Parasites & Vectors

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.

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.

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.

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.

Accurately determining the mating status of the agricultural fruit fly pest Ceratitis capitata, commonly known as Medfly, is essential for timely and effective eradication efforts. To overcome the limitations of subjective DAPI-based staining assessments of females captured in Jackson dry traps and Multilure liquid traps, we developed a multi-tier molecular diagnostic method that unequivocally detects mating status using DNA probes targeting the male-specific Y114 locus on the Y-chromosome of the species. Our protocol integrates morphological evaluation with increasingly sensitive molecular assays through the following steps: 1) A preliminary quality assessment of the specimens physical condition, DNA preservation, and mating status using conventional PCR followed by agarose electrophoresis (cPCR); 2) Quantification and real-time detection of sperm presence via quantitative PCR (qPCR); and 3) Detection of trace sperm amounts through droplet digital PCR (ddPCR). This PCR-based framework is designed for samples collected in the field, enabling accurate analysis of specimens exposed to adverse environmental conditions and varying levels of preservation after 2- and 3-weeks weathering times in traps. It allows quantitative determination of mating status even when sperm concentrations are extremely low, such as during transient copulation, and achieves detection limits down to approximately 14 spermatozoa in a mated female. By accounting for variable specimen quality and the performance characteristics of each molecular platform, this tiered approach ensures highly sensitive and unequivocal detection of mated females. The methodology can be used to assist eradication efforts across the C. capitata geographic range through the timely detection of mated females, halting their expansion and establishment into novel regions reducing control and eradication costs.

IntroductionChanges in vector control strategies alter the selection pressures exerted on natural populations of Anopheles gambiae s.l. and may influence the dynamics of insecticide resistance mechanisms. However, the consequences of discontinuing indoor residual spraying (IRS) campaigns on the evolution of Ace-1-mediated resistance remain poorly documented under natural conditions. This study aimed to assess the spatiotemporal evolution of resistance to pirimiphos-methyl following the cessation of IRS and to investigate evidence consistent with the existence of a biological cost associated with Ace-1-mediated resistance. MethodsNatural populations of An. gambiae s.l. were collected between 2017 and 2023 in three districts in Burkina Faso that had undergone IRS campaigns (Kampti, Solenzo, and Kongoussi). Susceptibility tests with pirimiphos-methyl (0.25%) were conducted out following WHO protocols, and a subsample of exposed mosquitoes was genotyped to detect the Ace-1 G119S mutation. Spatiotemporal trends in mortality, allele frequencies and genotypes were analyzed according to the pre-IRS, IRS, and post-IRS periods. The association between the Ace-1 genotype and survival following exposure to pirimiphos-methyl was assessed using logistic regression, while the concordance between phenotypic and molecular indicators of resistance was examined using Spearmans correlation. ResultsThe susceptibility of An. gambiae s.l. populations to pirimiphos-methyl was gradually restored after the discontinuation of IRS at all sites. At the same time, the frequencies of the Ace-1 119S resistance allele declined sharply, particularly in Kampti and Solenzo, while they remained low in Kongoussi throughout the study period. Mosquitoes carrying resistant genotypes had a significantly higher probability of survival after exposure to pirimiphos-methyl than susceptible homozygotes, with resistant homozygotes (RR) exhibiting the greatest survival advantage (OR = 27.62; 95% CI: 6.91-110.45; p < 0.001). A significant negative correlation was observed between the frequency of the Ace-1 119S allele and phenotypic mortality ({rho} = -0.48; p = 0.033), indicating a concordance between the two indicators of resistance. The progressive decline in allele frequencies, the decreasing prevalence of resistant genotypes, and the concomitant restoration of susceptibility are field observations consistent with the existence of biological costs associated with Ace-1-mediated resistance. ConclusionThis study provides field evidence consistent with the existence of biological costs associated with Ace-1-mediated resistance in natural populations of An. gambiae s.l. These results underscore the value of an adaptive resistance management strategy based on alternating selection pressures and could guide future vector control strategies, particularly if indoor residual spraying campaigns or other interventions relying on organophosphates were reintroduced.

IntroductionDengue, one of the most important arboviral infections worldwide, is transmitted primarily by the mosquito Aedes aegypti, a vector closely associated with human habitations. Because vector control is the primary prevention strategy for this disease novel tools are urgently needed. We evaluated an Attractive Lethal OviTrap (ALOT) that targets epidemiologically relevant gravid female mosquitoes for public health impact against dengue disease. MethodsWe conducted a proof-of-concept field efficacy trial in the Amazonian city of Iquitos, Peru, to quantify the reduction of vector density, parity, and sex ratio and symptomatic human dengue infection over a 3.5 year follow-up period. After three baseline entomological surveys in core (753 houses), and buffer (1,549 houses) areas where traps were placed in and around homes, and a control (1,233 houses) area without traps, entomological surveys were carried out every 2 months, and household residents were monitored for dengue disease 3 times a week. Trap maintenance was conducted at 2-3 week intervals by study staff during the first 2.5 years and by residents in the final year. ResultsTotal and female Ae. aegypti abundance demonstrated a strong initial effect with 52% and 47% fewer mosquitoes after trap placement, respectively (Total: RR = 0.48, 95% CI: 0.40 - 0.58, p < 0.001; Females: RR = 0.53, 95% CI: 0.43 - 0.66, p < 0.001). The effect diminished significantly, however, over the study period (interaction coefficient = 0.035 per month, p < 0.001; interaction coefficient = 0.030 per month, p < 0.001). Female-to-male ratio decreased after trap deployment and impact on non-Aedes abundance was like that of Aedes. Cumulative symptomatic dengue incidence was 1.40% (95% CI: 1.00%-1.70%) in the ALOT area versus 3.20% (95% CI: 2.60%-3.80%) in the control area, a 56% relative reduction (log-rank {chi}{superscript 2}=30.0, p<0.001). Restricted mean survival time analysis showed participants in the intervention area on average remained dengue-free for 15.5 additional days (95% CI: 11.1-20.0, p<0.001). ConclusionsThe ALOT strategy functioned as predicted, decreasing but not eliminating female vector densities, with a clear public health impact, lowering dengue disease in areas where traps were deployed. Our study also illustrates the challenges associated with conducting large-scale vector control trials and the need to consider programmatic implementation early in the process of bringing a new product to market. Author SummaryDengue is one of the most important mosquito transmitted infections worldwide. Aedes aegypti, the most important species involved, has a unique biology living in containers associated with households. New methods to control this mosquito are needed and we present our evaluation of a novel trap called the Attractive Lethal OviTrap (ALOT) that targets older female mosquitoes that are the most important for dengue disease transmission. We conducted a trial in the Amazonian city of Iquitos, Peru, to measure reductions in mosquito numbers, age, and sex ratio and symptomatic human dengue infections over a 3.5 years. After baseline entomological surveys prior to placing ALOT traps in homes in a treatment area, which included a central core area with 753 houses and a protective buffer area with 1,549 houses areas. We also had a control area without any traps in 1,233 houses. Mosquito surveys were carried out every 2 months, and household residents were monitored for dengue disease 3 times per week. Trap maintenance was conducted at 2 - 3 week intervals by study staff during the first 2.5 years and by residents in the final year. The number of all Ae. aegypti were initially reduced by 52% after trap placement whereas females were reduced by 47%. The effect diminished significantly, however, over the study period. Female-to-male ratio decreased after trap deployment and impact on other mosquito species was like that of Aedes. Dengue incidence was 56% less in the area with traps compared to the control area, estimating that participants in the intervention area on average remained dengue-free for 15.5 additional days. The ALOT strategy functioned as predicted, decreasing but not eliminating female mosquitos and had a clear public health impact, lowering dengue disease in areas where traps were deployed. Our study also illustrates the challenges associated with conducting large-scale vector control trials and the need to consider programmatic implementation early in the process of bringing a new product to market.

Japanese encephalitis virus (JEV) is an emerging mosquito-borne flavivirus with potential for geographic expansion, yet the risk of establishment in North America remains poorly characterized. We assessed vector competence of three North American Culex species (Cx. pipiens, Cx. quinquefasciatus, and Cx. tarsalis) for the JEV Nakayama strain, isolated from human brain in 1934 in Japan, across five constant temperatures (15, 20, 25, 30, and 33{degrees}C) at 4, 7, and 14 days post-feeding, quantifying infection, dissemination, and transmission rates. Vector competence was low but non-zero across all species. Cx. pipiens showed higher infection rates than the other species, whereas Cx. quinquefasciatus and Cx. tarsalis were minimally susceptible under these experimental conditions. Temperature had limited effects on infection and no detectable effects on dissemination or transmission. These findings suggest limited transmission potential of JEV Nakayama in North America, with Cx. pipiens as a relatively permissive vector.

Background Tsetse are vectors of trypanosomes responsible for African animal trypanosomosis (AAT) and human African trypanosomiasis (HAT). While Cote dIvoire has successfully eliminated HAT as a public health problem and approaches elimination of transmission, AAT remains a major obstacle to agriculture and livestock production. Understanding the spatial distribution of tsetse is essential for prioritizing and sustaining disease control and elimination efforts. Methodology/Principal Findings Using 1,702 occurrence records from the national tsetse atlas we modeled the habitat suitability of the nine tsetse species present in Cote dIvoire. We identified suitable habitats in unsampled areas and quantified environmental constraints on tsetse distribution. Resampling the data to a 1km x 1km grid produced spatially explicit outputs at a resolution more relevant for operational planning. An ensemble modeling approach was employed integrating four algorithms--Random Forest, XGBoost, Maximum Entropy (MaxEnt), and Generalized Additive Models (GAM)-- with satellite-derived environmental and anthropogenic predictors--which achieved high predictive accuracy, area under the curve and True Skill Statistics 0.80 and 0.83, respectively. Distance to waterbodies, soil moisture, distance to protected areas, maximum land surface temperature, and sheep density were key drivers of habitat suitability. Importantly, the models identified suitable habitats in 11 administrative regions not covered by the atlas, providing an improved national tsetse risk profile. Conclusions/Significance These results provide a detailed assessment of the ecological suitability of tsetse across Cote dIvoire and their persistence in agroecological mosaics with high human and livestock densities. We offer a high-resolution blueprint for vector and disease control, particularly in areas where field data are currently lacking. We provide a robust framework for evidence-based decision-making within the Progressive Control Pathway (PCP) for AAT by enabling the identification of priority areas and resource allocation optimization to improve livestock productivity through more effective AAT control and reduce the risk of resurgence of HAT.

Prolonged use of the few insecticide classes available for long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) has driven widespread physiological resistance of malaria vector mosquitoes to this limited arsenal of active ingredients. However, recent innovations like next-generation LLINs (NG-LLINs) containing two complementary insecticides and new insecticide classes for IRS offer new opportunities for pre-emptive resistance management by deploying more diversified actives as mixtures, combinations, rotations or mosaics. Here a deterministic model of mosquito foraging behaviour was formulated to predict the probabilities of deterrence, mortality or successful feeding across repeated feeding attempts in scenarios with different combinations of NG-LLINs and/or IRS micro-mosaics with varying levels of insecticide diversification between neighbouring houses. Final fates were classified based on whether or not the mosquito eventually died or successfully fed, and whether the latter occurred indoors or outdoors after exposure to zero, one or several IRS insecticides. The primary outcome was the probability that a single F mosquito carrying a novel resistance trait to a new IRS insecticide successfully feeds, survives and reproduces, thereby establishing those traits within the population. The secondary outcome was the selection coefficient governing the spread of such novel resistance traits from the F generation onwards. For highly anthropophagic and endophagic vectors like Anopheles funestus, combining NG-LLINs with IRS micro-mosaics using two insecticides may reduce emergence rates for novel resistance traits against IRS insecticides by approximately 2 to 2.5-fold, mainly through direct killing by NG-LLINs, although exposure to both IRS actives when forced to visit multiple houses also contributes to a lesser extent. However, such resistance management benefits are fundamentally constrained by outdoor feeding behaviours that limit or completely prevent indoor insecticide exposure. Increasing IRS micro-mosaic insecticide diversity beyond two actives is unlikely to further dampen resistance emergence rates because few mosquitoes survive long enough without feeding to encounter several IRS treatments. Once a resistance trait becomes established in the vector population, selection coefficients remain consistently high enough to force the spread of those traits, regardless of intervention combination. For more exophagic, zoophagic vectors like An. arabiensis, NG-LLINs plus IRS micro-mosaics are not expected to provide any meaningful resistance management benefit because frequent outdoor feeding, often on animals, allows them to largely avoid insecticide exposure altogether. Exclusively indoor-focused vector control strategies may not satisfactorily slow insecticide resistance emergence and spread, so new outdoor protection measures that close these coverage gaps with complementary insecticides will be needed.

Abstract Background: Urogenital schistosomiasis remains a major neglected tropical disease in rural communities, particularly in reservoir-adjacent settings where sustained human-water contact facilitates persistent transmission. Despite ongoing control efforts, communities located around reservoirs may experience persistent transmission partly due to socioeconomic water exposure and presence of infected intermediate host snails in aquatic habitats. This study assessed the prevalence, intensity, and epidemiological correlates of Schistosoma haematobium infection in reservoir-adjacent communities in Kano State, Nigeria to better understand local transmission. Methods: A community-based cross-sectional study was conducted among 447 consenting participants from six communities located around three water reservoirs in Kano State. Sociodemographic characteristics, water-contact behaviour, sanitation practices, treatment-seeking behaviour, and knowledge related to urogenital schistosomiasis were obtained using interviewer-administered questionnaires. Urine samples collected between 10:00 and 14:00 were examined using microscopy following centrifugation/sedimentation. Schistosoma haematobium isolates were characterised by molecular methods, using PCR and Sanger dideoxy sequencing. Bulinus snails were collected from the reservoirs by handpicking and scooping methods. Nucleotide sequences were analysed using NCBI BLAST to confirm species identity. Epidemiological data on Schistosoma haematobium infection were analysed using chi-square tests and univariate and multivariate logistic regression analyses to assess associations with explanatory variables. Results: The overall prevalence of S. haematobium infection was 34.23% (153/447), indicating moderate endemicity. Molecular study confirmed the occurrence of pure S. haematobium. Community-specific prevalence ranged from 32.35% to 37.84%. Heavy-intensity infection (>50 eggs/10 mL urine) occurred in 17.65% of infected participants. Infection was most common among children and adolescents and was higher in males than females, although the sex difference was not statistically significant. Water-contact behaviour was the strongest correlate of infection: participants reporting water contact had a markedly higher prevalence than those without such exposure (39.12% vs 3.28%; OR = 18.96, 95% CI: 4.56-78.73). Fetching water (aOR = 2.57; 95% CI = 1.53 -4.34; p < 0.001) and Swimming (aOR = 2.37; 95% CI = 1.42 -3.94; p = 0.001) were the specific activities most strongly associated with higher odds of S. haematobium infection. Age (aOR = 0.91; 95% CI = 0.87 - 0.95; p < 0.001) significantly reduced the odds of infection. In terms of gender, female participants had significantly lower odds of infection compared with males (aOR = 0.52; 0.29 0.92; p = 0.03). Knowledge of transmission was poor, with only a small proportion of participants correctly identifying contact with contaminated water as the cause of infection. A rare ectopic detection of S. mansoni eggs in urine was also observed. Presence of S. haematobium-infected Bulinus intermediate hosts in the water reservoirs was reported. Conclusions: Urogenital schistosomiasis remains actively transmitted in reservoir-adjacent communities in Kano State, with infection strongly associated with frequent human-water contact and poor disease knowledge. These findings support the need for integrated control strategies combining praziquantel-based preventive chemotherapy, targeted health education, improved water and sanitation, and behaviour-focused interventions to reduce reinfection and interrupt transmission. Keywords: Urogenital schistosomiasis, Schistosoma haematobium, water reservoirs, prevalence, risk factors

Insecticide-treated net (ITN) use - defined as the proportion of a population that use ITNs - is a measure of ITN uptake that is used in the estimation of malaria burden and evaluation of intervention programs. However, binary classification of individuals as users or non-users does not account for variations in ITN-related protection attributed to deleterious factors such as chemical and physical degradation, and increased insecticide resistance in vector populations. In this paper, we present a parsimonious model for malaria dynamics in mosquito-human populations in the presence of varying ITN use conditions. Using this model, we propose a new standardised measure of ITN coverage termed the "efficacy-adjusted use" defined as the equivalent level of use, assuming fully efficacious nets, that would be required to achieve the same level of theoretical EIR reduction. This more nuanced measure is used as a proxy for studying ITN-attributed protection across 44 African countries. We find that estimated protection levels in current ITN paradigms is significantly lower than indicated by crude ITN use metrics, with insecticide resistance having the largest deleterious effect. Furthermore, recent adoption of next-generation nets is estimated to have mitigated a 13% reduction in protection compared to a counterfactual pyrethroid only scenario.

The black-legged tick (Ixodes scapularis), a key vector of Lyme disease, anaplasmosis, and babesiosis, exhibits regionally distinct patterns of seasonal activity driven by climate. Consequently, the relative timing of larval and nymphal activity varies across geographic locations, influencing pathogen transmission dynamics. Early-emerging nymphs may increase pathogen transmission, whereas early-emerging larvae may reduce transmission. In addition, synchrony between the two life-stages facilitates co-feeding transmission, which contributes to pathogen maintenance and coinfection risk. Temperature is thought to be an important driver of tick phenology, but existing mechanistic models that incorporate temperature fail to accurately capture the timing of larval and nymphal tick activity. To address this limitation, we developed a mechanistic model that includes two additional factors: humidity-dependent questing and low rates of overwinter development. To assess the value of these factors for explaining real-world patterns, we fitted alternative models to tick collection data from the National Ecological Observatory Network. In doing so, we found that explicitly incorporating humidity is necessary to reproduce observed tick phenology, with larval ticks being especially sensitive to relative humidity compared to other life stages. In addition, we found that accounting for humidity had a larger effect at Mid-Atlantic sites than at Northeastern sites, underscoring the importance of region-specific interactions between temperature and humidity in shaping I. scapularis phenology. By more accurately capturing tick seasonality compared to existing mechanistic models, our model illustrates the importance of accounting for factors beyond temperature for investigating how climate variability influences seasonal tick activity and pathogen transmission.

Parasitic infections pose multifaceted threats to farmed fish, extending beyond direct pathogenicity to facilitate infections of bacteria, viruses, and microparasites. This synergistic interaction often leads to co-infections that significantly exacerbate disease outbreaks and mortality, presenting a severe challenge to aquaculture sustainability. Recently, a novel trypanosomiasis caused by the Trypanosoma carassii spectrum has emerged in cage-cultured Larimichthys crocea along the southeast coast of China, resulting in widespread prevalence and high mortality rates. Although this pathogen is hypothesized to originate from freshwater fish, its transmission route in marine environments has remained elusive. In this study, we investigated potential vectors and intermediate hosts of T. carassii spectrum, including leeches and monogenean in natural marine settings, and simulated transmission pathways using an established laboratory model involving T. carassii spectrum, Micropterus salmoides and the leech Poecilobdella manillensis. First, our field surveys in the coast of Ningde, Fujian Province, revealed a nearly 100% co-infection rate of T. carassii spectrum and the monogenean Neobenedenia girellae in diseased juvenile L. crocea. PCR analysis detected T. carassii spectrum traces in some N. girellae specimens, and subsequent experiments confirmed that N. girellae ingests the trypanosome while feeding on host blood. Furthermore, bacterial co-pathogens, such as Vibrio harveyi, were also detected within N. girellae. We also document two fatal leech infestations: Zeylanicobdella arugamensis in hybrid groupers (Epinephelus moara [female] X Epinephelus lanceolatus [male]) in Zhangpu, and Limnotrachelobdella okae in E. lanceolatus and E. fuscoguttatus in Raoping. These leeches tested negative for trypanosomes but carried pathogenic bacteria that co-infected the host fish; nonetheless, they are established vectors for trypanosome transmission. In a laboratory cohabitation model simulating T. carassii spectrum transmission, infected M. salmoides were housed with healthy conspecifics under three conditions: Group A (with the leech P. manillensis), Group B (no leeches), and Group C (no leeches, with physical separation between infected and healthy fish). After 14 days, blood smear microscopy and PCR analysis revealed infection rates in healthy fish of 58.33% in Group A, 40.00% in Group B, and 0% in Group C. Conclusively, T. carassii spectrum can be transmitted via leeches (with higher efficiency) and may also spread through direct contact under high-density aquaculture conditions, whereas N. girellae may act as an incidental vector, further research is warranted to clarify transmission dynamics in natural marine ecosystems. Additionally, our findings highlight the role of ectoparasites, including N. girellae and leeches, as potential reservoirs and vectors for bacterial pathogens of fish. In high-density intensive aquaculture, this vectorial capacity transforms parasites from primary pathogens into key drivers of polymicrobial disease outbreaks.

Predicting species distributions under climate change typically relies on thermal limits for survival. However, recent evidence suggests that sublethal temperatures that reduce fertility can constrain distributions more strongly than temperatures causing mortality alone. Despite the importance of mosquitoes as vectors of human disease, thermal fertility limits remain largely uncharacterized in these insects. Here, we show that fertility in Aedes aegypti is highly sensitive to thermal stress and exhibits distinct sex-specific responses. Adult males were more vulnerable to both heat and cold exposure than females. At 38{degrees}C, survival remained high in both sexes despite significant reproductive impairment, indicating that sterility can occur independently of mortality. Similarly, exposure to 2{degrees}C induced male sterility whereas females maintained reproductive function. Morphological analyses of reproductive organs showed temperature-associated damage consistent with the observed fertility loss. Incorporating these fertility thresholds into ecological niche models generated different predictions of climatic suitability compared with models based solely on lethal temperature limits. Our findings demonstrate that sublethal effects on reproduction can substantially influence estimates of mosquito climatic suitability and highlight the importance of incorporating fertility based thermal limits into projections of vector distributions and disease risk under future climate change.

BackgroundMalaria control is increasingly challenged by the urban-adapted vector Anopheles stephensi, yet molecular and cellular tools for this species remain scarce, restricting functional genomic studies and the development of genetic control strategies. To help address this gap, we established a new embryo-derived Anopheles stephensi cell line. ResultsWe generated and characterised a novel embryo-derived Anopheles stephensi (SDA-500) cell line capable of sustained growth in vitro. Species identity was confirmed by mitochondrial COI barcoding, and karyotypic analysis revealed a diploid chromosome complement with the presence of a Y chromosome, confirming that at least some cells are of male origin. Transfection conditions were optimized, with TransIT-PRO showing higher efficiency than Lipofectamine-based reagents. Using a dual-luciferase reporter assay, of several promoters tested the Anopheles gambiae polyubiquitin promoter exhibited the strongest and most consistent transcriptional activity in SDA-500 cells. ConclusionsThe SDA-500 cell line provides a stable and genetically validated in vitro platform that supports efficient transgene expression. This resource provides a useful system for functional genomics and molecular manipulation in Anopheles stephensi and is expected to facilitate studies of mosquito biology and contribute to the development of novel malaria control strategies.

Here we report a newly developed method utilizing long-range PCR and long-read Pacific Biosciences HiFi sequencing that successfully obtained two full-length and annotated mitochondrial genomes from Culex quinquefasciatus Say, 1823 and Culex tarsalis Coquillett, 1896, both from Maricopa County, Arizona, USA. Given the substantial burden of West Nile virus in Maricopa County over the past decade, and that these vectors are primarily responsible for spillover to human populations in the county, it is critical to better understand their distribution over time and space. This study begins to approach this need by contributing a novel approach that has resulted in the first West Nile virus vector mitochondrial genomes from Arizona. Our circular Cx. quinquefasciatus mitogenome is 15,587 bp in length, making it the first USA-based mitogenome sequenced through the AT-rich control region. The Cx. tarsalis mitochondrial genome is 16,416 bp long, longer than recently published California-based CTarK1 and Texas-based PQ585801 mitogenomes. The increased length of the Cx. tarsalis mitogenome is a result of a 905 bp insertion in the AT-rich control region, not present in the species publicly available mitogenomes. A maximum likelihood-based phylogenetic reconstruction supports the species designation of these newly-sequenced mitogenomes. The newly developed methodology offers a unique approach to study medically-important vector species around the globe, providing a solution to study populations through pooled vector pathogen surveillance programs.