Anopheles Stephensi Research Articles

Eco-friendly synthesis of silver nanoparticles using Phyllanthus niruri leaf extract: Assessment of antimicrobial activity, effectiveness on tropical neglected mosquito vector control, and biocompatibility using a fibroblast cell line model

Abstract Mosquitoes are rapidly advancing as vectors of several severe diseases. The increasing resistance of mosquitoes and the environmental harm caused by insecticides pose significant challenges for eradicating mosquito vectors. In this study, 18 plant extracts were tested for larvicidal properties against Aedes aegypti, Culex quinquefasciatus, and Anopheles stephensi larvae. Phyllanthus niruri (Pn) showed enhanced larvicidal activity in both laboratory and field trials. The biosynthesis of silver nanoparticles (AgNPs) using Pn leaf methanol extract (Pn-LME) was confirmed by UV-visible spectroscopy, X-ray diffraction, transmission electron microscopy, scanning electron microscopy, and Fourier-transform infrared spectroscopy. Among various concentrations, 3 mM AgNPs exhibited significant LC90 values of 0.83, 1.46, and 9.11 ppm compared to 9.25, 93.48, and 14.60 ppm of Pn-LME against A. stephensi, C. quinquefasciatus, and A. aegypti, respectively. This indicates the high mortality of mosquito vectors at low AgNP concentrations. Additionally, Pn-AgNPs showed enhanced antibacterial activity and no cytotoxicity in normal fibroblast cells (L929). Field trials demonstrated a 98.70% decrease in mosquito larval density at A. stephensi breeding sites, a 96.55% reduction at C. quinquefasciatus sites, and a 97.85% reduction at Ae. aegypti sites. This study presents an eco-friendly and cost-effective AgNP bio-pesticide synthesized from Pn leaves for controlling and preventing the transmission of filarial, dengue, and malaria vectors.

A quantitative and systematic analysis of Anopheles stephensi bionomics and control approaches

Anopheles stephensi is a mosquito endemic to South Asia and the Arabian Peninsula that has recently been detected in eight African countries, posing a significant threat to global malaria control efforts. A challenge with An. stephensi is that it requires unique surveillance and control tools when compared to other malaria vectors. Through a systematic literature review, we investigated the efficacy of trapping methods and controls for An. stephensi mosquitoes, with a focus on studies of its behavior and biology. Data from 83 articles (native range: Afghanistan, India, Iran, Iraq, Pakistan, and Qatar; invasive range: Djibouti, Ethiopia, Sri Lanka, Saudi Arabia, and Sudan) met our study inclusion criteria. Data from these studies revealed that using host-seeking animal baited traps increased the number of mosquitoes collected per trap per day in the native range when compared to host-seeking human baited traps. However, these differences were not present in data collection rate assessments from the invasive range. We also found that An. stephensi equally used a large variety of breeding habitats in the native range, but that it tended to prefer water reservoirs and wastewater in the invasive range. Finally, we found that temephos, fenthion, Bacillus thuringiensis israelensis, and Beauveri bassiana were more effective at reducing larvae in their native range compared to the approaches found in our systematic literature search, but the relative effectiveness of these approaches in the invasive range was less clear. Understanding proven historical surveillance and control approaches is essential to the advancement of invasive An. stephensi mitigation efforts, but continued investigations in the invasive range are critical to reducing the impacts of malaria morbidity and mortality.

Host 5-HT affects Plasmodium transmission in mosquitoes via modulating mosquito mitochondrial homeostasis.

Malaria parasites hijack the metabolism of their mammalian host during the blood-stage cycle. Anopheles mosquitoes depend on mammalian blood to lay eggs and to transmit malaria parasites. However, it remains understudied whether changes in host metabolism affect parasite transmission in mosquitoes. In this study, we discovered that Plasmodium infection significantly decreased the levels of the tryptophan metabolite, 5-hydroxytryptamine (5-HT), in both humans and mice. The reduction led to the decrease of 5-HT in mosquitoes. Oral supplementation of 5-HT to Anopheles stephensi enhanced its resistance to Plasmodium berghei infection by promoting the generation of mitochondrial reactive oxygen species. This effect was due to the accumulation of dysfunctional mitochondria caused by 5-HT-mediated inhibition of mitophagy. Elevating 5-HT levels in mouse serum significantly suppressed parasite infection in mosquitoes. In summary, our data highlight the critical role of metabolites in animal blood in determining the capacity of mosquitoes to control parasite infection.

Predation Efficiency of Non-Blood Sucking Mosquito Larvae of Toxorhynchites splendens (Weidman) Determined by Kruskal–Wallis One-Way Analysis of Variance by Ranks over Immature Stages of Malaria (Anopheles stephensi), Filariasis (Culex quinquefasciatus)

Predation Efficiency of Non-Blood Sucking Mosquito Larvae of Toxorhynchites splendens (Weidman) Determined by Kruskal–Wallis One-Way Analysis of Variance by Ranks over Immature Stages of Malaria (Anopheles stephensi), Filariasis (Culex quinquefasciatus)

Urban malaria and its determinants in Eastern Ethiopia: the role of Anopheles stephensi and urbanization

BackgroundMalaria prevention and control strategies have been hampered by urbanization and the spread of Anopheles stephensi. The spread of this vector into Africa further complicates the already complex malaria situation, that could put about 126 million Africans at risk of infection. Hence, this study aimed to assess the determinants of urban malaria, focusing on the role of urbanization and the distribution of An. stephensi in Eastern Ethiopia.MethodsA matched case control study was conducted among febrile urban residents of Dire Dawa (malaria positive as cases and negative as a control). A capillary blood sample was collected for parasite identification using microscopic examination and an interviewer administered questionnaire was used to collect additional data. Centers for Disease Control and Prevention miniature light traps (CDC-LT) and Prokopack aspirator were used to collect adult mosquito vectors from the selected cases and control houses to identify the mosquito vector species. Then, the data were exported to STATA for analysis. Conditional logistic regression was done to identify determinants, and principal component Analysis (PCA) was done for some independent variables.ResultsThis study enrolled 132 cases and 264 controls from urban setting only. Of the 132 cases, 90 cases were positive for Plasmodium falciparum, 34 were positive for Plasmodium vivax and 8 had mixed infections. All cases and controls were similar with regard to their respective age and sex. Travel history (AOR: 13.1, 95% CI 2.8–61.4), presence of eves and holes on walls (AOR: 2.84, 95% CI 1.5–5.5), history of malaria diagnosis (AOR: 2.4, 95% CI 1.1–5.3), owning any livestock (AOR: 7.5, 95% CI 2.4–22.8), presence of stagnant water in the area (AOR: 3.2, 95% CI 1.7–6.1), sleeping under bed net the previous night (AOR: 0.21, 95% CI 0.1–0.6) and knowledge on malaria and its prevention (AOR: 2.2, 95% CI 1.2–4.1) were determinants of urban malaria infection. About 34 adult Anopheles mosquitoes were collected and identified from those selected cases and control houses and 27 of them were identified as An. stephensi.ConclusionAmong the cases, the dominant species were P. falciparum. This study identified travel history, house condition, past infection, livestock ownership, stagnant water, bed net use, and malaria knowledge as determinants of infection. This study also found the dominance of the presence of An. stephensi among the collected mosquito vectors. This suggests that the spread of An. stephensi may be impacting malaria infection in the study area. Hence, strengthening urban-targeted malaria interventions should be enhanced to prevent and control further urban malaria infection and spread.

Insecticidal effect of Cannabis

Currently, there is a need for the development of vector insect control products that are ecologically viable, utilizing active principles available in nature. Natural compounds with insecticidal potential already identified in Cannabis are promising prospects for the control and management of vector arthropods of diseases. In this context, the work aims to investigate possible disparities in the toxicity effects of Cannabis in different formulations and at different stages of the vectors' life. This is a systematic review. Experimental studies describing the toxic effects of Cannabis on eggs, larvae, pupae, and adult vector insects were included. Articles in which the insect was characterized as a mechanical vector, literature review research, and ethnobotanical studies were excluded. The study revealed the potential insecticidal effect of Cannabis when applied in different formulations on Ctenocephalides felis felis, Aedes albopictus, Anopheles stephensi, Anopheles gambiae, and Culex quinquefasciatus at different stages of development. Cannabis proved to be a promising plant in terms of mortality and evidenced effects on insect fertility, birth rates, and adult emergence. The study revealed a potential insecticidal effect of Cannabis. However, further investigations are crucial to elucidate the role of this plant in vector insect control strategies.

Dihydroartemisinin suppresses the susceptibility of Anopheles stephensi to Plasmodium yoelii by activating the Toll signaling pathway

BackgroundMalaria is a serious public health concern. Artemisinin and its derivatives are first-line drugs for the treatment of Plasmodium falciparum malaria. In mammals, artemisinin exhibits potent anti-inflammatory and immunoregulatory properties. However, it is unclear whether artemisinin plays a regulatory role in the innate immunity of mosquitoes, thereby affecting the development of Plasmodium in Anopheles when artemisinin and its metabolites enter mosquitoes. This study aims to determine the effect of dihydroartemisinin (DHA), a first-generation semisynthetic derivative of artemisinin, on innate immunity and malaria vector competence of Anopheles stephensi.MethodsAnopheles stephensi was fed Plasmodium-infected mice treated with DHA via gavage, Plasmodium-infected blood containing DHA in vitro, or DHA-containing sugar, followed by Plasmodium yoelii infection. The engorged female mosquitoes were separated and dissected 8 and 17 days after infection. Plasmodium oocysts and sporozoites were counted and compared between the control and DHA-treated groups. Additionally, total RNA and proteins were extracted from engorged mosquitoes 24 and 72 h post infection (hpi). Real-time polymerase chain reaction (PCR) and western blotting were performed to detect the transcriptional levels and protein expression of immune molecules in mosquitoes. Finally, the Toll signaling pathway was inhibited via RNA interference and the infection density was analyzed to confirm the role of the Toll signaling pathway in the effect of DHA on the vector competence of mosquitoes.ResultsDHA treatment via different approaches significantly reduced the number of Plasmodium oocysts and sporozoites in mosquitoes. The transcriptional levels of anti-Plasmodium immune genes (including TEP1, LRIM1, and APL1C), Toll pathway genes (including Tube, MyD88, and Rel1), and the effector defensin 1 were upregulated by DHA treatment at 24 and 72 hpi. TEP1 and Rel1 protein expression was significantly induced under DHA treatment. However, Rel1 knockdown in DHA-treated mosquitoes abrogated DHA-mediated refractoriness to Plasmodium infection.ConclusionsDHA treatment effectively inhibited the development of P. yoelii in A. stephensi by upregulating mosquitoes’ Toll signaling pathway, thereby influencing the susceptibility of Anopheles to Plasmodium.Graphical

Identifying the sex chromosome and sex determination genes in the cattle tick, Rhipicephalus (Boophilus) microplus.

Rhipicephalus (Boophilus) microplus is globally one of the most economically important ectoparasites of cattle costing the agriculture industry billions of dollars annually. Resistance to chemical control measures has prompted the development of novel methods of control. Recent advancements in genetic control measures for human and other animal vectors have utilized sex determination research to manipulate sex ratios, which have shown promising results in mosquitoes namely Aedes aegypti and Anopheles stephensi. Here, we use R. (B.) microplus as a model to provide foundational research to allow similar avenues of investigation in ticks using R. (B.) microplus as a model. Karyotypes for R. (B.) microplus show an XX:XO sex determining system with the largest chromosome being the sex chromosome. Using flow cytometric methods, the size of the sex chromosome was estimated at 526.91 Mb. All measures to identify the sex chromosome within the cattle tick genome assembly associated sex chromosomal characteristics to two chromosomes. This discrepancy between the assembly and karyotypes of the tick led to generating a new genome assembly with a single adult male specimen. The two chromosomes in question aligned with a single scaffold within the new genome that had a length of 513.29 Mb and was the first time the sex chromosome was identified in an Ixodid genome assembly.

Stable introduction of Wolbachia wPip into invasive Anopheles stephensi for potential malaria control.

The spread and invasion of the urban malaria vector Anopheles stephensi has emerged as a significant threat to ongoing malaria control and elimination efforts, particularly in Africa. The successful use of the maternally inherited endosymbiotic bacterium Wolbachia for arbovirus control has inspired the exploration of similar strategies for managing malaria vectors, necessitating the establishment of a stable Wolbachia-Anopheles symbiosis. In this study, we successfully transferred Wolbachia wPip into An. stephensi, resulting in the establishment of a stable transinfected HP1 line with 100% maternal transmission efficiency. We demonstrate that wPip in the HP1 line induces nearly complete unidirectional cytoplasmic incompatibility (CI) and maintains high densities in both somatic and germline tissues. Despite a modest reduction in lifespan and female reproductive capacity, our results suggest the Wolbachia infection in the HP1 line has little impact on life history traits, body size, and male mating competitiveness, as well as the ability of its larvae to tolerate rearing temperatures up to 38°C, although wPip densities moderately decrease when larvae are exposed to a constant 33°C and diurnal cyclic temperatures of 27-36°C and 27-38°C. These findings highlight the potential of the HP1 line as a robust candidate for further development in malaria control.

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.

A cub and sushi domain-containing protein with esterase-like activity confers insecticide resistance in the Indian malaria vector Anopheles stephensi

Chemical insecticides (organophosphates and pyrethroids) in the form of IRS (Indoor Residual Sprays) and LLINs (Long Lasting insecticidal nets) are the cornerstone for vector control, globally. However, their incessant use has resulted in widespread development of resistance in mosquito vectors, warranting continuous monitoring and investigation of the underlying mechanisms of resistance. Here, we identified a previously uncharacterized- Cub and Sushi Domain containing Insecticide Resistance (CSDIR) protein and generated evidence for its role in mediating insecticide resistance in the Anopheles stephensi. A strong binding affinity of the CSDIR protein towards different classes of insecticide molecules-malathion (KD 6.43 μM) and deltamethrin (KD 46.7μM) were demonstrated using MD simulation studies and Surface Plasmon Resonance (SPR) experiments. Further, the recombinant CSDIR913-1190 protein exhibited potent esterase-like activity (α-naphthyl acetate (α-NA)- 1.356±0.262 mM/min/mg and β-naphthyl acetate (β -NA)- 1.777±0.220 mM/min/mg). Interestingly, dsRNA-mediated gene silencing of the CSDIR transcripts caused >60% mortality in resistant An. stephensi upon 1-hour exposure to deltamethrin and malathion insecticides, compared to the control group. A significant reduction in the esterase-like activity was also observed against α-NA (P=0.004) and β-NA (P=0.025) in CSDIR silenced mosquitoes compared to the control group. Using computational analysis and experimental data, our results provided significant evidence of the involvement of the CSDIR protein in mediating insecticide resistance in Anopheles mosquitoes. Thereby making the CSDIR protein, a novel candidate for exploration of novel insecticide molecules. These data would also be helpful in further understanding the development of metabolic resistance by the Anopheles vector.

Fusarium begoniae metabolites: a promising larvicidal, pupicidal potential, histopathological alterations and detoxifications enzyme profiles of medically important mosquito vector Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi.

The online version contains supplementary material available at 10.1007/s13205-024-04061-z.

Anopheles jamesii Supports Plasmodium vivax Sporogony under Laboratory Conditions.

Anopheles jamesii, considered to be a non-malaria vector, is an abundant mosquito species found in coastal India and several countries in Asia. In Goa, western India, An. jamesii is the dominant anopheline species by numbers, and in recent epidemiological surveys, they have tested positive for Plasmodium carriage. To assess An. jamesii's capacity as a malaria vector, we carried out six controlled membrane feeding assays with the F1 generation of field-caught An. jamesii and Plasmodium vivax-infected patient blood samples. As a control, the established local vector, Anopheles stephensi, was fed in parallel to compare the oocyst and sporozoite loads. The average oocyst load of An. jamesii was 3.29, while for An. stephensi it was 10.23. Furthermore, An. jamesii's blood feeding rate (21.7% versus 85%), oocyst positivity rate (60% versus 90.6%), and sporozoite positivity (45.16% versus 83%) were significantly lower (P <0.05, unpaired t test) than those of An. stephensi, suggesting a recent adaptation to support Plasmodium sporogony.

Molecular characterization, expression and in-silico analysis of fibrinogen-related protein 1 (frep1) in malaria vector Anopheles stephensi.

Fibrinogen-related protein 1 (frep1) is a member of the pattern-recognizing receptor family (PRR) which generates an innate immune response after recognizing the pattern associated molecular pattern (PAMP) that occurs on the surface of microorganisms. The main objective of this study is to characterize frep1 and its in-silico analysis in Anopheles stephensi. The DNA was extracted from female Anopheles stephensi. PCR was performed for complete analysis of frep1 using specific primers. The gene sequence of frep1 was identified by Sanger sequencing. The bioinformatics structure analysis approach revealed the presence of 3 exons and 4 introns in the frep1. The sequence of frep1 was submitted to NCBI GeneBank with accession number ON817187.1. Quantitative real-time PCR was performed to analyze frep1 expression. At the developmental stage, frep1 is highly expressed in the L1 stage, egg, and adult female mosquito. In addition, frep1 is highly expressed in the tissue fat body, midgut, and salivary gland. After blood-fed, an upregulation of frep1 at 48h in the midgut, and downregulation in fat body were observed at different time intervals. The genomic data of frep1 is encoded by 12,443bp. The frep1 has a significant role in the early metamorphosis. Its expression in fat body and midgut suggests it could be important for fat metabolism and post-blood digestion. The conserved domain could be targeted for vector control. Further study is required to elucidate its function against malaria parasites to confirm its agonist role in malaria transmission.

Larvicidal potential of Trachyspermum ammi essential oil and Delphinium speciosum extract against malaria, dengue, and filariasis mosquito vectors

Mosquito-borne diseases, such as malaria, dengue, and Zika, pose major public health challenges globally, affecting millions of people. The growing resistance of mosquito populations to synthetic insecticides underscores the critical need for effective and environmentally friendly larvicides. Although chemical pesticides can initially be effective, they often lead to negative environmental consequences and health hazards for non-target species, including humans. This study aimed to evaluate the larvicidal effects of Trachyspermum ammi essential oil and Delphinium speciosum extract on the larvae of three major mosquito species: Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus. Mosquito larvae of Ae. aegypti, An. stephensi, and Cx. quinquefasciatus were reared under controlled laboratory conditions. The larvicidal activity of T. ammi essential oil and D. speciosum extract was evaluated through standard bioassays, using various concentrations of essential oils (10, 20, 40, 80, and 160 ppm) and extracts (160, 320, 640, 1280, and 2560 ppm) to determine the lethal concentration (LC50) values after 24 h of exposure. Fresh plant materials were collected, with the essential oil extracted via hydro-distillation, and the extract prepared using methanol solvent extraction. The chemical composition of T. ammi essential oil was examined using gas chromatography-mass spectrometry (GC-MS). Additionally, the preliminary analysis of the chemical compounds in D. speciosum extract was carried out using thin layer chromatography (TLC) and nuclear magnetic resonance spectroscopy (NMR) techniques. The results indicated that the essential oil of T. ammi exhibited more effective larvicidal activity compared to the D. speciosum extract. Specifically, the essential oil demonstrated LC50 values of 18 ppm for Cx. quinquefasciatus and 19 ppm for Ae. aegypti. In contrast, the D. speciosum extract showed the strongest larvicidal effect against An. stephensi, with an LC50 of 517 ppm. Concentrations of 40 ppm of the essential oil and 1280 ppm of the extract resulted in 100% mortality across all three species. Both the essential oil of T. ammi and the D. speciosum extract exhibited concentration-dependent larvicidal activity, and these results were statistically significant (p < 0.001) compared to the no-treatment group. GC-MS analysis revealed thymol (88.95%), o-cymen-5-ol (4.11%), and γ-terpinene (2.10%) as the major constituents of the T. ammi essential oil. Additionally, TLC verified the presence of alkaloids in both chloroform and methanolic extracts. Proton NMR identified a diterpene structure for these alkaloids. These findings suggest that T. ammi essential oil is a promising candidate for natural mosquito control strategies. Given its efficacy, further research is warranted to explore its potential in integrated vector management programs.

Investigating the association between household exposure to Anopheles stephensi and malaria in Sudan and Ethiopia: A case-control study protocol.

Endemic African malaria vectors are poorly adapted to typical urban ecologies. However, Anopheles stephensi, an urban malaria vector formerly confined to South Asia and the Persian Gulf, was recently detected in Africa and may change the epidemiology of malaria across the continent. Little is known about the public health implications of An. stephensi in Africa. This study is designed to assess the relative importance of household exposure to An. stephensi and endemic malaria vectors for malaria risk in urban Sudan and Ethiopia. Case-control studies will be conducted in 3 urban settings (2 in Sudan, 1 in Ethiopia) to assess the association between presence of An. stephensi in and around households and malaria. Cases, defined as individuals positive for Plasmodium falciparum and/or P. vivax by microscopy/rapid diagnostic test (RDT), and controls, defined as age-matched individuals negative for P. falciparum and/or P. vivax by microscopy/RDT, will be recruited from public health facilities. Both household surveys and entomological surveillance for adult and immature mosquitoes will be conducted at participant homes within 48 hours of enrolment. Adult and immature mosquitoes will be identified by polymerase chain reaction (PCR). Conditional logistic regression will be used to estimate the association between presence of An. stephensi and malaria status, adjusted for co-occurrence of other malaria vectors and participant gender. Findings from this study will provide evidence of the relative importance of An. stephensi for malaria burden in urban African settings, shedding light on the need for future intervention planning and policy development.

Feeding Behavior and Plasmodium Detection in Anopheles stephensi, a Malaria Vector in District Khyber, Khyber Pakhtunkhwa, Pakistan.

Anopheles stephensi is a significant malaria vector in Pakistan, and understanding its feeding behavior is necessary to control the spread of malaria. However, limited information is available on the host preferences of A. stephensi in Pakistan. Therefore, we aimed to explore the feeding behavior of A. stephensi, a malaria vector, in the District Khyber, Khyber Pakhtunkhwa, Pakistan. A total of 7462 mosquitoes were collected between March and September 2021, with 1674 (22.4%) identified as A. stephensi (952 female and 722 male). Among the female A. stephensi, 495 (52%) were blood-fed. DNA was extracted from the blood-fed female A. stephensi mosquitoes using the Ammonium Acetate Precipitation Method followed by PCR analysis, blood meal sources were identified. Nested PCR on 191 pooled samples was used to detect Plasmodium falciparum and Plasmodium vivax. Cattle blood meals were predominant (73%), followed by human (20%) and chicken (7%), with no dog blood meals detected. All individual mosquito samples were negative for Plasmodium falciparum, while two pooled samples (out of 191) tested positive for P. vivax. A. stephensi in Khyber District primarily displayed anthropophagic feeding behavior, with a small portion of the population infected with P. vivax. The results underscore the importance of targeted vector control strategies, environmental management, community engagement and continuous monitoring to suppress malaria transmission.

A regional One Health approach to the risk of invasion by Anopheles stephensi in Mauritius.

Anopheles stephensi is an invasive malaria vector in Africa that threatens to put an additional 126 million people at risk of malaria if it continues to spread. The island nation of Mauritius is highly connected to Asia and Africa and is at risk of introduction due to this connectivity. For early detection of An. stephensi, the Vector Biology and Control Division under the Ministry of Health in Mauritius, leveraged a well-established Aedes program, as An. stephensi is known to share Aedes habitats. These efforts triggered multisectoral coordination and cascading benefits of integrated vector and One Health approaches. Beginning June 2021, entomological surveys were conducted at points of entry (seaport, airport) and on ships transporting livestock in collaboration with the Civil Aviation Department, the Mauritian Port Authority and National Veterinary Services. A total of 18, 39, 723 mosquito larval surveys were respectively conducted in the airport, seaport, and other localities in Mauritius while two, 20, and 26 adult mosquito surveys were respectively conducted in the airport, seaport, and twenty-six animal assembly points. Alongside adult mosquito surveys, surveillance of vectors of veterinary importance (e.g.- Culicoides spp.) was also carried out in collaboration with National Parks and Conservation Service and land owners. A total of 8,428 adult mosquitoes were collected and 1,844 larval habitats were positive for mosquitoes. All collected mosquitoes were morphologically identified and 151 Anopheles and 339 Aedes mosquitoes were also molecularly characterized. Mosquito species detected were Aedes albopictus, Anopheles arabiensis, An. coustani, An. merus, Culex quinquefasciatus, Cx. thalassius and Lutzia tigripes. Anopheles stephensi was not detected. The One Health approach was shared with the French Agricultural Research Centre for International Development (CIRAD), strengthening collaboration between Mauritius and Réunion Island on vector surveillance at entry points and insecticide resistance monitoring. The Indian Ocean Commission (IOC) was also alerted to the risk of An. stephensi, leading to regional efforts supporting trainings and development of a response strategy to An. stephensi bringing together stakeholders from Comoros, Madagascar, Mauritius, Réunion Island and Seychelles. Mauritius is a model system showing how existing public health entomology capabilities can be used to enhance vector surveillance and control and create multisectoral networks to respond to any emerging public and veterinary health vector-borne disease threat.

The expansion of an invasive malaria vector: Anopheles stephensi emergence in Arba Minch town in the southern Rift Valley of Ethiopia

Urban areas in malaria-endemic countries in East Africa are experiencing a significant increase in malaria cases, with the establishment of an “exotic” urban malaria vector, Anopheles stephensi, increasing the risk of urban malaria. To this end, the present study aimed to investigate the emergence of this species in Arba Minch, Ethiopia. Following the detection of An. stephensi in other parts of Ethiopia, 76 artificial containers (55 discarded tyres, 18 concrete water storage, and three plastic containers) were sampled in 21 locations in Arba Minch town, for immature Anopheles mosquito stages, using the standard dipping technique. Larvae were reared into adults which were morphologically identified at the species level 2–3 days after emergence. Morphological identification results were confirmed by species-specific polymerase chain reaction. Of the examined containers, 67 (88%) had at least one Anopheles larva. Thirty-two of the adults emerged were morphologically identified as An. stephensi, with 26 (81%) confirmed by molecular analysis. This is the first study to report An. stephensi from Arba Minch, one of South Ethiopia’s largest towns, highlighting the need for increased vigilance. The planned and ongoing study in and around Arba Minch will contribute to understanding the bionomics and role of An. stephensi in malaria parasite transmission, helping develop a strategy to address the impending risk of urban malaria in Ethiopia.

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.