Insecticide 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.

Knowledge on the spread and distribution of insecticide resistance in major malaria vectors such as Anopheles funestus is key to implement successful resistance management strategies across Africa. Here, by assessing the susceptibility status of an inland population of An. funestus Giles (Kpome) and investigating molecular basis of resistance, we show that multiple resistance and consistent plasmodium infection rate are present in Anopheles funestus populations from Kpome. The insecticide susceptibility level of collected Anopheles funestus was assessed. Synergist (PBO) was used to screen resistance mechanisms. The TaqMan technique was used for genotyping of insecticide resistant alleles and detecting plasmodium infection levels. The nested PCR was used to further assess the plasmodium infection rate. The TaqMan analysis of plasmodial infections revealed an infection rate (18.2 %) of An. funestus in this locality. The WHO bioassays revealed a multiple phenotypic resistance profile for An. funestus in Kpome. This population is highly resistant to pyrethroids (permethrin and deltamethrin), organochlorines (DDT), and carbamates (bendiocarb). A reduced susceptibility was observed with dieldrin. Mortalities did not vary after pre-exposure to PBO for DDT indicating that cytochrome P450s play little role in DDT resistance in Kpome. In contrast, we noticed, a significant increase in mortalities when PBO was combined to permethrin suggesting the direct involvement of P450s in pyrethroid resistance. A high frequency of the L119F-GSTe2 DDT resistance marker was observed in the wild DDT resistant population (9 %RS and 91 %RR) whereas the A296S mutation was detected at a low frequency (1 %RS and 99 %SS). The presence of multiple resistance in An. funestus populations in the inland locality of Kpome is established in this study as recently documented in the costal locality of Pahou. Data from both localities suggest that resistance could be widespread in Benin and this highlights the need for further studies to assess the geographical distribution of insecticide resistance across Benin and neighboring countries as well as a more comprehensive analysis of the resistance mechanisms involved.

Insecticide resistance in the mosquito vector is the one of the main obstacles against effective malaria control. In order to implement insecticide resistance management strategies, it is important to understand the genetic factors involved. In this context, we investigated the molecular basis of DDT resistance in the main malaria vector from Benin. Anopheles gambiae mosquitoes were collected from four sites across Benin and identified to species/molecular form. Mosquitoes from Cotonou (M-form), Tori-Bossito (S-form) and Bohicon (S-form) were exposed to DDT 4% at a range of exposure times (30 min to 300 min). Another batch of mosquitoes from Cotonou and Malanville were exposed to DDT for 1 hour and the survivors 48 hours post exposure were used to quantify metabolic gene expression. Quantitative PCR assays were used to quantify mRNA levels of metabolic enzymes: GSTE2, GSTD3, CYP6P3 and CYP6M2. Expression (fold-change) was calculated using the ∆∆Ct method and compared to susceptible strains. Detection of target-site mutations (L1014F, L1014S and N1575Y) was performed using allelic discrimination TaqMan assays. DDT resistance was extremely high in all populations, regardless of molecular form, with no observed mortality after 300 min exposure. In both DDT-survivors and non-exposed mosquitoes, GSTE2 and GSTD3 were over-expressed in the M form at 4.4-fold and 3.5-fold in Cotonou and 1.5-fold and 2.5-fold in Malanville respectively, when compared to the susceptible strain. The CYP6M2 and CYP6P3 were over-expressed at 4.6-fold and 3.8-fold in Cotonou and 1.2-fold and 2.5-fold in Malanville respectively. In contrast, no differences in GSTE2 and CYP6M2 were observed between S form mosquitoes from Tori-Bossito and Bohicon compared to susceptible strain. The 1014 F allele was fixed in the S-form and at high frequency in the M-form (0.7-0.914). The frequency of 1575Y allele was 0.29-0.36 in the S-form and nil in the M-form. The 1014S allele was detected in the S form of An. gambiae in a 1014 F/1014S heterozygous specimen. Our results show that the kdr 1014 F, 1014S and 1575Y alleles are widespread in Benin and the expression of two candidate metabolic markers (GSTE2 and CYP6M2) are over-expressed specifically in the M-form.

Large-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. Anopheles 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). Anopheles 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. The 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.

Background: The implementation of anti-larval strategies in the fight against malaria vectors requires fundamental knowledge of their oviposition sites. The aim of this study was to assess the spatial and temporal distribution of Anopheles breeding sites as well as the influence of abiotic and biotic factors on the proliferation of larvae in urban and non-urban areas of Benin. Methods: Sampling of Anopheles larvae was carried out during the rainy and dry seasons in urbanized and non-urbanized areas of the cities of Cotonou, Bohicon, Parakou, and Natitingou in Benin. The Anopheles larval breeding sites were georeferenced and characterized by their nature, type, physicochemical (pH, temperature, dissolved oxygen, conductivity, turbidity, salinity) and biological attributes (larval density and coliform density). Results: A total of 198 positive breeding sites for Anopheles larvae were identified, comprising 163 (82.3%) in the rainy season and 35 (17.7%) in the dry season. Out of these larval habitats, 61.9% were located in urbanized areas, and were predominantly puddles. Principal component analysis revealed a high positive correlation of larval density with temperature and dissolved oxygen, and with salinity in the coastal zone. In addition, cross-sectional analysis of the microbiological results with larval density showed a significant negative correlation between larval productivity and faecal coliform load. Conclusions: This study indicated the presence of multiple larval habitats of Anopheles in the urban areas which were created through human activities, and associations between larval density and intrinsic factors of the habitats such as temperature, dissolved oxygen and faecal coliform load. This type of information may be useful for the implementation of appropriate control strategies in urban areas, including regulation of the human activities that lead to the creation of breeding sites, proper environmental management and targeted larvicidal use. Graphical

The Culex quinquefasciatus mosquito, a major pest and vector of filariasis and arboviruses in the tropics, has developed multiple resistance mechanisms to the main insecticide classes currently available in public health. Among them, the insensitive acetylcholinesterase (ace-1^R allele) is widespread worldwide and confers cross-resistance to organophosphates and carbamates. Fortunately, in an insecticide-free environment, this mutation is associated with a severe genetic cost that can affect various life history traits. Salivary proteins are directly involved in human-vector contact during biting and therefore play a key role in pathogen transmission. An original proteomic approach combining 2D-electrophoresis and mass spectrometry was adopted to compare the salivary expression profiles of two strains of C. quinquefasciatus with the same genetic background but carrying either the ace-1^R resistance allele or not (wild type). Four salivary proteins were differentially expressed (>2 fold, P<0.05) in susceptible (SLAB) and resistant (SR) mosquito strains. Protein identification indicated that the D7 long form, a major salivary protein involved in blood feeding success, presented lower expression in the resistant strain than the susceptible strain. In contrast, three other proteins, including metabolic enzymes (endoplasmin, triosephosphate isomerase) were significantly over-expressed in the salivary gland of ace-1^R resistant mosquitoes. A catalogue of 67 salivary proteins of C. quinquefasciatus sialotranscriptome was also identified and described. The “resistance”-dependent expression of salivary proteins in mosquitoes may have considerable impact on biting behaviour and hence on the capacity to transmit parasites/viruses to humans. The behaviour of susceptible and insecticide-resistant mosquitoes in the presence of vertebrate hosts and its impact on pathogen transmission urgently requires further investigation. All proteomic data will be deposited at PRIDE (http://www.ebi.ac.uk/pride/).