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Abstract In temperate regions of the United States, female Anopheles mosquitoes respond to low temperatures and short photoperiods by entering an overwintering dormancy or diapause. Diapause in Anopheles results in reduced frequency of blood-feeding and reproductive arrest, indicating a period when pathogen transmission by these mosquitoes is unlikely. However, it is unclear precisely how late into the fall and how early in the spring these mosquitoes are biting, reproducing, and potentially transmitting pathogens. This is further complicated by the lack of clear markers of diapause in Anopheles (e.g., changes in egg follicle length). Our goal was to characterize the seasonal reproductive activity of female Anopheles in central Ohio, United States and evaluate egg follicle length as an indicator of Anopheles diapause. We used traditional mosquito traps and aspirators to collect Anopheles from urban woodlots and culverts, respectively, from late September 2021 through mid-May 2022 in central Ohio. By measuring their egg follicle length, reproductive status, and blood-feeding status, we found that egg follicle length is not a reliable indicator of Anopheles diapause. We also found that a small proportion of An. punctipennis (Say), An. perplexens (Ludlow), and An. quadrimaculatus (Say) continued to bite and reproduce into early November 2021 and that females of these species terminated reproductive dormancy and began biting by mid-March 2022. This period of reproductive activity extends beyond current mosquito surveillance and control in Ohio. Our findings suggest that within temperate regions of North America, Anopheles have the capacity to transmit pathogens throughout the spring, summer, and fall. 

Culexmosquitoes transmit several pathogens to humans and animals, including viruses that cause West Nile fever and St. Louis encephalitis and filarial nematodes that cause canine heartworm and elephantiasis. Additionally, these mosquitoes have a cosmopolitan distribution and provide interesting models for understanding population genetics, overwintering dormancy, disease transmission, and other important and ecological questions. However, unlikeAedesmosquitoes that produce eggs that can be stored for weeks at a time, no obvious “stopping” point exists in the development ofCulexmosquitoes. Therefore, these mosquitoes require nearly continuous care and attention. Here, we describe some general considerations when rearing laboratory colonies ofCulexmosquitoes. We highlight different methods so that readers may choose what works best for their experimental needs and laboratory infrastructure. We hope that this information will enable additional scientists to conduct laboratory research on these important disease vectors. 

Culexlarvae are well adapted to growing and developing in containers, and therefore collecting and rearing field-collectedCulexto adulthood in the laboratory is relatively straightforward. What is substantially more challenging is simulating natural conditions that encourageCulexadults to mate, blood feed, and reproduce in laboratory settings. In our experience, this is the most difficult hurdle to overcome when establishing new laboratory colonies. Here, we detail how to collectCulexeggs from the field and establish a colony in the laboratory. Successfully establishing a new colony ofCulexmosquitoes in the laboratory will allow researchers to evaluate physiologically, behaviorally, and ecologically relevant aspects of their biology and better understand and manage these important disease vectors. 

After overcoming the significant obstacle of getting adultCulexmosquitoes to reproduce and blood feed in the laboratory, maintaining a laboratory colony is much more achievable. However, great care and attention to detail are still required to ensure that the larvae have adequate food without being overwhelmed by bacterial growth. Additionally, achieving the appropriate densities of larvae and pupae is essential, as overcrowding delays development, prevents pupae from successfully emerging as adults, and/or reduces adult fecundity and alters sex ratios. Finally, adult mosquitoes should have constant access to H₂O and nearly constant access to sugar sources to ensure that both males and females have adequate nutrition and can produce the maximum number of offspring. Here, we describe our methods for maintaining the Buckeye strain ofCulex pipiensand how other researchers might modify them to suit their specific needs. 

Understanding the molecular and physiological processes underlying biting behavior in vector mosquitoes has important implications for developing novel strategies to suppress disease transmission. Here, we conduct small-RNA sequencing and qRT-PCR to identify differentially expressed microRNAs (miRNAs) in the head tissues of two subspecies of Culex pipiens that differ in biting behavior and the ability to produce eggs without blood feeding. We identified eight differentially expressed miRNAs between biting C. pipiens pipiens (Pipiens) and non-biting C. pipiens molestus (Molestus); six of these miRNAs have validated functions or predicted targets related to energy utilization (miR8-5-p, miR-283, miR-2952-3p, miR-1891), reproduction (miR-1891), and immunity (miR-2934-3p, miR-92a, miR8-5-p). Although miRNAs regulating physiological processes associated with blood feeding have previously been shown to be differentially expressed in response to a blood meal, our results are the first to demonstrate differential miRNA expression in anticipation of a blood meal before blood is actually imbibed. We compare our current miRNA results to three previous studies of differential messenger RNA expression in the head tissues of mosquitoes. Taken together, the combined results consistently show that biting mosquitoes commit to specific physiological processes in anticipation of a blood meal, while non-biting mosquitoes mitigate these anticipatory costs. 

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