Evolution of the Ace-1 and Gste2 Mutations and Their Potential Impact on the Use of Carbamate and Organophosphates in IRS for Controlling Anopheles gambiae s.l., the Major Malaria Mosquito in Senegal.

Widespread of insecticide resistance amongst the species of the Anopheles gambiae complex continues to threaten vector control in Senegal. In this study, we investigated the presence and evolution of the Ace-1 and Gste2 resistance genes in natural populations of Anopheles gambiae s.l., the main malaria vector in Senegal. Using historical samples collected from ten sentinel health districts, this study focused on three different years (2013, 2017, and 2018) marking the periods of shift between the main public health insecticides families (pyrethroids, carbamates, organophosphates) used in IRS to track back the evolutionary history of the resistance mutations on the Ace-1 and Gste2 loci. The results revealed the presence of four members of the Anopheles gambiae complex, with the predominance of An. arabiensis followed by An. gambiae, An. coluzzii, and An. gambiae-coluzzii hybrids. The Ace-1 mutation was only detected in An. gambiae and An. gambiae-coluzzii hybrids at low frequencies varying between 0.006 and 0.02, while the Gste2 mutation was found in all the species with a frequency ranging between 0.02 and 0.25. The Ace-1 and Gste2 genes were highly diversified with twenty-two and thirty-one different haplotypes, respectively. The neutrality tests on each gene indicated a negative Tajima's D, suggesting the abundance of rare alleles. The presence and spread of the Ace-1 and Gste2 resistance mutations represent a serious threat to of the effectiveness and the sustainability of IRS-based interventions using carbamates or organophosphates to manage the widespread pyrethroids resistance in Senegal. These data are of the highest importance to support the NMCP for evidence-based vector control interventions selection and targeting.

Evolution of the Pyrethroids Target-Site Resistance Mechanisms in Senegal: Early Stage of the Vgsc-1014F and Vgsc-1014S Allelic Frequencies Shift.

The evolution and spread of insecticide resistance mechanisms amongst malaria vectors across the sub-Saharan Africa threaten the effectiveness and sustainability of current insecticide-based vector control interventions. However, a successful insecticide resistance management plan relies strongly on evidence of historical and contemporary mechanisms circulating. This study aims to retrospectively determine the evolution and spread of pyrethroid resistance mechanisms among natural Anopheles gambiae s.l. populations in Senegal. Samples were randomly drawn from an existing mosquito sample, collected in 2013, 2017, and 2018 from 10 sentinel sites monitored by the Senegalese National Malaria Control Programme (NMCP). Molecular species of An. gambiae s.l. and the resistance mutations at the Voltage-gated Sodium Channel 1014 (Vgsc-1014) locus were characterised using PCR-based assays. The genetic diversity of the Vgsc gene was further analyzed by sequencing. The overall species composition revealed the predominance of Anopheles arabiensis (73.08%) followed by An. gambiae s.s. (14.48%), Anopheles coluzzii (10.94%) and Anopheles gambiae-coluzii hybrids (1.48%). Both Vgsc-1014F and Vgsc-1014S mutations were found in all studied populations with a spatial variation of allele frequencies from 3% to 90%; and 7% to 41%, respectively. The two mutations have been detected since 2013 across all the selected health districts, with Vgsc-L1014S frequency increasing over the years while Vgsc-1014F decreasing. At species level, the Vgsc-1014F and Vgsc-1014S alleles were more frequent amongst An. gambiae s.s. (70%) and An. arabiensis (20%). The Vgsc gene was found to be highly diversified with eight different haplotypes shared between Vgsc-1014F and Vgsc-1014S. The observed co-occurrence of Vgsc-1014F and Vgsc-1014S mutations suggest that pyrethroid resistance is becoming a widespread phenomenon amongst malaria vector populations, and the NMCP needs to address this issue to sustain the gain made in controlling malaria.

Mapping the breeding sites of Anopheles gambiae s. l. in areas of residual malaria transmission in central western Senegal.

Despite the deployment of several effective control interventions in central-western Senegal, residual malaria transmission is still occurring in some hotspots. To better tailor targeted control actions, it is critical to unravel the underlying environmental and geographical factors that cause the persistence infection in hotspot villages. "Hotspots villages" were defined in our study as those reporting more than six indigenous malaria cases during the previous year. A total of ten villages, including seven hotspots and three non-hotspots, were surveyed. All potential mosquito breeding sites identified in and around the ten study villages were regularly monitored between 2013 and 2017. Monitoring comprised the detection of anopheline larvae and the collection of epidemiological, hydrogeological, topographical, and biogeographical data. The number of larval breeding sites described and monitored during the study period ranged from 50 to 62. Breeding sites were more numerous in hotspot sites in each year of monitoring, with 90.3% (56/62) in 2013, 90.9% (50/55) in 2014, 90.3% (56/62) in 2015 and 86% (43/50) in 2017 (Fisher exact test; p = 1). In the non-hotspot areas, the data for the same years were, respectively, 9.7% (6/62), 9.1% (5/55), 9.7% (6/62) and 14% (7/50) (p = 1). The Hotspot villages were characterized mostly by saline or moderately saline hydro-morphic and halomorphic soils allowing water retention and a potential larval breeding sites. By contrast, non-hotspot villages were characterized mainly by a high proportion of extremely permeable sandy-textured soils, which due to their porosity had low water retention. The annual number of confirmed malaria cases was correlated with the frequency and extent of breeding sites. Malaria cases were significantly more frequent in the hamlets located near breeding sites of An. gambiae s.l., gradually decreasing with increasing remoteness. This study shows that the characteristics of larval breeding sites, as measured by their longevity, stability, proximity to human habitation, and their positivity in Anopheles larvae are likely determining factors in the persistence of malaria hotspots in central-western Senegal. The results of this study shed more light on the environmental factors underlying the residual transmission and should make it possible to better target vector control interventions for malaria elimination in west-central Senegal.