Study area
EVALMOUS study was carried out in 15 villages of Sô‐Ava district (6° 28′ 00″ N 2° 25′ 00″ E) located in Nokoue Lake in Southern Benin. Sô‐Ava is one of the eight districts of Atlantic department with about 100 000 inhabitants and a surface area of 218 km².
Mosquito collection
Mosquito collection was carried out in all villages selected for EVALMOUS study, using human landing catches method (HLCs) as described by Djènontin et al, 2010 [10]. Mosquito collection was carried out both indoors and outdoors in three sites per village for one night during the rainy season between July and August 2016. Mosquitos were collected from 7 p.m. to 9 a.m. Collector teams were rotated among the collection points to minimize sampling bias.
Laboratory analysis
Mosquitoes were identified to species level using morphological criteria according to the identification keys [11, 12]. All mosquitoes identified as Anopheles gambiaesensu lato (s. l.) were stored in individual tubes with silica gel and preserved at -20°C in the laboratory.
Heads and thoraces of anopheline females were tested by enzyme‐linked immunosorbent assays (ELISA) for detection of Plasmodium falciparum circumsporozoite protein (CSP), as previously described [13].
Genomic DNA was extracted from the abdomen of all mosquito using the extraction buffer Livak [14]. The species was then identified by diagnostic Polymerase Chain Reaction (PCR) using the Scott protocol [15] and molecular characterization according to Santolamazza protocol [16, 17]. The knockdown resistance mutation (kdr‐west, L1014F) was detected by Taqman allelic discrimination assays as described by Bass et al [18].
Pregnant women sleeping behaviour and bed net use
Data regarding the time at which each pregnant woman entered and exited their house, the time each bed net user entered and exited their sleeping space the night preceding the survey were collected by questionnaire.
Data analysis
Every hour from 7 p.m. to 9 a.m, the proportion of malaria vectors collected indoors was determined, as well as the proportion of pregnant women under bed nets at each hour from 7 p.m. to 9 a.m the night preceding the survey.
The mosquito collection period was divided into 3 different timeframes (7 p.m. – 10 p.m.; 10 p.m. – 6 a.m.; 6 a.m. – 9 a.m.). The proportion of mosquito collected at each timeframe (aggressiveness) was calculated, as well as the 95% confidence interval.
CSP index in malaria vectors Anopheles gambiaes.l. in each village was calculated as the number of positive vector divided by the total tested. Endophagy was calculated as the number of vectors collected indoors divided by the total collected. The 95% confidence interval of the CSP index and endophagy were also estimated. Allelic frequency of kdr gene in Anopheles gambiaes.l. at each period was calculated and compared using Chi-square tests.