BackgroundInsecticide resistance monitoring is essential to help national programmers to implement more effective and sustainable malaria control strategies in endemic countries. This study reported the spatial and seasonal variations of insecticide resistance in malaria vectors in Benin, West Africa.MethodsAnopheles gambiae s.l populations were collected from October 2008 to June 2010 in four sites selected on the basis of different use of insecticides and environment. WHO susceptibility tests were carried out to detect resistance to DDT, fenitrothion, bendiocarb, permethrin and deltamethrin. The synergist piperonyl butoxide was used to assess the role of non-target site mechanisms in pyrethroid resistance. Anopheles gambiae mosquitoes were identified to species and to molecular M and S forms using PCR techniques. Molecular and biochemical assays were carried out to determine kdr and Ace.1R allelic frequencies and activity of the detoxification enzymes.ResultsThroughout the surveys very high levels of mortality to bendiocarb and fenitrothion were observed in An. gambiae s.l. populations. However, high frequencies of resistance to DDT and pyrethroids were seen in both M and S form of An. gambiae s.s. and Anopheles arabiensis. PBO increased the toxicity of permethrin and restored almost full susceptibility to deltamethrin. Anopheles gambiae s.l. mosquitoes from Cotonou and Malanville showed higher oxidase activity compared to the Kisumu susceptible strain in 2009, whereas the esterase activity was higher in the mosquitoes from Bohicon in both 2008 and 2009. A high frequency of 1014F kdr allele was initially showed in An. gambiae from Cotonou and Tori-Bossito whereas it increased in mosquitoes from Bohicon and Malanville during the second year. For the first time the L1014S kdr mutation was found in An. arabiensis in Benin. The ace.1R mutation was almost absent in An. gambiae s.l.ConclusionPyrethroid and DDT resistance is widespread in malaria vector in Benin and both metabolic and target site resistance are implicated. Resistance was not correlated with a change of malaria species and/or molecular forms. The 1014S kdr allele was first identified in wild population of An. arabiensis hence confirming the expansion of pyrethroid resistance alleles in Africa.

BackgroundLarge-scale implementation of Indoor Residual Spraying and Insecticide Treated Nets has been implemented in Plateau Department, Benin between 2011 and 2014. The purpose of this study was to monitor the frequency and mechanisms of pyrethroid resistance in malaria vectors following the implementation of vector control tools for malaria prevention.MethodsAnopheles larvae were collected in 13 villages twice a year from 2012 to 2014. WHO tube tests were used to assess the phenotypic resistance of each population to 0.05 % deltamethrin. Sibling species within Anopheles gambiae complex were identified by PCR techniques. Taqman and biochemical assays were performed to identify the presence of kdr mutations in individual mosquitoes and to detect any increase in the activity of enzymes putatively involved in insecticide metabolism (oxidases, esterase and glutathione-S-transferases). Quantitative real time PCR was used to measure the expression of three metabolic genes involved in pyrethroid resistance (CYP6P3, CYP6M2 and GSTD3).ResultsAnopheles populations showed < 90 % mortality to deltamethrin in all villages and at all time points. The 1014 F kdr allele frequency was close to fixation (> 0.9) over the sampling periods in both An. gambiae and An. coluzzii. Biochemical assays showed higher activities of alpha esterase and GST in field malaria vector populations compared to susceptible mosquitoes. qPCR assays showed a significant increase of CYP6P3, CYP6M2 GSTD3 expression in An. gambiae after a three-year implementation of LLINs.ConclusionThe study confirmed that deltamethrin resistance is widespread in malaria vectors in Southern Benin. We suspect that the increase in deltamethrin resistance between 2012 and 2014 resulted from an increased expression of metabolic detoxification genes (CYP6M2 and CYP6P3) rather than from kdr mutations. It is urgent to evaluate further the impact of metabolic resistance on the efficacy of vector control interventions using pyrethroid insecticides.

BackgroundThis 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.MethodsAdult 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.ResultsAnopheles 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.ConclusionsThis 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 Several studies carried out in Benin have shown the involvement of Anopheles gambiae sensu lato (s.l.), the Anopheles funestus group in malaria transmission, but none of them reported the contribution of the Anopheles nili group to the transmission of this disease. The current study investigated the question through an entomological cross-sectional survey performed in Northern Benin. Methods Mosquito samplings were performed in September and October 2017 in 4 villages located in two districts: Bambaba and Wodara (Kérou district) and, Péhunco 2 and Béké (Péhunco district). The collections were carried out indoors and outdoors using human landing catches (HLC) to assess the human biting rate (HBR) and pyrethrum spray catches (PSC) to evaluate the blood feeding rate and the blood meal origin using the ELISA test. All collected mosquitoes were morphologically identified and, the polymerase chain reaction (PCR) technique was used for molecular identification of sibling species of An. gambiae s.l., An. funestus group and An. nili group sporozoite index (SI) was also assessed by the ELISA test. Results Overall, An. gambiae s.l., An. funestus group and An. nili group were the three vectors found in the study area. A significantly higher human biting rate (HBR) was recorded in An. nili group (5 bites/human/night) compared to An. funestus group (0.656 bites/human/night) in the Kérou district (p < 0.0001). Anopheles gambiae s.l. displayed the highest HBR (26.19 bites/human/night) in the same district. The entomological inoculation rate (EIR) was 1.875 infected bites/human/month in An. nili group against 13.05 infected bites/human/month in An. gambiae s.l. and 0.938 infected bites/human/month in An. funestus group in Kérou. In Péhunco, the EIR was 1.02 infected bites/human/month in An. gambiae s.l. PCR results showed that An. nili sensu stricto (s.s.) and An. funestus s.s. were the only species of the An. nili and An. funestus groups, respectively. The anthropophagic character of An. gambiae s.l. was also highlighted. Conclusion This study provides useful information on the contribution of An. nili group as secondary vector to malaria transmission in northern Benin. Broader studies must also be carried out in a larger study area to assess the involvement of other Anopheles species to malaria transmissi...

Background The fight against malaria faces various biological obstacles, including the resistance of parasites to anti-malarial drugs and the resistance of mosquito vectors to insecticides. The resistance of Anopheles gambiae sensu lato (s.l.) to pyrethroids, the only class of insecticides used to impregnate mosquito nets, is known in Benin; the expansion of this resistance is influenced by the existence of gene flow between species, otherwise by the presence or absence of the kdr mutation in them. The objective of this study is to determine the spatial distribution of An. gambiae and the level of expression of the pyrethroid resistance kdr gene in seven agro-ecological zones of Benin. Methods The study was conducted in 18 localities belonging to seven agro-ecological zones where environmental parameters varied. The sites represent the main areas of eco-epidemiological malaria in Benin. Anopheles gambiae larvae were collected in natural breeding sites using ladles and dipping method and reared under standard conditions. These larvae were reared under standard conditions of temperature and humidity (26 to 30 °C and 60 to 90%) at the insectarium of the Centre de Recherche Entomologique de Cotonou (CREC). Adult female mosquitoes having emerged are morphologically and molecularly identified. Homozygous resistant ( 1014F/1014F ), homozygous sensitive ( 1014L/1014L ) and heterozygous ( 1014F/1014L ) genotypes of the L1014F kdr gene mutation are determined by PCR. Results A total of 677 An. gambiae was subjected at the PCR. The results revealed the presence of three vector species of the An. gambiae complex, of which 409 Anopheles coluzzii , 259 An. gambiae , 5 hybrids ( An. coluzzii / An. gambiae ) and 4 Anopheles arabiensis in the different agro-ecological zones. The four An. arabiensis were only found in Dassa, a locality in the cotton zone of central Benin. The frequency of distribution of the L1014F allele of the kdr gene varies from 84.48 to 100% in An. gambiae , from 80 to 100% in An. coluzzii and from 0 to 75% in An. arabiensis in the different agro-ecological zones. Moreover, a significant difference is generally observed in the distribution of the L1014F allele (P < 0.05). By comparing in pairs the distribution frequencies of this allele in the two species by agro-ecological zone, only a signific...