Characterizing the seasonal abundance and reproductive activity of overwintering Anopheles (Diptera: Culicidae) mosquitoes.

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.

MicroRNA Expression Prior to Biting in a Vector Mosquito Anticipates Physiological Processes Related to Energy Utilization, Reproduction and Immunity.

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.

Characterizing seasonal changes in the reproductive activity of Culex mosquitoes throughout the fall, winter, and spring in Ohio.

BACKGROUND: Culex mosquitoes 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 female Culex mosquitoes that were in diapause during the fall and winter and when they terminated diapause and began blood feeding in the spring. METHODS: Mosquitoes 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. Culex mosquitoes were morphologically identified to species, and the ovaries of females were dissected to determine their diapause and parity statuses. RESULTS: By early October 2021, roughly 95% of Culex pipiens collected in culverts were in diapause and 98% of Cx. erraticus were in diapause. Furthermore, gravid and blood-fed Culex salinarius, Cx. pipiens, and Cx. restuans were collected in late November in 2019 and 2021 in standard mosquito traps. In the winter of 2021, the proportions of non-diapausing Culex decreased within culverts. The last non-diapausing Cx. erraticus was collected in late December 2021 while the final non-diapausing Cx. pipiens was collected in mid-January 2022, both in culverts. Roughly 50% of Cx. pipiens terminated 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 of Cx. pipiens, Cx. restuans, and Cx. territans were collected by the 1st week of May in 2022, indicating that multiple species of Culex produced a second generation that reached adulthood by this time. CONCLUSIONS: We collected blood-fed and gravid Culex females 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-diapausing Cx. pipiens and Cx. erraticus throughout December has important implications for the winter survival of WNV vectors and our overall understanding of diapause. Finally, determining when Culex terminate diapause in the spring may allow us to optimize mosquito management programs and reduce the spread of WNV before it is transmitted to humans.

Establishing a Culex Colony from Field-Collected Eggs.

Culex larvae are well adapted to growing and developing in containers, and therefore collecting and rearing field-collected Culex to adulthood in the laboratory is relatively straightforward. What is substantially more challenging is simulating natural conditions that encourage Culex adults 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 collect Culex eggs from the field and establish a colony in the laboratory. Successfully establishing a new colony of Culex mosquitoes 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.

Rearing and Maintaining a Culex Colony in the Laboratory.

After overcoming the significant obstacle of getting adult Culex mosquitoes 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 of Culex pipiens and how other researchers might modify them to suit their specific needs.

Points to Consider When Establishing and Rearing Culex Mosquitoes in the Laboratory.

Culex mosquitoes 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, unlike Aedes mosquitoes that produce eggs that can be stored for weeks at a time, no obvious "stopping" point exists in the development of Culex mosquitoes. Therefore, these mosquitoes require nearly continuous care and attention. Here, we describe some general considerations when rearing laboratory colonies of Culex mosquitoes. 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.

Conserved molecular pathways underlying biting in two divergent mosquito genera.

Mosquitoes transmit a wide variety of devastating pathogens when they bite vertebrate hosts and feed on their blood. However, three entire mosquito genera and many individual species in other genera have evolved a nonbiting life history in which blood is not required to produce eggs. Our long-term goal is to develop novel interventions that reduce or eliminate the biting behavior in vector mosquitoes. A previous study used biting and nonbiting populations of a nonvector mosquito, Wyeomyia smithii, as a model to uncover the transcriptional basis of the evolutionary transition from a biting to a nonbiting life history. Herein, we ask whether the molecular pathways that were differentially expressed due to differences in biting behavior in W. smithii are also differentially expressed between subspecies of Culex pipiens that are obligate biting (Culex pipiens pipiens) and facultatively nonbiting (Culex pipiens molestus). Results from RNAseq of adult heads show dramatic upregulation of transcripts in the ribosomal protein pathway in biting C. pipiens, recapitulating the results in W. smithii, and implicating the ancient and highly conserved ribosome as the intersection to understanding the evolutionary and physiological basis of blood feeding in mosquitoes. Biting Culex also strongly upregulate energy production pathways, including oxidative phosphorylation and the citric acid (TCA) cycle relative to nonbiters, a distinction that was not observed in W. smithii. Amino acid metabolism pathways were enriched for differentially expressed genes in biting versus nonbiting Culex. Relative to biters, nonbiting Culex upregulated sugar metabolism and transcripts contributing to reproductive allocation (vitellogenin and cathepsins). These results provide a foundation for developing strategies to determine the natural evolutionary transition between a biting and nonbiting life history in vector mosquitoes.