Parasites & Vectors

Tunisia is considered a high-risk country for arboviral transmission, and the re-emergence of malaria. Vector control programs predominantly rely on insecticide-based interventions, however knowledge on the insecticide resistance status of local vectors is limited. Here, we determined the mosquito species composition and investigated the resistance status of disease vectors to insecticides, collected from two distinct ecological settings; urban setting and rural setting. Known insecticide resistance loci were molecularly analysed. A total of 210 mosquitoes were collected, representing three main genera: Culex (n = 119), Aedes (n = 79), and Anopheles (n = 1). Within the Culex genus, Cx. pipiens pipiens and Cx. pipiens hybrids were the predominant species in Monastir and Kairouan respectively. Amongst Aedes, Ae. albopictus was the most abundant species in Monastir, while Ae. caspius predominated in Kairouan. The knockdown (kdr) mutation L1014F was detected at both sites across the three Cx. pipiens biotypes. In contrast, Cx. perexiguus showed no kdr mutation in Kairouan and a low frequency in Monastir. Among Aedes species, kdr mutations were detected exclusively in Ae. albopictus, restricted to domain III (mutation I1532T). No mutations in the chs gene were identified in both populations. Aedes albopictus collected in Monastir exhibited a moderate copy number variation for both carboxylesterases 3 and 6. No mutations were detected in the Anopheles labranchia. Our study results indicate the suitability of diflubenzuron for Culex and Ae. albopictus control in central Tunisia, emerging pyrethroid resistance in both vector species, and possibly reduced organophosphate efficacy against Ae. albopictus.

BackgroundAnopheles gambiae sensu lato (s.l.) is the primary vector of malaria in sub-Saharan Africa. Although insecticide-based vector control has been central to prevention, the widespread emergence of insecticide resistance poses a serious biological threat to control efforts. Effective resistance monitoring is essential for sustaining vector control but remains highly limited in malaria-endemic hotspots. Here, we assessed pyrethroid and DDT resistance intensity and the frequency of the L1014F knockdown resistance (kdr) mutation in Anopheles gambiae s.l. populations from Sokoto, north-western Nigeria. MethodologyResistance status and intensity to five insecticides were determined in adult Anopheles reared from larvae collected in 2021 and 2022 using the World Health Organization (WHO) tube test and Centers for Disease Control and Prevention (CDC) bottle bioassay, respectively. A subset of resistant mosquitoes was analyzed using PCR-based diagnostic assays to identify species within the Anopheles gambiae complex and to genotype for the West African kdr mutation (L1014F). ResultsHigh knockdown times (KDT) were observed, with KDT50 ranging from 38 to 91 minutes and KDT95 from 104 to 678 minutes, indicating increased resistance levels across all insecticides. In 2021, resistance was detected to DDT, lambda-cyhalothrin, and permethrin, while susceptibility to alpha-cypermethrin (98%) and suspected resistance to deltamethrin (91%) were recorded. In 2022, a general increase in resistance to all insecticides was observed, with mortality rates ranging from 41% to 81%. High resistance intensity was observed against DDT, while permethrin and alpha-cypermethrin exhibited low resistance intensity in both years, failing to reach 10x the diagnostic dose. Deltamethrin and lambda-cyhalothrin showed low to moderate resistance intensity. The 1014F kdr mutant genotype was widely distributed (68.1%) across species and years. Allele frequencies were higher in An. gambiae s.s. (0.83) than in An. arabiensis (0.71), with significant deviations from Hardy-Weinberg equilibrium (p < 0.05), except for An. gambiae s.s. in 2021 (p = 0.7). ConclusionThese findings reveal a concerning increase in key insecticide resistance among Anopheles populations in Sokoto, underpinned by strong genetic mechanisms. This underscores the urgent need for integrated vector management strategies to sustain effective vector control efforts in the region.

Entomological surveillance is essential for the control of lymphatic filariasis (LF). This xenomonitoring study, conducted in 2022-2023, evaluated the effectiveness of three mosquito trapping methods to identify the most suitable tool for surveillance following the discontinuation of mass drug administration. The surveys took place in two Malian villages: Konfra (seasonal watercourse) and Siradjouba (permanent watercourse). Three techniques were compared: Pyrethrum spray catch (PSC) conducted at the end of each visit inside 30 randomly selected houses, and different collection points were chosen based on the characteristics of the study area to place the Ifakara Type C tent (Ifakara) and the gravid trap (GT). Collections were conducted simultaneously at both sites. All Anopheles gambiae and Culex spp. collected in 2022-2023 were morphologically identified and then analyzed in the laboratory for their infectious status. Data was processed using SPSS v25; Fishers exact test was used to compare proportions, with a significance threshold of p < 0.05. A total of 4,732 mosquitoes were captured (95.05% in 2022). In 2022, the Anopheles were predominant in Konfra (91.38%), while Culex predominated in Siradjouba (63.41%). In 2023, Anopheles was found only in Konfra (3 specimens), while Culex was predominant in Siradjouba (64.07%). In 2022, PSC collected 90.57% of Anopheles, Ifakara 9.43%, and GT 0%. For Culex, GT collected 53.67%, PSC 36.62%, and Ifakara 9.71%. In 2023, the few Anopheles (3) came from PSC; Culex were mainly collected by the Ifakara tent (43.29%). For FL xenomonitoring, the combination of PSC and the GT appear to be effective collection methods.

Understanding how vector ecology intersects with host behaviour is essential for predicting zoonotic disease risk in tropical ecosystems. We conducted a two-year field study (November 2016-October 2018) in a riverine forest in Sabah, Malaysian Borneo, to characterize mosquito communities and the spatial distribution of potential simian-malaria vectors. Mosquitoes were sampled on 44 nights using CO{square}-baited light traps repeatedly deployed along a 500 m transect at three distances from the river (0, 250, 500 m) and two vertical strata (ground and canopy), yielding 244 trap collections across repeated sampling nights. In total, 9,928 mosquitoes were collected, dominated by Culex spp. (9,079; 91.4%), whereas Anopheles spp. were rare (153; 1.5%); most remaining specimens were unidentified to genus, and species-level identification was limited primarily to Anopheles. Nevertheless, female Anopheles (n=57) were more frequently detected near the river and less commonly at intermediate distance, and tended to be captured more often in ground traps. Zero-inflated negative binomial GLMMs based on the full mosquito dataset indicated significant effects of river distance and height on mosquito abundance, while night-time temperature and humidity showed no detectable effects. The zero-inflated structure of the data further suggested that many zero captures reflected true absence rather than sampling variability. The Anopheles assemblage was dominated by An. balabacensis, and molecular screening of 57 females detected simian Plasmodium DNA in two individuals. Overall, these findings suggest that river-edge habitats may represent localized areas where vectors persist and where primates and human activities overlap, creating repeated opportunities for host-vector contact even when vector densities are low.

Aedes aegypti mosquitoes are the primary vectors for dengue, yellow fever, chikungunya and zika virus transmission, posing significant public health risks. In Kenya, these viruses drive disease outbreaks especially, dengue and chikungunya with coastal Kenya being the most affected. Low-level circulation of the Zika virus has been reported in parts of the Kenyan coast, with confirmed cases reported in Lamu County between August to September 2024. Except for yellow fever, there are no approved vaccines or therapeutics, hence vector control remains the most effective means of protection. However, prolonged exposure to insecticides can lead to resistance, threatening these interventions. Therefore, monitoring resistance in mosquito populations is critical to allow for appropriate interventions using effective chemical classes to prevent disease outbreaks. This study aimed to establish the levels of resistance to pyrethroids, and associated markers, among Ae. aegypti populations in sections of Lamu County where there had been a recent localized outbreak of Zika. Mosquito eggs were collected from Mkomani, Kashmir, and Kandahar villages in Lamu County, reared and tested for susceptibility to three pyrethroid insecticides (0.75% permethrin, 0.05% Alpha-cypermethrin and 0.05% deltamethrin) using WHO tube assays. Genotyping of knockdown resistance (kdr) mutations L982W, S989P, A1007G, V1016G/I, and F1534C was done using Sanger sequencing. Association between resistant phenotypes and genotypes were inferred. The results varied between the three pyrethroids with high resistance to permethrin observed (6-15% mortality), for deltamethrin mortality ranged between 53-57%, while for alphacypermethrin 88%-99% mortality was observed. Two mutation types and six genotypes were identified at F1534. No other kdr mutations were detected. The CC genotype was significantly associated with 0.75% permethrin resistance in Ae. aegypti populations (OR = 2.87, 95% CI: 1.34-6.17, P = 0.0036). The current data show that Ae. aegypti from Kandahar, Kashmir and Mkomani villages in Lamu County have developed very high resistance to permethrin, and varying resistance to other pyrethroids, thus threatening pyrethroid-based control strategies in this region, highlighting the need for alternative strategies to control the vector for arboviruses.

Despite extensive efforts to understand the population biology and ecology of the African malaria mosquitoes questions regarding their movement pattern, survival, and population size persist, reflecting methodological limitations. Site fidelity, in which mosquitoes return to feeding sites, resting sites, or oviposition sites remain debated. Mark release recapture (MRR) studies are vital to address such questions. Using locality- and date-specific DNA tags in fluorescent spray, we carried out a continuous MRR in a Malian village from September to December 2019 with three days interval between capture and release across seven zones. A total of 12,937 Anopheles gambiae s.l. (7,455 females) were captured during 35 indoor collections. Handling related mortality was 3.4%., A. coluzzii predominated (89.7%), followed by A. gambiae (9.4%), and A. arabiensis (0.9%). Overall recapture rate was 1.05% (N=129). Contrary to the site-fidelity hypothesis, the distribution of recaptured mosquitoes across zones (regardless of their zone of release) was similar to the distribution of the captured mosquitoes (r=0.97, P<0.001), with 70% recaptured in a different zone. There was no difference in distance moved between sexes, but males average distance increased over time since release, whereas females distance remained unchanged. Simulated movements (across released points), with equal probability to reach any of the village houses predicted actual distance moved by mosquitoes. The regression of observed distance from each zone over predicted had a slope of 1 (r2=94%, P=0.006), suggesting that the layout of the capture area greatly affected the results. The average days post release (minimum age of wild captured mosquitoes) for recaptures was 6.4 d with the longest being 30 d. No seasonal and sex related difference in minimum age were detected. The corrected probability of daily survival (PDS) was 94% and the daily increase in sporozoite rate was 4.9%. Limiting the recapture duration period showed that PDS increased with recapture duration from 74% to 86% (12 to 30 d, uncorrected). Thus, larger recapture area and longer recapture duration are needed to obtain accurate estimates of movement range and of daily survival.

BackgroundThe invasive mosquito Aedes koreicus has established populations in several European countries during the past decade, raising increasing public health concerns due to its potential role as a vector of pathogens. While species identification is primarily based on morphological characters, Ae. koreicus exhibits distinct morphological variants originating from mainland Korea and Jeju Island, which complicates surveillance and may lead to misidentification, particularly in regions where closely related species co-occur. To date, the genetic basis and population-level relevance of these morphological forms in Europe remain poorly understood. MethodsWe investigated the co-occurrence of two morphological forms of Ae. koreicus in Hungary, representing the first confirmed European location where both forms were detected sympatrically and even within the same breeding sites. Adult mosquitoes were morphologically characterised using diagnostic traits, and individuals representing both morphotypes were subjected to comprehensive genetic analyses. We sequenced multiple mitochondrial markers (COX1, COX2, COX3, ATP6, ND1, ND3) and the nuclear ITS2 region using Oxford Nanopore long-read sequencing. Phylogenetic reconstructions and haplotype network analyses were applied to assess genetic differentiation between morphotypes and to compare them with Aedes japonicus as a closely related reference species. ResultsAcross all analysed mitochondrial and nuclear markers, no genetic differentiation was detected between specimens identified as the "mainland" or "Jeju-do" morphological forms of Ae. koreicus. Phylogenetic and haplotype network analyses consistently grouped individuals independently of their morphotype, indicating a shared genetic background at the population level. In contrast, Ae. japonicus formed a clearly distinct genetic lineage, confirming the robustness of the applied markers for interspecific discrimination. ConclusionsOur results demonstrate that the observed morphological variability in Ae. koreicus populations are not underpinned by detectable genetic differentiation using commonly applied mitochondrial and genomic markers. These findings highlight the limitations of using morphology alone to infer population origin or structure and emphasise the need for heightened awareness of intraspecific variability in routine surveillance. Accurate morphological identification remains critical, particularly in citizen science-based monitoring programmes and AI-based automatization platforms, such as Mosquito Alert, to avoid confusion with morphologically similar invasive species. Further studies integrating genomic and ecological approaches are required to elucidate the mechanisms underlying morphological variability in this emerging vector species.

BackgroundAnopheles stephensi, an invasive malaria vector originally endemic to South Asia, has rapidly expanded across East Africa. Its emerging malaria threats in urban Ethiopia threaten the elimination efforts, especially in areas once deemed low risk. A systematic review was conducted to synthesize the evidence regarding its bionomics and epidemiological impact, highlighting implications for urban malaria control strategies. This systematic review provides the first Ethiopia-specific quantitative evidence synthesis, addressing a vital knowledge gap necessary for guiding national malaria elimination programmes. MethodsWe conducted a PRISMA 2020-compliant systematic review and meta-analysis registered with PROSPERO (CRD420251176953). Searches of PubMed, Scopus, Web of Science, and regional repositories (2016-February 2026) identified studies reporting An. stephensi bionomics and epidemiological role in Ethiopia. Eligible studies required [&ge;]50% quality score on JBI appraisal tools. Random-effects meta-analysis estimated pooled proportions of An. stephensi among total Anopheles, with subgroup analyses by geography, habitat, and behavioural traits. Publication bias was assessed using Eggers and Beggs tests. ResultsEighteen studies (9 epidemiological, 11 bionomical) met inclusion criteria. The pooled proportion of An. stephensi was 0.51 (95% CI: 0.28-0.75) in epidemiological studies and 0.46 (95% CI: 0.26-0.66) in bionomics studies, with extreme heterogeneity (I{superscript 2} > 99%). Geographic analysis indicated significant variation: south-eastern Ethiopia showed a dominance of 0.73 (95% CI: 0.28-1.18) and eastern Ethiopia 0.57 (95% CI: 0.32-0.82), while central Ethiopia remained lower at 0.13 (95% CI: 0.12-0.14). These findings demonstrate genuine ecological differences rather than methodological objects, with substantial implications for region-specific vector management strategies. Extreme heterogeneity reflected genuine ecological variation across Ethiopia. No evidence of publication bias was detected. ConclusionAn. stephensi has very rapidly emerged as a major malaria vector in Ethiopia, with pooled proportions increasing from <10% at first detection (2016) to 51-73% in recent surveys (2024-2025), suggesting ongoing vector displacement parallel to invasion patterns documented in Djibouti. Geographic stratification indicates an urgent need for region-specific urban vector management integrating larval source management, resistance monitoring, and community engagement, particularly in south-eastern Ethiopia, where near-complete vector replacement has occurred. Author SummaryMalaria is usually thought of as a rural disease, but a new mosquito species called An. stephensi is changing that picture in Ethiopia. Originally found in South Asia, this mosquito has spread quickly across East Africa and is now common in Ethiopian towns and cities. We reviewed and combined results from published studies to understand how this species behaves and how much it contributes to malaria transmission. Our analysis shows that An. stephensi has become one of the dominant malaria vectors in several regions of Ethiopia, especially in the east and south-east, where it has almost replaced other mosquito species. This rapid change means that malaria risk is increasing in urban areas that were previously considered low risk. These findings highlight the urgent need for new control strategies that focus on city environments, such as managing breeding sites, monitoring insecticide resistance, and involving communities in prevention efforts. By understanding how An. stephensi is spreading and adapting, we can better protect urban populations and support Ethiopias malaria elimination goals.

ObjectivesTo estimate the pooled prevalence of Wolbachia infection in biting midges (Ceratopogonidae) across Southwest Asia and to evaluate ecological and biological factors associated with infection patterns. Study DesignSystematic review and meta-analysis. MethodsA comprehensive search of international and regional databases (PubMed, Scopus, Web of Science, Embase, SID, MagIran) was conducted without date restriction. Eligible studies included those using molecular techniques to detect Wolbachia in Ceratopogonidae collected from Southwest Asia. Pooled prevalence was calculated using a random-effects model. Subgroup and meta-regression analyses were performed to assess variations by country, species, altitude, habitat type, and sex. Heterogeneity and publication bias were evaluated using I{superscript 2}, Cochrans Q, and Eggers tests in accordance with PRISMA guidelines. ResultsTwenty-four studies comprising 14,832 midges from six countries were included. The pooled prevalence of Wolbachia infection was 32.6% (95% CI: 28.4-36.9%; I{superscript 2}=78.3%). Iran showed the highest prevalence (38.2%), and Culicoides imicola was the most frequently infected species (36.8%). Higher prevalence was associated with lower altitudes (<500 m; P=0.012), rural habitats (P=0.034), and female midges (P=0.008). Limited evidence suggested the presence of cytoplasmic incompatibility and reduced bluetongue virus competence in infected midges. ConclusionsWolbachia infection is common among Ceratopogonidae in Southwest Asia and is influenced by ecological and biological factors. These findings highlight the potential of Wolbachia as a biocontrol tool in regional vector management, underscoring the need for further experimental and strain-level studies.

In many leishmaniasis foci, reservoirs that maintain infection remain unknown. Here, we developed a field-applicable toolkit based on the analysis of individual blood fed sand flies (IBF) to identify reservoirs. Sand flies were given a Leishmania donovani-infected first blood meal (iBM1) by feeding artificially on a membrane or naturally on a clinically ill animal followed by two subsequent uninfected blood meals (BMS+). Bulk-RNAseq was used to identify two target parasite genes, sherp and a novel hypothetical gene (HPB), which exhibited a significantly higher expression in BMS+ compared to iBM1 sand flies. DNA and RNA were co-extracted from IBF. DNA was used to detect Leishmania infection and the blood meal source; RNA was used to assess expression of target genes by qRT-PCR. Linear discriminant analysis (LDA) of target gene expression classified sand fly specimens based on their iBM1 or BMS+ status. Co-extraction yielded a mean of >800ng per IBF for DNA and RNA. We detected [&ge;]1 parasite/s by kDNA qPCR and ssu rRNA RT-qPCR. LDA identified iBM1 parasites with a predictive accuracy of [~]87% and [~]82%, in membrane or naturally fed sand flies, respectively. This toolkit provides an innovative approach to identification of leishmaniasis reservoirs informing targeted control strategies.

Control of arboviruses remains heavily reliant on insecticide-based vector control targeting adult Aedes aegypti, especially during outbreaks, but the effectiveness of these tools can be compromised by insecticide resistance. While the mechanisms underlying resistance have been widely studied in Latin American and South East Asian Ae. aegypti, knowledge from African populations is limited, particularly regarding metabolic resistance. To address this knowledge gap, we sequenced the transcriptomes of Ae. aegypti collected in Angola, from both unexposed individuals and survivors of exposure to the organophosphate fenitrothion, alongside two insecticide-susceptible laboratory reference strains. Many overexpressed genes belonged to the major detoxification enzyme families, including 96 cytochrome P450 monooxygenases (CYP450s), 18 glutathione S-transferases (GSTs), and 35 carboxylesterases, with multiple genes previously detected as upregulated in Latin American and Asian populations. These included frequently reported, functionally-validated, metabolic resistance genes such as CYP9J24, CYP9J26, and CYP6BB2. However, expression of auxiliary resistance families including hexamerins, heat shock proteins, and odorant binding proteins were linked to the insecticide resistance phenotype, whilst numerous cuticular genes differentiated the Angolan population from both susceptible laboratory strains. A novel candidate, CYP6AG7, that was overexpressed after fenitrothion exposure was experimentally validated, and surprisingly metabolised fenitrothion into its toxic oxon form, which it did not subsequently break down. The antioxidant response element (ARE) motif, to which the transcription factor Maf-S binds, was detected in all CYP450 overexpressed in the fenitrothion treatment suggesting their potential coordinated induction. Analysis of genetic differentiation revealed several resistance-linked genes under potential selection, and SNP screening identified both known and novel non-synonymous mutations in the voltage-gated sodium channel (VGSC) gene, the target for pyrethroid insecticides. This is the first RNAseq dataset for Ae. aegypti from Africa in the context of insecticide resistance, providing insight into the complexity of resistance mechanisms, including some shared, and others potentially novel, compared to better studied populations from other geographical regions.

BackgroundAttractive Targeted Sugar Baits (ATSBs) are a promising, environmentally friendly approach for mosquito control, but the direct field application, scalability and long-term effectiveness of ATSBs across diverse mosquito species remain significant challenges. Methodology/Principal FindingsWe assessed the efficacy of a genetically engineered RNA interference (RNAi) yeast strain (Sh.463_56.10R) formulated in three sugar baits, soda (Coca-ColaTM), 10% sucrose, and a commercial mosquito bait (BaitStabTM), on Aedes, Anopheles and Culex mosquitoes. All RNAi yeast bait formulations induced significantly higher mean mortality (87-100%) compared to the control groups (0-9%; P<0.0001), but mosquitoes exhibited a higher feeding preference for RNAi yeast-soda baits, which induced mortality rates of 94-100% (P < 0.0001) recorded across all mosquito species. Additionally, to assess the competitiveness of the RNAi yeast-soda bait to other tropical sugar sources, semi-field choice assays were conducted in Trinidad, West Indies using competing flowering plants and fruits typically found in residential environments. The RNAi yeast-soda ATSB continued to perform well in the presence of competing floral and fruit sugar sources during both Aedes albopictus and Culex quiquefasciatus trials, though the presence of several fruits and flowers did reduce A. aegypti mortality, suggesting that further field testing will be necessary. The residual activity of the Sh.463_56.10R + soda formulation was retained for at least 14 months, with sustained 100% mortality in C. quinquefasciatus and 93-100% mortality in Aedes aegypti, Anopheles gambiae and Anopheles stephensi. The RNAi yeast-soda ATSB also performed well in semi-field studies performed with a prototype soda bottle feeder. Conclusions/SignificanceThis study demonstrates the potential of soda-baited RNAi yeast as a potent, long-lasting, and scalable platform for ATSB-based mosquito control as a component of integrated vector management programs. Author SummaryMosquito-borne diseases continue to affect millions of people worldwide, and current mosquito control methods face challenges such as low public uptake, insecticide resistance and environmental concerns. Here we evaluated a new and environmentally friendly approach to mosquito control using ATSBs. We tested genetically engineered species-specific yeast producing RNAi molecules capable of killing mosquitoes that feed on it. We mixed the yeast with three different sugar baits, including soda (Coca-ColaTM), 10% sucrose, and the commercial mosquito bait BaitStabTM formulation, and evaluated how well they worked against different mosquitoes. The results showed that the RNAi yeast mixed with soda was the most effective, killing up to 100% of mosquitoes in laboratory and outdoor tests. The bait remained effective in the presence of many competing natural tropical fruit and floral sugar sources. Remarkably, the bait, which can be delivered in a soda bottle feeder, stayed active for at least 14 months under simulated field conditions. These findings suggest that soda-based RNAi yeast baits could provide a practical, long-lasting and scalable tool for mosquito control and may help strengthen future strategies to reduce mosquito-borne diseases.

This data paper presents a curated, georeferenced dataset of the frequencies of the two main target site mutations (V1016G and F1534C) associated with resistance to pyrethroid insecticides in Aedes albopictus in Italy. Populations were collected in 102 out 107 Italian provinces between 2023 and 2025. Specimens were sampled by members of the Mosquito Insecticide Resistance Italian Network (MosqIRIT) as part of RN2 activities within the INF-ACT project. Genotyping was performed on 3,517 individuals by specific allele-specific PCR assays. Each record includes metadata on sampling site, administrative location, developmental stage, collection method, and mutation-specific genotype frequencies. To support spatial analysis modelling effort, the dataset integrates geographic, eco-climatic, and demographic data. This resource will support mosquito control programs, pyrethroid resistance monitoring and managing, as well as ecological modelling, and is compliant with the FAIR data program.

BackgroundAedes vittatus, an emerging invasive species and arboviral vector with high ecological plasticity and dispersal capacity, is expanding in the Caribbean, being detected in the Dominican Republic, Cuba, and Jamaica and recently in Yucatan, Mexico. ObjectivesThis study aimed to provide evidence of the establishment of Ae. vittatus in Haiti. MethodsLarval surveys were done across 12 localities around Cap-Haitien, in the North-East department of Haiti, the specimens were identified morphologically and confirmed by COI DNA barcoding. FindingsWe report, for the first time, the detection of the invasive vector Aedes (Fredwardsius) vittatus in Haiti, confirmed morphologically and molecularly, and being established in half of the municipalities sampled. Phylogenetic analyses group Haitian specimens with Caribbean and American populations, closely related to Mexico, indicating regional expansion. Main conclusionGiven its role as a vector of chikungunya, dengue, Zika and yellow fever, strengthened entomological surveillance and early detection in North America and the Caribbean are urgently needed.

Insecticide treated nets (ITNs) remain one the frontline tool in malaria control in Africa. The synergist piperonyl butoxide (PBO) has been incorporated into ITNs to combat pyrethroid resistance and these pyrethroid-PBO ITNs are now in widespread use across Africa. PBO is known to inhibit the degradation of pyrethroids and other insecticide classes, but little is known about other biological or behavioural effects of PBO exposure. This work assessed the impact of PBO on Anopheles gambiae s.l. male longevity as well as female host-seeking and blood feeding behaviours using laboratory assays. PBO had an immediate impact on blood feeding of female An. gambiae s.l. whereas the effect was not observed 24h post-exposure. Kaplan-Meier survival curves showed a significant difference in mosquito longevity post-exposure to PBO with male mosquitoes living nearly three times as long if exposed to control papers compared to PBO. In olfactometer assays, after exposure to control papers, control mosquitoes were significantly more attracted to host-odours, however, this attraction preference was reversed following PBO exposure. This study shows that in experimental assays, PBO has other effects on mosquito physiology and this is likely to contribute to the impact of pyrethroid - PBO ITNs in vector control programmes.

Conventional mosquito surveillance typically relies on contemporaneous data, making it challenging to anticipate future vector surges. To support proactive vector management, this study evaluates a multi-model forecasting framework designed to generate probabilistic 1- to 4-week-ahead forecasts of Aedes aegypti relative abundance per trap night. The framework was validated using multi-year surveillance data across four US jurisdictions spanning varied environments (from subtropical to temperate and arid). We found that an ensemble approach aggregating statistical and machine learning models generally achieved the best performance across all locations and forecast horizons. Relative forecast performance improved as the forecast horizon extended from 1 to 4 weeks ahead. The most challenging data to forecast were primarily restricted to low mosquito activity periods or atypical population peaks with unusual timing or magnitude. While full integration into routine vector management workflows represents a long-term process requiring operational adaptation, this work advances forecasting research and establishes a baseline for translating these approaches into real-time applications for public health authorities, with downstream effects in mitigating the risks of mosquito-borne diseases.

BackgroundControl of Aedes aegypti, the primary vector of dengue and other Aedes-borne viruses, is challenged by insecticide resistance, limited efficacy of existing tools and the large and widespread epidemics. Targeted Indoor Residual Spraying (TIRS), a modification of traditional indoor residual spraying focused on Ae. aegypti resting sites, has demonstrated promising results, yet its indirect community-wide effects remain underexplored. Methodology/Principal FindingsWe conducted an entomological cluster-randomized controlled trial in Iquitos, Peru, to evaluate the direct and indirect entomological impacts of TIRS using pirimiphos-methyl. Thirty clusters were randomized to receive either TIRS (15 clusters, 898 structures) or standard Ministry of Health vector control activities (15 clusters, 1,018 structures). Aedes aegypti indoor densities were assessed in the 45 days pre-intervention and at four time points up to 255 days post-intervention using Prokopack aspiration. Generalized linear mixed models with a negative binomial link were used to estimate incidence rate ratios (IRRs) and calculate efficacy (1-IRR) for houses that received TIRS (direct effect) and untreated houses in TIRS clusters (indirect effect). Direct efficacy reached 96% at 15 days post-spraying and remained significant (40%) at 255 days post-spraying. Indirect efficacy reached 69% at 15 days and declined to 7% by 255 days post-spraying. Despite only 57% household-level TIRS coverage, both direct and indirect impacts on Ae. aegypti were significant during early post-intervention surveys, and after 8 months in TIRS clusters. Conclusions/SignificanceTIRS provided substantial and sustained reductions in indoor Ae. aegypti density, including measurable indirect effects in untreated homes within intervention clusters. These findings demonstrate the entomological value of TIRS even at moderate coverage levels and highlight its potential for both preventive and reactive vector control programs and should be considered for implementation by Ministries of Health in dengue-endemic urban settings as well as by the U.S. military when deployed to tropical or subtropical locations.

Locally acquired arboviral infections are an increasing public health concern in the United States. Aedes aegypti and Culex quinquefasciatus, vectors of dengue virus and West Nile virus, respectively, are established in regions where local transmission has been reported. Seasonal variation in mosquito abundance affects vector density, human-vector contact, and arbovirus transmission risk. The aim of this study is to investigate seasonal variations in Ae. aegypti and Cx. quinquefasciatus population dynamics in Miami-Dade County, Florida; Maricopa County, Arizona; and Clark County, Nevada. Monthly relative abundance, average mosquitoes collected per trap-night, normalized abundance ratios, and seasonal-trend decomposition were used to evaluate species- and location-specific seasonal patterns. Mosquito population dynamics differed by species and location. In Miami-Dade County, the two species showed seasonal turnover, with Cx. quinquefasciatus peaking during winter and spring and Ae. aegypti peaking during summer. In Maricopa and Clark counties, both species peaked primarily between August and September. Species dominance also differed by site, with Ae. aegypti more abundant than Cx. quinquefasciatus in Maricopa County and Cx. quinquefasciatus more abundant than Ae. aegypti in Clark and Miami-Dade counties. Seasonal decomposition showed that Ae. aegypti peaked earlier in Miami-Dade County than in Maricopa and Clark counties, whereas Cx. quinquefasciatus showed a spring peak in Miami-Dade County and bimodal seasonal patterns in Maricopa and Clark counties. These results suggest that mosquito population dynamics are species- and location-specific and support the use of local surveillance data to guide the timing of mosquito control and arbovirus preparedness.

Laboratory models are invaluable tools for studying parasite biology and pathogenesis, especially for helminth infections. However, the complex life cycles and frequently narrow host specificity of helminths present challenges to maintaining access to critical parasite material in a laboratory setting. This is especially true of Necator americanus, the most common species of hookworm that infects humans globally. Here we report the successful laboratory adaptation of an African strain of N. americanus, originally isolated from infected individuals in Beposo, Ghana. The Beposo strain has been successfully passaged across 9 generations in Golden Syrian hamsters maintained on oral dexamethasone. Differential susceptibility to mebendazole and albendazole was evaluated using an egg hatch assay, and DNA sequencing of the beta-tubulin isotype 1 gene did not identify known resistance-associated mutations in the endemic strain. Sequencing of the mitochondrial COX1 gene revealed that specimens of N. americanus from Ghana, along with reported sequences from Togo, are distinct from those from South America and Asia. Complementary microsatellite-based population analysis revealed substantial genetic variation in the founding parasite population. To further characterize the novel Beposo strain, a draft hybrid genome assembly was generated from genomic DNA extracted from a single adult male worm via an optimized Oxford Nanopore Technologies MinION library preparation approach tailored to low-input sample types. This high-quality assembly, including a complete mitogenome, is 202.8Mb in 950 contigs with an N50 >449 kb. It contains >95% of conserved nematode orthologs in complete single copy and is estimated by homology-based gene prediction to contain 12,804 genes. This study represents the first comprehensive characterization of a strain of N. americanus originating in Africa that has been successfully adapted to a laboratory animal model.

Background: The lifespan of insecticide-treated nets (ITNs) varies widely across settings, reflecting both intrinsic product characteristics and external factors related to use, care, and environment. While the resistance to damage (RD) score captures intrinsic product durability, there is no standardized metric to quantify contextual risks. This study presents a proof of concept for the Risk Index (RI), a composite measure of site-level risk factors for ITN physical durability and survival. Methods: We conducted a secondary analysis of durability monitoring data from 44 sites across 15 countries in sub-Saharan Africa, covering 14 ITN products. The RI was calculated as a weighted composite of 12 indicators spanning net handling, net care attitudes, and use environment. Associations between RI and median ITN survival were assessed using weighted linear regression and multivariable mixed-effects models adjusting for RD score, with country included as a random effect. Results: RI scores ranged from 25.1 to 83.7 across study sites. In bivariable analysis, a 10-point decrease in RI was associated with a 4.0-month increase in median ITN survival (95% CI: 1.7-6.3; p=0.001). In multivariable analysis adjusting for RD, this association remained significant but attenuated to 2.2 months (95% CI: 0.1-4.2; p=0.037). Independently, a 10-point increase in RD score was associated with a 3.5-month increase in survival (95% CI: 1.3-5.7; p=0.001). No interaction was observed between RI and RD. Predicted survival differed by approximately one year between the lowest- and highest-risk settings. Conclusion: The RI provides a standardized measure of contextual risk factors affecting ITN lifespan, independent of ITN product type. When used alongside a product's RD score, the RI enables improved interpretation of expected site-level variation in net performance. This combined framework offers a practical basis for incorporating behavioural and environmental risk into vector control planning and for tailoring ITN strategies to local conditions.