Journal of Medical Entomology

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.

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.

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.

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.

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.

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.

Insects can experience a loss of fertility under acute heat stress, but impacts vary among species and depend on life stage and sex. We examined the effects of sublethal heat shocks on the fertility of three mosquito species (Aedes aegypti, Aedes notoscriptus and Culex quinquefasciatus) when males or females were exposed at the pupal and adult stages. Reproductive performance was assessed through egg-laying success, fecundity, egg hatchability and viable offspring production. Female heat shock had limited effects on fertility in all three species, though exposure of adult females to 41 {degrees}C for 1 hr reduced viable offspring production in Ae. notoscriptus. Male heat shock reduced egg hatchability in Cx. quinquefasciatus and viable offspring production in Ae. aegypti depending on the life stage exposed. We investigated several other aspects of heat shock exposure on Ae. aegypti fertility. In this species, male fertility partially recovered within 24-48 hours following heat shock exposure, while effects of heat shocks on female fertility occurred regardless of whether heat exposure occurred pre- or post-mating. In addition, maternal heat shock exposure enhanced offspring fecundity and viable offspring production under subsequent sublethal heat stress. Overall, our findings demonstrate that mosquito fertility is largely robust to short-term heat shocks, but there are also complex trait, sex, stage, temporal and species-specific effects, with implications for predicting mosquito population persistence and species persistence under future climate extremes.

West Nile virus (WNV; Flaviviridae) and Eastern Equine Encephalitis virus (EEEV; Togaviridae) represent the two most significant mosquito-borne zoonoses in the southeastern United States. While both viruses utilize avian amplifying hosts and mosquito vectors, it is unclear if they occur within distinct ecological niches. This study assessed the cumulative effects of both landscape composition and weather variables on the spatial-temporal distribution of WNV and EEEV outbreaks in Georgia over a twenty-four-year period (2001-2025). We used a modeling framework that directly accounts for both spatial and temporal effects but additionally integrates key EO variables (Earth observation). These environmental effects were investigated using Bernoulli generalized linear mixed-effects models (GLMMs) and executed with an integrated nested Laplace approximation (INLA). Our findings revealed disproportional distribution ranges of Culex quinquefasciatus, Aedes albopictus and Culiseta melanura in highly urbanized Georgian counties such as Chatham, Dekalb, and Fulton. Results showed that WNV transmission was heavily influenced by urban land cover (Posterior Mean: +0.78, 95% CrI: [0.61,0.95]) but negatively associated with lagged precipitation (Posterior Mean: -0.18, 95% CrI: [-0.29, -0.07]), confirming drought-driven amplification. Conversely, EEEV transmission was strongly influenced by wetland cover (Posterior Mean: +1.12, 95% CrI: [0.94,1.30]) and precipitation (Posterior Mean: +0.51, 95% CrI: [0.39,0.63]). These results underscore the need for improved mosquito surveillance across all Georgian counties in the face of growing vector-borne disease risks.

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.

Elucidating the biological and molecular mechanisms that govern male fertility and mating behavior in mosquitoes is critical for optimizing genetic and sterile insect technique-based vector control strategies. Here, we examined age-related changes in male reproductive capacity in Anopheles culicifacies, using female egg output as an indirect indicator of male fertility. Our results demonstrated that male reproductive age follows a non-linear pattern of fertility. Morphometric analysis from emergence to day 13 post-eclosion revealed a strong correlation between seminal vesicle capacity and female fecundity, suggesting that age-dependent gonadal development directly influences reproductive potential. At the molecular level, we identified AcLITAF6 as a key regulator of male reproductive homeostasis. RNAi-mediated knockdown of AcLITAF6 impaired apoptosis-associated and phagocytic clearance, reduced sperm viability, and decreased female productive outcomes. Conclusively, we reveal a previously unrecognized role of LITAF in sperm quality control and male reproductive fitness, highlighting AcLITAF6 as a potential target for mosquito population suppression strategies.

Background Spatial Emanators (SEs) are a promising tool for complementing existing malaria vector control interventions. In 2025, the World Health Organization (WHO) issued a conditional recommendation for SEs, and two transfluthrin-based products were added to the WHO Prequalification List. As SEs are scaled up, additional products are needed to ensure reliable supply, competitive pricing, and continued innovation. Methods This study evaluated the efficacy of SumiOne SE, containing 10% metofluthrin, in experimental huts in Siaya County, western Kenya. SumiOne SE efficacy was evaluated in six experimental huts, three intervention and three control, using human landing catches (HLCs) and aspiration. Collections were conducted in two 12-night phases, alternating between HLC and aspiration every six nights. Outcomes included reductions in human landing rates, density, exophily and blood feeding inhibition of Anopheles mosquitoes. Results SumiOne SE significantly reduced indoor densities of An. funestus with 63% protective effect (IRR = 0.37, 95% CI: 0.20-0.69, p = 0.0087). Overall An. funestus density (indoor and outdoor combined) was reduced by 58% (IRR=0.42, 95% CI: 0.22-0.79; p=0.0222). The rate of exophily of An. funestus exiting was 34% (95% CI: 27-42) in the intervention huts compared to 2% (95% CI: 1-4) in the control huts (p< 0.0001). Blood feeding inhibition for An. funestus was 46% with 44% (95% CI: 36-51) of the collected mosquitoes being blood fed in the intervention huts compared to 81% (95% CI: 76-86) blood fed in the control huts, (p<0.0001). Conclusion These findings support SumiOne SEs potential as a complementary vector control tool, alongside existing core interventions such as Insecticide Treated Nets and Indoor Residual Spraying, particularly in contexts where additional protection is needed.

While mosquito invasions and associated pathogens are increasingly prominent in Europe, evidence gaps remain for many northerly regions where proactive management interventions may be most effective. At the regional periphery of Europe, there remains a significant knowledge gap concerning mosquito vector distributions in Northern Ireland. This study aimed to address this major gap by systematically surveying larval and adult mosquito populations in key habitats (ponds, reedbeds, saltmarsh, wet grasslands, wet woodlands, and urban gardens). We further identified environmental drivers of important vector species abundance and screened collected specimens with molecular techniques to identify presence of cryptic human-biting vector species. In Northern Ireland, mosquitoes (including potential bridge vectors of West Nile and Usutu viruses) were found to be widely distributed. Between May and August 2025, a total of 2430 mosquitoes (951 adults and 1479 larvae) were collected over 196 trap nights and 76 larval collections. Collections encompassed 17 species, with Culex pipiens/torrentium being the most widely distributed and abundant taxon recorded. Mosquito abundances were greatest in reedbed habitats, related negatively with rainfall and peaked in June and July. A subset of Culex pipiens/torrentium mosquitoes were identified to species/biotype level using PCR, with the majority (89.3%) identified as Culex pipiens pipiens and 9.3% as Culex torrentium. Importantly, the first detections of Culex pipiens/molestus hybrids (1.8%) on the island of Ireland were found. This work underscores the wide, yet previously undocumented abundance and distribution of potential zoonotic vectors in Northern Ireland and provides an important baseline for management planning and future research, while contributing to broader understandings of environmental population drivers and distributions.

Aedes aegypti, an important vector of several arboviral diseases, is highly adapted to human-associated environments and commonly exploits anthropogenic water-holding containers for breeding. Understanding the ecological factors governing oviposition-site selection is essential for developing sustainable vector control strategies. Previous studies have established that microbial communities in breeding waters influence mosquito oviposition-site selection through the production of volatile organic compounds. Importantly, rapid urbanization in India has increased the availability of artificial breeding habitats for Ae. aegypti, potentially facilitating the transmission and persistence of arboviral diseases. However, the composition of bacterial communities in these larval habitats and their potential role in shaping the oviposition behavior of Ae. aegypti remain poorly characterized in the Indian context. Here, we investigated the bacterial diversity and community composition of Aedes larval habitats during the 2024 Zika and chikungunya outbreaks in Pune, India. Water samples, collected from artificial urban containers and natural freshwater habitats, were subjected to 16S rRNA amplicon sequencing. Artificial breeding habitats exhibited significantly higher bacterial diversity and were enriched with Enterobacter spp. compared with natural habitats. Oviposition bioassays further demonstrated a strong preference of gravid Ae. aegypti females for Enterobacter-conditioned water. GCMS analysis of bacterial suspension identified indole as a prominent volatile metabolite associated with Enterobacter, suggesting its possible role in mediating oviposition site selection. Together, these findings provide insights into the microbial and chemical ecology of Ae. aegypti breeding habitats and establish a basis for investigating microbial volatile cues as potential targets for vector surveillance and environmentally sustainable attract-and-kill 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.

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.

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.

Personal insect repellents can offer an effective means of preventing mosquito bites. Despite a wide variety of products that claim to repel mosquitoes, relatively few studies have systematically compared their performance through standardized tests. In Australia, chemical-based repellents must be registered with the Australian Pesticides and Veterinary Medicines Authority (APVMA); however, many unregistered products with unverified effectiveness remain commercially available. Here, we tested 55 insect repellents and 5 non-repellent perfumes and deodorants sold in Australia for their protection against Aedes aegypti mosquito landings (as a proxy for bites) using arm-in-cage assays across eight human subjects. We tested 43 topical repellents (containing 18 different primary active ingredients), four wristbands, three clothing stickers, three ultrasonic devices, one garment, one repellent applied to fabric, two perfumes, two deodorants and one antiperspirant. Thirteen repellents, including all wristbands, stickers and ultrasonic devices did not provide complete protection against mosquito landings for any human subject, while a further sixteen provided complete protection for 1 hr or less on average. Repellents containing DEET, oil of lemon eucalyptus, picaridin and IR3535 as the primary active ingredient were the only products that provided more than 2 hours of complete protection, but with significant variation among products with the same active ingredient. Only 7/30 essential oil-based topical repellents and perfumes provided complete protection for greater than 1 hr. Nearly half (25/52) of the repellents subject to registration with the APVMA were unregistered despite being advertised or labelled as mosquito repellents, and these tended to be less effective than registered products. Only 5/28 repellents met or exceeded their claimed protection time. This study highlights the prevalence of unregistered and ineffective mosquito repellents on the Australian market while providing evidence for products and active ingredients that do protect against mosquito bites under laboratory conditions.

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.

Abstract Background Chikungunya re-emerged in Mauritius in 2025 after a 19-year period with no outbreak. As precipitation and temperature play a key role in mosquito proliferation, the climatic conditions prevailing in Mauritius in 2025 may have influenced the emergence and spread of the outbreak. Our aim in this study was to investigate the spatial distribution of the 2025 chikungunya outbreak in Mauritius, and to determine the association between chikungunya incidence and selected predictor variables using Geographic Information Systems (GIS). Methods The variables considered were mean altitude, mean air temperature and mean precipitation, population density, road density, number of houses near rivers and the relative deprivation index (RDI) per village council area (VCA) and municipal ward (MW). Rainfall and temperature data were obtained from the climate bulletins of the Mauritius Meteorological Services and were also extracted from the ERA5 Monthly Averaged Data on Single levels available from the platform of Copernicus Climate Data Store. The U.S. Geological Survey Earth Explorer platform was used to access elevation data for Mauritius. In the absence of publicly available, spatially disaggregated official case data, reported chikungunya cases were reconstructed from multiple publicly accessible sources supplemented by communications with professionals involved in the outbreak. Information was collected contemporaneously as the outbreak evolved during 2025 and was supplemented by searches of archived material. Cases were georeferenced and aggregated to VCAs and MWs. All data was mapped using QGIS version 3.40.13.Page 2 of 17 Results The main findings were that the 2025 chikungunya outbreak was significantly associated with elevation and precipitation. Temporal analysis demonstrated that rainfall in the preceding month was the strongest climatic predictor of monthly chikungunya cases. Spatial analysis, however, showed that mean precipitation had a significant (p-value < 0.001) and negative association with chikungunya incidence, indicating lower disease occurrence in areas of high precipitation. Mean elevation was significantly (p-value < 0.001) and negatively associated with chikungunya incidence, indicating lower disease occurrence in higher-altitude areas. Temperature did not have any significant relationship with chikungunya incidence. Conclusions Rainfall had opposite associations depending on the analytical scale: preceding-month rainfall was positively associated with temporal case variation, whereas spatially averaged precipitation was negatively associated with incidence. Thus, rainfall exerted distinct temporal and spatial influences on chikungunya transmission, while higher elevation was associated with lower chikungunya incidence. These findings highlight the importance of integrating climatic and spatial analyses to improve surveillance Keywords Chikungunya; Elevation; Precipitation; Temperature; Geographical Information Systems; Mauritius.