Distribution and insecticide resistance profile of the major malaria vector Anopheles funestus group across the African continent.

There has been significant progress in malaria control in the last 2 decades, with a decline in mortality and morbidity. However, these gains are jeopardised by insecticide resistance, which negatively impacts the core interventions, such as insecticide-treated nets (ITN) and indoor residual spraying (IRS). While most malaria control and research efforts are still focused on Anopheles gambiae complex mosquitoes, Anopheles funestus remains an important vector in many countries and, in some cases, contributes to most of the local transmission. As countries move towards malaria elimination, it is important to ensure that all dominant vector species, including An. funestus, an important vector in some countries, are targeted. The objective of this review is to compile and discuss information related to A. funestus populations' resistance to insecticides and the mechanisms involved across Africa, emphasising the sibling species and their resistance profiles in relation to malaria elimination goals. Data on insecticide resistance in An. funestus malaria vectors in Africa were extracted from published studies. Online bibliographic databases, including Google Scholar and PubMed, were used to search for relevant studies. Articles published between 2000 and May 2023 reporting resistance of An. funestus to insecticides and associated mechanisms were included. Those reporting only bionomics were excluded. Spatial variation in species distribution and resistance to insecticides was recorded from 174 articles that met the selection criteria. It was found that An. funestus was increasingly resistant to the four classes of insecticides recommended by the World Health Organisation for malaria vector control; however, this varied by country. Insecticide resistance appears to reduce the effectiveness of vector control methods, particularly IRS and ITN. Biochemical resistance due to detoxification enzymes (P450s and glutathione-S-transferases [GSTs]) in An. funestus was widely recorded. However, An. funestus in Africa remains susceptible to other insecticide classes, such as organophosphates and neonicotinoids. This review highlights the increasing insecticide resistance of An. funestus mosquitoes, which are important malaria vectors in Africa, posing a significant challenge to malaria control efforts. While An. funestus has shown resistance to the recommended insecticide classes, notably pyrethroids and, in some cases, organochlorides and carbamates, it remains susceptible to other classes of insecticides such as organophosphates and neonicotinoids, providing potential alternative options for vector control strategies. The study underscores the need for targeted interventions that consider the population structure and geographical distribution of An. funestus, including its sibling species and their insecticide resistance profiles, to effectively achieve malaria elimination goals.

Des progrès importants ont été réalisés dans le contrôle du paludisme au cours des deux dernières décennies, qui se traduisent par une baisse de la mortalité et de la morbidité. Cependant, ces gains sont compromis par la résistance aux insecticides, ce qui a un impact négatif sur les interventions de base, telles que les moustiquaires imprégnées d'insecticides et la pulvérisation intradomicilliare (PID). Alors que la plupart des efforts de contrôle et de recherche sur le paludisme sont toujours axés sur les moustiques du complexes Anopheles gambiae, Anopheles funestus reste un vecteur important dans de nombreux pays et, dans certains cas, contribue à la majeure partie de la transmission locale. Au moment où certains pays se dirigent vers l'élimination du paludisme, il serait important de prendre en considération toutes les espèces vectrices dominantes, y compris An. funestus. L'objectif de cette revue est de compiler et de discuter des informations liées à la résistance des populations d'An. funestus aux insecticides et les mécanismes impliqués à travers l'Afrique, en mettant l'accent sur les sous espèces et leurs profils de résistance en relation avec les objectifs d'élimination du paludisme. Les données sur la résistance aux insecticides chez An. funestus vecteurs du paludisme en Afrique ont été extraites d'études publiées dans des bases de données bibliographiques comme Google Scholar et PubMed. Les articles publiés entre 2000 et mai 2023, rapportant la résistance de An. funestus aux insecticides et les mécanismes associés ont été inclus. Ceux portant uniquement sur la bionomie ont été exclus. Au total 174 articles portant sur la variation spatiale de la résistance des espèces du groupe An. funestus aux insecticides répondaient aux critères de sélection. De ces analyses, il ressort qu'An. funestus était de plus en plus résistant aux quatre classes d'insecticides recommandées par l'Organisation Mondiale de la Santé (OMS) pour le contrôle des vecteurs du paludisme ce qui semble réduire l'efficacité des méthodes de contrôle des vecteurs, en particulier les moustiquaires imprégnées d'insecticide et la pulvérisation intradomiciliaire. avec des variations en fonction des pays. Les mécanismes de résistance aux insecticides de type biochimique liée aux enzymes de détoxification (P450S et GST) ont été largement rapportés chez An. funestus. De nombreux gènes P450 associés à la résistance métabolique ont été mis en évidence chez An. funestus collecté sur le terrain. Cependant, An. funestus en Afrique reste sensible à d'autres classes d'insecticides, telles que les organophosphorés et les néonicotinoïdes. La résistance aux insecticides. Cette revue met en évidence la résistance croissante aux insecticides chez les moustiques du groupe Funestus, un vecteur important du paludisme en Afrique, posant ainsi un défi important aux efforts de contrôle du paludisme. Tandis que An. funestus a montré une résistance aux classes d'insecticide recommandées, notamment les pyréthroïdes et, dans certains cas, les organochlorés et les carbamates, il reste sensible à d'autres classes d'insecticides tels que les organophosphorés et les néonicotinoïdes, offrant des options alternatives potentielles de contrôle des vecteurs. L'étude souligne la nécessité d'interventions ciblées qui considèrent la structure de la population et la distribution géographique d'An. funestus, y compris ses sous espèces et leurs profils de résistance aux insecticides, pour atteindre efficacement les objectifs d'élimination du paludisme.

Advances in the genetic characterization of the malaria vector, Anopheles funestus, and implications for improved surveillance and control.

Anopheles mosquitoes present a major public health challenge in sub-Saharan Africa; notably, as vectors of malaria that kill over half a million people annually. In parts of the east and southern Africa region, one species in the Funestus group, Anopheles funestus, has established itself as an exceptionally dominant vector in some areas, it is responsible for more than 90% of all malaria transmission events. However, compared to other malaria vectors, the species is far less studied, partly due to difficulties in laboratory colonization and the unresolved aspects of its taxonomy and systematics. Control of An. funestus is also increasingly difficult because it has developed widespread resistance to public health insecticides. Fortunately, recent advances in molecular techniques are enabling greater insights into species identity, gene flow patterns, population structure, and the spread of resistance in mosquitoes. These advances and their potential applications are reviewed with a focus on four research themes relevant to the biology and control of An. funestus in Africa, namely: (i) the taxonomic characterization of different vector species within the Funestus group and their role in malaria transmission; (ii) insecticide resistance profile; (iii) population genetic diversity and gene flow, and (iv) applications of genetic technologies for surveillance and control. The research gaps and opportunities identified in this review will provide a basis for improving the surveillance and control of An. funestus and malaria transmission in Africa.

Putative pleiotropic effects of the knockdown resistance (L1014F) allele on the life-history traits of Anopheles gambiae.

BACKGROUND: Existing mechanisms of insecticide resistance are known to help the survival of mosquitoes following contact with chemical compounds, even though they could negatively affect the life-history traits of resistant malaria vectors. In West Africa, the knockdown resistance mechanism kdrR (L1014F) is the most common. However, little knowledge is available on its effects on mosquito life-history traits. The fitness effects associated with this knockdown resistance allele in Anopheles gambiae sensu stricto (s.s.) were investigated in an insecticide-free laboratory environment. METHODS: The life-history traits of Kisumu (susceptible) and KisKdr (kdr resistant) strains of An. gambiae s.s. were compared. Larval survivorship and pupation rate were assessed as well as fecundity and fertility of adult females. Female mosquitoes of both strains were directly blood fed through artificial membrane assays and then the blood-feeding success, blood volume and adult survivorship post-blood meal were assessed. RESULTS: The An. gambiae mosquitoes carrying the kdrR allele (KisKdr) laid a reduced number of eggs. The mean number of larvae in the susceptible strain Kisumu was three-fold overall higher than that seen in the KisKdr strain with a significant difference in hatching rates (81.89% in Kisumu vs 72.89% in KisKdr). The KisKdr larvae had a significant higher survivorship than that of Kisumu. The blood-feeding success was significantly higher in the resistant mosquitoes (84%) compared to the susceptible ones (34.75%). However, the mean blood volume was 1.36 µL/mg, 1.45 µL/mg and 1.68 µL/mg in Kisumu, homozygote and heterozygote KisKdr mosquitoes, respectively. After blood-feeding, the heterozygote KisKdr mosquitoes displayed highest survivorship when compared to that of Kisumu. CONCLUSIONS: The presence of the knockdown resistance allele appears to impact the life-history traits, such as fecundity, fertility, larval survivorship, and blood-feeding behaviour in An. gambiae. These data could help to guide the implementation of more reliable strategies for the control of malaria vectors.

Interplay Between Oxytetracycline and the Homozygote kdr (L1014F) Resistance Genotype on Fecundity in Anopheles gambiae (Diptera: Culicidae) Mosquitoes.

The insecticide resistance in Anopheles gambiae mosquitoes has remained the major threat for vector control programs but the fitness effects conferred by these mechanisms are poorly understood. To fill this knowledge gap, the present study aimed at testing the hypothesis that antibiotic oxytetracycline could have an interaction with insecticide resistance genotypes and consequently inhibit the fecundity in An. gambiae. Four strains of An. gambiae: Kisumu (susceptible), KisKdr (kdr (L1014F) resistant), AcerKis (ace-1 (G119S) resistant) and AcerKdrKis (both kdr (L1014F) and ace-1 (G119S) resistant) were used in this study. The different strains were allowed to bloodfeed on a rabbit previously treated with antibiotic oxytetracycline at a concentration of 39·10-5 M. Three days later, ovarian follicles were dissected from individual mosquito ovaries into physiological saline solution (0.9% NaCl) under a stereomicroscope and the eggs were counted. Fecundity was substantially lower in oxytetracycline-exposed KisKdr females when compared to that of the untreated individuals and oxytetracycline-exposed Kisumu females. The exposed AcerKis females displayed an increased fecundity compared to their nontreated counterparts whereas they had reduced fecundity compared to that of oxytetracycline-exposed Kisumu females. There was no substantial difference between the fecundity in the treated and untreated AcerKdrKis females. The oxytetracycline-exposed AcerKdrKis mosquitoes had an increased fecundity compared to that of the exposed Kisumu females. Our data indicate an indirect effect of oxytetracycline in reducing fecundity of An. gambiae mosquitoes carrying kdrR (L1014F) genotype. These findings could be useful for designing new integrated approaches for malaria vector control in endemic countries.

Proteomic Investigation on Anopheles gambiae in Burkina Faso Related to Insecticide Pressures from Different Climatic Regions.

In Sub-Saharan Africa, An. gambiae sensu lato (s.l.) Giles 190, largely contributes to malaria transmission. Therefore, the authors carry out a proteomic analysis to compare its metabolic state, depending on different pesticide pressures by selecting areas with/without cotton crops. The proteomes data are available via ProteomeXchange with identifier PXD016300. From a total of 1.182 identified proteins, 648 are retained for further statistical analysis and are attributed to biological functions, the most important of which being energy metabolism (120 proteins) followed by translation-biogenesis (74), cytoskeleton (71), stress response (62), biosynthetic process (60), signalling (44), cellular respiration (38), cell redox homeostasis (25), DNA processing (17), pheromone binding (10), protein folding (9), RNA processing (9), other proteins (26) and unknown functions (83). In the Sudano-Sahelian region, 421 (91.3%) proteins are found in samples from areas both with and without cotton crops. By contrast, in the Sahelian region, only 271 (55.0%) are common to both crop areas, and 233 proteins are up-regulated from the cotton area. The focus is placed on proteins with putative roles in insecticide resistance, according to literature. This study provides the first whole-body proteomic characterisation of An. gambiae s.l. in Burkina Faso, as a framework to strengthen vector control strategies.

Bacterial communities associated with the midgut microbiota of wild Anopheles gambiae complex in Burkina Faso.

Plasmodium falciparum is transmitted by mosquitoes from the Anopheles gambiae sensu lato (s.l) species complex and is responsible for severe forms of malaria. The composition of the mosquitoes' microbiota plays a role in P. falciparum transmission, so we studied midgut bacterial communities of An. gambiae s.l from Burkina Faso. DNA was extracted from 17 pools of midgut of mosquitoes from the Anopheles gambiae complex from six localities in three climatic areas, including cotton-growing and cotton-free localities to include potential differences in insecticide selection pressure. The v3-v4 region of the 16S rRNA gene was targeted and sequenced using Illumina Miseq (2 × 250 nt). Diversity analysis was performed using QIIME and R software programs. The major bacterial phylum was Proteobacteria (97.2%) in all samples. The most abundant genera were Enterobacter (32.8%) and Aeromonas (29.8%), followed by Pseudomonas (11.8%), Acinetobacter (5.9%) and Thorsellia (2.2%). No statistical difference in operational taxonomic units (OTUs) was found (Kruskal-Wallis FDR-p > 0.05) among the different areas, fields or localities. Richness and diversity indexes (observed OTUs, Chao1, Simpson and Shannon indexes) showed significant differences in the cotton-growing fields and in the agroclimatic zones, mainly in the Sudano-Sahelian area. OTUs from seven bacterial species that mediate refractoriness to Plasmodium infection in An. gambiae s.l were detected. The beta diversity analysis did not show any significant difference. Therefore, a same control strategy of using bacterial species refractoriness to Plasmodium to target mosquito midgut bacterial community and affect their fitness in malaria transmission may be valuable tool for future malaria control efforts in Burkina Faso.

Anopheline species composition and the 1014F-genotype in different ecological settings of Burkina Faso in relation to malaria transmission.

BACKGROUND: A three-year longitudinal study was conducted in four sentinel sites from different ecological settings in Burkina Faso, between 2008 and 2010 to identify longitudinal changes in insecticide resistance within Anopheles gambiae complex species based on larval collection. During this study, adult mosquitoes were also collected indoor and outdoor using several methods of collection. The present study reports the diversity of malaria vectors and the 1014F-genotype from this adult collection and investigates the association between this 1014F-genotype and sporozoite rate. METHODS: Adult mosquitoes were collected from July to August (corresponding to the start of rainy season) and October to November (corresponding to the end of rainy season) over 3 years (2008-2010) at four sites across the country, using pyrethrum spray catches (PSC), exit traps and pit shelters. Anopheles gambiae complex mosquitoes were identified to species and genotyped for the L1014F kdr mutation by PCR using genomic DNA. The circumsporozoite antigen of Plasmodium falciparum was detected in mosquitoes using sandwich ELISA. RESULTS: Overall 9212 anopheline mosquitoes were collected during the study period. Of those, 6767 mosquitoes were identified as Anopheles gambiae sensu lato (s.l.). Anopheles arabiensis, Anopheles coluzzii, Anopheles gambiae and or Anopheles funestus were incriminated as vectors of P. falciparum in the study area with an average sporozoite rate of 5%, (95% CI 4.14-5.99%). The kdr1014F-genotype frequencies were 11.44% (95% CI 2.5-39.85%), 19.2% (95% CI 4.53-53.73%) and 89.9 (95% CI 63.14-97.45%), respectively for An. arabiensis, An. coluzzii and An. gambiae. The proportion of the 1014F-genotype varied between sporozoite-infected and uninfected An. gambiae s.l. group. There was no significant difference in the 1014F-genotype frequency between infected and uninfected mosquitoes. CONCLUSION: The current study shows the diversity of malaria vectors and significant interaction between species composition and kdr1014F-genotype in An. gambiae complex mosquitoes from Burkina Faso. In this study, no associations were found between the 1014F-genotype and P. falciparum infection in the major malaria vector An. gambiae s.l.

Detection of G119S ace-1 (R) mutation in field-collected Anopheles gambiae mosquitoes using allele-specific loop-mediated isothermal amplification (AS-LAMP) method.

BACKGROUND: Malaria vectors have developed resistance to the four families of insecticides available for public health purposes. For example, the kdr mutation is associated with organochlorines and pyrethroids resistance. It is of particular concern that organophosphate and carbamate resistance associated with the G119S ace-1 (R) mutation has recently increased in West Africa in extent and frequency, and is now spreading through the Anopheles gambiae malaria vector population. There is an urgent need to improve resistance management using existing insecticides and new tools to quickly assess resistance level for rapid decision-making. METHODS: DNA extracted from field-collected mosquitoes was used to develop the method. Specific primers were designed manually to match the mutation region and an additional mismatched nucleotide in the penultimate position to increase specificity. Other primers used are common to both wild and mutant types. The allele specific (AS)-LAMP method was compared to the PCR restriction fragment length polymorphism (PCR-RFLP) and real-time PCR (RT-PCR) methods using the genomic DNA of 104 field-collected mosquitoes. RESULTS: The primers designed for LAMP were able to distinguish between the wild type (ace-1 (S) ) and mutated type allele (ace-1 (R) ). Detection time was 50 min for the wild type homozygous and 64 min for the heterozygous. No amplification of the resistant allele took place within the 75-min test period when using the wild type primers. For the ace-1 (R) resistant type, detection time was 51 min for the resistant homozygous and 55 min for the heterozygous. No amplification of the wild type allele took place within the 75-min test period when using the resistant type primers. Gel electrophoresis of LAMP products confirmed that amplification was primer-DNA specific, i.e., primers could only amplify their target specific DNA. AS-LAMP, PCR-RFLP, and RT-PCR showed no significant difference in the sensitivity and specificity of their ace-1 (R) detection ability. CONCLUSIONS: The AS-LAMP method could detect the ace-1 (R) mutation within 60 min, which is faster than conventional PCR-RFLP. This method may be used to quickly detect the ace-1 (R) mutation for rapid decision-making, even in less well-equipped laboratories.

Laboratory evaluation of Fendona 6SC treated bednets and Interceptor long-lasting nets against Anopheles gambiae s.l. in Burkina Faso.

Insecticide-treated bednets play a cornerstone role in the efforts to control malaria. Bednets entomological efficacy is the determinant factor of their use to control malaria. In this study, we compared under laboratory conditions, the efficacy of two long-lasting nets (PermaNet versus Interceptor) and two treatments kits K-O TAB (deltamethrin) versus Fendona 6SC (alpha-cypermethrin) against Anopheles gambiae s.l. malaria vectors. The efficacy of washed and unwashed bednets was assessed by contact bioassays using World Health Organization (WHO) cones. Three to five-days-old mosquitoes were exposed to the netting for 3 min; the median and 95% knockdown time, the after 24 h mortality was recorded for each type of bednet. The mortality after 24 h was equivalent for the Fendona 6SC treated bednets and the K-O TAB treated bednets [79.4% confidence limits (CL) (73.9-84.6) and 74% CL (68.3-80.0), respectively]. However, the Fendona 6SC treated bednets were superior in 50% knockdown time to the K-O TAB treated bednets [7.8 min, CL (6.5-9.0) and 15. 2 min, CL (14.0-16.4), respectively]. Washed Interceptor and PermaNet bednets showed similar efficacy in terms of 50% knockdown times. Mortality after 24 h was similar from the fifth to the twentieth wash, but PermaNet performed better than Interceptor for the first four washes and for unwashed bednets. This study showed that Fendona 6SC kit and the Interceptor bednets have exhibited consistent comparable efficacy in the laboratory compared to the well known and in use K-O TAB kit and PermaNet bednets.

Blood-Feeding Patterns and Resting Behavior of Aedes aegypti from Three Health Districts of Ouagadougou City, Burkina Faso.

Recent dengue outbreaks have occurred in Ouagadougou and Bobo-Dioulasso, the two major cities of Burkina Faso. Dengue is a viral disease transmitted primarily by Aedes aegypti, a highly anthropophilic mosquito that thrives in human-transformed environments and breeds predominantly in artificial containers. In 2018, we investigated the resting and blood-feeding habits of Ae. aegypti in urban settings of Ouagadougou. In a 3-month cross-sectional study starting in August 2018, indoors and outdoors resting adult mosquitoes were collected using Prokopack aspirators in three health districts (HD). All mosquitoes were morphologically identified, and DNA was extracted from blood-fed Ae. aegypti females. A multiplex polymerase chain reaction with specific primers was used to identify the origin of the blood meal. A total of 4,256 adult Ae. aegypti mosquitoes, including 1,908 females, were collected. A preference for exophily was recorded in Bogodogo and Nongremassom, although an unexpectedly higher proportion of blood-fed females were found indoors than outdoors. Respectively, 96.09%, 91.03%, and 95.54% of the blood meals successfully analyzed in Baskuy, Bogodogo, and Nongremassom were from a single human host, with the remainder from domestic mammals as single or multiple hosts. Modeling total Ae. aegypti and blood-fed female counts showed that among other predictors, human density, outdoor environment, and house type affect their total densities. Our study revealed an exophilic tendency as well as a pronounced anthropophilic preference of Ae. aegypti adults, critical findings to consider when planning accurate entomological surveillance and effective interventions against Ae. aegypti in urban settings.

First report of V1016I, F1534C and V410L kdr mutations associated with pyrethroid resistance in Aedes aegypti populations from Niamey, Niger.

BACKGROUND: Ae. aegypti is the vector of important µ arboviruses, including dengue, Zika, chikungunya and yellow fever. Despite not being specifically targeted by insecticide-based control programs in West Africa, resistance to insecticides in Ae. aegypti has been reported in countries within this region. In this study, we investigated the status and mechanisms of Ae. aegypti resistance in Niamey, the capital of Niger. This research aims to provide baseline data necessary for arbovirus outbreak prevention and preparedness in the country. METHODS: Ovitraps were used to collect Ae. aegypti eggs, which were subsequently hatched in the insectary for bioassay tests. The hatched larvae were then reared to 3-5-day-old adults for WHO tube and CDC bottle bioassays, including synergist tests. The kdr mutations F1534C, V1016I, and V410L were genotyped using allele-specific PCR and TaqMan qPCR methods. RESULTS: Ae. aegypti from Niamey exhibited moderate resistance to pyrethroids but susceptibility to organophosphates and carbamates. The kdr mutations, F1534C, V1016I and V410L were detected with the resistant tri-locus haplotype 1534C+1016L+410L associated with both permethrin and deltamethrin resistance. Whereas the homozygote tri-locus resistant genotype 1534CC+1016LL+410LL was linked only to permethrin resistance. The involvement of oxidase and esterase enzymes in resistance mechanisms was suggested by partial restoration of mosquitoes' susceptibility to pyrethroids in synergist bioassays. CONCLUSION: This study is the first report of Ae. aegypti resistance to pyrethroid insecticides in Niamey. The resistance is underpinned by target site mutations and potentially involves metabolic enzymes. The observed resistance to pyrethroids coupled with susceptibility to other insecticides, provides data to support evidence-based decision-making for Ae. aegypti control in Niger.

Extensive variation and strain-specificity in dengue virus susceptibility among African Aedes aegypti populations.

African populations of the mosquito Aedes aegypti are usually considered less susceptible to infection by human-pathogenic flaviviruses than globally invasive populations found outside Africa. Although this contrast has been well documented for Zika virus (ZIKV), it is unclear to what extent it is true for dengue virus (DENV), the most prevalent flavivirus of humans. Addressing this question is complicated by substantial genetic diversity among DENV strains, most notably in the form of four genetic types (DENV1 to DENV4), that can lead to genetically specific interactions with mosquito populations. Here, we carried out a survey of DENV susceptibility using a panel of seven field-derived Ae. aegypti colonies from across the African range of the species and a colony from Guadeloupe, French West Indies as non-African reference. We found considerable variation in the ability of African Ae. aegypti populations to acquire and replicate a panel of six DENV strains spanning the four DENV types. Although African Ae. aegypti populations were generally less susceptible than the reference non-African population from Guadeloupe, in several instances some African populations were equally or more susceptible than the Guadeloupe population. Moreover, the relative level of susceptibility between African mosquito populations depended on the DENV strain, indicating genetically specific interactions. We conclude that unlike ZIKV susceptibility, there is no clear-cut dichotomy in DENV susceptibility between African and non-African Ae. aegypti. DENV susceptibility of African Ae. aegypti populations is highly heterogeneous and largely governed by the specific pairing of mosquito population and DENV strain.

Global genomics of Aedes aegypti unveils widespread and novel infectious viruses capable of triggering a small RNA response.

The mosquito Aedes aegypti is a prominent vector for arboviruses, but the breadth of mosquito viruses that infects this specie is not fully understood. In the broadest global survey to date of over 200 Ae. aegypti small RNA samples, we detected viral small interfering RNAs (siRNAs) and Piwi interacting RNAs (piRNAs) arising from mosquito viruses. We confirmed that most academic laboratory colonies of Ae. aegypti lack persisting viruses, yet two commercial strains were infected by a novel tombus-like virus. Ae. aegypti from North to South American locations were also teeming with multiple insect viruses, with Anphevirus and a bunyavirus displaying geographical boundaries from the viral small RNA patterns. Asian Ae. aegypti small RNA patterns indicate infections by similar mosquito viruses from the Americas and reveal the first wild example of dengue virus infection generating viral small RNAs. African Ae. aegypti also contained various viral small RNAs including novel viruses only found in these African substrains. Intriguingly, viral long RNA patterns can differ from small RNA patterns, indicative of viral transcripts evading the mosquitoes' RNA interference (RNAi) machinery. To determine whether the viruses we discovered via small RNA sequencing were replicating and transmissible, we infected C6/36 and Aag2 cells with Ae. aegypti homogenates. Through blind passaging, we generated cell lines stably infected by these mosquito viruses which then generated abundant viral siRNAs and piRNAs that resemble the native mosquito viral small RNA patterns. This mosquito small RNA genomics approach augments surveillance approaches for emerging infectious diseases.

Biology and Behaviour of Aedes aegypti in the Human Environment: Opportunities for Vector Control of Arbovirus Transmission.

Aedes aegypti is a ubiquitous vector of arboviruses mostly in urbanised areas throughout the tropics and subtropics and a growing threat beyond. Control of Ae. aegypti is difficult and costly, and no vaccines are available for most of the viruses it transmits. With practical control solutions our goal, ideally suitable for delivery by householders in affected communities, we reviewed the literature on adult Ae. aegypti biology and behaviour, within and close to the human home, the arena where such interventions must impact. We found that knowledge was vague or important details were missing for multiple events or activities in the mosquito life cycle, such as the duration or location of the many periods when females rest between blood feeding and oviposition. The existing body of literature, though substantial, is not wholly reliable, and evidence for commonly held "facts" range from untraceable to extensive. Source references of some basic information are poor or date back more than 60 years, while other information that today is accepted widely as "fact" is not supported by evidence in the literature. Many topics, e.g., sugar feeding, resting preferences (location and duration), and blood feeding, merit being revisited in new geographical regions and ecological contexts to identify vulnerabilities for exploitation in control.

Adhesion and virulence properties of native Metarhizium fungal strains from Burkina Faso for the control of malaria vectors.

BACKGROUND: Local strains of the entomopathogenic fungus Metarhizium pingshaense in Burkina Faso have demonstrated remarkable virulence against malaria vectors, positioning them as promising candidates for inclusion in the future arsenal of malaria control strategies. However, the underlying mechanisms responsible for this virulence remain unknown. To comprehend the fungal infection process, it is crucial to investigate the attachment mechanisms of fungal spores to the mosquito cuticle and explore the relationship between virulence and attachment kinetics. This study aims to assess the adhesion and virulence properties of native Metarhizium fungal strains from Burkina Faso for controlling malaria vectors. METHODS: Fungal strains were isolated from 201 insects and 1399 rhizosphere samples, and four strains of Metarhizium fungi were selected. Fungal suspensions were used to infect 3-day-old female Anopheles coluzzii mosquitoes at three different concentrations (106, 107, 108 conidia/ml). The survival of the mosquitoes was measured over 14 days, and fungal growth was quantified after 1 and 24 h to assess adhesion of the fungal strains onto the mosquito cuticle. RESULTS: All four fungi strains increased mosquito mortality compared to control (Chi-square test, χ2 = 286.55, df = 4, P < 0.001). Adhesion of the fungal strains was observed on the mosquito cuticle after 24 h at high concentrations (1 × 108 conidia/ml), with one strain, having the highest virulent, showing adhesion after just 1 h. CONCLUSION: The native strains of Metarhizium spp. fungi found in Burkina Faso have the potential to be effective biocontrol agents against malaria vectors, with some strains showing high levels of both virulence and adhesion to the mosquito cuticle.

Association of 410L, 1016I and 1534C kdr mutations with pyrethroid resistance in Aedes aegypti from Ouagadougou, Burkina Faso, and development of a one-step multiplex PCR method for the simultaneous detection of 1534C and 1016I kdr mutations.

BACKGROUND: Since 2000, Burkina Faso has experienced regular dengue cases and outbreaks, making dengue an increasingly important health concern for the country. Previous studies in Burkina Faso reported that resistance of Aedes aegypti to pyrethroid insecticides was associated with the F1534C and V1016I kdr mutations. The current study reports high resistance of Ae. aegypti populations to pyrethroid insecticides, likely supported by mutations in the voltage-gated sodium channel, here evidenced by genotyping the kdr SNPs V410L, V1016I and F1534C. We also describe a new multiplex PCR-based diagnostic of F1534C and V1016I kdr SNPs. METHODS: Larvae of Ae. aegypti were collected from three health districts of Ouagadougou in 2018. The resistance status of Ae. aegypti to permethrin (15 µg/ml) and deltamethrin (10 µg/ml) was tested using bottles and to malathion (5%) using WHO tube tests. All bioassays used 1-h exposure and mortality recorded 24 h post-exposure. Bioassay results were interpreted according to WHO thresholds for resistance diagnosis. The kdr mutations were screened using AS-PCR and TaqMan methods in exposed and non-exposed Aedes mosquitoes. RESULTS: Females from all health districts were resistant to permethrin and deltamethrin (< 20% mortality) but were fully susceptible to 5% malathion. The F1534C and V1016I kdr mutations were successfully detected using a newly developed multiplex PCR in perfect agreement with TaqMan method. The 1534C/1016I/410L haplotype was correlated with permethrin resistance but not with deltamethrin resistance; however, the test power was limited by a low frequency of dead individuals in deltamethrin exposure. CONCLUSIONS: Resistance to pyrethroid insecticides is associated with kdr mutant haplotypes, while the absence of substantial resistance to malathion suggests that it remains a viable option for dengue vector control in Ouagadougou.

Methanol and acetone extracts from the leaves of selected aromatic plants affect survival of field collected Anopheles arabiensis (Diptera: Culicidae) from Kisumu, Kenya.

The massive and inappropriate use of synthetic insecticides is causing significant and increasing environmental disruption. Therefore, developing effective natural mosquitocidal compounds could be an alternative tool for malarial vector control. The present study investigates the larvicidal and adulticidal effect of methanol and acetone extracts of leaves from Lippia chevalieri, Lippia multiflora, Cymbopogon schoenanthus, and Lantana camara against Anopheles arabiensis, to control the most widespread vector transmitting malaria in sub-Saharan. Africa. Extracts were evaluated following WHO modified test procedure against third- to fourth-instar larvae and, non-blood-fed females from 3- to 5-day-old field populations of An. arabiensis under laboratory conditions using WHO larval and CDC bottle bioassays, respectively. Mortality was recorded after 24-h exposure and several compounds were identified in the extracts. The methanolic and acetonic extracts of L. camara were effective against larvae showing lethal concentrations to 50% (LC50) of the population, at 89.48 and 58.72 ppm, respectively. The acetonic extracts of C. schoenanthus and L. chevalieri showed higher toxicities LC50s of 0.16% and 0.22% against female adults, respectively. The methanolic extracts of L. multiflora and L. chevalieri LC50s were effective at 0.17% and 0.27%, respectively, against female adults. These results indicate that the plant extracts tested may represent effective means to control An. arabiensis when used to treat the surface of the marshes.

Dating the origin and spread of specialization on human hosts in Aedes aegypti mosquitoes.

The globally invasive mosquito subspecies Aedes aegypti aegypti is an effective vector of human arboviruses, in part because it specializes in biting humans and breeding in human habitats. Recent work suggests that specialization first arose as an adaptation to long, hot dry seasons in the West African Sahel, where Ae. aegypti relies on human-stored water for breeding. Here, we use whole-genome cross-coalescent analysis to date the emergence of human-specialist populationsand thus further probe the climate hypothesis. Importantly, we take advantage of the known migration of specialists out of Africa during the Atlantic Slave Trade to calibrate the coalescent clock and thus obtain a more precise estimate of the older evolutionary event than would otherwise be possible. We find that human-specialist mosquitoes diverged rapidly from ecological generalists approximately 5000 years ago, at the end of the African Humid Period-a time when the Sahara dried and water stored by humans became a uniquely stable, aquatic niche in the Sahel. We also use population genomic analyses to date a previously observed influx of human-specialist alleles into major West African cities. The characteristic length of tracts of human-specialist ancestry present on a generalist genetic background in Kumasi and Ouagadougou suggests the change in behavior occurred during rapid urbanization over the last 20-40 years. Taken together, we show that the timing and ecological context of two previously observed shifts towards human biting in Ae. aegypti differ; climate was likely the original driver, but urbanization has become increasingly important in recent decades.

Extensive variation and strain-specificity in dengue virus susceptibility among African Aedes aegypti populations.

African populations of the mosquito Aedes aegypti are usually considered less susceptible to infection by human-pathogenic flaviviruses than globally invasive populations found outside Africa. Although this contrast has been well documented for Zika virus (ZIKV), it is unclear to what extent it is true for dengue virus (DENV), the most prevalent flavivirus of humans. Addressing this question is complicated by substantial genetic diversity among DENV strains, most notably in the form of four genetic types (DENV1 to DENV4), that can lead to genetically specific interactions with mosquito populations. Here, we carried out a continent-wide survey of DENV susceptibility using a panel of field-derived Ae. aegypti colonies from across the African range of the species and a colony from Guadeloupe, French West Indies as non-African reference. We found considerable variation in the ability of African Ae. aegypti populations to acquire and replicate a panel of six DENV strains spanning the four DENV types. Although African Ae. aegypti populations were generally less susceptible than the reference non-African population from Guadeloupe, in several instances some African populations were equally or more susceptible than the Guadeloupe population. Moreover, the relative level of susceptibility between African mosquito populations depended on the DENV strain, indicating genetically specific interactions. We conclude that unlike ZIKV susceptibility, there is no clear-cut dichotomy in DENV susceptibility between African and non-African Ae. aegypti. DENV susceptibility of African Ae. aegypti populations is highly heterogeneous and largely governed by the specific pairing of mosquito population and DENV strain.

Development of an allele-specific, loop-mediated, isothermal amplification method (AS-LAMP) to detect the L1014F kdr-w mutation in Anopheles gambiae s. l.

BACKGROUND: Malaria control relies heavily on treated bed nets and indoor residual spraying with pyrethroid insecticides. Unfortunately, the resistance to pyrethroid insecticides, mainly due to the kdr mutation, is spreading in the main malaria vector Anopheles gambiae s.l., decreasing the insecticides' efficacy. To manage the insecticide resistance rapidly and flexibly, simple and effective tools for the early detection of resistant mosquitoes are needed. This study aimed to develop an allele-specific, loop-mediated, isothermal amplification (AS-LAMP) method to detect the West African-type kdr mutation (kdr-w; L1014F) in field-collected mosquitoes. METHODS: DNA fragments of the wild-type and the mutated kdr gene were used to select the primers and develop the method. The primers were designed with the mutation at the 5' end of the backward inner primer (BIP). The AS-LAMP method was compared to the AS-PCR method using the genomic DNA of 120 field-collected mosquitoes. RESULTS: The AS-LAMP method could discriminate between the wild-type homozygote, the heterozygote, and the kdr-w homozygote within 75 min. The AS-LAMP method has the advantage of being faster and at least as sensitive and specific as the AS-PCR method. CONCLUSIONS: The AS-LAMP method can be used to detect the kdr mutation for quick decision-making, even in less well-equipped laboratories.