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Background The selection and the spread of insecticide resistance in malaria vectors to the main classes of insecticides used in vector control tools are a major and ongoing challenge to malaria vector control programmes. This study aimed to determine the intensity of vector resistance to insecticides in three regions of Benin with different agro-ecological characteristics. Methods Larvae of Anopheles gambiae sensu lato (s.l.) were collected from September to November 2017 in different larval sites in three northern Benin communes: Parakou, Kandi and Malanville. Two to five-day-old, non-blood-fed, female mosquitoes were exposed to papers impregnated with deltamethrin, permethrin and bendiocarb at dosages of 1 × the diagnostic dose, 5 × and 10 × to determine the intensity of resistance in these vectors. Molecular frequencies of the kdr L1014F and ace-1R G119S insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled at each study site. Results Resistance to pyrethroids (permethrin and deltamethrin) was recorded in all three communes with mortality rates below 60% using the diagnostic dose (1x). The results obtained after exposure of An. gambiae to permethrin 10 × were 99% in Kandi, 98% in Malanville and 99% in Parakou. With deltamethrin 10x, mortality rates were 100% in Kandi, 96% in Malanville and 73% in Parakou. For the diagnostic dose of bendiocarb, suspected resistance was recorded in the communes of Malanville (97%) and Kandi (94%) while sensitivity was observed in Parakou (98%).Using the 10 × dose, mortality was 98% in Kandi, 100% in Malanville and 99% in Parakou. The frequencies of the kdr L1014F allele varied between 59 and 83% depending on the sites and species of the An. gambiae complex, while the frequency of the ace-1R G119S gene varied between 0 and 5%. Biochemical tests showed high levels of oxidase and esterase activity compared to the susceptible colony strain of An. gambiae sensu stricto (Kisumu strain). Conclusion Anopheles gambiae showed a generalized loss of susceptibility to permethrin and deltamethrin but also showed moderate to high intensity of resistance in different regions of Benin. This high intensity of resistance is a potential threat to the effectiveness of vector control.

Background Insecticide resistance is threatening the effectiveness of efforts to control malaria vectors in Benin. This study explores the levels and mechanisms of insecticide resistance in An. gambiae s.l. to pyrethroids. Methods Larvae were collected from August 2017 to July 2018 in five communes in southern Benin (Adjohoun, Allada, Bohicon, Cotonou, and Porto-Novo) representing diverse ecological regions, and were reared in Benin’s insectary. Two- to five-day-old female mosquitoes from each district were exposed to multiple doses of deltamethrin and permethrin (1×, 2×, 5×, and 10×) using the WHO insecticide resistance intensity bioassay. The effect of pre-exposure to the synergist, piperonyl butoxide (PBO), was also tested at different pyrethroid doses. Molecular allele frequencies of kdr (1014F) and ace-1R (119S) insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled from each study area. Results An. gambiae s.l. were resistant to pyrethroid-only exposure up to 10× the diagnostic doses in all the study sites for both deltamethrin and permethrin. Mortality was significantly higher in An. gambiae s.l. pre-exposed to PBO followed by exposure to deltamethrin or permethrin compared to mosquitoes exposed to deltamethrin or permethrin only (p kdr resistance allele (1014F) frequency was high (75–100%), while the ace-1 resistance allele (G119S) frequency was low (0–3%). Analysis of the metabolic enzymatic activity of An. gambiae s.l. showed overexpression of nonspecific esterases and glutathione S-transferases (GST) in all study sites. In contrast to the PBO results, oxidase expression was low and was similar to the susceptible An. gambiae s.s. Kisumu strain in all sites. Conclusion There is high-intensity resistance to pyrethroids in southern Benin. However, pre-exposure to PBO significantly increased susceptibility to the pyrethroids in the different An. gambiae s.l. populations sampled. The use of PBO insecticide-treated bed nets may help maintain the gains in An. gambiae (s.l.) control in southern Benin. Graphical Abstract

BackgroundChanges in the natural habitats of insect groups are determined the genetic polymorphisms between individuals. The objective of this study was to establish the genetic structure of theAnopheles coluzziipopulations in four localities of Benin.MethodsInsecticide surveys and larval sampling were conducted on 4 study localities, including Cotonou, Ketou, Zagnanado, and Sô-Ava. Molecular characterizations were performed on theAnophelesmosquitoes collected with the allelic and genotypic frequencies ofkdrgene determined. The multiple comparison Chi square test for proportions was performed with R version 3.3.3. Next, the observed heterozygosity, expected heterozygosity, and indices of fixation, and genetic differentiation were estimated. Finally, the Hardy–Weinberg equilibrium (EHW) was determined to assess whether panmixia exists in the different populations of mosquitoes of the agroecological zones under study.ResultsCarbamates, pyrethroids, organophosphorus and organochlorines use have been reported in all localities except Sô-Ava.Anopheles coluzziiwas strongly represented across all study localities. TheL1014Fallele was observed in the localities of Kétou, Cotonou and Zagnanado. Likewise, insecticide selection pressure of homozygous resistant individuals (L1014F/L1014F) was significantly higher in Kétou, Cotonou and Zagnanado (pvalue L1014Fallele despite the reported absence of pesticide use was observed. All mosquito populations were found to be deficient in heterozygosity across the study sites (FIS0). No genetic differentiation (FST0) was observed in the localities of Zagnanado and Kétou.ConclusionThe survey on the use of insecticides showed that insecticide selection pressures differ across the investigated localities. It would be desirable to rotate or apply formulations of combined products with different modes of action. Doing so would enable a better management of resistant homozygous individuals, and mitigate the resistance effect of commonly used insecticides.

Monitoring vector resistance to insecticides is a major component of resistance management. Adults of Anopheles gambiae s.l. from larvae collected at thirteen sites were tested with papers impregnated with bendiocarb, permethrin; deltamethrin; pirimiphos-methyl and induced bottles of insecticide including bendiocarb; deltamethrin, permethrin and pirimiphos-methyl. Molecular analyses were performed and detoxification enzyme levels were determined for each study site. This study revealed a generalized distribution of resistance of An. gambiae s.l. to pyrethroids (permethrin and deltamethrin) and a clear resistance to carbamates (bendiocarb) especially in the northern region of the country. This vector is still susceptible to organophosphates, especially pirimiphos-methyl, but resistance is a dynamic phenomenon and it would be necessary to monitor the susceptibility of these anopheles to pyrimiphos-methyl and to find alternative vector control methods to slow down the spread of resistance genes.

Background: It is generally accepted that one of the reasons for the declining effectiveness of vector control tools (LLINs, IRS) is the misuse of these tools. Some people sleep outside while the walls of houses are treated with insecticide to protect people from mosquito bites.Methods:To check whether communities that misuse these tools are more exposed to malaria, we used the technique of direct and participatory observation of sleeping habits and we clocked every 30 min, the activities and positions of the communities from 7pm to 7am. The survey was conducted in 3 departments Northern Benin. These were mainly treatments related to variance analysis and descriptive statistics. Softwares used included SPSS 21, SAS 9.2 and Arcgis 10.4.Results: The study shows that 95% of the villages population included in the study stay in their rooms from 7:00 pm to 7:00 am, and only 5% of household members are outdoors during same time intervals during the harmattan season. On the other hand, during hot weather, there are more households outdoors (37%). Less than 20% of household members are outside their rooms during the harmattan season. This number decreases gradually until 10:00 pm when all of them are inside their rooms.Student Newman Keuls test shows that the household members being inside or outside their rooms does not depend on the urbanization situation of the village, but rather on the period.65% exposed to mosquito bites gave irritation as the main reason for their behavior and 15% because of heat and lack of space. Overall, because of household member mobility and malaria vector behaviors, human exposure to An. gambiae was higher outside than inside, despite all the observed food preferences in the vectors.Conclusion:To curtail residual malaria transmission, it must be prioritized to raise awareness in the best use of the tools and provide other complementary control tools for use outside the home.

Background: It is generally accepted that one of the reasons for the declining effectiveness of vector control tools (LLINs, IRS) is the misuse of these tools. Some people sleep outside while the walls of houses are treated with insecticide to protect people from mosquito bites.Methods:To check whether communities that misuse these tools are more exposed to malaria, we used the technique of direct and participatory observation of sleeping habits and we clocked every 30 min, the activities and positions of the communities from 7pm to 7am. The survey was conducted in 3 departments Northern Benin. These were mainly treatments related to variance analysis and descriptive statistics. Softwares used included SPSS 21, SAS 9.2 and Arcgis 10.4.Results: The study shows that 95% of the villages population included in the study stay in their rooms from 7:00 pm to 7:00 am, and only 5% of household members are outdoors during same time intervals during the harmattan season. On the other hand, during hot weather, there are more households outdoors (37%). Less than 20% of household members are outside their rooms during the harmattan season. This number decreases gradually until 10:00 pm when all of them are inside their rooms.Student Newman Keuls test shows that the household members being inside or outside their rooms does not depend on the urbanization situation of the village, but rather on the period. 65% exposed to mosquito bites gave irritation as the main reason for their behavior and 15% because of heat and lack of space. Overall, because of household member mobility and malaria vector behaviors, human exposure to An. gambiae was higher outside than inside, despite all the observed food preferences in the vectors.Conclusion:To curtail residual malaria transmission, it must be prioritized to raise awareness in the best use of the tools and provide other complementary control tools for use outside the home.

Background Since 2008, Indoor Residual Spraying (IRS) has been performed in Benin in 19 districts, including 4 in southern Benin, 9 in Atacora, and 8 in Atacora, Alibori and Donga in northern Benin. However, Benin still struggles with questions about IRS cost–benefit and epidemiological impact. Lessons learned and challenges from 10 years of IRS in Benin to be shared with the stakeholders involved in vector control implementation for decision-making. Methods Entomological parameters have been assessed entomological parameters in IRS communes since 2008. In all IRS intervention communes, decreases in human biting rate (HBR) of Anopheles gambiae, blood feeding inhibition and entomological inoculation rate (EIR) as compared to control district have been measured. Results EIR was reduced by 80–90%, which is encouraging, but should be observed with caution because: (i) the reduction may be insufficient to decrease epidemiological indicators given that the residual EIR in IRS districts is still higher than it is in some regions of stable malaria; (ii) the reduction in EIR is based on comparisons with control communes, but it is difficult to select control areas with the same environmental characteristics as intervention areas; (iii) despite the reduction, half of all mosquitoes that entered IRS-treated houses succeeded in taking human blood meals. Further, there are behaviours among Benin’s population that limit IRS efficacy, including recent data showing that > 90% of people are not protected by IRS between 7 and 10 p.m. This is due to the fact that they remain outdoors and that most people are not protected from mosquito bites after 10 p.m. because they either sleep outdoors without IRS protection or indoors without an ITN. Moreover, people have large amounts of clothing hanging on walls where mosquitoes can rest instead of IRS-treated walls. Finally, other components are important to consider in implementing IRS among which: (i) Vector resistance management strategies are sometimes poorly understood; this is actually different from the need to replace one insecticide with another after the emergence of resistance; (ii) African countries should prepare to finance IRS themselves. Conclusion To curtail residual malaria transmission, additional interventions able to target vectors escaping IRS should be prioritized.

Background Despite the success of indoor residual insecticide spraying (IRS) in Africa, particularly in Benin, some gaps of information need to be filled to optimize the effectiveness of this intervention in the perspective of the country’s effort to eliminate malaria. In anticipation to the 2018 IRS campaign in two targeted regions of northern Benin, this study aimed, to collect baseline information on vector composition, spatio-temporal variation and peak malaria transmission in the Alibori and Donga, two targeted regions of northern Benin. Information collected will help to better plan the implementation and later on the impact assessment of this IRS campaign. Methods The study was carried out in four districts of the two IRS targeted regions of northern Benin. Human landing catches and pyrethrum spray catches protocols were used to assess the biting rate (HBR) and, biting/resting behaviour of malaria vector populations. After morphological identification of collected Anopheles, the heads and thoraxes of Anopheles gambiae sensu lato (s.l.) were analysed by the ELISA CSP tests to estimate the sporozoite index (SI). The entomological inoculation rate was calculated as the product of mosquito biting rate (HBR) and the SI. Results The biting rates of An. gambiae s.l., the major vector in this study sites, varied significantly from region to region. It was higher: in rural than in urban areas, in rainy season than in dry season, indoors than outdoors. Overall, SI was comparable between sites. The highest EIRs were observed in the Donga region (16.84 infectious bites/man/month in Djougou district and 17.64 infectious bites/man/month in Copargo district) and the lowest in the Alibori region (10.74 infectious bites/man/month at Kandi district and 11.04 infectious bites/man/month at Gogounou district). Conclusion This study showed the heterogeneous and various nature of malaria epidemiology in Northern Benin. Indeed, the epidemiological profile of malaria transmission in the Alibori and Donga regions is made of a single season of transmission interrupted by a dry season. This period of transmission is relatively longer in Donga region than in Alibori. This information can be used to guide the extension of IRS in the Alibori and in the Donga, by primarily targeting areas with short periods of transmission, and easy to cover.

Background The current study shows the results of three years of IRS entomological monitoring (2016, before intervention; 2017 and 2018, after intervention) performed in Alibori and Donga, northern Benin. Methods Mosquito collections were performed on a monthly basis using human landing catches and pyrethrum spray catches in six districts including four treated with Actellic 300 CS (Kandi, Gogounou, Djougou and Copargo) and two untreated (Bembèrèkè and Kouandé) which served as control sites. Key transmission indicators of Anopheles gambiae (s.l.) as well as the residual activity of Actellic 300 CS assessed through WHO cone tests, were determined. Results The residual efficacy duration of Actellic 300 CS after the two IRS campaigns (2017 and 2018) was 4–5 months (May–September). The parity rate and the sporozoite index of An. gambiae (s.l.) were 36.62% and 0.71%, respectively, after the first spray round in treated areas compared to 57.24% and 3.7%, respectively, in the control areas (P vs 9.75 ib/m in the control areas, corresponding to a reduction of 84.61%. A decrease in the parity rate (46.26%), sporozoite index (85.75%) and EIR (87.27%) was observed for An. gambiae (s.l.) after the first round of IRS (June–October 2017) compared to the pre-intervention period (June–October 2016). The density of An. gambiae (s.l.) ranged between 0.38–0.48 per house in treated areas vs 1.53–1.76 An. gambiae (s.l.) per house respectively after the first and second IRS rounds. Conclusions This study showed the positive impact of IRS in reducing key entomological parameters of malaria transmission in Alibori and Donga. However, the considerable blood-feeding rate of An. gambiae (s.l.) in spray areas, stress the need for the population to sleep under long-lasting insecticidal nets (LLINs) in addition, to prevent from mosquito bites which did not succeed in resting on sprayed walls.

Background: Climate variability influence the diversity and abundance of malaria vectors and thereby on malaria transmission dynamics. Examine its effect on Anopheles parameters involved in transmission may predict the potential malaria hotspot as a right target for its control intervention strategies. Here, we investigated the influence of meteorological parameters on the aggressiveness and infectivity of Anopheles in two health districts zones where IRS has been extended in Northern Benin. Methods: Mosquito collections were carried out using human landing catches to evaluate rates of aggression and infectivity in twelve villages. Concomitantly, meteorological data from synoptic stations of Benin and neighbouring countries were collected in 2016-2017. Results: The spatial distribution of infective bites of An. gambiae is characterized by an intense aggression in the rural villages of the study area. Analysis of variances showed significant HBR difference according to the period but not according to the locality. However, the same analysis carried out with the infectivity rate shows no significant difference according to the period and the locality. In addition, the number of infective bites per man per month is higher in August and October, and the climatic parameters that have mainly favoured aggression are wind speed, humidity, sunshine and temperature. Indeed, the peak of wind speed is concentrated around 1.2 km / h and in September (5 km / h) whereas the aggressiveness score of Anopheles in the region is greater than 10 infective bites per man a year. Conclusion Malaria transmission by Anopheles is influenced by climatic factors. The climate observed in the districts where IRS was extended in northern Benin has a real impact on Anopheles density and weakens current and future vector control strategies. This could lead to a series of modifications observed in anopheline populations just after IRS implementation ranging from a tendency to exophagy, from a decrease in the rate of blood-feeding to changes in the time, and change in aggressiveness. These phenomena most likely contribute to the sustainability of malaria transmission despite vector control measures. Keywords: Infectivity, aggression, Climate, Anopheles gambiae ( s.l. ), IRS, Benin.

Background This study aims to provide baseline data on the resistance status to insecticides, the frequency of mechanisms involved and the impact of the association with the synergist piperonyl butoxide (PBO) on resistant Anopheles gambiae (s.l.) populations in two regions of northern Benin, prior to an indoor residual spraying campaign and introduction of next generation long-lasting insecticidal nets (LLINs) incorporating PBO. Methods Adult Anopheles gambiae (s.l.) originating from larvae collected in two study regions (Alibori within the Kandi-Gogounou-Segbana districts and Donga within the Djougou-Copargo-Ouake districts) were tested with impregnated papers (bendiocarb 0.1%, pirimiphos-methyl 0.25%, permethrin 0.75% and deltamethrin 0.05%). The synergist PBO was used to check for the involvement of detoxification enzymes in pyrethroid resistant populations. Molecular analyses were performed for the identification of species within the Anopheles gambiae (s.l.) complex and kdr L1014F and G119S Ace-1 mutations. Biochemical assays assessed the activity of detoxification enzymes. Results Anopheles gambiae (s.l.) was resistant to pyrethroids, with a mortality range of 25–83% with deltamethrin and 6–55% with permethrin. A significant increase in mortality was observed after pre-exposure to PBO for both deltamethrin (63–99%) and permethrin (56–99%). With bendiocarb, An. gambiae (s.l.) were susceptible in Kandi (99% mortality), with possible resistance (92–95%) recorded in Djougou, Copargo, Gogounou, Ouake and Segbana. All study populations were fully susceptible to pirimiphos-methyl. The frequencies of resistant mutations varied according to species and sites: 0.67–0.88 for L1014F kdr and 0–0.06 for G119S Ace-1. Three study locations (Djougou, Gogounou and Kandi) showed high oxidase activity and four sites (Djougou, Ouake, Copargo and Kandi) showed elevated esterase activity. Conclusions This study confirms resistance to pyrethroids and suggests emerging bendiocarb resistance in An. gambiae (s.l.) populations in northern Benin. However, recovery of susceptibility to pyrethroids after PBO exposure, and susceptibility to organophosphates in the An. gambiae (s.l.) populations indicate that next generation LLINs incorporating PBO synergist combined with an indoor residual spraying (IRS) campaign with organophosphate insecticides may be regarded as alternative control tools.

Background: Malaria transmission is based on four essential elements: the vector, the parasite, humans and the environment. However, of the four elements, the environment is not sufficiently exploited. Methods: In the research presence carried out in 6 localities in North Benin, we used a microscopic vision of the health geographer, focusing on certain components of geography, entomology and meteorology to show what to show spatial disparities in malaria transmission using Arcgis 10.4, Global Mapper and SPSS 21.0 for regression and correlation analysis. Results: The results of our research show that the slopes are unstable. Also, the lower the altitude, the lower the water kinetics and consequently a lot of water stagnation favourable for the development of mosquito breeding sites. The explanatory power of the regression model means that 54.3% of the variation in positive mosquito breeding is explained by human population density. Conclusions: Benin must make significant progress in the elimination of malaria using a new effort to understand the ecology of vector mosquitoes based on spatial disparities in the fight against malaria.

Background: Climate variability influence the diversity and abundance of malaria vectors and thereby on malaria transmission dynamics. Examine its effect on Anopheles parameters involved in transmission may predict the potential malaria hotspot as a right target for its control intervention strategies. Here, we investigated the influence of meteorological parameters on the aggressiveness and infectivity of Anopheles in two health districts zones where IRS has been extended in Northern Benin. Mosquito collections were carried out using human landing catches to evaluate rates of aggression and infectivity in twelve villages. Concomitantly, meteorological data from synoptic stations of Benin and neighbouring countries were collected in 2016-2017. The spatial distribution of infective bites of An. gambiae is characterized by an intense aggression in the rural villages of the study area. Analysis of variances showed significant HBR difference according to the period but not according to the locality. However, the same analysis carried out with the infectivity rate shows no significant difference according to the period and the locality. In addition, the number of infective bites per man per month is higher in August and October, and the climatic parameters that have mainly favoured aggression are wind speed, humidity, sunshine and temperature. Indeed, the peak of wind speed is concentrated around 1.2 km / h and in September (5 km / h) whereas the aggressiveness score of Anopheles in the region is greater than 10 infective bites per man a year. Malaria transmission by Anopheles is influenced by climatic factors. The climate observed in the districts where IRS was extended in northern Benin has a real impact on Anopheles density and weakens current and future vector control strategies. This could lead to a series of modifications observed in anopheline populations just after IRS implementation ranging from a tendency to exophagy, from a decrease in the rate of blood-feeding to changes in the time, and change in aggressiveness. These phenomena most likely contribute to the sustainability of malaria transmission despite vector control measures.