Molecular surveillance supports differing roles for Culex pipiens and Culex restuans (Diptera: Culicidae) in West Nile virus transmission in Chicago, Illinois.

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.