Assessing the potential of nano-formulated chlorfenapyr and clothianidin insecticides-treated sugar baits against Anopheles funestus, Anopeles coluzzii and Culex quinquefasciatus mosquitoes.

Mosquitoes serve as vectors for various diseases like malaria, dengue fever, yellow fever, and lymphatic filarial diseases causing significant global health problems, highlighting the importance of vector control. The study was conducted to assess the effectiveness of nanoformulated clothianidin and chlorfenapyr insecticides treated with ATSB in controlling three mosquito strains. The development of a natural thiolated polymer-coated ATSB nano formulation involved incorporating nano-carriers to deliver insecticides. Field- collected mosquito strains were subjected to laboratory-based bioassays using 1 % and 1.5 % concentrations of each conventionally used and nanoformulated insecticide with ATSB solution. Adult mosquitoes were left overnight to contact with N-ATSB and efficacy was recorded after 36 and 72 h. The results showed that nanoformulated chlorfenapyr was significantly more effective as compared to clothianidin against An. funestus and Cx. quinquefasciatus but the results were not significantly different against An. coluzzii (100 %). An. coluzzii was found to be the most susceptible strain followed by An. funestus and showed 100 % and ∼ 98 % mortality against nanoformulated chlorfenapyr (1.5 %). Nanoformulated clothianidin induced more than 92 % and ∼ 100 % mortality against An. funestus and An. coluzzii respectively. However, Cx. quinquefasciatus significantly showed less mortality against nanoformulated clothianidin (88 %) and chlorfenapyr (>95 %) as compared to Anopheline strains. Furthermore, results indicate that nanoformulated insecticides significantly caused greater and prolonged fatality as compared to conventional form, suggesting effective and suitable strategies for vector management.

Sibling species of the major malaria vector Anopheles gambiae display divergent preferences for aquatic breeding sites in southern Nigeria.

BACKGROUND: When integrated with insecticide-treated bed nets, larval control of Anopheles mosquitoes could fast-track reductions in the incidence of human malaria. However, larval control interventions may deliver suboptimal outcomes where the preferred breeding places of mosquito vectors are not well known. This study investigated the breeding habitat choices of Anopheles mosquitoes in southern Nigeria. The objective was to identify priority sites for mosquito larval management in selected urban and periurban locations where malaria remains a public health burden.  METHODS: Mosquito larvae were collected in urban and periurban water bodies during the wet-dry season interface in Edo, Delta, and Anambra States. Field-collected larvae were identified based on PCR gel-electrophoresis and amplicon sequencing, while the associations between Anopheles larvae and the properties and locations of water bodies were assessed using a range of statistical methods. RESULTS: Mosquito breeding sites were either man-made (72.09%) or natural (27.91%) and mostly drainages (48.84%) and puddles (25.58%). Anopheles larvae occurred in drainages, puddles, stream margins, and a concrete well, and were absent in drums, buckets, car tires, and a water-holding iron pan, all of which contained culicine larvae. Wild-caught Anopheles larvae comprised Anopheles coluzzii (80.51%), Anopheles gambiae sensu stricto (s.s.) (11.54%), and Anopheles arabiensis (7.95%); a species-specific PCR confirmed the absence of the invasive urban malaria vector Anopheles stephensi among field-collected larvae. Anopheles arabiensis, An. coluzzii, and An. gambiae s.s. displayed preferences for turbid, lowland, and partially sunlit water bodies, respectively. Furthermore, An. arabiensis preferred breeding sites located outside 500 m of households, whereas An. gambiae s.s. and An. coluzzii had increased detection odds in sites within 500 m of households. Anopheles gambiae s.s. and An. coluzzii were also more likely to be present in natural water bodies; meanwhile, 96.77% of An. arabiensis were in man-made water bodies. Intraspecific genetic variations were little in the dominant vector An. coluzzii, while breeding habitat choices of populations made no statistically significant contributions to these variations. CONCLUSION: Sibling malaria vectors in the An. gambiae complex display divergent preferences for aquatic breeding habitats in southern Nigeria. The findings are relevant for planning targeted larval control of An. coluzzii whose increasing evolutionary adaptations to urban ecologies are driving the proliferation of the mosquito, and An. arabiensis whose adults typically evade the effects of treated bed nets due to exophilic tendencies.

Evidence of Transmission of Plasmodium vivax 210 and Plasmodium vivax 247 by Anopheles gambiae and An. coluzzii, Major Malaria Vectors in Benin/West Africa.

Current diagnostic and surveillance systems in Benin are not designed to accurately identify or report non-Plasmodium falciparum (Pf) human malaria infections. This study aims to assess and compare the prevalence of circumsporozoite protein (CSP) antibodies of Pf and P. vivax (Pv) in Anopheles gambiae s.l. in Benin. For that, mosquito collections were performed through human landing catches (HLC) and pyrethrum spray catches (PSC). The collected mosquitoes were morphologically identified, and Pf, Pv 210, and Pv 247 CSP antibodies were sought in An. gambiae s.l. through the ELISA and polymerase chain reaction (PCR) techniques. Of the 32,773 collected mosquitoes, 20.9% were An. gambiae s.l., 3.9% An. funestus gr., and 0.6% An. nili gr. In An. gambiae s.l., the sporozoite rate was 2.6% (95% CI: 2.1-3.1) for Pf, against 0.30% (95% CI: 0.1-0.5) and 0.2% (95% CI: 0.1-0.4), respectively, for Pv 210 and Pv 247. P. falciparum sporozoite positive mosquitoes were mostly An. gambiae (64.35%), followed by An. coluzzii (34.78%) and An. arabiensis (0.86%). At the opposite, for the Pv 210 sporozoite-positive mosquitoes, An. coluzzii and An. gambiae accounted for 76.92% and 23.08%, respectively. Overall, the present study shows that P. falciparum is not the only Plasmodium species involved in malaria cases in Benin.

Bendiocarb and Malathion Resistance in Two Major Malaria Vector Populations in Cameroon Is Associated with High Frequency of the G119S Mutation (Ace-1) and Overexpression of Detoxification Genes.

The spread of pyrethroid resistance in malaria vectors is a major threat affecting the performance of current control measures. However, there is still not enough information on the resistance profile of mosquitoes to carbamates and organophosphates which could be used as alternatives. The present study assessed the resistance profile of Anopheles gambiae s.l. to bendiocarb and malathion, at the phenotypic and molecular levels, in different eco-epidemiological settings in Cameroon. Anopheles gambiae s.l. mosquitoes were collected from four eco-epidemiological settings across the country and their susceptibility level to bendiocarb and malathion was determined using WHO tubes bioassays. The ace-1 target site G119S mutation was screened by PCR. Reverse Transcription quantitative PCR 3-plex TaqMan assays were used to quantify the level of expression of eight genes associated with metabolic resistance. Resistance to malathion and/or bendiocarb was recorded in all study sites except in mosquitoes collected in Kaélé and Njombé. The Ace-1 (G119S) mutation was detected in high frequencies (>40%) in Kékem and Santchou. Both An. gambiae and An. coluzzii were detected carrying this mutation. The cytochrome P450s gene Cyp6p3 associated with carbamate resistance and the glutathione S-transferase gene Gste2 associated with organophosphate resistance were found to be overexpressed. Genes associated with pyrethroid (Cyp6m2, Cyp9k1, Cyp6p3) and organochlorine (Gste2, Cyp6z1, Cyp6m2) and cuticle resistance (Cyp4g16) were also overexpressed. The rapid spread of resistance to organophosphates and carbamates could seriously compromise future control strategies based on IRS. It is therefore becoming important to assess the magnitude of bendiocarb and malathion resistance countrywide.

Increased prevalence of insecticide resistance in Anopheles coluzzii populations in the city of Yaoundé, Cameroon and influence on pyrethroid-only treated bed net efficacy.

In Cameroon, pyrethroid-only long-lasting insecticidal nets (LLINs) are still largely used for malaria control. The present study assessed the efficacy of such LLINs against a multiple-resistant population of the major malaria vector, Anopheles coluzzii, in the city of Yaoundé via a cone bioassay and release-recapture experimental hut trial. Susceptibility of field mosquitoes in Yaoundé to pyrethroids, DDT, carbamates and organophosphate insecticides was investigated using World Health Organization (WHO) bioassay tube tests. Mechanisms of insecticide resistance were characterised molecularly. Efficacy of unwashed PermaNet® 2.0 was evaluated against untreated control nets using a resistant colonised strain of An. coluzzii. Mortality, exophily and blood feeding inhibition were estimated. Field collected An. coluzzii displayed high resistance with mortality rates of 3.5% for propoxur (0.1%), 4.16% for DDT (4%), 26.9% for permethrin (0.75%), 50.8% for deltamethrin (0.05%), and 80% for bendiocarb (0.1%). High frequency of the 1014F west-Africa kdr allele was recorded in addition to the overexpression of several detoxification genes, such as Cyp6P3, Cyp6M2, Cyp9K1, Cyp6P4 Cyp6Z1 and GSTe2. A low mortality rate (23.2%) and high blood feeding inhibition rate (65%) were observed when resistant An. coluzzii were exposed to unwashed PermaNet® 2.0 net compared to control untreated net (p < 0.001). Furthermore, low personal protection (52.4%) was observed with the resistant strain, indicating reduction of efficacy. The study highlights the loss of efficacy of pyrethroid-only nets against mosquitoes exhibiting high insecticide resistance and suggests a switch to new generation bed nets to improve control of malaria vector populations in Yaoundé.

TITLE: Augmentation de la prévalence de la résistance aux insecticides chez les populations d'Anopheles coluzzii de la ville de Yaoundé (Cameroun) et influence sur l'efficacité des moustiquaires traitées uniquement aux pyréthrinoïdes. ABSTRACT: Au Cameroun, moustiquaires insecticides de longue durée (MILDA) contenant uniquement des pyréthrinoïdes sont encore largement utilisées pour lutter contre le paludisme. La présente étude a évalué l'efficacité de ces MILDA contre une population multi-résistante du principal vecteur du paludisme, Anopheles coluzzii, de la ville de Yaoundé, en utilisant un test de bio-efficacité et la technique de lâchage-recapture dans des cases-pièges expérimentales. La sensibilité des moustiques collectés sur le terrain à Yaoundé aux pyréthrinoïdes, au DDT, aux carbamates et aux insecticides organophosphorés a été étudiée à l'aide de tests en tube de bio-essai de l'Organisation Mondiale de la Santé (OMS). Les mécanismes de résistance aux insecticides ont été caractérisés au niveau moléculaire. L'efficacité du PermaNet® 2.0 non lavé a été évaluée par rapport à une moustiquaire contrôle non traitée en utilisant une souche colonisée résistante d'An. coluzzii. La mortalité, le taux d'inhibition de gorgement ont été estimées. Les An. coluzzii collectés sur le terrain ont montré une résistance élevée avec des taux de mortalité de 3,5 % pour le propoxur (0,1 %), 4,16 % pour le DDT (4 %), 26,9 % pour la perméthrine (0,75 %), 50,8 % pour la deltaméthrine (0,05 %) et 80% pour le bendiocarbe (0,1 %). Une fréquence élevée de l'allèle 1014F kdr ouest-africain a été enregistrée en plus de la surexpression de plusieurs gènes de détoxification tels que Cyp6P3, Cyp6M2, Cyp9K1, Cyp6P4 Cyp6Z1 et GSTe2. Un faible taux de mortalité (23,2 %) et un taux élevé d'inhibition de gorgement (65 %) ont été observés lorsque les An. coluzzii ont été exposés à une moustiquaire PermaNet® 2.0 non lavée par rapport à une moustiquaire témoin non traitée (p < 0,001). De plus, une faible protection individuelle (52,4 %) a été observée avec la souche résistante indiquant une réduction de leur efficacité. L'étude met en évidence la perte d'efficacité des moustiquaires aux pyréthrinoïdes contre les moustiques présentant une résistance élevée aux insecticides et suggère le remplacement de ces moustiquaires par des moustiquaires de nouvelle génération pour améliorer le contrôle des populations de vecteurs du paludisme à Yaoundé.

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.

Multiple insecticide resistance target sites in adult field strains of An. gambiae (s.l.) from southeastern Senegal.

BACKGROUND: High coverage of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) are the cornerstones of vector control strategy in Senegal where insecticide resistance by the target vectors species is a great of concern. This study explores insecticide susceptibility profile and target-site mutations mechanisms within the Anopheles gambiae complex in southeastern Senegal. METHODS: Larvae of Anopheles spp. were collected in two sites from southeastern Senegal Kedougou and Wassadou/Badi in October and November 2014, and reared until adult emergence. Wild F0 adult mosquitoes were morphologically identified to species. Susceptibility of 3-5-day-old An. gambiae (s.l.) samples to 11 insecticides belonging to the four insecticide classes was assessed using the WHO insecticide susceptibility bioassays. Tested samples were identified using molecular techniques and insecticide resistance target-site mutations (kdr, ace-1 and rdl) were determined. RESULTS: A total of 3742 An. gambiae (s.l.) were exposed to insecticides (2439 from Kedougou and 1303 from Wassadou-Badi). Tests with pyrethroid insecticides and DDT showed high level of resistance in both Kedougou and Wassadou/Badi. Resistance to pirimiphos-methyl and malathion was not detected while resistance to bendoicarb and fenitrothion was confirmed in Kedougou. Of the 745 specimens of An. gambiae (s.l.) genotyped, An. gambiae (s.s.) (71.6%) was the predominant species, followed by An. arabiensis (21.7%), An. coluzzii (6.3%) and hybrids (An. gambiae (s.s.)/An. coluzzii; 0.4%). All target site mutations investigated (Vgsc-1014F, Vgsc-1014S, Ace-1 and Rdl) were found at different frequencies in the species of the Anopheles gambiae complex. Vgsc-1014F mutation was more frequent in An. gambiae (s.s.) and An. coluzzii than An. arabiensis. Vgsc-1014S was present in An. gambiae (s.l.) populations in Wassadou but not in Kedougou. Ace-1 and rdl mutations were more frequent in An. gambiae (s.s.) in comparison to An. arabiensis and An. coluzzii. CONCLUSIONS: Resistance to all the four insecticide classes tested was detected in southeastern Senegal as well as all target site mutations investigated were found. Data will be used by the national Malaria Control Programme.

Fitness cost of insecticide resistance on the life-traits of a Anopheles coluzzii population from the city of Yaoundé, Cameroon.

Background: Pyrethroid resistance is rapidly expanding in An. gambiae s.l. populations across Sub-Saharan Africa. Yet there is still not enough information on the fitness cost of insecticide resistance . In the present study, the fitness cost of insecticide resistance on Anopheles coluzzii population from the city of Yaoundé was investigated.   Methods: A resistant An. coluzzii colony was established from field collected mosquitoes resistant to both DDT and pyrethroid and selected for 12 generations with deltamethrin 0.05%. The Ngousso laboratory susceptible strain was used as control. A total of 100 females of each strain were blood fed and allowed for individual eggs laying, and then different life traits parameters such as fecundity, fertility, larval development time, emergence rate and longevity were measured. The TaqMan assay was used to screen for the presence of the L1014F and L1014S kdr mutations. Results:  Field collected mosquitoes from the F0 generation had a mortality rate of 2.05% for DDT, 34.16% for permethrin and 50.23% for deltamethrin. The mortality rate of the F12 generation was 30.48% for deltamethrin, 1.25% for permethrin  and 0% for DDT. The number of eggs laid per female was lower in the resistant colony compared to the susceptible (p <0.0001). Insecticide resistant larvae were found with a significantly long larval development time (10.61±0.33 days) compare to susceptible (7.57±0.35 days). The number of emerging females was significantly high in the susceptible group compared to the resistant . The adults lifespan was also significantly high for susceptible (21.73±1.19 days) compared to resistant (14.63±0.68 days). Only the L1014F- kdr allele was detected in resistant population.. Conclusion: The study suggests that pyrethroid resistance is likely associated with a high fitness cost on An.coluzzii populations. The addition of new tools targeting specifically larval stages could improve malaria vectors control and insecticide resistance management.

High Plasmodium infection and multiple insecticide resistance in a major malaria vector Anopheles coluzzii from Sahel of Niger Republic.

BACKGROUND: Information on insecticide resistance and the mechanisms driving it in the major malaria vectors is grossly lacking in Niger Republic, thus hindering control efforts. To facilitate evidence-based malaria control, the role of Anopheles coluzzii population from southern Niger, in malaria transmission, its insecticides resistance profile and the molecular mechanisms driving the resistance were characterized. METHODS: Blood fed female Anopheles gambiae sensu lato resting indoor were collected at Tessaoua, Niger. Source of blood was established using PCR and infection with Plasmodium determined using TaqMan assay. Resistance profile was established with the major public health insecticides, and resistance intensity determined with deltamethrin. Synergist assays were conducted with piperonyl butoxide and diethyl maleate. Presence of L1014F and L1014S knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) was investigated using TaqMan genotyping, and strength of selection pressure acting on the Anopheles populations determined by assessing the genetic diversity of a fragment spanning exon-20 of the VGSC from alive and dead females. RESULTS: High human blood index (96%) and high Plasmodium falciparum infection (~ 13%) was observed in the An. coluzzii population. Also, a single mosquito was found infected with Plasmodium vivax. High pyrethroid and organochloride resistance was observed with mortalities of less than 20% for deltamethrin, permethrin, α-cypermethrin, and DDT. A high LD50 (156.65 min) was obtained for deltamethrin, with a resistance ratio of ~ 47.18 compared to the susceptible Ngoussou colony. Moderate carbamate resistance was observed, and a full susceptibility to organophosphates recorded. Synergist bioassays with piperonyl butoxide and diethyl maleate significantly recovered deltamethrin and DDT susceptibility, respectively implicating CYP450 s (mortality = 82%, χ2 = 84.51, p < 0.0001) and glutathione S-transferases (mortality = 58%, χ2 = 33.96, p < 0.001) in resistance. A high frequency of 1014F kdr mutation (82%) was established, with significant difference in genotype distribution associated with permethrin resistance [odds ratio = 7.71 (95% CI 2.43-14.53, χ2 = 13.67, p = 0.001]. Sequencing of intron-1 of the voltage-gated sodium channel (VGSC) revealed a low genetic diversity. CONCLUSION: High pyrethroid resistance highlight the challenges to the effectiveness of the pyrethroids-based ITNs and indoor residual spraying (IRS) against An. coluzzii in Niger. The pyrethroids-synergists LLINs and organophosphate-based IRS maybe the alternatives for malaria control in southern Niger.

Vector bionomics and malaria transmission in an area of sympatry of An. arabiensis, An. coluzzii and An. gambiae.

Despite extensive genetic studies on their variability and differentiation, few is known about the specific and relative role of An. coluzzii, An. gambiae and An. arabiensis in areas of sympatry. Indeed, their behavioral dissimilarities and divergent population dynamics can impact on malaria transmission level and intensity. This study was undertaken in four sympatric sites belonging to two different ecosystems with differential insecticide pressure to study the bionomics of these species and their relative role in malaria transmission. Mosquitoes were collected monthly from July to December 2011 when landing on human volunteers and by pyrethrum spray catches. Specimens belonging to the An. gambiae complex were further identified using molecular tools. Plasmodium falciparum infection and blood-feeding preferences were studied using the ELISA techniques. Overall, the three species were in sympatry in each of the four sites with the predominance of An. gambiae. Mosquito populations' dynamics varied temporally depending on the rainy season for each zone. The anthropophilic rates varied between 45.7 and 78.1% for An. arabiensis, 81.8 and 100% for An. coluzzii and 80 and 96.7% for An. gambiae. Plasmodium infection rates were higher in An. gambiae (range: 2.17%-6.54%) while for An. arabiensis and An. coluzzii it varied respectively between 0-1.24% and 0-3.66%. Malaria transmission occured in each of the four sites both indoors and outdoors and was due mainly to An. gambiae. An. arabiensis and An. coluzzii played a limited role due both to a low anthropophilic rate and a lower biting rate for An. coluzzii in comparison with An. gambiae. This study showed that, while present in sympatric areas, species from the An. gambiae complex could exhibit differential involvement in malaria transmission. Even less involved in malaria transmission, the occurrence of ecological and environmental changes tending to a good adaptation of An. coluzzii could lead to a great risk for malaria transmission in time and space in human populations.

Experimental huts trial of the efficacy of pyrethroids/piperonyl butoxide (PBO) net treatments for controlling multi-resistant populations of Anopheles funestus s.s. in Kpomè, Southern Benin.

Background: Insecticides resistance in Anopheles mosquitoes limits Long-Lasting Insecticidal Nets (LLIN) used for malaria control in Africa, especially Benin. This study aimed to evaluate the bio-efficacy of current LLINs in an area where An. funestuss.l. and An. gambiae have developed multi-resistance to insecticides, and to assess in experimental huts the performance of a mixed combination of pyrethroids and piperonyl butoxide (PBO) treated nets on these resistant mosquitoes. Methods: The study was conducted at Kpomè, Southern Benin. The bio-efficacy of LLINs against An. funestus and An. gambiae was assessed using the World Health Organization (WHO) cone and tunnel tests. A released/recapture experiment following WHO procedures was conducted to compare the efficacy of conventional LLINs treated with pyrethroids only and LLINs with combinations of pyrethroids and PBO. Prior to huts trials, we confirmed the level of insecticide and PBO residues in tested nets using high performance liquid chromatography (HPLC). Results: Conventional LLINs (Type 2 and Type 4) have the lowest effect against local multi-resistant An. funestus s.s. and An. coluzzii populations from Kpomè. Conversely, when LLINs containing mixtures of pyrethroids and PBO (Type 1 and Type 3) were introduced in trial huts, we recorded a greater effect against the two mosquito populations (P < 0.0001). Tunnel test with An. funestus s.s. revealed mortalities of over 80% with this new generation of LLINs (Type 1 and Type 3),while conventional LLINs produced 65.53 ± 8.33% mortalities for Type 2 and 71.25 ±7.92% mortalities for Type 4. Similarly, mortalities ranging from 77 to 87% were recorded with the local populations of An. coluzzii. Conclusion: This study suggests the reduced efficacy of conventional LLINs (Pyrethroids alone) currently distributed in Benin communities where Anopheles populations have developed multi-insecticide resistance. The new generation nets (pyrethroids+PBO) proved to be more effective on multi-resistant populations of mosquitoes.

The current insecticide resistance status of Anopheles gambiae (s.l.) (Culicidae) in rural and urban areas of Bouaké, Côte d'Ivoire.

BACKGROUND: Several studies were carried out in experimental hut station in areas surrounding the city of Bouaké, after the crisis in Côte d'Ivoire. They reported increasing resistance levels to insecticide for malaria transmiting mosquitoes. The present work aims to evaluate the current resistance level of An. gambiae (s.l.) in rural and urban areas in the city of Bouaké. METHODS: Larvae of Anopheles gambiae (s.l.) were collected from five different study sites and reared to adult stages. The resistance status was assessed using the WHO bioassay test kits for adult mosquitoes, with eight insecticides belonging to pyrethroids, organochlorines, carbamates and organophosphates classes. Molecular assays were performed to identify the molecular forms of An. gambiae (s.l.), the L1014F kdr and the ace-1R alleles in individual mosquitoes. The synergist PBO was used to investigate the role of enzymes in resistance. Biochemical assays were performed to detect potential increased activities in mixed function oxidase (MFO) levels, non-specific esterases (NSE) and glutathione S-transferases (GST). RESULTS: High resistance levels to pyrethroids, organochlorines, and carbamates were observed in Anopheles gambiae (s.l.) from Bouaké. Mortalities ranged between 0 and 73% for the eight tested insecticides. The pre-exposure to PBO restored full or partial susceptibility to pyrethroids in the different sites. The same trend was observed with the carbamates in five sites, but to a lesser extent. With DDT, pre-exposure to PBO did not increase the mortality rate of An. gambiae (s.l.) from the same sites. Tolerance to organophosphates was observed. An increased activity of NSE and higher level of MFO were found compared to the Kisumu susceptible reference strain. Two molecular forms, S form [(An. gambiae (s.s)] and M form (An. coluzzi) were identified. The kdr allele frequencies vary from 85.9 to 99.8% for An. gambiae (s.s.) and from 81.7 to 99.6% for An. coluzzii. The ace-1R frequencies vary between 25.6 and 38.8% for An. gambiae (s.s.) and from 28.6 to 36.7% for An. coluzzii. CONCLUSION: Resistance to insecticides is widespread within both An. gambiae (s.s.) and An. coluzzii. Two mechanisms of resistance, i.e. metabolic and target-site mutation seemed to largely explain the high resistance level of mosquitoes in Bouaké. Pyrethroid resistance was found exclusively due to the metabolic mechanism.

The implementation of long-lasting insecticidal bed nets has differential effects on the genetic structure of the African malaria vectors in the Anopheles gambiae complex in Dielmo, Senegal.

BACKGROUND: Mosquitoes belonging to the Anopheles gambiae complex are the main vectors of malaria in sub-Saharan Africa. Among these, An. gambiae, Anopheles coluzzii and Anopheles arabiensis are the most efficient vectors and are largely distributed in sympatric locations. However, these species present ecological and behavioural differences that impact their vectorial capacity and complicate vector-control efforts, mainly based on long-lasting insecticidal bed nets (LLINs) and indoor residual spraying (IRS). In this study, the genetic structure of these three species in a Senegalese village (Dielmo) was investigated using microsatellite data in samples collected in 2006 before implementation of LLINs, in 2008, when they were introduced, and in 2010, 2 years after the use of LLINs. RESULTS: In this study 611 individuals were included, namely 136 An. coluzzii, 101 An. gambiae, 6 An. coluzzii/An. gambiae hybrids and 368 An. arabiensis. According to the species, the effect of the implementation of LLINs in Dielmo is differentiated. Populations of the sister species An. coluzzii and An. gambiae regularly experienced bottleneck events, but without significant inbreeding. The Fst values suggested in 2006 a breakdown of assortative mating resulting in hybrids, but the introduction of LLINs was followed by a decrease in the number of hybrids. This suggests a decrease in mating success of hybrids, ecological maladaptation, or a lesser probability of mating between species due to a decrease in An. coluzzii population size. By contrast, the introduction of LLINs has favoured the sibling species An. arabiensis. In this study, some spatial and temporal structuration between An. arabiensis populations were detected, especially in 2008, and the higher genetic diversity observed could result from a diversifying selection. CONCLUSIONS: This work demonstrates the complexity of the malaria context and shows the need to study the genetic structure of Anopheles populations to evaluate the effectiveness of vector-control tools and successful management of malaria vector control.

Profiles of soluble proteins in chemosensory organs of three members of the afro-tropical Anopheles gambiae complex.

In female mosquitoes, host-seeking and preference as well as several other important behaviors are largely driven by olfaction. Species of the Afrotropical Anopheles gambiae complex display divergent host-preference that are associated with significant differences in their vectorial capacity for human malaria. Olfactory sensitivity begins with signal transduction and activation of peripheral sensory neurons that populate the antennae, maxillary palps and other appendages. We have used shotgun proteomics to characterize the profile of soluble proteins of antennae and maxillary palps of three different species: An. coluzzii, An. arabiensis and An. quadriannulatus that display remarkable differences in anthropophilic behavior. This analysis revealed interspecific differences in the abundance of several proteins that comprise cuticular components, glutathione S-transferase and odorant binding proteins, the latter of which known to be directly involved in odor recognition.

Malaria transmission and the sensitivity of aggressive mosquitoes to insecticides in a poorly urbanized area of the Deido health district in Douala (Cameroon).

The design of an appropriate strategy for mosquito control in urban areas must take into account the local ecoclimate and its particularities. This study sought to determine the dynamics of malaria transmission and the level of insecticide susceptibility of aggressive mosquitoes in Ndogbong, a poorly urbanized district of Douala. Mosquitoes were captured on volunteers in 4 houses and outdoors for 2 consecutive nights a month, from April to September 2014, and identified by molecular and morphologic techniques. The infectivity of vectors was determined by a circumsporozoite protein (CSP) ELISA test. The susceptibility of vectors to DDT, deltamethrin, and permethrin was also assessed. Overall, 3794 mosquitoes belonging to 6 species were captured in 96 person-nights. The aggressive mosquito fauna comprised : Culex pipiens (57.83%), Cx. poicilipes (1.45%), Anopheles gambiae s.l. (39.01%), Aedes albopictus (1.51%), and An. paludis (0.10%). An. coluzzii accounted for 97% of members of the Gambiae complex captured and was the only species found to be infected with P. falciparum, with an average infection rate of 1.9% (95% CI : 0.82-4.41). The mean entomological inoculation rate was estimated at 0.3 ib/p/n. Susceptibility testing on female An. gambiae s.l. and Cx. pipiens revealed mortality rates lower than 85 % and thus suggested strong resistance to insecticides. An. Coluzzii develops well in poorly urbanized built-up areas of Africa and appears to be the major malaria vector in Ndogbong, especially during the rainy season. The high resistance to insecticides observed requires the development of new formulations for insecticides.

Multiple insecticide resistance in the major malaria vector Anopheles funestus in southern Ghana: implications for malaria control.

BACKGROUND: Understanding the dynamics of insecticide resistance in African malaria vectors is crucial for successful implementation of resistance management strategies in the continent. This study reports a high and multiple insecticide resistance in Anopheles funestus from southern Ghana which could compromise the Malaria Operational Plan in this country, if not tackled. Adult Anopheles mosquitoes were collected in Obuasi and Adawukwa, in southern Ghana. Plasmodium infection rates, susceptibility to the main insecticides used in public health and the molecular basis of insecticide resistance were established. RESULTS: An. funestus (sensu stricto) (s.s.) was the predominant mosquito species found resting inside the houses in Obuasi, while at Adawukwa it was found together with An. coluzzii. Parasite rates were high in An. funestus (s.s.) populations from both localities, with Plasmodium infection rates greater than 12.5 %. Both, An. funestus (s.s.) and An. coluzzii, from the two sites exhibited high resistance to the insecticide from various classes including the pyrethroids, carbamates and DDT, but remained fully susceptible to the organophosphates. A preliminary characterization of the underlying molecular mechanisms of resistance in An. funestus (s.s.) populations from both sites revealed that CYP6P9a, CYP6P9b, CYP6M7 and GSTe2 genes are upregulated, markedly higher in Obuasi (between 3.35 and 1.83 times) than in Adawukwa population. The frequency of L119F-GSTe2 and A296S-RDL resistance markers were also higher in Obuasi (42.5 and 68.95 % higher), compared with An. funestus (s.s.) populations from Adawukwa. These findings suggest that the similar resistance pattern observed in both An. funestus (s.s.) populations are driven by different mechanisms. CONCLUSIONS: Resistance to multiple insecticides in public health use is present in malaria vectors from Ghana with major resistance genes already operating in the field. This should be taken into consideration in the design of resistance management strategies to avoid operational failure.

The larvicidal effects of black pepper (Piper nigrum L.) and piperine against insecticide resistant and susceptible strains of Anopheles malaria vector mosquitoes.

BACKGROUND: Insecticide resistance carries the potential to undermine the efficacy of insecticide based malaria vector control strategies. Therefore, there is an urgent need for new insecticidal compounds. Black pepper (dried fruit from the vine, Piper nigrum), used as a food additive and spice, and its principal alkaloid piperine, have previously been shown to have larvicidal properties. The aim of this study was to investigate the larvicidal effects of ground black pepper and piperine against third and fourth instar Anopheles larvae drawn from several laboratory-reared insecticide resistant and susceptible strains of Anopheles arabiensis, An. coluzzii, An. gambiae, An. quadriannulatus and An. funestus. METHODS: Larvae were fed with mixtures of standard larval food and either ground black pepper or piperine in different proportions. Mortality was recorded 24 h after black pepper and 48 h after piperine were applied to the larval bowls. RESULTS: Black pepper and piperine mixtures caused high mortality in the An. gambiae complex strains, with black pepper proving significantly more toxic than piperine. The An. funestus strains were substantially less sensitive to black pepper and piperine which may reflect a marked difference in the feeding habits of this species compared to that of the Gambiae complex or a difference in food metabolism as a consequence of differences in breeding habitat between species. CONCLUSIONS: Insecticide resistant and susceptible strains by species proved equally susceptible to black pepper and piperine. It is concluded that black pepper shows potential as a larvicide for the control of certain malaria vector species.

Insecticide susceptibility of natural populations of Anopheles coluzzii and Anopheles gambiae (sensu stricto) from Okyereko irrigation site, Ghana, West Africa.

BACKGROUND: The increasing spread of insecticide resistance in malaria vectors has been well documented across sub-Saharan Africa countries. The influence of irrigation on increasing vector resistance is poorly understood, and is critical to successful and ethical implementation of food security policies. This study investigated the insecticide resistance status of An. gambiae (s.l.) mosquitoes collected from the irrigated rice area of Okyereko, a village containing about 42 hectares of irrigated field within an irrigation project plan in the Central Region of Ghana. Large amounts of insecticides, herbicides and fertilizers are commonly used in the area to boost the annual production of the rice. METHODS: Mosquito larvae were collected and adults were assayed from the F1 progeny. The resistance status, allele and genotype were characterized using WHO susceptibility testing and PCR methods respectively. RESULTS: The An. gambiae (s.l.) populations from Okyereko are highly resistant to DDT and pyrethroid insecticides, with possible involvement of metabolic mechanisms including the elevation of P450 and GST enzyme as well as P-gp activity. The population was mostly composed of An. coluzzii specimens (more than 96 %) with kdr and ace-1 frequencies of 0.9 and 0.2 %, respectively. CONCLUSION: This study brings additional information on insecticide resistance and the characterization of An. gambiae (s.l.) mosquitoes from Okyereko, which can be helpful in decision making for vector control programmes in the region.

Swarming behaviour in natural populations of Anopheles gambiae and An. coluzzii: review of 4 years survey in rural areas of sympatry, Burkina Faso (West Africa).

The swarming behaviour of natural populations of Anopheles gambiae and An. coluzzii (formerly known as An. gambiae S and M forms, respectively) were investigated through longitudinal surveys conducted between July 2006 and October 2009 in two rural areas of south-western Burkina Faso where these forms are sympatric. In both sites, the majority of swarms were recorded above visual markers localised among houses. In Soumousso, a wooded area of savannah, 108 pairs caught in copula from 205 swarms were sampled; in VK7, a rice growing area, 491 couples from 250 swarms were sampled. If segregated swarms were the norm in both sites, many visual markers were shared by the two forms of An. gambiae. Furthermore, mixed swarms were collected annually in frequencies varying from one site to another, though no mixed inseminations were recorded, corroborating the low hybrid rate previously reported in the field. The occurrence of inter-specific mate-recognition mechanisms, which allow individuals to avoid hybridisation, is discussed.

Larval competition between An. coluzzii and An. gambiae in insectary and semi-field conditions in Burkina Faso.

Competition in mosquito larvae is common and different ecological context could change competitive advantage between species. Here, larval competition between the widely sympatric African malaria mosquitoes, Anopheles coluzzii and Anopheles gambiae were investigated in controlled insectary conditions using individuals from laboratory colonies and under ambient conditions using wild mosquitoes in a semi-field enclosure in western Burkina Faso. Larvae of both species were reared in trays at the same larval density and under the same feeding regimen in either single-species or mixed-species populations at varying species ratios reflecting 0%, 25%, 50% and 75% of competitor species. In the insectaries, where environmental variations are controlled, larvae of the An. coluzzii colony developed faster and with lower mortality than larvae of the An. gambiae colony (8.8±0.1 days and 21±3% mortality vs. 9.5±0.1 days and 32±3% mortality, respectively). Although there was no significant effect of competition on these phenotypic traits in any species, there was a significant trend for higher fitness of the An. coluzzii colony when competing with An. gambiae under laboratory conditions (i.e. lower development time and increased wing length at emergence, Cuzik's tests, P<0.05). In semi-field experiments, competition affected the life history traits of both species in a different way. Larvae of An. gambiae tended to reduce development time when in competition with An. coluzzii (Cuzick's test, P=0.002) with no impact either on mortality or size at emergence. On the other hand, An. coluzzii showed a significant trend for reduced larval mortality with increasing competition pressure (Cuzick's test, P=0.037) and production of smaller females when grown together with An. gambiae (Cuzick's test, P=0.002). Our results hence revealed that competitive interactions between larvae of the two species are context dependent. They further call for caution when exploring ecological processes using inbred laboratory colonies in this system of utmost medical importance.