MOSQUITO SPECIES DISTRIBUTION IN TIRES IN RURAL AND URBAN LANDSCAPES ACROSS NORTHERN KENTUCKY

The U.S. Tire Manufacturers Association (2021) estimates that 274 million scrap vehicle tires are generated annually in the United States. Due to challenges in recycling tires, a majority of discarded vehicle tires end up in tire collection yards and landfills, along the sides of roads, or in natural areas across human-dominated landscapes in the United States (Yee 2008, Moise et al. 2022). Discarded tires pose both environmental and public health threats. Discarded vehicle tires are productive artificial container habitats for mosquito larval development of numerous species (Yee 2008, Crespo et al. 2024). The rubber of the tires insulates the internal habitat from the elements, hides mosquito larvae from potential predators, and increases overwintering success for invasive mosquito species (Yee 2008, Wilke et al. 2019, Susong et al. 2022). Discarded vehicle tires degrade under ultraviolet light, shedding microplastics and leaching organic substances, including polycyclic aromatic hydrocarbons, and soluble metals, most importantly zinc, into the environment (Wik and Dave 2009, Villena et al. 2017, Cunningham et al. 2022). These toxic substances enter freshwater habitats and can have negative effects on many species of aquatic organisms including mosquito developmental stages within the tire habitat (Wik and Dave 2009; Villena et al. 2017, 2022; Cunningham et al. 2022).

Most discarded tires are found throughout human-dominated landscapes from urban to rural habitats (Joy and Sullivan 2005, Sallam et al. 2020, Crespo et al. 2024). Proximity to these tires has been shown to increase the risk of vector-borne disease transmission in humans, including for St. Louis encephalitis, La Crosse encephalitis, dengue and chikungunya viruses, and West Nile virus (Yee 2008, Yee et al. 2015). Therefore, understanding mosquito species composition in discarded tires across different habitats is important for targeted mosquito control. The purpose of this study was to compare mosquito species compositions in discarded tires in urban and rural habitats in the Northern Kentucky region.

Six rural sites and 6 urban sites were selected in forested parks across northern Kentucky for weekly mosquito collections (Fig. 1). Each site contained 2 to 3 discarded tires that were cleared of debris and supplemented weekly with grass infusion. Grass infusion was made by steeping ten grams of dried grass clippings wrapped in cheese cloth in 185 liters of water in a 50-gallon (189.3 liters) rain barrel. Samples were collected at each site once per week from June through August 2021. During each sampling event, mosquito larvae and pupae were collected from the tires for 1 h per site. At each site, adult mosquitoes were also collected using 1 light trap and 1 gravid trap. Larvae, pupae, and adult mosquitoes were brought to the laboratory for species identification. Larvae were placed in ethanol, then identified to species. Pupae were reared to adulthood. Adults were euthanized before identification, and all were identified to species, using taxonomic keys (Ross and Horsfall 1965, Darsie and Ward 2016).

Generalized linear mixed-effects models (GLMERs) were used to analyze the fixed main effect of habitat (urban or rural park) and the random effects of site by date on the total number of juveniles collected in the tires and the total number of adult collected in the adult traps. The same analysis was conducted on the most commonly collected juvenile species (Aedes albopictus (Skuse), Ae. japonicus (Theobald), Ae. triseriatus (Say), Culex restuans Theobald, and Cx. pipiens L.) and adult species (Cx. restuans/pipiens complex, and Ae. japonicus). Overdispersion was assessed for each model (Korner-Nievergelt et al. 2019). No overdispersion was detected for the models. All analyses were carried out using an alpha level of 0.05 in R (version 3.5.1, R Core Team 2018) using RStudio (RStudio, Inc. Boston, MA).

Across all sites, a total of 9,906 juvenile mosquitoes were collected belonging to 9 species across 5 genera. Of the collected juveniles, Ae. japonicus was the predominate species collected (44.7%), followed by Cx. restuans (21.5%) and Ae. triseriatus (17.7%). The remaining species were collected in low numbers: Cx. pipiens (9.2%), Ae. albopictus (2.9%), Toxorhynchites rutilus (Coquillett) (0.005%), Anopheles crucians Wiedemann (<0.001%), An. punctipennis (Say) (<0.001%), and Orthopodomyia signifera (Coquillett) (<0.001%). There was no significant difference in the total number of juvenile mosquitoes collected from tires between rural and urban sites (Fig. 2, z = –1.23, P = 0.219). Significantly more Ae. triseriatus juveniles were collected from tires in urban sites compared to rural sites (z = 2.381, P = 0.017), and significantly more Ae. albopictus juveniles were collected from tires in urban sites compared to rural sites (z = 3.868, P < 0.001). There were no significant differences for the number of juveniles collected from tires in urban and rural sites for Ae. japonicus (z = –0.742, P = 0.458), Cx. restuans (z = –1.313, P = 0.189), or Cx. pipiens (z = 1.771, P = 0.077).

Across all sites, 2,197 adults were collected belonging to 8 species across 4 genera. Of the collected adults, the Culex restuans/pipiens complex was the predominant species collected (77.8%), followed by Ae. japonicus (16.4%). The remaining species were collected in low numbers: Ae. triseriatus (3.2%), Ae. albopictus (1.6%), An. punctipennis (<0.01%), Ae, vexans (Meigen) (<0.001%), and Or. signifera (<0.001%). There was a significant difference in the total number of adult mosquitoes collected from traps between rural and urban sites with more adults being collected in the urban sites (Fig. 2, z = 6.879, P < 0.001). There were no significant differences in number of adults collected at urban and rural sites for Ae. japonicus (z = 0.865, P = 0.387), Ae. triseriatus (z = 0.047, P = 0.963), Ae. albopictus (z = 1.554, P = 0.120), or the Cx. restuans/pipiens complex (z = 0.668, P = 0.504).

There were significantly more Ae. albopictus and Ae. triseriatus juveniles collected from tires in urban areas compared to rural areas. Aedes triseriatus is a common woodland inhabiting mosquito throughout the eastern United States (Yee 2008). Though Ae. triseriatus is considered a woodland species, there was no significant difference in the number of adults found between urban and rural park sites in this study. This could be because of the fact that even at the selected urban park sites, there is still a large amount of forest habitat available. However, the high number of Ae. triseriatus collected from the tires may suggest that Ae. triseriatus is more common in urban areas than previously thought. Conversely, Ae. albopictus tends to be more common in urban habitats compared to rural habitats, but there was no significant difference in the number of adults found between urban and rural park sites in this study. This may suggest that Ae. albopictus is expanding its range into more rural habitats as it continues to become more established in the region. The use of grass infusion in the tires many have skewed the results because of the varying level of attractiveness for different species. For example, Culex species have been shown to preferentially oviposit in containers with grass infusion (Burkett-Cadena and Mullen 2007), whereas Ae. albopictus has been shown to either not have a strong preference for specific infusions or prefer other infusions over grass infusions (Grim et al. 2007, Obenauer et al. 2010). These results suggest that multiple means of mosquito sampling provide a clearer picture of mosquito species distribution, which can aid in more targeted mosquito control during mosquito-borne disease outbreaks.

Tires are often discarded out of sight in both urban and rural environments. This increases the amount of pollution in an area, both physically from the tires being present in the environment and from chemical leaching, which has harmful impacts on the environment, particularly for aquatic species (Wik and Dave 2009, Cunningham et al. 2022). Mosquito larvae developing in tire habitats can be challenging to control because of being difficult to find in forested habitats and because of the difficulty of using insecticides (Wilke et al. 2021). While many studies have focused on productivity of tires in urban habitats (Yee 2008, Wilke et al. 2021, Crespo et al. 2024), the results of this study suggest that tires serve as a productive habitat across a human land-use gradient, including in rural habitats, and therefore should be monitored and controlled to reduce mosquito-borne disease transmission in both urban and rural environments.

We thank the City of Covington Solid Waste and Recycling and Boone County Solid Waste and Recycling for providing discarded tires and setting up tires at our locations. We thank Brandon Riley, Amy Harouna, Mitchell Culbertson, Regina Utz, Noah Weidig, Zach Bischoff, and Phat Nguyen for their contributions to the study. We thank Joshua Cooper for his assistance with designing the map. Support was provided by the Northern Kentucky University Center for Integrative Natural Science and Mathematics (CINSAM) Research Grant and the CINSAM's UR-STEM Program.