Malaria Vector Anopheles Coluzzii Research Articles

Exploring the molecular mechanisms of increased intensity of pyrethroid resistance in Central African population of a major malaria vector Anopheles coluzzii.

Molecular mechanisms driving the escalation of pyrethroid resistance in the major malaria mosquitoes of Central Africa remain largely uncharacterized, hindering effective management strategies. Here, resistance intensity and the molecular mechanisms driving it were investigated in a population of Anopheles coluzzii from northern Cameroon. High levels of pyrethroid and organochloride resistance were observed in An. coluzzii population, with no mortality for 1× permethrin; only 11% and 33% mortalities for 5× and 10× permethrin diagnostic concentrations, and <2% mortalities for deltamethrin and DDT, respectively. Moderate bendiocarb resistance (88% mortality) and full susceptibility to malathion were observed. Synergist bioassays with piperonyl butoxide recovered permethrin susceptibility, with mortalities increasing to 53.39%, and 87.30% for 5× and 10× permethrin, respectively, implicating P450 monooxygenases. Synergist bioassays with diethyl maleate (DEM) recovered permethrin and DDT susceptibilities (mortalities increasing to 34.75% and 14.88%, respectively), implicating glutathione S-transferases. RNA-seq-based genome-wide transcriptional analyses supported by quantitative PCR identified glutathione S-transferase, GSTe2 (RNA-seqFC = 2.93 and qRT-PCRFC = 8.4, p < 0.0043) and CYP450, CYP6Z2 (RNA-seqFC = 2.39 and qRT-PCRFC = 11.7, p < 0.0177) as the most overexpressed detoxification genes in the pyrethroid-resistant mosquitoes, compared to mosquitoes of the susceptible Ngousso colony. Other overexpressed genes include P450s, CYP6M2 (FC = 1.68, p < 0.0114), CYP4G16 (FC = 2.02, p < 0.0005), and CYP4G17 (FC = 1.86, p < 0.0276). While high frequency of the 1014F kdr mutation (50%) and low frequencies of 1014S (6.61%) and 1575Y (10.29%) were observed, no ace-1 mutation was detected in bendiocarb-resistant populations, suggesting the preeminent role of metabolic mechanism. Overexpression of metabolic resistance genes (including GSTe2 and CYP6Z2 known to confer resistance to multiple insecticides) in An. coluzzii from the Sudan Savannah of Cameroon highlights the need for alternative management strategies to reduce malaria burden in northern Cameroon.

Impact of blood meals taken on ivermectin-treated livestock on survival and egg production of the malaria vector Anopheles coluzzii under laboratory conditions.

Treatment of livestock with endectocides such as ivermectin is viewed as a complementary vector control approach to address residual transmission of malaria. However, efficacy of this treatment may vary between animal species. Hence, our purpose was to investigate the effects of ivermectin treatments of common livestock species on life history traits of the opportunistic malaria vector Anopheles coluzzii. Sheep, goats and pigs were treated using injectable veterinary ivermectin formulation at the species-specific doses (recommended dose for all species and high dose in pig). Mosquito batches were exposed to treated and control (not injected) animals at different days after treatment. Daily mosquito mortality was recorded and fecundity assessed through the count of gravid females and the number of eggs they developed. The recommended dose of ivermectin induced a significant decrease in mosquito survival for up to 7 days after injection (DAI), with a decrease of 89.7%, 66.7%, and 48.4% in treated pigs, goats and sheep, respectively, compared to control animals. In treated pigs, the triple therapeutic dose decreased mosquito survival of 68.97% relatively to controls up to 14 DAI. The average number in gravid females Anopheles that survived after feeding on treated animals were reduced when blood-meals were taken on sheep (2.57% and 42.03% at 2 and 7 DAI), or on goats (decrease of the 28.28% and 73.64% respectively at 2 and 7 DAI). This study shows that ivermectin treatments to animals negatively impacts An. coluzzii life history traits and could reduce vector densities in areas where livestock live near humans. However, due to short-term efficacy of single dose treatments, repeated treatments and potentially increased dosages would be required to span the transmission season. The use of long-acting ivermectin formulations is discussed as a mean for extending efficacy while remaining cost effective.

Chemical composition and Larvicidal efficacy of Ficus sycomorus leaf extract against major malaria vector Anopheles coluzzii

Resistance to synthetic insecticides used in control of malaria vectors is a major threat to malaria control globally. Natural insecticides of plant origin provide environmentally safe alternativess. This study characterized the phytochemical contents of the Ficus sycomorus leaves and insecticidal activities of its methanolic extract on Anopheles colluzzii larvae. The active secondary metabolites from methanol extract of F. sycomorus leaves were characterized using gas chromatography - mass spectrometry (GC-MS). A modified WHO standard protocol for larvicidal bioassay was used to determine the activity of methanol extract of the F. sycomorus. Molecular species identification showed that 75 % of the larvae were Anopheles coluzzii (Coetzee &amp; Wilkerson); 6.25 % Anopheles arabiensis and 12.5 % Anopheles gambiae s.s. The extract reduced the survival of An. coluzzii larvae by approximately 52 % at LC50 of 0.225 mg/ml (95% CI: 0.197-0.234; R2 =0.9445) after 24 h, and the mortality was dose dependent (with highest mortality of 51.67 % at 1 mg/ml). Phytochemical screening revealed the presence of alkaloids, flavonoids, tannins, saponins, terpenoids, phytosterols and phenols, with alkaloid and flavonoids having the highest concentration in the extract. The GC-MS revealed that the extract contained: tetrapentacontane, 66.6 %; stigmasterol, 2.81 %; squalene, 1.01 %; bis(2-ethylhexyl) phthalate, 4.04 %; and 4,8,12,16-tetramethylheptadecan-4-olide, 0.51 %. The toxicity of the extract may be due to different insecticidal phytochemicals, probably working independently, synergistically, or additively. Overall, the F. sycomorus leaf extract is a promising larvicide against malarial vector An. coluzzii and should be exploited as an alternative, environmentally safe larvicide.

Chemical composition and Larvicidal efficacy of Ficus sycomorus leaf extract against major malaria vector Anopheles coluzzii

Resistance to synthetic insecticides used in control of malaria vectors is a major threat to malaria control globally. Natural insecticides of plant origin provide environmentally safe alternativess. This study characterized the phytochemical contents of the Ficus sycomorus leaves and insecticidal activities of its methanolic extract on Anopheles colluzzii larvae. The active secondary metabolites from methanol extract of F. sycomorus leaves were characterized using gas chromatography - mass spectrometry (GC-MS). A modified WHO standard protocol for larvicidal bioassay was used to determine the activity of methanol extract of the F. sycomorus. Molecular species identification showed that 75 % of the larvae were Anopheles coluzzii (Coetzee &amp; Wilkerson); 6.25 % Anopheles arabiensis and 12.5 % Anopheles gambiae s.s. The extract reduced the survival of An. coluzzii larvae by approximately 52 % at LC50 of 0.225 mg/ml (95% CI: 0.197-0.234; R2 = 0.9445) after 24 h, and the mortality was dose dependent (with highest mortality of 51.67 % at 1 mg/ml). Phytochemical screening revealed the presence of alkaloids, flavonoids, tannins, saponins, terpenoids, phytosterols and phenols, with alkaloid and flavonoids having the highest concentration in the extract. The GC-MS revealed that the extract contained: tetrapentacontane, 66.6 %; stigmasterol, 2.81 %; squalene, 1.01 %; bis(2-ethylhexyl) phthalate, 4.04 %; and 4,8,12,16-tetramethylheptadecan-4-olide, 0.51 %. The toxicity of the extract may be due to different insecticidal phytochemicals, probably working independently, synergistically, or additively. Overall, the F. sycomorus leaf extract is a promising larvicide against malarial vector An. coluzzii and should be exploited as an alternative, environmentally safe larvicide.

Molecular drivers of insecticide resistance in the Sahelo-Sudanian populations of a major malaria vector Anopheles coluzzii

BackgroundInformation on common markers of metabolic resistance in malaria vectors from countries sharing similar eco-climatic characteristics can facilitate coordination of malaria control. Here, we characterized populations of the major malaria vector Anopheles coluzzii from Sahel region, spanning four sub-Saharan African countries: Nigeria, Niger, Chad and Cameroon.ResultsGenome-wide transcriptional analysis identified major genes previously implicated in pyrethroid and/or cross-resistance to other insecticides, overexpressed across the Sahel, including CYP450s, glutathione S-transferases, carboxylesterases and cuticular proteins. Several, well-known markers of insecticide resistance were found in high frequencies—including in the voltage-gated sodium channel (V402L, I940T, L995F, I1527T and N1570Y), the acetylcholinesterase-1 gene (G280S) and the CYP4J5-L43F (which is fixed). High frequencies of the epidemiologically important chromosomal inversion polymorphisms, 2La, 2Rb and 2Rc, were observed (~80% for 2Rb and 2Rc). The 2La alternative arrangement is fixed across the Sahel. Low frequencies of these inversions (<10%) were observed in the fully insecticide susceptible laboratory colony of An. coluzzii (Ngoussou). Several of the most commonly overexpressed metabolic resistance genes sit in these three inversions. Two commonly overexpressed genes, GSTe2 and CYP6Z2, were functionally validated. Transgenic Drosophila melanogaster flies expressing GSTe2 exhibited extremely high DDT and permethrin resistance (mortalities <10% in 24h). Serial deletion of the 5′ intergenic region, to identify putative nucleotide(s) associated with GSTe2 overexpression, revealed that simultaneous insertion of adenine nucleotide and a transition (T->C), between Forkhead box L1 and c-EST putative binding sites, were responsible for the high overexpression of GSTe2 in the resistant mosquitoes. Transgenic flies expressing CYP6Z2 exhibited marginal resistance towards 3-phenoxybenzylalcohol (a primary product of pyrethroid hydrolysis by carboxylesterases) and a type II pyrethroid, α-cypermethrin. However, significantly higher mortalities were observed in CYP6Z2 transgenic flies compared with controls, on exposure to the neonicotinoid, clothianidin. This suggests a possible bioactivation of clothianidin into a toxic intermediate, which may make it an ideal insecticide against populations of An. coluzzii overexpressing this P450.ConclusionsThese findings will facilitate regional collaborations within the Sahel region and refine implementation strategies through re-focusing interventions, improving evidence-based, cross-border policies towards local and regional malaria pre-elimination.

Integration of whole genome sequencing and transcriptomics reveals a complex picture of the reestablishment of insecticide resistance in the major malaria vector Anopheles coluzzii.

Insecticide resistance is a major threat to gains in malaria control, which have been stalling and potentially reversing since 2015. Studies into the causal mechanisms of insecticide resistance are painting an increasingly complicated picture, underlining the need to design and implement targeted studies on this phenotype. In this study, we compare three populations of the major malaria vector An. coluzzii: a susceptible and two resistant colonies with the same genetic background. The original colonised resistant population rapidly lost resistance over a 6-month period, a subset of this population was reselected with pyrethroids, and a third population of this colony that did not lose resistance was also available. The original resistant, susceptible and re-selected colonies were subject to RNAseq and whole genome sequencing, which identified a number of changes across the transcriptome and genome linked with resistance. Firstly, an increase in the expression of genes within the oxidative phosphorylation pathway were seen in both resistant populations compared to the susceptible control; this translated phenotypically through an increased respiratory rate, indicating that elevated metabolism is linked directly with resistance. Genome sequencing highlighted several blocks clearly associated with resistance, including the 2Rb inversion. Finally, changes in the microbiome profile were seen, indicating that the microbial composition may play a role in the resistance phenotype. Taken together, this study reveals a highly complicated phenotype in which multiple transcriptomic, genomic and microbiome changes combine to result in insecticide resistance.

Ecological plasticity to ions concentration determines genetic response and dominance of Anopheles coluzzii larvae in urban coastal habitats of Central Africa

In Central Africa, the malaria vector Anopheles coluzzii is predominant in urban and coastal habitats. However, little is known about the environmental factors that may be involved in this process. Here, we performed an analysis of 28 physicochemical characteristics of 59 breeding sites across 5 urban and rural sites in coastal areas of Central Africa. We then modelled the relative frequency of An. coluzzii larvae to these physicochemical parameters in order to investigate environmental patterns. Then, we assessed the expression variation of 10 candidate genes in An. coluzzii, previously incriminated with insecticide resistance and osmoregulation in urban settings. Our results confirmed the ecological plasticity of An. coluzzii larvae to breed in a large range of aquatic conditions and its predominance in breeding sites rich in ions. Gene expression patterns were comparable between urban and rural habitats, suggesting a broad response to ions concentrations of whatever origin. Altogether, An. coluzzii exhibits a plastic response to occupy both coastal and urban habitats. This entails important consequences for malaria control in the context of the rapid urban expansion in Africa in the coming years.

Transcriptomic analysis reveals pronounced changes in gene expression due to sub-lethal pyrethroid exposure and ageing in insecticide resistance Anopheles coluzzii

BackgroundMalaria control is heavily reliant on the use of insecticides that target and kill the adult female Anopheline vector. The intensive use of insecticides of the pyrethroid class has led to widespread resistance in mosquito populations. The intensity of pyrethroid resistance in some settings in Africa means mosquitoes can contact bednets treated with this insecticide class multiple times with minimal mortality effects. Furthermore, both ageing and diel cycle have been shown to have large impacts on the resistance phenotype. Together, these traits may affect other aspects of vector biology controlling the vectorial capacity or fitness of the mosquito.ResultsHere we show that sublethal exposure of a highly resistant Anopheles coluzzii population originally from Burkina Faso to the pyrethroid deltamethrin results in large and sustained changes to transcript expression. We identify five clear patterns in the data showing changes to transcripts relating to: DNA repair, respiration, translation, behaviour and oxioreductase processes. Further, we highlight differential regulation of transcripts from detoxification families previously linked with insecticide resistance, in addition to clear down-regulation of the oxidative phosphorylation pathway both indicative of changes in metabolism post-exposure. Finally, we show that both ageing and diel cycle have major effects on known insecticide resistance related transcripts.ConclusionSub-lethal pyrethroid exposure, ageing and the diel cycle results in large-scale changes in the transcriptome of the major malaria vector Anopheles coluzzii. Our data strongly supports further phenotypic studies on how transcriptional changes such as reduced expression of the oxidative phosphorylation pathway or pyrethroid induced changes to redox state might impact key mosquito traits, such as vectorial capacity and life history traits.

High pyrethroid/DDT resistance in major malaria vector Anopheles coluzzii from Niger-Delta of Nigeria is probably driven by metabolic resistance mechanisms.

Entomological surveillance of local malaria vector populations is an important component of vector control and resistance management. In this study, the resistance profile and its possible mechanisms was characterised in a field population of the major malaria vector Anopheles coluzzii from Port Harcourt, the capital of Rivers state, in the Niger-Delta Region of Nigeria. Larvae collected in Port-Harcourt, were reared to adulthood and used for WHO bioassays. The population exhibited high resistance to permethrin, deltamethrin and DDT with mortalities of 6.7% ± 2.4, 37.5% ± 3.2 and 6.3% ± 4.1, respectively, but were fully susceptible to bendiocarb and malathion. Synergist bioassays with piperonylbutoxide (PBO) partially recovered susceptibility, with mortalities increasing to 53% ± 4, indicating probable role of CYP450s in permethrin resistance (χ2 = 29.48, P < 0.0001). Transcriptional profiling revealed five major resistance-associated genes overexpressed in the field samples compared to the fully susceptible laboratory colony, Ngoussou. Highest fold change (FC) was observed with GSTe2 (FC = 3.3 in permethrin exposed and 6.2 in unexposed) and CYP6Z3 (FC = 1.4 in exposed and 4.6 in unexposed). TaqMan genotyping of 32 F0 females detected the 1014F and 1575Y knockdown resistance (kdr) mutations with frequencies of 0.84 and 0.1, respectively, while 1014S mutation was not detected. Sequencing of a fragment of the voltage-gated sodium channel, spanning exon 20 from 13 deltamethrin-resistant and 9 susceptible females revealed only 2 distinct haplotypes with a low haplotype diversity of 0.33. The findings of high pyrethroid resistance but with a significant degree of recovery after PBO synergist assay suggests the need to move to PBO-based nets. This could be complemented with carbamate- or organophosphate-based indoor residual spraying in this area.

Infection\xa0of highly insecticide-resistant malaria vector Anopheles coluzzii with entomopathogenic bacteria Chromobacterium violaceum reduces its survival, blood feeding propensity and fecundity

BackgroundThis is now a concern that malaria eradication will not be achieved without the introduction of novel control tools. Microbiological control might be able to make a greater contribution to vector control in the future. The interactions between bacteria and mosquito make mosquito microbiota really promising from a disease control perspective. Here, the impact of Chromobacterium violaceum infections, isolated from both larvae and adult of wild-caught Anopheles gambiae sensu lato mosquitoes in Burkina Faso, was evaluated on mosquito survival, blood feeding and fecundity.MethodsTo assess entomopathogenic effects of C. violaceum infection on mosquitoes, three different types of bioassays were performed in laboratory. These bioassays aimed to evaluate the impact of C. violaceum infection on mosquito survival, blood feeding and fecundity, respectively. During bioassays mosquitoes were infected through the well-established system of cotton ball soaked with 6% glucose containing C. violaceum.ResultsChromobacterium violaceum kills pyrethroid resistant Anopheles coluzzii (LT80 of 8.78 days ± 0.18 at 108 bacteria cell/ml of sugar meal). Interestingly, this bacterium had other negative effects on mosquito lifespan by significantly reducing (~ 59%, P < 0.001) the mosquito feeding willingness from day 4-post infection (~ 81% would seek a host to blood feed) to 9- day post infection (22 ± 4.62% would seek a host to blood feed). Moreover, C. violaceum considerably jeopardized the egg laying (~ 16 eggs laid/mosquito with C. violaceum infected mosquitoes vs ~ 129 eggs laid/mosquito with control mosquitoes) and hatching of mosquitoes (a reduction of ~ 22% of hatching rate with C. violaceum infected mosquitoes). Compared to the bacterial uninfected mosquitoes, mosquitoes infected with C. violaceum showed significantly higher retention rates of immature eggs and follicles.ConclusionThese data showed important properties of Burkina Faso C. violaceum strains, which are highly virulent against insecticide-resistant An. coluzzii, and reduce both mosquito blood feeding and fecundity propensities. However, additional studies as the sequencing of C. violaceum genome and the potential toxins secreted will provide useful information render it a potential candidate for the biological control strategies of malaria and other disease vectors.

Genome variation and population structure among 1142 mosquitoes of the African malaria vector species Anopheles gambiae and Anopheles coluzzii.

Mosquito control remains a central pillar of efforts to reduce malaria burden in sub-Saharan Africa. However, insecticide resistance is entrenched in malaria vector populations, and countries with a high malaria burden face a daunting challenge to sustain malaria control with a limited set of surveillance and intervention tools. Here we report on the second phase of a project to build an open resource of high-quality data on genome variation among natural populations of the major African malaria vector species Anopheles gambiae and Anopheles coluzzii. We analyzed whole genomes of 1142 individual mosquitoes sampled from the wild in 13 African countries, as well as a further 234 individuals comprising parents and progeny of 11 laboratory crosses. The data resource includes high-confidence single-nucleotide polymorphism (SNP) calls at 57 million variable sites, genome-wide copy number variation (CNV) calls, and haplotypes phased at biallelic SNPs. We use these data to analyze genetic population structure and characterize genetic diversity within and between populations. We illustrate the utility of these data by investigating species differences in isolation by distance, genetic variation within proposed gene drive target sequences, and patterns of resistance to pyrethroid insecticides. This data resource provides a foundation for developing new operational systems for molecular surveillance and for accelerating research and development of new vector control tools. It also provides a unique resource for the study of population genomics and evolutionary biology in eukaryotic species with high levels of genetic diversity under strong anthropogenic evolutionary pressures.

Species and sex-specific chemosensory gene expression in Anopheles coluzzii and An. quadriannulatus antennae

BackgroundOlfactory cues drive mosquito behaviors such as host-seeking, locating sugar sources and oviposition. These behaviors can vary between sexes and closely related species. For example, the malaria vector Anopheles coluzzii is highly anthropophilic, whereas An. quadriannulatus is not. These behavioral differences may be reflected in chemosensory gene expression.MethodsThe expression of chemosensory genes in the antennae of both sexes of An. coluzzii and An. quadriannulatus was compared using RNA-seq. The sex-biased expression of several genes in An. coluzzii was also compared using qPCR.ResultsThe chemosensory expression is mostly similar in the male antennae of An. coluzzii and An. quadriannulatus, with only a few modest differences in expression. A handful of chemosensory genes are male-biased in both species; the highly expressed gustatory receptor AgGr33, odorant binding proteins AgObp25, AgObp26 and possibly AgObp10. Although the chemosensory gene repertoire is mostly shared between the sexes, several highly female-biased AgOrs, AgIrs, and one AgObp were identified, including several whose expression is biased towards the anthropophilic An. coluzzii. Additionally, the expression of several chemosensory genes is biased towards An. coluzzii in both sexes.ConclusionsChemosensory gene expression is broadly similar between species and sexes, but several sex- biased/specific genes were identified. These may modulate sex- and species-specific behaviors. Although the male behavior of these species remains poorly studied, the identification of sex- and species-specific chemosensory genes may provide fertile ground for future work.

Neuronal Odor Coding in the Larval Sensory Cone of &lt;i&gt;Anopheles Coluzzii&lt;/i&gt;: Complex Responses from a Simple System

Anopheles mosquitoes are the sole vectors of malaria and other diseases that represent significant threats to global public health. While adult female mosquitoes are responsible for disease transmission, the pre-adult larval stages of the malaria vector Anopheles coluzzii and other mosquitoes rely on a broad spectrum of sensory cues to navigate their aquatic habitats efficiently to avoid predators and search for food. Of these, mosquito larvae rely heavily on volatile chemical signals that directly activate their olfactory apparatus. Because most studies on mosquito olfaction focus on adults, a paucity of attention has been given to the larval olfactory system, in which the peripheral components are associated with the sensory cone of the larval antennae. To address this, we have investigated the electrophysiological response profile of the larval sensory cone in Anopheles mosquitoes. We found that the larval sensory cone is particularly tuned to alcohols, thiazoles and heterocyclics. Furthermore, these responses can be assigned to discrete groups of sensory cone neurons with distinctive, dose-dependent odorant-response profiles that also provide larvae with the ability to discriminate among compounds with similar chemical structures. A correlation analysis was conducted to determine the relationship between specific larval chemosensory receptors and the response profiles of sensory cone neuron groups. These studies reveal that the larval sensory cone is a highly sophisticated organ that is sensitive to a broad range of compounds and is capable of a remarkable degree of chemical discrimination. Taken together, this study presents critical insights into olfactory coding processes in An. coluzzii larvae that further our understanding of larval chemical ecology and will contribute to the development of novel larval-based strategies and tools for mosquito control and the reduction of vector-borne disease transmission.

High insecticide resistance in the major malaria vector Anopheles coluzzii in Chad Republic

BackgroundThe Sahel region of Chad Republic is a prime candidate for malaria pre-elimination. To facilitate pre-elimination efforts in this region, two populations of Anopheles coluzzii from Central Chad Republic were characterized, their insecticide resistance profile and the possible molecular mechanisms driving the resistance in the field investigated.MethodsBloodfed female Anopheles gambiae s.l. resting indoor, were collected at N’djamena and Massakory, Chad in 2018 and characterized for species composition, and infection rate was determined using the TaqMan assay. Susceptibility to various insecticides was assessed using WHO tube bioassays. Cone bioassays were conducted using various long-lasting insecticidal nets (LLINs). Results were analysed using Chi Square test. Knockdown resistance (kdr) and ace-1 markers were investigated by TaqMan genotyping.ResultsAnopheles coluzzii was the major vector found in N’djamena (100%) and Massakory (~ 94%). No Plasmodium was found in 147 bloodfed F0An. coluzzii (82 from N’djamena and 65 from Massakory). High intensity pyrethroid resistance was observed with mortalities of < 2% for permethrin, deltamethrin and etofenprox, and with < 50% and < 60% dead following exposure to 10× diagnostic doses of deltamethrin and permethrin, respectively. For both sites, < 10% mortalities were observed with DDT. Synergist bioassays with piperonylbutoxide significantly recovered pyrethroid susceptibility in Massakory populations, implicating CYP450s (mortality = 13.6% for permethrin, χ2 = 22.8, df = 1, P = 0.0006; mortality = 13.0% for deltamethrin, χ2 = 8.8, df = 1, P < 0.00031). Cone-bioassays established complete loss of efficacy of the pyrethroid-based LLINs; and a 100% recovery of susceptibility following exposure to the roof of PermaNet®3.0, containing piperonylbutoxide. Both populations were susceptible to malathion, but high bendiocarb resistance was observed in Massakory population. The absence of ace-1 mutation points to the role of metabolic resistance in the bendiocarb resistance. Both 1014F and 1014S mutations were found in both populations at around 60% and < 20% respectively. Sequencing of intron-1 of the voltage-gated sodium channel revealed a low genetic diversity suggesting reduced polymorphism.ConclusionsMultiple resistance in An. coluzzii populations from Chad highlight challenges associated with deployment of LLINs and indoor residual spraying (IRS) in the Sahel of this country. The pyrethroid-synergists LLINs (e.g. PermaNet®3.0) and organophosphate-based IRS maybe the alternatives for malaria control in this region.

Windborne long-distance migration of malaria mosquitoes in the Sahel.

Over the past two decades efforts to control malaria have halved the number of cases globally, yet burdens remain high in much of Africa and the elimination of malaria has not been achieved even in areas where extreme reductions have been sustained, such as South Africa1,2. Studies seeking to understand the paradoxical persistence of malaria in areas in which surface water is absent for 3-8months of the year have suggested that some species of Anopheles mosquito use long-distance migration3. Here we confirm this hypothesis through aerial sampling of mosquitoes at 40-290m above ground level and provide-to our knowledge-the first evidence of windborne migration of African malaria vectors, and consequently of the pathogens that they transmit. Ten species, including the primary malaria vector Anopheles coluzzii, were identified among 235anopheline mosquitoes that were capturedduring 617nocturnal aerial collections in the Sahel of Mali. Notably, females accounted for more than 80% of all ofthe mosquitoesthat we collected. Of these, 90% had taken a blood meal before their migration, which implies that pathogens areprobably transported over long distances by migrating females. The likelihood of capturing Anopheles species increased with altitude (the height of the sampling panel above ground level) and during the wet seasons, but variation between years and localities was minimal. Simulated trajectories of mosquito flights indicated that there would be mean nightly displacements of up to 300km for 9-h flight durations. Annually, the estimated numbers of mosquitoes at altitude that cross a 100-km line perpendicular to the prevailing wind direction included 81,000Anopheles gambiae sensu stricto, 6million A.coluzzii and 44million Anopheles squamosus. These results provide compelling evidence that millions of malaria vectors that have previously fed on blood frequently migrate over hundreds of kilometres, and thus almost certainly spread malaria over these distances. The successful elimination of malaria may therefore depend on whether the sources of migrant vectors can be identified and controlled.

Age-dependent regulation of host seeking in Anopheles coluzzii

Behavioural attraction of the malaria vector Anopheles coluzzii to human host odour increases during adult maturation. We have previously demonstrated that the onset of host seeking in An. coluzzii coincides with an increased sensitivity of the CO2-sensitive neurons and abundance of chemosensory receptor gene transcripts in the maxillary palp. In this study, we extend our analysis to the antenna. Functional characterisation of the near-complete repertoire of odorant receptors (Ors) expressed in this tissue, to fractioned human odour, reveals a subset of salient human odorants to be detected by Ors at physiological relevant concentrations. When presented as a blend in their ratio of natural emission, these odorants elicit attraction by host-seeking mosquitoes, emphasising that Ors alone can mediate this behaviour. However, the same blend inhibits attraction in teneral mosquitoes. This switch in behavioural response indicates a change in valence during adult maturation. Quantitative analysis of Or transcript abundance and in vivo electrophysiological analysis reveal that the olfactory system of female An. coluzzii undergoes concerted changes that correlate with the onset of host seeking. We conclude that changes in Or abundance modulate peripheral olfactory coding, resulting in ecologically relevant behavioural effects.

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

BackgroundInformation 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.MethodsBlood 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.ResultsHigh 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.ConclusionHigh 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.

Interaction of RNA viruses of the natural virome with the African malaria vector, Anopheles coluzzii

Mosquitoes are colonized by a little-studied natural virome. Like the bacterial microbiome, the virome also probably influences the biology and immunity of mosquito vector populations, but tractable experimental models are lacking. We recently discovered two novel viruses in the virome of wild Anopheles and in colonies of the malaria vector Anopheles coluzzii: Anopheles C virus and Anopheles cypovirus. Here, we describe biological interactions between these two viruses and An. coluzzii mosquitoes. Viral abundance varies reproducibly during mosquito development. DNA forms of these viruses were not detected, and thus viral persistence is likely based on vertical transmission of RNA genomes. At least Anopheles C virus is vertically transmitted by an intraembryonic route. Relative abundance of the two viruses is inversely correlated in individual mosquitoes. One possible mechanism for this could be interactions with host immunity, and functional genomic analysis indicated differential influence of at least the Toll and JAK/STAT immune signaling pathways upon the viruses. The nonrandom distributions and interactions with host immunity suggest that these and other members of the natural virome may constitute a source of unrecognized heterogeneity in mosquito vector populations.

MicroRNAs from saliva of anopheline mosquitoes mimic human endogenous miRNAs and may contribute to vector-host-pathogen interactions

During blood feeding haematophagous arthropods inject into their hosts a cocktail of salivary proteins whose main role is to counteract host haemostasis, inflammation and immunity. However, animal body fluids are known to also carry miRNAs. To get insights into saliva and salivary gland miRNA repertoires of the African malaria vector Anopheles coluzzii we used small RNA-Seq and identified 214 miRNAs, including tissue-enriched, sex-biased and putative novel anopheline miRNAs. Noteworthy, miRNAs were asymmetrically distributed between saliva and salivary glands, suggesting that selected miRNAs may be preferentially directed toward mosquito saliva. The evolutionary conservation of a subset of saliva miRNAs in Anopheles and Aedes mosquitoes, and in the tick Ixodes ricinus, supports the idea of a non-random occurrence pointing to their possible physiological role in blood feeding by arthropods. Strikingly, eleven of the most abundant An. coluzzi saliva miRNAs mimicked human miRNAs. Prediction analysis and search for experimentally validated targets indicated that miRNAs from An. coluzzii saliva may act on host mRNAs involved in immune and inflammatory responses. Overall, this study raises the intriguing hypothesis that miRNAs injected into vertebrates with vector saliva may contribute to host manipulation with possible implication for vector-host interaction and pathogen transmission.

Investigation of the Role of ABC Transporters in Pyrethroids Resistance in the Major Malaria Vector Anopheles coluzzii from Northern Nigeria

Resistance to currently available insecticides in the major malaria vectors like Anopheles coluzzii is seriously reducing the effectiveness of core vector control tools. Overexpression of ATP binding cassette transporters (ABC transporters) has been implicated in insecticides resistance in the major malaria vector Anopheles funestus and An. gambiae. To identify the potential role of ABC transporters in pyrethroids resistance in this work, we use verapamil-a p-glycoprotein inhibitor in a synergist bioassay with pyrethroids on one An. coluzzii populations from northern Nigeria. Genomic DNA extraction and SINE 200 PCR established that the Anopheles s. l. from Auyo, Jigawa State was An. coluzzii. Pre-exposure to verapamil followed by Permethrin results in 0.12% knockdown after 1 hour exposure and 10.7% mortality after 24 hours, which was higher than that for permethrin alone, but not statistically significant. This bioassay result reveals that ABC transporters possibly do not contribute to the resistance.