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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.

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.

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.

Herein, we determine life‐history consequences of selection on blood feeding in a polymorphic population of the pitcher‐plant mosquito, Wyeomyia smithii Coq. (Diptera: Culicidae). All populations of W. smithii produce an initial batch of eggs without ever taking a blood meal (biting); southern populations require a blood meal for the second and subsequent batches of eggs, but are polymorphic for propensity to bite. To determine correlated life‐history responses to direct selection on blood feeding, we compared fecundity, adult longevity, and reproductive allocation between a line selected specifically for increased blood feeding and its unselected, control line maintained in parallel for 11 generations. Previous studies have focused on the fitness benefits of blood feeding in terms of overall fecundity. Herein, we evaluate a novel fitness benefit of blood feeding that reduces the risk of reproductive failure by spreading that risk across multiple reproductive events in a population confronted with an unpredictably variable larval environment. We propose that “spreading the risk” reinforces selection on blood feeding in other arthropods in which the separation of fecundity from reproductive allocation in time or space has previously been neglected. Importantly, heritable variation for “spreading the risk” should enhance vectorial capacity and make more difficult vector control through larval source reduction. Coq. (Diptera: Culicidae). Previous studies have focused on the fitness benefits of variable larval environment.

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.

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.

Understanding how communities respond to changes in temperature is a major challenge for community ecology. Temperature influences the relative degree to which top-down and bottom-up forces structure ecological communities. In greenhouse experiments using the aquatic community found in pitcher plants ( Sarracenia purpurea ), I tested how temperature affected the relative importance of top-down (mosquito predation) and bottom-up (ant carcasses) forces on protozoa and bacteria populations. While bottom-up effects did not vary consistently with temperature, the top-down effects of predators on protozoa increased at higher temperatures. These results suggest that temperature could change the relative importance of top-down and bottom-up effects in ecological communities. Specifically, higher temperature may increase the strength of top-down effects by raising predator metabolic rate and concomitant processes (e.g., activity, foraging, digestion, growth) relative to cooler temperatures. These findings apply broadly to an understanding of trophic interactions in a variable environment and are especially relevant in the context of ongoing climate change.

Carnivory in pitcher plants generally involves digestion of prey, by the plant itself, by symbionts, or both. While symbionts appear to be important in the digestion of prey in Sarracenia purpurea, the importance of pitcher-derived enzymes is less well documented. Our goal was to reduce microbial numbers in pitcher fluid in order to measure the acid phosphatase activity attributable to the pitchers themselves. Preliminary experiments indicated that various antibiotics were minimally effective at reducing microbial populations and that antibiotic-resistant microbes were easily cultured from pitcher fluid. Consequently, we measured the abundance of culturable microbes in every sample taken for the measurement of acid phosphatase activity. Pitchers fed with one sterilized ant had higher levels of acid phosphatase activity than unfed pitchers. Older pitchers were more responsive to feeding than young pitchers. Pitchers with high levels of microbes (on Day 5) had higher acid phosphatase activity than pitchers with low levels of microbes. However, fed pitchers were not more likely to have higher microbe levels and microbe levels were not related to pitcher age. When fluid samples from inside the pitcher were compared to appropriate controls incubated outside the pitcher, acid phosphatase activity was higher inside the pitcher. Results from the feeding experiments are consistent with a primary role of microbes in the digestion of prey in pitchers of S. purpurea. However, the relationship between pitcher age and enzyme activity is not a function of microbes in the pitcher fluid and may depend on enzymes produced by the plant. Our methods would not detect microbes embedded on the inner surface of the pitcher; and if they survived the alcohol rinse and antibiotics, we cannot rule out microbes as the source of the relationship between pitcher age and acid phosphatase activity.

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.

The effects of resource pulses on natural communities are known to vary with the type of pulse. However, less is known about mechanisms that determine the responses of different species to the same pulse. We hypothesized that these differences are related to the size of the species, as increasing size may be correlated with increasing competitive ability and decreasing tolerance to predation. A factorial experiment quantified the magnitude and timing of species’ responses to a resource pulse using the aquatic communities found in the leaves of the carnivorous pitcher plant, Sarracenia purpurea. We added prey to leaves and followed the abundances of bacteria and bacterivores (protozoa and rotifers) in the presence and absence of a top predator, larvae of the mosquito Wyeomyia smithii. Resource pulses had significant positive effects on species abundances and diversity in this community; however, the magnitude and timing of responses varied among the bacterivore species and was not related to body size. Larger bacterivores were significantly suppressed by predators, while smaller bacterivores were not; predation also significantly reduced bacterivore species diversity. There were no interactions between the effects of the resource pulse and predation on protozoa abundances. Over 67 days, some species returned to pre-pulse abundances quickly, others did not or did so very slowly, resulting in new community states for extended periods of time. This study demonstrates that species-specific differences in responses to resource pulses and predation are complex and may not be related to simple life history trade-offs associated with size.