Distribution and dynamics of Anopheles gambiae s.l. larval habitats in three Senegalese cities with high urban malaria incidence.

Urban malaria has become a challenge for most African countries due to urbanization, with increasing population sizes, overcrowding, and movement into cities from rural localities. The rapid expansion of cities with inappropriate water drainage systems, abundance of water storage habitats, coupled with recurrent flooding represents a concern for water-associated vector borne diseases, including malaria. This situation could threaten progress made towards malaria elimination in sub-Saharan countries, including Senegal, where urban malaria has presented as a threat to national elimination gains. To assess drivers of urban malaria in Senegal, a 5-month study was carried out from August to December 2019 in three major urban areas and hotspots for malaria incidence (Diourbel, Touba, and Kaolack) including the rainy season (August-October) and partly dry season (November-December). The aim was to characterize malaria vector larval habitats, vector dynamics across both seasons, and to identify the primary eco- environmental entomological factors contributing to observed urban malaria transmission. A total of 145 Anopheles larval habitats were found, mapped, and monitored monthly. This included 32 in Diourbel, 83 in Touba, and 30 in Kaolack. The number of larval habitats fluctuated seasonally, with a decrease during the dry season. In Diourbel, 22 of the 32 monitored larval habitats (68.75%) were dried out by December and considered temporary, while the remaining 10 (31.25%) were classified as permanent. In the city of Touba 28 (33.73%) were temporary habitats, and of those 57%, 71% and 100% dried up respectively by October, November, and December. However, 55 (66.27%) habitats were permanent water storage basins which persisted throughout the study. In Kaolack, 12 (40%) permanent and 18 (60%) temporary Anopheles larval habitats were found and monitored during the study. Three malaria vectors (An. arabiensis, An. pharoensis and An. funestus s.l.) were found across the surveyed larval habitats, and An. arabiensis was found in all three cities and was the only species found in the city of Diourbel, while An. arabiensis, An. pharoensis, and An. funestus s.l. were detected in the cities of Touba and Kaolack. The spatiotemporal observations of immature malaria vectors in Senegal provide evidence of permanent productive malaria vector larval habitats year-round in three major urban centers in Senegal, which may be driving high urban malaria incidence. This study aimed to assess the presence and type of anopheline larvae habitats in urban areas. The preliminary data will better inform subsequent detailed additional studies and seasonally appropriate, cost-effective, and sustainable larval source management (LSM) strategies by the National Malaria Control Programme (NMCP).

Dengue 1 outbreak in Rosso, northern Senegal, October 2021: entomologic investigations.

Senegal has experienced periodic epidemics of dengue in urban areas with increased incidence in recent years. However, few data are available on the local ecology of the epidemic vectors. In October 2021, a dengue outbreak was reported in northern Senegal to the Institute Pasteur de Dakar. Entomologic investigations then were undertaken to identify the areas at risk of transmission and to identify the vector(s). Adult mosquitoes were collected indoors and outdoors at selected households, while containers with water were inspected for mosquito larvae. All the Aedes aegypti (L.) collected were tested for dengue virus NS1 protein using a rapid diagnostic test (RDT), and positive samples were confirmed by real-time RT-PCR. The qRT-PCR positive samples were subjected to whole genome sequencing using Nanopore technology. The majority of the larvae-positive containers (83.1%) were used for water storage. The Breteau and Container indices exceeded the WHO-recommended thresholds for the risk of dengue virus transmission except at 2 localities. Ae. aegypti, the only reputed dengue vector, was collected resting indoors as well as outdoors and biting during the day and night. The NS1 protein was detected in 22 mosquito pools, including one pool of females emerging from field-collected larvae. All NS1-positive results were confirmed by RT-PCR. Virus serotyping showed that the outbreak was caused by DENV-1. This study demonstrates the need for continuous control of adult and aquatic stages of Ae. aegypti to prevent future dengue epidemics in Senegal. RDTs appear to be a promising tool for dengue diagnostics and surveillance.

Differential insecticide resistance in Anopheles arabiensis populations in the seaside area of Mbour and its suburbs in Senegal.

Regular monitoring of insecticide resistance status is an important step in implementing appropriate and adapted insecticide-based strategies for vector control. In Senegal, Indoor Residual Spraying (IRS) and a national distribution campaign for long-lasting insecticide-treated net (LLIN) have been implemented since 2007 and 2009, respectively to prevent malaria transmission. To expand and ensure the sustainability of these strategies, we conducted a study on the status of insecticide resistance in malaria vectors in the seaside area of Mbour and its suburbs where no data were previously available. Anopheles larvae were sampled from four study sites (two in both coastal and inland areas) and reared to adulthood in the insectarium. Non-blood-fed females aged 3-5 days were then tested for susceptibility to permethrin, deltamethrin, lambdacyhalothrin, bendiocarb and pirimiphos-methyl. PCR amplification was used to identify sibling species of the An. gambiae complex and genotyping for the presence of resistance knockdown (kdr) L1014S, L1014F and Ace-1 G119S. Anopheles arabiensis was the only species present in the area. At all four sites, mosquitoes were resistant to deltamethrin, permethrin, and lambdacyhalothrin, and exhibited varying degrees of resistance to bendiocarb and pirimiphos-methyl. Overall, high levels of leucine-serine/phenylalanine substitutions at position 1014 (L1014S/L1014F) were observed, with frequencies ranging from 76.4 to 85.2 % for L1014F, and from 43.2 to 66.7 % for L1014S, compared to 8.1 to 28.3 for the Ace-1 G119S mutation. These results indicate a high level of phenotypic and genotypic resistance to insecticides, which is alarming, as it could have a significant impact on the operational effectiveness of current vector control tools that rely on pyrethroids. However, in the case of bendiocarb and pirimiphos-methyl, while some level of tolerance was observed, their potential use requires regular monitoring to prevent operational failure, as their deployment could potentially lead to an increase in resistance to them.

Host feeding preferences of malaria vectors in an area of low malaria transmission.

Studying the behaviour and trophic preferences of mosquitoes is an important step in understanding the exposure of vertebrate hosts to vector-borne diseases. In the case of human malaria, transmission increases when mosquitoes feed more on humans than on other animals. Therefore, understanding the spatio-temporal dynamics of vectors and their feeding preferences is essential for improving vector control measures. In this study, we investigated the feeding behaviour of Anopheles mosquitoes at two sites in the Sudanian areas of Senegal where transmission is low following the implementation of vector control measures. Blood-fed mosquitoes were collected monthly from July to November 2022 by pyrethrum spray catches in sleeping rooms of almost all houses in Dielmo and Ndiop villages, and blood meals were identified as from human, bovine, ovine, equine and chicken by ELISA. Species from the An. gambiae complex were identified by PCR. The types and numbers of potential domestic animal hosts were recorded in each village. The Human Blood Index (HBI) and the Manly Selection Ratio (MSR) were calculated to determine whether hosts were selected in proportion to their abundance. Spatio-temporal variation in HBI was examined using the Moran's index. A total of 1251 endophilic Anopheles females were collected in 115 bedrooms, including 864 blood fed females of 6 species. An. arabiensis and An. funestus were predominant in Dielmo and Ndiop, respectively. Of the 864 blood meals tested, 853 gave a single host positive result mainly on bovine, equine, human, ovine and chicken in decreasing order in both villages. Overall, these hosts were not selected in proportion to their abundance. The human host was under-selected, highlighting a marked zoophily for the vectors. Over time and space, the HBI were low with no obvious trend, with higher and lower values observed in each of the five months at different points in each village. These results highlight the zoophilic and exophagic behaviour of malaria vectors. This behaviour is likely to be a consequence of the distribution and use of LLINs in both villages and may increase risk of residual outdoor transmission. This underlines the need to study the feeding host profile of outdoor resting populations and how domestic animals may influence malaria epidemiology in order to tailor effective malaria vector control strategies in the two villages.

A High Proportion of Malaria Vector Biting and Resting Indoors despite Extensive LLIN Coverage in Côte d'Ivoire.

Malaria is still a leading cause mortality in Côte d'Ivoire despite extensive LLINs coverage. We present the results of an entomological survey conducted in a coastal and in an inland village with the aim to estimate Anopheles gambiae sensu lato (s.l.) female's abundance indoor/outdoor and Plasmodium falciparum infection rate and analyze the occurrence of blood-feeding in relation to LLINs use. Pyrethrum spray (PSC) and window exit traps (WT) collections were carried out to target endophagic/endophilic and endophagic/exophilic females, respectively. Data on LLINs use in sampled houses were collected. (1) high levels of malaria transmission despite LLINs coverage >70% (~1 An. gambiae s.l. predicted mean/person/night and ~5% Plasmodium falciparum infection rate); (2) 46% of females in the PSC sample were blood-fed, suggesting that they fed on an unprotected host inside the house; (3) 81% of females in WT were unfed, suggesting that they were leaving the house to find an available host. Model estimates that if everyone sleeps under LLINs the probability for a mosquito to bite decreases of 48% and 95% in the coastal and inland village, respectively. The results show a high proportion of mosquito biting and resting indoors despite extensive LLINs. The biological/epidemiological determinants of accounting for these results merit deeper investigations.

Characterization of the swarming behavior of Anopheles coluzzii and Anopheles gambiae (Diptera: Culicidae) populations in a hybrid zone of Senegal.

Anopheles gambiae and Anopheles coluzzii, often found in sympatry and synchronous, have undergone a premating reproductive isolation across their distribution range. However, in the Western coast of Africa, unexpected hybridization zones have been observed, and little is known about swarming behavior of these cryptic taxa. Here, we characterized the swarming behavior of An. coluzzii and An. gambiae to investigate its role in the high hybridization level in Senegal. The study was conducted in the south and central Senegal during the 2018 rainy season. Mating swarms of malaria vectors were surveyed at sunset and collected using an insect net. Meanwhile, indoor resting populations of malaria vectors were collected by pyrethrum spray catches. Upon collection, specimens were identified morphologically, and then members of the An. gambiae complex were identified at the species level by polymerase chain reaction (PCR). An. gambiae swarmed mainly over bare ground, whereas An. coluzzii were found swarming above various objects creating a dark-light contrast with the bare ground. The swarms height varied from 0.5 to 2.5 m. Swarming starting time was correlated with sunset whatever the months for both species, and generally lasted about 10 min. No mixed swarm of An. gambiae and An. coluzzii was found even in the high hybridization area. These results indicated a premating isolation between An. coluzzii and An. gambiae. However, the high hybridization rate in the sympatric area suggests that heterogamous mating is occurring, thus stressing the need for further extensive studies.

Detection of Crimean-Congo Haemorrhagic Fever Virus from Livestock Ticks in Northern, Central and Southern Senegal in 2021.

Crimean-Congo haemorrhagic fever virus (CCHFV) occurs sporadically in Senegal, with a few human cases each year. This active circulation of CCHFV motivated this study which investigated different localities of Senegal to determine the diversity of tick species, tick infestation rates in livestock and livestock infections with CCHFV. The samples were collected in July 2021 from cattle, sheep and goats in different locations in Senegal. Tick samples were identified and pooled by species and sex for CCHFV detection via RT-PCR. A total of 6135 ticks belonging to 11 species and 4 genera were collected. The genus Hyalomma was the most abundant (54%), followed by Amblyomma (36.54%), Rhipicephalus (8.67%) and Boophilus (0.75%). The prevalence of tick infestation was 92%, 55% and 13% in cattle, sheep and goats, respectively. Crimean-Congo haemorrhagic fever virus (CCHFV) was detected in 54/1956 of the tested pools. The infection rate was higher in ticks collected from sheep (0.42/1000 infected ticks) than those from cattle (0.13/1000), while all ticks collected from goats were negative. This study confirmed the active circulation of CCHFV in ticks in Senegal and highlights their role in the maintenance of CCHFV. It is imperative to take effective measures to control tick infestation in livestock to prevent future CCHFV infections in humans.

Assessment of the Risk of Exotic Zika Virus Strain Transmission by Aedes aegypti and Culex quinquefasciatus from Senegal Compared to a Native Strain.

Zika virus (ZIKV) shows an enigmatic epidemiological profile in Africa. Despite its frequent detection in mosquitoes, few human cases have been reported. This could be due to the low infectious potential or low virulence of African ZIKV lineages. This study sought to assess the susceptibility of A. aegypti and C. quinquefasciatus to ZIKV strains from Senegal, Brazil, and New Caledonia. Vertical transmission was also investigated. Whole bodies, legs/wings and saliva samples were tested for ZIKV by real-time PCR to estimate infection, dissemination and transmission rates as well as the infection rate in the progeny of infected female A. aegypti. For A. aegypti, the Senegalese strain showed at 15 days post-exposure (dpe) a significantly higher infection rate (52.43%) than the Brazilian (10%) and New Caledonian (0%) strains. The Brazilian and Senegalese strains were disseminated but not detected in saliva. No A. aegypti offspring from females infected with Senegalese and Brazilian ZIKV strains tested positive. No infection was recorded for C. quinquefasciatus. We observed the incompetence of Senegalese A. aegypti to transmit ZIKV and the C. quinquefasciatus were completely refractory. The effect of freezing ZIKV had no significant impact on the vector competence of Aedes aegypti from Senegal, and vertical transmission was not reported in this study.

High Levels of Admixture in Anopheles gambiae Populations from Côte d'Ivoire Revealed by Multilocus Genotyping.

Anopheles coluzzii and An. gambiae-the two most recently radiated species of the An. gambiae complex and the major Afrotropical malaria vector species-are identified by markers in the X-centromeric IGS rDNA region. Putative IGS-hybrids are rarely found in the field, except in restricted areas where genomic studies have led to the hypothesis that the observed IGS-patterns are due to cryptic taxa rather than to hybridization between the two species. We investigated the genome-wide levels of admixture in two villages in Côte d'Ivoire where high levels of IGS-hybrids have been detected, confirming unparalleled high frequencies in the coastal village. Genotyping of 24 Ancestry Informative Markers (AIMs) along the three chromosomes produced discordant results between the IGS-marker and the multilocus genotype obtained for AIMs across the whole genome (29%) as well as AIMs on chromosome-X (considered to be fundamental for species reproductive isolation) only (21%). Results highlight a complicated pattern of admixture that deserves deeper genomic analyses to understand better possible underlying causes (from extensive processes of hybridization to the existence of different cryptic taxa), and stress the need of developing advanced diagnostics for An. coluzzii, An. gambiae and putative new taxa, instrumental for assessing taxon-specific epidemiological characters.

Evaluation of the Efficacy of Fludora® Fusion WP-SB 56.25 (Mixture of Clothianidin and Deltamethrin) against Anopheles coluzzii Laboratory and An. arabiensis Wild Colonies.

For malaria control, the application of long-lasting insecticidal nets and indoor residual spraying has led to a significant reduction in morbidity and mortality. However, the sustainability of these gains is hampered by the increase in insecticide resistance. It is therefore judicious to evaluate new insecticide formulations. In comparison to clothianidin and deltamethrin, the efficacy and residual effect of Fludora® Fusion was evaluated using an Anopheles coluzzii laboratory and An. arabiensis wild colonies in huts from August 2016 to June 2017 on cement and mud walls. Mortality was recorded at 24, 48, 72, and 96 h post exposure. Like deltamethrin and clothianidin, Fludora® Fusion showed delayed mortality rates above the WHO's 80% threshold over a period of 11 months with the laboratory strain. With the wild strain, while residual efficacy was observed at 2 months for the three insecticides, no residual efficacy was observed at 8 months at 24 h in both substrates. However, the increased efficacy was observed with increased holding periods (72 h and 96 h). These findings suggest that Fludora® Fusion could be an alternative candidate since this duration covers the transmission period in most areas in Senegal.

Evaluation of the residual efficacy and physical durability of five long-lasting insecticidal nets (LLINs) in Senegal.

BACKGROUND: The preventive and curative strategies of malaria are based on promoting the use of long-lasting insecticidal nets (LLINs) and treating confirmed cases with artemisinin-based combination therapy. These strategies have led to a sharp decline in the burden of malaria, which remains a significant public health problem in sub-Saharan countries. The objective of this study was to determine and compare the residual efficacy of LLINs recommended by the World Health Organization. METHODS: The study was conducted in six villages in two sites in Senegal located in the Sahelo-Sudanian area of the Thiès region, 70 km from Dakar and in Mbagame, a semi-urban zone in the Senegal River Valley. A census was conducted of all sleeping places in each household to be covered by LLINs. Five brands of LLIN were distributed, and every six months, retention rates, net use, maintenance, physical integrity, insecticide chemical content, and biological efficacy were examined for each type of LLIN. RESULTS: A total of 3012 LLINs were distributed in 1249 households in both sites, with an average coverage rate of 94% (95% CI 92.68-95.3). After 36 months, the average retention rate was 12.5% and this rate was respectively 20.5%, 15.1%, 10%, 7%, and 3% for Olyset Net®, Dawa Plus® 2.0, PermaNet® 2.0, NetProtect® and Life Net®, respectively. The proportion of LLINs with holes and the average number of holes per mosquito net increased significantly during each follow-up, with a large predominance of size 1 (small) holes for all types of LLINs distributed. During the three-year follow-up, bioassay mortality rates of a susceptible strain of insectary reared Anopheles coluzzii decreased in the following net types: in Dawa Plus® 2.0 (100% to 51.7%), PermaNet® 2.0 (96.6% to 83%), and Olyset Net® (96.6% to 33.3%). Mortality rates remained at 100% in Life Net® over the same time period. After 36 months, the average insecticide content per brand of LLIN decreased by 40.9% for Dawa Plus® 2.0, 31% for PermaNet® 2.0, 39.6% for NetProtect® and 51.9% for Olyset Net® and 40.1% for Life Net. CONCLUSIONS: Although some net types retained sufficient insecticidal activity, based on all durability parameters measured, none of the net types survived longer than 2 years.

Dengue vectors in Africa: A review.

Dengue fever is a mosquito-borne-disease of growing public health importance in Africa. The continuous increase of number and frequency of outbreaks of dengue fever, especially in urban area in Africa underline the need to review the current data available on vectors involved in dengue virus transmission in Africa. Here, we summarized the available data on vectors involved in the transmission of dengue virus in the sylvatic and urban environments, vertical transmission, vector competence studies, and vector control strategies used in Africa. The virus was isolated mainly from Aedes furcifer, Ae. luteocephalus, and Ae. taylori in the sylvatic environment and from Ae. aegypti and Ae. albopictus in the urban areas. Prospective and urgently needed studies on vectors biology, behavior, and alternative control strategies are suggested.

Blood Feeding Patterns of Aedes aegypti Populations in Senegal.

Aedes aegypti plays an important role in the transmission of several arboviruses of medical importance. The availability of information on the blood-feeding preferences of mosquito vectors is a critical step in the understanding of the transmission of human pathogens and implementation of control strategies. In Senegal, no data currently exist on the feeding pattern of Ae. aegypti in urban areas. To fill this gap, Ae. aegypti blood-fed females were collected in five localities by aspiration and using BG Sentinel 2 traps. Collections were carried out monthly between July and November 2019 inside and outside human dwellings. The origin of the blood meal of Ae. aegypti females were identified by an ELISA technique. A total of 1,710 blood-engorged females were examined and showed that Ae. aegypti preferentially fed on human with 78.6% of the identified blood meals. The other blood meals were from animals including dog, cat, horse, cattle, sheep, and rat. This is the first report on the feeding behavior of Ae. aegypti in urban settings in West Africa. It demonstrated that this species is highly anthropophilic.

Is Côte D'Ivoire a new high hybridization zone for the two major malaria vectors, Anopheles coluzzii and An. gambiae (Diptera, Culicidae)?

Anopheles gambiae and An. coluzzii are very closely related and recently differentiated species representing the main malaria vectors in the Afrotropical region and responsible of up to >3 infective bites/person/night in Côte D'Ivoire, where prevention and control has stagnated in recent years. The aim of the present study was to genetically and ecologically characterize An. gambiae and An. coluzzii populations from two villages of Côte D'Ivoire, lying in the coastal forest belt and 250 km inland in the Guinean savannah mosaic belt, respectively. Results reveal high frequencies of both species in both study sites and high frequencies of hybrids (4-33%) along the whole year of sampling. Consistently with observations for the well-known high hybridization zone at the far-west of the species range, hybrid frequencies were higher in the coastal village and highest when the two species occurred at more balanced frequencies, supporting the "frequency-dependent hybridization" ecological speciation theory. Pilot genotyping revealed signatures of genomic admixture in both chromosome-X and -3. Coupled with previous reports of hybrids in the region, the results point to the coastal region of Côte D'Ivoire as a possible regions of high hybridization. Preliminary characterization of parameters relevant for malaria transmission and control (e.g. possibly higher sporozoite rates and indoor biting preferences in hybrids than in the parental species) highlight the possible relevance of the breakdown of reproductive barriers between An. gambiae and An. coluzzii not only in the field of ecological evolution, but also in malaria epidemiology and control.

Comparison of the efficiency and performance of two systems and three membranes for blood feeding mosquitoes.

OBJECTIVE: Due to different concerns in using appropriate mosquito blood feeding techniques, this work focused on evaluating the effectiveness of two artificial mosquito feeding systems (Rutledge and Hemotek) and three different membranes (Parafilm, mouse and chicken skins). Female mosquitoes from an An. coluzzii strain aged between 2 and 5 days were exposed to blood with the two systems at time intervals (5, 10, 15, 20, 25 and 30 min) with blood used on the day of collection, the next day and 2 days after. RESULTS: Our results showed that the Hemotek system gave better blood feeding rates than the Rutledge system. Among the three membranes, the blood feeding rates with chicken and mouse skins were higher than those provided by the Parafilm membrane. Likewise, blood stored 1 day after collection gave higher levels than blood used on the day of collection and 2 days after. Regardless of the system, the lowest blood feeding rates were observed at 5 min compared to the other exposure times.

Entomological Survey Confirms Changes in Mosquito Composition and Abundance in Senegal and Reveals Discrepancies among Results by Different Host-Seeking Female Traps.

Mosquitoes-borne diseases are major public health issues particularly in Africa. Vector control interventions and human-made environmental/climatic changes significantly affect the distribution and abundance of vector species. We carried out an entomological survey targeting host-seeking mosquitos in two different ecological contexts-coastal and inland-in Senegal, by CDC-light and BG-sentinel traps. Results show high predominance of Culex quinquefasciatus (90%) and of Anopheles arabiensis within malaria vectors (46%), with mean numbers of females/trap/nights =8 and <1, respectively, reinforcing previous evidence of changes in species composition and abundance, highlighting thus increasing risk of transmission of filariasis and emerging arboviruses in the Senegambia region. From the methodological perspective, results show a higher specificity of BG traps for Cx. quinquefasciatus and of CDC traps for An. gambiae s.l. and highlight that, despite both traps target the host-seeking fraction of the population, they provide different patterns of species abundance, temporal dynamics and host-seeking activity, leading to possible misinterpretation of the species bionomics. This draws attention to the need of taking into account trapping performance, in order to provide realistic quantification of the number of mosquitoes per units of space and time, the crucial parameter for evaluating vector-human contact, and estimating risk of pathogen transmission.

Resting Behavior of Blood-Fed Females and Host Feeding Preferences of Aedes aegypti (Diptera: Culicidae) Morphological Forms in Senegal.

Aedes aegypti (Linnaeus) is the main vector of most arboviruses in tropical and subtropical urban areas. In West Africa, particularly in Senegal, domestic and wild populations have been described. Both Ae. aegypti aegypti (Aaa) and Ae. aegypti formosus (Aaf) were found in progenies of Ae. aegypti families from several localities of Senegal. However, nothing is known about their resting and trophic behavior, which are key data for vector control. To fill this gap, blood-fed mosquitoes were collected monthly indoors and outdoors with BackPack aspirators and BG-Sentinel 2 traps between July and November 2019 from four urban sites. The enzyme-linked immunosorbent assay technique was used to analyze blood-fed Aaa and Aaf specimens. Both forms were found resting in all investigated places with the highest proportions found in scrap metals (51.7% for Aaa and 44.1% for Aaf) and used tires (19.2% for Aaa and 26.1% for Aaf). Blood-fed Aaf females showed lower occupation of the indoors environment compared to Aaa. Overall, the percentages of single bloodmeals from human were 80.5% (916/1138) for Aaa and 71.1% (263/370) for Aaf. A low frequency of other domestic hosts, including bovine, ovine, and cat were detected for both forms. This study provides the first data on resting and trophic behavior of Aaa and Aaf in Senegal. Both forms showed differences in their resting behavior but fed primarily on human and highlight the risk of arboviruses transmission in urban areas.

Insecticide resistance status and mechanisms in Aedes aegypti populations from Senegal.

Aedes aegypti is the main epidemic vector of arboviruses in Africa. In Senegal, control activities are mainly limited to mitigation of epidemics, with limited information available for Ae. aegypti populations. A better understanding of the current Ae. aegypti susceptibility status to various insecticides and relevant resistance mechanisms involved is needed for the implementation of effective vector control strategies. The present study focuses on the detection of insecticide resistance and reveals the related mechanisms in Ae. aegypti populations from Senegal. Bioassays were performed on Ae. aegypti adults from nine Senegalese localities (Matam, Louga, Barkedji, Ziguinchor, Mbour, Fatick, Dakar, Kédougou and Touba). Mosquitoes were exposed to four classes of insecticides using the standard WHO protocols. Resistance mechanisms were investigated by genotyping for pyrethroid target site resistance mutations (V1016G, V1016I, F1534C and S989P) and measuring gene expression levels of key detoxification genes (CYP6BB2, CYP9J26, CYP9J28, CYP9J32, CYP9M6, CCEae3a and GSTD4). All collected populations were resistant to DDT and carbamates except for the ones in Matam (Northern region). Resistance to permethrin was uniformly detected in mosquitoes from all areas. Except for Barkédji and Touba, all populations were characterized by a susceptibility to 0.75% Permethrin. Susceptibility to type II pyrethroids was detected only in the Southern regions (Kédougou and Ziguinchor). All mosquito populations were susceptible to 5% Malathion, but only Kédougou and Matam mosquitoes were susceptible to 0.8% Malathion. All populations were resistant to 0.05% Pirimiphos-methyl, whereas those from Louga, Mbour and Barkédji, also exhibited resistance to 1% Fenitrothion. None of the known target site pyrethroid resistance mutations was present in the mosquito samples included in the genotyping analysis (performed in > 1500 samples). In contrast, a remarkably high (20-70-fold) overexpression of major detoxification genes was observed, suggesting that insecticide resistance is mostly mediated through metabolic mechanisms. These data provide important evidence to support dengue vector control in Senegal.

Insecticide Resistance Profiling of Anopheles coluzzii and Anopheles gambiae Populations in the Southern Senegal: Role of Target Sites and Metabolic Resistance Mechanisms.

The emergence and spread of insecticide resistance among the main malaria vectors is threatening the effectiveness of vector control interventions in Senegal. The main drivers of this resistance in the Anopheles gambiae complex (e.g., An. gambiae and Anopheles coluzzii) remains poorly characterized in Senegal. Here we characterized the main target site and metabolic resistances mechanisms among the An. gambiae and An. coluzzii populations from their sympatric and allopatric or predominance area in Senegal. Larvae and pupae of An. gambiae s.l. were collected, reared to adulthood, and then used for insecticides susceptibility and synergist assays using the WHO (World Health Organisation) test kits for adult mosquitoes. The TaqMan method was used for the molecular characterization of the main target site insecticide resistance mechanisms (Vgsc-1014F, Vgsc-1014S, N1575Y and G119S). A RT-qPCR (Reverse Transcriptase-quantitative Polymerase Chaine Reaction) was performed to estimate the level of genes expression belonging to the CYP450 (Cytochrome P450) family. Plasmodium infection rate was investigated using TaqMan method. High levels of resistance to pyrethroids and DDT and full susceptibility to organophosphates and carbamates where observed in all three sites, excepted a probable resistance to bendiocarb in Kedougou. The L1014F, L1014S, and N1575Y mutations were found in both species. Pre-exposure to the PBO (Piperonyl butoxide) synergist induced a partial recovery of susceptibility to permethrin and full recovery to deltamethrin. Subsequent analysis of the level of genes expression, revealed that the CYP6Z1 and CYP6Z2 genes were over-expressed in wild-resistant mosquitoes compared to the reference susceptible strain (Kisumu), suggesting that both the metabolic resistance and target site mutation involving kdr mutations are likely implicated in this pyrethroid resistance. The presence of both target-site and metabolic resistance mechanisms in highly pyrethroid-resistant populations of An. gambiae s.l. from Senegal threatens the effectiveness and the sustainability of the pyrethroid-based tools and interventions currently deployed in the country. The Kdr-west mutation is widely widespread in An. coluzzii sympatric population. PBO or Duo nets and IRS (Indoor Residual Spraying) with organophosphates could be used as an alternative measure to sustain malaria control in the study area.

Morphology and taxonomic status of Aedes aegypti populations across Senegal.

Aedes aegypti is the primary vector of dengue, Zika, yellow fever and chikungunya viruses to humans. In Africa, two subspecies, Ae. aegypti aegypti (Aaa) and Ae. aegypti formosus (Aaf) have been described. Until very recently, it was considered that the two forms were sympatric in East Africa and that only Aaf was present in Central and West Africa. However, recent data suggests that Aaa was also common in Senegal without any clear evidence of genetic differences with Aaf. This study was carried out in different Ae. aegypti populations from Senegal to better clarify their taxonomic status. The larvae, pupae and eggs were collected between July and September 2018 and reared individually to adult stage. For each population, F1 progeny from eggs laid by a single female F0 were reared as sibling samples. The number of pale scales on the first abdominal tergite (T1) and the basal part of the second tergite (T2) were counted. Individuals with no pale scale on T1 were classified as Aaf while those with at least one pale scale on this tergite were classified as Aaa. The morphological variations within families of Aaf were studied across 4 generations. In total, 2400 individuals constituting 240 families were identified, of which 42.5% were heterogeneous (families with both forms). Multivariate statistical analysis of variance including T1 and T2 data together showed that populations were significantly different from each other. Statistical analysis of T1 alone showed a similarity between populations from the southeast while variations were observed within northwest population. The analysis of family composition across generations showed the presence of Aaa and Aaf forms in each generation. The classification of Ae. aegypti into two subspecies is invalid in Senegal. Populations exhibit morphological polymorphism at the intra-family level that could have biological and epidemiological impacts.