Journal of Medical Entomology

Aedes aegypti is the primary vector species for dengue, Zika, and chikungunya viruses, and is adapted to thrive in urban environments. Their proximity to human populations and the adverse health outcomes associated with arboviral infections make mosquito control essential for reducing the risk of outbreaks and disease spread. Source reduction and larvicide applications are central components of mosquito control because they reduce adult mosquito emergence by targeting immature life stages. Therefore, our objective was to identify urban aquatic habitats used by Ae. aegypti and assess temporal variation in their contribution to immature mosquito production. To distinguish habitat use from habitat productivity, we considered larval presence as an indicator of oviposition or early-stage survival and pupal presence as evidence that a habitat supported development through most of the immature life cycle. Between July 2018 and October 2019, 2,482 inspections in Miami-Dade County, Florida, identified 2,756 aquatic habitats from which 19,466 Ae. aegypti larvae and 3,648 pupae were collected. Unique temporal trends were observed for both larval and pupal presence and abundance at county and habitat resolutions. County-level trends showed the highest larval and pupal production during Summer and the lowest pupal production during Winter. However, temporal patterns differed across habitats, supporting a context-dependent interpretation of habitat conduciveness, in which the same habitat type may support different levels of immature mosquito production depending on local environmental conditions. Our results show that habitats with frequent larval occurrence did not consistently have high pupal presence, indicating that larval presence does not necessarily reflect habitat productivity. Control strategies that prioritize habitats repeatedly associated with pupal production by season may improve year-round mosquito control, outbreak preparedness, and resource allocation.

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

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.

Aedes albopictus is a major arboviral vector whose global expansion, driven by human activities and climate change, poses a growing public health concern for a number of neglected tropical diseases in both tropical and temperate regions. As a poikilotherm, its biology and population dynamics are strongly influenced by temperature, thereby shaping disease transmission. To thwart and control its geographic expansion, effective vector control strategies are increasingly critical. Densoviruses (DVs), such as AalDV2, are being explored as mosquito viral biocontrol agents due to their restricted host range and ability to disseminate through oviposition sites. However, the influence of environmental parameters on the interactions between Ae. albopictus and AalDV2 remains poorly understood. This makes their performance under realistic, fluctuating thermal regimes difficult to estimate. In this study, we investigated the combined effects of temperature and AalDV2 exposure on Ae. albopictus survival and development across its full life cycle. Mosquitoes were reared under fluctuating temperature regimes (26-28 {degrees}C and 32-34 {degrees}C, 12:12 day[ndash]night cycles) and exposed to AalDV2 or a control treatment. Chronic exposure to 32-34 {degrees}C significantly reduced overall survival, decreasing median lifespan by approximately 10 days (HR=2.21, p=0.0018), with a deleterious effect increasing over time. It extended aquatic lifespan and increased pupal mortality. It also reduced adult lifespan in both sexes with a stronger effect in females. AalDV2 exposure had no significant effect on overall survival, stage-specific mortality, or adult lifespan. However, a significant interaction between viral exposure and thermal stress was detected on aquatic lifespan: AalDV2-exposed females showed further extended larval and pupal development specifically under the 32-34 {degrees}C regime, without any effect on survival. These results indicate that the biocontrol potential of AalDV2 cannot be assessed independently of thermal context: while lethal effects were absent under both fluctuating regimes, the prolongation of aquatic development by the virus under thermal stress may have indirect consequences for mosquito population dynamics that warrant further investigation.

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

BackgroundA female Aedes aegypti mosquito typically mates only once, receiving all of the reproductive materials required for her lifetime from a single male. During mating, males transfer chemical signals that permanently suppress female receptivity to subsequent mating attempts. Successful mating can be disrupted by satyrization, a form of interspecific reproductive interference in which Aedes albopictus males inseminate Aedes aegypti females without producing viable offspring, effectively rendering them sterile. Aedes aegypti populations from areas where both species co-occur can develop resistance to satyrization, which may arise through premating mechanisms including altered mating interactions and/or postmating mechanisms including reduced female sensitivity to male-derived chemical compounds. Methodology/Principal FindingsWe compared two Aedes aegypti strains derived from field collections in Lee County, Florida: one from a site where Aedes albopictus is established (Fort Myers) and one from a site with no known history of Aedes albopictus co-occurrence (Upper Captiva Island). Using laboratory mating assays, fluorescent dye transfer experiments, and male reproductive homogenate injections, we show that females from the co-occurring population exhibit reduced susceptibility to satyrization. This resistance is associated with two distinct mechanisms: reduced physical interactions with heterospecific males and reduced physiological sensitivity to Aedes albopictus homogenate following injection. Notably, co-occurring females retain sensitivity to signals from conspecific males, indicating that resistance mechanisms are species-selective rather than a generalized reduction in mating responsiveness. ConclusionsAedes aegypti females from a population co-occurring with Aedes albopictus show reduced susceptibility to satyrization under laboratory conditions, mediated by both premating and postmating mechanisms. The species selectivity of these responses suggests that co-occurring populations can discriminate between conspecific and heterospecific signals. Understanding how this discrimination is achieved and how it varies across populations will be important for interpreting interspecific mating dynamics and for evaluating vector control strategies that depend on mating interference, including sterile male release programs.

Mosquito-borne diseases, such as malaria, remain an important public health problem globally, including on the island of Madagascar. Malaria vector control strategies are typically aimed at adult Anopheles mosquitoes in predominantly rural settings. However, very little information is available on larval populations of Anopheles and other vector genera, such as Aedes and Culex, in urban areas, where urbanization creates a multitude of potential larval habitats. Here, the objectives are to identify different types of existing larval habitats and the diversity of vector species in the capital city of Antananarivo, and to study the temporal dynamics and spatial distribution of mosquito vectors of public health interest. From 2022-2023, longitudinal larval surveillance was carried out across all six districts of Antananarivo and consisted of prospecting 2,856 potential larval habitats, of which 653 (23%) were found to contain Aedes, Culex, or Anopheles larvae. Anopheles larvae were reared and morphologically identified using taxonomic keys, and molecular identification by PCR and Sanger sequencing was used for Anopheles sibling species determination. A total of 27,418 larval specimens were collected from eight species: Culex quinquefasciatus (53%), Aedes albopictus (26%), Cx. pipiens (14%), Anopheles gambiae s.l. (3%, all molecularly identified as An. arabiensis), Cx. univittatus (1%), Cx. antennatus (1%), Cx. poicilipes (1%), and An. coustani (1%). Geospatial analysis reveals that 55% of larvae came from artificial larval habitats within the city, which harbor lower species diversity than natural larval habitats. Twenty-one different types of larval habitats were observed, and Cx. quinquefasciatus, a vector of West Nile virus, was present in all of them. Aedes albopictus, vector of arboviral pathogens, prefers artificial habitats such as tires and flower pots, whereas the common rural malaria vector, An. arabiensis preferred natural sites like brick pits and rice fields. An. coustani, often considered a livestock-associated rural vector, was found to thrive in brick pits, wells, ponds, and in aquatic agriculture. Notably, of the 1,270 Anopheles larvae collected, none were found in an artificial container. Culex quinquefasciatus, Cx. pipiens, and Ae. albopictus reached peak abundance in March during the rainy season, while malaria vectors An. arabiensis and An. coustani showed unexpected abundance peaks during the dry periods, in September and April. Two districts (arrondissements 2 and 5) showed higher abundance and diversity of mosquito vectors than the other districts. The data presented here reveal the spatiotemporal dynamics of malaria and arboviral mosquito vectors in urban Madagascar and highlight the heterogeneity of habitats across the urban ecosystem, with important practical implications for the surveillance and control of mosquito-borne diseases.

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.

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.

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

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.

Diapause is a critical adaptive strategy that enables temperate mosquito species to survive adverse environmental conditions and maintain population persistence across seasons. In Culex pipiens, diapause plays a key role in overwintering and influences the seasonal dynamics of arbovirus transmission. However, diapause expression is often assessed using single traits, limiting our understanding of its integrated physiological basis and variation among populations. In this study, we investigated the behavioural, morphological, and reproductive signatures of diapause across three laboratory strains of Culex pipiens (Mogden, Pirbright, and Pirbright Hybrid) reared under diapause-inducing (10 {degrees}C), cold (14 {degrees}C), and control (26-27 {degrees}C) conditions. We quantified blood-feeding behaviour, wing size as a proxy for somatic growth, and spermatheca size as an indicator of reproductive development. Diapause-inducing conditions resulted in a coordinated phenotype characterised by strong suppression of blood-feeding, increased somatic size, and marked inhibition of reproductive development. Mosquitoes reared at 10 {degrees}C exhibited near-complete feeding inhibition and significantly reduced spermatheca size, consistent with reproductive arrest, while those reared at 14 {degrees}C showed intermediate phenotypes. In contrast, control mosquitoes displayed active feeding and fully developed reproductive structures. Wing size increased progressively with decreasing temperature, with the largest individuals observed under diapause-inducing conditions. When analysed together, wing size and spermatheca development exhibited opposing responses across temperature treatments, revealing a strong negative association and indicating a trade-off between somatic growth and reproductive investment. This integrated response supports the interpretation of diapause as a coordinated life-history strategy involving resource reallocation towards survival. Additionally, diapause expression varied among strains, with the Mogden strain showing reduced sensitivity compared with Pirbright and hybrid populations, highlighting the role of genetic background in diapause plasticity. These findings demonstrate that diapause in Culex pipiens is a multi-trait, plastic phenotype with important implications for overwintering success and the seasonal dynamics of arbovirus transmission in temperate regions.

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.

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

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.

IntroductionProgress against malaria has stalled since 2015, with insecticide resistance a key driver. Spatial emanators release volatile insecticides into the air, exposing mosquitoes through a route distinct from the tarsal contact used by treated nets, indoor residual spraying, and standard bioassays. Transfluthrin is currently monitored using the WHO bottle bioassay, which combines contact and vapour exposure and cannot isolate airborne effects. A scalable, vapour-only method is needed to characterise susceptibility to volatile pyrethroids. MethodsWe adapted the WHO tube bioassay to deliver non-contact transfluthrin vapour exposure without contacting treated papers. Non-blood-fed female Anopheles were exposed to acetone control or transfluthrin papers at 0.005, 0.1 and 2 mg/paper. Knockdown was recorded over 60 minutes and mortality at 24 hours. Testing spanned three laboratories (LSTM, AIRID and KEMRI) using susceptible reference and resistant lab colonies. Dose response models estimated EC{square}{square} for knockdown (EC{square}{square}KD) and mortality (EC{square}{square}Mort), with resistance ratios (RR) against the local susceptible reference. ResultsThe assay generated clear concentration- and time-dependent responses. At LSTM, Kisumu gave an EC{square}{square}KD of 0.052 and EC{square}{square}Mort of 0.054 mg/paper; Siaya was comparable (RR{square}{square}KD 1.17, RR{square}{square}Mort 0.89), whereas Tiassale 13 and KDR showed reduced susceptibility (RR{square}{square}KD 2.42 and 2.85; RR{square}{square}Mort 5.56 and 6.65). At AIRID, the resistant Cove strain did not reach 50% knockdown or mortality. At KEMRI, Siaya showed reduced susceptibility (RR{square}{square}KD 3.36/RR{square}{square}Mort 5.65). Kisumu at AIRID and KEMRI was comparable to LSTM (EC{square}{square}KD 0.007/EC{square}{square}Mort 0.030; EC{square}{square}KD 0.044/EC{square}{square}Mort 0.023). Despite inter-laboratory variation, susceptible strains remained distinguishable from resistant ones. Lower RRs than for contact pyrethroids suggest contact-based phenotypes may not predict vapour susceptibility. ConclusionThe assay provides a practical benchtop method using standard WHO hardware, distinguishing susceptible from resistant strains across three laboratories. Twenty-four-hour mortality is the recommended endpoint, 60-minute knockdown secondary. Not yet validated, it provides a foundation for further validation and adaptation to other actives.

BackgroundAccurate estimation of mosquito age is critical for malaria surveillance, as only older female Anopheles mosquitoes can transmit the parasite. Traditional age-grading methods, include the Polovodova technique in which dilatations on the ovarian pedicel to tally completed gonotrophic cycles (GC) are counted. Despite their widespread use, these techniques remain poorly validated under realistic operational conditions, with limited evidence on their precision and accuracy across different parity rates, GC, and species. In this study, the accuracy and precision of the Polovodova method was evaluated across multiple age-graders and species under both laboratory and field conditions. MethodsA blinded validation study was conducted using laboratory-reared Anopheles arabiensis and Anopheles funestus (N > 3,600) spanning four gonotrophic cycles, and field-collected mosquitoes from Ulanga district, Tanzania (N = 600). Three blinded researchers performed dissections and readings, while a fourth unblinded researcher served as the reference reader (R0) to obtain ground truth (for parity) or assumed truth (for GC) for laboratory mosquitoes. Accuracy and precision for parity and gonotrophic cycle classification were assessed using pairwise inter-rater comparisons, percent agreement, Fleiss kappa and Cohens kappa O_SCPCAP(C_SCPCAPO_SCPLOWKC_SCPLOWO_SCPCAP)C_SCPCAP statistics. For field mosquitoes, which had no age-referenced group, only precision was assessed. FindingsThe Polovodova method accurately classified parity in both species, correctly identifying 98-100% of nulliparous and 94-96% of parous mosquitoes compared with the reference dataset. Inter-rater reliability for parity was almost perfect in An. arabiensis and An. funestus (k = 0.81-0.83), with 82-96% pairwise agreement. GC classification was highly accurate for early stages (GC0-GC2: 85-100%) but declined at later stages GC4(34-46%), reflecting systematic underestimation of mosquito age. Overall GC reliability was moderate (k = 0.56-0.59). Field classifications showed almost perfect agreement (k = 0.87-0.88), supporting operational applicability. ConclusionsThe Polovodova method provides robust estimates of parity and early gonotrophic-cycle status in Anopheles mosquitoes, but late-cycle classifications are more prone to reader disagreement and systematic underestimation. Operational use should therefore rely on trained multiple-reader workflows, including adjudication of discordant or late-cycle specimens, rather than single-reader classification. These findings provide empirical benchmarks for quality-assured Polovodova age-grading in malaria vector surveillance and intervention evaluation.

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.