Anopheles Stephensi Mosquitoes Research Articles

Larvicidal Efficacies of Nanoliposomes Containing Alpha-pinene, Citral, Camphor, and Thymol Against Aedes aegypti and Anopheles stephensi Mosquito Vectors.

Diseases transmitted by Aedes aegypti or Anopheles stephensi, such as Zika virus, dengue fever, and malaria, pose substantial risks to public health, particularly in tropical areas. Plant-derived compounds have emerged as promising alternatives due to their inherent safety and potential efficiency against mosquitoes. This study aimed to improve the efficacy of certain natural compounds, including α-pinene, citral, camphor, and thymol, by developing nanoliposomal formulations. The ‎nanoliposomes containing α-pinene, citral, camphor, and thymol were prepared using the ethanol injection method and then characterized. Using WHO-recommended guidelines, their larvicidal efficacy was investigated against Aedes aegypti and Anopheles stephensi. The ‎nanoliposomes particle sizes were 105 ± 7, 86 ± 5, 149 ± 5, and 135 ± 8nm, and zeta potentials ‎were - 25.1 ± 0.5, -17.2 ± 1.2, -16.4 ± 1.6, and - 21.3 ± 1.7 mV, respectively. In addition, the ATR-FTIR (Attenuated Total Reflectance-Fourier Transform InfraRed) analysis verified the successful loading of the compound. Nanoliposomal compounds exhibited superior performance compared to their non-formulated counterparts in larvicidal bioassays. The nanoliposomes containing thymol showed the highest efficacy, with a Lethal Concentration 50 (LC50) of 20µg/mL against Ae. aegypti. Nanoliposomes containing citral exhibited an LC50 of 20µg/mL against An. stephensi. The results suggest that nanoliposomes have the potential to serve as an effective vehicle to improve the efficiency of plant-based larvicides. This could play a significant role in developing sustainable mosquito control strategies.

Activity of Essential Oils From Pentanema indicum (L.) Y. Ling and Chromolaena odorata (L.) R.M.King & H. Rob Against Three Mosquito Species

ABSTRACTMosquitoes act as biological carriers spreading numerous vector borne diseases globally. The present research aimed to study the effect of essential oils (EOs) from Pentanema indicum (L.) Y. Ling and Chromolaena odorata (L.) R.M. King & H. Rob against Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus mosquitoes. Concentrations of 3.125, 6.25, 12.5 25 and 50 ppm were tested for their mosquitocidal properties. The essential oil (EO) from P. indicum demonstrated promising ovicidal activity, achieving 100% efficacy against Ae. aegypti and Cx. quinquefasciatus and 76% efficacy against An. stephensi at a concentration of 50 ppm within 24 h. Similarly, C. odorata exhibited significant activity at higher concentrations, with ovicidal rates of 72% for Ae. aegypti, 79% for An. stephensi and 64% for Cx. quinquefasciatus at 300 ppm after 72 h. In terms of larvicidal activity, P. indicum showed potent toxicity with LC50 values of 4.28 ppm for Ae. aegypti, 14.01 ppm for An. stephensi and 5.47 ppm for Cx. quinquefasciatus. Additionally, C. odorata exhibited considerable mosquito larvicidal activity at higher concentrations, with LC50 values of 95.28 ppm for Ae. aegypti, 98.95 ppm for An. stephensi and 89.05 ppm for Cx. quinquefasciatus after 24 h. However, no significant pupicidal activity was observed for both EOs. GC–MS analysis identified β‐Pinene (14.31%), D‐Limonene (13.48%) and Caryophyllene (25.48%) as the main components in P. indicum. The EO from C. odorata contained α‐Pinene (12.10%), Geijerene (11.62%) and Caryophyllene (10.64%) as the primary components; probably these compounds were responsible for the observed activities. Toxicity studies indicated that the EOs from both plants had no adverse effects on natural predators. The EOs were then formulated for field application and tested in a laboratory setting; they demonstrated promising mosquitocidal activity without harming natural predators. This study suggests that EOs from P. indicum and C. odorata offer an effective alternative to synthetic pesticides.

Comparative larvicidal, pupicidal, adulticidal activity of Artemisia nilagirica (C.B. Cl) Pamp extract in controlling Culex quinquefasciatus, Anopheles stephensi, Aedes aegypti and Aedes albopictus.

Vector-borne diseases cause increase in burden, poverty, social liability and death all over the world. Mosquitoes serve as the vector for malaria, dengue, filariasis, yellow fever and also play a major role in transmission of chikungunya and Zika virus. The development of mosquitocidal resistance and associated health problems with the use of synthetic insecticides, have paved the way to control mosquito population by using plant-based botanicals. This study was carried out to evaluate the larvicidal, pupicidal and adulticidal properties of six solvent extracts of Artemisia nilagirica (C.B.Cl) against four infectious vector mosquitoes Anopheles stephensi, Aedes aegypti, Aedes albopictus and Culex quinquefasciatus, by assessing LC50 and LC90 mortality values. Among all six leaf solvent extracts, chloroform extract had higher toxicity (LC50= 127.27ppm and LC90=544.45ppm) against fourth instar larva of C. quinquefasciatus and aqueous extract had lowest lethal effects (LC50=583.33ppm and LC90= 927.27ppm) against fourth instar larva of A. aegypti. Moderate results were found in n- hexane, petroleum ether, methanol and ethanol plant extracts. Phytochemical analysis by GC-MS method confirms presence of significant 12 bioactive compounds like Bi-cyclo (3.1.1) heptanes-2, 4, 6 trimethyl, 3, 7, 11, 15- Tetramethyl-1.2 hexadecan-1-ol, Thiophene, Tetrahydro-2-methyl 1,3 propane diamine and camphor, which were responsible for insecticidal activity. Altogether, current study would serve as an initial step towards replacement of synthetic insecticides to plant- based bio-pesticide against dreadful vector mosquitoes in future.

Characterization of the Sodium Multi-Vitamin Transporter in the Mosquito Anopheles stephensi and Its Capacity to Mobilize Pantothenate and Biotin.

Pantothenate (Pan), or vitamin B5, is essential for the synthesis of co-enzyme A (CoA), acetyl-CoA, and numerous downstream physiological processes. We previously demonstrated that Pan is not only essential for mosquito survival, but also for the development of malaria parasites within the mosquito, suggesting that targeting Pan and CoA biosynthesis may be a novel approach for malaria control. However, little is known about how Pan is acquired and mobilized within the mosquito. In this work, we examined Pan levels in the important human malaria vector Anopheles stephensi, including the abundance of Pan during immature development and adulthood. We also assessed the distribution of Pan in various adult tissues and examined the impact of provisioning Pan to the mosquito via a sugar or blood meal on mosquito survival and reproduction. Furthermore, we examined how Pan was mobilized in the mosquito via a putative Pan transporter, the A. stephensi sodium multi-vitamin transporter. We demonstrated that this transporter is capable of mobilizing both Pan and biotin (vitamin B7) in a dose dependent manner. We also assessed the distribution of A. stephensi sodium multi-vitamin transporter in the mosquito and its capacity to transport vitamins. This work establishes the basic physiology of Pan uptake and mobilization in the mosquito, providing essential information for Pan based malaria control strategies.

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.

Host preference of Anopheles stephensi mosquitoes for blood feeding in south of Iran: Insights from Multiplex-PCR analysis.

The study aims to determine the host preference for blood feeding among potential hosts of Anopheles stephensi in Iran, using the Multiplex-PCR method. An. stephensi is the primary malaria vector in urban areas of South Asia and the Middle East, including southern Iran, where approximately 30.21% of malaria cases are urban. This trend has become more evident during the recent outbreaks in Iran, driven by infections of Plasmodium falciparum, Plasmodium vivax, and as well as mixed infections. Hormozgan province, one of the most endemic areas in Iran, was selected for its critical public health significance. This study builds on the validated efficiency of Multiplex-PCR for blood meal analysis by applying it to mosquitoes in southern Iran. In 2021, mosquitoes were collected monthly from three coastal villages in Bandar Abbas county, Hormozgan province, using WHO-recommended collection methods. Blood-fed An. stephensi mosquitoes were dissected, and their stomach contents analysed via Multiplex-PCR to identify human and animal blood sources. Of 77 An. stephensi samples analysed, humans were the most common host was humans (29.9%), followed by mammals (19%), dogs (2.6%), and birds (1.3%). Mixed blood meals were detected in 34% of samples, including 23% with human and other hosts. Informal observations suggest that domestic animals such as goats, sheep, and chickens are commonly present near homes in these areas. Approximately 50% of An. stephensi blood meals were sourced from humans, with 29% exclusively from humans and 23% from mixed hosts. Domestic animals such as goats, sheep, and chickens appear to attract mosquitoes, highlighting their potential role in malaria dynamics. Zooprophylaxis, alongside existing measures like insecticide residual spraying, insecticide-treated bed nets, and personal protection strategies, may strengthen urban malaria control. Further research on the ecological and behavioural drivers of mosquito host selection in urban settings is warranted.

Influence of Household Roof Types on the Development of Plasmodium vivax in Anopheles stephensi Mosquitoes.

Urbanization and microclimate variation in cities can influence mosquito behavior and parasite development, thus affecting malaria transmission. This study investigates how the impact of microclimate variations due to household roof types can aid in the survival of Anopheles stephensi and the development of Plasmodium vivax in an urban slum setting. Understanding these vital environmental interactions is essential for devising effective control strategies to achieve malaria elimination. Anopheles stephensi (F1) mosquitoes were membrane-fed with blood collected from P. vivax-infected patients before (day 0) and during (day 1) antimalarial treatment. The parasite development and mosquito survival were monitored in simulated microclimatic conditions of a variety of household roof types (thatched, asbestos, tiled) against standard laboratory conditions. Mosquito dissections were undertaken to detect oocysts and sporozoites in An. stephensi mosquitoes (oocyst: day 3-5, sporozoites: day 7-11). The maximum number of oocysts were detected in infected mosquitoes in thatched-roof conditions, whereas the largest oocyst was in the asbestos roof type. Circumsporozoite-ELISA results indicated the presence of sporozoites in infected mosquitoes for up to 29 days under standard conditions, 18 days in thatched-roof and asbestos roof conditions, and 14 days in tiled conditions. The univariate binary logistic regression model indicated a significant influence of microclimatic conditions of thatched roofs on parasite development. The Kaplan-Meier survival analysis revealed that the median survival of P. vivax-infected An. stephensi in thatched-roof conditions was 14 days, followed by asbestos (11 days) and tiled (10 days) roof conditions. In conclusion, thatched-roof houses were favorable for the development and survival of P. vivax-infected An. stephensi.

Pathogenicity and sub-lethal activity of orally administered entomopathogenic fungi against two adult mosquito species, Aedes aegypti (Diptera: Culicidae) and Anopheles stephensi (Diptera: Culicidae)

Entomopathogenic fungi (EPF) are known for their efficacy in controlling adult mosquito populations by penetrating through their cuticle. However, the effect of oral administration of EPF on the biological parameters of Aedes aegypti and Anopheles stephensi remains largely unexplored. This study aimed to assess the effect of orally administrated EPF isolates on the survival, feeding behavior, fecundity, fertility, follicle development and host-searching behavior in response to yeast-generated CO2 of Ae. aegypti and An. stephensi. An initial screening of 50 isolates involved exposure of adult Ae. aegypti and An. stephensi by integument inoculation. Subsequently, the entomopathogenic effect of the five highly virulent isolates was confirmed through oral administration revealing Beauveria pseudobassiana 42–51 as a potent mosquito killer. B. pseudobassiana 42–51 was administered orally to evaluate sub-lethal effects. The results showed a 63 % and 43 % reduction in blood feeding of Ae. aegypti and An. stephensi, respectively. Furthermore, a decrease in egg hatching rate was observed, with a reduction of 83% for Ae. aegypti and 74% for An. stephensi on the seventh day following fungal administration, showing decreased hatchability in both species. Poor and abnormal follicle development was observed in both mosquito species. Also, the host-searching behavior was evaluated by attraction to CO2 utilizing a Y-tube olfactometer. A tendency of reduction in the attraction rate towards the odor was observed three days post-fungal administration. These findings underscore the significant impact of oral administration of B. pseudobassiana 42–51 on mosquitoes, highlighting not only its lethal effects but also sub lethal impacts on their biology. Moreover, this fungus may exhibit the potential to simultaneously control both mosquito species and serve as a biocontrol agent for the management of vector-borne diseases.

Mean daily temperatures can predict the thermal limits of malaria transmission better than rate summation.

Temperature shapes the distribution, seasonality, and magnitude of mosquito-borne disease outbreaks. Mechanistic models predicting transmission often use mosquito and pathogen thermal responses from constant temperature experiments. However, mosquitoes live in fluctuating environments. Rate summation (nonlinear averaging) is a common approach to infer performance in fluctuating environments, but its accuracy is rarely validated. We measured three mosquito traits that impact transmission (bite rate, survival, fecundity) in a malaria mosquito (Anopheles stephensi) across temperature gradients with three diurnal temperature ranges (0, 9 and 12°C). We compared thermal suitability models with temperature-trait relationships observed under constant temperatures, fluctuating temperatures, and those predicted by rate summation. We mapped results across An. stephenesi's native Asian and invasive African ranges. We found: 1) daily temperature fluctuation significantly altered trait thermal responses; 2) rate summation partially captured decreases in performance near thermal optima, but also incorrectly predicted increases near thermal limits; and 3) while thermal suitability characterized across constant temperatures did not perfectly capture suitability in fluctuating environments, it was more accurate for estimating and mapping thermal limits than predictions from rate summation. Our study provides insight into methods for predicting mosquito-borne disease risk and emphasizes the need to improve understanding of organismal performance under fluctuating conditions.

Loop-Mediated Isothermal Amplification Assay to Detect Invasive Malaria Vector Anopheles stephensi Mosquitoes.

Spread of the Anopheles stephensi mosquito, an invasive malaria vector, threatens to put an additional 126 million persons per year in Africa at risk for malaria. To accelerate the early detection and rapid response to this mosquito species, confirming its presence and geographic extent is critical. However, existing molecular species assays require specialized laboratory equipment, interpretation, and sequencing confirmation. We developed and optimized a colorimetric rapid loop-mediated isothermal amplification assay for molecular An. stephensi species identification. The assay requires only a heat source and reagents and can be used with or without DNA extraction, resulting in positive color change in 30-35 minutes. We validated the assay against existing PCR techniques and found 100% specificity and analytical sensitivity down to 0.0003 ng of genomic DNA. The assay can successfully amplify single mosquito legs. Initial testing on samples from Marsabit, Kenya, illustrate its potential as an early vector detection and malaria mitigation tool.

A Pan Plasmodium lateral flow recombinase polymerase amplification assay for monitoring malaria parasites in vectors and human populations

Robust diagnostic tools and surveillance are crucial for malaria control and elimination efforts. Malaria caused by neglected Plasmodium parasites is often underestimated due to the lack of rapid diagnostic tools that can accurately detect these species. While nucleic-acid amplification technologies stand out as the most sensitive methods for detecting and confirming Plasmodium species, their implementation in resource-constrained settings poses significant challenges. Here, we present a Pan Plasmodium recombinase polymerase amplification lateral flow (RPA–LF) assay, capable of detecting all six human infecting Plasmodium species in low resource settings. The Pan Plasmodium RPA-LF assay successfully detected low density clinical infections with a preliminary limit of detection between 10–100 fg/µl for P. falciparum. When combined with crude nucleic acid extraction, the assay can serve as a point-of-need tool for molecular xenomonitoring. This utility was demonstrated by screening laboratory-reared Anopheles stephensi mosquitoes fed with Plasmodium-infected blood, as well as field samples of An. funestus s.l. and An. gambiae s.l. collected from central Africa. Overall, our proof-of-concept Pan Plasmodium diagnostic tool has the potential to be applied for clinical and xenomonitoring field surveillance, and after further evaluation, could become an essential tool to assist malaria control and elimination.

Investigating the lipid profile of Anopheles stephensi mosquitoes across developmental life stages

Holometabolous insects undergo a distinct transition in their development, tightly correlated with shifting feeding patterns from larval stages and some adult phases to non-feeding phases as pupae and during other adult phases. Furthermore, the intricate life cycle of mosquitoes involves a sequence of developmental stages influenced by aquatic and terrestrial factors, demanding precise energy resource orchestration. Lipids serve multifaceted roles, encompassing energy storage, membrane structure, and participation in signal transduction and molecular recognition processes. A significant gap in the current research landscape is the need for a comprehensive study exploring the lipid repertoire throughout the developmental stages of Anopheles stephensi mosquitoes. We undertook an analysis of the An. stephensi metabolome across all life stages. We hypothesized that An. stephensi mosquitoes will have unique lipid metabolite markers for each life stage. A specific extraction and LC-MS based lipidomic approach was used to test this hypothesis. Our findings demonstrated that our methods were successful, with lipids comprising 62.15 % of the analyzed metabolome. Additionally, phospholipids (PL), lysophospholipids (LPL), sphingomyelin (SM), and triglycerides (TG) were abundant and dynamic across all life stages. Interestingly, comparison between the L1 and L2 lipidome revealed a dominant pattern of specific TGs in decreased abundance between these two life stages. Lastly, 20-hydroxyecdysone (20E), was found to be present in similar abundance across all 4 larval stages. These data indicate that there may be lipid metabolome pathways serving unique roles during mosquito development that may be used to explore laboratory management of colonies, parasite resistance, and environmental adaptation.

Infectivity of Plasmodium parasites to Aedes aegypti and Anopheles stephensi mosquitoes maintained on blood-free meals of SkitoSnack

BackgroundAedes and Anopheles mosquitoes are responsible for tremendous global health burdens from their transmission of pathogens causing malaria, lymphatic filariasis, dengue, and yellow fever. Innovative vector control strategies will help to reduce the prevalence of these diseases. Mass rearing of mosquitoes for research and support of these strategies presently depends on meals of vertebrate blood, which is subject to acquisition, handling, and storage issues. Various blood-free replacements have been formulated for these mosquitoes, but none of these replacements are in wide use, and little is known about their potential impact on competence of the mosquitoes for Plasmodium infection.MethodsColonies of Aedes aegypti and Anopheles stephensi were continuously maintained on a blood-free replacement (SkitoSnack; SS) or bovine blood (BB) and monitored for engorgement and hatch rates. Infections of Ae. aegypti and An. stephensi were assessed with Plasmodium gallinaceum and P. falciparum, respectively.ResultsReplicate colonies of mosquitoes were maintained on BB or SS for 10 generations of Ae. aegypti and more than 63 generations of An. stephensi. The odds of engorgement by SS- relative to BB-maintained mosquitoes were higher for both Ae. aegypti (OR = 2.6, 95% CI 1.3–5.2) and An. stephensi (OR 2.7, 95% CI 1.4–5.5), while lower odds of hatching were found for eggs from the SS-maintained mosquitoes of both species (Ae. aegypti OR = 0.40, 95% CI 0.26–0.62; An. stephensi OR = 0.59, 95% CI 0.36–0.96). Oocyst counts were similar for P. gallinaceum infections of Ae. aegypti mosquitoes maintained on SS or BB (mean ratio = [mean on SS]/[mean on BB] = 1.11, 95% CI 0.85–1.49). Similar oocyst counts were also observed from the P. falciparum infections of SS- or BB-maintained An. stephensi (mean ratio = 0.76, 95% CI 0.44–1.37). The average counts of sporozoites/mosquito showed no evidence of reductions in the SS-maintained relative to BB-maintained mosquitoes of both species.ConclusionsAedes aegypti and An. stephensi can be reliably maintained on SS over multiple generations and are as competent for Plasmodium infection as mosquitoes maintained on BB. Use of SS alleviates the need to acquire and preserve blood for mosquito husbandry and may support new initiatives in fundamental and applied research, including novel manipulations of midgut microbiota and factors important to the mosquito life cycle and pathogen susceptibility.Graphical

Molecular Confirmation of Anopheles stephensi Mosquitoes in the Al Hudaydah Governorate, Yemen, 2021 and 2022.

We detected malaria vector Anopheles stephensi mosquitoes in the Al Hudaydah governorate in Yemen by using DNA sequencing. We report 2 cytochrome c oxidase subunit I haplotypes, 1 previously found in Ethiopia, Somalia, Djibouti, and Yemen. These findings provide insight into invasive An. stephensi mosquitoes in Yemen and their connection to East Africa.

Basophil-Derived IL-4 and IL-13 Protect Intestinal Barrier Integrity and Control Bacterial Translocation during Malaria.

Our previous work demonstrated that basophils regulate a suite of malaria phenotypes, including intestinal mastocytosis and permeability, the immune response to infection, gametocytemia, and parasite transmission to the malaria mosquito Anopheles stephensi. Given that activated basophils are primary sources of the regulatory cytokines IL-4 and IL-13, we sought to examine the contributions of these mediators to basophil-dependent phenotypes in malaria. We generated mice with basophils depleted for IL-4 and IL-13 (baso IL-4/IL-13 (-)) and genotype controls (baso IL-4/IL-13 (+)) by crossing mcpt8-Cre and Il4/Il13fl/fl mice and infected them with Plasmodium yoelii yoelii 17XNL. Conditional deletion was associated with ileal mastocytosis and mast cell (MC) activation, increased intestinal permeability, and increased bacterial 16S levels in blood, but it had no effect on neutrophil activation, parasitemia, or transmission to A. stephensi. Increased intestinal permeability in baso IL-4/IL-13 (-) mice was correlated with elevated plasma eotaxin (CCL11), a potent eosinophil chemoattractant, and increased ileal MCs, proinflammatory IL-17A, and the chemokines MIP-1α (CCL3) and MIP-1β (CCL4). Blood bacterial 16S copies were positively but weakly correlated with plasma proinflammatory cytokines IFN-γ and IL-12p40, suggesting that baso IL-4/IL-13 (-) mice failed to control bacterial translocation into the blood during malaria infection. These observations suggest that basophil-derived IL-4 and IL-13 do not contribute to basophil-dependent regulation of parasite transmission, but these cytokines do orchestrate protection of intestinal barrier integrity after P. yoelii infection. Specifically, basophil-dependent IL-4/IL-13 control MC activation and prevent infection-induced intestinal barrier damage and bacteremia, perhaps via regulation of eosinophils, macrophages, and Th17-mediated inflammation.

A Pfs48/45-based vaccine to block Plasmodium falciparum transmission: phase 1, open-label, clinical trial

BackgroundThe stalling global progress in malaria control highlights the need for novel tools for malaria elimination, including transmission-blocking vaccines. Transmission-blocking vaccines aim to induce human antibodies that block parasite development in the mosquito and mosquitoes becoming infectious. The Pfs48/45 protein is a leading Plasmodium falciparum transmission-blocking vaccine candidate. The R0.6C fusion protein, consisting of Pfs48/45 domain 3 (6C) and the N-terminal region of P. falciparum glutamate-rich protein (R0), has previously been produced in Lactococcus lactis and elicited functional antibodies in rodents. Here, we assess the safety and transmission-reducing efficacy of R0.6C adsorbed to aluminium hydroxide with and without Matrix-M™ adjuvant in humans.MethodsIn this first-in-human, open-label clinical trial, malaria-naïve adults, aged 18–55 years, were recruited at the Radboudumc in Nijmegen, the Netherlands. Participants received four intramuscular vaccinations on days 0, 28, 56 and 168 with either 30 µg or 100 µg of R0.6C and were randomised for the allocation of one of the two different adjuvant combinations: aluminium hydroxide alone, or aluminium hydroxide combined with Matrix-M1™ adjuvant. Adverse events were recorded from inclusion until 84 days after the fourth vaccination. Anti-R0.6C and anti-6C IgG titres were measured by enzyme-linked immunosorbent assay. Transmission-reducing activity of participants’ serum and purified vaccine-specific immunoglobulin G was assessed by standard membrane feeding assays using laboratory-reared Anopheles stephensi mosquitoes and cultured P. falciparum gametocytes.ResultsThirty-one participants completed four vaccinations and were included in the analysis. Administration of all doses was safe and well-tolerated, with one related grade 3 adverse event (transient fever) and no serious adverse events occurring. Anti-R0.6C and anti-6C IgG titres were similar between the 30 and 100 µg R0.6C arms, but higher in Matrix-M1™ arms. Neat participant sera did not induce significant transmission-reducing activity in mosquito feeding experiments, but concentrated vaccine-specific IgGs purified from sera collected two weeks after the fourth vaccination achieved up to 99% transmission-reducing activity.ConclusionsR0.6C/aluminium hydroxide with or without Matrix-M1™ is safe, immunogenic and induces functional Pfs48/45-specific transmission-blocking antibodies, albeit at insufficient serum concentrations to result in transmission reduction by neat serum. Future work should focus on identifying alternative vaccine formulations or regimens that enhance functional antibody responses.Trial registrationThe trial is registered with ClinicalTrials.gov under identifier NCT04862416.

Spatiotemporal distribution and bionomics of Anopheles stephensi in different eco-epidemiological settings in Ethiopia

BackgroundMalaria is a major public health concern in Ethiopia, and its incidence could worsen with the spread of the invasive mosquito species Anopheles stephensi in the country. This study aimed to provide updates on the distribution of An. stephensi and likely household exposure in Ethiopia.MethodsEntomological surveillance was performed in 26 urban settings in Ethiopia from 2021 to 2023. A kilometer-by-kilometer quadrant was established per town, and approximately 20 structures per quadrant were surveyed every 3 months. Additional extensive sampling was conducted in 50 randomly selected structures in four urban centers in 2022 and 2023 to assess households’ exposure to An. stephensi. Prokopack aspirators and CDC light traps were used to collect adult mosquitoes, and standard dippers were used to collect immature stages. The collected mosquitoes were identified to species level by morphological keys and molecular methods. PCR assays were used to assess Plasmodium infection and mosquito blood meal source.ResultsCatches of adult An. stephensi were generally low (mean: 0.15 per trap), with eight positive sites among the 26 surveyed. This mosquito species was reported for the first time in Assosa, western Ethiopia. Anopheles stephensi was the predominant species in four of the eight positive sites, accounting for 75–100% relative abundance of the adult Anopheles catches. Household-level exposure, defined as the percentage of households with a peridomestic presence of An. stephensi, ranged from 18% in Metehara to 30% in Danan. Anopheles arabiensis was the predominant species in 20 of the 26 sites, accounting for 42.9–100% of the Anopheles catches. Bovine blood index, ovine blood index and human blood index values were 69.2%, 32.3% and 24.6%, respectively, for An. stephensi, and 65.4%, 46.7% and 35.8%, respectively, for An. arabiensis. None of the 197 An. stephensi mosquitoes assayed tested positive for Plasmodium sporozoite, while of the 1434 An. arabiensis mosquitoes assayed, 62 were positive for Plasmodium (10 for P. falciparum and 52 for P. vivax).ConclusionsThis study shows that the geographical range of An. stephensi has expanded to western Ethiopia. Strongly zoophagic behavior coupled with low adult catches might explain the absence of Plasmodium infection. The level of household exposure to An. stephensi in this study varied across positive sites. Further research is needed to better understand the bionomics and contribution of An. stephensi to malaria transmission.Graphical

Quantification of sporozoite expelling by Anopheles mosquitoes infected with laboratory and naturally circulating P. falciparum gametocytes

It is currently unknown whether all Plasmodium falciparum-infected mosquitoes are equally infectious. We assessed sporogonic development using cultured gametocytes in the Netherlands and naturally circulating strains in Burkina Faso. We quantified the number of sporozoites expelled into artificial skin in relation to intact oocysts, ruptured oocysts, and residual salivary gland sporozoites. In laboratory conditions, higher total sporozoite burden was associated with shorter duration of sporogony (p<0.001). Overall, 53% (116/216) of infected Anopheles stephensi mosquitoes expelled sporozoites into artificial skin with a median of 136 expelled sporozoites (interquartile range [IQR], 34–501). There was a strong positive correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.8; p<0.0001) and a weaker positive correlation between salivary gland sporozoite load and number of sporozoites expelled (ρ = 0.35; p=0.0002). In Burkina Faso, Anopheles coluzzii mosquitoes were infected by natural gametocyte carriers. Among salivary gland sporozoite positive mosquitoes, 89% (33/37) expelled sporozoites with a median of 1035 expelled sporozoites (IQR, 171–2969). Again, we observed a strong correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.9; p<0.0001) and a positive correlation between salivary gland sporozoite load and the number of sporozoites expelled (ρ = 0.7; p<0.0001). Several mosquitoes expelled multiple parasite clones during probing. Whilst sporozoite expelling was regularly observed from mosquitoes with low infection burdens, our findings indicate that mosquito infection burden is positively associated with the number of expelled sporozoites. Future work is required to determine the direct implications of these findings for transmission potential.

Quantification of sporozoite expelling by Anopheles mosquitoes infected with laboratory and naturally circulating P. falciparum gametocytes.

It is currently unknown whether all Plasmodium falciparum-infected mosquitoes are equally infectious. We assessed sporogonic development using cultured gametocytes in the Netherlands and naturally circulating strains in Burkina Faso. We quantified the number of sporozoites expelled into artificial skin in relation to intact oocysts, ruptured oocysts, and residual salivary gland sporozoites. In laboratory conditions, higher total sporozoite burden was associated with shorter duration of sporogony (p<0.001). Overall, 53% (116/216) of infected Anopheles stephensi mosquitoes expelled sporozoites into artificial skin with a median of 136 expelled sporozoites (interquartile range [IQR], 34-501). There was a strong positive correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.8; p<0.0001) and a weaker positive correlation between salivary gland sporozoite load and number of sporozoites expelled (ρ = 0.35; p=0.0002). In Burkina Faso, Anopheles coluzzii mosquitoes were infected by natural gametocyte carriers. Among salivary gland sporozoite positive mosquitoes, 89% (33/37) expelled sporozoites with a median of 1035 expelled sporozoites (IQR, 171-2969). Again, we observed a strong correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.9; p<0.0001) and a positive correlation between salivary gland sporozoite load and the number of sporozoites expelled (ρ = 0.7; p<0.0001). Several mosquitoes expelled multiple parasite clones during probing. Whilst sporozoite expelling was regularly observed from mosquitoes with low infection burdens, our findings indicate that mosquito infection burden is positively associated with the number of expelled sporozoites. Future work is required to determine the direct implications of these findings for transmission potential.

Detection of Invasive Anopheles stephensi Mosquitoes through Molecular Surveillance, Ghana.

The invasive Anopheles stephensi mosquito has rapidly expanded in range in Africa over the past decade. Consistent with World Health Organization guidelines, routine entomologic surveillance of malaria vectors in Accra, Ghana, now includes morphologic and molecular surveillance of An. stephensi mosquitoes. We report detection of An. stephensi mosquitoes in Ghana.