Martin James Donnelly

Background: The establishment of the invasive malaria vector Anopheles stephensi in Ethiopia threatens existing malaria control efforts. Unlike native vectors, An. stephensi thrives in both urban and rural areas, lacks clear seasonal peaks of abundance, and often breeds in artificial water containers. SumiLarv ^™ 2MR containing the larvicide pyriproxyfen (PPF) offers a promising method for controlling the immature stages of An. stephensi in such habitats. This habitat-randomised controlled trial evaluated the efficacy of SumiLarv ^™ 2MR against An. stephensi in Danan town, Somali region, Ethiopia. Methods: Entomological surveillance identified 102 large artificial containers (“ birkas ”) which were randomly allocated in a 1:1 ratio to receive either SumiLarv ^™ 2MR (treatment arm) or no treatment (control arm). SumiLarv ^™ 2MR was introduced into the birkas at a dose of one disc per 200 L of water. Morphological identification was performed on adult mosquitoes emerging from control containers, while molecular analysis was conducted on both morphologically identified adult mosquitoes and dead pupae collected from the control and treatment arm. The residual efficacy of PPF content of the remaining discs was also analysed. Inhibition of adult mosquito emergence was assessed every 2 weeks for 40 weeks to determine the duration of efficacy. Results: The number of SumiLarv ^™ 2MR remained constant in the treatment arm throughout the study period. No significant differences were observed in water volume, pH, or temperature between the two study arms. Introduction of SumiLarv ^™ 2MR resulted in complete (100%) inhibition of adult emergence throughout the 40 weeks’ study period (t = 6.26, p < 0.05). Morphological identification of a sub-samples of adult mosquitoes emerging from control containers at different time points confirmed the presence of An. stephensi (99.85%) and An. gambiae s.l. (0.15%). Molecular analysis of morphologically identified adults from control containers and dead pupae from treatment containers confirmed that 98.91% were An.stephensi . Post-trial analysis showed that approximately 82.20% of the original PPF had been released from SumiLarv ^™ 2MR during study period. Conclusions: The high and sustained efficacy of SumiLarv ^™ 2MR demonstrated in this study suggests that it is a suitable tool for controlling An. stephensi in large artificial water storage containers.

Background: Anopheles stephensi, an urban-adapted Asian malaria vector, is spreading across Africa amid rapid urbanization, yet its contribution to malaria transmission remains unclear, especially in typical situations where other vector species co-occur. Here, we quantified the association between peri-domestic An. stephensi exposure and individual malaria risk in Metehara town, central Ethiopia, alongside the role of native vectors.Methods: An age-matched case-control study (1:2 ratio) was conducted from October 2023 to November 2025. Cases were febrile outpatients with microscopy-confirmed Plasmodium falciparum or Plasmodium vivax infection. Controls were afebrile, microscopy-negative outpatients recruited from the same health facility within 72 hours of case enrolment. Field teams conducted household and entomological assessment within 48 hours of enrolment. The primary exposure variable was the presence of An. stephensi in or around participants’ households.Findings: Of the 1806 enrolled participants, 486 cases and 915 controls were analyzed. Most cases were P. falciparum (83·1%), followed by mixed (9·7%) and P. vivax (7·2%) infections. Adult anophelines were detected more often in case households (58·0%) than in controls (40·3%), the majority being An. arabiensis (79·6%). Anopheles stephensi was more common in case households at both adult (6·6% versus 4·2%) and immature stages (12·7% versus 6·3%) than controls. In adjusted models, the presence of adult (adjusted odds ratio [AOR]=2·80, 95% CI 1·64–4·79) and immature An. stephensi (AOR=2·46, 95% CI 1·61–3·74) was independently associated with case status, with effect sizes comparable to those observed for native adult malaria vectors (AOR=2·71, 95% CI 2·02–3·62). Combined exposure to adult and/or immature stages of An. stephensi showed similar associations (AOR = 2·80, 95% CI: 1·98–3·96), with a magnitude of risk comparable to that of native vectors (AOR = 2·61, 95% CI 1·99-3·41).Interpretation: In this urban setting, co-endemic for native malaria vectors, peri-domestic exposure to An. stephensi was an independent predictor of malaria case status, with an effect comparable to that of native vectors. These findings reinforce the importance of integrated, urban-focused vector surveillance and control strategies across Africa.

The invasive urban malaria vector Anopheles stephensi threatens 126 million city dwellers in Africa. Controlling An. stephensi requires greater understanding of its origin, invasion dynamics, and insecticide resistance mechanisms. Analysis of 645 whole genomes sampled across Africa, the Middle East, and Asia supports an invasion scenario in which an initial South Asian introduction established a bridgehead population in Djibouti, which seeded distinct invasion fronts in Sudan, Ethiopia/Kenya, and Yemen. These incursions show contrasting rates and routes of spread shaped by landscape topology. Insecticide resistance is predominantly mediated by metabolic detoxification genes, with resistance haplotypes and copy-number amplifications introduced from South Asia. These findings, alongside a companion genomic resource, enable genomic surveillance of An. stephensi spread and resistance to aid control strategies.

The online version contains supplementary material available at 10.1186/s12864-026-12721-y.

Culex tritaeniorhynchus is the primary vector of Japanese encephalitis virus (JEV) and is thought to exist as 2 forms: the continental type (Ct-C) and the Japanese type (Ct-J), distinguished by variations in the cytochrome oxidase subunit 1 (COI) region of mitochondrial DNA (mtDNA). However, the extent of genetic variation within and between the 2 types remains largely unexamined. To address this gap in the literature, we performed mitochondrial genome sequencing and additionally developed a rapid and cost-effective identification method that may facilitate efficient vector management and research. The method for type identification is based on a restriction fragment length polymorphism (RFLP) assay, targeting type-specific sequence variations in the COI region. Phylogenetic analysis using mtDNA confirmed that Ct-C and Ct-J represent genetically distinct taxa within the genus Culex. The proposed molecular identification method has been validated to effectively differentiate between the 2 types in the ROK. Early detection of cryptic invasions is critical for effective vector management. The results of this study confirm that the 2 types of Cx. tritaeniorhynchus, the primary vector of JEV, represent genetically distinct lineages. These findings may serve as a foundational reference for developing vector control strategies aimed at suppressing JEV in East Asia.

While substantial progress has been made toward malaria elimination in Southeast Asia, major challenges remain. The principal mosquito vectors, Anopheles dirus and Anopheles minimus, inhabit diverse ecological niches and exhibit a wide range of behavioural and physiological insecticide resistance phenotypes, complicating vector control efforts. In Africa, genomic surveillance has transformed our understanding of vector evolution and resistance, supported by open-access tools and data. Comparable resources for Southeast Asian vectors remain limited. Here, we release Adir1.0, a curated, analysis-ready catalog of 540 An. dirus whole-genome sequences, accessible for interactive cloud-based analysis via the malariagen-data-python API. Alongside, we provide and updated Amin API to enhance functionality and usability, enabling integrated analyses across historical and new datasets. We demonstrate these resources by performing the first exploratory population genomic analysis of An. dirus from Bangladesh, Thailand, and Cambodia, revealing population structure, candidate regions of physiological resistance, and genomic structural variation. Together, these resources provide a foundation for genomic surveillance studies to inform vector control and malaria elimination strategies in Southeast Asia.

Insecticide resistance poses a major threat to malaria control efforts in Africa, yet our understanding of the genomic basis of adaptation in malaria vectors remains incomplete. Here we present a comprehensive atlas of recent positive selection in natural populations of the major African malaria vectors; the An. gambiae species complex and An. funestus . By analyzing whole-genome sequence data from 4,306 mosquitoes collected across twenty-one countries, we identify and characterize both known and novel genomic regions under strong positive selection. We develop an innovative approach for identifying and localising selection signals, and find extensive evidence for parallel evolution between species, with all important loci in An. funestus also under selection in An. gambiae s.l. We confirm intense selection at established resistance loci including known target site proteins ( Vgsc, Rdl, Ace1 ) and metabolic enzymes ( Cyp6p , Cyp9k1 , Gste , Coeaexf ) and describe novel signals at a diacylglycerol kinase on the X chromosome, and in An. gambiae s.l , at the metabolic regulator Keap1 . To support real-time monitoring of emerging variants threatening vector control, we present an open-source web resource containing detailed selection scan results for all cohorts. The resource is driven by an automated computational workflow, which enables continuous updates as new whole-genome data are generated. The selection atlas provides a foundation for understanding vector adaptation to insecticide-based interventions and we hope will inform evidence-based resistance management strategies that are critical for sustaining malaria control.

In response to the emerging threat of insecticide resistance in malaria vectors, insecticides are being repurposed for vector control or developed de novo . Good stewardship of these finite new resources is essential if disease control programmes are to remain effective. This is dependent on timely data to help guide evidence-based decision-making for National Malaria Control programmes (NMCPs). By embedding genomics into cluster randomized control trials (cRCTs), we can perform surveillance and early detection of insecticide resistance variants to new and repurposed chemicals in natural field conditions, supporting effective stewardship. The LLIN Evaluation Uganda Project (LLINEUP) trial evaluated the efficacy of pyrethroid-piperonyl butoxide (PBO) and pyrethroids-only long-lasting insecticidal nets (LLINs). It was conducted in Uganda between 2017-2020 and was the largest cRCT to date, covering 40% of the country in 104 health sub-districts. We embedded genomic surveillance within LLINEUP to detect and track insecticide resistance variants. At baseline and throughout the trial, we sampled Anopheles mosquitoes with Prokopack aspirators and performed Illumina whole-genome sequencing. We show that An. funestus populations were relatively unaffected by the interventions, compared to An. gambiae s.l., which were markedly reduced six months following LLIN deployment. Standard approaches for describing genetic diversity and population structure e.g. fixation index ( F _ST ), Principal Component Analysis (PCA) and Neighbour-Joining (NJ) trees, were consistent with the density observations and suggestive of a single large An. funestus population in Uganda with little genetic differentiation. Genome-wide selection scans revealed strong signals of selection at the Resistance to pyrethroid-1 (RP1 ) locus and Cyp9k1 , both loci previously implicated in pyrethroid resistance. We report two additional loci, eye diacylglycerol kinase ( Dgk ) (≅13.5Mb on the X chromosome) and O-mannosyl-transferase ( TMC-like ) (≅67.9Mb on 3RL) that showed signals of selection. Known DDT and permethrin resistance-associated variants at the Gste2 locus, L119F and L119V, were also identified. Over the trial period, changes in haplotype frequencies were observed in regions under selection, with more pronounced shifts in the PBO arm. Notably, there were significant reductions in the frequencies of swept haplotypes (measured by delta (Δ) H12) in the Dgk and Cyp6p9a regions, while significant increases in haplotype frequency were observed at Gste2 and Cyp9k1 loci. Our findings reveal the differential impact of the trial on An. gambiae s.l. and An. funestus densities and the differing responses of An. funestus populations to pyrethroid and pyrethroid-PBO selection pressure. These insights underscore the potential value of tailored, species- and region-specific vector control strategies, supported by regional genetic surveillance, to better control insecticide resistance evolution and spread. By embedding genomic surveillance in cRCTs we can facilitate the discovery of putative resistance variants and can provide evidence of their impact on vector control tool efficacy; both of crucial importance to evidence-based deployment of vector control tools by NMCPs.

Progress in malaria control in sub-Saharan Africa is stalling, partly due to the spread of insecticide resistance in Anopheles vectors. Monitoring the evolution of insecticide resistance alleles and their spatial heterogeneity is important for malaria control programmes, and genomic surveillance has emerged as a pivotal tool for this purpose. Earlier genomics research has typically employed convenience-based sampling, and research has yet to be performed to optimise sampling regimens for malaria vector genomics. In an earlier study, we developed a spatially explicit sampling framework that considers the underlying ecology to enable sampling mosquitoes with reduced bias. We applied this framework to sample and perform whole-genome sequencing on 485 individual specimens of An. gambiae s.l mosquitoes from Obuasi, central Ghana, an area with extensive gold mining activities. In this region, An. gambiae s.l have been documented as highly resistant to multiple insecticides, including pyrethroids used for the treatment of bed-nets. High resolution of fine-scale population structure enabled the detection of isolation-by-distance in An. coluzzii in Obuasi, whilst finding that at this scale, geographic distance, rather than the underlying habitat, drives population structure. We examine methods to estimate mosquito kinship, demonstrating that polymorphic chromosomal inversions significantly confound established analytical tools, while revealing how the mosquito’s chromosome architecture generates high variance in genetic relatedness among kin. Using genome-wide selection scans and clustering, we discover the novel mutations Gste2- F120L and Cyp9K1 -N224I in detoxification enzymes that are driving selective sweeps, likely in response to insecticidal pressure. We find that the frequencies of some resistance variants are associated with artisanal-gold mines, and elucidate the continued evolution of the Voltage-gated sodium channel , the target of pyrethroid insecticides. Overall, we show that sampling vectors strategically may enhance our ability to perform effective genomic surveillance.