Study areas: This study was conducted in the villages of Karadié (7.60W, 13.24N) and Koula (7.65W, 13.12N) in the district of Koulikoro and Kolondialan (7.51W, 13.49N) and N'Galamadibi (7.48W, 13.48N) in the district of Banamba (Figure 1). The climate in both districts is typical Sudano -sahelian savannah with two seasons: a long dry season from November to May and a wet season from june to october with a mean annual rainfall of 900–1200 mm. The monthly mean temperature during the rainy season varies from 29 to 33ºC. An. gambiae s.l. is the predominant malaria vector (> 98%). Malaria transmission occurs mostly during the rainy season (June to October) with the mean monthly mosquito human biting rate reaching its peak in August and September.
LLINs, SMC for Children of 3 to 59 months old, and IPT with SP for pregnant women are control interventions implemented in both districts. Besides this, PMI has supported ten IRS campaigns in the districts of Koulikoro (2008 to 2016), while it has never been implemented in the district of Banamba. Agriculture, livestock and trade are the main economic activities.
Data collection: In each selected village, vector data were collected every month from June to November 2016 by Pyrethrum Spray Catch (PSC) and Entry Windows Traps (EWT).
Pyrethroid spray catches (PSC). In each study village, 30 sentinel houses were randomly selected from a list of total households. The sampling took into account the type of housing (e.g. thatch roof vs metal roof). Indoor-resting mosquitoes were collected by PSC which consisted of spraying pyrethrum in the selected houses between 8:00 to 11:00 in the morning. Two teams of 2 entomologists (a total of 4) were sampling 10-15 houses per day.
Entry window trapping (EWT). This method consisted of mounting on inside bedrooms windows the traps in order to trap the mosquitoes entering the rooms. Ten traps were mounted in ten randomly selected rooms. The catches took place during three successive nights per month. One of the local guides of the different villages was responsible for closing the traps very early in the morning (around 6:00 am) to prevent the trapped mosquitoes to come out with a small curtain sewn on the opening of the traps. The trapped mosquitoes were removed from the traps using a mouth aspirator, held in carton cups, and the number recorded on a data sheet.
Insecticide susceptibility bioassay (WHO, 2016). Anopheles gambiae larvae were collected in different types of breeding habitats in and around each site and reared to adults at the insectary (F0 generation). At the emergence, adult stages of 2-5 day-old young females were exposed to insecticides-impregnated papers (permethrin [0.75%], deltamethrin [0.05%] and pirimiphos-methyl [0.25%]). Approximately 20-25 mosquitoes per tube with 2-6 replicates were exposed to diagnostic concentrations of insecticides on impregnated filter papers for 1hour, and then transferred to a clean holding tube supplied with 10% sugar. Mortalities post exposures were determined after 24 hours according to who standard procedures. All the tests were carried out at 27 ± 1°C and a relative humidity of 70-80%.
Samples processing. Collected mosquitoes were identified into species (An. gambiae s.s. An. coluzzii and An. arabiensis) by Polymerase Chain Reaction technique . The sporozoïte infection rate  and human blood index (HBI)  were established using Enzyme Immuno-Sorbent Assay (ELISA) technique.
Statistical analysis. The data were entered in Excel and analyzed in SPSS version 22, STATA version 10, and GraphPad Prism 7. The following entomological parameters were calculated: vector density, man biting rate (MBR), sporozoite index (SI), entomological inoculation rate (EIR), human blood index HBI) and parity rate (PR). The density of malaria vector was calculated as the average number of indoor resting mosquitoes per room; the MBR as the average number of mosquito bites (fed + half gravid over rooms’ sleepers) per person per time unit; the SI corresponds to the percentage of anopheles of a given species carrying sporozoites; the HBI as the proportion of females mosquito having human blood in their guts; the PR was calculated as the proportion of parous females relative to the total number of mosquitoes dissected (Pare and nulliparous); and the EIR was calculated as the total number of infectious bites per human per time unit. The Pearson correlation test was used to determine the correlation between density and rainfall. The Chi-square test was used to compare the MBR, HBI, and PR.
Mosquito’s mortality rates were calculated by dividing the number of survivor mosquitoes by the number exposed to the insecticide.
- Mortality in the range of 98–100% indicates susceptibility of the mosquitoes.
- A mortality of less than 98% is suggestive of the existence of resistance and further investigation is needed.
- If mortality is less than 90%, confirmation of the existence of resistant genes in the test population with additional bioassays is not necessary, provided that at least 100 mosquitoes of each species were tested.