Longitudinal survey of insecticide resistance in a village of central region of Burkina Faso reveals co-occurrence of 1014F, 1014S and 402L mutations in Anopheles coluzzii and Anopheles arabiensis.

BACKGROUND: Pyrethroid resistance is one of the major threats for effectiveness of insecticide-treated bed nets (ITNs) in malaria vector control. Genotyping of mutations in the voltage gated sodium channel (VGSC) gene is widely used to easily assess the evolution and spread of pyrethroid target-site resistance among malaria vectors. L1014F and L1014S substitutions are the most common and best characterized VGSC mutations in major African malaria vector species of the Anopheles gambiae complex. Recently, an additional substitution involved in pyrethroid resistance, i.e. V402L, has been detected in Anopheles coluzzii from West Africa lacking any other resistance alleles at locus 1014. The evolution of target-site resistance mutations L1014F/S and V402L was monitored in An. coluzzii and Anopheles arabiensis specimens from a Burkina Faso village over a 10-year range after the massive ITN scale-up started in 2010. METHODS: Anopheles coluzzii (N = 300) and An. arabiensis (N = 362) specimens collected both indoors and outdoors by different methods (pyrethrum spray catch, sticky resting box and human landing collections) in 2011, 2015 and 2020 at Goden village were genotyped by TaqMan assays and sequencing for the three target site resistance mutations; allele frequencies were statistically investigated over the years. RESULTS: A divergent trend in resistant allele frequencies was observed in the two species: 1014F decreased in An. coluzzii (from 0.76 to 0.52) but increased in An. arabiensis (from 0.18 to 0.70); 1014S occurred only in An. arabiensis and slightly decreased over time (from 0.33 to 0.23); 402L increased in An. coluzzii (from 0.15 to 0.48) and was found for the first time in one An. arabiensis specimen. In 2020 the co-occurrence of different resistance alleles reached 43% in An. coluzzii (alleles 410L and 1014F) and 32% in An. arabiensis (alleles 1014F and 1014S). CONCLUSIONS: Overall, an increasing level of target-site resistance was observed among the populations with only 1% of the two malaria vector species being wild type at both loci, 1014 and 402, in 2020. This, together with the co-occurrence of different mutations in the same specimens, calls for future investigations on the possible synergism between resistance alleles and their phenotype to implement local tailored intervention strategies.

First detection of kdr L1014F allele in Anopheles ziemanni and Anopheles pharoensis in Cameroon and distribution of the allele in members of the Anopheles gambiae complex.

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.

Intense malarial transmission during the dry season in irrigated rice-growing areas: a case study in Sakassou, Côte d'Ivoire.

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

The role of the major chemosensory organs in the host-seeking activity of Anopheles coluzzii (Diptera: Culicidae).

Anopheles coluzzii (Coetzee & Wilkerson) and its sibling species Anopheles gambiae s.s. (Giles) are highly anthropophilic and among the major malaria vectors in sub-Saharan Africa. Mosquitoes use various senses to find hosts, but rely primarily on olfaction. Therefore, the mosquito olfactory system has been studied extensively, including a variety of studies comparing chemosensory gene expression between An. coluzzii and its zoophilic sibling species Anopheles quadriannulatus (Theobald). These studies revealed species-specific chemosensory gene expression in the antennae and maxillary palps, which raised the question of a potential role for the palps in determining species-specific host preferences. To answer this question, we mechanically ablated the antennae, maxillary palps, and labella, and ran both control and ablated mosquitoes through a dual-port olfactometer. While we aimed to identify the organs responsible for vertebrate host choice, the ablated mosquitoes exclusively responded to human odor, so we were unable to do so. However, we were able to refine our understanding of the roles of these organs in host-seeking activation (leaving the release cage) as well as odor response (entering an odor port). As expected, the antennae are the most important organs to both behaviors: activation was roughly halved and vertebrate odor response was abolished in antennae-ablated mosquitoes. Maxillary palp ablation had little impact on activation, but reduced odor response to a similar degree as the exclusion of CO2. Finally, while labellar ablation dramatically reduced activation (probably associated with the inability to feed), it had little impact on odor response, suggesting that any labellar role in host choice is likely not olfactory.

Multisensory integration in Anopheles mosquito swarms: The role of visual and acoustic information in mate tracking and collision avoidance.

Male mosquitoes form aerial aggregations, known as swarms, to attract females and maximize their chances of finding a mate. Within these swarms, individuals must be able to recognize potential mates and navigate the dynamic social environment to successfully intercept a mating partner. Prior research has almost exclusively focused on the role of acoustic cues in mediating the male mosquito's ability to recognize and pursue flying females. However, the role of other sensory modalities in this behavior has not been explored. Moreover, how males avoid collisions with one another in the dense swarm while pursuing females remains poorly understood. In this study, we combined free-flight and tethered flight simulator experiments to demonstrate that swarming Anopheles coluzzii mosquitoes integrate visual and acoustic information to track conspecifics and avoid collisions. Our tethered experiments revealed that acoustic stimuli gated mosquito steering responses to visual objects simulating nearby mosquitoes, especially in males that exhibited attraction to visual objects in the presence of female flight tones. Additionally, we observed that visual cues alone could trigger changes in mosquitoes' wingbeat amplitude and frequency. These findings were corroborated by our free-flight experiments, which revealed that mosquitoes modulate their flight responses to nearby conspecifics in a similar manner to tethered animals, allowing for collision avoidance within swarms. Together, these results demonstrate that both males and females integrate multiple sensory inputs to mediate swarming behavior, and for males, the change in flight kinematics in response to multimodal cues allows them to simultaneously track females while avoiding collisions.

Lethal and sublethal impacts of membrane-fed ivermectin are concentration dependent in Anopheles coluzzii.

BACKGROUND: Ivermectin is a well-tolerated anthelminthic drug with wide clinical and veterinary applications. It also has lethal and sublethal effects on mosquitoes. Mass drug administration with ivermectin has therefore been suggested as an innovative vector control tool in efforts to curb emerging insecticide resistance and reduce residual malaria transition. To support assessments of the feasibility and efficacy of current and future formulations of ivermectin for vector control, we sought to establish the relationship between ivermectin concentration and its lethal and sublethal impacts in a primary malaria vector. METHODS: The in vitro effects of ivermectin on daily mortality and fecundity, measured by egg production, were assessed up to 14 days post-blood feed in a laboratory colony of Anopheles coluzzii. Mosquitoes were fed ivermectin in blood meals delivered by membrane feeding at one of six concentrations: 0 ng/ml (control), 10 ng/ml, 15 ng/ml, 25 ng/ml, 50 ng/ml, 75 ng/ml, and 100 ng/ml. RESULTS: Ivermectin had a significant effect on mosquito survival in a concentration-dependent manner. The LC50 at 7 days was 19.7 ng/ml. The time to median mortality at ≥ 50 ng/ml was ≤ 4 days, compared to 9.6 days for control, and 6.3-7.6 days for ivermectin concentrations between 10 and 25 ng/ml. Fecundity was also affected; no oviposition was observed in surviving females from the two highest concentration treatment groups. While females exposed to 10 to 50 ng/ml of ivermectin did oviposit, significantly fewer did so in the 50 ng/ml treatment group compared to the control, and they also produced significantly fewer eggs. CONCLUSIONS: Our results showed ivermectin reduced mosquito survival in a concentration-dependent manner and at ≥ 50 ng/ml significantly reduced fecundity in An. coluzzii. Results indicate that levels of ivermectin found in human blood following ingestion of a single 150-200 µg/kg dose would be sufficient to achieve 50% mortality across 7 days; however, fecundity in survivors is unlikely to be affected. At higher doses, a substantial impact on both survival and fecundity is likely. Treating human populations with ivermectin could be used as a supplementary malaria vector control method to kill mosquito populations and supress their reproduction; however strategies to safely maintain mosquitocidal blood levels of ivermectin against all Anopheles species require development.

The Anopheles coluzzii range extends into Kenya: detection, insecticide resistance profiles and population genetic structure in relation to conspecific populations in West and Central Africa.

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

The paradox of plant preference: The malaria vectors Anopheles gambiae and Anopheles coluzzii select suboptimal food sources for their survival and reproduction.

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

First report of natural infection of Anopheles gambiae s.s. and Anopheles coluzzii by Wolbachia and Microsporidia in Benin: a cross-sectional study.

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.

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

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

The Anopheles coluzzii range extends into Kenya: Detection, insecticide resistance profiles and population genetic structure in relation to conspecific populations in West and Central Africa.

Background: Anopheles coluzzii is a primary vector of malaria found in West and Central Africa, but its presence has hitherto never been documented in Kenya. A thorough understanding of vector bionomics is important as it enables the implementation of targeted and effective vector control interventions. Malaria vector surveillance efforts in the country have tended to focus on historically known primary vectors. In the current study, we sought to determine the taxonomic status of samples collected from five different malaria epidemiological zones in Kenya as well asdescribe the population genetic structure and insecticide resistance profiles in relation to other An. coluzzi populations. Methods: Mosquitoes were sampled as larvae from Busia, Kwale, Turkana, Kirinyaga and Kiambu counties, representing the range of malaria endemicities in Kenya, in 2019 and 2021 and emergent adults analysed using Whole Genome Sequencing data processed in accordance with the Anopheles gambiae 1000 Genomes Project phase 3. Where available, historical samples from the same sites were included for WGS. Results: This study reports the detection of Anopheles coluzzii for the first time in Kenya. The species was detected in Turkana County across all three time points sampled and its presence confirmed through taxonomic analysis. Additionally, we found a lack of strong population genetic differentiation between An. coluzzii from Kenya and those from the more northerly regions of West and Central Africa, suggesting they represent a connected extension to the known species range. Mutations associated with target-site resistance to DDT and pyrethroids and metabolic resistance to DDT were found at high frequencies of ~60%. The profile and frequencies of the variants observed were similar to An. coluzzii from West and Central Africa but the ace-1 mutation linked to organophosphate and carbamate resistance present in An. coluzzii from coastal West Africa was absent in Kenya. Conclusions: These findings emphasise the need for the incorporation of genomics in comprehensive and routine vector surveillance to inform on the range of malaria vector species, and their insecticide resistance status to inform the choice of effective vector control approaches.

Mosquitoes escape looming threats by actively flying with the bow wave induced by the attacker.

To detect and escape looming threats, night-flying insects must rely on other senses than vision alone. Nocturnal mosquitoes can evade looming objects in the dark, but how they achieve this is still unknown. Here, we show how night-active female malaria mosquitoes escape from rapidly looming objects that simulate defensive actions of blood-hosts. First, we quantified the escape performance of flying mosquitoes from an event-triggered mechanical swatter, showing that mosquitoes use swatter-induced airflow to increase their escape success. Secondly, we used high-speed videography and deep-learning-based tracking to analyze escape flights in detail, showing that mosquitoes use banked turns to evade the threat. By combining escape kinematics data with numerical simulations of attacker-induced airflow and a mechanistic movement model, we unraveled how mosquitoes control these banked evasive maneuvers: they actively steer away from the danger, and then passively travel with the bow wave produced by the attacker. Our results demonstrate that night-flying mosquitoes can detect looming objects when visual cues are minimal, suggesting that they use attacker-induced airflow both to detect the danger and as a fluid medium to move with away from the threat. This shows that escape strategies of flying insects are more complex than previous visually induced escape flight studies suggest. As most insects are of similar or smaller sizes than mosquitoes, comparable escape strategies are expected among millions of flying insect species. The here-observed escape maneuvers are distinct from those of mosquitoes escaping from odor-baited traps, thus providing new insights for the development of novel trapping techniques for integrative vector management.

The impact of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr long-lasting insecticidal nets on density of primary malaria vectors Anopheles gambiae s.s. and Anopheles coluzzii in Benin: a secondary analysis of a cluster randomised controlled trial.

BACKGROUND: Long-lasting insecticidal nets (LLINs) may have different impacts on distinct mosquito vector species. We assessed the efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr LLINs on the density of Anopheles gambiae s.s. and An. coluzzii compared to pyrethroid-only nets in a three-arm cluster randomised control trial in Benin. METHODS: Indoor and outdoor collections of adult mosquitoes took place in 60 clusters using human landing catches at baseline and every 3 months for 2 years. After morphological identification, around 15% of randomly selected samples of An. gambiae s.l. were dissected to determine parity, species (using PCR). RESULTS: Overall, a total of 46,613 mosquito specimens were collected at baseline and 259,250 in the eight quarterly collections post-net distribution. Post-net distribution, approximately 70% of the specimens of An. gambiae s.l. speciated were An. coluzzii, while the rest were mostly composed of An. gambiae s.s. with a small proportion (< 1%) of hybrids (An. gambiae/coluzzii). There was no evidence of a significant reduction in vector density indoors in either primary vector species [An. coluzzii: DR (density ratio) = 0.62 (95% CI 0.21-1.77), p = 0.3683 for the pyrethroid-pyriproxyfen LLIN and DR = 0.56 (95% CI 0.19-1.62), p = 0.2866 for the pyrethroid-chlorfenapyr LLIN, An. gambiae s.s.: DR = 0.52 (95% CI 0.18-1.46), p = 0.2192 for the pyrethroid-pyriproxyfen LLIN and DR = 0.53 (95% CI 0.19-1.46), p = 0.2222 for the pyrethroid-chlorfenapyr]. The same trend was observed outdoors. Parity rates of An. gambiae s.l. were also similar across study arms. CONCLUSIONS: Compared with pyrethroid-only LLINs, pyrethroid-chlorfenapyr LLINs and pyrethroid-pyriproxyfen LLINs performed similarly against the two primary mosquito species An. gambiae s.s. and An. coluzzii in Benin.

Characterization of the swarming behavior of Anopheles coluzzii and Anopheles gambiae (Diptera: Culicidae) populations in a hybrid zone of Senegal.

Anopheles gambiae and Anopheles coluzzii, often found in sympatry and synchronous, have undergone a premating reproductive isolation across their distribution range. However, in the Western coast of Africa, unexpected hybridization zones have been observed, and little is known about swarming behavior of these cryptic taxa. Here, we characterized the swarming behavior of An. coluzzii and An. gambiae to investigate its role in the high hybridization level in Senegal. The study was conducted in the south and central Senegal during the 2018 rainy season. Mating swarms of malaria vectors were surveyed at sunset and collected using an insect net. Meanwhile, indoor resting populations of malaria vectors were collected by pyrethrum spray catches. Upon collection, specimens were identified morphologically, and then members of the An. gambiae complex were identified at the species level by polymerase chain reaction (PCR). An. gambiae swarmed mainly over bare ground, whereas An. coluzzii were found swarming above various objects creating a dark-light contrast with the bare ground. The swarms height varied from 0.5 to 2.5 m. Swarming starting time was correlated with sunset whatever the months for both species, and generally lasted about 10 min. No mixed swarm of An. gambiae and An. coluzzii was found even in the high hybridization area. These results indicated a premating isolation between An. coluzzii and An. gambiae. However, the high hybridization rate in the sympatric area suggests that heterogamous mating is occurring, thus stressing the need for further extensive studies.

Can the use of larviciding with biological compounds contribute in increasing Anopheles gambiae s.l. susceptibility to pyrethroid in a population expressing high resistance intensity?

BACKGROUND: Larviciding using non-insecticide compounds is considered appropriate for controlling outdoor biting mosquitoes and for managing insecticide resistance. However, there is still not enough information on the influence of larviciding in managing pyrethroid resistance. In the present study, we checked whether the introduction of larviciding using the biolarvicide VectoMax G in the city of Yaoundé is contributing in restoring the susceptibility of An. coluzzii populations to pyrethroids. METHODOLOGY: The susceptibility status of field An. coluzzii population was evaluated at different time points before and during larviciding treatments. In addition, An. coluzzii larvae collected in the city of Yaoundé, were split into four groups and exposed to different selection regimes for many generations as follow; (i): deltamethrin 0.05%_only, (ii): Vectomax_only, (iii): Vectomax+deltamethrin 0.05%, (iv): VectoMax+deltamethrin 0.05% + susceptible. Life traits parameters were measured in the progeny and compared between colonies. The control was the susceptible laboratory strain "Ngousso". Kdr allele frequency and the profile of expression of different detoxification genes and oxidative stress genes was checked using qPCR analysis. Gene's expression was compared between the first and the last generation of each colony and in field populations collected before and during larviciding. RESULTS: An increase in mosquito susceptibility to deltamethrin and permethrin was recorded for the field populations after larviciding implementation. Resistance intensity to deltamethrin was found to decrease from high to low in field populations. Only the colony vectomax+deltamethrin+susceptible presented a high susceptibility to deltamethrin after 21 generations. The kdr gene frequency was found to be unchanged in the field population and laboratory colonies. A significant decrease in the overexpression profile of Gste2 was detected in field population after larviciding implementation. Other genes showing a similar pattern though not significant were Cyp6z1, Cyp6p1 and Cyp6g16. Concerning fitness only the colony vectomax+deltamethrin+susceptible was found to display a fitness profile similar to the susceptible colony with high fecundity, high hatching rate, short development time and long adult survival rate. CONCLUSION: The profile of the field population supported reversal of phenotypic resistance to pyrethroids however no reduction in the frequency of the kdr allele was recorded. Some detoxification genes were detected less overexpressed. The study suggest that reversal may take longer to achieve in a population expressing a very high resistance profile and under continuous insecticide selection pressure.

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

BACKGROUND: Information on common markers of metabolic resistance in malaria vectors from countries sharing similar eco-climatic characteristics can facilitate coordination of malaria control. Here, we characterized populations of the major malaria vector Anopheles coluzzii from Sahel region, spanning four sub-Saharan African countries: Nigeria, Niger, Chad and Cameroon. RESULTS: Genome-wide transcriptional analysis identified major genes previously implicated in pyrethroid and/or cross-resistance to other insecticides, overexpressed across the Sahel, including CYP450s, glutathione S-transferases, carboxylesterases and cuticular proteins. Several, well-known markers of insecticide resistance were found in high frequencies-including in the voltage-gated sodium channel (V402L, I940T, L995F, I1527T and N1570Y), the acetylcholinesterase-1 gene (G280S) and the CYP4J5-L43F (which is fixed). High frequencies of the epidemiologically important chromosomal inversion polymorphisms, 2La, 2Rb and 2Rc, were observed (~80% for 2Rb and 2Rc). The 2La alternative arrangement is fixed across the Sahel. Low frequencies of these inversions (<10%) were observed in the fully insecticide susceptible laboratory colony of An. coluzzii (Ngoussou). Several of the most commonly overexpressed metabolic resistance genes sit in these three inversions. Two commonly overexpressed genes, GSTe2 and CYP6Z2, were functionally validated. Transgenic Drosophila melanogaster flies expressing GSTe2 exhibited extremely high DDT and permethrin resistance (mortalities <10% in 24h). Serial deletion of the 5' intergenic region, to identify putative nucleotide(s) associated with GSTe2 overexpression, revealed that simultaneous insertion of adenine nucleotide and a transition (T->C), between Forkhead box L1 and c-EST putative binding sites, were responsible for the high overexpression of GSTe2 in the resistant mosquitoes. Transgenic flies expressing CYP6Z2 exhibited marginal resistance towards 3-phenoxybenzylalcohol (a primary product of pyrethroid hydrolysis by carboxylesterases) and a type II pyrethroid, α-cypermethrin. However, significantly higher mortalities were observed in CYP6Z2 transgenic flies compared with controls, on exposure to the neonicotinoid, clothianidin. This suggests a possible bioactivation of clothianidin into a toxic intermediate, which may make it an ideal insecticide against populations of An. coluzzii overexpressing this P450. CONCLUSIONS: These findings will facilitate regional collaborations within the Sahel region and refine implementation strategies through re-focusing interventions, improving evidence-based, cross-border policies towards local and regional malaria pre-elimination.

Infection of the malaria vector Anopheles coluzzii with the entomopathogenic bacteria Chromobacterium anophelis sp. nov. IRSSSOUMB001 reduces larval survival and adult reproductive potential.

BACKGROUND: Vector control tools are urgently needed to control malaria transmission in Africa. A native strain of Chromobacterium sp. from Burkina Faso was recently isolated and preliminarily named Chromobacterium anophelis sp. nov. IRSSSOUMB001. In bioassays, this bacterium showed a promising virulence against adult mosquitoes and reduces their blood feeding propensity and fecundity. The current study assessed the entomopathogenic effects of C. anophelis IRSSSOUMB001 on larval stages of mosquitoes, as well as its impacts on infected mosquitoes reproductive capacity and trans-generational effects. METHODS: Virulence on larvae and interference with insemination were assayed by co-incubation with C. anophelis IRSSSOUMB001 at a range of 104 to 108 cfu/ml. Trans-generational effects were determined by measuring body size differences of progeny from infected vs. uninfected parent mosquitoes using wing size as a proxy. RESULTS: Chromobacterium anophelis IRSSSOUMB001 killed larvae of the pyrethroid-resistant Anopheles coluzzii with LT80 of ~ 1.75 ± 0.14 days at 108 cfu/ml in larval breeding trays. Reproductive success was reduced as a measure of insemination rate from 95 ± 1.99% to 21 ± 3.76% for the infected females. There was a difference in wing sizes between control and infected mosquito offsprings from 2.55 ± 0.17 mm to 2.1 ± 0.21 mm in infected females, and from 2.43 ± 0.13 mm to 1.99 ± 0.15 mm in infected males. CONCLUSIONS: This study showed that C. anophelis IRSSSOUMB001 was highly virulent against larvae of insecticide-resistant Anopheles coluzzii, and reduced both mosquito reproduction capacity and offspring fitness. Additional laboratory, field, safety and social acceptance studies are needed to draw firm conclusions about the practical utility of this bacterial strain for malaria vector control.

The interplay between malaria vectors and human activity accounts for high residual malaria transmission in a Burkina Faso village with universal ITN coverage.

BACKGROUND: Mosquito and human behaviour interaction is a key determinant of the maximum level of protection against malaria that can be provided by insecticide-treated nets (ITNs). Nevertheless, scant literature focuses on this interaction, overlooking a fundamental factor for efficient malaria control. This study aims to estimate malaria transmission risk in a Burkina Faso village by integrating vector biting rhythms with some key information about human habits. METHODS: Indoor/outdoor human landing catches were conducted for 16 h (16:00-08:00) during 8 nights (September 2020) in Goden village. A survey about net usage and sleeping patterns was submitted to half the households (October-December 2020). A subsample of collected specimens of Anopheles gambiae sensu lato was molecularly processed for species identification, Plasmodium detection from heads-thoraxes and L1014F pyrethroid-resistance allele genotyping. Hourly mosquito abundance was statistically assessed by GLM/GAM, and the entomological inoculation rate (EIR) was corrected for the actual ITN usage retrieved from the questionnaire. RESULTS: Malaria transmission was mainly driven by Anopheles coluzzii (68.7%) followed by A. arabiensis (26.2%). The overall sporozoite rate was 2% with L1014F estimated frequency of 0.68 (N = 1070 out of 15,201 A. gambiae s.l. collected). No major shift in mosquito biting rhythms in response to ITN or differences between indoor and outdoor catches were detected. Impressive high biting pressure (mean 30.3 mosquitoes/person/hour) was exerted from 20:00 to 06:00 with a peak at 4:00. Human survey revealed that nearly all inhabitants were awake before 20:00 and after 7:00 and at least 8.7% had no access to bednets. Adjusting for anthropological data, the EIR dropped from 6.7 to 1.2 infective bites/person/16 h. In a scenario of full net coverage and accounting only for the human sleeping patterns, the daily malaria transmission risk not targetable by ITNs was 0.69 infective bites. CONCLUSIONS: The high mosquito densities and interplay between human/vector activities means that an estimated 10% of residual malaria transmission cannot be prevented by ITNs in the village. Locally tailored studies, like the current one, are essential to explore the heterogeneity of human exposure to infective bites and, consequently, to instruct the adoption of new vector control tools strengthening individual and community protection.

Trophic preferences of Anopheles coluzzii (Diptera: Culicidae): what implications for malaria vector control in Benin?

The main objective of the present study is to assess the preferences in terms of vertebrate hosts of Anopheles coluzzii, the main malaria vector in the pastoral area of Malanville, Benin, where rice cultivation and livestock are the main source of income for the populations. Adult mosquitoes were collected through pyrethrum spray catch, and human landing catch in two communes in Benin: Malanville, a pastoral area, and Porto-Novo, a nonpastoral area. Molecular species identification was performed through PCR within the Anopheles gambiae complex. Blood meal origin and P. falciparum sporozoite infection were determined using ELISA blood meal and circumsporozoite protein tests, respectively. Overall, 97% of females of An. gambiae s.l. were An. coluzzii, with biting behavior more pronounced outdoors in the pastoral area. In Malanville, the main vertebrate hosts on which females An. coluzzii blood fed were goats (44%), humans (24.29%), bovines (22%), and pigs (1.4%). Our results also showed that single-host blood meals (human: 24.29% or animal: 68%) were mostly observed compared to mixed blood meals (8.58%). The human biting rate (HBR) and P. falciparum sporozoite rate (SR) of An. coluzzii were 66.25 bites/man/night and 0.77%, respectively. However, in the nonpastoral zone (Porto-novo), 93.98% of samples were An. coluzzii. The latter blood-fed mostly (86.84%) on humans, with an estimated HBR of 21.53 b/m/n and SR of 5.81%. The present study revealed an opportunistic and zoophagic behavior of An. coluzzii in the Malanville area with an overall low mean SR.

Molecular characterization and genetic authentication assay for Anopheles 'hemocyte-like' cell lines 4a-3A and 4a-3B.

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.