Parasites & Vectors

The effectiveness of malaria vector control is increasingly threatened by widespread insecticide resistance, particularly to pyrethroids. In response, the World Health Organization recommends dual-active ingredients (dual-A.I.) insecticide-treated nets (ITNs) in resistant settings. This study evaluated the efficacy of several dual-A.I. ITNs in two experimental hut trial sites an area with high insecticide resistant malaria vectors in Tanzania with differing vector species composition and bionomics. Five ITNs were evaluated: a pyrethroid-only net (MAGNet(R)), three pyrethroid-piperonyl butoxide (PBO) nets (OlysetTM Plus, Veeralin(R), and PermaNet(R) 3.0), one dual-A.I. net (Interceptor(R) G2; chlorfenapyr + alpha-cypermethrin), and an untreated control net. Mosquitoes were collected from huts, morphologically identified, and monitored for mortality up to 72 h. WHO cone and tunnel tests using susceptible An. gambiae s.s. were conducted for quality control, while field-collected mosquitoes were used for bio-efficacy monitoring. Molecular species identification was performed using PCR assays, and genomic DNA was extracted from An. funestus s.l. and An. gambiae s.l. collected from the trials. All ITNs met WHO bio-efficacy criteria in cone and tunnel tests. An. gambiae s.l. predominated in Mwagagala (77%) but was rare in Magu (4%), where An. funestus complex was dominant. In Magu, Interceptor(R) G2 and Veeralin(R) achieved significantly higher mortality than MAGNet(R) at 24 h (53% and 45% vs 24%) and 72 h (61% and 53% vs 24%). In contrast, no significant mortality differences were observed among ITNs in Mwagagala. High resistance intensity was detected in both vector groups (mortality <20% in bioassays). In An. funestus from Magu, the 4.3 kb-SV resistant genotype was fixed, while G454A-CYP9K1 resistance was highly prevalent (RR: 0.70-1.00). Interceptor(R) G2 and Veeralin(R) outperformed the pyrethroid-only reference net against An. funestus complex but not against predominantly An. arabiensis populations.

Aedes albopictus (Skuse), the Asian tiger mosquito, is a highly competent arboviral vector whose range has expanded substantially across the United States over the past four decades. Despite predictive models placing Nebraska within the species' climatically suitable range, its establishment status in the state has remained poorly characterized. Here, we report results from a nine-year mosquito surveillance program (2017-2025) conducted across 44 Nebraska counties in collaboration with the Nebraska Department of Health and Human Services. Ae. albopictus was detected in five counties, with sustained, annually increasing populations documented in Richardson, Douglas, and Lancaster counties. Richardson County recorded continuous detections during 2017-2025, with proportional representation rising to 60.50% of collected mosquitoes by 2025. In Douglas and Lancaster counties, temporal advancement of first seasonal detection in 2024 and 2025 provide evidence consistent with successful overwintering rather than annual reintroduction. A cumulative degree-day model predicted adult emergence in mid-May across all county-year combinations, consistently preceding trap deployment by two to seven weeks and revealing a systematic early-season surveillance gap. Generalized linear mixed-effects models indicated that trap-level detection persistence, rather than urban location, was the primary predictor of yearly Ae. albopictus positivity, suggesting that current invasion dynamics are driven by focal source populations. These findings provide strong evidence for the establishment of Ae. albopictus in eastern Nebraska and highlight the need for earlier seasonal surveillance and standardized criteria to define establishment in northward-expanding vector populations.

BackgroundResistance of the arbovirus vector Aedes albopictus to pyrethroid insecticides is an emerging threat to vector control programs in Europe. Three knock-down resistance (Kdr) mutations affecting the voltage-gated sodium channels targeted by pyrethroids are known to confer resistance: V1016G, I1532T and F1534C. As these Kdr mutations are actively circulating in Europe, their monitoring is crucial for resistance surveillance programs. However, current Kdr genotyping methods are labor-intensive and costly, limiting their applicability for large-scale high-throughput surveillance. MethodologyNovel digital droplet PCR (ddPCR) TaqMan assays were developed, allowing the quantification of these Kdr mutations from pooled mosquito samples from field populations. The specificity of these assays was validated by using mosquitoes of known genotypes together with synthetic DNA constructs carrying haplotype combinations previously unseen in the field. The assays accuracy was assessed by comparing Kdr frequencies measured from pooled mosquitoes to those derived from individual genotypes. These assays were then used in a pilot surveillance study in mainland France, integrating deltamethrin bioassays, pooled Kdr mutation tracking and vector control interventions data. FindingsThe developed ddPCR assays demonstrated high accuracy and specificity, with matching Kdr mutation frequencies between pooled samples and individual genotyping. The pilot surveillance study confirmed the low prevalence of Kdr V1016G and I1532T mutations in most French populations, though some populations exhibited a moderate decrease in susceptibility to deltamethrin. Deltamethrin spraying intensity was weakly correlated with Kdr I1532T mutation frequency while no correlation was observed with deltamethrin susceptibility, suggesting that insecticide selective pressure from curative vector control activities is at most a minor driver of resistance. ConclusionThese novel ddPCR assays provide a simple, cost-effective and high-throughput method for quantifying the frequency of Kdr mutations in Ae. albopictus populations. Their implementation in routine country-wide surveillance programs will enhance the detection of emerging resistance, and inform vector control strategies, preventing arboviral disease transmission. Author summaryAedes albopictus, the Asian tiger mosquito, is an emerging global threat due to its ability to transmit the dengue, chikungunya and Zika viruses among others. Recurrent use of insecticides to prevent infections selects resistant mosquitoes. Kdr (knock-down resistance) mutations confer resistance to pyrethroid insecticides, like deltamethrin. In order to track the arrival of these mutations in a population, individual PCR tests are routinely used, which is very inefficient and costly. The digital droplet PCR tests presented in this study can instead be performed on pools of mosquitoes from the field, greatly increasing the output while reducing the costs. This makes them ideal for use in the surveillance of these mutations in field populations. We first confirmed that the tests are as efficient in pools as they are on single mosquitoes, then we applied these tests in a pilot study on French field mosquitoes to demonstrate their applicability for use in surveillance. Finally, we explored the link between deltamethrin sprayings, bioassay mortality and Kdr mutations frequency, which is non-existent. This means the current management of mosquito populations via insecticide use is sensible. We believe these tests have a place in streamlining the future surveillance programs of these mutations in the field.

Mosquitoes in the Toxorhynchites genus (Theobald, 1901) are specialized predators of container-breeding mosquito larvae, including major Aedes disease vectors. Despite their biological control potential, species boundaries and phylogenetic relationships remain poorly understood. Utilizing country-wide sampling across Panama, we evaluated the taxonomic status and diversity of local Toxorhynchites species. We collected 147 specimens from artificial and natural containers, yielding 97 cytochrome c oxidase subunit I barcodes from 101 sequenced individuals. A Panama-only Neighbor-Joining analysis revealed four distinct, well-supported clusters (I - IV) exhibiting high mean inter-cluster genetic divergence (4.8 - 20.2%), alongside morphological and ecological segregation. A subsequent global Neighbor-Joining analysis incorporating BOLD and GenBank sequences recovered Toxorhynchites as a strongly supported monophyletic group (99.0% bootstrap support) and revealed deep phylogenetic divergence separating Old World Toxorhynchites (Toxorhynchites) (Clades A and B) from New World Toxorhynchites (Lynchiella) (Clades C - G) lineages. Panamanian Clusters I and II formed two distinct groupings within Clade G, nesting as sisters to Subclade G1 characterized by severe taxonomic discordance involving sequences labeled as Tx. moctezuma, Tx. theobaldi, and Tx. rutilus. Cluster III nested within Tx. hypoptes (1.5% divergence) in Clade E, whereas Cluster IV associated with Tx. haemorrhoidalis s.l. (>5.0% divergence) in Clade C. The high degree of taxonomic uncertainty or cryptic diversity uncovered within Clades C, D, and G underscores an urgent need for formal taxonomic revision to accurately delimit species boundaries. Due to its widespread peri-urban distribution, Cluster I (within the Tx. moctezuma s.l. complex) shows the greatest promise for mass-rearing and Aedes biocontrol applications in Panama.

The Culex pipiens complex comprises globally distributed mosquito species that serve as vectors for various arboviruses and filariasis. Female mosquitoes within the Cx. pipiens complex are morphologically very similar, making species identification based on morphologically characteristics challenging. To date, Cx. pallens and Cx. pipiens f. molestus have been reported to inhabit the Republic of Korea (ROK). Conversely, Cx. quinquefasciatus, predominantly distributed in tropical regions, has not been documented in the country since a single record in the 1950s. Recently, however, Cx. quinquefasciatus was reported from the Jeju region of the ROK based on molecular identification. This study aimed to re-evaluate the occurrence of Cx. quinquefasciatus in the ROK by conducting molecular identification using the ace-2 and CQ11 markers on Cx. pipiens complex specimens collected nationwide, including from Jeju, where Cx. quinquefasciatus had previously been reported. Our analysis identified only Cx. quinquefasciatus-Cx. pallens hybrid individuals among the Cx. pipiens complex specimens from the Jeju region, highlighting the need to re-evaluate the analytical methods used in previous studies that reported the presence of pure Cx. quinquefasciatus. These findings provide essential evidence for future surveillance of the distribution, introduction, and establishment of Cx. quinquefasciatus and underscore the need for continuous monitoring.

Invasive mosquito species pose substantial risks to human and animal health. Since 2004, Culex coronator, a mosquito vector species of public health concern, has shown rapid range expansion within the United States, spreading from a historically limited distribution in southern Texas to across the Gulf Coast region and into eastern and mid-Atlantic states. However, changes in environmental suitability associated with this expansion across historical, contemporary, and future climate conditions have not been evaluated. Here, we used species distribution models (SDMs) to compare predictions of abiotic suitability for Cx. coronator under historic (1960-1989) and recent (2000-2024) climate conditions calibrated on the historical range in the United States. We also created a contemporary SDM based on occurrence records prior to and following species range expansion (1960-2024), and further, to predict potential distributions under current and future climate conditions. Models calibrated on the historical range predicted only modest changes in suitability along the Gulf Coast region and failed to identify large areas of the humid subtropical eastern United States that are now occupied. In contrast, the contemporary model predicted widespread suitability across much of the southern and eastern United States. Future projections under the mid-range SSP3 scenario predicted increasing suitability at higher latitudes and elevations. Across all models, suitability was consistently low in arid and semi-arid regions, including along the historical western range limit, suggesting that moisture availability may constrain Cx. coronator distributions. Together, these results highlight the need to incorporate updated occurrence records when modeling invasive mosquito species to strengthen surveillance and control strategies.

Background: Unexplained spatial heterogeneity in field-observed Human Blood Index (HBI) hinders vector-borne disease control. We synthesized micro-morphological sensory structure densities across primary disease vectors to formulate a novel Proboscis Sensory Index (PSI) from micro-morphological sensory structure densities to evaluate its capacity to predict vector anthropophily, zooprophylactic modulation, and spatial transmission risk across Northern Nigeria. Methods: A systematic review of 42 studies (N = 18,647 blood-fed mosquitoes across 126 georeferenced sites, 2005 to 2025) synthesized baseline species-specific PSI metrics from electron microscopy sensilla counts. A multi-variable binomial Generalized Linear Mixed Model (GLMM) evaluated interactions between PSI, livestock, human density, and environmental covariates. Predictive accuracy was validated against field-derived Entomological Inoculation Rates (EIR) and historical outbreak records. Results: The overall pooled HBI was 0.642 (95% CI: 0.588 to 0.694), exhibiting significant variation across the Sudan (0.728), Guinea (0.615), and Sahel (0.584) savannas. Species specific PSI values graded from Anopheles gambiae s.s. (8.91 {+/-} 0.34), Anopheles funestus (8.12 - 0.38), and Anopheles arabiensis (7.68 {+/-} 0.41) to Culex quinquefasciatus (4.87 {+/-} 0.52). In GLMM analysis, PSI was the strongest predictor of anthropophily ({beta} = 0.89, aOR = 2.44, 95% CI: 2.32 to 2.56, p < 0.001), with fixed effects explaining 78% of total field variance (Marginal R2 = 0.78). High PSI species maintained elevated anthropophily regardless of livestock availability, whereas low-PSI species displayed steep zooprophylactic diversion ({Delta}HBI = -0.71, p < 0.001) under high cattle density. Incorporating PSI reduced localized EIR prediction error by 64.2% (RMSE = 15.0 vs. 41.8 infective bites/person/month), and high-resolution (1 km2) predictive mapping correctly identified 87.1% (27/31) of historically documented outbreak Local Government Areas. Conclusion: Micro-sensory organ density dictates vector host preference and livestock-mediated zooprophylactic potential. Integrating PSI into spatial models resolves localized transmission heterogeneity, guiding targeted vector control prioritizing indoor residual spraying and dual-ingredient nets in high PSI foci, while leveraging endectocides and larval management in low-PSI pastoral corridors.

Experimental hut trials (EHTs) are WHO-recommended for the entomological evaluation of insecticide-treated nets (ITNs), but several hut designs are in operational use, and structural differences between them may confound efficacy predictions and limit cross-site comparability. We developed a Bayesian hierarchical framework comprising a host-seeking model, which jointly estimates biting deterrence and preprandial mortality while accounting for night-to-night variation and overdispersion, and a postprandial mortality model, which expresses hut and net effects as hazard ratios through a complementary log-log link. We applied it to a comparative trial of four hut designs (East African, West African, Ifakara and Rapley) conducted at a single site in Tanzania, evaluating eight ITNs when new and after twenty washes. Posterior estimates parameterise a vectorial capacity framework to predict reductions in transmission potential. Hut design influenced baseline mosquito behaviour and all three modes of action. Relative to the Rapley reference, baseline feeding rates were substantially lower in the East African and West African huts and closer to Rapley in the Ifakara hut. Preprandial mortality was amplified in the Ifakara hut. Comparing to previous analysis provides evidence that combining mortality before and after feeding into a single endpoint does not reliably reflect impact, supporting the decomposition of entomological outcomes into separate modes of action. Expressing modes of action as mechanism-specific parameters allows the estimates to be carried directly into transmission models. For every net, the predicted reduction in vectorial capacity was greatest in the Ifakara hut and smallest in the West African and Rapley huts. The effect of hut design on predicted impact exceeded that of washing the nets twenty times. Results indicate that the hut design under which trial data were collected should be considered when forecasting population-level effect.

Background Trypanosoma cruzi, the causative agent of Chagas disease, is subdivided into distinct genetic groups known as Discrete Typing Units (DTUs), each with distinct genetic traits that influence epidemiology and transmission dynamics. Several triatomine species serve as potential vectors of T. cruzi in the United States. However, despite the growing number of Chagas disease cases in the country, little is known about the genetic diversity and population structure of T. cruzi in natural vector populations. Methodology/Principal Findings We applied a multilocus metabarcoding approach to improve DTU resolution and characterize the genetic diversity and structure of T. cruzi in triatomines collected across five states of the southern United States. Five single-copy nuclear markers and one mitochondrial marker were amplified and processed by high-throughput sequencing to assess genetic diversity. We recovered 35 nuclear and 15 mitochondrial haplotypes from 70 infected specimens. Overall, genetic diversity was low ({pi} < 0.01 at all nuclear loci), with DTUs TcI and the North American lineage of TcIV detected, TcI being the most prevalent. Geographic structuring was particularly evident in TcI strains, which exhibited a distinctive haplotype profile in Florida populations, potentially linked to the recently revalidated vector species Triatoma ambigua. Mitochondrial introgression from TcIV into TcI suggests inter-DTU genetic exchange in these populations. Multiple haplotypes within individual insects detected across single-copy nuclear markers, support multiclonal infection as common feature of T. cruzi in natural vectors. Conclusions/Significance These findings provide new insights into the genetic landscape and evolution of T. cruzi in the United States. Evolutionary connectivity through mitochondrial introgression and frequent multiclonality highlights the importance of deep sequencing approaches for resolving T. cruzi genetic diversity, with direct implications for understanding for transmission dynamics, disease monitoring and control.

Background: Angola is one of the most affected countries in sub-Saharan Africa and was off-track to meet the WHO Global Technical Strategy targeting of a 75% reduction in malaria incidence and mortality by 2025. Recent studies highlighted major knowledge gaps regarding malaria vectors in Angola, particularly secondary vectors. A malaria control project initiated in 2007 in Balombo (Benguela Province, central Angola) has monitored malaria dynamics for 18 years, revealing increased Plasmodium prevalence and more frequent detection of non-falciparum species, mainly Plasmodium malariae (in co-infection with P. falciparum) in villagers of the Balombo region. This study aimed to characterize Anopheles populations in this region by assessing species composition, behavioural patterns, and infection rate. And to a less extent, to compare the performance of two indoor and outdoor mosquito trapping methods. Methods: Surveys were conducted during the rainy (December 2023 and 2024) and dry seasons (July 2024) in villages around the town of Balombo. Adult mosquitoes were collected indoors and outdoors using CDC Light Traps and BG-Pro traps in randomly selected houses. Female Anopheles were morphologically and molecularly identified. Primary and secondary vectors were screened for Plasmodium spp. using qPCR-high resolution melting, and positive samples were sequenced. Results: A strong seasonal effect was observed in Anopheles populations, with higher abundance, diversity, and Plasmodium- infection rates during the rainy season. An. funestus remained the main vector, but Plasmodium spp. was also detected in five other species: An. arabiensis, An. gambiae, An. marshallii, An. rufipes, and An. maculipalpis. This is the first report of P. malariae in Anopheles mosquitoes and the first mention of the role of secondary vectors in malaria transmission in Benguela Province. Conclusion: This study provides a thorough and updated inventory of Anopheles species in the Balombo region. Molecular biology confirmed the presence of P. falciparum and P. malariae in several species of Anopheles underscoring the existence of a complex transmission system in villages of Balombo. Infected exophagic and exophilic Anopheles species, exhibiting opportunistic behaviour, likely contribute, as secondary vectors, to the increase of Plasmodiumspp. transmission in the study sites. Therefore, confirming the need to implement complementary interventions targeting outdoor malaria transmission.

There is a growing body of research demonstrating the utility of invertebrate models for studies of fungal pathogenesis. In this study we evaluated Tenebrio molitor larvae as a model for studying talaromycosis, the infection caused by Talaromyces marneffei. T. marneffei is a thermally dimorphic opportunistic pathogen of humans, which transitions from hyphae to yeast when exposed to human body temperature (37{degrees}C). Using a combination of virulence assays and histopathology techniques, we have found T. molitor larvae to be useful simple hosts for modelling the yeast-associated disease at 37{degrees}C, as well as for studying the influence of temperature on T. marneffei biology. Infection establishment was found to be temperature-dependent: Larval infections could be established at both 37{degrees}C and 25{degrees}C, however 10-fold higher doses of conidia were required to cause significant disease at 25{degrees}C. Infections were also established more quickly when directly injecting larvae with the yeast cells, indicating that the yeast form has an increased capacity for host damage. T. marneffei in vivo yeast cell development was observed within larval tissues and hemolymph primarily at 37{degrees}C, but also at 25{degrees}C along with filamentous hyphal growth. The larval host environment therefore strongly supports yeast development, even partially in the absence of the 37{degrees}C signal, emphasising the combined importance of temperature and host environment for maintenance of the pathogenic morphology. This work provides a new model to assist future studies of this neglected tropical disease and improves our understanding of the complex relationship between morphology and pathogenicity in T. marneffei.

Tick-borne disease risk reflects the combined influence of vector abundance, pathogen prevalence, human exposure, and seasonal activity, yet these components are rarely integrated at the county level. We developed species-specific ecological risk scores for three medically important tick species (Acari: Ixodidae): Amblyomma americanum (L.), Dermacentor variabilis (Say), and Ixodes scapularis Say, across all 102 Illinois counties using data from 2019 to 2022. County-level predicted tick abundance and pathogen prevalence were scaled and combined using equal weights to generate primary vector-level risk scores. Robustness to the equal-weight assumption was evaluated by systematically varying the relative weights assigned to abundance and pathogen prevalence, while a separate sensitivity analysis incorporated reported tick-bite incidence where available. Associations with human disease were evaluated using negative binomial regression of tick-borne disease case counts on the corresponding risk scores, with county population included as an offset. Monthly phenology indicators were constructed by integrating human disease cases, reported tick bites, and sampled tick abundance and were evaluated in relation to temperature and precipitation. Seasonal risk was estimated by combining county-level vector risk with species-specific monthly phenology, generating monthly risk surfaces that were deployed through an interactive R Shiny application. Higher risk scores were significantly associated with greater disease case rates for each tick species. County rankings were highly stable under moderate changes in abundance and prevalence weighting. Seasonal activity differed among species, with A. americanum and D. variabilis showing concentrated warm-season risk and I. scapularis exhibiting a broader seasonal profile. These county-level seasonal risk maps identify where and when relative tick-borne disease risk is elevated in Illinois, supporting targeted prevention, surveillance, and clinical awareness.

The extrinsic incubation period (EIP), defined as the time between a mosquito acquiring a virus and becoming capable of transmitting it, is a key component of arbovirus transmission and varies with temperature. For Zika virus (ZIKV), empirical estimates of EIP are derived from heterogeneous laboratory studies and are typically analysed without accounting for interval censoring inherent in vector competence experiments. We applied Bayesian interval-censored survival models to pooled individual-level observations from eight published studies, comparing 20 candidate models representing four parametric survival distributions and five temperature-response functions in Ae. aegypti and Ae. albopictus. Model performance was evaluated using approximate leave-one-out cross-validation. A lognormal survival model with a quadratic temperature response and study-level random intercept provided the best predictive performance. Median EIP declined non-linearly with increasing temperature, from 59.5 days (95% CrI 27.0-124.0) at 20{degrees}C to 8.4 days (3.8-17.0) at 32{degrees}C in Ae. aegypti. Across the temperature range examined, estimated EIPs for Ae. albopictus were approximately 1.5-fold longer than those for Ae. aegypti. Credible intervals widened at temperature extremes, reflecting between-study heterogeneity and limited data availability. These results provide a statistical framework for estimating temperature-dependent ZIKV EIP while accounting for censoring and uncertainty, supporting improved parameterisation of mechanistic models of arbovirus transmission. Author summaryWhen a mosquito feeds on an infected host, the virus must replicate and spread to the mosquitos salivary glands before it can be transmitted. This delay, known as the extrinsic incubation period (EIP), is a key determinant of mosquito-borne disease transmission and varies with temperature. For Zika virus (ZIKV), estimates of EIP come from laboratory studies that differ in experimental design, mosquito species, and viral dose, making them difficult to combine. We analysed data from eight studies using a Bayesian statistical approach that accounts for uncertainty in the time mosquitoes become infectious, because mosquitoes are typically tested only at discrete time points after infection. Our analysis showed that ZIKV EIP decreases non-linearly with increasing temperature, ranging from approximately 60 days at 20{degrees}C to approximately 8 days at 32{degrees}C in Aedes aegypti. Across temperatures, Aedes albopictus had consistently longer EIPs than Aedes aegypti. Compared with Bayesian EIP models developed for other arboviruses, including dengue, yellow fever, and West Nile virus, ZIKV showed a stronger non-linear temperature response. These estimates and their uncertainty provide improved parameters for models predicting when and where Zika transmission is most likely.

Attractive targeted sugar baits (ATSBs) are a novel vector intervention targeting the sugar feeding behaviour of mosquitos by providing toxic sugar meals. ATSBs have shown strong efficacy in terms of mosquito population reduction in entomological trials but their epidemiological impact remains incompletely understood. This modelling study explores the impact of ATSBs on entomological and epidemiological outcomes over a wide range of transmission settings. We quantify the required killing probabilities to achieve target incidence reductions and demonstrate the sensitivity of ATSB efficacy to mosquito characteristics such as sugar feeding frequency and sac rate. Furthermore, we show that ATSBs can maintain reduced incidence levels previously achieved by long-term insecticide-treated net campaigns. Despite mixed results from clinical trials, these results highlight the high potential of ATSBs as a complementary vector control strategy, while underscoring the critical importance of entomological parameters in predicting public health impact. Realizing this potential will require accurate characterization of mosquito behavior and optimized deployment strategies to ensure sufficient population-level killing probabilities of mosquitoes.

BackgroundLarval habitat management is increasingly recognized as a complementary strategy for malaria vector control. While bacterial communities are abundant in mosquito breeding sites, their role in shaping oviposition behavior of Anopheles gambiae (Diptera: Culicidae) remains poorly understood. This study evaluated the relative attractiveness or repellency of water infused with bacterial isolates obtained from mosquito larval habitats to gravid Anopheles gambiae. MethodsWe isolated and taxonomically characterized bacteria from 30 larval habitats in coastal Kenya using 16S rRNA sequencing. Thirty-two representative isolates were tested in two-choice oviposition bioassays with gravid An. gambiae s.s., and volatile organic compounds (VOCs) emitted were analyzed by GC-MS. Statistical analyses included Students t-tests, Mann-Whitney U tests, and calculation of the Oviposition Activity Index (OAI). ResultsProteobacteria dominated larval habitats (78%), with frequent genera including Aeromonas, Acinetobacter, Pseudomonas, Enterobacter, and Cronobacter. Most isolates exerted strong deterrent effects, with Aeromonas hydrophila, Enterobacter hormaechei, and Pseudomonas mendocina completely inhibiting oviposition (OAI = -1; 100% repellency). Other isolates such as Neobacillus drentensis and Aeromonas veronii reduced egg deposition by >85%. In contrast, a small subset (Aquitalea pelogenes, Pseudomonas oleovorans, Cronobacter sakazakii) showed weak, non-significant attraction (OAI = 0.15-0.23). GC-MS revealed that highly repellent isolates emitted abundant benzenoids, ketones, phenols, ethers, oxygenated heterocycles, and aldehydes. ConclusionsBacterial volatiles strongly modulate oviposition site selection in An. gambiae, with most isolates acting as potent repellents. These findings highlight microbial VOCs as promising candidates for novel vector control strategies targeting gravid mosquitoes. Limitations include the laboratory-based bioassay design and lack of field validation of VOC activity. Future work should identify specific bioactive compounds and evaluate their efficacy under natural conditions. Research HighlightsO_LIBacteria from mosquito larval habitats influence oviposition of Anopheles gambiae. C_LIO_LISeveral bacterial isolates significantly repel gravid mosquitoes. C_LIO_LIGC-MS analysis revealed VOCs including benzenoids, ketones, phenols, and aldehydes. C_LIO_LIBacterial volatiles show potential for novel mosquito control strategies. C_LI

BackgroundMalaria caused by Plasmodium knowlesi and lymphatic filariasis caused by Brugia malayi are mosquito-borne infections with non-human primates as reservoirs. P. knowlesi has emerged as a significant cause of human malaria in Southeast Asia over the past two decades. Belitung district, Indonesia, was until recently assumed to have eliminated B. malayi until infections were detected in humans and long-tailed macaques. To investigate whether the local reservoir of B. malayi is also a reservoir for malaria we screened macaques from 4 areas in Belitung for malaria parasites. Methods and findingsBlood samples from 163 long-tailed macaques (Macaca fascicularis) that had been tested for B. malayi were examined by quantitative PCR assays specific for Plasmodium spp., P. knowlesi, P. inui, P. coatneyi and P. cynomolgi. A total of 130 macaques (79.8%) tested positive in the pan-Plasmodium qPCR assay. Plasmodium inui was most prevalent (41.7%), followed by P. knowlesi (38.7%), P. coatneyi (24.5%) and P. cynomolgi (13.5%). Multiple species infections, with 2-3 Plasmodium species were detected in 37% of macaques. Notably, 20 (91%) of 22 B. malayi-positive macaques were co-infected with at least one Plasmodium species. We sequenced the complete mitochondrion from 9 samples diagnosed by qPCR as mono-infections. Phylogenetic analysis confirmed 7 as P. knowlesi, and the other two as P. inui and P. coatneyi. Phylogenetic and pairwise analysis revealed that P. knowlesi isolates from Belitung were closely related to each other and to P. knowlesi from humans and monkeys from Thailand, Malaysia and Indonesia. ConclusionsMolecular evidence shows high prevalence of zoonotic malaria parasites in macaques from Belitung, emphasizing the risk of human transmission. Increased surveillance, improved diagnostics, and targeted interventions are needed to prevent zoonotic spillover of P. knowlesi as it has been observed for B. malayi in Belitung. Author summaryWe examined macaques for Plasmodium parasites in Belitung Island, Indonesia, a region classified as free of locally transmitted human malaria. Plasmodium knowlesi is a monkey parasite that commonly infects humans and has emerged as a concern in Southwest Asia over the last 20 years. In Belitung, macaques are infected with Brugia malayi, a filarial nematode, that causes lymphatic filariasis in humans. Blood samples from 163 macaques were screened for Plasmodium DNA and 80% carried malaria parasites. Plasmodium inui was most common, followed by P. knowlesi, P. coatneyi, and P. cynomolgi. Many macaques were infected with multiple parasites simultaneously, but no host was infected with all four Plasmodium species. Ninety one percent of B. malayi-positive macaques were co-infected with malaria parasites, including P. knowlesi, indicating multi-parasite infections that could potentially spread to humans. Analysis of the mitochondrial genome of 7 P. knowlesi isolates from Belitung showed that they were most similar to each other and both human and monkey samples from Malaysia, Thailand and Indonesia. The study highlights the need for enhanced malaria monitoring and prevention, given the complex epidemiology of co-infecting parasites and risk to humans and animals.

Background: Empirical surveillance for Aedes-borne arboviruses is inherently reactive, detecting transmission after it has commenced. For small island settings where dengue and chikungunya circulate sporadically, characterising when and where environmental conditions could support local transmission is critical for preparedness. In Mauritius, Aedes albopictus is the sole primary vector for dengue and chikungunya viruses, but previous suitability assessments have relied on Aedes aegypti parameterisation. Methods: We estimated monthly Index P for dengue and chikungunya across 160 localities in Mauritius from January 2014 to October 2024. Index P, a mechanistic transmission suitability measure derived from the Ross-Macdonald framework that climate-dependent transmission potential attributable to one adult female mosquito. Mean temperature and relative humidity were derived from ERA5-Land reanalysis dataset via Google Earth Engine and incorporated within the Mosquito-borne Viral Suitability Estimator (MVSE) framework. Index P was also parameterised with Ae. albopictus-specific biological priors and virus-specific vector competence values for both dengue and chikungunya. Results: Transmission suitability for both viruses was concentrated within the austral summer (November to April), with near-zero values in winter, below the indicative transmission threshold (Index P [&ge;] 0.5). Chikungunya exhibited consistently higher, more spatially widespread and longer-lasting suitability than dengue: all districts exceeded the transmission suitability threshold for chikungunya (Index P = 0.71), while median dengue Index P = 0.24, remaining below this threshold, during the same study period. Dengue peak suitability was concentrated in western coastal localities, consistent with the greater thermal sensitivity of its extrinsic incubation period in Ae. albopictus. Conclusions: These findings indicate that dengue and chikungunya have distinct, virus-specific climate-suitability profiles in Mauritius, and should not be treated as interchangeable for preparedness purposes. This provides an important Ae. albopictus-parameterised evidence base for Mauritius, enabling seasonal and geographic targeting of surveillance and vector control ahead of, rather than in response to local transmission.

Extensive use of insecticides is increasing resistance risks that could severely reduce the control of mosquito vectors that transmit infectious agents of Neglected Tropical Diseases. Addressing resistance threats by discovering new insecticides can be challenging because of the limited throughput and translatability of existing in vitro screening pipelines. Acquiring multiple phenotypic endpoints of larvae could alleviate these restrictions by more thoroughly profiling drug effects on whole organisms, providing leads to compounds with novel mechanisms of action, and increasing screening throughput compared to adult-stage assays. Here, we establish a pair of assays for profiling motility and development traits in larval-stage mosquitoes at a higher scale than standard larvicidal screening techniques. We optimized assay parameters and developed novel image processing approaches that enable relatively high throughput screening of chemical compounds on single larvae within a screen with condition replicates. We tested the assay with an insect growth regulator (S-methoprene), a slow-acting pyrrole (chlorfenapyr), and two microbial larvicides (Spinosad and Lysinibacillus sphaericus). These measurements aligned well with known insecticide mechanisms, and dose response curves established assay baselines for comparison in future screens. Finally, we present ways in which the assay design can be modified across different imaging technologies, showing the flexibility of the screening approach.

Onchocerciasis (river blindness) is targeted for elimination through mass administration (MDA) of ivermectin (IVM) to endemic populations. In areas where loiasis, caused by the blood-borne filarial parasite Loa loa, is co-endemic, IVM MDA faced significant challenges because individuals harboring more than 30,000 L. loa microfilaria (mf)/mL of blood are at high risk of developing serious adverse events (SAEs) that can sometimes be fatal. An alternative strategy was developed for safe IVM distribution: the so-called 'Test and Not Treat' (TaNT), which identifies and excludes individuals with high mf density from IVM MDA and treats only those with minimal risk. TaNT requires a point-of-care diagnostic device to rapidly quantify L. loa parasites in field conditions. The NTDscope is a handheld device which captures bright-field videos of whole blood in capillaries. An onboard algorithm detects and counts the live microfilaria of L. loa via movement of red blood cells. This paper describes (i) a new detection algorithm; and (ii) a May 2025 field trial in Cameroon using the NTDscope with new algorithm (550 patients). Results for the TaNT use case (i.e. flagging cases with L. loa mf densities > 30,000 mf/mL): 94% to 97% sensitivity, and 94% to 97% specificity. The results indicate that the NTDscope and new algorithm provide a greater margin of safety than the predecessor device, and can potentially offer rapid, effective field detection of high mf infections to enable scalability of the TaNT strategy.

Alpha-gal syndrome (AGS) is a tick bite-associated allergic condition induced by the lone star tick (Amblyomma americanum). Illinois had no confirmed AGS case data as of January 2026, when mandatory reporting began under the state's TICK Act, leaving practitioners without data to guide screening or resource allocation. We constructed a county-level proxy risk score using a Bayesian conditional autoregressive spatial model applied to three Illinois surveillance sources from 2019-2022: tick abundance, ehrlichiosis cases, and tick establishment status, all linked by a shared vector. Ehrlichiosis was modeled as a population-adjusted rate, ticks as a relative-intensity index, and establishment status as a fixed ecological component. The combined risk score identified a high-risk cluster in far southern Illinois that remained stable across alternative weighting scenarios. This approach is transferable to jurisdictions lacking direct AGS surveillance and offers a starting point for clinician education pending validation against confirmed case data.