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.

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.

Clock-talk: have we forgotten about geographic variation?

Wyeomyia smithii, the pitcher-plant mosquito, has evolved from south to north and from low to high elevations in eastern North America. Along this seasonal gradient, critical photoperiod has increased while apparent involvement of the circadian clock has declined in concert with the evolutionary divergence of populations. Response to classical experiments used to test for a circadian basis of photoperiodism varies as much within and among populations of W. smithii as have been found in the majority of all other insects and mites. The micro-evolutionary processes revealed within and among populations of W. smithii, programmed by a complex underlying genetic architecture, illustrate a gateway to the macro-evolutionary divergence of biological timing among species and higher taxa in general.

Latent Genetic Effects of Past Selection on Blood Feeding: History Matters.

Conventional wisdom is that selection decreases genetic variation in populations, variation that should enable and be essential for population persistence in an ever-changing world. Basically, we find the opposite. Response to selection on biting in the pitcher-plant mosquito, Wyeomyia smithii, increases from 20 to 80% in 19 generations, but reverts back to the original 20% after seven generations of relaxed (not reversed) selection. At the same time, biting in the control line remains at the original 20% through 30 generations without blood feeding. Imposition of selection on biting in both lines elicits a rapid response in the previously selected line, but, importantly, not in the control line. Genetic variation for biting has increased, not decreased, as a consequence of long-term directional selection, contrary to expectations. Convergent phenotypes belie the underlying difference in future adaptive potential. Selection events over time in the background of individuals or populations will determine outcomes of applied research, be it in the fields of medicine, agriculture, or conservation. In short, history matters.

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.

Evolutionary transition from blood feeding to obligate nonbiting in a mosquito.

The spread of blood-borne pathogens by mosquitoes relies on their taking a blood meal; if there is no bite, there is no disease transmission. Although many species of mosquitoes never take a blood meal, identifying genes that distinguish blood feeding from obligate nonbiting is hampered by the fact that these different lifestyles occur in separate, genetically incompatible species. There is, however, one unique extant species with populations that share a common genetic background but blood feed in one region and are obligate nonbiters in the rest of their range: Wyeomyia smithii Contemporary blood-feeding and obligate nonbiting populations represent end points of divergence between fully interfertile southern and northern populations. This divergence has undoubtedly resulted in genetic changes that are unrelated to blood feeding, and the challenge is to winnow out the unrelated genetic factors to identify those related specifically to the evolutionary transition from blood feeding to obligate nonbiting. Herein, we determine differential gene expression resulting from directional selection on blood feeding within a polymorphic population to isolate genetic differences between blood feeding and obligate nonbiting. We show that the evolution of nonbiting has resulted in a greatly reduced metabolic investment compared with biting populations, a greater reliance on opportunistic metabolic pathways, and greater reliance on visual rather than olfactory sensory input. W. smithii provides a unique starting point to determine if there are universal nonbiting genes in mosquitoes that could be manipulated as a means to control vector-borne disease.

Natural Variation and Genetics of Photoperiodism in Wyeomyia smithii.

Seasonal change in the temperate and polar regions of Earth determines how the world looks around us and, in fact, how we live our day-to-day lives. For biological organisms, seasonal change typically involves complex physiological and metabolic reorganization, the majority of which is regulated by photoperiodism. Photoperiodism is the ability of animals and plants to use day length or night length, resulting in life-historical transformations, including seasonal development, migration, reproduction, and dormancy. Seasonal timing determines not only survival and reproductive success but also the structure and organization of complex communities and, ultimately, the biomes of Earth. Herein, a small mosquito, Wyeomyia smithii, that lives only in the water-filled leaves of a carnivorous plant over a wide geographic range, is used to explore the genetic and evolutionary basis of photoperiodism. Photoperiodism in W. smithii is considered in the context of its historical biogeography in nature to examine the startling finding that recent rapid climate change can drive genetic change in plants and animals at break-neck speed, and to challenge the ponderous 80+ year search for connections between daily and seasonal time-keeping mechanisms. Finally, a model is proposed that reconciles the seemingly disparate 24-h daily clock driven by the invariant rotation of Earth about its axis with the evolutionarily flexible seasonal timer orchestrated by variable seasonality driven by the rotation of Earth about the Sun.

Keeping time without a spine: what can the insect clock teach us about seasonal adaptation?

Seasonal change in daylength (photoperiod) is widely used by insects to regulate temporal patterns of development and behaviour, including the timing of diapause (dormancy) and migration. Flexibility of the photoperiodic response is critical for rapid shifts to new hosts, survival in the face of global climate change and to reproductive isolation. At the same time, the daily circadian clock is also essential for development, diapause and multiple behaviours, including correct flight orientation during long-distance migration. Although studied for decades, how these two critical biological timing mechanisms are integrated is poorly understood, in part because the core circadian clock genes are all transcription factors or regulators that are able to exert multiple effects throughout the genome. In this chapter, we discuss clocks in the wild from the perspective of diverse insect groups across eco-geographic contexts from the Antarctic to the tropical regions of Earth. Application of the expanding tool box of molecular techniques will lead us to distinguish universal from unique mechanisms underlying the evolution of circadian and photoperiodic timing, and their interaction across taxonomic and ecological contexts represented by insects.This article is part of the themed issue 'Wild clocks: integrating chronobiology and ecology to understand timekeeping in free-living animals'.

Replicate phylogenies and post-glacial range expansion of the pitcher-plant mosquito, Wyeomyia smithii, in North America.

Herein we tested the repeatability of phylogenetic inference based on high throughput sequencing by increased taxon sampling using our previously published techniques in the pitcher-plant mosquito, Wyeomyia smithii in North America. We sampled 25 natural populations drawn from different localities nearby 21 previous collection localities and used these new data to construct a second, independent phylogeny, expressly to test the reproducibility of phylogenetic patterns. Comparison of trees between the two data sets based on both maximum parsimony and maximum likelihood with Bayesian posterior probabilities showed close correspondence in the grouping of the most southern populations into clear clades. However, discrepancies emerged, particularly in the middle of W. smithii's current range near the previous maximum extent of the Laurentide Ice Sheet, especially concerning the most recent common ancestor to mountain and northern populations. Combining all 46 populations from both studies into a single maximum parsimony tree and taking into account the post-glacial historical biogeography of associated flora provided an improved picture of W. smithii's range expansion in North America. In a more general sense, we propose that extensive taxon sampling, especially in areas of known geological disruption is key to a comprehensive approach to phylogenetics that leads to biologically meaningful phylogenetic inference.

Footprints in time: comparative quantitative trait loci mapping of the pitcher-plant mosquito, Wyeomyia smithii.

Identifying regions of the genome contributing to phenotypic evolution often involves genetic mapping of quantitative traits. The focus then turns to identifying regions of 'major' effect, overlooking the observation that traits of ecological or evolutionary relevance usually involve many genes whose individual effects are small but whose cumulative effect is large. Herein, we use the power of fully interfertile natural populations of a single species of mosquito to develop three quantitative trait loci (QTL) maps: one between two post-glacially diverged populations and two between a more ancient and a post-glacial population. All demonstrate that photoperiodic response is genetically a highly complex trait. Furthermore, we show that marker regressions identify apparently 'non-significant' regions of the genome not identified by composite interval mapping, that the perception of the genetic basis of adaptive evolution is crucially dependent upon genetic background and that the genetic basis for adaptive evolution of photoperiodic response is highly variable within contemporary populations as well as between anciently diverged populations.

Conservation of the photoperiodic neuroendocrine axis among vertebrates: evidence from the teleost fish, Gasterosteus aculeatus.

Photoperiod, or length of day, has a predictable annual cycle, making it an important cue for the timing of seasonal behavior and development in many organisms. Photoperiod is widely used among temperate and polar animals to regulate the timing of sexual maturation. The proper sensing and interpretation of photoperiod can be tightly tied to an organism's overall fitness. In photoperiodic mammals and birds the thyroid hormone pathway initiates sexual maturation, but the degree to which this pathway is conserved across other vertebrates is not well known. We use the threespine stickleback Gasterosteus aculeatus, as a representative teleost to quantify the photoperiodic response of key genes in the thyroid hormone pathway under controlled laboratory conditions. We find that the photoperiodic responses of the hormones are largely consistent amongst multiple populations, although differences suggest physiological adaptation to various climates. We conclude that the thyroid hormone pathway initiates sexual maturation in response to photoperiod in G. aculeatus, and our results show that more components of this pathway are conserved among mammals, birds, and teleost fish than was previously known. However, additional endocrinology, cell biology and molecular research will be required to define precisely which aspects of the pathway are conserved across vertebrates.

Testing for causality in covarying traits: genes and latitude in a molecular world.

Many traits are assumed to have a causal (necessary) relationship with one another because of their common covariation with a physiological, ecological or geographical factor. Herein, we demonstrate a straightforward test for inferring causality using residuals from regression of the traits with the common factor. We illustrate this test using the covariation with latitude of a proxy for the circadian clock and a proxy for the photoperiodic timer in Drosophila and salmon. A negative result of this test means that further discussion of the adaptive significance of a causal connection between the covarying traits is unwarranted. A positive result of this test provides a point of departure that can then be used as a platform from which to determine experimentally the underlying functional connections and only then to discuss their adaptive significance.

Circadian clock genes, ovarian development and diapause.

Insects, like most organisms, have an internal circadian clock that oscillates with a daily rhythmicity, and a timing mechanism that mediates seasonal events, including diapause. In research published in BMC Biology, Ikeno et al. show that downregulation of the circadian clock genes period and cycle affects expression of ovarian diapause in the insect Riptortus pedestris. They interpret these important results as support for Erwin Bünning's (1936) hypothesis that the circadian clock constitutes the basis of photoperiodism. However, their observations could also be the result of pleiotropic effects of the individual clock genes.See research article http://www.biomedcentral.com/1741-7007/8/116.

Resolving postglacial phylogeography using high-throughput sequencing.

The distinction between model and nonmodel organisms is becoming increasingly blurred. High-throughput, second-generation sequencing approaches are being applied to organisms based on their interesting ecological, physiological, developmental, or evolutionary properties and not on the depth of genetic information available for them. Here, we illustrate this point using a low-cost, efficient technique to determine the fine-scale phylogenetic relationships among recently diverged populations in a species. This application of restriction site-associated DNA tags (RAD tags) reveals previously unresolved genetic structure and direction of evolution in the pitcher plant mosquito, Wyeomyia smithii, from a southern Appalachian Mountain refugium following recession of the Laurentide Ice Sheet at 22,000-19,000 B.P. The RAD tag method can be used to identify detailed patterns of phylogeography in any organism regardless of existing genomic data, and, more broadly, to identify incipient speciation and genome-wide variation in natural populations in general.

What season is it anyway? Circadian tracking vs. photoperiodic anticipation in insects.

The daily rhythm of 24 h and the annual rhythm of 12 mo constitute the 2 major, highly predictable rhythms of the biosphere. The internal circadian clock enables organisms to track daily changes in their environment; the photoperiodic timer, alone or in concert with a circannual clock, enables organisms to anticipate and prepare in advance for seasonal changes in their environment. The circadian clock entrains to dawn and dusk and tracks light and temperature on a day-to-day basis, while the photoperiodic timer serves as a long-term, physiological go/no-go switch that commits an animal to development, reproduction, dormancy, or migration on a seasonal or even lifetime basis. In 1936, Erwin Bünning proposed that circadian rhythms formed the basis (Grundlage) for photoperiodic response to day length. Historical inertia generated by correlative evidence from early physiological studies and a proliferating number of descriptive models has resulted in the widespread assumption that the circadian clock constitutes the necessary, causal basis of photoperiodism in general. This historical inertia has also restricted the search for genes involved in insect photoperiodism to genes central to the circadian clock in Drosophila and has led investigators to conclude that any behavior, process, or gene expression that varies with day length represents photoperiodism or a gene involved in photoperiodism. The authors discuss how blinders imposed by the circadian imperative have retarded progress toward identifying the genetic basis of photoperiodism and how the insights gained from geographic variation in photoperiodic response have been used to show the independent evolution of the circadian clock and photoperiodism. When geographic variation is found in circadian genes, the most immediate and parsimonious search for adaptive significance should be in circadian function, not in extrapolation to photoperiodism. Finally, the authors propose that circadian-unbiased, forward genetic approaches should be used to identify genes involved in photoperiodism within extant populations and among populations over evolutionary time.

Microarrays reveal early transcriptional events during the termination of larval diapause in natural populations of the mosquito, Wyeomyia smithii.

BACKGROUND: The mosquito Wyeomyia smithii overwinters in a larval diapause that is initiated, maintained and terminated by day length (photoperiod). We use a forward genetic approach to investigate transcriptional events involved in the termination of diapause following exposure to long-days. METHODS/PRINCIPAL FINDINGS: We incorporate a novel approach that compares two populations that differentially respond to a single day length. We identify 30 transcripts associated with differential response to day length. Most genes with a previously annotated function are consistent with their playing a role in the termination of diapause, in downstream developmental events, or in the transition from potentially oxygen-poor to oxygen-rich environments. One gene emerges from three separate forward genetic screens as a leading candidate for a gene contributing to the photoperiodic timing mechanism itself (photoperiodic switch). We name this gene photoperiodic response gene 1 (ppdrg1). WsPpdrg1 is up-regulated under long-day response conditions, is located under a QTL for critical photoperiod and is associated with critical photoperiod after 25 generations of recombination from a cross between extreme phenotypes. CONCLUSIONS: Three independent forward genetic approaches identify WsPpdrg1 as a gene either involved in the photoperiodic switch mechanism or very tightly linked to a gene that is. We conclude that continued forward genetic approaches will be central to understanding not only the molecular basis of photoperiodism and diapause, but also the evolutionary potential of temperate and polar animal populations when confronted with rapid climate change.

Light, time, and the physiology of biotic response to rapid climate change in animals.

Examination of temperate and polar regions of Earth shows that the nonbiological world is exquisitely sensitive to the direct effects of temperature, whereas the biological world is largely organized by light. Herein, we discuss the use of day length by animals at physiological and genetic levels, beginning with a comparative experimental study that shows the preeminent role of light in determining fitness in seasonal environments. Typically, at seasonally appropriate times, light initiates a cascade of physiological events mediating the input and interpretation of day length to the output of specific hormones that ultimately determine whether animals prepare to develop, reproduce, hibernate, enter dormancy, or migrate. The mechanisms that form the basis of seasonal time keeping and their adjustment during climate change are reviewed at the physiological and genetic levels. Future avenues for research are proposed that span basic questions from how animals transition from dependency on tropical cues to temperate cues during range expansions, to more applied questions of species survival and conservation biology during periods of climatic stress.

Environmental control of ovarian dormancy in natural populations of Drosophila melanogaster.

Drosophila melanogaster from Australia, Europe and North America enter an adult ovarian dormancy in response to short days and low temperatures. The independent effects of temperature and day length in the determination of dormancy have been examined only in one long-established laboratory line (Canton-S). In all other studies of natural or laboratory populations, dormancy has been assessed at either a single short day or a single moderately low temperature. Herein, we determine the relative roles of temperature, photoperiod, and their interaction in the control of ovarian dormancy in D. melanogaster from two natural populations representing latitudinal extremes in eastern North America (Florida at 27 degrees N and Maine at 44 degrees N). In both natural populations, temperature is the main determinant of dormancy, alone explaining 67% of the total variation among replicate isofemale lines, whereas photoperiod has no significant effect. We conclude that ovarian dormancy in D. melanogaster is a temperature-initiated syndrome of winter-tolerant traits that represents an adaptive phenotypic plasticity in temperate seasonal environments.

Complications of complexity: integrating environmental, genetic and hormonal control of insect diapause.

Understanding gene interaction and pleiotropy are long-standing goals of developmental and evolutionary biology. We examine the genetic control of diapause in insects and show how the failure to recognize the difference between modular and gene pleiotropy has confounded our understanding of the genetic basis of this important phenotype. This has led to complications in understanding the role of the circadian clock in the control of diapause in Drosophila and other insects. We emphasize three successive modules - each containing functionally related genes - that lead to diapause: photoperiodism, hormonal events and diapause itself. Understanding the genetic basis for environmental control of diapause has wider implications for evolutionary response to rapid climate change and for the opportunity to observe evolutionary change in contemporary time.

Evolution of photoperiodic time measurement is independent of the circadian clock in the pitcher-plant mosquito, Wyeomyia smithii.

For over 70 years, researchers have debated whether the ability to use day length as a cue for the timing of seasonal events (photoperiodism) is related to the endogenous circadian clock that regulates the timing of daily events. Models of photoperiodism include two components: (1) a photoperiodic timer that measures the length of the day, and (2) a photoperiodic counter that elicits the downstream photoperiodic response after a threshold number of days has been counted. Herein, we show that there is no geographical pattern of genetic association between the expression of the circadian clock and the photoperiodic timer or counter. We conclude that the photoperiodic timer and counter have evolved independently of the circadian clock in the pitcher-plant mosquito Wyeomyia smithii and hence, the evolutionary modification of photoperiodism throughout the range of W. smithii has not been causally mediated by a corresponding evolution of the circadian clock.