Genome-wide identification of PAR domain protein 1 (PDP1) targets through ChIP-seq reveals the regulation of diapause-specific characteristics in Culex pipiens.

Insects use seasonal diapause as an alternative strategy to endure adverse seasons. This developmental trajectory is induced by environmental cues like short-day lengths in late summer and early fall, but how insects measure day length is unknown. The circadian clock has been implicated in regulating photoperiodic or seasonal responses in many insects, including the Northern house mosquito, Culex pipiens, which enters adult diapause. To investigate the potential control of diapause by circadian control, we employed ChIP-sequencing to identify the downstream targets of a circadian transcription factor, PAR domain protein 1 (PDP1), that contribute to the hallmark features of diapause. We identified the nearest genes in a 10 kb region of the anticipated PDP1 binding sites, listed prospective targets and searched for PDP1-specific binding sites. By examining the functional relevance to diapause-specific behaviours and modifications such as metabolic pathways, lifespan extension, cell cycle regulation and stress tolerance, eight genes were selected as targets and validated using ChIP-qPCR. In addition, qRT-PCR demonstrated that the mRNA abundance of PDP1 targets increased in the heads of diapausing females during the middle of the scotophase (ZT17) compared with the early photophase (ZT1), in agreement with the peak and trough of PDP1 abundance. Thus, our investigation uncovered the mechanism by which PDP1 might generate a diapause phenotype in insects.

Consuming royal jelly alters several phenotypes associated with overwintering dormancy in mosquitoes.

Introduction: Females of the Northern house mosquito, Culex pipiens, enter an overwintering dormancy, or diapause, in response to short day lengths and low environmental temperatures that is characterized by small egg follicles and high starvation resistance. During diapause, Culex pipiens Major Royal Jelly Protein 1 ortholog (CpMRJP1) is upregulated in females of Cx. pipiens. This protein is highly abundant in royal jelly, a substance produced by honey bees (Apis mellifera), that is fed to future queens throughout larval development and induces the queen phenotype (e.g., high reproductive activity and longer lifespan). However, the role of CpMRJP1 in Cx. pipiens is unknown. Methods: We first conducted a phylogenetic analysis to determine how the sequence of CpMRJP1 compares with other species. We then investigated how supplementing the diets of both diapausing and nondiapausing females of Cx. pipiens with royal jelly affects egg follicle length, fat content, protein content, starvation resistance, and metabolic profile. Results: We found that feeding royal jelly to females reared in long-day, diapause-averting conditions significantly reduced the egg follicle lengths and switched their metabolic profiles to be similar to diapausing females. In contrast, feeding royal jelly to females reared in short-day, diapause-inducing conditions significantly reduced lifespan and switched their metabolic profile to be similar nondiapausing mosquitoes. Moreover, RNAi directed against CpMRJPI significantly increased egg follicle length of short-day reared females, suggesting that these females averted diapause. Discussion: Taken together, our data show that consuming royal jelly reverses several key seasonal phenotypes of Cx. pipiens and that these responses are likely mediated in part by CpMRJP1.

Trade-offs between winter survival and reproduction in female insects.

In temperate environments most species of insects enter an arrested state of development, known as diapause, that enables them to survive the adverse environmental conditions associated with winter. Although diapause is restricted to a single life stage within species of insects, there are examples of insects that overwinter in the egg, larval, pupal and adult stages. Here we offer a targeted, non-systematic literature review examining how overwintering impacts subsequent reproduction in female insects. Several factors, including the lifestage at which insects overwinter, the type of energy investment strategy females use for breeding, elements of the winter environment, and contributions from male insects can influence trade-offs that female insects face between overwintering survival and post-diapause reproduction. Additionally, climate change and elements of the urban environment, including light pollution and higher temperatures in cities, can exacerbate or ameliorate trade-offs faced by reproducing female insects. Better understanding trade-offs between overwintering survival and reproduction in insects not only enhances our understanding of the underlying physiological mechanisms and ecological processes governing diapause and reproduction, but also provide opportunities to better manage insect pests and/or support beneficial insects.

Phenotypic variation in biting behavior associated with differences in expression of olfactory genes in the vector mosquito, Aedes albopictus (Diptera: Culicidae).

We evaluated miRNA and mRNA expression differences in head tissues between avid-biting vs. reluctant-biting Aedes albopictus (Skuse) females from a single population over a 20-min timescale. We found no differences in miRNA expression between avid vs. reluctant biters, indicating that translational modulation of blood-feeding behavior occurs on a longer timescale than mRNA transcription. In contrast, we detected 19 differentially expressed mRNAs. Of the 19 differentially expressed genes at the mRNA level between avid-biting vs. reluctant-biting A. albopictus, 9 are implicated in olfaction, consistent with the well-documented role of olfaction in mosquito host-seeking. Additionally, several of the genes that we identified as differentially expressed in association with phenotypic variation in biting behavior share similar functions with or are inferred orthologues of, genes associated with evolutionary variation in biting behaviors of Wyeomyia smithii (Coq.) and Culex pipiens (Lin.). A future goal is to determine whether these genes are involved in the evolutionary transition from a biting to a non-biting life history.

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.

Light pollution disrupts circadian clock gene expression in two mosquito vectors during their overwintering dormancy.

Artificial light at night (ALAN) is an increasingly important form of environmental disturbance as it alters Light:Dark cycles that regulate daily and seasonal changes in physiology and phenology. The Northern house mosquito (Culex pipiens) and the tiger mosquito (Aedes albopictus) enter an overwintering dormancy known as diapause that is cued by short days. These two species differ in diapause strategy: Cx. pipiens diapause as adult females while Ae. albopictus enter a maternally-programmed, egg diapause. Previous studies found that ALAN inhibits diapause in both species, but the mechanism is unknown. As the circadian clock is implicated in the regulation of diapause in many insects, we examined whether exposure to ALAN altered the daily expression of core circadian cloc genes (cycle, Clock, period, timeless, cryptochrome 1, cryptochrome 2, and Par domain protein 1) in these two species when reared under short-day, diapause-inducing conditions. We found that exposure to ALAN altered the abundance of several clock genes in adult females of both species, but that clock gene rhythmicity was maintained for most genes. ALAN also had little effect on clock gene abundance in mature oocytes that were dissected from female Ae. albopictus that were reared under short day conditions. Our findings indicate that ALAN may inhibit diapause initiation through the circadian clock in two medically-important mosquitoes.

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.

Potential for urban warming to postpone overwintering dormancy of temperate mosquitoes.

Cities are generally hotter than surrounding rural areas due to the Urban Heat Island (UHI) effect. These increases in temperature advance plant and animal phenology, development, and reproduction in the spring. However, research determining how increased temperatures affect the seasonal physiology of animals in the fall has been limited. The Northern house mosquito, Culex pipiens, is abundant in cities and transmits several pathogens including West Nile virus. Females of this species enter a state of developmental arrest, or reproductive diapause, in response to short days and low temperatures during autumn. Diapausing females halt reproduction and blood-feeding, and instead accumulate fat and seek sheltered overwintering sites. We found that exposure to increased temperatures in the lab that mimic the UHI effect induced ovarian development and blood-feeding, and that females exposed to these temperatures were as fecund as non-diapausing mosquitoes. We also found that females exposed to higher temperatures had lower survival rates in winter-like conditions, despite having accumulated equivalent lipid reserves relative to their diapausing congeners. These data suggest that urban warming may inhibit diapause initiation in the autumn, thereby extending the active biting season of temperate mosquitoes.

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.

Characterizing the relative abundance of circadian transcription factors in diapausing and nondiapausing Northern house mosquitoes.

The Northern house mosquito (Culex pipiens) is a major vector of West Nile virus. To survive harsh conditions in winter adult females of Cx. pipiens enter a state of arrested reproductive development called diapause. Diapause is triggered by the short daylengths of late summer and early fall. The methods by which Cx. pipiens measures daylength are still unknown. However, it is suspected that clock genes, which provide information on daylength, may also regulate diapause. The proteins produced by these genes often cycle in abundance throughout the day in diapausing and nondiapausing insects. Two clock genes suspected to control diapause are cycle (cyc) and Par domain protein1 (Pdp1) as they encode circadian transcription factors that may regulate genes that are involved in diapause. Using Western blotting we measured the relative protein abundance of CYC and PDP1 throughout the day in the whole bodies and the heads of Cx. pipiens reared under either long-day, diapause-averting conditions or short-day, diapause-inducing conditions. We found that in whole bodies there was no significant oscillation of CYC or PDP1 abundance in both long day and short day-reared mosquitoes. In the heads of long day-reared mosquitoes both CYC and PDP1 cycled. In contrast, only PDP1 abundance showed diel differences in abundance in the heads of short day-reared mosquitoes. These data bring us one step closer to understanding the role that CYC and PDP1 may play in regulating diapause and other biological processes.

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.

Critical Photoperiod and Its Potential to Predict Mosquito Distributions and Control Medically Important Pests.

Diapause, a period of arrested development that allows mosquitoes to survive inhospitable conditions, is triggered by short daylengths in temperate mosquitoes. Different populations of mosquitoes initiate diapause in response to a specific photoperiod, or daylength, resulting in population-specific differences in annual cycles of abundance. The photoperiod that causes approximately 50% of a population to initiate diapause is known as the critical photoperiod (CPP). The autumn daylength corresponding to the CPP in the field likely marks the day beyond which the photoperiods would trigger and maintain 50% or more diapause incidence in a population, although temperature, diet, and other factors can impact diapause initiation. In the Northern Hemisphere, northern populations of mosquitoes experience lower temperatures earlier in the year and must be triggered into diapause by longer daylengths than southern populations. CPP is genetically based, but also adapts over time responding to the population's environment. Therefore, CPP has been shown to lengthen with increasing latitude and altitude. While the positive correlation between CPP and latitude/altitude has been established in a few mosquito species, including Aedes albopictus (Skuse, Diptera: Culicidae), Aedes triseriatus, Aedes sierrensis, and Wyeomyia smithii (Coquillett, Diptera: Culicidae), we do not know when most other species initiate their seasonal responses. As several of these species transmit important diseases, characterizing the CPP of arthropod vectors could improve existing control by ensuring that surveillance efforts align with the vector's seasonally active period. Additionally, better understanding when mosquitoes and other vectors initiate diapause can reduce the frequency of chemical applications, thereby ameliorating the negative impacts to nontarget insects.

Hello Darkness, My Old Friend: A Tutorial of Nanda-Hamner Protocols.

Plants and animals use circadian and photoperiodic timekeeping mechanisms to respond to daily and seasonal changes in light:dark and appropriately coordinate their development. Although the mechanisms that may connect the circadian and photoperiodic clock are still unclear in many species, researchers have been using Nanda-Hamner protocols for decades to elucidate how seasonal time is measured and determine whether seasonal responses have a circadian basis in a given species. In this brief tutorial we describe how to design and interpret the results of Nanda-Hamner experiments, and provide suggestions on how to use both Nanda-Hamner protocols and modern molecular experiments to better understand the mechanisms of seasonal timekeeping.

Artificial light at night alters the seasonal responses of biting mosquitoes.

Urban light pollution caused by artificial light at night (ALAN) profoundly affects the ecology, behavior, and physiology of plants and animals. Further, this widespread environmental pollutant has the potential to negatively impact human and animal health by changing the seasonal dynamics of disease-transmitting insects. In response to short days, females of the Northern house mosquito enter an overwintering dormancy, or diapause. While in diapause, female mosquitoes divert energy away from reproduction, cease blood-feeding, and no longer transmit disease. We demonstrate that exposure to dim ALAN (~4 lx) causes female mosquitoes to avert diapause and become reproductively active, as these females acquired less fat content, developed larger egg follicles, imbibed vertebrate blood, and produced viable eggs and larvae. Our findings suggest that mosquitoes in highly light-polluted areas such as cities may be actively reproducing and biting later in the season, thereby extending the period of disease risk for urban residents. Our results suggest that ALAN should be considered when modeling mosquito abundance, disease risk, and when deciding how long mosquito surveillance and control should persist in temperate regions.

Determining the effects of nutrition on the reproductive physiology of male mosquitoes.

Nutrition affects multiple aspects of insect physiology such as body size and fecundity, but we lack a detailed understanding of how nutrition influences the reproductive physiology of male insects such as mosquitoes. Given that female mosquitoes are vectors of many deadly diseases and can quickly proliferate, understanding how male nutrition impacts female fecundity could be of critical importance. To uncover the relationship between nutrition in adult male mosquitoes and its impacts on reproductive physiology, we reared larvae of the Northern house mosquito, Culex pipiens, on a standard lab diet and divided adult males among three different dietary treatments: low (3%), moderate (10%), and high (20%) sucrose. We found that although overall body size did not differ among treatments, one-week-old males raised on the 3% sucrose diet had significantly smaller male accessory glands (MAGs) compared to males that consumed the 10% and the 20% sucrose diets. Diet affected whole-body lipid content but did not affect whole-body protein content. Using nuclear magnetic resonance (NMR) spectroscopy, we found that diet altered the metabolic composition of the MAGs, including changes in lactic acid, formic acid, and glucose. We also observed changes in protein and lipid abundance and composition in MAGs. Females who mated with males on the 3% diet were found to produce significantly fewer larvae than females who had mated with males on the 10% diet. Taken together, our results demonstrate that the diet of adult male mosquitoes clearly affects male reproductive physiology and female fecundity.

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9.

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.

Circadian transcription factors differentially regulate features of the adult overwintering diapause in the Northern house mosquito, Culex pipiens.

The short days of late summer and early fall are the environmental cues that most temperate insects and other animals use to predict winter's arrival. Although it is still unclear precisely how insects measure daylength, there is mounting evidence that the circadian clock regulates seasonal responses including photoperiodic diapause. Females of the Northern house mosquito, Culex pipiens, enter an adult reproductive diapause in response to short daylengths. While in this state, females divert their resources from reproduction to survival, arresting egg follicle development and increasing fat content. Here, we characterized the expression profile of two circadian transcription factors, vrille (vri) and Par domain protein 1 (Pdp1), as well as genes downstream of the clock, takeout (to) and Nocturnin (Noc) and under different seasonal conditions. We saw that while vri mRNA oscillated under both long day and short day conditions, Pdp1 expression oscillated only under long day conditions and was constitutively upregulated in diapausing females. We saw similar expression profiles for to and Noc, suggesting that PDP1 might regulate their expression or that Pdp1, to and Noc might be regulated by the same transcription factor. We suppressed vri and Pdp1 using RNA interference. dsRNA against vri provided inconsistent results, sometimes stimulating autogenous egg follicle development in both long and short day-reared females, and other times had no effect. In contrast, knocking down Pdp1 prevented short day-reared females from accumulating fat reserves, but increased expression of to and Noc. Taken together, these data suggest that the circadian transcription factors Vri and Pdp1 may independently regulate signaling pathways underlying arrested egg follicle development and fat accumulation in diapausing females of Cx. pipiens.