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Cues involved in mate seeking and recognition prevent hybridization and can be involved in speciation processes. In malaria mosquitoes, females of the two sibling species Anopheles gambiae s.s. and An. coluzzii mate in monospecific male swarms and hybrids are rare. Long-range sex pheromones driving this behavior have been debated in literature but to date, no study has proven their existence or their absence. Here, we attempted to bring to light their existence. To put all the odds in our favor, we used different chemical ecology methods such as behavioral and electrophysiological assays as well chemical analyses, and we worked with mosquitoes at their optimal physiological mating state i.e. with swarming males during their natural swarming windows. Despite all our efforts, our results support the absence of long-range sex pheromones involved in swarm detection and recognition by females. We briefly discuss the implications of this finding in ecology, evolution and for control strategies.

Pyriproxyfen (PPF) is an insect growth regulator used in the co-treatment of long-lasting insecticidal nets for its ability to sterilize female mosquitoes. To evaluate the efficacy of PPF-treated nets on mosquito reproductivity, most studies observe oviposition (egg-laying) rates in the laboratory. This technique has several technical disadvantages. Our study assessed if ovarial dissection could serve as an effective proxy for evaluating sterility in Anopheles gambiae mosquitoes. Blood-fed females were exposed to untreated or PPF-treated nets in cylinder assays and followed over several days to observe oviposition rates or egg development by dissection. For identifying PPF-exposed mosquitoes, both techniques demonstrated high sensitivity (oviposition: 99.1%; dissection: 100.0%), but for identifying non-exposed mosquitoes, specificity was significantly higher in the dissection group (52.5% vs. 18.9%). To assess whether dissection could be applied to nets treated with a pyrethroid or co-treated with a pyrethroid and PPF in tunnel tests, a blinded investigator performed dissections to predict the PPF exposure status across different treatment groups. The exposure status of dissected females was predicted with >90% accuracy. We report that dissection is a sensitive technique to assess sterility in female Anopheles gambiae mosquitoes and can be used as a predictor of PPF exposure.

Objective: We evaluated the susceptibility status of Anopheles gambiae in two communities of Ikorodu, Lagos, Nigeria to DDT, deltamethrin, lambda cyhalothrin and bendiocarb. Methods: Anopheles immature stages were collected from their habitats in the surveyed community and allowed to emerge before exposure adult females to discriminating doses of WHO insecticides including DDT, deltamethrin, lambda cyhalothrin, bendiocarb and malathion. PBO synergistic bioassay was conducted for insecticides where the mosquito samples showed resistance. PCR assay was used for the detection of kdr mutation in the mosquitoes. Results: Resistance to DDT (40% and 86%) and lambda cyhalothrin (75% and 84%) in Oke-Ota and Majidun respectively. Suspected resistance to deltamethrin (94.9%) and bendiocarb (93.5%) was recorded in Oke-Ota community and the mosquitoes were susceptible to malathion in both communities. KDR mutation (L1014F) from resistance samples from both locations though with a low frequency that significantly departs from Hardy-Weinberg’s probability (P> 0.01). PBO synergized bioassay was able to increase knockdown, percentage mortality and restore full susceptibility to deltamethrin and bendiocarb. Conclusion: Results from this study indicates that the metabolic resistance mechanism is highly implicated in the resistance to different classes of insecticide in Ikorodu and this should be taken into consideration when implementing vector control activities in this area. Keywords: Malaria; Anopheles gambiae; insecticide resistance; knockdown resistance; metabolic resistance.

Der Überträger von Malaria, Anopheles gambiae, fordert jedes Jahr das Leben von etwa einer halben Million Menschen. Dennoch haben Mücken Mechanismen entwickelt, die die Ausbreitung von Krankheitserregern einschränken können. Der NF-κB-ähnliche Signalweg REL2 wurde als Schlüsselmechanismus für die Abtötung von Plasmodium falciparum in Mücken beschrieben, der sie bei experimenteller Aktivierung resistent gegen Plasmodium-Parasiten macht. Der Weg blieb jedoch schlecht charakterisiert. Zur Charakterisierung des REL2-Signalwegs wurden Loss-of-Function-Mutanten des Gens das Transkriptionsfaktor REL2 kodiert verwendet. Hier wurde die Fitness der Mücken mit REL2-Mangel, ihre Anfälligkeit für eine Infektion mit P. falciparum und Veränderungen in ihren mikrobiellen Gemeinschaften untersucht. Es wurde eine entscheidende Rolle des REL2-Signalwegs bei der Kontrolle der Proliferation des Mitteldarmmikrobioms und folglich des Überlebens der Mücken identifiziert. Es konnte kein konsistenter Phänotyp in der Anfälligkeit von REL2-Mutanten gegenüber Plasmodium beobachtet werden. Die Interaktion des Signalwegs und des Parasiten war indirekt, als Folge der REL2- und Mikrobiom-Interaktion. Gewebespezifische Transkriptomsequenzierung wurde verwendet um REL2-Genziele zu identifizieren. Die Ergebnisse zeigten die gewebespezifische Funktion des REL2-Signalwegs, wobei der Mitteldarm der Hauptort seiner Aktivität ist. Die identifizierten REL2-Genziele gehörten zu verschiedenen Domänen der Mückenphysiologie, einschließlich des Stoffwechsels. Mit weiteren Experimenten wurde die Rolle des REL2-Signalwegs bei der Blutmahlzeitverdauung und dem Management von Zucker- und Lipidressourcen beobachtet. Diese Studie ist die erste, die die Hauptgewebe der REL2-Aktivität und die Genziele des Signalwegs in Mücken identifiziert. Seine Hauptfunktion ist die Regulierung der mikrobiellen Gemeinschaften der Mücken im Mitteldarm, aber der Signalweg ist auch an der Feinabstimmung von Stoffwechselprozessen beteiligt. Malaria vector, Anopheles gambiae, claims lives of around half a million people each year. But mosquitoes have developed an array of mechanisms that can limit pathogen proliferation. NF-κB-like signaling pathway REL2 has been described as a key mechanism for Plasmodium falciparum killing in mosquitoes, rendering them resistant to Plasmodium parasites when experimentally activated. However, the pathway remained poorly characterized. With an aim to characterize the REL2-dependent Plasmodium killing, loss-of-function mutants of the gene encoding the NF-κB-like transcription factor REL2 were used. The fitness of the REL2 deficient mosquitoes, their susceptibility to P. falciparum infection, and changes in their microbial communities were investigated. A critical role of REL2 pathway in controlling the midgut microbiome proliferation, and consequently the mosquito survival, has been identified. Interestingly, no consistent phenotype could be observed in the susceptibility of REL2 mutants to Plasmodium. Rather, the interaction of the pathway and parasite seems to be indirect, as a consequence of the REL2 and microbiome interaction. Furthermore, tissue-specific transcriptome sequencing was used to identify REL2 gene targets. The sequencing results revealed the tissue-specific function of the REL2-pathway, with the midgut being the main site of its activity. Identified REL2 gene targets belonged to various domains of mosquito physiology, including metabolism. With further experiments, the role of the REL2 pathway in blood meal digestion and management of sugar and lipid resources has been observed. Taken together, this study is the first to identify the main tissues of the REL2 activity and also gene targets of the pathway in mosquitoes. Its main function was found to be the regulation of mosquito microbial communities in the midgut, but the pathway also seems involved in fine-tuning of metabolic processes.

Microbiome composition has been associated with insecticide resistance in malaria vectors. However, the contribution of major symbionts to the increasingly reported resistance escalation remains unclear. This study explores the possible association of a specific endosymbiont, Asaia spp., with elevated levels of pyrethroid resistance driven by cytochrome P450s enzymes and voltage-gated sodium channel mutations in Anopheles funestus and Anopheles gambiae. Molecular assays were used to detect the symbiont and resistance markers (CYP6P9a/b, 6.5 kb, L1014F, and N1575Y). Overall, genotyping of key mutations revealed an association with the resistance phenotype. The prevalence of Asaia spp. in the FUMOZ_X_FANG strain was associated with the resistance phenotype at a 5X dose of deltamethrin (OR = 25.7; p = 0.002). Mosquitoes with the resistant allele for the markers tested were significantly more infected with Asaia compared to those possessing the susceptible allele. Furthermore, the abundance correlated with the resistance phenotype at 1X concentration of deltamethrin (p = 0.02, Mann-Whitney test). However, for the MANGOUM_X_KISUMU strain, findings rather revealed an association between Asaia load and the susceptible phenotype (p = 0.04, Mann-Whitney test), demonstrating a negative link between the symbiont and permethrin resistance. These bacteria should be further investigated to establish its interactions with other resistance mechanisms and cross-resistance with other insecticide classes.

The crude extracts of leaves of Aristolochia elegans Mast (Aristolochiaceae) collected from Rombo district in Kilimanjaro, Tanzania (3˚11ʹ15.4ʹʹS 37˚37ʹ05.5ʹʹE), were obtained by maceration of sample at room temperature followed by phytochemical analysis by GC-MS. The GC-MS analysis of the extracts showed that the main content was Caryophyllene among all extract investigated, followed by Calarene and Bicyclogermacrene. The crude extracts were then evaluated for larvicidal activity using World Health Organization (WHO) procedures against the third Instar larvae of Anopheles gambiae and monitored at an interval of 24 h, from 24 to 72 h and activity was recorded for various concentrations of the ranging from 25 to 200.0 μg/mL. Mortality effect of investigated extracts against laboratory-reared larvae had variations indicated by their LC50. LC50 for ethanol leaf extract were 165 µg/mL and 135 µg/mL after 48 and 72 h, respectively. LC50 for dichloromethane leaf extract were 145 µg/mL and 136 µg/mL after 48 and 72 h, respectively. For n-hexane extract, LC50 were 34 µg/mL and 33 µg/mL after 48 and 72 h, respectively. The remarkable differences in the larvicidal activity of crude extracts against the larvae of A. gambiae were due to qualitative and quantitative variations of the components, exemplified by the presence of sesquiterpenes with caryophyllene skeleton. The findings indicate that n-hexane leaf extract has potentials for use in the control of A. gambiae larvae and may be useful in the search for novel and effective natural compounds as larvicides.

Each year, over 200 million people are infected with the malaria parasite, nearly half a million of whom succumb to the disease. Emerging genetic technologies could, in theory, eliminate the burden of malaria throughout the world by intentionally eradicating the mosquitoes that transmit the disease. In this paper, we offer an ethical examination of the intentional eradication of Anopheles gambiae, the main malaria vector of sub-Saharan Africa. In our evaluation, we focus on two main considerations: the benefit of alleviating the malaria burden, and the loss of value that would accompany the eradication of the species. We outline a typology of the different ways in which species are valued or could be valuable, then use that typology to appraise the value of the species in question. We argue that Anopheles gambiae has minor (and redundant) instrumental value, little final subjective value and no objective final value.

This study was aimed to investigate thecurrent insecticide resistance status in malaria vector mosquitoes from Dogbo district in South-western Benin, West Africa. Larvae and pupae ofAnopheles gambiae s.l.populations were collected from the breeding sites from March to July 2020 during the first rainy season in Couffo department using the dipping method. WHO susceptibility tests were conducted on unfed female mosquitoes aged 2-5 days old. WHO bioassays were performed with impregnated papers of permethrin (0.75%), deltamethrin (0.05%), cyfluthrin (0.15%), lambdacyhalothrin (0.05%) and dichlorodiphenyltrichloroethane (DDT) (4%).An. gambiaes.l.populations from Dogbo district were resistant to permethrin, deltamethrin, cyfluthrin, lambdacyhalothrin and DDT. There is cross-resistance to both pyrethroid and DDT insecticides. However, the physical barrier of Long Lasting Insecticidal Nets (LLINs) which are regularly distributed free by Beninese National Malaria Control Programme throughout the entire country to increase coverage of LLINs is still important despite the insecticide resistance observed.

The current study was aimed to compare the susceptibility to deltamethrin in female adultAnopheles gambiae s.l.from Dogbo district with their F1 progeny susceptibility in South-Western Benin, West Africa. Larvae and pupae ofAnopheles gambiae s.l.populations were collected from the breeding sites in Couffo department in 2020 and reared up for obtaining F1 progeny. Female adultAn. gambiae s.l.mosquitoes were also collected from window traps put on windows of rooms in Dogbo district surveyed.An. gambiae s.l.mosquitoes were collected from March to July and August to November 2020 during the rainy season in the locations of Ayomi, Dévé, Honton, Lokogohoué, Madjrè and Totchangni. Female adultAn. gambiaespecies collected from window traps were morphologically identified using morphological keys and then transferred into mosquito cages for WHO bioassays performed with impregnated papers of deltamethrin (0.05%). WHO bioassays were also performed with F1 progeny. The physiological age of female adultAn. gambiae s.l.collected from window traps was determined through dissection using Detinova method. The results showed that the mortality rates recorded with female adultAn. gambiae s.l.mosquitoes collected from window traps were higher than those obtained with their F1 progenyAn. gambiae s.l.mosquitoes obtained after reproduction of parent mosquitoes. The current study clearly shows that changes in mosquito physiology occur with senescence.

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.

The essential oil (EO) of plants of the Poaceae family has diverse chemical constituents with several biological properties. But, data on the chemical constituents and toxicity are still unavailable for some species belonging to this family, such as Euclasta condylotricha Steud (Eu. condylotricha). In this study, the chemical composition of the EOs of Eu. condylotricha flowers was evaluated by gas chromatography coupled with mass spectrometry (GC–MS). The EOs larvicidal property was assessed against third instar larvae of three Anopheles gambiae laboratory strains (Kisumu, Acerkis and Kiskdr) according to the WHO standard protocol. The percentage yields of the EOs obtained from hydro distillation of Eu. condylotricha flowers varied 0.070 to 0.097%. Gas Chromatography-Mass Spectrometry (GC-MS) applied to the EOs revealed fifty-five (55) chemical constituents, representing 94.95% to 97.78% of the total essential oils. Although different chemical profiles of the dominant terpenes were observed for each sample, EOs were generally dominated by sesquiterpenoids with juvenile hormones as the major compounds. The primary compounds were juvenile hormone C16 (JH III) (35.97–48.72%), Methyl farnesoate 10,11-diol (18.56–28.73%), tau-Cadinol (18.54%), and β-Eudesmene (12.75–13.46%). Eu. condylotricha EOs showed a strong larvicidal activity with LC50 values ranging from 35.21 to 52.34 ppm after 24 hours of exposition. This study showed that Eu. Condylotricha flowers essential oils are potent sources of juvenile hormones that could be a promising tool for developing an eco-friendly malaria vector control strategy.

In eukaryotes, cellular differentiation is often orchestrated by programmed arrays of activation and repression of genes underlying the specific phenotypes of cell-types. To complete its life cycle, the single-celled Apicomplexan parasite Plasmodium falciparum, the most deadly of the human malaria parasites, must repeatedly differentiate and convert into unique cell types that can exploit niches within their human and mosquito hosts. One of these key developmental transitions occurs in the human host and includes sexual-stage commitment, followed by gametocyte development, which results in male and female gametocytes that can infect the female anopheline mosquito once taken up during the blood meal. The following gamete stage undergoes fusion and meiosis, followed by morphological changes as they invade the midgut wall to form the oocyst. The sexual stages are responsible for malaria transmission and the spread of antimalarial resistance, making it an important target for malaria eradication. Previous studies have established the understanding of a few mechanisms involved in sexual differentiation; the transcription factor, AP2-G, is a master regulator of sexual commitment. Uncovering genes and regulators underlying P. falciparum sexual stage development, including host-parasite interactions are the subjects of this thesis. In paper I, we describe the temporal landscape of differential transcriptional programs underlying the P. falciparum male and female gametocyte development and highlight a bifurcation point for the onset of differential male and female transcriptional programs. Furthermore, we predict novel candidate driver genes underlying sexual cell fate determination in P. falciparum. Finally, we traced the potential sex-specific regulatory mechanisms among the ApiAP2 family of transcription factors including prediction of target genes based on co-expression and the presence of upstream binding sites, rendering a motif-driven gene regulatory network. In paper II, we study the P. falciparum development in the mosquito midgut, from unfertilized female gamete through the first 20 hours of development, including the zygote and ookinete stages. Using pseudotime analysis, we demonstrate that the transcriptional trajectory of the parasite is controlled by three main transcriptional programs including five unique transcriptional stages. Further, we employ structural-based functional predictions to predict intrinsically disordered proteins encoding upregulated genes through the mid to late ookinete with antigenic properties, which may serve as suitable targets for transmission suppression strategies. In paper III, we utilize single-cell RNA sequencing combined with functional assays to demonstrate an increased complexity of the Anopheles mosquito immune cells, previously defined as hemocytes, Based on panels of genetic markers and morphological traits. We define additional marker genes that enable deeper characterization of Anopheles immune cell subtypes and uncover transcriptional programs involved in immune cell differentiation and maturation. In summary, this thesis aims to uncover previously unknown aspects of eukaryotic cell fate determination and life cycle stage differentiation with a focus on P. falciparum sexual stage development. Furthermore, we aim to enable deeper characterization of the Anopheles mosquito immune cell subtypes, their differentiation, development, and fate during steady-state and upon immune activation.

Background Pyrethroids are the most widely used insecticides for the control of malaria transmitting Anopheles gambiae mosquitoes and rapid increase in resistance to this insecticide class is of major concern. Pyrethroids target the Voltage Gated Sodium Channels (VGSCs), that have a key role in the normal function of the mosquitoes' nervous system. VGSC mutations L995F and L995S have long been associated with pyrethroid resistance and screening for their presence is routine in insecticide resistance management programs. Recently, a VGSC haplotype containing two amino acid substitutions associated with resistance in other species, V402L and I1527T, was identified. These two VGSC mutations are found in tight linkage and are mutually exclusive to the classical L995F/S mutations. Results We identify the presence of the V402L-I1527 T haplotype in resistant An. coluzzii colonized strains and in field populations from Burkina Faso at frequencies higher than previously reported; in some cases almost reaching fixation. Functional validation of V402L in insecticide resistance using a CRISPR/Cas9 genome modified line showed that it confers reduced mortality after exposure to all tested pyrethroids and DDT, but at lower levels compared to L995F. In contrast however to L995F, no fitness costs were identified for mosquitoes carrying V402L under laboratory conditions. Conclusion The V402L substitution confers pyrethroid resistance in An. gambiae in the absence of any other VGSC substitution and/or alternative resistance mechanisms. The lower fitness cost associated with this kdr mutation may provide a selective advantage over the classical kdr in some settings and genotyping at this locus should be added in the list of resistant alleles for routine screening. This article is protected by copyright. All rights reserved.

Background Long-lasting insecticide nets (LLINs) are a core malaria intervention. LLINs should retain efficacy against mosquito vectors for a minimum of three years. Efficacy and durability of Olyset® Plus, a permethrin and piperonyl butoxide (PBO) treated LLIN, was evaluated versus permethrin treated Olyset® Net. In the absence of WHO guidelines of how to evaluate PBO nets, and considering the manufacturer’s product claim, Olyset® Plus was evaluated as a pyrethroid LLIN. Methods This was a household randomized controlled trial in a malaria endemic rice cultivation zone of Kirinyaga County, Kenya between 2014 and 2017. Cone bioassays and tunnel tests were done against Anopheles gambiae Kisumu. The chemical content, fabric integrity and LLIN survivorship were monitored. Comparisons between nets were tested for significance using the Chi-square test. Exact binomial distribution with 95% confidence intervals (95% CI) was used for percentages. The WHO efficacy criteria used were ≥ 95% knockdown and/or ≥ 80% mortality rate in cone bioassays and ≥ 80% mortality and/or ≥ 90% blood-feeding inhibition in tunnel tests. Results At 36 months, Olyset® Plus lost 52% permethrin and 87% PBO content; Olyset® Net lost 24% permethrin. Over 80% of Olyset® Plus and Olyset® Net passed the WHO efficacy criteria for LLINs up to 18 and 12 months, respectively. At month 36, 91.2% Olyset® Plus and 86.4% Olyset® Net survived, while 72% and 63% developed at least one hole. The proportionate Hole Index (pHI) values representing nets in good, serviceable and torn condition were 49.6%, 27.1% and 23.2%, respectively for Olyset® Plus, and 44.9%, 32.8% and 22.2%, respectively for Olyset® Net but were not significantly different. Conclusions Olyset® Plus retained efficacy above or close to the WHO efficacy criteria for about 2 years than Olyset® Net (1–1.5 years). Both nets did not meet the 3-year WHO efficacy criteria, and showed little attrition, comparable physical durability and survivorship, with 50% of Olyset® Plus having good and serviceable condition after 3 years. Better community education on appropriate use and upkeep of LLINs is essential to ensure effectiveness of LLIN based malaria interventions. Graphical Abstract

Background: Anopheles gambiae (An. gambiae) is considered the most effective malaria vector worldwide and is widely distributed in Africa. The purpose of this study is to determine the potential impact of climate change on An. gambiae and predict the present and future potential suitable habitat globally. Methods: In this study, environmental variables, global occurrence data of An. gambiae, and the maximum entropy model (MaxEnt) were used to evaluate the contribution of environmental factors and predict the habitat suitability of An. gambiae under the current and future scenarios. Results: Among all environmental variables, isothermality (Bio3, 34.5%) contributed the most to An. gambiae distribution. Under current climate conditions, the potential suitable areas for An. gambiae are mainly located near the equator (approximately 30°N-30°S), with a total area of 16.58 million km2, including central and northern South America, a fraction of areas near the equator of North America, central and southern Africa, some tropical regions of southern Asia, and small areas of Oceania. The areas of potential suitable habitats would be reduced to varying degrees in future climate scenarios. Conclusions: Potential suitable habitats for An. gambiae may not be limited to Africa. Necessary surveillance and preventive measures should be undertaken in high-risk regions, including those outside Africa, to monitor and control the spread of An. gambiae.

Assessing the genetic diversity of metabolic resistance genes, such as cytochrome P450s, helps to understand the dynamics and evolution of resistance in the field. Here, we analyzed the polymorphisms of CYP6M2 and CYP6P4, associated with pyrethroid resistance in Anopheles coluzzii and Anopheles gambiae, to detect potential resistance markers. Field-caught resistant mosquitos and susceptible lab strains were crossed, and F4 was exposed to permethrin for 15 min and 90 min to discriminate highly susceptible (HS) and highly resistant (HR) mosquitos, respectively. Significant permethrin mortality reduction was observed after pre-exposure to PBO, suggesting the gene involvement of P450s. qPCR analysis revealed significant overexpression of CYP6M2 (FC = 19.57 [95% CI 13.96–25.18] for An. coluzzii; 10.16 [7.86–12.46] for An. gambiae) and CYP6P4 (FC = 6.73 [6.15–7.30] An. coluzzii; 23.62 [26.48–20.76] An. gambiae). Full-gene and ≈1 kb upstream were sequenced. For CYP6M2, the upstream region shows low diversity in HR and HS (overall Hd = 0.49, π = 0.018), whereas the full-gene shows allelic-variation but without evidence of ongoing selection. CYP6P4 upstream region showed a lower diversity in HR (Hd = 0.48) than HS (Hd = 0.86) of An. gambiae. These results highlighted that CYP6P4-associated resistance is potentially driven by modification in upstream region. However, further work is needed to determine the real causative variants that will help design rapid detection tools.

Methylene blue (MB) is the oldest synthetic anti-infective. Its high potency against asexual and sexual stages of malaria parasites is well documented. This study aimed to investigate possible additional activities of MB in interfering with parasite transmission and determine target stages in Anopheles vectors and humans. MB’s transmission-blocking activity was first evaluated by an ex vivo direct membrane feeding assay (DMFA) using Plasmodium falciparum field isolates. To investigate anti-mosquito stage activity, Plasmodium berghei-infected Anopheles stephensi mosquitoes were fed a second blood meal on mice that had been treated with methylene blue, 3, 6- and 15-days after the initial infectious blood meal. Anti-sporozoite and liver stage activities were evaluated in vitro and in vivo via sporozoite invasion and liver stage development assays, respectively. MB exhibited a robust inhibition of P. falciparum transmission in An. gambiae, even when added shortly before the DMFA but only a moderate effect against P. berghei oocyst development. Exposure of mature P. berghei and P. falciparum sporozoites to MB blocked hepatocyte invasion, yet P. berghei liver stage development was unaffected by MB. Our results indicate previously underappreciated rapid specific activities of methylene blue against Plasmodium transmission stages, preventing the establishment of both mosquito midgut and liver infections as the first essential steps in both hosts.

The use of chemical insecticides causes important damages to environment and human health and there is a need to search for alternative solutions. This study aims to investigate on the effect of coconut oil onAnopheles gambiae sensu latolarvae tolerance in malaria vector control in Dogbo district in south-western Benin, West Africa. Larvae ofAnopheles gambiae s.l.mosquitoes were collected from breeding sites using the dipping method in May 2020 during the rainy season in Dogbo district. A batch of 25 larvae of fourth instar were exposed to a mixture of coconut oil with distilled water saturated with oxygen containing in each of five glass jars or test cups of same dimensions contained each 48 ml distilled water saturated with oxygen plus 2 ml of coconut oil and one control jar containing no trace of coconut oil. Larval mortality was recorded after 24 hours, 48 hours and 72hours exposure. The results show that the use of coconut oil causes full-grown Anopheles larvae to die by suffocation. After the application of this mixture, the larvae of four instars cannot breathe. The use of coconut oil is effective method for disturbing the siphonal respiration of mosquito larvae. Coconut oil is effective method for mosquito larvae control.

The use of chemical insecticides causes important damages to environment and human health and there is a need to search for alternative solutions. This study aims to investigate on the effect of aqueous extract of lemon inAnopheles gambiaes.l.larvae tolerance in couffo department in south-western Republic of Benin, West Africa. Larvae ofAnopheles gambiae s.lmosquitoes were collected from breeding sites using the dipping method in July 2020 during the rainy season in Dogbo district. A batch of 15 larvae of fourth instar were exposed to a mixture of aqueous extract of lemon with distilled water saturated with oxygen containing in each of five glass jars or test cups of same dimensions contained each 48 ml distilled water saturated with oxygen plus 2 ml of aqueous extract of lemon (Citrus limon)and one control jar containing no trace of aqueous extract of lemon. Larval mortality was recorded after 24hours, 48hours and 72hours exposure. The results show that the use of aqueous extract of lemon causes full-grown Anopheles larvae to die by suffocation. After the application of this mixture, the larvae of four instars cannot breathe. The use of aqueous extract of lemon is effective method for disturbing the siphonal respiration of mosquito larvae. Aqueous extract of lemon is effective method for mosquito larvae control.

Aim: In a view to determining the capacity of Clerodendrum polycephalum to control mosquitoes, the methanol extract of the leaf was investigated for insecticidal activities using three species of mosquitoes, Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Methodology: The leaves of C. polycephalum were collected, dried and extracted with methanol. Mosquito larvae were exposed to different concentrations for 24/48h. and sublethal concentrations (L25, 50, 75) of the extract to determine larvicidal activity and monitor growth and development respectively. Twenty (20) blood-fed female Aedes aegyti mosquitoes were allowed to lay eggs on treated filter papers for antioviposition bioassay. The crude extract was separated into N-hexane, Dichloromethane, ethyl-acetate, ethanol fractions using Vacuum Liquid Chromatography to determine the active fraction. Results: Results showed that larval mortalities were in the order C. quiquefasciatus>A. gambiae>A. aegypti with effective concentration ranging from 250 – 8000 ppm. Mortalities at 48hr were significantly different (p

Background There is evidence that the knockdown resistance gene (Kdr) L1014F and acetylcholinesterase-1 gene (Ace-1R) G119S mutations involved in pyrethroid and carbamate resistance in Anopheles gambiae influence malaria transmission in sub-Saharan Africa. This is likely due to changes in the behaviour, life history and vector competence and capacity of An. gambiae. In the present study, performed as part of a two-arm cluster randomized controlled trial evaluating the impact of household screening plus a novel insecticide delivery system (In2Care Eave Tubes), we investigated the distribution of insecticide target site mutations and their association with infection status in wild An. gambiae sensu lato (s.l.) populations. Methods Mosquitoes were captured in 40 villages around Bouaké by human landing catch from May 2017 to April 2019. Randomly selected samples of An. gambiae s.l. that were infected or not infected with Plasmodium sp. were identified to species and then genotyped for Kdr L1014F and Ace-1R G119S mutations using quantitative polymerase chain reaction assays. The frequencies of the two alleles were compared between Anopheles coluzzii and Anopheles gambiae and then between infected and uninfected groups for each species. Results The presence of An. gambiae (49%) and An. coluzzii (51%) was confirmed in Bouaké. Individuals of both species infected with Plasmodium parasites were found. Over the study period, the average frequency of the Kdr L1014F and Ace-1R G119S mutations did not vary significantly between study arms. However, the frequencies of the Kdr L1014F and Ace-1R G119S resistance alleles were significantly higher in An. gambiae than in An. coluzzii [odds ratio (95% confidence interval): 59.64 (30.81–131.63) for Kdr, and 2.79 (2.17–3.60) for Ace-1R]. For both species, there were no significant differences in Kdr L1014F or Ace-1R G119S genotypic and allelic frequency distributions between infected and uninfected specimens (P > 0.05). Conclusions Either alone or in combination, Kdr L1014F and Ace-1R G119S showed no significant association with Plasmodium infection in wild An. gambiae and An. coluzzii, demonstrating the similar competence of these species for Plasmodium transmission in Bouaké. Additional factors including behavioural and environmental ones that influence vector competence in natural populations, and those other than allele measurements (metabolic resistance factors) that contribute to resistance, should be considered when establishing the existence of a link between insecticide resistance and vector competence. Graphical Abstract

Background Mosquitoes are important vectors of disease-causing organisms such as filarial worms, malaria parasites, and arboviruses endemic to sub-Saharan Africa including Nigeria. Malaria is a disease caused by a plasmodium parasite, transmitted by the bite of infected mosquitoes and is no doubt a public health concern. There is limited information on the genetic diversity of mosquitoes in Nigeria. This is necessary because information about the genetic diversity of mosquitoes is a very important step towards vector control and management with the aim to mitigate or eliminate burden resulting from malaria and other diseases caused by mosquitoes. In the present study, we investigated the genetic variability and relatedness of mosquitoes based on the DNA sequences of the mitochondrial cytochrome oxidase subunit I (COI) and ribosomal intergenic non-coding spacer gene regions (IGS). Mosquitoes were collected from five different states in Northcentral Nigeria, they were morphologically identified using standard keys and genomic DNA was extracted. The specific regions of interests were amplified, and the PCR products were then sequenced. Results PCR was able to successfully amplify the expected amplicon sizes of COI and IGS sequences (710 and 169) base pairs, respectively. For COI sequence, pairwise genetic distance between mosquito species ranged from 0.00 to 0.17 in the COI sequences. The pairwise genetic distance among Culex, Aedes and Anopheles species in the IGS sequences ranged from 0.000 to 0.118. Phylogenetic analysis of the sequences of the mitochondrial cytochrome oxidase subunit showed that there was genetic diversity amongst the different mosquito species sampled. It effectively showed marked differences between Culicine and Anopheline mosquitoes. Conclusions The ribosomal IGS primers used for this study only amplified Anopheles spp. However, it revealed that there is diversity among the Anopheles gambiae and Anopheles arabiensis samples collected. This study concludes that the mitochondrial COI and ribosomal IGS gene regions are reliable markers for mosquito genetic diversity study and will surely yield a reliable result for molecular diversity assessment of mosquito species.

Background Mosquitoes are key vectors for the transmission of several diseases. Anopheles gambiae is known to transmit pathogens of malaria and filariasis. Due to several anthropogenic factors such as climate change and population growth leading to diverse land use, their distribution and disease spreading pattern may change. This study estimated the potential distribution and climatic suitability of An. gambiae under the present-day and future conditions across Southwest Nigeria using Ecological Niche Modelling (ENM). The future scenarios assessed were based on two general circulation models (GCMs), namely community climate system model 4 (CCSM4) and geophysical fluid dynamics laboratory-climate model 3 (GFDL-CM3), in two representative concentration pathways (RCP 2.6 and RCP 8.5). Methodology The occurrence data were obtained from literatures that have reported the presence of An. gambiae mosquito species in locations within the study area. Ecological niche modelling data were processed and analysed using maximum entropy algorithm implemented in MaxEnt. Result Fifty-five (55) unique occurrences of An. gambiae were used in the model calibration after data cleaning. Data analysis for the present-day habitat suitability shows that more than two-thirds (81.71%) of the study area was observed to be suitable for An. gambiae population. However, the two future GCMs showed contrasting results. The CCSM4 models indicated a slight increase in both RCPs with 2.5 and 8.5 having 81.77 and 82.34% suitability, respectively. The reverse was the case for the GFDL-CM3 models as RCPs 2.5 and 8.5 had 78.86 and 76.86%. Conclusion This study revealed that the study area is climatically suitable for An. gambiae and will continue to be so in the future irrespective of the contrasting results from the GCMs used. Since vector population is often linked with their disease transmission capacity, proper measures must be put in place to mitigate disease incidences associated with the activities of An. gambiae.

Background Irrigation schemes may result in subsequent changes in malaria disease dynamics. Understanding the mechanisms and effects of irrigation on malaria vector bionomics and transmission intensity is essential to develop new or alternative surveillance and control strategies to reduce or control malaria risk. This study was designed to assess the effect of rice irrigation on malaria vector bionomics and transmission intensity in the Gambella Region, Ethiopia. Methods Comparative cross-sectional study was conducted in Abobo District of the Gambella Region, Ethiopia. Accordingly, clusters (kebeles) were classified into nearby and faraway clusters depending on their proximity to the irrigation scheme. Adult mosquito survey was conducted in February, August and November 2018 from three nearby and three faraway clusters using Centers for Disease Control and Prevention (CDC) light traps (LTs). During the November survey, human landing catch (HLC) and pyrethrum spray catch (PSC) were also conducted. The collected mosquitoes were morphologically identified to species and tested for Plasmodium infection using circumsporozoite protein enzyme-linked immunosorbent assay (CSP-ELISA). Furthermore, species-specific polymerase chain reaction (PCR) was performed to identify member species of the Anopheles gambiae complex. Chi-square and t-tests were used to analyze the data using the SPSS version 20 software package. Results A total of 4319 female anopheline mosquitoes comprising An. gambiae sensu lato, An. funestus group, An. pharoensis, An. coustani complex and An. squamosus were collected. Overall, 84.5% and 15.5% of the anopheline mosquitoes were collected from the nearby and faraway clusters, respectively. Anopheles gambiae s.l. was the predominant (56.2%) anopheline species in the area followed by An. pharoensis (15.7%). The density of anopheline mosquitoes was significantly higher in the nearby clusters in both HLCs [t(3) = 5.14, P = 0.0143] and CDC LT catches [t(271.97) = 7.446, P Conclusions Significantly higher mosquito population density was observed in areas close to the irrigation sites. Sporozoite infection rate in the mosquito population was also markedly higher from the nearby clusters. Therefore, the irrigation scheme could increase the risk of malaria in the area. Graphical abstract

BackgroundUntil recently, relatively little research has been done on how mosquitoes behave around the occupied bed net in the indoor environment. This has been partly remedied in the last few years through laboratory and field studies, most of these using video methods and mosquito flight tracking. Despite these recent advances, understanding of the mosquito-bed net environment system, and the principles that underlie mosquito behaviour within it, is limited. This project aimed to further understand this system by studying the effects of gently moving air (such as might be introduced through room design to make the indoor environment more comfortable and conducive to ITN use) and warmer vs. cooler ambient conditions on mosquito activity around ITNs and other bed nets.MethodsThe activity of colonized femaleAnopheles gambiaearound an occupied untreated bed net set up in a mosquito-proof tent in a large laboratory space was recorded under different ambient conditions using a laser detection-video recording system. Conditions tested were ‘cool’ (23–25 °C) and ‘warm’ (27–30 °C) air temperatures and the presence or absence of a cross-flow produced by a small central processing unit (CPU) fan pointed at the side of the net so that it produced a ‘low-’ or ‘high-’ speed cross-draught (approx. 0.1 and 0.4 m/s, respectively). Near-net activity in recordings was measured using video image analysis.ResultsIn cool, still air conditions, more than 80% of near-net activity byAn. gambiaeoccurred on the net roof. Introduction of the low-speed or high-speed cross-draught resulted in an almost total drop off in roof activity within 1 to 2 min and, in the case of the high-speed cross-draught, a complementary increase in activity on the net side. In warm, still conditions, near-net activity appeared to be lower overall than in cool, still air conditions and to be relatively less focussed on the roof. Introduction of the high-speed cross-draught in warm conditions resulted in a decrease in roof activity and increase in side activity though neither effect was statistically significant.ConclusionsResults are interpreted in terms of the flow of the stimulatory odour plume produced by the net occupant which, consistent with established principles of fluid dynamics, appears to rise quickly and remain more intact above the net occupant in cool, still air than in warm, still air. Cross-draught effects are ascribed to the changes they cause in the flow of the host odour plume as opposed to mosquito flight directly. The implications of these results for house designs that promote indoor air movement, on bed net design, and on other vector control measures are discussed. How mosquitoes approach a net is influenced both by indoor temperature and ventilation and their interaction. This system is in need of further study.

Effective management of insect disease vectors requires a detailed understanding of their ecology and behavior. In Anopheles gambiae sensu lato (s.l.) (Diptera: Culicidae) mating occurs during swarming, but knowledge of their mating behavior under natural conditions is limited. Mosquitoes mate in flight over specific landmarks, known as swarm markers, at particular locations. Swarms consist of males; the females usually approach the swarm and depart following copulation. The number of mating pairs per swarm is closely associated with swarm size. The shape and height of swarm markers vary and may depend on the environmental conditions at the swarm’s location. Male–male interactions in mosquito swarms with similar levels of attractive flight activity can offer a mating advantage to some individuals. Flight tone is used by mosquitoes to recognize the other sex and choose a desirable mate. Clarifying these and other aspects of mosquito reproductive behavior can facilitate the development of population control measures that target swarming sites. This review describes what is currently known about swarming behavior in Anopheles gambiae s.l., including swarm characteristics; mating within and outside of swarms, insemination in females, and factors affecting and stimulating swarming.

Background Understanding the ecology and behaviour of disease vectors, including the olfactory cues used to orient and select hosts and egg-laying sites, are essential for the development of novel, insecticide-free control tools. Selected graminoid plants have been shown to release volatile chemicals attracting malaria vectors; however, whether the attraction is selective to individual plants or more general across genera and families is still unclear. Methods To contribute to the current evidence, we implemented bioassays in two-port airflow olfactometers and in large field cages with four live graminoid plant species commonly found associated with malaria vector breeding sites in western Kenya: Cyperus rotundus and C. exaltatus of the Cyperaceae family, and Panicum repens and Cynodon dactylon of the Poaceae family. Additionally, we tested one Poaceae species, Cenchrus setaceus, not usually associated with water. The volatile compounds released in the headspace of the plants were identified using gas chromatography/mass spectrometry. Results All five plants attracted gravid vectors, with the odds of a mosquito orienting towards the choice-chamber with the plant in an olfactometer being 2–5 times higher than when no plant was present. This attraction was maintained when tested with free-flying mosquitoes over a longer distance in large field cages, though at lower strength, with the odds of attracting a female 1.5–2.5 times higher when live plants were present than when only water was present in the trap. Cyperus rotundus, previously implicated in connection with an oviposition attractant, consistently elicited the strongest response from gravid vectors. Volatiles regularly detected were limonene, β-pinene, β-elemene and β-caryophyllene, among other common plant compounds previously described in association with odour-orientation of gravid and unfed malaria vectors. Conclusions The present study confirms that gravid Anopheles gambiae sensu stricto use chemical cues released from graminoid plants to orientate. These cues are released from a variety of graminoid plant species in both the Cyperaceae and Poaceae family. Given the general nature of these cues, it appears unlikely that they are exclusively used for the location of suitable oviposition sites. The utilization of these chemical cues for attract-and-kill trapping strategies must be explored under natural conditions to investigate their efficiency when in competition with complex interacting natural cues. Graphical abstract

An evaluation of fly pests of cattle was conducted in Federal College of Animal Health and Production Technology (FCAH&PT) Farm, Chaha Village, Jos South Local Government Area, Plateau State, Nigeria between August and October, 2020. Samples were collected from the farm weekly for three months. A total of nine hundred and nineteen (919) adult dipterous flies comprising fifteen species in eight genera across the three sub-orders were caught and identified using aerial sweeping hand net, light trapping and aerial insecticide spray. Phlebotomus papaltasi (44.18%) was the most prevalent while Simulium damnosum (0.22%) was the least. Others were Musca domestica (17.73%), Stomoxys calcitrans (11.43%), Culicoides species (10.99%), Musca autumnalis (10.23%), mosquitoes – Anopheles gambiae/Culex quequinfasciatus (4.89%) and Tabanus species (0.33%). The haematophagous flies (72.04%) out-numbered the non-haematophagous flies (27.96%) and the number of females collected/caught 780 (84.87%) were higher than the males 139 (15.13%). The highest collections were made in October (353 representing 38.41%) while September had the lowest (263 /28.62%). The study established the presence of fly pests in the farm and recommends that appropriate control measures should be put in place to forestall any disease outbreak.

The decline in malaria across Africa has been largely attributed to vector control using long-lasting insecticidal nets (LLINs). However, this intervention has prompted widespread insecticide resistance (IR) and been associated with changes in mosquito behaviour that reduce their contact with LLINs. The relative importance and rate at which IR and behavioural adaptations emerge are poorly understood. We conducted surveillance of mosquito behaviour and IR at 12 sites in Burkina Faso to assess the magnitude and temporal dynamics of insecticide, biting and resting behaviours in vectors in the 2-year period following mass LLIN distribution. Insecticide resistance was present in all vector populations and increased rapidly over the study period. In contrast, no longitudinal shifts in LLIN-avoidance behaviours (earlier or outdoor biting and resting) were detected. There was a moderate but statistically significant shift in vector species composition from Anopheles coluzzii to Anopheles gambiae which coincided with a reduction in the proportion of bites preventable by LLINs; possibly driven by between-species variation in behaviour. These findings indicate that adaptations based on insecticide resistance arise and intensify more rapidly than behavioural shifts within mosquito vectors. However, longitudinal shifts in mosquito vector species composition were evident within 2 years following a mass LLIN distribution. This ecological shift was characterized by a significant increase in the exophagic species (An. gambiae) and coincided with a predicted decline in the degree of protection expected from LLINs. Although human exposure fell through the study period due to reducing vector densities and infection rates, such ecological shifts in vector species along with insecticide resistance were likely to have eroded the efficacy of LLINs. While both adaptations impact malaria control, the rapid increase of the former indicates this strategy develops more quickly in response to selection from LLINS. However, interventions targeting both resistance strategies will be needed.

The scale up of indoor residual spraying (IRS) and insecticide treated nets have contributed significantly to global reductions in malaria prevalence over the last two decades. However, widespread pyrethroid resistance has necessitated the use of new and more expensive insecticides for IRS. Partial IRS with pirimiphos-methyl in experimental huts and houses in a village-wide trial was evaluated against Anopheles gambiae s.l. in northern Ghana. Four different scenarios in which either only the top or bottom half of the walls of experimental huts were sprayed, with or without also spraying the ceiling were compared. Mortality of An. gambiae s.l. on partially sprayed walls was compared with the standard procedures in which all walls and ceiling surfaces are sprayed. A small-scale trial was then conducted to assess the effectiveness, feasibility, and cost of spraying only the upper walls and ceiling as compared to full IRS and no spraying in northern Ghana. Human landing catches were conducted to estimate entomological indices and determine the effectiveness of partial IRS. An established transmission dynamics model was parameterized by an analysis of the experimental hut data and used to predict the epidemiological impact and cost effectiveness of partial IRS for malaria control in northern Ghana. In the experimental huts, partial IRS of the top (IRR 0.89, p = 0.13) or bottom (IRR 0.90, p = 0.15) half of walls and the ceiling was not significantly less effective than full IRS in terms of mosquito mortality. In the village trial, the annual entomological inoculation rate was higher for the unsprayed control (217 infective bites/person/year (ib/p/yr)) compared with the fully and partially sprayed sites, with 28 and 38 ib/p/yr, respectively. The transmission model predicts that the efficacy of partial IRS against all-age prevalence of malaria after six months would be broadly equivalent to a full IRS campaign in which 40% reduction is expected relative to no spray campaign. At scale, partial IRS in northern Ghana would have resulted in a 33% cost savings ($496,426) that would enable spraying of 36,000 additional rooms. These findings suggest that partial IRS is an effective, feasible, and cost saving approach to IRS that could be adopted to sustain and expand implementation of this key malaria control intervention.

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.

Diseases transmitted by mosquitoes constitute serious health, economic and social problems, especially in developing countries such as Nigeria. This study aimed to examine the salivary glands of Anopheles mosquitoes for sporozoites and the culicine mosquitoes for filarial worms in Kunga community of Jos North Local Government Area, Plateau State, Nigeria. Adult mosquitoes were collected by Pyrethrum Spray Catch (PSC) and identified with morphological keys. The salivary glands of the female mosquitoes were then dissected to check for Plasmodium sporozoites and third-stage infective nematode larvae (L3i). A total of 75 mosquitoes were collected from 20 households. Out of which 18 representing 24.0% were Anopheles gambiae and 57 representing 76.0% were Culex quinquefasciatus. There was a significant difference (P < 0.05) in the species composition of the mosquitoes with sex. All the 18 (100%) of An. gambiae mosquitoes collected were females, while 55 (96.49%) out of the 57 Culex quinquefasciatus were females and 2 (3.51%) were males. The population of mosquitoes in relation to house types showed no significant variation (P > 0.05). Although there was a significant difference (P < 0.05) in the infectivity status of the mosquitoes, the distribution of the infection status by mosquito species showed that 12 females of An. gambiae representing 66.7% were infected with Plasmodium sporozoites, while 19 females represented 34.5% of Cx. quinquefasciatus were infected with L3i. The lack of good drainage and sewage systems in the study area could be one of the factors contributing to the mosquito burden and therefore health education will go a long way in sensitizing the inhabitants on the importance of environmental and personal hygiene.

Background Malaria is often persistent in communities surrounded by mosquito breeding habitats. Anopheles gambiae sensu lato exploit a variety of aquatic habitats, but the biotic determinants of its preferences are poorly understood. This study aimed to identify and quantify macroinvertebrates in different habitat types with determined water physico-chemical parameters to establish those preferred by An. gambiae s.l. larvae as well as their predators and competitors. Methods A field survey was conducted in Kibuye and Kayonjo villages located in the vicinity of the River Sezibwa, north-eastern Uganda to identify Anopheline larval habitats shared by aquatic insects. Habitats were geo-recorded and as streams, ponds, temporary pools and roadside ditches. From October to December 2017, random microhabitats/quadrats were selected from each habitat type, their water physico-chemical parameters (electrical conductivity, total dissolved solids, temperature and pH) were measured, and they were sampled for macroinvertebrates using standard dippers. All collected arthropod macroinvertebrates were then morphologically identified to family level and enumerated. Results Principal component analysis showed that the four larval habitat types were characterized by distinct physico-chemical parameter profiles. Ponds and streams had the highest number and diversity of macroinvertebrate insect taxa and sustained few An. gambiae s.l. larvae. Anopheles gambiae s.l. were more common in roadside ditches and particularly abundant in temporary pools which it commonly shared with Dytiscidae (predaceous diving beetles) and Culex spp. Cluster correlation analysis conducted on the abundance of these taxa within quadrats suggested that An. gambiae s.l. and Dytiscidae have the most similar patterns of microhabitat use, followed by Cybaeidae (water spiders). Whilst Culex spp. co-occurred with An. gambiae s.l. in some habitats, there was only partial niche overlap and no clear evidence of competition between the two mosquito taxa. Conclusions Ponds and streams are habitats that host the largest diversity and abundance of aquatic insect taxa. Anopheles gambiae s.l. larvae distinctively preferred temporary pools and roadside ditches, where they were exposed to few predators and no apparent competition by Culex spp. Further studies should aim to test the impact of Dytiscidae and Cybaeidae on An. gambiae s.l. dynamics experimentally. Graphical Abstract

The objectives of this study were to determine the chemical composition and to evaluate the mosquito repellent property of essential oils extracted from the bark of the stem of Sterculia tragacantha Lindl on Anopheles gambiae, vector of malaria. The essential oil of the stem bark of S. tragacantha Lindl is extracted by hydrodistillation and their chemical compositions were identified by GC (Ir) and GC-MS. This oil has been tested on sensitive « kisumu » strains of A. gambiae adults, according to the World Health Organization (WHO) guidelines for laboratory testing of the repellency of chemicals. The extraction yield obtained from this oil is 0.12%. By means of GC (Ir) and GC-MS, 29 compounds representing 88.4% of the HE were identified. β-elemol (40.54%) is the major compound of this oil. It is rich in sesquiterpenes (79.47%) with a predominance of oxygenated sesquiterpenes (55.86%). As for the oxygenated monoterpenes, they represent 8.93%. The repellency test against adult female Anopheles, revealed that the EO of S. tragacantha (10%) has repellent properties against mosquitoes. Indeed, it induces an average reduction of 98.2% of the blood meal (TIRS) of mosquitoes on guinea pigs with a complete protection time (CPT) of 5 h compared to 3 h 30 min for the natural reference substance (Cymbopogon citratus). In view of its extractive value and prolonged repellent properties, S. tragacantha could be a good alternative in the vector borne diseases control if used as natural repellents for skin application. Key words: Anopheles gambiae, Sterculia tragacantha Lindl, essential oil, chemical composition, mosquito repellent.

The current study was aimed to investigate on dynamics of propoxur resistance inAnopheles gambiae s.l. populations from N’ dali district in northern Benin (West Africa) and also to investigate on dynamics of malathion resistance inAnopheles gambiae s. l. populations from Toffo district in southern Benin. Larvae and pupae ofAnopheles gambiae s. l .mosquitoes were collected from the breeding sites in Borgou and Atlantic departments in 2015 and 2019. WHO susceptibility tests were conducted on unfed female mosquitoes aged 2-5 days old. WHO bioassays were performed with impregnated papers with propoxur 0.1% and with malathion 5%. PCR techniques were used to detect species andAce-1mutations in 2015.Anopheles gambiaes. l. populations from N’dali were resistant to propoxur in 2015 and were still remained resistant to this product in 2019. RegardingAnopheles gambiaes. l. populations from Toffo, they were susceptible to malathion in 2015 whereas the malathion resistance status of these mosquitoes requires further investigation in 2019. PCR revealed that all specimens tested wereAnopheles gambiae s. s.The presence ofAce-1Rat very low frequency (0.01) was observed inAnopheles gambiaes. l. populations from both districts. This study shows that propoxur resistance detected inAn. gambiaes. l. populations from N’ dali needs to be monitored for insecticide resistance in this area.

Background Insecticide resistance has become a widespread problem causing a decline in the effectiveness of vector control tools in sub-Saharan Africa. In this situation, ongoing monitoring of vector susceptibility to insecticides is encouraged by the WHO to guide national malaria control programmes. Our study was conducted from April to November 2018 in Tchonka (Sud-Kivu, Democratic Republic of the Congo) and reported primary data on the resistance status of Anopheles funestus and Anopheles gambiae. Methods Insecticide susceptibility bioassays were performed on wild populations of A. funestus and A. gambiae using WHO insecticide-impregnated papers at discriminating concentration. In addition, PCR was performed to identify mosquito species and to detect kdr and ace-1R mutations involved in insecticide resistance. Results Bioassay results show resistance to all tested insecticides except pirimiphos-methyl, propoxur, fenitrothion and malathion with a mortality rate ranging from 95.48 to 99.86%. The addition of piperonyl butoxide (PBO) increased the susceptibility of vectors to deltamethrin and alpha-cypermethrin by exhibiting a mortality ranging from 91.50 to 95.86%. The kdr mutation was detected at high frequencies (approximately 0.98) within A. gambiae while ace-1R was not detected. Conclusions This study provides useful data on the insecticide resistance profiles of malaria vector populations to better manage vector control. Our results highlight that, despite the high level of resistance, organophosphorus compounds and pyrethroids + PBO remain effective against the vectors.

A cross sectional study on mosquito fauna of Anambra East Local Government Area was carried out between June 2017 and August 2018. Pyrethrum knockdown and Human Landing Catch Collection methods were used to collect adult mosquitoes from indoor and outdoor locations respectively, in four randomly selected towns in the LGA. The mosquitoes were identified using standard taxonomic keys. A total of 681 adult mosquitoes were collected: 182 (26.7%) in Aguleri, 135 (19.8%) in Umuleri, 273 (40.1%) in Igbariam and 91 (13.4%) in Nando. Out of these, 551 (80.9%) mosquitoes were collected from indoor location and 130 (19.1%) mosquitoes from outdoor location (P < 0.05). Six (6) Genera of mosquito collected were: Anopheles (39.4%), Aedes (3.7%), Culex (37.9%), Mansonia (18.4%), Eretmapodite (0.6%), and Coquilletidia (0.15%) (P < 0.05). Only Culex mosquitoes showed significant difference in indoor abundance, 43.6% and outdoor abundance 13.8% (P < 0.05). Sixteen species of mosquitoes were identified. The most abundant species of mosquito was Cx. quinquefasciatus, with 35.9% abundance and the least were Coquellettidia species and Cx. annuloris, 0.15% abundance each (P < 0.05). The most abundant Species of mosquito in indoor location was Anopheles gambiae s. l, with 42.3% abundance and the least were Ae. albopictus and Cx. annuloris, with 0.2% abundance each (P < 0.05). The most abundant species of mosquito in the outdoor locations was Mansonia uniformis, with 34.6% abundance and the least were An. funestus and Coquellettidia, with 0.80% abundance (P < 0.05). The presence of the different mosquito species found in this study area is of great medical importance. Most of the species are already known vectors of disease pathogens. Therefore, personal protection, provision of adequate mosquito management options, regular surveillance, and public health sensitizations are hereby recommended.

Culex quinquefasciatus, a member of the Culex pipiens complex, is widespread in Saudi Arabia and other parts of the world. It is a vector for lymphatic filariasis, Rift Valley fever, and West Nile virus. Studies have shown the deleterious effect of RNA interference (RNAi)-mediated knockdown of various lethal genes in model and agricultural pest insects. RNAi was proposed as a tool for mosquito control with a focus on Aedes aegypti and Anopheles gambiae. In this study, we examined the effect of RNAi of selected target genes on both larval mortality and adult emergence of Cx. quinquefasciatus through two delivery methods: soaking and nanoparticles. Ten candidate genes were selected for RNAi based on their known lethal effect in other insects. Disruption of three genes, chitin synthase-1, inhibitor of apoptosis 1, and vacuolar adenosine triphosphatase, resulted in the highest mortality among the selected genes using the two treatment methods. Silencing the other seven genes resulted in a medium to low mortality in both assays. These three genes are also active against a wide range of insects and could be used for RNAi-based mosquito control in the future.

A study of molecular characterization of Anopheles gambiae sensu lato, from three senatorial districts in Enugu State, was conducted between May, 2019 and June, 2020. The aim was to determine the siblings’ species of the mosquito complex and the distribution of the S (Savannah) and M (Mopti) molecular forms within the An. gambiae sensu stricto in the area. Three randomly selected communities; Awkunanaw (Enugu East), Obukpa (Enugu North) and Awgu (Enugu West senatorial districts) were used as sampling sites. Anopheles gambiae s.l. larvae were collected by dip method and reared to adult for the study. A total of 450(100%) An. gambiae s.l. mosquitoes were subjected to Polymerase Chain Reaction (PCR). Of this number, 150(33.33%) were randomly selected from bulk of An. gambiae s.l. mosquitoes collected from each of the three communities. All the 450(100%) were identified as An. gambiae s.s. The Restriction Fragment Length Polymorphism (RFLP) was done on the 450(100%) An. gambiae s.s. A total of 300(66.67%) from Awkunanaw and Obukpa Nsukka communities were An. gambiae S-form while 150(33.33%) from Awgu were identified as An. gambiae M-form. The study has revealed that An. gambiae s.s. is the dominant malaria vector within the complex in the area. Integrated vector control is recommended.

A study of insecticide resistant status and detection of G119S and L1014F mutation genes in Anopheles gambiae sensu lato. in Enugu State was conducted between May, 2019 and June, 2020. The aim was to investigate the resistance status of An. gambiae s.l. to four classes of insecticides used in vector control in the area and to identify the target site resistant genes present in resistant mosquitoes. Three randomly selected communities; Awkunanaw (Enugu East), Obukpa (Enugu North) and Awgu (Enugu West senatorial districts) were used as sampling sites. Anopheles gambiae s.l. larvae were collected by dip method and reared to adult for the study. WHO Tube Bioassay was used to determine resistance status. Polymerase Chain Reaction (PCR) was employed for detection of resistance mechanisms. The average mortality of An. gambiae s.l. tested with DDT was 28.3%, deltamethrin 63.53%, bendiocarb 88.33%, while malathion had the highest average mortality rate of 100%. The Acetycholinesterase Resistance Gene (GII9S Ace-1) mutation was detected at low frequencies in 75(100%) randomly selected An. gambiae s.l. from the three senatorial districts while the Knockdown Resistance Gene (LI0I4F kdr) mutation was not detected. Implementation of insecticide resistance management strategies in the state to control the spread of resistance is recommended.

CRISPR-based gene-drives targeting the gene doublesex in the malaria vector Anopheles gambiae effectively suppressed the reproductive capability of mosquito populations reared in small laboratory cages. To bridge the gap between laboratory and the field, this gene-drive technology must be challenged with vector ecology. Here we report the suppressive activity of the gene-drive in age-structured An. gambiae populations in large indoor cages that permit complex feeding and reproductive behaviours. The gene-drive element spreads rapidly through the populations, fully supresses the population within one year and without selecting for resistance to the gene drive. Approximate Bayesian computation allowed retrospective inference of life-history parameters from the large cages and a more accurate prediction of gene-drive behaviour under more ecologically-relevant settings. Generating data to bridge laboratory and field studies for invasive technologies is challenging. Our study represents a paradigm for the stepwise and sound development of vector control tools based on gene-drive., Experimental analysis of gene drive population dynamics has mostly been limited to small cage trials. Here the authors, to fill the gap between lab based studies and field studies, use large indoor cages and see population suppression without the emergence of resistant alleles

Background The selection and the spread of insecticide resistance in malaria vectors to the main classes of insecticides used in vector control tools are a major and ongoing challenge to malaria vector control programmes. This study aimed to determine the intensity of vector resistance to insecticides in three regions of Benin with different agro-ecological characteristics. Methods Larvae of Anopheles gambiae sensu lato (s.l.) were collected from September to November 2017 in different larval sites in three northern Benin communes: Parakou, Kandi and Malanville. Two to five-day-old, non-blood-fed, female mosquitoes were exposed to papers impregnated with deltamethrin, permethrin and bendiocarb at dosages of 1 × the diagnostic dose, 5 × and 10 × to determine the intensity of resistance in these vectors. Molecular frequencies of the kdr L1014F and ace-1R G119S insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled at each study site. Results Resistance to pyrethroids (permethrin and deltamethrin) was recorded in all three communes with mortality rates below 60% using the diagnostic dose (1x). The results obtained after exposure of An. gambiae to permethrin 10 × were 99% in Kandi, 98% in Malanville and 99% in Parakou. With deltamethrin 10x, mortality rates were 100% in Kandi, 96% in Malanville and 73% in Parakou. For the diagnostic dose of bendiocarb, suspected resistance was recorded in the communes of Malanville (97%) and Kandi (94%) while sensitivity was observed in Parakou (98%).Using the 10 × dose, mortality was 98% in Kandi, 100% in Malanville and 99% in Parakou. The frequencies of the kdr L1014F allele varied between 59 and 83% depending on the sites and species of the An. gambiae complex, while the frequency of the ace-1R G119S gene varied between 0 and 5%. Biochemical tests showed high levels of oxidase and esterase activity compared to the susceptible colony strain of An. gambiae sensu stricto (Kisumu strain). Conclusion Anopheles gambiae showed a generalized loss of susceptibility to permethrin and deltamethrin but also showed moderate to high intensity of resistance in different regions of Benin. This high intensity of resistance is a potential threat to the effectiveness of vector control.

The performance of the three-part mosquito lure ATRAKTA (1-octen-3-ol, ammonium bicarbonate, and lactic acid) was evaluated in two DynaTrap commercial mosquito traps (models DT160 and DT700) as well as in one model of CDC trap (model 512). Lures were evaluated fresh from the factory, after being aged in functioning traps under field conditions, and after prolonged storage in the packaging (aged for 30 days aged in functioning traps before being tested in the DynaTrap models; and two years stored in the packaging before being tested in CDC traps). The primary study questions were whether the addition of lures would increase efficacy of various trap types and whether lures would retain effectiveness after a lengthy stay on the shelf or in traps. To do this, traps with no lures, new lures and old lures were used to trap three mosquito species (Aedes albopictus, Culex quinquefasciatus, and Anopheles gambiae) in the field in West Africa Mali, the first two species are also common North American nuisance mosquitoes. The addition of ATRAKTA lures aged 30 days to both DynaTrap® models, and ATRAKTA lures aged two years in the packaging to the CDC trap significantly increased catches of female Cx. quinquefasciatus and Ae. albopictus mosquitoes. Aged lures did not significantly lose their attraction in comparison to lures fresh from the factory. The addition of lures to traps resulted in slight increases in catches of An. gambiae, but these were not statistically significant. No effect of any lures on males was observed.

Background Whole genome re-sequencing provides powerful data for population genomic studies, allowing robust inferences of population structure, gene flow and evolutionary history. For the major malaria vector in Africa, Anopheles gambiae, other genetic aspects such as selection and adaptation are also important. In the present study, we explore population genetic variation from genome-wide sequencing of 765 An. gambiae and An. coluzzii specimens collected from across Africa. We used t-SNE, a recently popularized dimensionality reduction method, to create a 2D-map of An. gambiae and An. coluzzii genes that reflect their population structure similarities. Results The map allows intuitive navigation among genes distributed throughout the so-called “mainland” and numerous surrounding “island-like” gene clusters. These gene clusters of various sizes correspond predominantly to low recombination genomic regions such as inversions and centromeres, and also to recent selective sweeps. Because this mosquito species complex has been studied extensively, we were able to support our interpretations with previously published findings. Several novel observations and hypotheses are also made, including selective sweeps and a multi-locus selection event in Guinea-Bissau, a known intense hybridization zone between An. gambiae and An. coluzzii. Conclusions Our results present a rich dataset that could be utilized in functional investigations aiming to shed light onto An. gambiae s.l genome evolution and eventual speciation. In addition, the methodology presented here can be used to further characterize other species not so well studied as An. gambiae, shortening the time required to progress from field sampling to the identification of genes and genomic regions under unique evolutionary processes.

Background Anopheles cell lines are used in a variety of ways to better understand the major vectors of malaria in sub-Saharan Africa. Despite this, commonly used cell lines are not well characterized, and no tools are available for cell line identification and authentication. Methods Utilizing whole genome sequencing, genomes of 4a-3A and 4a-3B ‘hemocyte-like’ cell lines were characterized for insertions and deletions (indels) and SNP variation. Genomic locations of distinguishing sequence variation and species origin of the cell lines were also examined. Unique indels were targeted to develop a PCR-based cell line authentication assay. Mitotic chromosomes were examined to survey the cytogenetic landscape for chromosome structure and copy number in the cell lines. Results The 4a-3A and 4a-3B cell lines are female in origin and primarily of Anopheles coluzzii ancestry. Cytogenetic analysis indicates that the two cell lines are essentially diploid, with some relatively minor chromosome structural rearrangements. Whole-genome sequence was generated, and analysis indicated that SNPs and indels which differentiate the cell lines are clustered on the 2R chromosome in the regions of the 2Rb, 2Rc and 2Ru chromosomal inversions. A PCR-based authentication assay was developed to fingerprint three indels unique to each cell line. The assay distinguishes between 4a-3A and 4a-3B cells and also uniquely identifies two additional An. coluzzii cell lines tested, Ag55 and Sua4.0. The assay has the specificity to distinguish four cell lines and also has the sensitivity to detect cellular contamination within a sample of cultured cells. Conclusions Genomic characterization of the 4a-3A and 4a-3B Anopheles cell lines was used to develop a simple diagnostic assay that can distinguish these cell lines within and across research laboratories. A cytogenetic survey indicated that the 4a-3A and Sua4.0 cell lines carry essentially normal diploid chromosomes, which makes them amenable to CRISPR/Cas9 genome editing. The presented simple authentication assay, coupled with screening for mycoplasma, will allow validation of the integrity of experimental resources and will promote greater experimental reproducibility of results. Graphical abstract

Background Malaria is transmitted when infected Anopheles mosquitoes take a blood meal. During this process, the mosquitoes inject a cocktail of bioactive proteins that elicit antibody responses in humans and could be used as biomarkers of exposure to mosquito bites. This study evaluated the utility of IgG responses to members of the Anopheles gambiae D7 protein family as serological markers of human–vector contact. Methods The D7L2, D7r1, D7r2, D7r3, D7r4 and SG6 salivary proteins from An. gambiae were expressed as recombinant antigens in Escherichia coli. Antibody responses to the salivary proteins were compared in Europeans with no prior exposure to malaria and lifelong residents of Junju in Kenya and Kitgum in Uganda where the intensity of malaria transmission is moderate and high, respectively. In addition, to evaluate the feasibility of using anti-D7 IgG responses as a tool to evaluate the impact of vector control interventions, we compared responses between individuals using insecticide-treated bednets to those who did not in Junju, Kenya where bednet data were available. Results We show that both the long and short forms of the D7 salivary gland antigens elicit a strong antibody response in humans. IgG responses against the D7 antigens reflected the transmission intensities of the three study areas, with the highest to lowest responses observed in Kitgum (northern Uganda), Junju (Kenya) and malaria-naïve Europeans, respectively. Specifically, the long form D7L2 induced an IgG antibody response that increased with age and that was lower in individuals who slept under a bednet, indicating its potential as a serological tool for estimating human–vector contact and monitoring the effectiveness of vector control interventions. Conclusions This study reveals that D7L2 salivary antigen has great potential as a biomarker of exposure to mosquito bites and as a tool for assessing the efficacy of vector control strategies such as bednet use. Graphical abstract

Transglutaminases are pleiotropic enzymes that in mosquitoes participate in the formation of the mating plug and the wound-induced antimalarial response. Moreover, one transglutaminase, TGase3, negatively regulates the infection-induced aggregation of hemocytes on the heart. Given that TGase3 is an inhibitor of periostial hemocyte aggregation, we used RNAi-based gene silencing followed by intravital video imaging to scrutinize whether any of the three transglutaminases encoded in the genome of the mosquito, Anopheles gambiae, play a role in modulating the heart rate of uninfected and infected mosquitoes. Initially, we confirmed that an infection decreases the heart rate. Then, we uncovered that silencing TGase1 does not impact heart physiology, but silencing TGase2 results in a constant heart rate regardless of infection status, eliminating the infection-induced decrease in the heart rate. Finally, silencing TGase3 decreases the heart rate in uninfected mosquitoes but increases the heart rate in infected mosquitoes. We conclude that TGase2 and TGase3 modulate heart physiology and demonstrate that factors not classically associated with insect circulatory physiology are involved in the functional integration of the immune and circulatory systems of mosquitoes.

The prevalence, intensity and some vector dynamics of malaria in two communities of Anambra East Local Government Area LGA was carried out using standard parasitological and entomological techniques for rapid assessment of malaria infection, level of parasitaemia and morphological features of mosquitoes. Results showed an overall malaria RDT (Rapid Diagnostic Test) prevalence of 22% and community based prevalence of 13 (30%) and 22(24%) for Umuoba -Anam and Nando respectively. Overall, age related prevalence in the Anambra West LGA showed that the age bracket 35 to 42 years had the highest malaria prevalence 3(42.9 %). Overall sex related prevalence in Anambra East LGA showed no statistical significance in prevalence among males (12.0%) and females (10.0%) (P500-2000 parasites/µl) parasitaemia. There was no high parasitaemia recorded in Umuoba-Anam. Overall, a total of 225 mosquitoes were encountered with anopheles making up 61%. A hundred and twenty one (53.8% of the total mosquito population in the study was found to be unfed with blood meal whereas 54(24%) and 50 (22.2%) were freshly and previously fed respectively. One hundred and twenty one (53.8%) of the mosquitoes appeared non-gravid against 82(36.4%) that are gravid. The mosquitoes encountered wereAnopheles gambiae31(13.8%),Anopheles funestus77(34.22%), andCulexSpecie117(52%). The two communities recorded variations in prevalence, intensity and vector dynamics. There should be intensified effort to continuously educate the masses on all the strategies of malaria prevention.

Mosquitoes are responsible for transmission of illnesses of public health importance including malaria, lymphatic filariasis, dengue, chikungunya, and many other diseases caused by viruses. Vector control using synthetic insecticides has been the cornerstone for management of vector-borne diseases. However, the chemical based interventions have not been sustainable due to emergency of resistance against insecticides among disease vectors. Plant based mosquitocidal products can be potential alternative tools in vector control. Therefore, the present study aimed at exploring the larvicidal properties of Dioscorea sansibarensis leaf extract against malaria and lymphatic filariasis vectors; Anopheles gambiae s.s. and Culex quinquefasciatus. The larvicidal activities of Dioscorea sansibarensis were assessed following WHO test procedures. Ethanol leaf extract of Dioscorea sansibarensis was evaluated against all the four instar larvae stages of An. gambiae s.s and Cx. quinquefasciatus susceptible laboratory colonies. The highest larvicidal potency was shown against the 4th instar stages of both species with the LC50 values of 60.915 ppm and 80.700 ppm for Cx. quinquefasciatus and An. gambiae s.s., respectively. The respective LC95 values for Cx. quinquefasciatus and An. gambiae s.s. were 168.898 ppm and 249.295 ppm. This implies that the extract can be applied as mosquito larvicide should its impact on non-targeted species be established. Keywords: Dioscorea sansibarensis; vectors, mosquitoes, Zanzibar yams, Dar es Salaam, Tanzania