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Most insects, including human-targeting mosquitoes, detect odors through odorant-activated ion channel complexes consisting of a divergent odorant-binding subunit (OR) and a conserved co-receptor subunit (Orco). As a basis for understanding how odorants activate these heteromeric receptors, we report here cryo2electron microscopy structures of two different heteromeric odorant receptor complexes containing ORs from disease-vector mosquitos Aedes aegypti or Anopheles gambiae. These structures reveal an unexpected stoichiometry of one OR to three Orco subunits. Comparison of structures in odorant-bound and unbound states indicates that odorant binding to the sole OR subunit is sufficient to open the channel pore, suggesting a mechanism of OR activation and a conceptual framework for understanding evolution of insect odorant receptor sensitivity. [ABSTRACT FROM AUTHOR]

This study explores the influence of small dams on the exposure to malaria vectors during the dry season in Kasungu district, Malawi, an area recently identified as high priority for malaria interventions by the National Malaria Control Programme. Small dam impoundments provide communities with a continuous supply of water for domestic and agricultural activities across sub‐Saharan Africa and are considered vital to food security and climate change resilience. However, these permanent water bodies also create ideal breeding sites for mosquitoes in typically arid landscapes. The study focuses on a specific dam impoundment and its vicinity, aiming to assess its spatial and temporal influence on indoor vector densities. From May to August 2021, CDC light traps were used to measure indoor mosquito densities for two consecutive nights per month in three communities located at increasing distances from the dam (0, ~1 and ~2 km). Simultaneously, drone imagery was captured for each community, enabling the identification of additional standing water within approximately 400 m of selected households. Larval sampling was carried out within the impoundment periphery and in additional water bodies identified in the drone imagery. Generalised linear mixed models (GLMMs) were employed to analyse the indoor Anopheles abundance data, estimating the effects of household structure (open/closed eaves), month, temperature and water proximity on malaria vector exposure. Throughout 685 trapping nights, a total of 1256 mosquitoes were captured, with 33% (412) being female Anopheles. Among these, 91% were morphologically identified as Anopheles funestus s.l., and 5% as Anopheles gambiae s.l. Catches progressively decline in each consecutive trapping month as the environment became drier. This decline was much slower in Malangano, the community next to the dam, with abundance being notably higher in June and July. Further, the majority of An. gambiae s.l. were caught in May, with none identified in July and August. Anopheles larvae were found both in the impoundment and other smaller water bodies such as irrigation wells in each survey month; however, the presence of these smaller water bodies did not have a significant impact on adult female mosquito catches in the GLMM. The study concludes that proximity to the dam impoundment was the primary driver of differences between survey communities with the abundance in Chikhombwe (~1 km away) and Chiponde (~2 km away) being 0.35 (95% confidence interval [CI], 0.19–0.66) and 0.28 (95% CI, 0.16–0.47) lower than Malangano, respectively, after adjusting for other factors. These findings underscore the importance of targeted interventions, such as larval source management or housing improvements, near small dams to mitigate malaria transmission risks during the dry season. Further research is needed to develop cost‐effective strategies for vector control within and around these impoundments. [ABSTRACT FROM AUTHOR]

Background: Malaria transmission in Tanzania is driven by mosquitoes of the Anopheles gambiae complex and Anopheles funestus group. The latter includes An. funestus s.s., an anthropophilic vector, which is now strongly resistant to public health insecticides, and several sibling species, which remain largely understudied despite their potential as secondary vectors. This paper provides the initial results of a cross-country study of the species composition, distribution and malaria transmission potential of members of the Anopheles funestus group in Tanzania. Methods: Mosquitoes were collected inside homes in 12 regions across Tanzania between 2018 and 2022 using Centres for Disease Control and Prevention (CDC) light traps and Prokopack aspirators. Polymerase chain reaction (PCR) assays targeting the noncoding internal transcribed spacer 2 (ITS2) and 18S ribosomal DNA (18S rDNA) were used to identify sibling species in the An. funestus group and presence of Plasmodium infections, respectively. Where DNA fragments failed to amplify during PCR, we sequenced the ITS2 region to identify any polymorphisms. Results: The following sibling species of the An. funestus group were found across Tanzania: An. funestus s.s. (50.3%), An. parensis (11.4%), An. rivulorum (1.1%), An. leesoni (0.3%). Sequencing of the ITS2 region in the nonamplified samples showed that polymorphisms at the priming sites of standard species-specific primers obstructed PCR amplification, although the ITS2 sequences closely matched those of An. funestus s.s., barring these polymorphisms. Of the 914 samples tested for Plasmodium infections, 11 An. funestus s.s. (1.2%), and 2 An. parensis (0.2%) individuals were confirmed positive for P. falciparum. The highest malaria transmission intensities [entomological inoculation rate (EIR)] contributed by the Funestus group were in the north-western region [108.3 infectious bites/person/year (ib/p/y)] and the south-eastern region (72.2 ib/p/y). Conclusions: Whereas An. funestus s.s. is the dominant malaria vector in the Funestus group in Tanzania, this survey confirms the occurrence of Plasmodium-infected An. parensis, an observation previously made in at least two other occasions in the country. The findings indicate the need to better understand the ecology and vectorial capacity of this and other secondary malaria vectors in the region to improve malaria control. [ABSTRACT FROM AUTHOR]

Macroinvertebrate predators such as backswimmers (Heteroptera: Notonectidae), dragonflies (Odonata: Aeshnidae), and predatory diving beetles (Coleoptera: Dytiscidae) naturally inhabit aquatic ecosystems. Some aquatic ecosystems inhabited by these macroinvertebrate predator taxa equally form malaria vector larval habitats. The presence of these predators in malaria vector larval habitats can negatively impact on development, adult body size, fecundity, and longevity of the malaria vectors, which form important determinants of their fitness and future vectorial capacity. These potential negative impacts caused by aquatic macroinvertebrate predators on malaria vectors warrant their consideration as biocontrol agents in an integrated program to combat malaria. However, the use of these macroinvertebrate predators in malaria biocontrol is currently constrained by technical bottlenecks linked to their generalist predatory tendencies and often long life cycles, demanding complex rearing systems. We reviewed the literature on the use of aquatic macroinvertebrate predators for biocontrol of malaria vectors from the An. gambiae s.l. complex. The available information from laboratory and semi-field studies has shown that aquatic macroinvertebrates have the potential to consume large numbers of mosquito larvae and could thus offer an additional approaches in integrated malaria vector management strategies. The growing number of semi-field structures available in East and West Africa provides an opportunity to conduct ecological experimental studies to reconsider the potential of using aquatic macroinvertebrate predators as a biocontrol tool. To achieve a more sustainable approach to controlling malaria vector populations, additional, non-chemical interventions could provide a more sustainable approach, in comparison with the failing chemical control tools, and should be urgently considered for integration with the current mosquito vector control campaigns. [ABSTRACT FROM AUTHOR]

Despite efforts to explore the genome of the malaria vector Anopheles gambiae, the Y chromosome of this species remains enigmatic. The large number of repetitive and heterochromatic DNA sequences makes the Y chromosome exceptionally difficult to fully assemble, hampering the progress of gene editing techniques and functional studies for this chromosome. In this study, we made use of a bioinformatic platform to identify Y-specific repetitive DNA sequences that served as a target site for a CRISPR/Cas9 system. The activity of Cas9 in the reproductive organs of males caused damage to Y-bearing sperm without affecting their fertility, leading to a strong female bias in the progeny. Cytological investigation allowed us to identify meiotic defects and investigate sperm selection in this new synthetic sex ratio distorter system. In addition, alternative promoters enable us to target the Y chromosome in specific tissues and developmental stages of male mosquitoes, enabling studies that shed light on the role of this chromosome in male gametogenesis. This work paves the way for further insight into the poorly characterised Y chromosome of Anopheles gambiae. Moreover, the sex distorter strain we have generated promises to be a valuable tool for the advancement of studies in the field of developmental biology, with the potential to support the progress of genetic strategies aimed at controlling malaria mosquitoes and other pest species. Author summary: Genetic elements known as sex ratio meiotic drive can manipulate the sex ratio of offspring, favouring the male or female sex. This fascinating phenomenon has inspired the development of synthetic sex ratio distorter systems in several organisms. In species where females and males harbour XX and XY sex chromosomes respectively, the X-chromosome can be 'shredded' during male gametogenesis, leading to the production of non-functional X-bearing sperm, while Y-bearing sperm are left intact and able to fertilise the eggs. These systems can produce offspring that are extremely biased towards males, which can be used as genetic tools to control harmful insect populations. In our study, we applied this molecular strategy to target the Y chromosome of Anopheles gambiae. Our aim was to investigate the cellular consequences of the shredding of this chromosome, the impact on meiosis and sperm selection, and the potential to achieve strong female bias in the offspring. The outcome of this study enhances our understanding of the molecular and biological mechanisms behind synthetic sex-ratio distorters in Anopheles mosquitoes, which could inform the development of vector control strategies that target sex ratio. Additionally, we present a genetic sexing strain able to produce mostly females, providing a valuable genetic tool for fundamental studies on this deadly vector. [ABSTRACT FROM AUTHOR]

In this study, the chemical composition, repellent, and oviposition deterrent effects of five plant essential oils (EOs) extracted from Lantana camara (Verbenaceae), Schinus terebinthifolia (Anacardiaceae), Callistemon viminalis (Myrtaceae), Helichrysum odoratissimum (Asteraceae), and Hyptis suaveolens (Lamiaceae) were evaluated against Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus. When tested at 33.3 µg/cm2, L. camara, S. terebinthifolia, C. viminalis, and H. odoratissimum were effective repellents against Ae. aegypti (89%, 91%, 90%, and 51% repellency, respectively), but they were less repellent against An. gambiae (66%, 86%, 59%, and 49% repellency, respectively). Interestingly, L. camara, S. terebinthifolia, C. viminalis, and H. odoratissimum exhibited 100% repellency against Cx. quinquefasciatus at 33.3 μg/cm2. In time-span bioassays performed at 333 μg/cm2, the EO of L. camara exhibited 100% repellence against Ae. aegypti and An. gambiae for up to 15 min and against Cx. quinquefasciatus for 75 min. The oviposition bioassays revealed that L. camara exhibited the highest activity, showing 85%, 59%, and 89% oviposition deterrence against Ae. aegypti, An. gambiae, and Cx. quinquefasciatus, respectively. The major compounds of L. camara, S. terebinthifolia, and C. viminalis were trans-β-caryophyllene (16.7%), α-pinene (15.5%), and 1,8-cineole (38.1%), respectively. In conclusion, the L. camara and S. terebinthifolia EOs have the potential to be natural mosquito repellents. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

High diversity and differential evolution profiles have been observed for DD34E/ Tc1 transposons; several families originating from these groups, such as DD34E/ ZB , DD34E/ SB , DD35E/ TR , DD36E/ IC, and DD38E/ IT , have been well defined. Even though Frisky , Tiang , Tsessebe, and Topi transposons have been identified in Anopheles gambiae , their taxonomic distribution and phylogenetic relationship in nature remain largely unknown. The evolutionary profiles of Frisky , Tiang , Tsessebe , and Topi homology transposons were investigated in the current study. In total, 254 homology transposons of Frisky , Tiang , Hob , Tsessebe, and Topi were obtained in 200 species by data mining. The phylogenetic tree revealed that these transposons were classified into five main clades (Frisky , Tiang , Hob , Tsessebe , and Topi) forming a monophyletic clade with 98% bootstrap support, belonging to the DD34E/ Tc1 group, and named as Skipper (SK). SK transposons show a wide distribution in animals; however, differential taxonomic distribution patterns were observed for the subfamilies of Frisky , Tiang , Hob , Tsessebe, and Topi ; extensive invasion of Frisky in animals was found, whereas Tiang , Hob , Tsessebe, and Topi were mainly detected in Diptera. SK elements share a similar structural organization and display high sequence identities across subfamilies. Evolutionary dynamics and structural analysis revealed that SK s in some species, such as Bombyx mori , Lordiphosa magnipectinata , Carassius gibelio , Triplophysa dalaica , and Silurus glanis , have recently evolved and present as intact copies, indicating that SK s in these genomes may be active. Together, these observations improve our understanding of the diversity of DD34E/ Tc1 transposons and their impacts on genome evolution in animals. [ABSTRACT FROM AUTHOR]

Ivermectin (IVM) has been proposed as a new tool for malaria control as it is toxic on vectors feeding on treated humans or cattle. Nevertheless, IVM may have a direct mosquitocidal effect when applied on bed nets or sprayed walls. The potential for IVM application as a new insecticide for long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) was tested in this proof-of-concept study in a laboratory and semi-field environment. Laboratory-reared, insecticide-susceptible Kisumu Anopheles gambiae were exposed to IVM on impregnated netting materials and sprayed plastered- and mud walls using cone bioassays. The results showed a direct mosquitocidal effect of IVM on this mosquito strain as all mosquitoes died by 24 h after exposure to IVM. The effect was slower on the IVM-sprayed walls compared to the treated nettings. Further work to evaluate possibility of IVM as a new insecticide formulation in LLINs and IRS will be required. [ABSTRACT FROM AUTHOR]

The Anopheles gambiae 1000 Genomes (Ag1000G) Consortium previously utilized deep sequencing methods to catalogue genetic diversity across African An. gambiae populations. We analyzed the complete datasets of 1142 individually sequenced mosquitoes through Microsoft Premonition's Bayesian mixture model based (BMM) metagenomics pipeline. All specimens were confirmed as either An. gambiae sensu stricto (s.s.) or An. coluzzii with a high degree of confidence (> 98% identity to reference). Homo sapiens DNA was identified in all specimens indicating contamination may have occurred either at the time of specimen collection, preparation and/or sequencing. We found evidence of vertebrate hosts in 162 specimens. 59 specimens contained validated Plasmodium falciparum reads. Human hepatitis B and primate erythroparvovirus-1 viral sequences were identified in fifteen and three mosquito specimens, respectively. 478 of the 1,142 specimens were found to contain bacterial reads and bacteriophage-related contigs were detected in 27 specimens. This analysis demonstrates the capacity of metagenomic approaches to elucidate important vector-host-pathogen interactions of epidemiological significance. A metagenomic analysis of 1,142 field-collected Anopheles gambiae mosquito specimens by the Microsoft Premonition Bayesian mixture model pipeline revealed a diverse set of vertebrate hosts, as well as the presence of Plasmodium parasites and other microbes. [ABSTRACT FROM AUTHOR]

Background: This study aimed to investigate the relationship between the physicochemical characteristics of An. gambiae s.s. and An. coluzzii breeding sites, the susceptibility profiles to commonly used insecticides in public health, and the underlying insecticide resistance mechanisms. Methods: Anopheles breeding sites surveys were conducted in Cotonou and Natitingou in September 2020, January and August 2021. Physicochemical properties and bacterial loads were determined in individual breeding sites. The WHO susceptibility assays were carried out using the female of the emerging adult mosquitoes. Anopheles species were identified through PCR techniques. Kdr L1014F/S, N1575Y and G119S mutations were investigated using TaqMan genotyping assays. Results: Molecular analysis showed that all mosquitoes analyzed in Cotonou were Anopheles coluzzii, while those of Natitingou were Anopheles gambiae s.s. Fecal coliforms were identified as playing a role in this distribution through their significant influence on the presence of An. coluzzii larvae. WHO susceptibility assay indicated a high level of resistance to deltamethrin in the two cities. The resistance levels to deltamethrin were higher in Cotonou (X2 = 31.689; DF = 1; P < 0.0001). There was a suspected resistance to bendiocarb in Cotonou, whereas the mosquito population in Natitingou was resistant. The kdr L1014F mutation was highly observed in both mosquito populations (frequence: 86–91%), while the Ace-1 mutation was found in a small proportion of mosquitoes. In Cotonou, salinity was the only recorded physicochemical parameter that significantly correlated with the resistance of Anopheles mosquitoes to deltamethrin (P < 0.05). In Natitingou, significant correlations were observed between the allelic frequencies of the kdr L1014F mutation and pH, conductivity, and TDS. Conclusion: These results indicate a high level of pyrethroid resistance in the anopheles populations of both Cotonou and Natitingou. Moreover, this study report the involvement of abiotic factors influencing Anopheles susceptibility profile. [ABSTRACT FROM AUTHOR]

Background: A low technology emanator device for slowly releasing vapour of the volatile pyrethroid transfluthrin was recently developed in Tanzania that provides robust protection against night biting Anopheles and Culex vectors of malaria and filariasis for several months. Here these same emanator devices were assessed in Dar es Salaam city, as a means of protection against outdoor-biting Aedes (Stegomia) aegypti, the most important vector of human arboviruses worldwide, in parallel with similar studies in Haiti and Brazil. Methods: A series of entomological experiments were conducted under field and semi-field conditions, to evaluate whether transfluthrin emanators protect against wild Ae. aegypti, and also compare the transfluthrin responsiveness of Ae. aegypti originating from wild-caught eggs to established pyrethroid-susceptible Ae. aegypti and Anopheles gambiae colonies. Preliminary measurements of transfluthrin vapour concentration in air samples collected near treated emanators were conducted by gas chromatography-mass spectrometry. Results: Two full field experiments with four different emanator designs and three different transfluthrin formulations consistently indicated negligible reduction of human landing rates by wild Ae. aegypti. Under semi-field conditions in large cages, 50 to 60% reductions of landing rates were observed, regardless of which transfluthrin dose, capture method, emanator placement position, or source of mosquitoes (mildly pyrethroid resistant wild caught Ae. aegypti or pyrethroid-susceptible colonies of Ae. aegypti and An. gambiae) was used. Air samples collected immediately downwind from an emanator treated with the highest transfluthrin dose (15g), contained 12 to 19 μg/m3 transfluthrin vapour. Conclusions: It appears unlikely that the moderate levels of pyrethroid resistance observed in wild Ae. aegypti can explain the modest-to-undetectable levels of protection exhibited. While potential inhalation exposure could be of concern for the highest (15g) dose evaluated, 3g of transfluthrin appears sufficient to achieve the modest levels of protection that were demonstrated entomologically. While the generally low levels of protection against Aedes reported here from Tanzania, and from similar entomological studies in Haiti and Brazil, are discouraging, complementary social science studies in Haiti and Brazil suggest end-users perceive valuable levels of protection against mosquitoes. It therefore remains unclear whether transfluthrin emanators have potential for protecting against Aedes vectors of important human arboviruses. [ABSTRACT FROM AUTHOR]

Background: Anopheles mosquito resistance to insecticide remains a serious threat to malaria vector control affecting several sub-Sahara African countries, including Côte d'Ivoire, where high pyrethroid, carbamate and organophosphate resistance have been reported. Since 2017, new insecticides, namely neonicotinoids (e.g.; clothianidin) and pyrroles (e.g.; chlorfenapyr) have been pre-qualified by the World Health Organization (WHO) for use in public health to manage insecticide resistance for disease vector control. Methods: Clothianidin and chlorfenapyr were tested against the field-collected Anopheles gambiae populations from Gagnoa, Daloa and Abengourou using the WHO standard insecticide susceptibility biossays. Anopheles gambiae larvae were collected from several larval habitats, pooled and reared to adulthood in each site in July 2020. Non-blood-fed adult female mosquitoes aged 2 to 5 days were exposed to diagnostic concentration deltamethrin, permethrin, alpha-cypermethrin, bendiocarb, and pirimiphos-methyl. Clothianidin 2% treated papers were locally made and tested using WHO tube bioassay while chlorfenapyr (100 µg/bottle) was evaluated using WHO bottle assays. Furthermore, subsamples of exposed mosquitoes were identified to species and genotyped for insecticide resistance markers including the knock-down resistance (kdr) west and east, and acetylcholinesterase (Ace-1) using molecular techniques. Results: High pyrethroid resistance was recorded with diagnostic dose in Abengourou (1.1 to 3.4% mortality), in Daloa (15.5 to 33.8%) and in Gagnoa (10.3 to 41.6%). With bendiocarb, mortality rates ranged from 49.5 to 62.3%. Complete mortality (100% mortality) was recorded with clothianidin in Gagnoa, 94.9% in Daloa and 96.6% in Abengourou, while susceptibility (mortality > 98%) to chlorfenapyr 100 µg/bottle was recorded at all sites and to pirimiphos-methyl in Gagnoa and Abengourou. Kdr-west mutation was present at high frequency (0.58 to 0.73) in the three sites and Kdr-east mutation frequency was recorded at a very low frequency of 0.02 in both Abengourou and Daloa samples and absent in Gagnoa. The Ace-1 mutation was present at frequencies between 0.19 and 0.29 in these areas. Anopheles coluzzii represented 100% of mosquitoes collected in Daloa and Gagnoa, and 72% in Abengourou. Conclusions: This study showed that clothianidin and chlorfenapyr insecticides induce high mortality in the natural and pyrethroid-resistant An. gambiae populations in Côte d'Ivoire. These results could support a resistance management plan by proposing an insecticide rotation strategy for vector control interventions. [ABSTRACT FROM AUTHOR]

Background: Mosquitoes of the Anopheles gambiae complex are one of the major vectors of malaria in sub-Saharan Africa. Their ability to transmit this disease of major public health importance is dependent on their abundance, biting behaviour, susceptibility and their ability to survive long enough to transmit malaria parasites. A deeper understanding of this behaviour can be exploited for improving vector surveillance and malaria control. Findings: Adult mosquitoes emerge from aquatic habitats at dusk. After a 24 h teneral period, in which the cuticle hardens and the adult matures, they may disperse at random and search upwind for a mate or to feed. Mating generally takes place at dusk in swarms that form over species-specific 'markers'. Well-nourished females may mate before blood-feeding, but the reverse is true for poorly-nourished insects. Females are monogamous and only mate once whilst males, that only feed on nectar, swarm nightly and can potentially mate up to four times. Females are able to locate hosts by following their carbon dioxide and odour gradients. When in close proximity to the host, visual cues, temperature and relative humidity are also used. Most blood-feeding occurs at night, indoors, with mosquitoes entering houses mainly through gaps between the roof and the walls. With the exception of the first feed, females are gonotrophically concordant and a blood meal gives rise to a complete egg batch. Egg development takes two or three days depending on temperature. Gravid females leave their resting sites at dusk. They are attracted by water gradients and volatile chemicals that provide a suitable aquatic habitat in which to lay their eggs. Conclusion: Whilst traditional interventions, using insecticides, target mosquitoes indoors, additional protection can be achieved using spatial repellents outdoors, attractant traps or house modifications to prevent mosquito entry. Future research on the variability of species-specific behaviour, movement of mosquitoes across the landscape, the importance of light and vision, reproductive barriers to gene flow, male mosquito behaviour and evolutionary changes in mosquito behaviour could lead to an improvement in malaria surveillance and better methods of control reducing the current over-reliance on the indoor application of insecticides. [ABSTRACT FROM AUTHOR]

Background: This study aimed to assess the spatial distribution of Anopheles mosquito larval habitats and the environmental factors associated with them, as a prerequisite for the implementation of larviciding. Methods: The study was conducted in December 2021, during the transition period between the end of the short rainy season (September–November) and the short dry season (December-February). Physical, biological, and land cover data were integrated with entomological observations to collect Anopheles larvae in three major towns: Mitzic, Oyem, and Bitam, using the "dipping" method during the transition from rainy to dry season. The collected larvae were then reared in a field laboratory established for the study period. After the Anopheles mosquitoes had emerged, their species were identified using appropriate morphological taxonomic keys. To determine the influence of environmental factors on the breeding of Anopheles mosquitoes, multiple-factor analysis (MFA) and a binomial generalized linear model were used. Results: According to the study, only 33.1% out of the 284 larval habitats examined were found to be positive for Anopheles larvae, which were primarily identified as belonging to the Anopheles gambiae complex. The findings of the research suggested that the presence of An. gambiae complex larvae in larval habitats was associated with various significant factors such as higher urbanization, the size and type of the larval habitats (pools and puddles), co-occurrence with Culex and Aedes larvae, hot spots in ambient temperature, moderate rainfall, and land use patterns. Conclusions: The results of this research mark the initiation of a focused vector control plan that aims to eradicate or lessen the larval habitats of An. gambiae mosquitoes in Gabon's Woleu Ntem province. This approach deals with the root causes of malaria transmission through larvae and is consistent with the World Health Organization's (WHO) worldwide objective to decrease malaria prevalence in regions where it is endemic. [ABSTRACT FROM AUTHOR]

Background: Anopheles funestus is a leading vector of malaria in most parts of East and Southern Africa, yet its ecology and responses to vector control remain poorly understood compared with other vectors such as Anopheles gambiae and Anopheles arabiensis. This study presents the first large-scale survey of the genetic and phenotypic expression of insecticide resistance in An. funestus populations in Tanzania. Methods: We performed insecticide susceptibility bioassays on An. funestus mosquitoes in nine regions with moderate-to-high malaria prevalence in Tanzania, followed by genotyping for resistance-associated mutations (CYP6P9a, CYP6P9b, L119F-GSTe2) and structural variants (SV4.3 kb, SV6.5 kb). Generalized linear models were used to assess relationships between genetic markers and phenotypic resistance. An interactive R Shiny tool was created to visualize the data and support evidence-based interventions. Results: Pyrethroid resistance was universal but reversible by piperonyl-butoxide (PBO). However, carbamate resistance was observed in only five of the nine districts, and dichloro-diphenyl-trichloroethane (DDT) resistance was found only in the Kilombero valley, south-eastern Tanzania. Conversely, there was universal susceptibility to the organophosphate pirimiphos-methyl in all sites. Genetic markers of resistance had distinct geographical patterns, with CYP6P9a-R and CYP6P9b-R alleles, and the SV6.5 kb structural variant absent or undetectable in the north-west but prevalent in all other sites, while SV4.3 kb was prevalent in the north-western and western regions but absent elsewhere. Emergent L119F-GSTe2, associated with deltamethrin resistance, was detected in heterozygous form in districts bordering Mozambique, Malawi and the Democratic Republic of Congo. The resistance landscape was most complex in western Tanzania, in Tanganyika district, where all five genetic markers were detected. There was a notable south-to-north spread of resistance genes, especially CYP6P9a-R, though this appears to be interrupted, possibly by the Rift Valley. Conclusions: This study underscores the need to expand resistance monitoring to include An. funestus alongside other vector species, and to screen for both the genetic and phenotypic signatures of resistance. The findings can be visualized online via an interactive user interface and could inform data-driven decision-making for resistance management and vector control. Since this was the first large-scale survey of resistance in Tanzania's An. funestus, we recommend regular updates with greater geographical and temporal coverage. [ABSTRACT FROM AUTHOR]

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

Background: Vector control using insecticides is a key prevention strategy against malaria. Unfortunately, insecticide resistance in mosquitoes threatens all progress in malaria control. In the perspective of managing this resistance, new insecticide formulations are being tested to improve the effectiveness of vector control tools. Methods: The efficacy and residual activity of Pirikool® 300 CS was evaluated in comparison with Actellic® 300 CS in experimental huts at the Tiassalé experimental station on three substrates including cement, wood and mud. The mortality, blood-feeding inhibition, exiting behaviour and deterrency of free-flying wild mosquitoes was evaluated. Cone bioassay tests with susceptible and resistant mosquito strains were conducted in the huts to determine residual efficacy. Results: A total of 20,505 mosquitoes of which 10,979 (53%) wild female Anopheles gambiae were collected for 112 nights. Residual efficacy obtained from monthly cone bioassay was higher than 80% with the susceptible, laboratory-maintained An. gambiae Kisumu strain, from the first to the tenth study period on all three types of treated substrate for both Actellic® 300CS and Pirikool® 300CS. This residual efficacy on the wild Tiassalé strain was over 80% until the 4th month of study on Pirikool® 300CS S treated substrates. Overall 24-h mortalities of wild free-flying An. gambiae sensu lato which entered in the experimental huts over the 8-months trial on Pirikool® 300CS treatment was 50.5%, 75.9% and 52.7%, respectively, on cement wall, wood wall and mud wall. The positive reference product Actellic® 300CS treatment induced mortalities of 42.0%, 51.8% and 41.8% on cement wall, wood wall and mud wall. Conclusion: Pirikool® 300CS has performed really well against resistant strains of An. gambiae using indoor residual spraying method in experimental huts. It could be an alternative product for indoor residual spraying in response to the vectors' resistance to insecticides. [ABSTRACT FROM AUTHOR]

Genetic surveillance of mosquito populations is becoming increasingly relevant as genetics-based mosquito control strategies advance from laboratory to field testing. Especially applicable are mosquito gene drive projects, the potential scale of which leads monitoring to be a significant cost driver. For these projects, monitoring will be required to detect unintended spread of gene drive mosquitoes beyond field sites, and the emergence of alternative alleles, such as drive-resistant alleles or non-functional effector genes, within intervention sites. This entails the need to distribute mosquito traps efficiently such that an allele of interest is detected as quickly as possible—ideally when remediation is still viable. Additionally, insecticide-based tools such as bednets are compromised by insecticide-resistance alleles for which there is also a need to detect as quickly as possible. To this end, we present MGSurvE (Mosquito Gene SurveillancE): a computational framework that optimizes trap placement for genetic surveillance of mosquito populations such that the time to detection of an allele of interest is minimized. A key strength of MGSurvE is that it allows important biological features of mosquitoes and the landscapes they inhabit to be accounted for, namely: i) resources required by mosquitoes (e.g., food sources and aquatic breeding sites) can be explicitly distributed through a landscape, ii) movement of mosquitoes may depend on their sex, the current state of their gonotrophic cycle (if female) and resource attractiveness, and iii) traps may differ in their attractiveness profile. Example MGSurvE analyses are presented to demonstrate optimal trap placement for: i) an Aedes aegypti population in a suburban landscape in Queensland, Australia, and ii) an Anopheles gambiae population on the island of São Tomé, São Tomé and Príncipe. Further documentation and use examples are provided in project's documentation. MGSurvE is intended as a resource for both field and computational researchers interested in mosquito gene surveillance. Author summary: Mosquito-borne diseases such as malaria and dengue fever continue to pose a major health burden throughout much of the world. The impact of currently-available tools, such as insecticides and antimalarial drugs, is stagnating, and gene drive-modified mosquitoes are considered a novel tool that could contribute to continuing reductions in disease transmission. Gene drive approaches are unique in the field of vector control in that they involve transgenes that could potentially spread on a wide scale, and consequently, surveillance is expected to be a major cost driver for the technology. This is needed to monitor for unintended spread of intact drive alleles, and the emergence of alternative alleles such as homing-resistance alleles and non-functional effector genes. Additionally, surveillance of insecticide-resistance alleles is of interest to support the impact of insecticide-based tools such as bednets. Here, we present MGSurvE, a computational framework that optimizes trap placement for genetic surveillance of mosquito populations in order to minimize the time to detection for an allele of interest. MGSurvE has been tailored to various features of mosquito ecology, and is intended as a resource for researchers to optimize the efficiency of limited surveillance resources. [ABSTRACT FROM AUTHOR]

Background: The direct membrane feeding assay (DMFA), whereby gametocyte-infected blood is collected from human donors and from which mosquitoes feed through a membrane, is proving essential for assessing parameters influencing Plasmodium transmission potential in endemic countries. The success of DMFAs is closely tied to gametocyte density in the blood, with relatively high gametocytaemia ensuring optimal infection levels in mosquitoes. As transmission intensity declines with control efforts, the occurrence of asymptomatic individuals with low gametocyte densities, who can significantly contribute to the infectious reservoir, is increasing. This poses a limitation to studies relying on the experimental infection of large numbers of mosquitoes with natural isolates of Plasmodium. A simple, field-applicable method is presented for improving parasite infectivity by concentrating Plasmodium falciparum gametocytes. Methods: Anopheles gambiae received one of the following 5 blood treatments through DMFA: (i) whole blood (WB) samples from naturally-infected donors; (ii) donor blood whose plasma was replaced with the same volume of Plasmodium-naive AB + serum (1:1 control); (iii) plasma replaced with a volume of malaria-naïve AB + serum equivalent to half (1:1/2), or to a quarter (1:1/4), of the initial plasma volume; and (v) donor blood whose plasma was fully removed (RBC). The experiment was repeated 4 times using 4 distinct wild parasite isolates. Seven days post-infection, a total of 1,095 midguts were examined for oocyst presence. Results: Substituting plasma with reduced amounts (1:1/2 and 1:1/4) of Plasmodium-naive AB + serum led to a 31% and 17% increase of the mosquito infection rate and to a 85% and 308% increase in infection intensity compared to the 1:1 control, respectively. The full removal of plasma (RBC) reduced the infection rate by 58% and the intensity by 64% compared to the 1:1 control. Reducing serum volumes (1:1/2; 1:1/4 and RBC) had no impact on mosquito feeding rate and survival when compared to the 1:1 control. Conclusions: Concentrating gametocytic blood by replacing natural plasma by lower amount of naive serum can enhance the success of mosquito infection. In an area with low gametocyte density, this simple and practical method of parasite concentration can facilitate studies on human-to-mosquito transmission such as the evaluation of transmission-blocking interventions. [ABSTRACT FROM AUTHOR]

Background The effect of clothing colour on the biting rates of different vector mosquito species is not well understood. Studies under tropical field conditions are lacking. This study aimed to determine the influence of clothing colours on mosquito biting rates in rural and suburban settings in West Africa. Methods We performed a simulated field study in a suburban and a rural site in Mali using Mosquito-Magnet traps utilizing CO2 and other attractants, which were covered with black, white, and black/white striped textile sheets covers. These targets operated continuously for 10 consecutive days with bright nights (around full moon) and 10 consecutive days with dark nights (around new moon). Trapped mosquitoes were collected and catch rates counted hourly. Mosquitoes were morphologically identified to the species complex level (Anopheles gambiae s.l. and Culex pipiens s.l.) or species level (Aedes aegypti). A subset of Anopheles specimens were further identified by molecular methods. Results Under bright-night conditions, An. gambiae s.l. was significantly more attracted to black targets than to white and striped targets; during dark nights, no target preference was noted. During bright nights, Cx. pipiens s.l. was significantly more attracted to black and striped targets than to white targets; a similar trend was noted during dark nights (not significant). For day-active Ae. aegypti , striped targets were more attractive than the other targets and black were more attractive than white targets. Conclusions The study firstly demonstrated that under field conditions in Mali, West Africa, mosquito catch rates were influenced by different clothing colours, depending on mosquito species and light conditions. Overall, light colours were least attractive to host-seeking mosquitoes. Using white or other light-coloured clothing can potentially reduce bite exposure and risk of disease transmission in endemic tropical regions. [ABSTRACT FROM AUTHOR]

The incorporation of phytoactive compounds in the management of malarial vectors holds promise for the development of innovative and efficient alternatives. Nevertheless, the molecular and physiological responses that these bioactive substances induce remain underexplored. This present study investigated the toxicity of different concentrations of aqueous and methanol extracts of Ocimum tenuiflorum against larvae of Anopheles gambiae (sensu stricto) and unraveled the possible underlying molecular pathways responsible for the observed physiological effects. FTIR and GCMS analyses of phytoactive compounds in aqueous and methanol crude extracts of O. tenuiflorum showed the presence of OH stretching vibration, C = C stretching modes of aromatics and methylene rocking vibration; ring deformation mode with high levels of trans-β-ocimene, 3,7-dimethyl-1,3,6-octatriene in aqueous extract and 4-methoxy-benzaldehyde, 1,3,5-trimethyl-cyclohexane and o-cymene in methanol extract. The percentage mortality upon exposure to methanol and aqueous extracts of O. tenuiflorum were 21.1% and 26.1% at 24 h, 27.8% and 36.1% at 48 h and 36.1% and 45% at 72 h respectively. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR), down-regulation of ABC transporter, overexpression of CYP6M2, Hsp70, and α-esterase, coupled with significantly increased levels of SOD, CAT, and GSH, were observed in An. gambiae (s.s.) exposed to aqueous and methanol extracts of O. tenuiflorum as compared to the control. Findings from this study have significant implications for our understanding of how An. gambiae (s.s.) larvae detoxify phytoactive compounds. [ABSTRACT FROM AUTHOR]

The health district of Sakassou is one of the 83 health districts in Côte d'Ivoire, located in a zone with very high malarial transmission rates, with an incidence rate of -40% Therefore, to guide vector control methods more effectively, it was crucial to have a good understanding of the vectors in the area. This study aimed to determine the level of malarial transmission during the dry season in Sakassou, Côte d'Ivoire. Female Anopheles mosquitoes were sampled using human landing catches (HLCs) and pyrethrum spraying catches (PSCs). The larvae were collected using the 'dipping' method. A total of 10,875 adult female mosquitoes of Anopheles gambiae were collected. The PCR analysis revealed that all individuals were Anopheles coluzzii. The geographical distribution of potential breeding sites of Anopheles showed the presence of An. coluzzii in all the wetlands of the city of Sakassou. During the dry season, the human-biting rate of An. coluzzii was 139.1 bites/person/night. An exophagic trend was displayed by an adult female of An. coluzzii. The entomological inoculation rate during the dry season was 1.49 infectious bites/person/night. This study demonstrated that An. coluzzii was the main vector of malarial transmission in Sakassou, and the intensity of transmission remains high throughout the dry season. [ABSTRACT FROM AUTHOR]

Background: This study investigates the effectiveness of new-generation mosquito nets, like Olyset® Plus and PermaNet® 3.0, and dual-action nets such as Interceptor® G2, against pyrethroid-resistant Anopheles gambiae mosquitoes following the 2023 mass distribution of long-lasting insecticidal nets in Benin. Methods: We tested wild mosquito populations from six communes in Benin against various pyrethroid (permethrin 0.75%, alphacypermethrin 0.05%, and deltamethrin 0.05%) using WHO tube tests. Additionally, we exposed mosquitoes to chlorfenapyr 100 µg/ml using the CDC bottle bioassay method. A subset of mosquitoes underwent biochemical and PCR tests to check the overexpression of metabolic enzymes and the Kdr L1014F mutation. We evaluated the effectiveness of Olyset® Plus, PermaNet® 3.0, and Interceptor® G2 nets using cone and tunnel tests on both laboratory and field populations of An. gambiae. Results: Overall, the highest mortality rate was 60% with pyrethroid and 98 to100% with chlorfenapyr. In cone tests, all three types of nets induced mortality rates above 80% in the susceptible laboratory strain of An. gambiae. Notably, Olyset® Plus showed the highest mortality rates for pyrethroid-resistant mosquitoes in cone tests, ranging from 81.03% (95% CI: 68.59–90.13) in Djougou to 96.08% (95% CI: 86.54–99.52) in Akpro-Missérété. PermaNet® 3.0 had variable rates, from 42.5% (95% CI: 27.04–59.11) in Djougou to 58.54% (95% CI: 42.11–73.68) in Porto-Novo. However, revealed good results for Interceptor® G2, with 94% (95% CI: 87.40–97.77) mortality and 89.09% blood sampling inhibition in local populations of An. gambiae. In comparison, Interceptor® had lower rates of 17% (95% CI: 10.23–25.82) and 60%, respectively. Conclusion: These results suggest that tunnel tests are effective for evaluating dual-active ingredient nets. Additionally, Interceptor® G2 and PBO nets like Olyset® Plus could be considered as alternatives against pyrethroid-resistant mosquitoes. [ABSTRACT FROM AUTHOR]

The control of arthropod disease vectors using chemical insecticides is vital in combating malaria, however the increasing insecticide resistance (IR) poses a challenge. Furthermore, climate variability affects mosquito population dynamics and subsequently IR propagation. We present a mathematical model to decipher the relationship between IR in Anopheles gambiae populations and climate variability. By adapting the susceptible-infected-resistant (SIR) framework and integrating temperature and rainfall data, our model examines the connection between mosquito dynamics, IR, and climate. Model validation using field data achieved 92% accuracy, and the sensitivity of model parameters on the transmission potential of IR was elucidated (e.g. μPRCC = 0.85958, p-value < 0.001). In this study, the integration of high-resolution covariates with the SIR model had a significant impact on the spatial and temporal variation of IR among mosquito populations across Africa. Importantly, we demonstrated a clear association between climatic variability and increased IR (width = [0–3.78], α = 0.05). Regions with high IR variability, such as western Africa, also had high malaria incidences thereby corroborating the World Health Organization Malaria Report 2021. More importantly, this study seeks to bolster global malaria combat strategies by highlighting potential IR 'hotspots' for targeted intervention by National malria control programmes. [ABSTRACT FROM AUTHOR]

Background: Anopheles coluzzii is a primary vector of malaria found in West and Central Africa, but its presence has hitherto never been documented in Kenya. A thorough understanding of vector bionomics is important as it enables the implementation of targeted and effective vector control interventions. Malaria vector surveillance efforts in the country have tended to focus on historically known primary vectors. The current study sought to determine the taxonomic status of samples collected from five different malaria epidemiological zones in Kenya as well as describe the population genetic structure and insecticide resistance profiles in relation to other An. coluzzii populations. Methods: Mosquitoes were sampled as larvae from Busia, Kwale, Turkana, Kirinyaga and Kiambu counties, representing the range of malaria endemicities in Kenya, in 2019 and 2021 and emergent adults analysed using Whole Genome Sequencing (WGS) data processed in accordance with the Anopheles gambiae 1000 Genomes Project phase 3. Where available, historical samples from the same sites were included for WGS. Comparisons were made with An. coluzzii cohorts from West and Central Africa. Results: This study reports the detection of An. coluzzii for the first time in Kenya. The species was detected in Turkana County across all three time points from which samples were analyzed and its presence confirmed through taxonomic analysis. Additionally, there was a lack of strong population genetic differentiation between An. coluzzii from Kenya and those from the more northerly regions of West and Central Africa, suggesting they represent a connected extension to the known species range. Mutations associated with target-site resistance to DDT and pyrethroids and metabolic resistance to DDT were found at high frequencies up to 64%. The profile and frequencies of the variants observed were similar to An. coluzzii from West and Central Africa but the ace-1 mutation linked to organophosphate and carbamate resistance present in An. coluzzii from coastal West Africa was absent in Kenya. Conclusions: These findings emphasize the need for the incorporation of genomics in comprehensive and routine vector surveillance to inform on the range of malaria vector species, and their insecticide resistance status to inform the choice of effective vector control approaches. [ABSTRACT FROM AUTHOR]

Background: The residual activity of a clothianidin + deltamethrin mixture and clothianidin alone in IRS covered more than the period of malaria transmission in northern Benin. The aim of this study was to show whether the prolonged residual efficacy of clothianidin-based products resulted in a greater reduction in vector populations and subsequent malaria transmission compared with the shorter residual efficacy of pirimiphos-methyl. Methods: Human bait mosquito collections by local volunteers and pyrethrum spray collections were used in 6 communes under IRS monitoring and evaluation from 2019 to 2021. ELISA/CSP and species PCR tests were performed on Anopheles gambiae sensu lato (s.l.) to determine the infectivity rate and subspecies by commune and year. The decrease in biting rate, entomological inoculation rate, incidence, inhibition of blood feeding, resting density of An. gambiae s.l. were studied and compared between insecticides per commune. Results: The An. gambiae complex was the major vector throughout the study area, acounting for 98.71% (19,660/19,917) of all Anopheles mosquitoes collected. Anopheles gambiae s.l. collected was lower inside treated houses (45.19%: 4,630/10,245) than outside (54.73%: 5,607/10,245) after IRS (p < 0.001). A significant decrease (p < 0.001) in the biting rate was observed after IRS in all departments except Donga in 2021 after IRS with clothianidin 50 WG. The impact of insecticides on EIR reduction was most noticeable with pirimiphos-methyl 300 CS, followed by the clothianidin + deltamethrin mixture and finally clothianidin 50 WG. A reduction in new cases of malaria was observed in 2020, the year of mass distribution of LLINs and IRS, as well as individual and collective protection measures linked to COVID-19. Anopheles gambiae s.l. blood-feeding rates and parous were high and similar for all insecticides in treated houses. Conclusion: To achieve the goal of zero malaria, the optimal choice of vector control tools plays an important role. Compared with pirimiphos-methyl, clothianidin-based insecticides induced a lower reductions in entomological indicators of malaria transmission. [ABSTRACT FROM AUTHOR]

Background: Clothianidin-based indoor residual spraying (IRS) formulations have become available for malaria control as either solo formulations of clothianidin or a mixture of clothianidin with the pyrethroid deltamethrin. While both formulations have been successfully used for malaria control, studies investigating the effect of the pyrethroid in IRS mixtures may help improve our understanding for development of future IRS products. It has been speculated that the irritant effect of the pyrethroid in the mixture formulation may result in shorter mosquito contact times with the treated walls potentially leading to a lower impact. Methods: We compared contact irritancy expressed as the number of mosquito take-offs from cement surfaces treated with an IRS formulation containing clothianidin alone (SumiShield® 50WG) to clothianidin-deltamethrin mixture IRS formulations against pyrethroid-resistant Anopheles gambiae sensu lato under controlled laboratory conditions using a modified version of the World Health Organisation cone bioassay. To control for the pyrethroid, comparison was made with a deltamethrin-only formulation. Both commercial and generic non-commercial mixture formulations of clothianidin and deltamethrin were tested. Results: The clothianidin solo formulation did not show significant contact irritancy relative to the untreated control (3.5 take-offs vs. 3.1 take-offs, p = 0.614) while all deltamethrin-containing IRS induced significant irritant effects. The number of take-offs compared to the clothianidin solo formulation (3.5) was significantly higher with the commercial clothianidin-deltamethrin mixture (6.1, p = 0.001), generic clothianidin-deltamethrin mixture (7.0, p < 0.001), and deltamethrin-only (8.2, p < 0.001) formulations. The commercial clothianidin-deltamethrin mixture induced similar contact irritancy as the generic clothianidin-deltamethrin mixture (6.1 take-offs vs. 7.0 take-offs, p = 0.263) and deltamethrin-only IRS (6.1 take-offs vs. 8.2, p = 0.071), showing that the irritant effect in the mixture was attributable to its deltamethrin component. Conclusions: This study provides evidence that the enhanced contact irritancy of the pyrethroid in clothianidin-deltamethrin IRS mixtures can shorten mosquito contact times with treated walls compared to the clothianidin solo formulation. Further trials are needed to directly compare the efficacy of these formulation types under field conditions and establish the impact of this enhanced contact irritancy on the performance of IRS mixture formulations containing pyrethroids. [ABSTRACT FROM AUTHOR]

Abstract: Background: Anopheles mosquitoes are ectothermic and involved in numerous pathogen transmissions. Their life history traits are influenced by several environmental factors such as temperature, relative humidity and photoperiodicity. Despite extensive investigations of these environmental conditions on vector population ecology, their impact on the different life stages of Anopheles at different seasons in the year remains poorly explored. This study reports the potential impact of these abiotic factors on the immature and adult stages of Anopheles gambiae sensu lato during different seasons. Methods: Environmental conditions were simulated in the laboratory using incubators to mimic the environmental conditions of two important periods of the year in Burkina Faso: the peak of rainy season (August) and the onset of dry season (December). Eggs from wild An. coluzzii and An. gambiae s.l. were reared separately under each environmental condition. For Anopheles coluzzii or An. gambiae s.l., eggs were equally divided into two groups assigned to the two experimental conditions. Four replicates were carried out for this experiment. Then, egg hatching rate, pupation rate, larval development time, larva-to-pupae development time, adult emergence dynamics and longevity of Anopheles were evaluated. Also, pupae-to-adult development time from wild L3 and L4 Anopheles larvae was estimated under semi-field conditions in December. Results: A better egg hatching rate was recorded overall with conditions mimicking the onset of the dry season compared to the peak of the rainy season. Larval development time and longevity of An. gambiae s.l. female were significantly longer at the onset of the dry season compared than at the peak of the rainy season. Adult emergence was spread over 48 and 96 h at the peak of the rainy season and onset of dry season conditions respectively. This 96h duration in the controlled conditions of December was also observed in the semi-field conditions in December. Conclusions: The impact of temperature and relative humidity on immature stages and longevity of An. gambiae s.l. adult females differed under both conditions. These findings contribute to a better understanding of vector population dynamics throughout different seasons of the year and may facilitate tailoring of control strategies. [ABSTRACT FROM AUTHOR]

Background: Anopheles gambiae, the major malaria mosquito in sub-Saharan Africa, feed largely indoors at night. Raising a house off the ground with no barriers underneath reduces mosquito-house entry. This experiment tested whether walling off the space under an elevated hut affects mosquito-hut entry. Methods: Four inhabited experimental huts, each of which could be moved up and down, were used in rural Gambia. Nightly collections of mosquitoes were made using light traps and temperature and carbon dioxide levels monitored indoors and outdoors using loggers. Each night, a reference hut was kept at ground level and three huts raised 2 m above the ground; with the space under the hut left open, walled with air-permeable walls or solid walls. Treatments were rotated every four nights using a randomized block design. The experiment was conducted for 32 nights. Primary measurements were mosquito numbers and indoor temperature in each hut. Results: A total of 1,259 female Anopheles gambiae sensu lato were collected in the hut at ground level, 655 in the hut with an open ground floor, 981 in the hut with air-permeable walls underneath and 873 in the hut with solid walls underneath. Multivariate analysis, adjusting for confounders, showed that a raised hut open underneath had 53% fewer mosquitoes (95% CI 47–58%), those with air-permeable walls underneath 24% fewer (95% CI 9–36%) and huts with solid walls underneath 31% fewer (95% CI 24–37%) compared with a hut on the ground. Similar results were found for Mansonia spp. and total number of female mosquitoes, but not for Culex mosquitoes where hut entry was unaffected by height or barriers. Indoor temperature and carbon dioxide levels were similar in all huts. Conclusion: Raising a house 2 m from the ground reduces the entry of An. gambiae and Mansonia mosquitoes, but not Culex species. The protective effect of height is reduced if the space underneath the hut is walled off. [ABSTRACT FROM AUTHOR]

Mosquitoes are prolific vectors of human pathogens, therefore a clear and accurate understanding of the organization of their antimicrobial defenses is crucial for informing the development of transmission control strategies. The canonical infection response in insects, as described in the insect model Drosophila melanogaster, is pathogen type-dependent, with distinct stereotypical responses to Gram-negative bacteria and Gram-positive bacteria/fungi mediated by the activation of the Imd and Toll pathways, respectively. To determine whether this pathogen-specific discrimination is shared by mosquitoes, we used RNAseq to capture the genome-wide transcriptional response of Aedes aegypti and Anopheles gambiae (s.l.) to systemic infection with Gram-negative bacteria, Gram-positive bacteria, yeasts, and filamentous fungi, as well as challenge with heat-killed Gram-negative, Gram-positive, and fungal pathogens. From the resulting data, we found that Ae. aegypti and An. gambiae both mount a core response to all categories of infection, and this response is highly conserved between the two species with respect to both function and orthology. When we compared the transcriptomes of mosquitoes infected with different types of bacteria, we observed that the intensity of the transcriptional response was correlated with both the virulence and growth rate of the infecting pathogen. Exhaustive comparisons of the transcriptomes of Gram-negative-challenged versus Gram-positive-challenged mosquitoes yielded no difference in either species. In Ae. aegypti, however, we identified transcriptional signatures specific to bacterial infection and to fungal infection. The bacterial infection response was dominated by the expression of defensins and cecropins, while the fungal infection response included the disproportionate upregulation of an uncharacterized family of glycine-rich proteins. These signatures were also observed in Ae. aegypti challenged with heat-killed bacteria and fungi, indicating that this species can discriminate between molecular patterns that are specific to bacteria and to fungi. [ABSTRACT FROM AUTHOR]

Background: Insecticide resistance (IR) is one of the major threats to malaria vector control programs in endemic countries. However, the mechanisms underlying IR are poorly understood. Thus, investigating gene expression patterns related to IR can offer important insights into the molecular basis of IR in mosquitoes. In this study, RNA-Seq was used to characterize gene expression in Anopheles gambiae surviving exposure to pyrethroids (deltamethrin, alphacypermethrin) and an organophosphate (pirimiphos-methyl). Results: Larvae of An. gambiae s.s. collected from Bassila and Djougou in Benin were reared to adulthood and phenotyped for IR using a modified CDC intensity bottle bioassay. The results showed that mosquitoes from Djougou were more resistant to pyrethroids (5X deltamethrin: 51.7% mortality; 2X alphacypermethrin: 47.4%) than Bassila (1X deltamethrin: 70.7%; 1X alphacypermethrin: 77.7%), while the latter were more resistant to pirimiphos-methyl (1.5X: 48.3% in Bassila and 1X: 21.5% in Djougou). RNA-seq was then conducted on resistant mosquitoes, non-exposed mosquitoes from the same locations and the laboratory-susceptible An. gambiae s.s. Kisumu strain. The results showed overexpression of detoxification genes, including cytochrome P450s (CYP12F2, CYP12F3, CYP4H15, CYP4H17, CYP6Z3, CYP9K1, CYP4G16, and CYP4D17), carboxylesterase genes (COEJHE5E, COE22933) and glutathione S-transferases (GSTE2 and GSTMS3) in all three resistant mosquito groups analyzed. Genes encoding cuticular proteins (CPR130, CPR10, CPR15, CPR16, CPR127, CPAP3-C, CPAP3-B, and CPR76) were also overexpressed in all the resistant groups, indicating their potential role in cross resistance in An. gambiae. Salivary gland protein genes related to 'salivary cysteine-rich peptide' and 'salivary secreted mucin 3' were also over-expressed and shared across all resistant groups. Conclusion: Our results suggest that in addition to metabolic enzymes, cuticular and salivary gland proteins could play an important role in cross-resistance to multiple classes of insecticides in Benin. These genes warrant further investigation to validate their functional role in An. gambiae resistance to insecticides. [ABSTRACT FROM AUTHOR]

In many countries, successful control of larval mosquito populations for malaria control is still hindered by poor knowledge of preferred breeding habitats and natural factors limiting larval abundance. In Africa, preferred breeding habitats for malaria mosquitoes vary regionally and for many regions, they are still not well known. Moreover, variables that limit abundance of larvae of malaria mosquitoes are still ambiguous and impacts of surrounding land use practices are unclear. To study this, we sampled mosquito larvae from 164 small ground habitats (SGHs) in the lake Manyara basin (LMB) in northern Tanzania, a malaria expansion area with diverse land use types. We found that Anopheles gambiae s.l. was the only malaria mosquito breeding in SGHs in the region and its abundance was positively affected by turbidity and proximity to human dwellings. SGHs appear to be the major breeding sites for An. gambiae s.l. in the LMB despite the presence of larger temporary ponds which support negligible numbers of this species. In this region, we recommend that control of malaria via control of population of larval An. gambiae s.l. should prioritize SGHs to optimize use of limited resources and avoid damage to the environment by targeting unimportant habitats. [ABSTRACT FROM AUTHOR]

Background: Entomological surveillance of lymphatic filariasis and malaria infections play an important role in the decision-making of national programs to control, or eliminate these both diseases. In areas where both diseases prevalence is low, a large number of mosquitoes need to be sampled to determine vectors infection rate. To do this, efficient mosquito collection methods must be used. This study is part in this framework, to assess appropriate mosquito collection methods for lymphatic filariasis xenomonitoring in a coexistence context with malaria in Burkina Faso. Methodology/Principal findings: Mosquito collections were performed between August and September 2018 in four villages (Koulpissi, Seiga, and Péribgan, Saptan), distributed in East and South-West health regions of Burkina Faso. Different collection methods were used: Human Landing Catches (HLC) executed indoor and outdoor, Window Exit-Trap, Double Net Trap (DNT) and Pyrethrum Spray Catches (PSC). Molecular analyses were performed to identify Anopheles gambiae s.l. sibling species and to detect Wuchereria bancrofti and Plasmodium falciparum infection in Anopheles mosquitoes. A total of 3 322 mosquitoes were collected among this, Anopheles gambiae s.l. was the vector caught in largest proportion (63.82%). An. gambiae s.l. sibling species molecular characterization showed that An. gambiae was the dominant specie in all villages. The Human Landing Catches (indoor and outdoor) collected the highest proportion of mosquitoes (between 61.5% and 82.79%). For the sampling vectors infected to W. bancrofti or P. falciparum, PSC, HLC and Window Exit-Trap were found the most effective collection methods. Conclusions/Significance: This study revealed that HLC indoor and outdoor remained the most effective collection method. Likewise, the results showed the probability to use Window Exit-Trap and PSC collection methods to sample Anopheles infected. Author summary: In Burkina Faso, the monitoring and evaluation scheme to assess the impact of lymphatic filariasis and malaria interventions is relied only on parasitological tests. While nowadays, the most simple and direct measure of vector borne diseases is xenomonitoring. When the both diseases prevalence is low in human population, sampling large numbers of mosquitoes is necessary for xenomonitoring. This study was undertaken in this context to assess the efficiency of four mosquito collection methods for xenomonitoring LF and malaria. Mosquito collections were performed between August and September 2018 in four villages, distributed in East and South-West health regions of Burkina Faso. Human Landing Catches (HLC), Window Exit-Trap, Double Net Trap (DNT) and Pyrethrum Spray Catches (PSC) were evaluated. The results showed that HLC remained the most effective collection method by collecting the highest number of Anopheles (2 388; 71.88% of total). Across the study, mosquito infection rate for W. bancrofti and P. falciparum were 0.004 and 0.13 respectively. To collect vectors infected it was found that Window Exit-Trap and PSC were efficient. In conclusion, HLC has been shown to be appropriate for collecting large number of mosquitoes. Likewise, Window Exit-Trap and PSC can be useful for malaria and lymphatic filariasis xenomonitoring. [ABSTRACT FROM AUTHOR]

Background: Anopheles gambiae continues to be widespread and an important malaria vector species complex in Uganda. New approaches to malaria vector control are being explored including population suppression through swarm reductions and genetic modification involving gene drives. Designing and evaluating these new interventions require good understanding of the biology of the target vectors. Anopheles mosquito swarms have historically been hard to locate in Uganda and therefore have remained poorly characterized. In this study we sought to identify and characterize An. gambiae s.l mosquito swarms in three study sites of high An. gambiae s.l prevalence within Central Uganda. Methods: Nine sampling visits were made to three villages over a 2-year period. Sampling targeted both wet and dry seasons and was done for 2 days per village during each trip, using sweep nets. All swarm data were analysed using the JMP 14 software (SAS Institute, Inc., Cary, NC, USA), parametrically or non-parametrically as appropriate. Results: Most of the An. gambiae s.s. swarms sampled during this study were single-species swarms. However, some mixed An. gambiae s.s. and Culex spp. mosquito swarms were also observed. Swarms were larger in the wet season than in the dry season. Mean swarm height ranged from 2.16 m to 3.13 m off the ground and only varied between villages but not by season. Anopheles gambiae mosquitoes were present in all three villages, preferred to swarm over bare ground markers, and could be effectively sampled by field samplers. Conclusions: This study demonstrated that An. gambiae s.l swarms could be effectively located and sampled in South Central Uganda and provided in-depth descriptions of hitherto poorly understood aspects of An. gambiae local swarm characteristics. Swarms were found close to inhabited households and were greater in size and number during the rainy season. Anopheles gambiae s.s swarms were significantly associated with bare ground markers and were sometimes at heights over 4 m above the ground, showing a necessity to develop tools suitable for swarm sampling at these heights. While mixed species swarms have been reported before elsewhere, this is the first documented instance of mixed genus swarms found in Uganda and should be studied further as it could have implications for swarm sampling explorations where multiple species of mosquitoes exist. [ABSTRACT FROM AUTHOR]

Mosquitoes of the genera Aedes, Anopheles and Culex vector a wide range of pathogens seriously affecting humans and livestock on a global scale. Over-reliance on insecticides and repellents has driven research into alternative, naturally-derived compounds to fulfil the same objectives. Steam distilled extracts of four plants with strong, yet attractive, volatile profiles were initially assessed for repellency in a dual-port olfactometer using Aedes aegypti as the model species. Picea sitchensis was found to be the most repellent, proving comparable to leading products when applied at 100% (p = 1.000). Key components of conifer-derived volatile profiles were then screened via electroantennography before those components eliciting an electrophysiological response were assayed individually in the olfactometer; according to WHO protocol. The most promising 5 were selected for reductive analyses to produce an optimised semiochemical blend. This combination, and a further two variations of the blend, were then progressed to a multi-species analysis using the BG-test whereby bite-attempt frequency on hands was assessed under different repellent treatments; assays were compared between Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Efficacy was found against all three species, although it was found that Ae. aegypti was the most susceptible to the repellent, with An. gambiae being the least. Here, a novel, naturally-derived blend is presented with weak spatial repellency, as confirmed in laboratory assays. Further work will be required to assess the full extent of the potential of the products, both in terms of field application and species screening; however, the success of the products developed demonstrate that plant metabolites have great capacity for use in the repellent sector; both to improve upon known compounds and to reduce the usage of toxic products currently on the market. [ABSTRACT FROM AUTHOR]

Background: Vector surveillance is among the World Health Organization global vector control response (2017–2030) pillars. Human landing catches are a gold standard but difficult to implement and potentially expose collectors to malaria infection. Other methods like light traps, pyrethrum spray catches and aspiration are less expensive and less risky to collectors. Methods: Three mosquito sampling methods (UV light traps, CDC light traps and Prokopack aspiration) were evaluated against human landing catches (HLC) in two villages of Rarieda sub-county, Siaya County, Kenya. UV-LTs, CDC-LTs and HLCs were conducted hourly between 17:00 and 07:00. Aspiration was done indoors and outdoors between 07:00 and 11:00 a.m. Analyses of mosquito densities, species abundance and sporozoite infectivity were performed across all sampling methods. Species identification PCR and ELISAs were done for Anopheles gambiae and Anopheles funestus complexes and data analysis was done in R. Results: Anopheles mosquitoes sampled from 608 trapping efforts were 5,370 constituting 70.3% Anopheles funestus sensu lato (s.l.), 19.7% Anopheles coustani and 7.2% An. gambiae s.l. 93.8% of An. funestus s.l. were An. funestus sensu stricto (s.s.) and 97.8% of An. gambiae s.l. were Anopheles arabiensis. Only An. funestus were sporozoite positive with 3.1% infection prevalence. Indoors, aspiration captured higher An. funestus (mean = 6.74; RR = 8.83, P < 0.001) then UV-LT (mean = 3.70; RR = 3.97, P < 0.001) and CDC-LT (mean = 1.74; RR = 1.89, P = 0.03) compared to HLC. UV-LT and CDC-LT indoors captured averagely 0.18 An. arabiensis RR = 5.75, P = 0.028 and RR = 5.87, P = 0.028 respectively. Outdoors, UV-LT collected significantly higher Anopheles mosquitoes compared to HLC (An. funestus: RR = 5.18, P < 0.001; An. arabiensis: RR = 15.64, P = 0.009; An. coustani: RR = 11.65, P < 0.001). Anopheles funestus hourly biting indoors in UV-LT and CDC-LT indicated different peaks compared to HLC. Conclusions: Anopheles funestus remains the predominant mosquito species. More mosquitoes were collected using aspiration, CDC-LTs and UV-LTs indoors and UV-LTs and CD-LTs outdoors compared to HLCs. UV-LTs collected more mosquitoes than CDC-LTs. The varied trends observed at different times of the night suggest that these methods collect mosquitoes with diverse activities and care must be taken when interpreting the results. [ABSTRACT FROM AUTHOR]

Background: Pyrethroid-based indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) have been employed as key vector control measures against malaria in Namibia. However, pyrethroid resistance in Anopheles mosquitoes may compromise the efficacy of these interventions. To address this challenge, the World Health Organization (WHO) recommends the use of piperonyl butoxide (PBO) LLINs in areas where pyrethroid resistance is confirmed to be mediated by mixed function oxidase (MFO). Methods: This study assessed the susceptibility of Anopheles gambiae sensu lato (s.l.) mosquitoes to WHO tube bioassays with 4% DDT and 0.05% deltamethrin insecticides. Additionally, the study explored the effect of piperonyl butoxide (PBO) synergist by sequentially exposing mosquitoes to deltamethrin (0.05%) alone, PBO (4%) + deltamethrin (0.05%), and PBO alone. The Anopheles mosquitoes were further identified morphologically and molecularly. Results: The findings revealed that An. gambiae sensu stricto (s.s.) (62%) was more prevalent than Anopheles arabiensis (38%). The WHO tube bioassays confirmed resistance to deltamethrin 0.05% in the Oshikoto, Kunene, and Kavango West regions, with mortality rates of 79, 86, and 67%, respectively. In contrast, An. arabiensis displayed resistance to deltamethrin 0.05% in Oshikoto (82% mortality) and reduced susceptibility in Kavango West (96% mortality). Notably, there was reduced susceptibility to DDT 4% in both An. gambiae s.s. and An. arabiensis from the Kavango West region. Subsequently, a subsample from PBO synergist assays in 2020 demonstrated a high proportion of An. arabiensis in Oshana (84.4%) and Oshikoto (73.6%), and 0.42% of Anopheles quadriannulatus in Oshana. Non-amplifiers were also present (15.2% in Oshana; 26.4% in Oshikoto). Deltamethrin resistance with less than 95% mortality, was consistently observed in An. gambiae s.l. populations across all sites in both 2020 and 2021. Following pre-exposure to the PBO synergist, susceptibility to deltamethrin was fully restored with 100.0% mortality at all sites in 2020 and 2021. Conclusions: Pyrethroid resistance has been identified in An. gambiae s.s. and An. arabiensis in the Kavango West, Kunene, and Oshikoto regions, indicating potential challenges for pyrethroid-based IRS and LLINs. Consequently, the data highlights the promise of pyrethroid-PBO LLINs in addressing resistance issues in the region. [ABSTRACT FROM AUTHOR]

Background: Understanding of malaria ecology is a prerequisite for designing locally adapted control strategies in resource-limited settings. The aim of this study was to utilize the spatial heterogeneity in malaria transmission for the designing of adaptive interventions. Methods: Field collections of clinical malaria incidence, asymptomatic Plasmodium infection, and malaria vector data were conducted from 108 randomly selected clusters which covered different landscape settings including irrigated farming, seasonal flooding area, lowland dryland farming, and highlands in western Kenya. Spatial heterogeneity of malaria was analyzed and classified into different eco-epidemiological zones. Results: There was strong heterogeneity and detected hot/cold spots in clinical malaria incidence, Plasmodium prevalence, and vector abundance. The study area was classified into four zones based on clinical malaria incidence, parasite prevalence, vector density, and altitude. The two irrigated zones have either the highest malaria incidence, parasite prevalence, or the highest malaria vector density; the highlands have the lowest vector density and parasite prevalence; and the dryland and flooding area have the average clinical malaria incidence, parasite prevalence and vector density. Different zones have different vector species, species compositions and predominant species. Both indoor and outdoor transmission may have contributed to the malaria transmission in the area. Anopheles gambiae sensu stricto (s.s.), Anopheles arabiensis, Anopheles funestus s.s., and Anopheles leesoni had similar human blood index and malaria parasite sporozoite rate. Conclusion: The multi-transmission-indicator-based eco-epidemiological zone classifications will be helpful for making decisions on locally adapted malaria interventions. [ABSTRACT FROM AUTHOR]

Background: Recently, bacterial endosymbiont, including Wolbachia and Microsporidia were found to limit the infection of Anopheles mosquitoes with Plasmodium falciparum. This study aimed to investigate the natural presence of key transmission-blocking endosymbionts in Anopheles gambiae and Anopheles coluzzii in Southern Benin. Methods: The present study was conducted in seven communes (Cotonou, Porto-Novo, Aguégués, Ifangni, Pobè Athiémé, and Grand-Popo) of Southern Benin. Anopheles were collected using indoor/outdoor Human Landing Catches (HLCs) and Pyrethrum Spray Catches (PSCs). Following morphological identification, PCR was used to identify An. gambiae sensu lato (s.l.) to species level and to screen for the presence of both Wolbachia and Microsporidia. Plasmodium falciparum sporozoite infection was also assessed using ELISA. Results: Overall, species composition in An. gambiae s.l. was 53.7% An. coluzzii, while the remainder was An. gambiae sensu stricto (s.s.). Combined data of the two sampling techniques revealed a mean infection prevalence with Wolbachia of 5.1% (95% CI 0.90–18.6) and 1.3% (95% CI 0.07–7.8) in An. gambiae s.s. and An. coluzzii, respectively. The mean infection prevalence with Microsporidia was 41.0% (95% CI 25.9–57.8) for An. gambiae s.s. and 57.0% (95% CI 45.4–67.9) for An. coluzzii. Wolbachia was only observed in Ifangni, Pobè, and Cotonou, while Microsporidia was detected in all study communes. Aggregated data for HLCs and PSCs showed a sporozoite rate (SR) of 0.80% (95% CI 0.09–2.87) and 0.69% (95% CI 0.09–2.87) for An. gambiae and An. coluzzii, respectively, with a mean of 0.74% (95% CI 0.20–1.90). Of the four individual mosquitoes which harboured P. falciparum, none were also infected with Wolbachia and one contained Microsporidia. Conclusions: The present study is the first report of natural infections of field-collected An. gambiae s.l. populations from Benin with Wolbachia and Microsporidia. Sustained efforts should be made to widen the spectrum of bacteria identified in mosquitoes, with the potential to develop endosymbiont-based control tools; such interventions could be the game-changer in the control of malaria and arboviral disease transmission. [ABSTRACT FROM AUTHOR]

Background: Malaria is a major public health problem in Angola, with Anopheles gambiae sensu lato (s.l.) and An. funestus s.l. being the primary vectors. This study aimed to clarify the information gaps concerning local Anopheles mosquito populations. Our objectives were to assess their abundance, geographical dispersion, and blood-feeding patterns. We also investigated their insecticide resistance. Molecular methods were used to identify sibling species, determine the origin of blood meals, measure Plasmodium falciparum infection rates, and detect the presence of knockdown resistance (kdr) mutations. Methods: Adult mosquitoes were collected indoors using CDC light traps from nine randomly selected households at two sentinel sites with distinct ecological characteristics. The samples were collected from 1 February to 30 June 2022. Anopheles mosquitoes were morphologically identified and subjected to molecular identification. Unfed Anopheles females were tested for the presence of P. falciparum DNA in head and thorax, and engorged females were screened for the source of the blood meals. Additionally, members of An. gambiae complex were genotyped for the presence of the L1014F and L1014S kdr mutations. Results: In total, 2226 adult mosquitoes were collected, including 733 Anopheles females. Molecular identification revealed the presence of Anopheles coluzzii, An. gambiae senso stricto (s.s.), An. arabiensis, and An. funestus s.s. Notably, there was the first record of An. coluzzii/An. gambiae s.s. hybrid and An. vaneedeni in Benguela Province. Plasmodium falciparum infection rates for An. coluzzii at the urban sentinel site and An. funestus s.s. at the rural site were 23.1% and 5.7%, respectively. The L1014F kdr mutation was discovered in both resistant and susceptible An. coluzzii mosquitoes, while the L1014S mutation was detected in An. gambiae s.s. for the first time in Benguela Province. No kdr mutations were found in An. arabiensis. Conclusions: This study provides valuable insights into the molecular characteristics of malaria vectors from the province of Benguela, emphasising the need for continuous surveillance of local Anopheles populations regarding the establishment of both kdr mutations for tailoring vector control interventions. [ABSTRACT FROM AUTHOR]

Background: Indoor residual spraying (IRS) was first implemented in the Atacora department, Benin from 2011 to 2012 using bendiocarb (carbamate) followed by annual spraying with pirimiphos-methyl (organophosphate) from 2013 to 2018. Before and after IRS implementation in Atacora, standard pyrethroid insecticide-treated bed nets were the main method of vector control in the area. This study investigated the knockdown resistance (kdr) gene (L1014F) and the acetylcholinesterase (ace-1) gene (G119S), before and during IRS implementation, and 4-years after IRS withdrawal from Atacora. This was done to assess how changes in insecticide pressure from indoor residual spraying may have altered the genotypic resistance profile of Anopheles gambiae s.l. Method: Identification of sibling species of An. gambiae s.l. and detection of the L1014F mutation in the kdr gene and G119S mutation in ace-1 genes was done using molecular analysis. Allelic and genotypic frequencies were calculated and compared with each other before and during IRS implementation and 4 years after IRS withdrawal. The Hardy–Weinberg equilibrium and genetic differentiation within and between populations were assessed. Results: Prevalence of the L1014F mutation in all geographic An. gambiae s.l. (An. gambiae s.s., Anopheles. coluzzii, Anopheles. arabiensis, and hybrids of "An. gambiae s.s. and An. coluzzii") populations increased from 69% before IRS to 87% and 90% during and after IRS. The G119S allele frequency during IRS (20%) was significantly higher than before IRS implementation (2%). Four years after IRS withdrawal, allele frequencies returned to similar levels as before IRS (3%). Four years after IRS withdrawal, the populations showed excess heterozygosity at the ace-1 gene and deficit heterozygosity at the kdr gene, whereas both genes had excess heterozygosity before and during IRS (FIS < 0). No genetic differentiation was observed within the populations. Conclusions: This study shows that the withdrawal of IRS with bendiocarb and pirimiphos-methyl may have slowed down the selection of individual mosquitoes with ace-1 resistance alleles in contrast to populations of An. gambiae s.l. with the L1014F resistance allele of the kdr gene. This may suggest that withdrawing the use of carbamates or organophosphates from IRS or rotating alternative insecticides with different modes of action may slow the development of ace-1 insecticide-resistance mutations. The increase in the prevalence of the L1014F mutation of the kdr gene in the population, despite the cessation of IRS, could be explained by the growing use of pyrethroids and DDT in agriculture and for other domestic use. More observational studies in countries where carbamates or organophosphates are still being used as public health insecticides may provide additional insights into these associations. [ABSTRACT FROM AUTHOR]

Background: There are several indications that pesticides used in agriculture contribute to the emergence and spread of resistance of mosquitoes to vector control insecticides. However, the impact of such an indirect selection pressure has rarely been quantified and the molecular mechanisms involved are still poorly characterized. In this context, experimental selection with different agrochemical mixtures was conducted in Anopheles gambiae. The multi-generational impact of agrochemicals on insecticide resistance was evaluated by phenotypic and molecular approaches. Methods: Mosquito larvae were selected for 30 generations with three different agrochemical mixtures containing (i) insecticides, (ii) non-insecticides compounds, and (iii) both insecticide and non-insecticide compounds. Every five generations, the resistance of adults to deltamethrin and bendiocarb was monitored using bioassays. The frequencies of the kdr (L995F) and ace1 (G119S) target-site mutations were monitored every 10 generations. RNAseq was performed on all lines at generation 30 in order to identify gene transcription level variations and polymorphisms associated with each selection regime. Results: Larval selection with agrochemical mixtures did not affect bendiocarb resistance and did not select for ace1 mutation. Contrastingly, an increased deltamethrin resistance was observed in the three selected lines. Such increased resistance was not majorly associated with the presence of kdr L995F mutation in selected lines. RNA-seq identified 63 candidate resistance genes over-transcribed in at least one selected line. These include genes coding for detoxification enzymes or cuticular proteins previously associated with insecticide resistance, and other genes potentially associated with chemical stress response. Combining an allele frequency filtering with a Bayesian FST-based genome scan allowed to identify genes under selection across multiple genomic loci, supporting a multigenic adaptive response to agrochemical mixtures. Conclusion: This study supports the role of agrochemical contaminants as a significant larval selection pressure favouring insecticide resistance in malaria vectors. Such selection pressures likely impact kdr mutations and detoxification enzymes, but also more generalist mechanisms such as cuticle resistance, which could potentially lead to cross-tolerance to unrelated insecticide compounds. Such indirect effect of global landscape pollution on mosquito resistance to public health insecticides deserves further attention since it can affect the nature and dynamics of resistance alleles circulating in malaria vectors and impact the efficacy of control vector strategies. [ABSTRACT FROM AUTHOR]

Background: Insecticide-treated nets (ITNs) contributed significantly to the decline in malaria since 2000. Their protective efficacy depends not only on access, use, and net integrity, but also location of people within the home environment and mosquito biting profiles. Anopheline mosquito biting and human location data were integrated to identify potential gaps in protection and better understand malaria transmission dynamics in Busia County, western Kenya. Methods: Direct observation of human activities and human landing catches (HLC) were performed hourly between 1700 to 0700 h. Household members were recorded as home or away; and, if at home, as indoors/outdoors, awake/asleep, and under a net or not. Aggregated data was analysed by weighting hourly anopheline biting activity with human location. Standard indicators of human-vector interaction were calculated using a Microsoft Excel template. Results: There was no significant difference between indoor and outdoor biting for Anopheles gambiae sensu lato (s.l.) (RR = 0.82; 95% CI 0.65–1.03); significantly fewer Anopheles funestus were captured outdoors than indoors (RR = 0.41; 95% CI 0.25–0.66). Biting peaked before dawn and extended into early morning hours when people began to awake and perform routine activities, between 0400–0700 h for An. gambiae and 0300–0700 h for An. funestus. The study population away from home peaked at 1700–1800 h (58%), gradually decreased and remained constant at 10% throughout the night, before rising again to 40% by 0600–0700 h. When accounting for resident location, nearly all bites within the peri-domestic space (defined as inside household structures and surrounding outdoor spaces) occurred indoors for unprotected people (98%). Using an ITN while sleeping was estimated to prevent 79% and 82% of bites for An. gambiae and An. funestus, respectively. For an ITN user, most remaining exposure to bites occurred indoors in the hours before bed and early morning. Conclusion: While use of an ITN was estimated to prevent most vector bites in this context, results suggest gaps in protection, particularly in the early hours of the morning when biting peaks and many people are awake and active. Assessment of additional human exposure points, including outside of the peri-domestic setting, are needed to guide supplementary interventions for transmission reduction. [ABSTRACT FROM AUTHOR]

The often tight association between parasites and their hosts means that under certain scenarios, the evolutionary histories of the two species can become closely coupled both through time and across space. Using spatial genetic inference, we identify a potential signal of common dispersal patterns in the Anopheles gambiae and Plasmodium falciparum host–parasite system as seen through a between-species correlation of the differences between geographic sampling location and geographic location predicted from the genome. This correlation may be due to coupled dispersal dynamics between host and parasite but may also reflect statistical artifacts due to uneven spatial distribution of sampling locations. Using continuous-space population genetics simulations, we investigate the degree to which uneven distribution of sampling locations leads to bias in prediction of spatial location from genetic data and implement methods to counter this effect. We demonstrate that while algorithmic bias presents a problem in inference from spatio-genetic data, the correlation structure between A. gambiae and P. falciparum predictions cannot be attributed to spatial bias alone and is thus likely a genetic signal of co-dispersal in a host–parasite system. [ABSTRACT FROM AUTHOR]

Background: Mosquitoes belonging to the Anopheles gambiae sensu lato complex play a major role in malaria transmission across Africa. This study assessed the relative importance of members of An. gambiae s.l. in malaria transmission in two rural villages in the Republic of the Congo. Methods: Adult mosquitoes were collected using electric aspirators from June to September 2022 in Djoumouna and Ntoula villages and were sorted by taxa based on their morphological features. Anopheles gambiae s.l. females were also molecularly identified. A TaqMan-based assay and a nested polymerase chain reaction (PCR) were performed to determine Plasmodium spp. in the mosquitoes. Entomological indexes were estimated, including man-biting rate, entomological inoculation rate (EIR), and diversity index. Results: Among 176 mosquitoes collected, An. gambiae s.l. was predominant (85.8%), followed by Culex spp. (13.6%) and Aedes spp. (0.6%). Three members of the An. gambiae s.l. complex were collected in both villages, namely An. gambiae sensu stricto (74.3%), Anopheles coluzzii (22.9%) and Anopheles arabiensis (2.8%). Three Plasmodium species were detected in An. gambiae s.s. and An. coluzzii (Plasmodium falciparum, P. malariae and P. ovale), while only P. falciparum and P. malariae were found in An. arabiensis. In general, the Plasmodium infection rate was 35.1% (53/151) using the TaqMan-based assay, and nested PCR confirmed 77.4% (41/53) of those infections. The nightly EIR of An. gambiae s.l. was 0.125 infectious bites per person per night (ib/p/n) in Djoumouna and 0.08 ib/p/n in Ntoula. The EIR of An. gambiae s.s. in Djoumouna (0.11 ib/p/n) and Ntoula (0.04 ib/p/n) was higher than that of An. coluzzii (0.01 and 0.03 ib/p/n) and An. arabiensis (0.005 and 0.0 ib/p/n). Conclusions: This study provides baseline information on the dominant vectors and dynamics of malaria transmission in the rural areas of the Republic of the Congo during the dry season. In the two sampled villages, An. gambiae s.s. appears to play a predominant role in Plasmodium spp. transmission. [ABSTRACT FROM AUTHOR]

Background: For decades, various agrochemicals have been successfully repurposed for mosquito control. However, preexisting resistance caused in larval and adult populations by unintentional pesticide exposure or other cross-resistance mechanisms poses a challenge to the efficacy of this strategy. A better understanding of larval adaptation to the lethal and sublethal effects of residual pesticides in aquatic habitats would provide vital information for assessing the efficacy of repurposed agrochemicals against mosquitoes. Methods: We reared field-collected mosquito larvae in water containing a concentration of agrochemical causing 100% mortality in susceptible mosquitoes after 24 h (lethal concentration). Using this experimental setup, we tested the effect of lethal concentrations of a pyrrole (chlorfenapyr, 0.10 mg/l), a pyrethroid (deltamethrin, 1.5 mg/l), and three neonicotinoids including imidacloprid (0.075 mg/l), acetamiprid (0.15 mg/l), and clothianidin (0.035 mg/l) on mortality rates, growth, and survival in third-instar larvae of the two sibling species Anopheles gambiae and Anopheles coluzzii collected from Yaoundé, Cameroon. Results: We found that An. gambiae and An. coluzzii larvae were susceptible to chlorfenapyr and were killed within 24 h by a nominal concentration of 0.10 mg/l. Consistent with strong resistance, deltamethrin induced low mortality in both species. Lethal concentrations of acetamiprid, imidacloprid, and clothianidin strongly inhibited survival, growth, and emergence in An. coluzzii larvae. By contrast, depending on the active ingredient and the population tested, 5–60% of immature stages of An. gambiae were able to grow and emerge in water containing a lethal concentration of neonicotinoids, suggesting cross-resistance to this class of insecticides. Conclusions: These findings corroborate susceptibility profiles observed in adults and suggest that unintentional pesticide exposure or other cross-resistance processes could contribute to the development of resistance to neonicotinoids in some Anopheles populations. [ABSTRACT FROM AUTHOR]

Background: Neonicotinoids are potential alternatives for controlling pyrethroid-resistant mosquitoes, but their efficacy against malaria vector populations of sub-Saharan Africa has yet to be investigated. The aim of the present study was to test the efficacy of four neonicotinoids against adult populations of the sibling species Anopheles gambiae and Anopheles coluzzii sampled along an urban-to-rural gradient. Methods: The lethal toxicity of three active ingredients for adults of two susceptible Anopheles strains was assessed using concentration–response assays, and their discriminating concentrations were calculated. The discriminating concentrations were then used to test the susceptibility of An. gambiae and An. coluzzii mosquitoes collected from urban, suburban and rural areas of Yaoundé, Cameroon, to acetamiprid, imidacloprid, clothianidin and thiamethoxam. Results: Lethal concentrations of neonicotinoids were relatively high suggesting that this class of insecticides has low toxicity against Anopheles mosquitoes. Reduced susceptibility to the four neonicotinoids tested was detected in An. gambiae populations collected from rural and suburban areas. By contrast, adults of An. coluzzii that occurred in urbanized settings were susceptible to neonicotinoids except acetamiprid for which 80% mortality was obtained within 72 h of insecticide exposure. The cytochrome inhibitor, piperonyl butoxide (PBO), significantly enhanced the activity of clothianidin and acetamiprid against An. gambiae mosquitoes. Conclusions: These findings corroborate susceptibility profiles observed in larvae and highlight a significant variation in tolerance to neonicotinoids between An. gambiae and An. coluzzii populations from Yaoundé. Further studies are needed to disentangle the role of exposure to agricultural pesticides and of cross-resistance mechanisms in the development of neonicotinoid resistance in some Anopheles species. [ABSTRACT FROM AUTHOR]

Anopheles gambiae and Anopheles coluzzii mosquitoes, two major malaria vectors in sub‐Saharan Africa, exhibit selectivity among plant species as potential food sources. However, it remains unclear if their preference aligns with optimal nutrient intake and survival. Following an extensive screening of the effects of 31 plant species on An. coluzzii in Burkina Faso, we selected three species for their contrasting effects on mosquito survival, namely Ixora coccinea, Caesalpinia pulcherrima, and Combretum indicum. We assessed the sugar content of these plants and their impact on mosquito fructose positivity, survival, and insemination rate, using Anopheles coluzzii and Anopheles gambiae, with glucose 5% and water as controls. Plants displayed varying sugar content and differentially affected the survival, sugar intake, and insemination rate of mosquitoes. All three plants were more attractive to mosquitoes than controls, with An. gambiae being more responsive than An. coluzzii. Notably, C. indicum was the most attractive but had the lowest sugar content and offered the lowest survival, insemination rate, and fructose positivity. Our findings unveil a performance–preference mismatch in An. coluzzii and An. gambiae regarding plant food sources. Several possible reasons for this negative correlation between performance and preference are discussed. [ABSTRACT FROM AUTHOR]

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