Characterizing the Microbiome and Prevalence of Wolbachia in Culex pipiens Complex and Culex restuans Mosquitoes in the Midwest United States

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.