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Abstract BackgroundCulexmosquitoes are the primary vectors of West Nile virus (WNV) across the USA. Understanding when these vectors are active indicates times when WNV transmission can occur. This study determined the proportion of femaleCulexmosquitoes that were in diapause during the fall and winter and when they terminated diapause and began blood feeding in the spring. MethodsMosquitoes were collected from parks using various traps and/or aspirated from culverts in Franklin County, Ohio, from October to mid-May from 2019 to 2022.Culexmosquitoes were morphologically identified to species, and the ovaries of females were dissected to determine their diapause and parity statuses. ResultsBy early October 2021, roughly 95% ofCulex pipienscollected in culverts were in diapause and 98% ofCx. erraticuswere in diapause. Furthermore, gravid and blood-fedCulex salinarius,Cx. pipiens, andCx. restuanswere collected in late November in 2019 and 2021 in standard mosquito traps. In the winter of 2021, the proportions of non-diapausingCulexdecreased within culverts. The last non-diapausingCx. erraticuswas collected in late December 2021 while the final non-diapausingCx. pipienswas collected in mid-January 2022, both in culverts. Roughly 50% ofCx. pipiensterminated diapause by mid-March 2022, further supported by our collections of gravid females in late March in all 3 years of mosquito collection. In fact, male mosquitoes ofCx. pipiens,Cx. restuans, andCx. territanswere collected by the 1st week of May in 2022, indicating that multiple species ofCulexproduced a second generation that reached adulthood by this time. ConclusionsWe collected blood-fed and gravidCulexfemales into late November in 2 of the 3 years of our collections, indicating that it might be possible for WNV transmission to occur in late fall in temperate climates like Ohio. The persistence of non-diapausingCx. pipiensandCx. erraticusthroughout December has important implications for the winter survival of WNV vectors and our overall understanding of diapause. Finally, determining whenCulexterminate diapause in the spring may allow us to optimize mosquito management programs and reduce the spread of WNV before it is transmitted to humans. Graphical Abstract