Anopheles Gambiae Research Articles

Unraveling the Molecular Architecture of Mosquito D1-Like Dopamine Receptors: Insights Into Ligand Binding and Structural Dynamics for Insecticide Development.

Vector-borne diseases pose a severe threat to human life, contributing significantly to global mortality. Understanding the structure-function relationship of the vector proteins is pivotal for effective insecticide development due to their involvement in drug resistance and disease transmission. This study reports the structural and dynamic features of D1-like dopamine receptors (DARs) in disease-causing mosquito species, such as Aedes aegypti, Culex quinquefasciatus, Anopheles gambiae, and Anopheles stephensi. Through molecular modeling and simulations, we describe the common structural fold of mosquito DARs within the G-protein-coupled receptor family, highlighting the importance of an orthosteric and enlarged binding pocket. The orthosteric binding pocket, resembling a cage-like structure, is situated ~15 Å deep within the protein, with two serine residues forming the roof and an aspartate residue, along with two conserved water molecules (W1 and W2), forming the floor. The side walls are composed of two phenylalanine residues on one side and a valine residue on the other. The antagonist binding site, an enlarged binding pocket (EBP) near the entrance cavity, can accommodate ligands of varying sizes. The binding energy of dopamine is observed to be ~2-3 kcal/mol higher than that of the antagonist molecules amitriptyline, asenapine, and flupenthixol in mosquito DARs. These antagonist molecules bind to EBP, which obstructs dopamine movement toward the active site, thereby inhibiting signal transduction. Our findings elucidate the molecular architecture of the binding pockets and the versatility of DARs in accommodating diverse ligands, providing a foundational framework for future drug and insecticide development.

Transmission of transgenic mosquito-killing fungi during copulation

Entomopathogenic fungi engineered to express insect-specific neurotoxins have demonstrated potential as microbial control agents against malaria mosquitoes. Currently, the primary application method is via direct contact of spores with indoor resting mosquitoes. However, many malaria-transmitting mosquitoes feed and rest outdoors. To target these, we have developed an alternative application method that exploits the lethality of transgenic fungi as a sexually transmitted mosquito disease. This approach has both a wider interdisciplinary significance and important implications for preventing mosquito-borne diseases.

MosGILT antibodies interfere with Plasmodium sporogony in Anopheles gambiae

Plasmodium, the causative agents of malaria, are obtained by mosquitoes from an infected human. Following Plasmodium acquisition by Anopheles gambiae, mosquito gamma-interferon-inducible lysosomal thiol reductase (mosGILT) plays a critical role in its subsequent sporogony in the mosquito. A critical location for this development is the midgut, a tissue we show expresses mosGILT. Using membrane-feeding and murine infection models, we demonstrate that antibodies against mosGILT reduce the number of P. falciparum and P. berghei oocysts in the midgut and the infection prevalence of both species in the mosquito. mosGILT antibodies act in the mosquito midgut, specifically impacting the Plasmodium oocyst stage. Targeting mosGILT can therefore interfere with the Plasmodium life cycle in the mosquito and potentially serve as a transmission-blocking vaccine.

Potential of emodepside for vector-borne disease control

BackgroundEmodepside is an anthelmintic used in veterinary medicine that is currently under investigation in human clinical trials for the treatment of soil-transmitted helminths and possibly Onchocerca volvulus. Emodepside targets the calcium-activated voltage-gated potassium slowpoke 1 (SLO-1) channels of presynaptic nerves of pharynx and body wall muscle cells of nematodes leading to paralysis, reduced locomotion and egg laying, starvation, and death. Emodepside also has activity against Drosophila melanogaster SLO-1 channels. Orthologous SLO-1 genes are present in Anopheles gambiae and Aedes aegypti, suggesting that emodepside may have activity against mosquitoes.MethodsBoth Anopheles dirus and Ae. aegypti were blood-fed emodepside across a range of concentrations (1–10,000 nM) and mosquito survival was monitored for 10 days. Co-feeding experiments were also performed with An. dirus blood fed ivermectin at the concentrations that kills 25% (LC25) and 50% (LC50) of mosquitoes with and without emodepside at clinical peak concentration in humans (Cmax) and five times the Cmax, and mosquito survival was monitored for 10 days.ResultsEmodepside had weak mosquito-lethal effects in An. dirus but none observed in Ae. aegypti at the concentrations evaluated. The An. dirus emodepside LC50 was 4,623 [4,159–5,066] ng/ml which is > 100-fold greater than the peak concentrations seen in human. The ivermectin and emodepside co-feed experiment with An. dirus did not indicate any altered effect of ivermectin on mosquito survival when emodepside co-fed at human Cmax or five times that of the human Cmax.ConclusionsEmodepside was not lethal to An. dirus at human-relevant concentrations and had no effect on Ae. aegypti survival. Thus, mass distribution of emodepside does not appear to be a potential tool for vector-borne disease control. Emodepside induced mortality in An. dirus does suggest that the SLO-1 channel could be a potential target for novel vector control and may warrant further investigation.

Semi-field experiments reveal contrasted predation and movement patterns of aquatic macroinvertebrate predators of Anopheles gambiae larvae

BackgroundMembers of the Anopheles gambiae complex are major malaria vectors in sub-Saharan Africa. Their larval stages inhabit a variety of aquatic habitats in which, under natural circumstances, they are preyed upon by different taxa of aquatic macroinvertebrate predators. Understanding the potential impact of predators on malaria vector larval population dynamics is important for enabling integrated local mosquito control programmes with a stronger emphasis on biocontrol approaches. This study experimentally evaluated the predation efficacy and foraging strategy of three common aquatic macroinvertebrate predators of An. gambiae, diving beetles (Coleoptera), backswimmers (Hemiptera), and dragonfly nymphs (Odonata) in a semi-field system in South-Eastern Tanzania.MethodsAn array of alternating small and large basins used as aquatic habitats was created in two compartments of a semi-field system and filled with well water. Field-collected adult diving beetles, backswimmers or dragonfly nymphs were randomly assigned to these habitats and Anopheles arabiensis larvae were added as prey in half of the habitats. The number of mosquito larvae consumed, predator mobility across habitats and mortality were recorded at 24, 48 and 72 h.ResultsThe presence of An. gambiae larvae in habitats significantly increased the survival of backswimmer and dragonfly nymphs, which are not mobile. In contrast, diving beetles survived well under any initial condition by preferentially flying away from habitats without prey to nearby larger habitats with prey. The larval predation rates of predacious diving beetle, backswimmer and dragonfly nymphs were stable over time at a mean of 3.2, 7.0 and 9.6 larvae consumed each day.ConclusionThis study demonstrates that aquatic macroinvertebrate predators display adaptive foraging behaviour in response to prey presence and aquatic habitat size. It also confirms the ability of these predators to significantly reduce An. gambiae larval densities in aquatic habitats, thus their potential for consideration as additional biocontrol tools for mosquito population reduction.

Identification of Potential Vectors and Detection of Rift Valley Fever Virus in Mosquitoes Collected Before and During the 2022 Outbreak in Rwanda

Rift Valley fever virus (RVFV) is an emerging mosquito-borne arbovirus of One Health importance that caused two large outbreaks in Rwanda in 2018 and 2022. Information on vector species with a role in RVFV eco-epidemiology in Rwanda is scarce. Here we sought to identify potential mosquito vectors of RVFV in Rwanda, their distribution and abundance, as well as their infection status. Since an outbreak of RVF occurred during the study period, data were obtained both during an interepidemic period and during the 2022 Rwanda RVF outbreak. Five districts of the eastern province of Rwanda were prospected using a combination of unbaited light traps and Biogents (BG Sentinel and Pro) traps baited with an artificial human scent during three periods, namely mid-August to mid-September 2021, December 2021, and April to May 2022. Trapped mosquitoes were morphologically identified and tested for viral evidence using both RT-PCR and virus isolation methods on a Vero cell line. A total of 14,815 adult mosquitoes belonging to five genera and at least 17 species were collected and tested as 765 monospecific pools. Culex quinquefasciatus was the most predominant species representing 72.7% of total counts. Of 527 mosquito pools collected before the 2022 outbreak, a single pool of Cx. quinquefasciatus showed evidence of RVFV RNA. Of 238 pools collected during the outbreak, RVFV was detected molecularly from five pools (two pools of Cx. quinquefasciatus, two pools of Anopheles ziemanni, and one pool of Anopheles gambiae sensu lato), and RVFV was isolated from the two pools of Cx. quinquefasciatus, from Kayonza and Rwamagana districts, respectively. Minimum infection rates (per 1000 mosquitoes) of 0.4 before the outbreak and 0.6–7 during the outbreak were noted. Maximum-likelihood phylogenetic analysis indicates that RVFV detected in these mosquitoes is closely related to viral strains that circulated in livestock in Rwanda and in Burundi during the same RVF outbreak in 2022. The findings reveal initial evidence for the incrimination of several mosquito species in the transmission of RVFV in Rwanda and highlight the need for more studies to understand the role of each species in supporting the spread and persistence of RVFV in the country.

CLIPA protein pairs function as cofactors for prophenoloxidase activation in Anopheles gambiae.

Insect prophenoloxidases (proPO) are activated during immune responses by a proPO activating protease (PAP) in the presence of a high molecular weight cofactor assembled from serine protease homologs (SPH) that lack proteolytic activity. PAPs and the SPHs have a similar architecture, with an amino-terminal clip domain and a carboxyl-terminal protease domain. The SPHs belong to CLIPA subfamily of SP-related proteins. In Manduca sexta, a well characterized biochemical model system for insect immunity, the functional SPH cofactor contains one molecule each from two SPH subfamilies, SPH-I and SPH-II. In Anopheles gambiae, three SPHI-SPHII pairs (CLIPs A4-A6, A4-A7Δ, and A4-A12) were previously reported as cofactors for CLIPB9-mediated activation of proPO2 and proPO7. In this study, we produced recombinant proteins for two splicing variants of CLIPA7, proCLIPA7s (s for short), proCLIPA7f (f for full-length) and proCLIPA14. We cleaved each along with proCLIPA4 using M. sexta PAP3 and found that the CLIPA pairs A4-A7s and A4-A14 are better than A4-A7f in generating highly active PO2 or PO7. CLIPA7f and CLIPA7s, products of alternative splicing, have different strengths as cofactors in combination with CLIPA4. Because mRNA for CLIPA7f is expressed at a significantly higher level than CLIPA7s, cofactors with the weaker combination A4-A7f may predominate in hemolymph, resulting in a potential dampening effect on proPO activation as a regulatory mechanism for altering the strength of the melanization response. A. gambiae CLIPB10xa is involved in proPO activation but its role as a PAP was not established using mosquito proPOs. Here we showed that factor Xa-treated proCLIPB10Xa activated proCLIPs A7s, A7f, A14, A4 (poorly), and proPO2. At higher concentrations, CLIPB10xa efficiently activated proPO2 in the absence of a cofactor, but at low concentrations it required a CLIPA cofactor, suggesting that highly active PO2 can be generated at low concentration of CLIPB10 in cooperation with an SPH cofactor in vivo. Using cofactors generated by PAP3, we demonstrated the order of efficacy for proPO2 activation by B10Xa is A4-A6 > A4-A14 or A4-A7s > A4-A7f > A4-A12. This agrees with their relative strengths as cofactors for proPO2 and proPO7 activation by M. sexta PAP3. In summary, we further developed an in vitro assay system to elucidate biochemical details of the complex process of proPO activation in A. gambiae.

Molecular docking and molecular dynamics simulation studies of inhibitor candidates against Anopheles gambiae 3-hydroxykynurenine transaminase and implications on vector control

Molecular docking and molecular dynamics simulation studies of inhibitor candidates against Anopheles gambiae 3-hydroxykynurenine transaminase and implications on vector control

Enhance fashion classification of mosquito vector species via self-supervised vision transformer

Vector-borne diseases pose a major worldwide health concern, impacting more than 1 billion people globally. Among various blood-feeding arthropods, mosquitoes stand out as the primary carriers of diseases significant in both medical and veterinary fields. Hence, comprehending their distinct role fulfilled by different mosquito types is crucial for efficiently addressing and enhancing control measures against mosquito-transmitted diseases. The conventional method for identifying mosquito species is laborious and requires significant effort to learn. Classification is subsequently carried out by skilled laboratory personnel, rendering the process inherently time-intensive and restricting the task to entomology specialists. Therefore, integrating artificial intelligence with standard taxonomy, such as molecular techniques, is essential for accurate mosquito species identification. Advancement in novel tools with artificial intelligence has challenged the task of developing an automated system for sample collection and identification. This study aims to introduce a self-supervised Vision Transformer supporting an automatic model for classifying mosquitoes found across various regions of Thailand. The objective is to utilize self-distillation with unlabeled data (DINOv2) to develop models on a mobile phone-captured dataset containing 16 species of female mosquitoes, including those known for transmitting malaria and dengue. The DINOv2 model surpassed the ViT baseline model in precision and recall for all mosquito species. When compared on a species-specific level, utilizing the DINOv2 model resulted in reductions in false negatives and false positives, along with enhancements in precision and recall values, in contrast to the baseline model, across all mosquito species. Notably, at least 10 classes exhibited outstanding performance, achieving above precision and recall rates exceeding 90%. Remarkably, when applying cropping techniques to the dataset instead of utilizing the original photographs, there was a significant improvement in performance across all DINOv2 models studied. This is demonstrated by an increase in recall to 87.86%, precision to 91.71%, F1 score to 88.71%, and accuracy to 98.45%, respectively. Malaria mosquito species can be easily distinguished from another genus like Aedes, Mansonia, Armigeres, and Culex, respectively. While classifying malaria vector species presented challenges for the DINOv2 model, utilizing the cropped images enhanced precision by up to 96% for identifying one of the top three malaria vectors in Thailand, Anopheles minimus. A proficiently trained DINOv2 model, coupled with effective data management, can contribute to the development of a mobile phone application. Furthermore, this method shows promise in supporting field professionals who are not entomology experts in effectively addressing pathogens responsible for diseases transmitted by female mosquitoes.

Comparison of EfficientNetB0 and EfficientNetB7 Models in Classifying Malaria Based on Blood Cells

Malaria is a disease caused by the bite of malaria mosquitoes, which spreads through blood. Malaria mosquitoes will spread the Plasmodium parasite through their bites. Early malaria identification is essential so the disease can be prevented immediately. Through data science, which utilizes the CNN model, the classification of blood infected with parasites can be predicted accurately. This research uses data obtained from Kaggle website with 27,558 image samples. The data is divided into two classes, parasite-infected and uninfected, which are then divided again into two types. The first class is training data divided into 80% of the total data and the other 20% as validation data. This research used two test scenarios to obtain a more effective classification model. The first scenario uses Hyperparameter Tuning and the EfficientNetB0 model with classification results of 95%. Meanwhile, the classification achievement for scenario two was 99% by utilizing EfficientNetB7.

Simplified Plasmodium falciparum membrane feeding assay using small Petri dishes and gel warmers

Studies on Plasmodium falciparum transmission require blood-feeding infectious gametocytes to mosquitoes using standard membrane-feeding assays (SMFAs). SMFAs are routinely performed using electric heating coils or glass membrane feeders connected to a circulatory water bath using tubing and clamps. Each of these approaches is expensive and requires a complex setup, hence restricting the number of assays that can be performed simultaneously. Furthermore, existing methods cannot be easily applied in low-resource field settings. This study presents a low-cost and simplified method for feeding mosquitoes with an infectious blood meal using 35 mm Petri dishes where temperature is maintained by using reusable gel warmers. The intensity and prevalence of infection in mosquitoes (Anopheles stephensi and Anopheles gambiae) fed via a Petri dish overlaid with gel warmers were comparable to mosquitoes fed using standard glass membrane feeders. The methodology described here can be easily applied in low-resource and field settings due to its low cost, ease of set up, and use of easily available supplies, such as Petri dishes, and reusable gel warmers. We believe a wide range of laboratories can easily adapt this method for P. falciparum transmission studies.

Pyrethroid-resistant malaria vector Anopheles gambiae restored susceptibility after pre-exposure to piperonyl-butoxide: results from country-wide insecticide resistance monitoring in Tanzania, 2023

BackgroundEffective vector control interventions, notably insecticide-treated nets (ITNs) and indoor residual spraying (IRS) are indispensable for malaria control in Tanzania and elsewhere. However, the emergence of widespread insecticide resistance threatens the efficacy of these interventions. Monitoring of insecticide resistance is, therefore, critical for the selection and assessment of the programmatic impact of insecticide-based interventions.MethodsThe study was conducted country-wide across 22 sentinel districts of Tanzania between May and July 2023 using standard World Health Organization susceptibility test with 1×, 5×, and 10× of deltamethrin, permethrin, and alpha-cypermethrin and discriminating concentrations of 0.25% pirimiphos-methyl. Synergist assays were conducted to explore the underlying mechanisms of the observed phenotypic pyrethroid-resistant mosquitoes. Three- to five-day-old wild adult females in the first filiar generation of Anopheles gambiae sensu lato (s.l.) were used for the susceptibility bioassays.ResultsAnopheles gambiae s.l. were resistant to all pyrethroids at the discriminating dose in most sentinel districts except in Rorya, which remains fully susceptible, and Ushetu, which remains susceptible to deltamethrin but not permethrin. In 5 sites (Bukombe, Ukerewe, Kilwa, Kibondo, and Kakonko), the An. gambiae s.l. species exhibited strong resistance to pyrethroids surviving the 10 X concentrations (mortality rate < 98%). However, they remained fully susceptible to pirimiphos-methyl in almost all the sites except in Kibondo and Shinyanga. Likewise, there was full restoration to susceptibility to pyrethroid following pre-exposure of An. gambiae s.l. to piperonyl-butoxide (PBO) in 13 out of 16 sites. The 3 sites that exhibited partial restoration include Kakonko, Tandahimba, and Newala.ConclusionThe evidence of widespread pyrethroid resistance of the major malaria vector justifies the decision made by the Tanzania National Malaria Control Programme to transition to PBO-based ITNs. Without this switch, the gains achieved in malaria control could be compromised. Equally important, the lack of full restoration to susceptibility observed in three sentinel districts upon pre-exposure to PBO merits close monitoring, as there could be other underlying resistance mechanisms besides oxidase metabolic resistance.

High pyrethroid resistance is associated with high frequencies of 1014F and 1014S kdr mutations in Anopheles arabiensis (Diptera: Culicidae) from Ouagadougou, Burkina Faso.

Malaria remains a major public health threat in Burkina Faso, as in most sub-Saharan Africacountries. Malaria control relies mainly on long-lasting insecticide-treated nets (LLINs) and indoor residual spraying. In Burkina Faso, an escalating of insecticide resistance has been observed over the last decades. This study aimed to investigate insecticide resistance and the underlying mechanisms in Anopheles gambiae complex in Ouagadougou. Anopheles gambiae s.l. larvae were collected from gutters and ponds, in Zogona, Tampouy and Tanghin, 3 localities in Ouagadougou from July to October 2018. The larvae were reared in the laboratory to adults stage and susceptibility profile to pyrethroid, carbamate, and organophosphate insecticides was assessed using WHO tube assays. Mosquito species and mutations linked with insecticide resistance, were identified through PCR. More than 95% of the collected An. gambiae s.l. were An. arabiensis. An. arabiensis displayed high resistance to permethrin and deltamethrin, with mortalities below 30%, but was fully susceptible to bendiocarb, fenitrothion, and malathion. A high-frequency of the pyrethroid resistance-associated kdr mutation 1014F (0.81) was recorded, while the frequency of 1014S mutation (0.18) was lower. However, the carbamate and organophosphate-associated Ace-1 119S mutation was not detected. Localities and breeding site type appear to influence pyrethroid resistance in the An. arabiensis population of Ouagadougou. The high resistance to pyrethroids in An. arabiensis of urban Ouagadougou is underpinned, at least in part by high-frequency kdr mutations. This result supports the switch to next-generation LLINs, in well-established pyrethroid resistance zones of Burkina Faso including Ouagadougout.

The status of insecticide resistance of Anopheles coluzzii on the islands of São Tomé and Príncipe, after 20 years of malaria vector control

BackgroundInsecticide-based malaria vector control has been implemented on the islands of São Tomé and Príncipe (STP) for more than 20 years. During this period malaria incidence was significantly reduced to pre-elimination levels. While cases remained low since 2015, these have significantly increased in the last year, challenging the commitment of the country to achieve malaria elimination by 2025. To better understand the reasons for increasing malaria cases, levels and underlying mechanisms of insecticide resistance in the local Anopheles coluzzii populations were characterized.MethodsMosquito larval collections were performed in the rainy and dry seasons, between 2022 and 2024, in two localities of São Tomé and one locality in Príncipe. Susceptibility to permethrin, α-cypermethrin, pirimiphos-methyl and DDT was assessed using WHO bioassays and protocols. Intensity of resistance and reversal by PBO pre-exposure were determined for pyrethroid insecticides. The kdr locus was genotyped by PCR assays in subsamples of the mosquitoes tested.ResultsAnopheles coluzzii populations were fully susceptible to pirimiphos-methyl, but high levels of resistance to pyrethroids and DDT were detected, particularly in São Tomé rainy season collections. Increasing the pyrethroid and DDT dosages to 5 × and 10 × did not restore full susceptibility in all populations. Pre-exposure to PBO resulted into partial reversal of the resistance phenotype suggesting the presence of cytochrome P450 oxidases-mediated metabolic resistance. The L1014F knockdown resistance mutation was present in An. coluzzii on both islands but at much higher frequency in São Tomé where it was associated with the resistant phenotype.ConclusionsFuture vector control interventions should consider the use of non-pyrethroid insecticides or combination with synergists to overcome the high levels of pyrethroid resistance. Alternative control methods not dependent on the use of insecticides should be additionally implemented to achieve malaria elimination in STP.

Risk of Aedes-borne diseases in and around the Tanzanian seaport of Tanga despite community members being more concerned about malaria

BackgroundIncreased global trade, while beneficial economically, can also increase the spread of vector-borne diseases, particularly those transmitted by Aedes mosquitoes spreading via trade routes. Given the heightened trade-induced activity at ports of entry, it is particularly crucial to assess the risk of mosquito-borne diseases in these settings. This study compared the risks of Aedes-borne disease in and around the eastern Tanzanian seaport of Tanga.MethodsA 200 m × 200 m grid-based system was used to sample mosquitoes within the port area, and in surrounding areas at 2 km, 2.5 km, and 5 km away, between June and December 2023. We characterized mosquito breeding habitats, collected mosquito larvae using standard dippers and tested susceptibility of raised adult Aedes aegypti populations to different insecticides. Adult mosquitoes were collected using BG sentinel traps (daytime) and Centers for Disease Control (CDC) light traps (night-time). Additionally, more than 200 port users and neighboring residents were surveyed to assess their experiences with and perceptions of mosquito biting and disease risks.ResultsThere were 2931 breeding sites, with (60.8%, n = 1782) positive for Aedes larvae. The percentage of water-holding containers infested with Aedes immatures, i.e., the container index (CI), was highest in the port area (66.2%), and lowest 5 km away (44.6%). The port area also had a greater proportion of temporary breeding sites (64.9%) than did the surrounding areas. The adult mosquito surveys revealed 20,449 mosquito species including: Culex quinquefasciatus (56.2%), Mansonia uniformis (38.6%), Ae. aegypti (5.1%), Anopheles gambiae (0.1%), and Anopheles funestus. Ae. aegypti were more abundant in the port area than in the surrounding areas (P < 0.001), whereas Culex sp., and Mansonia sp., were significantly outside (P < 0.001). Adult Anopheles sp., were found only in the port area, but Anopheles larvae were found both within and outside the port areas. Tests on Ae. aegypti sp., revealed susceptibility to bendiocarb and DDT, and resistance to permethrin. Awareness of mosquito-borne diseases among respondents was high for malaria (64.8%), but low for dengue (26.3%) and Chikungunya (1.7%). Most respondents reported being bothered by mosquitoes mostly at night (53.4%) or in the evening (40.7%). In addition to insecticidal bednets, which are used primarily against malaria, preventive measures for Aedes-borne diseases are limited.ConclusionsThis study identified significant potential risk of Aedes species, specifically Ae. aegypti sp., and associated diseases, but low perception of risk and inadequate personal protection measures in the study area. This low perception of risk highlights the need to improve public knowledge of the transmission and control of Aedes-borne diseases.Graphical

Genomic analyses revealed low genetic variation in the intron-exon boundary of the doublesex gene within the natural populations of An. gambiae s.l. in Burkina Faso

BackgroundThe recent success of a population control gene drive targeting the doublesex gene in Anopheles gambiae paved the way for developing self-sustaining and self-limiting genetic control strategies targeting the sex determination pathway to reduce and/or distort the reproductive capacity of insect vectors. However, targeting these genes for genetic control requires a better understanding of their genetic variation in natural populations to ensure effective gene drive spread. Using whole genome sequencing (WGS) data from the Ag1000G project (Ag3.0, 3.4 and 3.8), and Illumina pooled amplicon sequencing, we investigated the genetic polymorphism of the intron-4–exon-5 boundary of the doublesex gene in the natural populations of An. gambiae sensu lato (s.l.).ResultsThe analyses showed a very low variant density at the gRNA target sequence of the Ag(QFS)1 gene drive (previously called dsxFCRISPRh) within the populations of West and East Africa. However, populations from the forest area in Central Africa exhibited four SNP at frequencies ranging from 0.011 to 0.26. The SNP (2R:48714641[C > T]) at high frequencies, i.e. 0.26 is identified within the An. coluzzii population from Angola. The analyses also identified 90 low frequency (1 − 5%) SNPs in the genomic region around the gRNA target sequence (intron-4–exon-5 boundary). Three of these SNPs (2R:48714472 A > T; 2R:48714486 C > A; 2R:48714516 C > T) were observed at frequencies higher than 5% in the UTR region of the doublesex gene. The results also showed a very low variant density and constant nucleotide diversity over a five-year survey in natural An. gambiae s.l. populations of Burkina Faso.ConclusionThese findings will guide the implementation of doublesex-targeted gene drives to support the current control tools in malaria elimination efforts. Our methods can be applied to efficiently monitor the evolution of any sequence of interest in a natural population via pooled amplicon sequencing, surpassing the need for WGS.

Maternal lipid mobilization is essential for embryonic development in the malaria vector Anopheles gambiae.

Lipid metabolism is an essential component in reproductive physiology. While lipid mobilization has been implicated in the growth of Plasmodium falciparum malaria parasites in their Anopheles vectors, the role of this process in the reproductive biology of these mosquitoes remains elusive. Here, we show that impairing lipolysis in Anopheles gambiae, the major malaria vectors, leads to embryonic lethality. Embryos derived from females in which we silenced the triglyceride lipase AgTL2 or the lipid storage droplet AgLSD1 develop normally during early embryogenesis but fail to hatch due to severely impaired metabolism. Embryonic lethality is efficiently recapitulated by exposing adult females to broad-spectrum lipase inhibitors prior to blood feeding, unveiling lipolysis as a potential target for inducing mosquito sterility. Our findings provide mechanistic insights into the importance of maternal lipid mobilization in embryonic health that may inform studies on human reproduction.

Larvicidal efficacy and residual toxicity of plant extracts against Anopheles gambiae s.l. (Diptera: Culicidae) under semi-field conditions in Benatsemay district, southern Ethiopia

Larvicidal efficacy and residual toxicity of plant extracts against Anopheles gambiae s.l. (Diptera: Culicidae) under semi-field conditions in Benatsemay district, southern Ethiopia

A systematic review of interventions targeting Anopheles stephensi

Background Anopheles stephensi, a malaria mosquito originally from South Asia and the Middle East, has been expanding across both Asia and Africa in recent decades. The invasion of this species into sub-Saharan Africa is of particular concern given its potential to increase malaria burden, especially in urban environments where An. stephensi thrives. Whilst surveillance of this vector in Africa has recently increased markedly there is a need to review the existing methods of An. stephensi control so that we can stop, rather than simply monitor, its spread in Africa. Methods We searched published papers in PubMed using An. stephensi and intervention-specific search terms. Forty-five full-text articles were screened for eligibility and all those that reported the use of interventions against An. stephensi, and the effect on malaria incidence, malaria prevalence or vector densities were included in the analysis. All data retrieved from the literature were from the native range of An. stephensi and from the period 1995 to 2018. Results Fourteen studies which met the inclusion criteria were included in the final analysis. The vector control interventions discussed were bio larvicides (n=3), repellents (n=1), Indoor Residual Spraying (n=2), Insecticide Treated Nets (n=3), insecticide-treated materials other than nets (n=3), the combined use of repellents and mosquito nets (n=1), and combination of biolarvicide and fish (n=1). Outcomes of the studies were primarily vector density (n=10) although some reported malaria incidence and/or prevalence (n=4). Conclusions Long-lasting insecticidal nets and indoor residual spraying are effective in controlling, An. stephensi-transmitted malaria and reducing vector density, with repellents offering a complementary approach, especially in urban areas where this vector thrives. The private sector can help scale up affordable repellent production in Africa. There is a need to address gaps in cost-effectiveness analysis and gather more epidemiological evidence to better assess the impact of malaria control strategies.

Biocontrol toxicity of Trichoderma harzianum (Hypocreales: Hypocreaceae) derived chemical molecules against malarial mosquito Anopheles stephensi with molecular docking studies.

In this study, the crude chemical constituents extracted from Trichoderma harzianum and their toxicity were evaluated against the larvae, pupae, and adults of Anopheles stephensi at 24 and 48h post-treatment. Additionally, the chemical constituents of the crude extracts were identified using gas chromatography-mass spectrometry (GC-MS) analysis, and their ability to bind with target proteins was confirmed through molecular docking studies. The results clearly demonstrated that the chemical compounds from T. harzianum exhibited promising mortality rates in larvae (98.66%), pupae (92%), and adult mosquitoes (81.33%) of A. stephensi 48h after treatment. The study assessed the impact of crude extracts on insect enzymes 24h post treatment, revealing significant alterations: a reduction in catalase activity and an increase in glutathione S-transferase levels compared to the control group. The treatment with crude chemical extracts resulted in mortality rates of 37.33% and 52% at 24 and 48h, respectively, on Artemia salina , indicating minimal effects. After 48h, the crude extract exhibited minimal toxicity on Eudrilus eugeniae, with a recorded mortality rate of 15% after 48h. GC-MS analysis of T. harzianum-derived crude extracts identified ten major chemical constituents. Among these, chemicals, 2,4-bis(1,1-dimethylethyl) phenol (19.02%) was recognized as the predominant chemical component. This 2,4-bis(1,1-dimethylethyl) phenol molecule demonstrates a high binding affinity with target proteins, which is a key factor contributing to its insecticidal activity. This study concludes that the chemical constituents derived from T. harzianum are promising candidates for an eco-friendly, effective, and target-specific alternative control method for A. stephensi mosquitos.