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Abstract Background Attractive targeted sugar baits (ATSBs) have the potential to significantly reduce infective female Anopheles mosquitoes in arid areas, such as in Northern Mali. Malaria is epidemic in the north due to the limited viability of Anopheles species in the desert climate. The goal of this study was to determine of the effect of ATSB on the number of older female An. gambiae and on the number of sporozoite-positive females in villages in northern Mali. Methods Villages were located in the north of Mali. In this study, 5677 ATSB stations were deployed, two on each home, in ten villages during late July and early August 2019. Ten villages served as controls. After a pre-treatment monitoring period in July, An. gambiae populations were monitored again from August to December using CDC-UV light traps, pyrethrum spray catches (PSC), and human landing catches (HLC). Mosquitoes were dissected to estimate their age, while ELISA detected sporozoite positivity. The monthly entomological inoculation rates (EIRs) were calculated for HLC indoors and outdoors. Data from villages were compared using t-tests, while bait station weighted density versus amount of collected females was checked with a Pearson’s correlation. Results A total of 2703 female An. gambiae were caught from treated villages, 4582 from control villages, a 41.0% difference. Dissection of 1759 females showed that ATSB significantly reduced the number of older females. The proportion of older females in treated villages was 0.93% compared to 9.4% in control villages. ELISA analysis of 7285 females showed that bait stations reduced the number of sporozoite-positive females. The infective females in treated villages was 0.30% compared to 2.73% in the controls. The greater the density of bait stations deployed, the fewer the older, infective females (P

Abstract Background Knockdown resistance (kdr) is one of the primary resistance mechanisms present in anopheline species. Although this mutation is largely spread across the Anopheles gambiae s.l. members, its prevalence in other species is still not well documented. Methods The present study investigated the distribution and allelic frequencies of kdr in An. gambiae s.l., An. pharoensis, and An. ziemanni samples collected in 2022 and 2023 in nine sites spread across five ecogeographical settings in Cameroon. Members of the An. gambiae complex were identified molecularly by polymerase chain reaction (PCR). kdr L1014F and L1014S alleles were screened by PCR and confirmed by sequencing. Results An. gambiae (49.9%), An. coluzzii (36.5%), and An. arabiensis (13%) were identified, and the frequency of the kdr L1014F was high in both An. gambiae and An. coluzzii in all sites. The kdr L1014F allele was detected for the first time in 8 out of 14 An. ziemanni samples examined and in 5 out of 22 An. pharoensis samples examined. The kdr L1014S allele was scarce and found only in the heterozygote “RS” state in An. arabiensis and An. gambiae in Yangah and Santchou. Conclusions The present study sheds light on the rapid expansion of the kdr L1014F allele in malaria vectors in Cameroon and stresses the need for surveillance activities also targeting secondary malaria vectors to improve the control of malaria transmission. Graphical Abstract

Abstract Background The widespread use of insecticide-treated nets (ITNs) has significantly contributed to the reduction in malaria cases and deaths observed across Africa. Unfortunately, this control strategy is threatened by the rapid spread of pyrethroid resistance in malaria vectors. Dual-active-ingredient insecticidal nets are now available to mitigate the impact of pyrethroid resistance. To facilitate evidence-based decisions regarding product selection in specific use settings, data are needed on the efficacy of these different nets against local mosquito populations. Methods Two experimental hut trials were performed in Za-Kpota, southern Benin in 2021 to evaluate the performance of Interceptor G2 (BASF), Royal Guard (Disease Control Technologies) and PermaNet 3.0 (Vestergaard Frandsen), all dual-active-ingredient bednets, in comparison to untreated or standard pyrethroid-treated bednets, against free-flying wild Anopheles gambiae mosquitoes. The performance of some of these next-generation nets was compared to the same type of nets that have been in use for up to 2 years. Mosquitoes collected in the huts were followed up after exposure to assess the sublethal effects of treatments on certain life-history traits. Results The predominant species in the study site was Anopheles gambiae sensu stricto (An. gambiae s.s.). Both Anopheles coluzzii and An. gambiae s.s. were resistant to pyrethroids (deltamethrin susceptibility was restored by piperonyl butoxide pre-exposure). In the experimental hut trials, the highest blood-feeding inhibition (5.56%) was recorded for the Royal Guard net, relative to the standard PermaNet 2.0 net (44.44% inhibition). The highest 72-h mortality rate (90.11%) was recorded for the Interceptor G2 net compared to the PermaNet 2.0 net (56.04%). After exposure, the risk of death of An. gambiae sensu lato (An. gambiae s.l.) was 6.5-fold higher with the Interceptor G2 net and 4.4-fold higher with the PermaNet 3.0 net compared to the respective untreated net. Lower mosquito mortality was recorded with an aged Interceptor G2 net compared to a new Interceptor G2 net. Oviposition rates were lower in mosquitoes collected from huts containing ITNs compared to those of untreated controls. None of the mosquitoes collected from huts equipped with Royal Guard nets laid any eggs. Conclusions The Royal Guard and Interceptor G2 nets showed a potential to significantly improve the control of malaria-transmitting vectors. However, the PermaNet 3.0 net remains effective in pyrethroid-resistant areas. Graphical Abstract

Abstract Indoor residual spraying (IRS) and insecticide-treated nets (ITNs) are the main methods used to control mosquito populations for malaria prevention. The efficacy of these strategies is threatened by the spread of insecticide resistance (IR), limiting the success of malaria control. Studies of the genetic evolution leading to insecticide resistance could enable the identification of molecular markers that can be used for IR surveillance and an improved understanding of the molecular mechanisms associated with IR. This study used a weighted gene co-expression network analysis (WGCNA) algorithm, a systems biology approach, to identify genes with similar co-expression patterns (modules) and hub genes that are potential molecular markers for insecticide resistance surveillance in Kenya and Benin. A total of 20 and 26 gene co-expression modules were identified via average linkage hierarchical clustering from Anopheles arabiensis and An. gambiae, respectively, and hub genes (highly connected genes) were identified within each module. Three specific genes stood out: serine protease, E3 ubiquitin-protein ligase, and cuticular proteins, which were top hub genes in both species and could serve as potential markers and targets for monitoring IR in these malaria vectors. In addition to the identified markers, we explored molecular mechanisms using enrichment maps that revealed a complex process involving multiple steps, from odorant binding and neuronal signaling to cellular responses, immune modulation, cellular metabolism, and gene regulation. Incorporation of these dynamics into the development of new insecticides and the tracking of insecticide resistance could improve the sustainable and cost-effective deployment of interventions.

Abstract Background The current rise of new innovative tools for mosquito control, such as the release of transgenic mosquitoes carrying a dominant lethal gene and Wolbachia-based strategies, necessitates a massive production of mosquitoes in the insectary. However, currently laboratory rearing depends on vertebrate blood for egg production and maintenance. This practice raises ethical concerns, incurs logistical and cost limitations, and entails potential risk associated with pathogen transmission and blood storage. Consequently, an artificial blood-free diet emerges as a desirable alternative to address these challenges. This study aims to evaluate the effects of a previously formulated artificial blood-free diet (herein referred to as BLOODless) on Anopheles gambiae (An. gambiae s.s.; IFAKARA) gonotrophic parameters and fitness compared with bovine blood. Methods The study was a laboratory-based comparative evaluation of the fitness, fecundity and fertility of An. gambiae s.s. (IFAKARA) reared on BLOODless versus vertebrate blood from founder generation (F0) to eighth generation (F8). A total of 1000 female mosquitoes were randomly selected from F0, of which 500 mosquitoes were fed with bovine blood (control group) and the other 500 mosquitoes were fed with BLOODless diet (experimental group). The feeding success, number of eggs per female, hatching rate and pupation rate were examined post-feeding. Longevity and wing length were determined as fitness parameters for adult male and female mosquitoes for both populations. Results While blood-fed and BLOODless-fed mosquitoes showed similar feeding success, 92.3% [95% confidence interval (CI) 89.7–94.9] versus 93.6% (95% CI 90.6–96.6), respectively, significant differences emerged in their reproductive parameters. The mean number of eggs laid per female was significantly higher for blood-fed mosquitoes (P

Abstract Background The recent reduction in malaria burden in Côte d’Ivoire is largely attributable to the use of long-lasting insecticidal nets (LLINs). However, this progress is threatened by insecticide resistance and behavioral changes in Anopheles gambiae sensu lato (s.l.) populations and residual malaria transmission, and complementary tools are required. Thus, this study aimed to assess the efficacy of the combined use of LLINs and Bacillus thuringiensis israelensis (Bti), in comparison with LLINs. Methods This study was conducted in the health district of Korhogo, northern Côte d'Ivoire, within two study arms (LLIN + Bti arm and LLIN-only arm) from March 2019 to February 2020. In the LLIN + Bti arm, Anopheles larval habitats were treated every fortnight with Bti in addition to the use of LLINs. Mosquito larvae and adults were sampled and identified morphologically to genus and species using standard methods. The members of the An. gambiae complex were determined using a polymerase chain reaction technique. Plasmodium infection in An. gambiae s.l. and malaria incidence in local people was also assessed. Results Overall, Anopheles spp. larval density was lower in the LLIN + Bti arm 0.61 [95% CI 0.41–0.81] larva/dip (l/dip) compared with the LLIN-only arm 3.97 [95% CI 3.56–4.38] l/dip (RR = 6.50; 95% CI 5.81–7.29; P

Abstract 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

Abstract 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 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 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 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 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.

A bstract 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. Graphical Abstract

Abstract 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. Graphical Abstract

Abstract 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 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 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. Graphical abstract

Abstract 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 (F IS

Abstract Background The male mosquito microbiome may be important for identifying ideal candidates for disease control. Among other criteria, mosquito-associated symbionts that have high localization in both male and female mosquitoes and are transmissible through both vertical and sexual routes are desirable. However, mosquito microbiome studies have mainly been female-focused. In this study, the microbiota of male and female Anopheles gambiae sensu lato (s.l.) were compared to identify shared or unique bacteria. Methods Late larval instars of Anopheles mosquitoes were collected from the field and raised to adults. Equal numbers of males and females of 1-day-old non-sugar-fed, 4–5-day-old sugar-fed and post-blood-fed females were randomly selected for whole-body analyses of bacteria 16S rRNA. Results Results revealed that male and female mosquitoes generally share similar microbiota except when females were blood-fed. Compared to newly emerged unfed mosquitoes, feeding on sugar and/or blood increased variability in microbial composition (⍺-diversity), with a higher disparity among females (39% P = 0.01) than in males (29% P = 0.03). Elizabethkingia meningoseptica and Asaia siamensis were common discriminants between feeding statuses in both males and females. While E. meningoseptica was particularly associated with sugar-fed mosquitoes of both sexes and sustained after blood feeding in females, A. siamensis was also increased in sugar-fed mosquitoes but decreased significantly in blood-fed females (LDA score > 4.0, P

Abstract Background Hemocytes are immune cells that patrol the mosquito hemocoel and mediate critical cellular defense responses against pathogens. However, despite their importance, a comprehensive transcriptome of these cells was lacking because they constitute a very small fraction of the total cells in the insect, limiting the study of hemocyte differentiation and immune function. Results In this study, an in-depth hemocyte transcriptome was built by extensive bulk RNA sequencing and assembly of hemocyte RNAs from adult A. gambiae female mosquitoes, based on approximately 2.4 billion short Illumina and about 9.4 million long PacBio high-quality reads that mapped to the A. gambiae PEST genome (P4.14 version). A total of 34,939 transcripts were annotated including 4,020 transcripts from novel genes and 20,008 novel isoforms that result from extensive differential splicing of transcripts from previously annotated genes. Most hemocyte transcripts identified (89.8%) are protein-coding while 10.2% are non-coding RNAs. The number of transcripts identified in the novel hemocyte transcriptome is twice the number in the current annotation of the A. gambiae genome (P4.14 version). Furthermore, we were able to refine the analysis of a previously published single-cell transcriptome (scRNAseq) data set by using the novel hemocyte transcriptome as a reference to re-define the hemocyte clusters and determine the path of hemocyte differentiation. Unsupervised pseudo-temporal ordering using the Tools for Single Cell Analysis software uncovered a novel putative prohemocyte precursor cell type that gives rise to prohemocytes. Pseudo-temporal ordering with the Monocle 3 software, which analyses changes in gene expression during dynamic biological processes, determined that oenocytoids derive from prohemocytes, a cell population that also gives rise to the granulocyte lineage. Conclusion A high number of mRNA splice variants are expressed in hemocytes, and they may account for the plasticity required to mount efficient responses to many different pathogens. This study highlights the importance of a comprehensive set of reference transcripts to perform robust single-cell transcriptomic data analysis of cells present in low abundance. The detailed annotation of the hemocyte transcriptome will uncover new facets of hemocyte development and function in adult dipterans and is a valuable community resource for future studies on mosquito cellular immunity.

Abstract Background Pyrethroids are the main insecticides used in vector control for malaria. However, their extensive use in the impregnation of long-lasting insecticidal nets (LLINs) and indoor residual spraying has led to the development of resistance, threatening its success as a tool for malaria control. Baseline data prior to large scale distribution of LLINs are important for the implementation of efficient strategies. However, no data on the susceptibility of malaria vectors is available in the Moyen-Ogooué Province in Gabon. The aim of this study was to assess the susceptibility to pyrethroids and organochlorides of malaria vectors from a semi-urban and rural areas of the province and to determine the frequency of insecticide resistance genes. Methods Larvae were collected from breeding sites in Lambaréné and Zilé and reared to adults. Three to five-day old female Anopheles gambiae sensu lato mosquitoes were used in cone tube assays following the WHO susceptibility tests protocol for adult mosquitoes. A subsample was molecularly identified using the SINE200 protocol and the frequency of Vgsc-1014 F and − 1014 S mutations were determined. Results Anopheles gambiae sensu stricto (s.s.) was the sole species present in both Lambaréné and Zilé. Mosquito populations from the two areas were resistant to pyrethroids and organochlorides. Resistance was more pronounced for permethrin and DDT with mortality lower than 7% for both insecticides in the two study areas. Mosquitoes were statistically more resistant (P

Abstract Background Over a decade of vector control by indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) distribution on the mainland, and only LLINs on islands had a minimal impact on disease burden in Nchelenge district, northern Zambia. Anopheles funestus and Anopheles gambiae are vectors known only from the mainland. Understanding vector bionomics in the district is necessary for planning and targeting effective vector control. This study aimed to provide information on abundance, seasonality, and Plasmodium falciparum sporozoite infectivity of malaria vectors in Nchelenge, including islands. Methods Mosquitoes were collected in 192 CDC indoor light traps set in 56 households between January 2015 and January 2016. Morphological and molecular species identifications and P. falciparum circumsporoites by ELISA were performed. Mosquito counts and relative abundances from the islands and mainland were compared, and household factors associated with vector counts were determined. Results A total of 5888 anophelines were collected during the study. Of these, 5,704 were female Anopheles funestus sensu lato (s.l.) and 248 female An. gambiae s.l. The highest proportion of An. funestus (n = 4090) was from Chisenga Island and An. gambiae (n = 174) was from Kilwa Island. The highest estimated counts per trap for An. funestus s.l. were from Chisenga island, (89.9, p

Abstract Background Ivermectin (IVM) mass drug administration is a candidate complementary malaria vector control tool. Ingestion of blood from IVM treated hosts results in reduced survival in mosquitoes. Estimating bio-efficacy of IVM on wild-caught mosquitoes requires they ingest the drug in a blood meal either through a membrane or direct feeding on a treated host. The latter, has ethical implications, and the former results in low feeding rates. Therefore, there is a need to develop a safe and effective method for IVM bio-efficacy monitoring in wild mosquitoes. Methods Insectary-reared Anopheles gambiae s.s. were exposed to four IVM doses: 85, 64, 43, 21 ng/ml, and control group (0 ng/ml) in three different solutions: (i) blood, (ii) 10% glucose, (iii) four ratios (1:1, 1:2, 1:4, 1:8) of blood in 10% glucose, and fed through filter paper. Wild-caught An. gambiae s.l. were exposed to 85, 43 and 21 ng/ml IVM in blood and 1:4 ratio of blood-10% glucose mixture. Survival was monitored for 28 days and a pool of mosquitoes from each cohort sacrificed immediately after feeding and weighed to determine mean weight of each meal type. Results When administered in glucose solution, mosquitocidal effect of IVM was not comparable to the observed effects when similar concentrations were administered in blood. Equal concentrations of IVM administered in blood resulted in pronounced reductions in mosquito survival compared to glucose solution only. However, by adding small amounts of blood to glucose solution, mosquito mortality rates increased resulting in similar effects to what was observed during blood feeding. Conclusion Bio-efficacy of ivermectin is strongly dependent on mode of drug delivery to the mosquito and likely influenced by digestive processes. The assay developed in this study is a good candidate for field-based bio-efficacy monitoring: wild mosquitoes readily feed on the solution, the assay can be standardized using pre-selected concentrations and by not involving treated blood hosts (human or animal) variation in individual pharmacokinetic profiles as well as ethical issues are bypassed. Meal volumes did not explain the difference in the lethality of IVM across the different meal types necessitating further research on the underlying mechanisms.

Abstract Background The objective of this study was to determine the susceptibility of wild Anopheles gambiae sensu lato (s.l.) from southern Benin to the new insecticides (chlorfenapyr (CFP), pyriproxyfen (PPF), and clothianidin (CTD)) and assess the efficacy of insecticide-treated bed nets (ITNs) that contain these new products. Methods Wild An. gambiae from the Benin communes of Allada, Ifangni, Akpro-Missérété, and Porto-Novo were tested for their susceptibility to CFP and PPF using the WHO bottle tests, and pyrethroids (alpha-cypermethrin, deltamethrin, and permethrin) and CTD using WHO tube tests. WHO cone tests were used to evaluate the efficacy of Interceptor® (which contains alpha-cypermethrin (ACM) only), Interceptor® G2, (CFP + ACM), and Royal Guard® nets (PPF + ACM). The ovaries of blood-fed An. gambiae from Ifangni exposed to a new PPF net were dissected, and egg development status was examined using Christopher’s stages to determine the fertility status of the mosquitoes. Using a standardized protocol, the oviposition rate and oviposition inhibition rate were calculated from live blood-fed An. gambiae placed in oviposition chambers after exposure to PPF. Results In all four mosquito populations, pyrethroid mortality ranged from 5 to 80%, while chlorfenapyr and clothianidin mortality ranged from 98 to 100%. At Ifangni, all mosquitoes exposed to Royal Guard® nets were infertile (100%) while the majority (74.9%) of mosquitoes exposed to Interceptor® nets had fully developed their eggs to Christopher’s stage V. The oviposition inhibition rate after exposure of the mosquitoes to the PPF was 99% for the wild population of An. gambiae s.l. and the susceptible laboratory strain, An. gambiae sensu stricto (Kisumu). Conclusions The results of this study suggest that pyrethroid-resistant An. gambiae from the selected communes in southern Benin are susceptible to chlorfenapyr, clothianidin, and pyriproxyfen. In addition, based on bioassay results, new and unused Interceptor® G2 and Royal Guard® nets were effective on Ifangni’s mosquito populations. Despite the availability of new effective insecticides, continued vigilance is needed in Benin. Therefore, monitoring of resistance to these insecticides will continue to periodically update the Benin national insecticide resistance database and management plan.

Abstract Background Pyrethroid-chlorfenapyr (CFP) and pyrethroid-piperonyl butoxide (PBO) nets are being scaled across endemic countries to improve control of malaria transmitted by pyrethroid-resistant mosquitoes. CFP is a pro-insecticide requiring activation by mosquito cytochrome P450 monooxygenase enzymes (P450s) while PBO improves pyrethroid potency by inhibiting the action of these enzymes in pyrethroid-resistant mosquitoes. The inhibitory action of PBO against P450s may thus reduce the efficacy of pyrethroid-CFP nets when applied inside the same household as pyrethroid-PBO nets. Methods Two experimental hut trials were performed to evaluate the entomological impact of two different types of pyrethroid-CFP ITN (Interceptor® G2, PermaNet® Dual) when applied alone and in combination with pyrethroid-PBO ITNs (DuraNet® Plus, PermaNet® 3.0) against a pyrethroid-resistant vector population in southern Benin. In both trials, all net types were tested as single and double net treatments. Bioassays were also performed to assess the resistance profile of the vector population at the hut site and investigate interactions between CFP and PBO. Results The vector population was susceptible to CFP but exhibited a high intensity of pyrethroid resistance that was overcame by PBO pre-exposure. Vector mortality was significantly lower in huts with combinations of pyrethroid-CFP nets plus pyrethroid-PBO nets compared to huts with two pyrethroid-CFP nets (74% vs. 85% for Interceptor® G2 and 57% vs. 83% for PermaNet® Dual, p

Abstract Background Vector bionomics are important aspects of vector-borne disease control programs. Mosquito-biting risks are affected by environmental, mosquito behavior and human factors, which are important for assessing exposure risk and intervention impacts. This study estimated malaria transmission risk based on vector–human interactions in northern Ghana, where indoor residual spraying (IRS) and insecticide-treated nets (ITNs) have been deployed. Methods Indoor and outdoor human biting rates (HBRs) were measured using monthly human landing catches (HLCs) from June 2017 to April 2019. Mosquitoes collected were identified to species level, and Anopheles gambiae sensu lato (An. gambiae s.l.) samples were examined for parity and infectivity. The HBRs were adjusted using mosquito parity and human behavioral observations. Results Anopheles gambiae was the main vector species in the IRS (81%) and control (83%) communities. Indoor and outdoor HBRs were similar in both the IRS intervention (10.6 vs. 11.3 bites per person per night [b/p/n]; z = −0.33, P = 0.745) and control communities (18.8 vs. 16.4 b/p/n; z = 1.57, P = 0.115). The mean proportion of parous An. gambiae s.l. was lower in IRS communities (44.6%) than in control communities (71.7%). After adjusting for human behavior observations and parity, the combined effect of IRS and ITN utilization (IRS: 37.8%; control: 57.3%) on reducing malaria transmission risk was 58% in IRS + ITN communities and 27% in control communities with ITNs alone (z = −4.07, P

Abstract Background Insecticide resistance in malaria vectors can be spatially highly heterogeneous, yet population structure analyses frequently find relatively high levels of gene flow among mosquito populations. Few studies have contemporaneously assessed phenotypic, genotypic and population structure analysis on mosquito populations and none at fine geographical scales. In this study, genetic diversity, population structure, and insecticide resistance profiles of Anopheles funestus and Anopheles arabiensis were examined across mosquito populations from and within neighbouring villages. Methods Mosquitoes were collected from 11 towns in southern Mozambique, as well as from different neighbourhoods within the town of Palmeira, during the peak malaria transmission season in 2016. CDC bottle bioassay and PCR assays were performed with Anopheles mosquitoes at each site to determine phenotypic and molecular insecticide resistance profiles, respectively. Microsatellite analysis was conducted on a subsample of mosquitoes to estimate genetic diversity and population structure. Results Phenotypic insecticide resistance to deltamethrin was observed in An. funestus sensu stricto (s.s.) throughout the area, though a high level of mortality variation was seen. However, 98% of An. funestus s.s. were CYP6P9a homozygous resistant. An. arabiensis was phenotypically susceptible to deltamethrin and 99% were kdr homozygous susceptible. Both Anopheles species exhibited high allelic richness and heterozygosity. Significant deviations from Hardy–Weinberg equilibrium were observed, and high linkage disequilibrium was seen for An. funestus s.s., supporting population subdivision. However, the FST values were low for both anophelines (− 0.00457 to 0.04213), Nm values were high (9.4–71.8 migrants per generation), AMOVA results showed almost 100% genetic variation among and within individuals, and Structure analysis showed no clustering of An. funestus s.s. and An. arabiensis populations. These results suggest high gene flow among mosquito populations. Conclusion Despite a relatively high level of phenotypic variation in the An. funestus population, molecular analysis shows the population is admixed. These data indicate that CYP6P9a resistance markers do not capture all phenotypic variation in the area, but also that resistance genes of high impact are likely to easily spread in the area. Conversely, other strategies, such as transgenic mosquito release programmes will likely not face challenges in this locality.

Abstract Background Knowing the species composition and insecticide resistance status of the target vector population is important to guide malaria vector control. The aim of this study was to characterize the malaria vector population in terms of species composition, insecticide susceptibility status and potential underlying resistance mechanisms in Ellibou, southern Côte d’Ivoire. Methods A 1-year longitudinal entomological survey was conducted using light traps and pyrethroid spray catches to sample adult mosquitoes in combination with larval sampling. The susceptibility status of Anopheles gambiae sensu lato (s.l.) to bendiocarb, deltamethrin, DDT and malathion was assessed using the World Health Organization insecticide susceptibility test. Additionally, An. gambiae specimens were screened for knockdown (kdr) and acetylcholineesterase (ace1) target site resistance alleles, and the expression levels of eight metabolic resistance genes, including seven cytochrome P450 monooxygenases (P450s) and one glutathione S-transferase (GST), measured with reverse transcription quantitative real-time polymerase chain reaction (qPCR). Results Overall, 2383 adult mosquitoes from 12 different taxa were collected with Culex quinquefasciatus and An. gambiae being the predominant taxa. Molecular identification of An. gambiae s.l. revealed the presence of Anopheles arabiensis, Anopheles coluzzii, An. gambiae sensu stricto (s.s.) and Anopheles coluzzii/An. gambiae s.s. hybrids. Anopheles gambiae mosquitoes were resistant to all insecticides except malathion. PCR diagnostics revealed the presence of ace1-G280S and the kdr L995F, L995S and N1570Y target-site mutations. Additionally, several genes were upregulated, including five P450s (i.e., CYP6P3, CYP6M2, CYP9K1, CYP6Z1, CYP6P1) and GSTE2. Conclusion This is the first documented presence of An. arabiensis in Côte d’Ivoire. Its detection – together with a recent finding further north of the country – confirms its existence in the country, which is an early warning sign, as An. arabiensis shows a different biology than the currently documented malaria vectors. Because the local An. gambiae population was still susceptible to malathion, upregulation of P450s, conferring insecticide resistance to pyrethroids, together with the presence of ace1, suggest negative cross-resistance. Therefore, organophosphates could be an alternative insecticide class for indoor residual spraying in the Ellibou area, while additional tools against the outdoor biting An. arabiensis will have to be considered.

Abstract Background Due to the rapid expansion of pyrethroid-resistance in malaria vectors in Africa, Global Plan for Insecticide Resistance Management (GPIRM) has recommended the development of long-lasting insecticidal nets (LLINs), containing insecticide mixtures of active ingredients with different modes of action to mitigate resistance and improve LLIN efficacy. This good laboratory practice (GLP) study evaluated the efficacy of the chlorfenapyr and deltamethrin-coated PermaNet® Dual, in comparison with the deltamethrin and synergist piperonyl butoxide (PBO)-treated PermaNet® 3.0 and the deltamethrin-coated PermaNet® 2.0, against wild free-flying pyrethroid-resistant Anopheles gambiae sensu lato (s.l.), in experimental huts in Tiassalé, Côte d’Ivoire (West Africa). Methods PermaNet® Dual, PermaNet® 3.0 and PermaNet® 2.0, unwashed and washed (20 washes), were tested against free-flying pyrethroid-resistant An. gambiae s.l. in the experimental huts in Tiassalé, Côte d’Ivoire from March to August 2020. Complementary laboratory cone bioassays (daytime and 3-min exposure) and tunnel tests (nightly and 15-h exposure) were performed against pyrethroid-susceptible An. gambiae sensu stricto (s.s.) (Kisumu strain) and pyrethroid-resistant An. gambiae s.l. (Tiassalé strain). Results PermaNet® Dual demonstrated significantly improved efficacy, compared to PermaNet® 3.0 and PermaNet® 2.0, against the pyrethroid-resistant An. gambiae s.l. Indeed, the experimental hut trial data showed that the mortality and blood-feeding inhibition in the wild pyrethroid-resistant An. gambiae s.l. were overall significantly higher with PermaNet® Dual compared with PermaNet® 3.0 and PermaNet® 2.0, for both unwashed and washed samples. The mortality with unwashed and washed samples were 93.6 ± 0.2% and 83.2 ± 0.9% for PermaNet® Dual, 37.5 ± 2.9% and 14.4 ± 3.9% for PermaNet® 3.0, and 7.4 ± 5.1% and 11.7 ± 3.4% for PermaNet® 2.0, respectively. Moreover, unwashed and washed samples produced the respective percentage blood-feeding inhibition of 41.4 ± 6.9% and 43.7 ± 4.8% with PermaNet® Dual, 51.0 ± 5.7% and 9.8 ± 3.6% with PermaNet® 3.0, and 12.8 ± 4.3% and − 13.0 ± 3.6% with PermaNet® 2.0. Overall, PermaNet® Dual also induced higher or similar deterrence, exophily and personal protection when compared with the standard PermaNet® 3.0 and PermaNet® 2.0 reference nets, with both unwashed and washed net samples. In contrast to cone bioassays, tunnel tests predicted the efficacy of PermaNet® Dual seen in the current experimental hut trial. Conclusion The deltamethrin-chlorfenapyr-coated PermaNet® Dual induced a high efficacy and performed better than the deltamethrin-PBO PermaNet® 3.0 and the deltamethrin-only PermaNet® 2.0, testing both unwashed and 20 times washed samples against the pyrethroid-susceptible and resistant strains of An. gambiae s.l. The inclusion of chlorfenapyr with deltamethrin in PermaNet® Dual net greatly improved protection and control of pyrethroid-resistant An. gambiae populations. PermaNet® Dual thus represents a promising tool, with a high potential to reduce malaria transmission and provide community protection in areas compromised by mosquito vector resistance to pyrethroids.

Abstract Background The objective of this study was to estimate malaria transmission and insecticide resistance status in malaria vectors in Adjrako village from Zè District in Southern Benin. The present study was carried out prior to investigations on infectivity of blood from asymptomatic carriers of Plasmodium falciparum to malaria vector mosquitoes. Methods Human landing collections (HLCs) were performed in Adjrako village during the rainy season (September—November 2021). In this village, host-seeking mosquitoes were collected during three nights per survey from 22:00 to 06:00 in six randomly selected houses. Malaria vectors were dissected in orders to determinate their parity. Plasmodium falciparum infection in malaria vectors was determined by qPCR and the entomological inoculation rate (EIR) was calculated. The World Health Organization (WHO) insecticide susceptibility test-kits were used to evaluate the susceptibility of Anopheles gambiae sensu lato (s.l.) to deltamethrin at 0.05% and bendiocarb at 0.1%. Results A total of 3260 females of mosquitoes belonging to 4 genera (Anopheles, Culex, Aedes and Mansonia) were collected. Most of the mosquitoes collected were An. gambiae sensu lato (s.l.). The entomological inoculation rate (EIR) for the three collection months was 8.7 infective bites per person and the parity rate was 84%. Mortality rates of An. gambiae s.l. exposed to 0.05% deltamethrin and 0.1% bendiocarb were 18% and 96%, respectively, indicating that this vector population was resistant to deltamethrin and possibly resistant to bendiocarb in the study area. Conclusion This study showed that malaria transmission is effective in the study area and that An. gambiae s.l. is the main malaria vector. The entomological parameters indicate this study area is potentially favourable for investigations on P. falciparum asymptomatic carriers.

Abstract Background Entomological surveillance provides critical information on vectors for appropriate malaria vector control and strategic decision-making. The widely documented insecticide resistance of malaria vectors in Côte d’Ivoire requires that any vector control intervention deployment be driven by entomological data to optimize its effectiveness and appropriate resource allocations. To achieve this goal, this study documents the results of monthly vector surveillance and insecticide susceptibility tests conducted in 2019 and a review of all previous entomological monitoring data used to guide vector control decision making. Furthermore, susceptibility to pirimiphos-methyl and clothianidin was assessed in addition to chlorfenapyr and pyrethroids (intensity and piperonyl butoxide (PBO) synergism) tests previously reported. Vector bionomic data were conducted monthly in four sites (Sakassou, Béoumi, Dabakala and Nassian) that were selected based on their reported high malaria incidence. Adult mosquitoes were collected using human landing catches (HLCs), pyrethrum spray catches (PSCs), and human-baited CDC light traps to assess vector density, behaviour, species composition and sporozoite infectivity. Results Pirimiphos-methyl and clothianidin susceptibility was observed in 8 and 10 sites, respectively, while previous data reported chlorfenapyr (200 µg/bottle) susceptibility in 13 of the sites, high pyrethroid resistance intensity and increased mortality with PBO pre-exposure at all 17 tested sites. Anopheles gambiae sensu lato was the predominant malaria vector collected in all four bionomic sites. Vector density was relatively higher in Sakassou throughout the year with mean biting rates of 278.2 bites per person per night (b/p/n) compared to Béoumi, Dabakala and Nassian (mean of 48.5, 81.4 and 26.6 b/p/n, respectively). The mean entomological inoculation rate (EIR) was 4.44 infective bites per person per night (ib/p/n) in Sakassou, 0.34 ib/p/n in Beoumi, 1.17 ib/p/n in Dabakala and 1.02 ib/p/n in Nassian. The highest EIRs were recorded in October in Béoumi (1.71 ib/p/n) and Nassian (3.22 ib/p/n), in July in Dabakala (4.46 ib/p/n) and in May in Sakassou (15.6 ib/p/n). Conclusion Based on all results and data review, the National Malaria Control Programme developed and implemented a stratified insecticide-treated net (ITN) mass distribution in 2021 considering new generation ITNs. These results also supported the selection of clothianidin-based products and an optimal spraying time for the first indoor residual spraying (IRS) campaign in Sakassou and Nassian in 2020.

Abstract Vector control plays a key role in reducing the public health burden of mosquito-borne diseases. Today’s vector control strategies largely rely on synthetic insecticides that can have a negative environmental impact when applied outdoors and often become inefficient because of the mosquitoes’ ability to develop resistance. An alternative and promising approach to circumvent these challenges involves the implementation of insecticides derived from nature (biopesticides) for vector control. Biopesticides can constitute naturally occurring organisms or substances derived from them that have lifespan-shortening effects on disease vectors such as mosquitoes. Here we present the discovery and evaluation of natural product-based biological control agents that can potentially be developed into biopesticides for mosquito control. We screened a natural product collection comprising 390 compounds and initially identified 26 molecules with potential ability to kill the larval stages of the yellow fever mosquito Aedes aegypti, which is responsible for transmitting viruses such as dengue, Zika, chikungunya and yellow fever. Natural products identified as hits in the screen were further evaluated for their suitability for biopesticide development. We show that a selection of the natural product top hits, bactobolin, maytansine and ossamycin, also killed the larval stages of the malaria-transmitting mosquito Anopheles gambiae as well as the adult form of both species. We have further explored the usefulness of crude extracts and preparations from two of the best candidates’ sources (organisms of origin) for mosquitocidal activity, that is extracts from the two bacteria Burkholderia thailandensis and Streptomyces hygroscopicus var. ossamyceticus. Graphical abstract

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

Abstract Background Despite the scale-up of insecticide-treated nets and indoor residual spraying, the bulk of malaria transmission in western Kenya still occurs indoors, late at night. House improvement is a potential long-term solution to further reduce malaria transmission in the region. Methods The impact of eave screening on mosquito densities was evaluated in two rural villages in western Kenya. One-hundred-and-twenty pairs of structurally similar, neighbouring houses were used in the study. In each pair, one house was randomly selected to receive eave screening at the beginning of the study while the other remained unscreened until the end of the sampling period. Mosquito sampling was performed monthly by motorized aspiration method for 4 months. The collected mosquitoes were analysed for species identification. Results Compared to unscreened houses, significantly fewer female Anopheles funestus (RR = 0.40, 95% CI 0.29–0.55), Anopheles gambiae Complex (RR = 0.46, 95% CI 0.34–0.62) and Culex species (RR = 0.53, 95% CI 0.45–0.61) were collected in screened houses. No significant differences in the densities of the mosquitoes were detected in outdoor collections. Significantly fewer Anopheles funestus were collected indoors from houses with painted walls (RR = 0.05, 95% CI 0.01–0.38) while cooking in the house was associated with significantly lower numbers of Anopheles gambiae Complex indoors (RR = 0.60, 95% CI 0.45–0.79). Nearly all house owners (99.6%) wanted their houses permanently screened, including 97.7% that indicated a willingness to use their own resources. However, 99.2% required training on house screening. The cost of screening a single house was estimated at KES6,162.38 (US$61.62). Conclusion Simple house modification by eave screening has the potential to reduce the indoor occurrence of both Anopheles and Culex mosquito species. Community acceptance was very high although education and mobilization may be needed for community uptake of house modification for vector control. Intersectoral collaboration and favourable government policies on housing are important links towards the adoption of house improvements for malaria control.

Abstract Background This study was designed to provide insecticide resistance data for decision-making in terms of resistance management plans in Togo. Methods The susceptibility status of Anopheles gambiae sensu lato (s.l.) to insecticides used in public health was assessed using the WHO tube test protocol. Pyrethroid resistance intensity bioassays were performed following the CDC bottle test protocol. The activity of detoxification enzymes was tested using the synergists piperonyl butoxide, S.S.S-tributlyphosphorotrithioate and ethacrinic acid. Species-specific identification of An. gambiae s.l. and kdr mutation genotyping were performed using PCR techniques. Results Local populations of An. gambiae s.l. showed full susceptibility to pirimiphos methyl at Lomé, Kovié, Anié, and Kpèlè Toutou. At Baguida, mortality was 90%, indicating possible resistance to pirimiphos methyl. Resistance was recorded to DDT, bendiocarb, and propoxur at all sites. A high intensity of pyrethroid resistance was recorded and the detoxification enzymes contributing to resistance were oxidases, esterases, and glutathione-s-transferases based on the synergist tests. Anopheles gambiae sensu stricto (s.s.) and Anopheles coluzzii were the main species identified. High kdr L1014F and low kdr L1014S allele frequencies were detected at all localities. Conclusion This study suggests the need to reinforce current insecticide-based malaria control interventions (IRS and LLINs) with complementary tools.

Abstract Background Every evening, chimpanzees (Pan troglodytes) build a sleeping platform so called “nest” by intertwining branches of tree. Most of chimpanzees’ communities studied have a preference for tree species in which they nest. As female mosquitoes are feeding on the blood of their host at nighttime, chimpanzees may prevent being disturbed and bitten by mosquitoes by selecting tree species having properties to repel them. Methods To test the hypothesis that chimpanzees choose tree species for their aromatic properties, data related to 1,081 nesting trees built between 2017 and 2019 in the Sebitoli community of Kibale National Park (Uganda) were analysed. The 10 most used trees were compared to the 10 most common trees in the habitat that were not preferred for nesting. Leaves from the 20 trees species were collected and hydro-distillated to obtain essential oils and one of the by-products for behavioural bioassays against females of the African mosquito, Anopheles gambiae. Results Sebitoli chimpanzees showed tree preferences: 10 species correspond to more than 80% of the nesting trees. Out of the essential oil obtained from the 10 nesting trees, 7 extracts for at least one concentration tested showed spatial repellency, 7 were irritant by contact and none were toxic. In the other hand, for the abundant trees in their habitat not used by chimpanzees, only 3 were repellent and 5 irritants. Discussion and conclusion This study contributes to evidence that chimpanzees, to avoid annoying mosquitoes, may select their nesting trees according to their repellent properties (linked to chemical parameters), a potential inspiration for human health.

Abstract Background Evolutionary pressures lead to the selection of efficient malaria vectors either resistant or susceptible to Plasmodium parasites. These forces may favour the introduction of species genotypes that adapt to new breeding habitats, potentially having an impact on malaria transmission. Thioester-containing protein 1 (TEP1) of Anopheles gambiae complex plays an important role in innate immune defenses against parasites. This study aims to characterize the distribution pattern of TEP1 polymorphisms among populations of An. gambiae sensu lato (s.l.) in western Kenya. Methods Anopheles gambiae adult and larvae were collected using pyrethrum spray catches (PSC) and plastic dippers respectively from Homa Bay, Kakamega, Bungoma, and Kisumu counties between 2017 and 2020. Collected adults and larvae reared to the adult stage were morphologically identified and then identified to sibling species by PCR. TEP1 alleles were determined in 627 anopheles mosquitoes using restriction fragment length polymorphisms-polymerase chain reaction (RFLP-PCR) and to validate the TEP1 genotyping results, a representative sample of the alleles was sequenced. Results Two TEP1 alleles (TEP1*S1 and TEP1*R2) and three corresponding genotypes (*S1/S1, *R2/S1, and *R2/R2) were identified. TEP1*S1 and TEP1*R2 with their corresponding genotypes, homozygous *S1/S1 and heterozygous *R2/S1 were widely distributed across all sites with allele frequencies of approximately 80% and 20%, respectively both in Anopheles gambiae and Anopheles arabiensis. There was no significant difference detected among the populations and between the two mosquito species in TEP1 allele frequency and genotype frequency. The overall low levels in population structure (F ST = 0.019) across all sites corresponded to an effective migration index (Nm = 12.571) and low Nei’s genetic distance values (

Abstract Background More than 90% of malaria cases occur in Africa where the disease is transmitted by Anopheles gambiae and Anopheles arabiensis. This study evaluated the anti-mosquito properties of Juniperus virginiana (JVO) and Pelargonium roseum (PRO) essential oils (EOs) against larvae and adults of An. gambiae sensu lato (s.l.) from East Africa in laboratory and semi-field conditions. Methods EOs was extracted from the aerial green parts of Asian herbs by hydrodistillation. Their constituents were characterized by gas chromatography-mass spectrometry (GC-MS). Larvicidal activities of JVO, PRO, and PRO components [citronellol (CO), linalool (LO), and geraniol (GO)] were investigated against An. gambiae sensu stricto (s.s.). The percentage of knockdown effects and mortality rates of all oils were also evaluated in the adults of susceptible An. gambiae s.s. and permethrin-resistant An. arabiensis. Results GC-MS analyses identified major constituents of JVO (sabinene, dl-limonene, β-myrcene, bornyl acetate, and terpinen-4-ol) and PRO (citronellol, citronellyl formate, L-menthone, linalool, and geraniol). Oils showed higher larvicidal activity in the laboratory than semi-field trials. The LC50 values for JVO/PRO were computed as 10.82–2.89/7.13–0.9 ppm and 10.75–9.06/13.63–8.98 ppm in laboratory and semi-field environments, respectively at exposure time of 24–72 h. The percentage of knockdown effects of the oils were also greater in An. gambiae s.s. than in An. arabiensis. Filter papers impregnated with JVO (100 ppm) and PRO (25 ppm) displayed 100% mortality rates for An. gambiae s.s. and 3.75% and 90% mortality rates, for An. arabiensis populations, respectively. Each component of CO, LO, and GO exhibited 98.13%, 97.81%, and 87.5%, respectively, and a mixture of the PRO components indicated 94.69% adult mortality to permethrin-resistant An. arabiensis. Conclusions The findings of this study show that PRO and its main constituents, compared to JVO, have higher anti-mosquito properties in terms of larvicidal, knockdown, and mortality when applied against susceptible laboratory and resistant wild populations of An. gambiae s.l. Consequently, these oils have the potential for the development of new, efficient, safe, and affordable agents for mosquito control.

Abstract Background Long-lasting insecticidal nets (LLINs) are a vital tool in the fight against malaria vectors. However, their efficacy in the field can be impacted by several factors, including patterns of usage, net age, mosquito resistance and the delayed mortality effect, all of which could influence malaria transmission. We have investigated the effectiveness of the various brands of LLINs available in markets and households in Cameroon on pyrethroid-resistant mosquitoes and assessed their post-exposure effect. Methods Following quality control assessment on a susceptible laboratory mosquito strain, we evaluated the immediate and delayed mortality effects of exposure to LLINs (both newly bough LLINst and used ones collected from households in Elende village, Cameroon, in 2019) using standard WHO cone tests on Anopheles gambiae and Anopheles funestus populations collected from the Centre region of Cameroon. Alive female mosquitoes were genotyped for various resistance markers at different time points post-exposure to evaluate the impact of insecticide resistance on the efficacy of bednets. Results The laboratory-susceptible strain experienced high mortality rates when exposed to all pyrethroid-only brands of purchased nets (Olyset® Net, Super Net, PermaNet® 2.0, Yorkool®, Royal Sentry®) (Mean±SEM: 68.66 ± 8.35% to 93.33 ± 2.90%). However, low mortality was observed among wild An. funestus mosquitoes exposed to the bednets (0 ± 0 to 28 ± 6.7%), indicating a reduced performance of these nets against field mosquitoes. Bednets collected from households also showed reduced efficacy on the laboratory strain (mortality: 19–66%), as well as displaying a significant loss of efficacy against the local wild strains (mortality: 0 ± 0% to 4 ± 2.6% for An. gambiae sensu lato and 0 ± 0% to 8 ± 3.2% for An. funestus). However, compared to the unexposed group, mosquitoes exposed to bednets showed a significantly reduced longevity, indicating that the efficacy of these nets was not completely lost. Mosquitoes with the CYP6P9a-RR and L119F-GSTe2 mutations conferring pyrethroid resistance showed greater longevity after exposure to the Olyset net than their susceptible counterparts, indicating the impact of resistance on bednet efficacy and delayed mortality. Conclusion These findings show that although standard bednets drastically lose their efficacy against pyrethroid-resistant field mosquitoes, they still are able to induce delayed mortality in exposed populations. The results of this study also provide evidence of the actual impact of resistance on the quality and efficacy of LLINs in use in the community, with mosquitoes carrying the CYP6P9a-RR and L119F-GSTe2 mutations conferring pyrethroid resistance living longer than their susceptible counterparts. These results highlight the need to use new-generation nets that do not rely solely on pyrethroids. Graphical Abstract

Abstract Background Anopheles gambiae densovirus (AgDNV) is an insect-specific, single-stranded DNA virus that infects An. gambiae sensu stricto (s.s.), the major mosquito species responsible for transmitting malaria parasites throughout sub-Saharan Africa. AgDNV is a benign virus that is very specific to its mosquito host and therefore has the potential to serve as a vector control tool via paratransgenesis (genetic modification of mosquito symbionts) to limit transmission of human pathogens. Prior to being engineered into a control tool, the natural transmission dynamics of AgDNV between An. gambiae mosquitoes needs to be fully understood. Additionally, improved knowledge of AgDNV infection in male mosquitoes is needed. In the study presented here, we examined the tissue tropism of AgDNV in the male reproductive tract and investigated both venereal and vertical transmission dynamics of the virus. Methods Anopheles gambiae s.s. adult males were infected with AgDNV via microinjection, and reproductive tissues were collected and assayed for AgDNV using qPCR. Next, uninfected females were introduced to AgDNV-infected or control males and, after several nights of mating, both the spermatheca and female carcass were assessed for venereally transmitted AgDNV. Finally, F1 offspring of this cross were collected and assayed to quantify vertical transmission of the virus. Results AgDNV infected the reproductive tract of male mosquitoes, including the testes and male accessory glands, without affecting mating rates. AgDNV-infected males venereally transmitted the virus to females, and these venereally infected females developed disseminated infection throughout the body. However, AgDNV was not vertically transmitted to the F1 offspring of this cross. Conclusions Infected male releases could be an effective strategy to introduce AgDNV-based paratransgenic tools into naïve populations of An. gambiae s.s. females. Graphical Abstract

Abstract Background The practice of agriculture in urban settings contributes to the rapid expansion of insecticide resistance in malaria vectors. However, there is still not enough information on pesticide usage in most urban settings. The present study aims to assess the evolution of Anopheles gambiae (s.l.) population susceptibility to insecticides and patterns of pesticide usage in agriculture in the city of Yaoundé, Cameroon. Methods WHO susceptibility tests and synergist PBO bioassays were conducted on adult An. gambiae (s.l.) mosquitoes aged 3 to 5 days emerging from larvae collected from the field. Seven insecticides (deltamethrin, permethrin, DDT, bendiocarb, propoxur, fenitrothion and malathion) were evaluated. The presence of target site mutation conferring knockdown (kdr) resistance was investigated using TaqMan assay, and mosquito species were identified using SINE-PCR. Surveys on 81 retailers and 232 farmers were conducted to assess general knowledge and practices regarding agricultural pesticide usage. Results High resistance intensity to pyrethroids was observed with a high frequency of the kdr allele 1014F and low frequency of the kdr 1014S allele. The level of susceptibility of An. gambiae (s.l.) to pyrethroids and carbamates was found to decrease with time (from > 34% in 2017 to

Abstract Background Malaria remains one of the most devastating diseases globally, and the control of mosquitoes as the vector is mainly dependent on chemical insecticides. Elevated temperatures associated with future warmer climates could affect mosquitoes' metabolic enzyme expression and increase insecticide resistance, making vector control difficult. Understanding how mosquito rearing temperatures influence their susceptibility to insecticide and expression of metabolic enzymes could aid in the development of novel tools and strategies to control mosquitoes in a future warmer climate. This study evaluated the effects of temperature on the susceptibility of Anopheles gambiae sensu lato (s.l.) mosquitoes to pyrethroids and their expression of metabolic enzymes. Methods Anopheles gambiae s.l. eggs obtained from laboratory-established colonies were reared under eight temperature regimes (25, 28, 30, 32, 34, 36, 38, and 40 °C). Upon adult emergence, 3- to 5-day-old female non-blood-fed mosquitoes were used for susceptibility tests following the World Health Organization (WHO) bioassay protocol. Batches of 20–25 mosquitoes from each temperature regime (25–34 °C) were exposed to two pyrethroid insecticides (0.75% permethrin and 0.05% deltamethrin). In addition, the levels of four metabolic enzymes (α-esterase, β-esterase, glutathione S-transferase [GST], and mixed-function oxidase [MFO]) were examined in mosquitoes that were not exposed and those that were exposed to pyrethroids. Results Mortality in An. gambiae s.l. mosquitoes exposed to deltamethrin and permethrin decreased at temperatures above 28 °C. In addition, mosquitoes reared at higher temperatures were more resistant and had more elevated enzyme levels than those raised at low temperatures. Overall, mosquitoes that survived after being exposed to pyrethroids had higher levels of metabolic enzymes than those that were not exposed to pyrethroids. Conclusions This study provides evidence that elevated temperatures decreased An. gambiae s.l. mosquitoes' susceptibility to pyrethroids and increased the expression of metabolic enzymes. This evidence suggests that elevated temperatures projected in a future warmer climate could increase mosquitoes' resistance to insecticides and complicate malaria vector control measures. This study therefore provides vital information, and suggests useful areas of future research, on the effects of temperature variability on mosquitoes that could guide vector control measures in a future warmer climate. Graphical Abstract

Abstract Background Gene co-expression networks (GCNs) can be used to determine gene regulation and attribute gene function to biological processes. Different high throughput technologies, including one and two-channel microarrays and RNA-sequencing, allow evaluating thousands of gene expression data simultaneously, but these methodologies provide results that cannot be directly compared. Thus, it is complex to analyze co-expression relations between genes, especially when there are missing values arising for experimental reasons. Networks are a helpful tool for studying gene co-expression, where nodes represent genes and edges represent co-expression of pairs of genes. Results In this paper, we establish a method for constructing a gene co-expression network for the Anopheles gambiae transcriptome from 257 unique studies obtained with different methodologies and experimental designs. We introduce the sliding threshold approach to select node pairs with high Pearson correlation coefficients. The resulting network, which we name AgGCN1.0, is robust to random removal of conditions and has similar characteristics to small-world and scale-free networks. Analysis of network sub-graphs revealed that the core is largely comprised of genes that encode components of the mitochondrial respiratory chain and the ribosome, while different communities are enriched for genes involved in distinct biological processes. Conclusion Analysis of the network reveals that both the architecture of the core sub-network and the network communities are based on gene function, supporting the power of the proposed method for GCN construction. Application of network science methodology reveals that the overall network structure is driven to maximize the integration of essential cellular functions, possibly allowing the flexibility to add novel functions.

Abstract Background There is a pressing need to improve understanding of how insecticide resistance affects the functional performance of insecticide-treated nets (ITNs). Standard WHO insecticide resistance monitoring assays are designed for resistance surveillance and do not necessarily provide insight into how different frequencies, mechanisms or intensities of resistance affect the ability of ITNs to reduce malaria transmission. Methods The current study presents some novel laboratory-based assays that attempt to better simulate realistic exposure of mosquitoes to ITNs and to quantify impact of exposure not only on instantaneous mortality, but also on blood-feeding and longevity, two traits that are central to transmission. The assays evaluated the performance of a standard ITN (Permanet® 2.0; Vestergaard Frandsen), a ‘next generation’ combination ITN with a resistance-breaking synergist (Permanet® 3.0) and an untreated net (UTN), against field-derived Anopheles gambiae sensu lato mosquitoes from Côte d’Ivoire exhibiting a 1500-fold increase in pyrethroid resistance relative to a standard susceptible strain. Results The study revealed that the standard ITN induced negligible instantaneous mortality against the resistant mosquitoes, whereas the resistance-breaking net caused high mortality and a reduction in blood-feeding. However, both ITNs still impacted long-term survival relative to the UTN. The impact on longevity depended on feeding status, with blood-fed mosquitoes living longer than unfed mosquitoes following ITN exposure. Exposure to both ITNs also reduced the blood-feeding success, the time spent on the net and blood-feeding duration, relative to the untreated net. Conclusion Although a standard ITN did not have as substantial instantaneous impact as the resistance-breaking net, it still had significant impacts on traits important for transmission. These results highlight the benefit of improved bioefficacy assays that allow for realistic exposure and consider sub- or pre-lethal effects to help assess the functional significance of insecticide resistance. Graphical Abstract

Abstract Background Plasmodium ovale is a neglected malarial parasite that can form latent hypnozoites in the human liver. Over the last decade, molecular surveillance studies of non-falciparum malaria in Africa have highlighted that P. ovale is circulating below the radar, including areas where Plasmodium falciparum is in decline. To eliminate malaria where P. ovale is endemic, a better understanding of its epidemiology, asymptomatic carriage, and transmission biology is needed. Methods We performed a pilot study on P. ovale transmission as part of an ongoing study of human-to-mosquito transmission of P. falciparum from asymptomatic carriers. To characterize the malaria asymptomatic reservoir, cross-sectional qPCR surveys were conducted in Bagamoyo, Tanzania, over three transmission seasons. Positive individuals were enrolled in transmission studies of P. falciparum using direct skin feeding assays (DFAs) with Anopheles gambiae s.s. (IFAKARA strain) mosquitoes. For a subset of participants who screened positive for P. ovale on the day of DFA, we incubated blood-fed mosquitoes for 14 days to assess sporozoite development. Results Molecular surveillance of asymptomatic individuals revealed a P. ovale prevalence of 11% (300/2718), compared to 29% (780/2718) for P. falciparum. Prevalence for P. ovale was highest at the beginning of the long rainy season (15.5%, 128/826) in contrast to P. falciparum, which peaked later in both the long and short rainy seasons. Considering that these early-season P. ovale infections were low-density mono-infections (127/128), we speculate many were due to hypnozoite-induced relapse. Six of eight P. ovale-infected asymptomatic individuals who underwent DFAs successfully transmitted P. ovale parasites to A. gambiae. Conclusions Plasmodium ovale is circulating at 4–15% prevalence among asymptomatic individuals in coastal Tanzania, largely invisible to field diagnostics. A different seasonal peak from co-endemic P. falciparum, the capacity to relapse, and efficient transmission to Anopheles vectors likely contribute to its persistence amid control efforts focused on P. falciparum. Graphical Abstract

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

Abstract Background Mosquitoes transmit a variety of diseases. Due to widespread insecticide resistance, new effective pesticides are urgently needed. Entomopathogenic fungi are widely utilized to control pest insects in agriculture. We hypothesized that certain fungal metabolites may be effective insecticides against mosquitoes. Methods A high-throughput cytotoxicity-based screening approach was developed to search for insecticidal compounds in our newly established global fungal extract library. We first determined cell survival rates after adding various fungal extracts. Candidate insecticides were further analyzed using traditional larval and adult survival bioassays. Results Twelve ethyl acetate extracts from a total of 192 fungal extracts displayed > 85% inhibition of cabbage looper ovary cell proliferation. Ten of these 12 candidates were confirmed to be toxic to Anopheles gambiae Sua5B cell line, and six showed > 85% inhibition of Anopheles mosquito cell growth. Further bioassays determined a LC50, the lethal concentration that kills 50% of larval or adult mosquitoes, of 122 µg/mL and 1.7 µg/mosquito, respectively, after 24 h for extract 76F6 from Penicillium toxicarium. Conclusions We established a high-throughput MTT-based cytotoxicity screening approach for the discovery of new mosquitocides from fungal extracts. We discovered a candidate extract from P. toxicarium that exhibited high toxicity to mosquito larvae and adults, and thus were able to demonstrate the value of our recently developed approach. The active fungal extracts discovered here are ideal candidates for further development as mosquitocides. Graphical abstract

Abstract Background Malaria vector control in the Democratic Republic of the Congo is plagued by several major challenges, including inadequate infrastructure, lack of access to health care systems and preventative measures, and more recently the widespread emergence of insecticide resistance among Anopheles mosquitoes. Across 26 provinces, insecticide resistance has been reported from multiple sentinel sites. However, to date, investigation of molecular resistance mechanisms among Anopheles vector populations in DRC has been more limited. Methods Adult Anopheles gambiae sensu lato (s.l.) and Anopheles funestus s.l. were collected from two sites in Sud-Kivu province and one site in Haut-Uélé province and PCR-screened for the presence of 11 resistance mutations, to provide additional information on frequency of resistance mechanisms in the eastern DRC, and to critically evaluate the utility of these markers for prospective country-wide resistance monitoring. Results L1014F-kdr and L1014S-kdr were present in 75.9% and 56.7% of An. gambiae s.l. screened, respectively, with some individuals harbouring both resistant alleles. Across the three study sites, L43F-CYP4J5 allele frequency ranged from 0.42 to 0.52, with evidence for ongoing selection. G119S-ace1 was also identified in all sites but at lower levels. A triple mutant haplotype (comprising the point mutation CYP6P4-I236M, the insertion of a partial Zanzibar-like transposable element and duplication of CYP6AA1) was present at high frequencies. In An. funestus s.l. cis-regulatory polymorphisms in CYP6P9a and CYP6P9b were detected, with allele frequencies ranging from 0.82 to 0.98 and 0.65 to 0.83, respectively. Conclusions This study screened the most up-to-date panel of DNA-based resistance markers in An. gambiae s.l. and An. funestus s.l. from the eastern DRC, where resistance data is lacking. Several new candidate markers (CYP4J5, G119S-ace1, the triple mutant, CYP6P9a and CYP6P9b) were identified, which are diagnostic of resistance to major insecticide classes, and warrant future, larger-scale monitoring in the DRC to inform vector control decisions by the National Malaria Control Programme.

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

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