano-Guinean climate
This area has two seasons: a rainy season (April to October) and a dry one (November to March). Its annual rainfall varies between 1200 and 1300mm [28]. Investigated communes in this area include Savè, Dassa, Bantè, Parakou, Nikki, Djougou and Corpago which are predominantly peri-urban or rural (Fig. 1).
Area of savannahs with a Sudanian climate
A dry season from November to May, and a rainy season from June to October characterize this area. Its climate is of the Sudanian type. The communes of Gogounou and Ségbana were investigated here [28] (Fig. 1).
Overall, in each of all these communes, the surveys were carried out in two villages selected at random. The geographical coordinates of the surveyed villages were recorded using smartphones in order to map the distribution and density of Ae. albopictus and Ae. aegypti.
Mosquito collection techniques
To determine the different mosquito species of the Aedes genus, even those present at low frequency in all the study communes, three sampling techniques were used between July 2021 and October 2022. These are:
Ovitrapping
The ovitraps used, were made with a painted black polyethylene bottle, which contained 50 cl of water. A rectangular hardboard plate (5 cm x 20 cm) was introduced in the black polyethylene bottle to serve as a support for mosquito egg laying. Twelve ovitraps were placed per site, with 4 sites surveyed per commune. These traps were approximately 100 metres apart from each other. They were fixed to a tree or a wall, about 1.5 cm from the ground, using a nail and a metal string, for about 5 to 7 days in the domestic (yard) and peri-domestic environments. The ovitraps were regularly inspected to avoid egg hatching as much as possible. However, when larvae were observed in the traps, they were collected and brought back to the insectary of the “Centre de Recherche Entomologique de Cotonou” (CREC) for rearing until adulthood. The adults were then identified, counted and released into cages. Between the 5th and the 7th day, the ovitraps were withdrawn, and the hardboard plate brought back to the insectary of CREC. The eggs laid were counted and put in water at the insectary to enable hatching and development until adulthood. Adults that emerged were then identified and counted. They were grouped into a pool of 10 individuals, taking into account their geographical origin, their sex, the date of collection, then stored at -80°C.
Collection of mosquito immature stage
Larvae and pupae were sampled in different types of larval habitats such as tin cans, coconut shells, water containers, tires of abandoned vehicles, jars, tree holes. These larvae and pupae were then transported to the CREC insectarium where they were reared until adulthood.
Human Landing Catch (HLC)
This method that collects adult mosquitoes was used in two sites (one central and one peripheral) selected in each commune. On each site, collections on human bait were carried out from 7 a.m. to 6 p.m. in two houses, with one collector seated inside and a second outside in each house, which makes a total of four collectors/site/day and 16 collectors/commune/day. Two teams of eight collectors each were therefore formed per commune. The first team collected from 7 a.m. to 1 p.m., and was replaced by the second one from 1 p.m to 6 p.m. Collectors used haemolysis tubes to capture mosquitoes that tended to bite their bare feet and legs.
Morphological and molecular identification
Adult mosquitoes from the three sampling methods (ovitrapping, collection of immature, and HLC) were morphologically identified using a binocular and the keys of Edwards [29], and Yiau-Min [30]. Specimens of Aedes spp were referenced, preserved on RNA Later, and grouped according to species, locality, date and location (indoor/outdoor).
The legs of specimens of Aedes albopictus were used for the extraction of their DNA using the protocol of Linton et al. [31]. Due to a high degree of interspecific variation [32, 33], the ITS2 nuclear ribosomal spacer gene was amplified by PCR using primers 5.8S and 28S [34, 35]. The PCR product of Ae. albopictus was 509 bp and 518 bp according to sequences published on GenBank M95127 [35] and L22060 [36], respectively. PCR was performed in a volume of 50 μl containing 1× PCR buffer, 2 mM MgCl2, 0.2 μM of each dNTP, 100 pM of each primer, 2 U of Taq DNA polymerase and 2 μl of DNA to be amplified. The amplification was carried out using a thermocycler according to the following programme: initial denaturation at 94°C for 10 min; 40 cycles of denaturation at 94°C for 1 min; initial hybridization at 50°C for 1 min and hybridization at 72°C for 1 min. Final hybridization was performed at 72°C for 10 min. The products were migrated on 1.5% agarose gels containing 0.5 μg/ml ethidium bromide. Gels were photographed with a polaroid camera under UV illumination with standard procedures [37].
Ethical considerations
The protocol of this study was reviewed and approved by the “Comité Institutionnel d’Ethique pour la Recherche en Santé du Centre de Recherche Entomologique de Cotonou” (CIERS-CREC) (Ethical approval N°06-22/CREC/CIERS-CREC /SG). The collectors used were selected from the different study sites and trained to collect mosquitoes before they bite. They were all vaccinated against yellow fever and subjected to regular check-ups at the nearest health facility. In the occurrence of fever, they were immediately taken care of.
Data analysis
Data were analyzed using R statistical software, version 4.1.2 [38]. The Chi-square test of comparison of proportions was used to compare the distribution of each species according to the different eco-climatic areas. Species richness (S), which corresponds to the number of collected species, and their relative abundance (Pi) were computed (Pi = ni/N, where ni = number of species i; N = total number of species encountered; i = 1:S) per study site.
The Shannon-Weaver index (H), which shows the diversity of species, was determined in all 23 sites according to the formula: \(\text{H}= -\sum _{i=1}^{S}{P}_{i}*{log}_{2}{(P}_{i})\) [39].
The equity index was also calculated:\(\text{E}=\frac{ H}{{{log}}_{2}S}\) [40].
All these parameters were determined with the combined number of Aedes species obtained with the three sampling techniques to assess the proportion of the main dengue vectors and estimate the level of risk.
The human biting rate (HBR) for Ae. aegypti and Ae. albopictus was calculated as the number of mosquito species collected divided by the number of collectors day. The Poisson method [41] was used to estimate the confidence intervals of HBRs and compare them between study sites and locations (indoors and outdoors).