Preliminary monitoring of knockdown resistance (kdr) mutation in Anopheles stephensi: insights from a malarious area in Southeastern Iran.

BACKGROUND: Anopheles stephensi is recognized as the main malaria vector in Iran. In recent years, resistance to several insecticide classes, including organochlorine, pyrethroids, and carbamate compounds, has been reported for this medically important malaria vector. The main objective of the present study was to evaluate the insecticide susceptibility status of An. stephensi collected from the southern part of Iran, and to clarify the mechanism of resistance, using bioassay tests and molecular methods comparing the sequence of susceptible and resistant mosquitoes. METHODS: Mosquito larvae were collected from various larval habitats across six different districts (Gabrik, Sardasht, Tidar, Dehbarez, Kishi and Bandar Abbas) in Hormozgan Provine, located in the southern part of Iran. From each district standing water areas with the highest densities of Anopheles larvae were selected for sampling, and adult mosquitoes were reared from them. Finally, the collected mosquito species were identified using valid keys. Insecticide susceptibility of An. stephensi was tested using permethrin 0.75%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, and DDT 4%, following the World Health Organization (WHO) test procedures for insecticide resistance monitoring. Additionally, knockdown resistance (kdr) mutation in the voltage-gated sodium channel (vgsc) gene was sequenced and analysed among resistant populations to detect possible molecular mechanisms of observed resistance phenotypes. RESULTS: The susceptibility status of An. stephensi revealed that resistance to DDT and permethrin was found in all districts. Furthermore, resistance to all tested insecticides in An. stephensi was detected in Gabrik, Sardasht, Tidar, and Dehbarez. Analysis of knockdown resistance (kdr) mutations at the vgsc did not show evidence for the presence of this mutation in An. stephensi. CONCLUSION: Based on the results of the current study, it appears that in An. stephensi from Hormozgan Province (Iran), other resistance mechanisms such as biochemical resistance due to detoxification enzymes may be involved due to the absence of the kdr mutation or non-target site resistance. Further investigation is warranted in the future to identify the exact resistance mechanisms in this main malaria vector across the country.

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 nanograms 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.

Leech, potato, and tomato carboxypeptidase inhibitors against Anopheles stephensi carboxypeptidase B1 and B2.

Carboxypeptidase B (CPB) in Anopheles spp. breaks down blood and releases free amino acids, which promote Plasmodium sexual development in the mosquito midgut. Our goal was to computationally assess the inhibitory effectiveness of carboxypeptidase inhibitors obtained from tomato, potato (CPiSt), and leech against the Anopheles stephensi CPBAs1 and CPBAs2 enzymes. The tertiary structures of CPB inhibitors were predicted and their interaction mode with CPBAs1 and CPBAs2 were examined using molecular docking. Next, this data was compared with four licensed medications that are known to reduce the Anopheles' CPB activity. Molecular dynamics simulations were used to evaluate the stability of complexes containing CPiSt and its mutant form. Both CPiSt and its mutant form showed promise as possible candidates for further evaluations in the paratransgenesis technique for malaria control, based on the similar bindings of CPiSt and CPiSt-Mut to the active sites of CPBAs1 and CPBAs2, as well as their binding affinity in comparison to the drugs.

Invasive Anopheles stephensi in Africa: insights from Asia.

Anopheles stephensi is a highly competent urban malaria vector species, endemic in South Asia and the Persian Gulf, which has colonised eight countries in sub-Saharan Africa (SSA) since 2013 and is now spreading uncontrollably. In urban areas of Africa, where malaria transmission has previously been low or non-existent, the invasion of An. stephensi represents a significant problem, particularly to immunologically naïve populations. Despite this rapidly advancing threat, there is a paucity of information regarding the bionomics of An. stephensi in SSA. Here, we offer a critical synthesis of literature from An. stephensi's native range, focusing on the future of An. stephensi in a rapidly urbanising Africa, and highlighting key questions that warrant prioritisation by the global malaria vector control community.

Polyols induce acute oxidative stress and mortality in Indian malaria vector Anopheles stephensi (Diptera: Culicidae): potential for use as sugar-cum-toxin source in toxic sugar baits.

BACKGROUND: Development of insecticide resistance in the major malaria vectors has necessitated the development of novel vector control tools. One such strategy involves the use of toxic sugar baits that targets the sugar-feeding behaviour of mosquito vectors. In this study, we investigated the potential of polyols, as a toxic food (sugar) source in toxic sugar baits against the malaria vector Anopheles stephensi Liston. We examined the acute toxicity of six polyols, namely, erythritol, glycerol, mannitol, propylene glycol (PG), sorbitol, and xylitol on adult female An. stephensi mosquitoes at two different concentrations - 2% and 10%. We also studied changes in fecundity, egg hatchability and mid-gut peroxide levels induced by polyol exposure. RESULTS: Among the six polyol compounds tested, PG was most toxic and lethal followed by glycerol and erythritol (P < 0.001) compared to the control (sucrose). PG induced acute mortality at different tested concentrations. In the erythritol- and glycerol-fed groups, a dose-dependent effect on mortality was observed. Glycerol evidently reduced fecundity and egg-hatchability in gonotrophic cycles G1 and G2. Sucrose was the preferred food source (48%), followed by erythritol (18%), PG (10%) and glycerol (8%). Ingestion of polyols increased peroxide levels in mosquito guts, which persisted for extended durations ultimately resulting in rapid mortality (P < 0.05). CONCLUSION: The present study highlights the usefulness of sugar polyols for the development of toxic sugar baits with minimal yet effective ingredients. Further research could be focused on field experiments and on the exploration of synergistic effects of different polyols for optimization of field applications. © 2024 Society of Chemical Industry.

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.

A decade of invasive Anopheles stephensi sequence-based identification: toward a global standard.

Anopheles stephensi is an invasive malaria vector in Africa that has been implicated in malaria outbreaks in the Horn of Africa. In 10 years, it has been detected as far east as Djibouti and as far west as Ghana. Early detections were mostly incidental, but now active surveillance in Africa has been updated to include An. stephensi. Morphological identification of An. stephensi from native vectors can be challenging, thus, sequence-based assays have been used to confirm identification during initial detections. Methods of sequence-based identification of An. stephensi have varied across initial detections to date. Here, we summarize initial detections, make suggestions that could provide a standardized approach, and discuss how sequences can inform additional genomic studies beyond species identification.

Searching for new molecules involved in Anopheles mosquitoes' response to Plasmodium infection.

Plasmodium parasites within mosquitoes are exposed to various physiological processes, such as blood meal digestion activity, the gonotrophic cycle, and host responses preventing the entry of parasites into the midgut wall. However, when in vitro-cultured ookinetes are injected into the hemocoel of mosquitoes, Plasmodium parasites are not affected by the vertebrate host's blood contents and do not pass through the midgut epithelial cells. This infection method might aid in identifying mosquito-derived factors affecting Plasmodium development within mosquitoes. This study investigated novel mosquito-derived molecules related to parasite development in Anopheles mosquitoes. We injected in vitro-cultured Plasmodium berghei (ANKA strain) ookinetes into female and male Anopheles stephensi (STE2 strain) mosquitoes and found that the oocyst number was significantly higher in males than in females, suggesting that male mosquitoes better support the development of parasites. Next, RNA-seq analysis was performed on the injected female and male mosquitoes to identify genes exhibiting changes in expression. Five genes with different expression patterns between sexes and greatest expression changes were identified as being potentially associated with Plasmodium infection. Two of the five genes also showed expression changes with infection by blood-feeding, indicating that these genes could affect the development of Plasmodium parasites in mosquitoes.

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

BACKGROUND: Malaria 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. METHODS: Entomological 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. RESULTS: Catches 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). CONCLUSIONS: This 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.

Biocontrol efficacy of cajeput oil against Anopheles stephensi L. mosquito and its effect on non-target species.

Chemical insecticides are effective at controlling mosquito populations, but their excessive use can pollute the environment and harm non-target organisms. Mosquitoes can also develop resistance to these chemicals over time, which makes long-term mosquito control efforts challenging. In this study, we assessed the phytochemical, biochemical, and insecticidal properties of the chemical constituents of cajeput oil. Results show that Melaleuca cajuputi essential oil may exhibit mosquito larvicidal properties against Anopheles stephensi larvae (second-fourth instar) at 24 h post-treatment. At 24 h post-exposure, the essential oil resulted in a significant decrease in detoxifying enzymes. All of these findings indicate that cajeput oil infects An. stephensi larvae directly affect the immune system, leading to decreased immune function. Cajeput oil significantly affects the second, third, and fourth instar larvae of An. stephensi, according to the bioassay results. Cajeput oil does not induce toxicity in non-target Eudrilus eugeniae earthworm species, as indicated by a histological study of earthworms. Phytochemical screening and GC-MS analysis of the essential oil revealed the presence of several major phytochemicals that contribute to mosquito larvicidal activity. The importance of cajeput oil as an effective candidate for biological control of the malarial vector An. stephensi is supported by this study.

Chemical Exposure of Synthetic Pyrethroid on Deltamethrin Under the Selection Pressure over the Generations: A Reproductive Potential Study of Anopheles stephensi.

Biochemical synthetic pyrethroids, deltamethrin are presently used insecticides for the control of mosquito vector-borne diseases in worldwide. Mosquito re-emergence with diseases becoming a serious problem due to development of insecticide resistance. The comprehensive knowledge on the underlying mechanisms of resistance against deltamethrin is required for implementation of an efficient vector control programme. The assessment of the biological fitness of a mosquito strain exposed to insecticide pressure is extremely vital because it provides information on the development of resistance. In the present study, the adult stage of malaria vector, Anopheles stephensi, was designated for the study of deltamethrin resistance (F40 generations). The non-blood-fed, laboratory-reared females to sub-lethal doses of deltamethrin (0.004%, 0.005%, 0.007%, or 0.01%) exposed to every generation for up to F40. The adult mosquito susceptibility was performed by WHO standard method for evaluation. After 24 h, mortality was recorded in both treated and control groups. Therefore, the biological fitness characteristics such as feeding, fecundity, hatchability, egg retention, immature duration, adult emergence, and adult life span were studied to assess the exposed deltamethrin under selection pressure as compared to the unexposed (control) population. The laboratory selection of An. stephensi exposed deltamethrin over the generations were diminished its biological fitness. Information on biological fitness including reproductive potential of mosquito strain under selection pressure against deltamethrin is incredibly necessary because it would facilitate in resistance management. Baseline information gives in this experiment will guide for future studies on the susceptibilities of wild malaria mosquito populations in India.

Possible potential spread of Anopheles stephensi, the Asian malaria vector.

BACKGROUND: Anopheles stephensi is native to Southeast Asia and the Arabian Peninsula and has emerged as an effective and invasive malaria vector. Since invasion was reported in Djibouti in 2012, the global invasion range of An. stephensi has been expanding, and its high adaptability to the environment and the ongoing development of drug resistance have created new challenges for malaria control. Climate change is an important factor affecting the distribution and transfer of species, and understanding the distribution of An. stephensi is an important part of malaria control measures, including vector control. METHODS: In this study, we collected existing distribution data for An. stephensi, and based on the SSP1-2.6 future climate data, we used the Biomod2 package in R Studio through the use of multiple different model methods such as maximum entropy models (MAXENT) and random forest (RF) in this study to map the predicted global An. stephensi climatically suitable areas. RESULTS: According to the predictions of this study, some areas where there are no current records of An. stephensi, showed significant areas of climatically suitable for An. stephensi. In addition, the global climatically suitability areas for An. stephensi are expanding with global climate change, with some areas changing from unsuitable to suitable, suggesting a greater risk of invasion of An. stephensi in these areas, with the attendant possibility of a resurgence of malaria, as has been the case in Djibouti. CONCLUSIONS: This study provides evidence for the possible invasion and expansion of An. stephensi and serves as a reference for the optimization of targeted monitoring and control strategies for this malaria vector in potential invasion risk areas.

Impact of climate change on temperature variations and extrinsic incubation period of malaria parasites in Chennai, India: implications for its disease transmission potential.

BACKGROUND: The global temperature has significantly risen in the past century. Studies have indicated that higher temperature intensifies malaria transmission in tropical and temperate countries. Temperature fluctuations will have a potential impact on parasite development in the vector Anopheles mosquito. METHODS: Year-long microclimate temperatures were recorded from a malaria-endemic area, Chennai, India, from September 2021 to August 2022. HOBO data loggers were placed in different vector resting sites including indoor and outdoor roof types. Downloaded temperatures were categorised by season, and the mean temperature was compared with data from the same study area recorded from November 2012 to October 2013. The extrinsic incubation period for Plasmodium falciparum and P. vivax was calculated from longitudinal temperatures recorded during both periods. Vector surveillance was also carried out in the area during the summer season. RESULTS: In general, temperature and daily temperature range (DTR) have increased significantly compared to the 2012-2013 data, especially the DTR of indoor asbestos structures, from 4.30 â„ƒ to 12.62 â„ƒ in 2021-2022, unlike the marginal increase observed in thatched and concrete structures. Likewise, the average DTR of outdoor asbestos structures increased from 5.02 â„ƒ (2012-2013) to 8.76 â„ƒ (2021-2022) although the increase was marginal in thatched structures and, surprisingly, showed no such changes in concrete structures. The key finding of the extrinsic incubation period (EIP) is that a decreasing trend was observed in 2021-2022 compared to 2012-2013, mainly in indoor asbestos structures from 7.01 to 6.35 days, which negatively correlated with the current observation of an increase in temperature. Vector surveillance undertaken in the summer season revealed the presence of Anopheles breeding in various habitats. Anopheles stephensi could be collected using CDC light traps along with other mosquito species. CONCLUSION: The microclimate temperature has increased significantly over the years, and mosquitoes are gradually adapting to this rising temperature. Temperature negatively correlates with the extrinsic incubation period of the parasite. As the temperature increases, the development of the parasite in An. stephensi will be faster because of a decrease in EIP, thus requiring relatively fewer days, posing a risk for disease transmission and a hindrance to malaria elimination efforts.

An Investigation into the Larvicidal Activity of Biologically Synthesized Silver and Copper Oxide Nanoparticles Against Mosquito Larvae.

This study is primarily focused on the synthesis of silver and copper oxide nanoparticles utilizing the extract of Ipomoea staphylina plant and their larvicidal activity against specific larvae. Notably, Anopheles stephensi and Aedes aegypti are significant disease vectors responsible for transmitting diseases such as malaria, dengue fever, Zika virus, and chikungunya (Anopheles stephensi), and dengue and Zika (Aedes aegypti). These mosquitoes have a substantial impact on urban areas, influencing disease transmission dynamics. In an effort to control these larvae, we have pursued the synthesis of a herbal-based nanomedicine derived from I. staphylina, a valuable herb in traditional medicine. Our successful synthesis of silver and CuO nanoparticles followed environmentally sustainable green chemistry methodologies. The I. staphylina plant extract played a dual role as a reducing agent and dopant, aligning with principles of sustainability. We employed X-ray diffraction (XRD) analysis to validate the nanoparticle structure and size, while field-emission scanning electron microscopy (FE-SEM) revealed well-defined nanostructures. Elemental composition was determined through energy-dispersive X-ray (EDX) analysis, and UV-visible spectroscopy provided insights into the bandgap energy (3.15 eV for silver, 1.2 eV for CuO nanoparticles). These nanoparticles exhibited robust larvicidal activity, with CuO nanoparticles surpassing silver nanoparticles in terms of LC50 and LC90 values. Moreover, the developmental toxicity of CuO and Ag NPs was evaluated in zebrafish embryos as part of non-target eco-toxicological studies conducted in a standard laboratory environment. These findings underscore the potential utility of these nanoparticles as highly effective and environmentally friendly natural pesticides, offering cost-effectiveness and ecological benefits.

Artificial nighttime lighting impacts Plasmodium falciparum mature stage V gametocytes infectivity in Anopheles stephensi.

BACKGROUND: Malaria is one of the most important vector-borne diseases of humans with an estimated 241 million cases worldwide in 2020. As an urban and periurban mosquito species, Anopheles stephensi is exposed to artificial human stimuli like light that can alter many aspects of mosquito behaviour, physiology and metabolism. Therefore, fluctuations in the light environment may influence the host, parasite and/or mosquito biology and hence modulate risk for disease transmission. In this study, the effect of artifitial light at night on mosquito infectivity by Plasmodium falciparum during the first hours of blood digestion was tested. METHODS: A total of three independent standard membrane feeding assays were performed to artificially fed septic and aseptic mosquitoes with P. falciparum infected blood. After blood feeding, females were transferred to incubators with different photoperiod cycles, so digestion occurred under day artificial light or dark. At 7 and 16 days post blood feeding, mosquitoes were dissected for midguts and salivary glands, respectively. Percentage of mosquitoes fed, percentage of prevalence and P. falciparum oocyst intensity between septic and aseptic mosquitoes in the two different photoperiod regimes, were compared using a Kruskal-Wallis test followed by a Dunn´s multiple comparison test . RESULTS: The exposition of mosquitoes to light after they took an infected blood meal has a negative effect on the successful progression of P. falciparum in the mosquito midgut. Antibiotic treatment significantly incremented the number of oocysts per midgut. Photophase significantly reduced the median oocyst intensity in both septic and aseptic mosquitoes. The percentage of oocyst reduction, understood as the percentage of reduction in the mean oocyst intensity of the parasite in the mosquito midgut between photophase and scotophase, was 51% in the case of aseptic mosquitoes and 80% for septic mosquitoes, both in the photophase condition. CONCLUSION: Although there are still many gaps in the understanding of parasite-mosquito interactions, these results support the idea that light can, not only, influence mosquito biting behaviour but also parasite success in the mosquito midgut. Hence, light can be considered an interesting additional mosquito-control strategy to reduce mosquito-borne diseases.

Automated differentiation of mixed populations of free-flying female mosquitoes under semi-field conditions.

Great advances in automated identification systems, or 'smart traps', that differentiate insect species have been made in recent years, yet demonstrations of field-ready devices under free-flight conditions remain rare. Here, we describe the results of mixed-species identification of female mosquitoes using an advanced optoacoustic smart trap design under free-flying conditions. Point-of-capture classification was assessed using mixed populations of congeneric (Aedes albopictus and Aedes aegypti) and non-congeneric (Ae. aegypti and Anopheles stephensi) container-inhabiting species of medical importance. Culex quinquefasciatus, also common in container habitats, was included as a third species in all assessments. At the aggregate level, mixed collections of non-congeneric species (Ae. aegypti, Cx. quinquefasciatus, and An. stephensi) could be classified at accuracies exceeding 90% (% error = 3.7-7.1%). Conversely, error rates increased when analysing individual replicates (mean % error = 48.6; 95% CI 8.1-68.6) representative of daily trap captures and at the aggregate level when Ae. albopictus was released in the presence of Ae. aegypti and Cx. quinquefasciatus (% error = 7.8-31.2%). These findings highlight the many challenges yet to be overcome but also the potential operational utility of optoacoustic surveillance in low diversity settings typical of urban environments.

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.

Aeroplane wing, a new recessive autosomal phenotypic marker in the malaria vector, Anopheles stephensi Liston.

A novel and distinct mutant with a phenotype, aeroplane wing (ae) is reported for the first time in the urban malaria vector Anopheles stephensi. The main aim of this study was to establish the mode of inheritance of the ae gene performing genetic crossings between the mutants and wild types. These mutants show extended open wings that are visible to naked eyes in both the sexes. Mutants were first noticed in a nutritionally stressed isofemale colony. Strategic genetic crosses revealed that the ae gene is a recessive, autosomal, and monogenic trait having full penetrance with uniform expression in its adult stage. Egg morphometric analysis confirmed that these mutants were intermediate variant. No significant differences were observed in the wing venation and size of ae mutants compared to their control parental lines. Further cytogenetic analysis on the ovarian polytene chromosome of ae mutant showed an inversion (3Li) on the 3L arm like its parental line. This ae mutant would be a prominent marker and could be useful to study the functions of related specific genes within its genome.

Anopheles stephensi ecology and control in Africa.

The encroachment and rapid spread of Anopheles stephensi across Africa presents a significant challenge to malaria control and elimination efforts. Understanding the ecology and behavior of An. stephensi will critically inform control measures and provide prerequisite knowledge for exploring new larval and adult control tools to contain its spread.