The increasing prevalence of insecticide resistance threatens the efficacy of Integrated Mosquito Management (IMM) programs, particularly in regions reliant on chemical control for vector-borne disease prevention. Cross-resistance between active ingredients severely complicates essential resistance management strategies like product rotation. The previous literature suggests that laboratory-induced S-methoprene-resistant Culex species may be somewhat cross-resistant to pyriproxyfen, another juvenile hormone analog. This is a critical concern in the Chicago, IL, USA metropolitan area, where pyriproxyfen is used against mosquitoes with reduced susceptibility to S-methoprene. To determine if S-methoprene-resistant Culex pipiens are cross-resistant to pyriproxyfen in nature, we assessed 31 field-collected populations with significant S-methoprene exposure but varying histories of pyriproxyfen use by dose-response bioassays. Culex pipiens from all 31 sites exhibited high resistance to S-methoprene (RR50 > 10), and 84% were at least moderately resistant to pyriproxyfen (RR50 > 5). Reduced susceptibility to pyriproxyfen was confirmed in pyriproxyfen-unexposed populations, demonstrating potential S-methoprene-mediated cross-resistance. The level of S-methoprene resistance and the level of pyriproxyfen exposure significantly correlated with the level of pyriproxyfen resistance. We report the first widespread, high-level pyriproxyfen resistance in any medically significant mosquito species, underscoring the critical need for routine resistance surveillance and the adoption of integrated resistance management tactics utilizing larvicides with distinct modes of action.

Despite the ecological importance of symbiotic relationships, few studies have explored how microbial communities vary across species complexes or within hybrid zones. We characterized the spatial and temporal variation in microbial communities in Culex pipiens complex and Culex restuans mosquitoes, the most important vectors for West Nile virus in the midwestern United States. Cx. pipiens complex and Cx. restuans mosquitoes were collected monthly from May to September 2023 from three geographic regions (Champaign County, IL, Cook County, IL and Dane County, WI). DNA from individual mosquitoes was sequenced using Illumina NovaSeq amplicon sequencing to identify the mosquito species and characterize their bacterial communities. Microbial composition differed between Cx. pipiens complex and Cx. restuans, driven by high relative abundance of Wolbachia in Cx. pipiens complex compared to Cx. restuans, which also contributed to greater within-species microbiome variability. Despite the limited sample sizes for some forms and hybrids, there were no detectable differences in the bacterial diversity or community composition among the five observed Cx. pipiens forms. Microbial diversity also varied regionally and over the sampling season, increasing later in the season. These findings demonstrate a microbial convergence within Cx. pipiens complex and reveal that spatiotemporal factors influence acquisition of environmentally acquired microbes highlighting the dynamic nature of mosquito-microbe interactions.

Recent mosquito-borne disease outbreaks have highlighted vulnerabilities in our mosquito abatement programmes. The possibility of future outbreaks motivates mosquito abatement districts to optimise their control strategies. Spatial dissemination of vector borne disease is caused by the movements of hosts and mosquitoes. It should be noted that the vector activity and the spread of mosquito-borne pathogens are entirely intertwined and there is much of overlap between it. In our current study, we have developed a mathematical model for the dynamics of Culex mosquito populations in a patchy environment, incorporating entomological data, weather-driven factors, and vector control interventions practiced by the Northwest Mosquito Abatement District (NWMAD), Cook County, Illinois, USA. By coupling a temperature-driven multi-patch Ordinary Differential Equation (ODE) model with mosquito abatement strategies utilised by the NWMAD, we have explored how spatial heterogeneity and different mosquito control strategies can potentially affect mosquito abundance. The present study has focused on two mosquito species within the genus Culex: Culex restuans and Culex pipiens. These species were selected because they are among the most abundant Culex mosquitoes in the study region and are recognised as important vectors. We also assess the effectiveness of various vector control strategies, including adulticide and larvicide interventions, under different temporal, spatial configurations. We further have evaluated how mosquito dispersal influences the effectiveness of various control strategies by comparing single and two-patch model outcomes. Our simulations demonstrate that models ignoring spatial connectivity via mosquito dispersal can substantially overestimate the efficacy of different intervention strategies or inaccurately represent the threshold levels required for vector persistence. Through numerical simulations, we have analysed the impact of continuous-pulsatile control measures on population dynamics, providing insight into optimal control strategies for managing Culex populations and mitigating the spread of mosquito-borne diseases while considering of mosquito movements in a weather-driven settings in the Cook County, Chicago, Illinois, USA.

Abstract West Nile virus (WNv) in eastern North America is transmitted primarily by Culex pipiens and Culex restuans , two cryptic vector species whose relative abundance shifts seasonally and across urbanization gradients - gradients that are themselves now shifting under continuing climate change and urban expansion. Because the two species are difficult to separate morphologically and are typically pooled in routine surveillance, this turnover is seldom tracked directly, and agencies lack species-resolved tools to anticipate when, where, and how vector communities will reorganize. Yet the timing of this turnover has been linked to the seasonal timing and intensity of human WNv cases, making it a potentially forecastable correlate of risk. We molecularly identified 9,789 Culex collected over six years, of which 5,808 came from a designed multi-year study in the Baltimore-Washington metropolitan region, and the remainder from Philadelphia and Chicago for cross-regional comparison. We combined negative-binomial generalized linear mixed models of species-specific abundance in Baltimore-Washington with Gradient Forest models of compositional turnover across all three regions. The two species diverged along both the urbanization and thermal gradients: Cx. pipiens abundance increased with impervious surface and with temperature, whereas Cx. restuans declined along both. Consequently, highly urbanized areas remained Cx. pipiens –rich across the season, whereas suburban, low-to-moderate-development landscapes exhibited the largest seasonal shifts in community composition. Accumulated degree-days (ADD) and weekly mean temperature were the most consistent drivers of turnover across regions, with the shift from Cx. restuans to Cx. pipiens fastest between 594 and 610 ADD. These patterns translate into three operational tools for WNv management: a climate-based degree-day window that lets agencies forecast the community shift from temperature data before it is detectable in trap composition, identification of suburban landscapes as priority targets for intensified surveillance and early intervention, and species-resolved environmental responses that help anticipate how community composition may shift within these regions as local climate and land-use conditions change. Together they offer a path from reactive surveillance to anticipatory, spatially targeted, and forward-looking management of WNv risk under environmental change. Open Research Statement All datasets and R scripts used to generate the results and figures in this study will be made publicly available on DRYAD (DOI pending) upon acceptance of the manuscript. Climate data are publicly available from the PRISM Climate Group (PRISM Group, Oregon State University). Landscape predictor datasets are publicly available from the sources cited in Methods (Dewitz 2023; WorldPop 2018; U.S. Census Bureau 2020; Zell & Sanford 2020; Gesch et al., 2018; Didan 2015).

The rapid spread of West Nile virus (WNV) is a growing concern. With no vaccines or specific medications available, prevention through mosquito control is the only solution to curb the spread. Mosquito traps, used to detect viral presence in mosquito populations, are essential tools for WNV surveillance. But how do we decide where to place a mosquito trap? And what makes a good trap location, anyway? We present a robust statistical approach to determine a mosquito trap's ability to predict human WNV cases in the Chicago metropolitan area and its suburbs. We then use this value to detect the landscape, demographic, and socioeconomic factors associated with a mosquito trap's predictive ability. This approach enables resource-limited mosquito control programs to identify better trap locations while reducing trap numbers to increase trap-based surveillance efficiency. The approach can also be applied to a wide range of different environmental surveillance programs.

The Chicago metropolitan area is a hotspot for human West Nile virus (WNV) cases. Despite extensive surveillance and research, predicting WNV cases in Chicago on a local scale is a major challenge. Most studies and mosquito surveillance efforts do not differentiate between the cryptic species Culex pipiens Linnaeus and Culex restuans (Theobald), key vectors of WNV, due to the challenge of distinguishing them morphologically. This obscures each species' respective role in transmission and may blunt the accuracy of local case forecasting. We used species-specific PCR diagnosis to identify Chicago mosquitoes across 21 sites in July and August of 2021 and 2022. We found that the percentage of collected Cx. restuans declined between July and August, with Culex pipiens often being the sole species recorded from sites in August. We found that the relative proportions of our target species varied by site, sometimes dramatically, across years. Following species identification, we pooled our specimens and determined infection prevalence using reverse transcription quantitative polymerase chain reaction (RT-qPCR). We found that both species were infected with WNV and observed no significant difference in their infection rates between months. We also assessed the accuracy of a human case prediction model, and we found no evidence to support the molecular separation of these species in routine Chicago WNV surveillance.

S-methoprene, an insect growth regulator, and Lysinibacillus sphaericus (Ls), an entomopathogenic bacterium, are important larvicides used to control Culex pipiens [L.] mosquitoes, the primary vector of West Nile virus, in the Chicago, IL USA region. Resistance to both agents has been documented globally including a report of resistance ratios greater than 100 to S-methoprene in the Chicago region. Laboratory studies have suggested the potential for unidirectional cross-resistance between S-methoprene and Ls, despite differing modes of action. Among wild populations of Cx. pipiens in the Chicago area, this study aimed 1) to assess resistance status to Ls, 2) confirm the presence of S-methoprene resistance ratios >100, 3) determine if higher S-methoprene resistance ratios are associated with higher Ls resistance ratios, or whether Ls resistance arises solely from Ls exposure, and (4) determine the relationship between Ls treatment history and resistance levels of that active ingredient. We assessed susceptibility to both S-methoprene and Ls in 32 Cx. pipiens populations: 19 with S-methoprene exposure but no Ls history, and 13 with multi-year exposure to both larvicide active ingredients. Ls susceptibility was evaluated using dose-response bioassays to estimate LC50, LC90, and resistance ratios. Susceptibility to S-methoprene was tested using diagnostic doses corresponding to resistance ratios of 10 and 100 at the LC50. Resistance ratios to S-methoprene exceeding 10 were detected in 30 of 32 sampled populations. Among the 13 sites with prior Ls exposure, 11 were observed with resistance ratios > 5. In contrast, none of the 19 populations without Ls exposure exhibited Ls resistance, despite exhibiting higher S-methoprene resistance ratios. This lack of overlap supports the conclusion that S-methoprene resistance does not confer cross-resistance to Ls in the studied region. Logistic regression revealed a strong association between Ls treatment history and resistance development. The probability of Ls resistance exceeded 80% after 10 Ls applications within an eight-year period. These findings emphasize the need to develop improved resistance management strategies for larvicidal insecticides.

Insecticide resistance (IR) is an increasing problem globally, making control of vector-borne diseases more difficult. Reduced susceptibility to permethrin in Culex pipiens, an important vector for West Nile virus, has been reported across the US based on a standardized laboratory method: the CDC bottle bioassay. This bioassay uses a rapid phenotypic outcome to reveal evidence for IR, but how this translates to the effectiveness of formulated products used in an operational setting is unclear. Therefore, other methods for IR monitoring are recommended to quantify IR or evaluate formulated products against field populations in real-world conditions. To compare some of the available methods, we collected populations of Cx. pipiens from six sites in the Northwest Mosquito Abatement District (Cook Co., Illinois), and used a susceptible laboratory strain of Cx. pipiens as a control, to test for IR to pyrethroids using CDC bottle bioassays, caged field trials, and topical applications. CDC bottle bioassays suggested that Cx. pipiens from this area exhibit IR to both etofenprox and Sumithrin®. Caged field trials with ultra-low volume Anvil® 10 + 10 (Sumithrin®) demonstrated resistance to the product and underscored the need for inclusion of a susceptible control to differentiate IR from inadequate product distribution. Topical applications revealed low to high levels of resistance to synergized and unsynergized pyrethroids (etofenprox, Sumithrin®, and deltamethrin) in all field populations. Based on these data, we provide a new decision-making tree for mosquito control professionals which will guide selection of the most optimal assay for IR surveillance based on their goals, needs, and resources.

Even as the incidence of mosquito-borne diseases like West Nile Virus (WNV) in North America has risen over the past several decades, effectively modelling mosquito population density or abundance has proven to be a persistent challenge. It is critical to capture the fluctuations in mosquito abundance across seasons in order to forecast the varying risk of pathogen transmission from one year to the next. We develop a process-based mechanistic weather-driven Ordinary Differential Equation (ODE) model to study the population biology of both aquatic and terrestrial stages of mosquito population. The progression of mosquito lifecycle through these stages is influenced by different factors, including temperature, daylight hours, intra-species competition and the availability of aquatic habitats. In our work, weather-driven parameters are derived from a combination of laboratory research and data from the literature. In our model, we include precipitation data as a substitute for evaluating additional mortality in the mosquito population. We compute the Basic offspring number of the associated model and perform sensitivity analysis. Finally, we employ our model to assess the effectiveness of various adulticides strategies to predict the reduction in mosquito population. This enhancement in modelling of mosquito abundance can be instrumental in guiding interventions aimed at reducing mosquito populations and mitigating mosquito-borne diseases such as the WNV. This model could help optimise the timing of adulticide applications and evaluate the impact of multiple spray events within a short period.

Vector mosquitoes are well-adapted to habitats in urban areas, including belowground infrastructure such as stormwater systems. As a major source of larval habitat in population centers, control of larval populations in stormwater catch basins is an important tool for control of vector-borne disease. Larval development and adult phenotypes driving vectorial capacity in mosquitoes are modulated by the larval gut microbiota, which is recruited from the aquatic environment in which larvae develop. Laboratory studies have quantified microbe-mediated impacts on individual mosquito phenotypes, but more work is needed to characterise how microbiota variation shapes population-level outcomes. Here, we evaluated the relationship between habitat microbiota variation and mosquito population dynamics by simultaneously characterising microbiota diversity, water quality, and mosquito productivity in a network of stormwater catch basins in the Chicago metropolitan area. High throughput sequencing of 16S rRNA gene amplicons from water samples collected from 60 basins over an entire mosquito breeding season detected highly diverse bacterial communities that varied with measures of water quality and over time. In situ measurements of mosquito abundance in the same basins further varied by microbiota composition and the relative abundance of specific bacterial taxa. Altogether, these results illustrate the importance of habitat microbiota in shaping ecological processes that affect mosquito populations. They also lay the foundation for future studies to characterise the mechanisms by which specific bacterial taxa impact individual and population-level phenotypes related to mosquito vectorial capacity.

Since the introduction of West Nile virus (WNV) to the United States over 20 years ago, thousands of cases of human disease and death have been reported. Yearly seasonal outbreaks continue to persist, and the city and suburbs of Chicago, Illinois, is considered a "hot spot" for WNV activity. To interrupt WNV transmission, ground ultra-low volume (ULV) adulticide applications are regularly used to reduce Culex pipiens L. and Culex restuans Theobold (Diptera: Culicidae) abundance and infection. The real-world effectiveness of adulticide applications has not been comprehensively assessed, and prior studies, including our own investigation, have yielded inconclusive or conflicting results. Therefore, we expanded our prior work and evaluated the effects of 5 sequential weekly truck-mounted ULV adulticide applications in large residential areas in the northern suburbs of Chicago, Illinois, in 2019 and 2020. Each day, Cx. pipiens and Cx. restuans host-seeking and gravid mosquitoes were collected to assess abundance, age structure, and WNV infection rates. Adulticide applications resulted in significant reductions of both host-seeking and gravid abundance on the night of treatment. The reduction in host-seeking mosquitoes was followed by a reduction in gravid mosquitoes trapped 3 and 4 days after adulticide application and an increase in the proportion of nulliparous mosquitoes. WNV infection rates were significantly reduced in treatment sites as compared to untreated sites when infection rates were higher in 2020. This large-scale study provides comprehensive evidence that ground ULV adulticide applications are an effective tool in an integrated mosquito management program for combating WNV vectors and infection risk.

Over the course of three years, 200 ft to 0.75 mi (60 m to 1.2 km) sections of 3 larger (>6 ft [1.8 m] diam) belowground storm sewer conveyance pipes in the northwestern Chicago suburbs were inspected for the presence of adult mosquitoes. Culex mosquitoes were by far the most common (555 of 556 [99.8%] total mosquitoes) collected within pipes during all four meteorological seasons (i.e. during months of October, January, May, August). These observations support prior work elsewhere, suggesting storm sewer pipes are consistent sites of refuge for adult Culex mosquitoes.

Over the course of three years, 200 ft to 0.75 mi (60 m to 1.2 km) sections of 3 larger (.6ft[1.8m] diam) belowground storm sewer conveyance pipes in the northwestern Chicago suburbs were inspected for the presence of adult mosquitoes. Culex mosquitoes were by far the most common (555 of 556 [99.8%] total mosquitoes) collected within pipes during all four meteorological seasons (i.e. during months of October, January, May, August). These observations support prior work elsewhere, suggesting storm sewer pipes are consistent sites of refuge for adult Culex mosquitoes.

The rapid spread of West Nile Virus (WNV) is a growing concern. With no vaccines or specific medications available, prevention through mosquito control is the only solution to curb the spread. Mosquito traps, used to detect viral presence in mosquito populations, are essential tools for WNV surveillance. But how do we decide where to place a mosquito trap? And what makes a good trap location, anyway? We present a robust statistical approach to determine a mosquito trap's ability to predict human WNV cases in the Chicago metropolitan area and its suburbs. We then use this value to detect the landscape, demographic, and socioeconomic factors associated with a mosquito trap's predictive ability. This approach enables resource-limited mosquito control programs to identify better trap locations while reducing trap numbers to increase trap-based surveillance efficiency. The approach can also be applied to a wide range of different environmental surveillance programs.

Insect growth regulators, like S-methoprene, are heavily relied upon worldwide for larval mosquito chemical control due to their target specificity and long-lasting effects. In this study, susceptibility to S-methoprene was evaluated in Culex pipiens, a globally important vector species. Populations from 14 sites throughout the Chicago area with a long history of S-methoprene use and two sites with minimal use in Wisconsin were examined. Using a bioassay methodology and probit analyses, LC50 and LC90 values were calculated and compared to a susceptible laboratory strain to develop resistance ratios, then categorized for resistance intensity. The resistance ratios observed required the addition of another category, termed 'extreme' resistance, indicating resistance ratios greater than 100. 'Low' to 'extreme' levels of resistance to S-methoprene were detected throughout Illinois populations, with resistance ratios ranging from 2.33 to 1010.52. Resistance was not detected in populations where S-methoprene pressure has been very limited. These 'extreme' resistance ratios observed have never been documented in a wild vector species mosquito population. The relationships between historical S-methoprene use, resistance detected with laboratory bioassays, and the potential for field product failure remain unclear. However, the profound resistance detected here demonstrates a potential critical threat to protecting public health from mosquito-borne diseases.

West Nile virus (WNV) invaded the continental United States over 20 years ago and continues to cause yearly seasonal outbreaks of human and veterinary disease. In the suburbs of Chicago, Illinois, ultra-low volume (ULV) truck-mounted adulticide spraying frequently is performed to reduce populations of Culex restuans Theobald and Cx. pipiens L. mosquitoes (Diptera: Culicidae) in an effort to lower the risk of WNV transmission. The effectiveness of this control method has not been rigorously evaluated, and evidence for Culex population reduction after ULV adulticide spraying has been inconclusive. Therefore, we evaluated the results of 5 sequential weekly truck-mounted adulticide applications of Zenivex® E20 (etofenprox) in 2 paired sites located in Cook County, IL, during the summer of 2018. Mosquito population abundance, age structure, and WNV infection prevalence were monitored and compared between paired treatment and nearby control sites. Adulticide treatment did not result in consistent short-term or long-term reductions in target WNV vector Culex abundance. However, there was a significant increase in the proportion of nulliparous females in the treated sites compared to control sites and a decrease in Cx. pipiens WNV infection rates at one of the treated sites. This evidence that ULV adulticide spraying altered the age structure and WNV infection prevalence in a vector population has important implications for WNV transmission risk management. Our findings also underscore the importance of measuring these important indicators in addition to abundance metrics when evaluating the efficacy of control methods.

Background West Nile virus (WNV), primarily vectored by mosquitoes of the genus Culex , is the most important mosquito-borne pathogen in North America, having infected thousands of humans and countless wildlife since its arrival in the USA in 1999. In locations with dedicated mosquito control programs, surveillance methods often rely on frequent testing of mosquitoes collected in a network of gravid traps (GTs) and CO 2 -baited light traps (LTs). Traps specifically targeting oviposition-seeking (e.g. GTs) and host-seeking (e.g. LTs) mosquitoes are vulnerable to trap bias, and captured specimens are often damaged, making morphological identification difficult. Methods This study leverages an alternative mosquito collection method, the human landing catch (HLC), as a means to compare sampling of potential WNV vectors to traditional trapping methods. Human collectors exposed one limb for 15 min at crepuscular periods (5:00–8:30 am and 6:00–9:30 pm daily, the time when Culex species are most actively host-seeking) at each of 55 study sites in suburban Chicago, Illinois, for two summers (2018 and 2019). Results A total of 223 human-seeking mosquitoes were caught by HLC, of which 46 (20.6%) were mosquitoes of genus Culex . Of these 46 collected Culex specimens, 34 (73.9%) were Cx. salinarius , a potential WNV vector species not thought to be highly abundant in upper Midwest USA. Per trapping effort, GTs and LTs collected > 7.5-fold the number of individual Culex specimens than HLC efforts. Conclusions The less commonly used HLC method provides important insight into the complement of human-biting mosquitoes in a region with consistent WNV epidemics. This study underscores the value of the HLC collection method as a complementary tool for surveillance to aid in WNV vector species characterization. However, given the added risk to the collector, novel mitigation methods or alternative approaches must be explored to incorporate HLC collections safely and strategically into control programs. Graphical Abstract

Culex pipiens complex is an important vector of epizootic and zoonotic pathogens, including West Nile virus. Chicago, Illinois and its suburbs have suffered high incidence of human West Nile virus infections in the past. This makes abatement programs in and around the Chicago area an essential service. The control of Cx. pipiens is often complicated by rapidly evolving resistance to pyrethroids, which are the most widely used chemical class in US mosquito abatement programs. The present study assessed Sumithrin® resistance in Cx. pipiens collected from five locations around Cook County, Illinois, neighboring the city limits of Chicago. According to CDC guidelines, samples from all five locations demonstrated some resistance to Sumithrin®. When assessed with Anvil®, a formulated product made of Sumithrin® synergized with piperonyl butoxide, susceptibility was rescued in mosquitoes from three out of the five locations, suggesting involvement of mixed-function oxidases and/or carboxylesterases in Sumithrin® resistance at these locations. Not all locations had susceptibility rescued by Anvil®, but these locations had relatively low knockdown resistance allele frequencies, suggesting that mechanisms other than knockdown resistance may be involved. Enzyme activities did not reveal any marked trends that could be related back to mortality in the bottle bioassays, which highlights the need for multiple types of assays to infer enzymatic involvement in resistance. Future directions in pyrethroid resistance management in Chicago area Cx. pipiens are discussed.

Mosquito adulticides are tools to manage populations and reduce human disease risks. We examined the spatial impact of policies that affect the ability to conduct adult mosquito control. We used the Northwest Mosquito Abatement District (NWMAD) to illustrate how various constraints can impact vector control. Almost 12.7% of the 233 mi(2) covered by NWMAD is owned by the Forest Preserve District (FPD) or has been designated as a Nature Preserve Area (NPA). Pesticide application is prohibited in both FPDs and NPAs. Additionally, NWMAD allows residents to opt out of having their property parcel sprayed for mosquitoes by being placed on a "do not spray" (DNS) list. As of February 2019, 162 residential and beehive parcels encompassing 1,059.2 acres are listed. As a result of this policy, 2,686 residential and beehive parcels (1.3% of all parcels) received reduced or no mosquito adulticide sprays in 2018. These parcels were distributed unevenly across the districty eight townships, with approximately 90% of residential DNS acreage in two townships. Nearly 14% of all NWMAD acreage is exempt from treatment, which could affect the ability to respond effectively to disease outbreaks.

The Asian tiger mosquito, Aedes albopictus (Skuse), is a public health threat because it can potentially transmit multiple pathogenic arboviruses, exhibits aggressive diurnal biting, and is highly invasive. As Ae. albopictus moved northward into the United States, the limits of expansion were predicted as locations with a mean January temperature warmer than -2.5°C. We postulated that the range of Ae. albopictus could exceed these temperature limits if eggs in diapause overwinter in tires that provide an insulating effect from extreme temperatures. Fifteen tires with Ae. albopictus and Aedes triseriatus (Say) eggs, a native cold hardy species, were placed outside at five locations along a latitudinal gradient in Wisconsin and Illinois during the winter of 2018-2019; notably, in January 2019, a regional arctic air event brought the lowest temperatures recorded in over 20 yr. External and internal tire temperatures were recorded at 3 hr intervals, and egg survival was recorded after six months. Aedes albopictus eggs survived only from tires at northernmost locations. The mean internal January temperature of tires that supported survival was -1.8°C, while externally the mean temperature was -5.3°C, indicating that tires provided an average of +3.5°C of insulation. Tires that supported egg survival also had over 100 mm of snow cover during January. In the absence of snow cover, tires across the study area provided an average +0.79°C [95% CI 0.34-1.11] insulation. This work provides strong argument for the inclusion of microhabitats in models of dispersal and establishment of Ae. albopictus and other vector species.