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Background: Internal circadian (circa, about; dies, day) clocks enable organisms to maintain adaptive timing of their daily behavioral activities and physiological functions. Eukaryotic clocks consist of core transcription-translation feedback loops that generate a cycle and post-translational modifiers that maintain that cycle at about 24 h. We use the pitcher-plant mosquito, Wyeomyia smithii (subfamily Culicini, tribe Sabethini), to test whether evolutionary divergence of the circadian clock genes in this species, relative to other insects, has involved primarily genes in the core feedback loops or the post-translational modifiers. Heretofore, there is no reference transcriptome or genome sequence for any mosquito in the tribe Sabethini, which includes over 375 mainly circumtropical species., Methods: We sequenced, assembled and annotated the transcriptome of W. smithii containing nearly 95 % of conserved single-copy orthologs in animal genomes. We used the translated contigs and singletons to determine the average rates of circadian clock-gene divergence in W. smithii relative to three other mosquito genera, to Drosophila, to the butterfly, Danaus, and to the wasp, Nasonia., Results: Over 1.08 million cDNA sequence reads were obtained consisting of 432.5 million nucleotides. Their assembly produced 25,904 contigs and 54,418 singletons of which 62 % and 28 % are annotated as protein-coding genes, respectively, sharing homology with other animal proteomes., Discussion: The W. smithii transcriptome includes all nine circadian transcription-translation feedback-loop genes and all eight post-translational modifier genes we sought to identify (Fig. 1). After aligning translated W. smithii contigs and singletons from this transcriptome with other insects, we determined that there was no significant difference in the average divergence of W. smithii from the six other taxa between the core feedback-loop genes and post-translational modifiers., Conclusions: The characterized transcriptome is sufficiently complete and of sufficient quality to have uncovered all of the insect circadian clock genes we sought to identify (Fig. 1). Relative divergence does not differ between core feedback-loop genes and post-translational modifiers of those genes in a Sabethine species (W. smithii) that has experienced a continual northward dispersal into temperate regions of progressively longer summer day lengths as compared with six other insect taxa. An associated microarray platform derived from this work will enable the investigation of functional genomics of circadian rhythmicity, photoperiodic time measurement, and diapause along a photic and seasonal geographic gradient.

The genetic relationship between the daily circadian clock and the seasonal photoperiodic timer remains a subject of intense controversy. In Wyeomyia smithii, the critical photoperiod (an overt expression of the photoperiodic timer) evolves independently of the rhythmic response to the Nanda-Hamner protocol (an overt expression of the daily circadian clock) over a wide geographical range in North America. Herein, we focus on these two processes within a single local population in which there is a negative genetic correlation between them. We show that antagonistic selection against this genetic correlation rapidly breaks it down and, in fact, reverses its sign, showing that the genetic correlation is due primarily to linkage and not to pleiotropy. This rapid reversal of the genetic correlation within a small, single population means that it is difficult to argue that circadian rhythmicity forms the necessary, causal basis for the adaptive divergence of photoperiodic time measurement within populations or for the evolution of photoperiodic time measurement among populations over a broad geographical gradient of seasonal selection.

The two major rhythms of the biosphere are daily and seasonal; the two major adaptations to these rhythms are the circadian clock, mediating daily activities, and the photoperiodic timer, mediating seasonal activities. The mechanistic connection between the circadian clock and the photoperiodic timer remains unresolved. Herein, we show that the rhythmic developmental response to exotic light:dark cycles, usually used to infer a causal connection between the circadian clock and the photoperiodic timer, has evolved independently of the photoperiodic timer in the pitcher-plant mosquito Wyeomyia smithii across the climatic gradient of eastern North America from Florida to Canada and from the coastal plain to the mountains. We conclude that the photoperiodic timing of seasonal events can evolve independently of the daily circadian clock.

Expression of the circadian rhythm gene timeless was investigated in the pitcher-plant mosquito, Wyeomyia smithii (Coq.), and was found to vary with time of day, instar of diapause, and latitude of origin. The temporal pattern of timeless expression differed between the two diapausing instars and was significantly higher in southern (38-40 degrees N) than in northern (50 degrees N) populations, when diapausing instar was held constant. Expression of timeless is therefore both developmentally and evolutionarily variable. This result provides the first example of a latitudinal difference in the expression of timeless, suggesting that, along with evidence from other insects, timeless has the potential to affect photoperiodic response and its adaptive evolution in temperate seasonal environments.

Because mortality accumulates with age, Fisher proposed that the strength of selection acting on survival should increase from birth up to the age of first reproduction. Hamilton later theorized that the strength of selection acting on survival should not change from birth to age at first reproduction. As organisms in nature do not live in uniform environments but, rather, experience periodic stress, we hypothesized that resistance to environmental stress should increase (Fisher) or remain constant (Hamilton) from birth to age at first reproduction. Using the pitcher-plant mosquito, Wyeomyia smithii, we imposed heat stress by simulating the passage of a warm-weather front at different pre-adult and adult stages. Contrary to either Fisher or Hamilton, stress tolerance declined from embryos to larvae to pupae to adults. Consequently, reproductive value appears to have been of little consequence in the evolution of stage-specific tolerance of heat stress in W. smithii.

Photoperiodism, the ability to assess the length of day or night, enables a diverse array of plants, birds, mammals, and arthropods to organize their development and reproduction in concert with the changing seasons in temperate climatic zones. For more than 60 years, the mechanism controlling photoperiodic response has been debated. Photoperiodism may be a simple interval timer, that is, an hourglasslike mechanism that literally measures the length of day or night or, alternatively, may be an overt expression of an underlying circadian oscillator. Herein, we test experimentally whether the rhythmic response in Wyeomyia smithii indicates a causal, necessary relationship between circadian rhythmicity and the evolutionary modification of photoperiodic response over the climatic gradient of North America, or may be explained by a simple interval timer. We show that a day-interval timer is sufficient to predict the photoperiodic response of W. smithii over this broad geographic range and conclude that rhythmic responses observed in classical circadian-based experiments alone cannot be used to infer a causal role for circadian rhythmicity in the evolution of photoperiodic time measurement. More importantly, we argue that the pursuit of circadian rhythmicity as the central mechanism that measures the duration of night or day has distracted researchers from consideration of the interval-timing processes that may actually be the target of natural selection linking internal photoperiodic time measurement to the external seasonal environment.

To date, all altered patterns of seasonal interactions observed in insects, birds, amphibians, and plants associated with global warming during the latter half of the 20th century are explicable as variable expressions of plastic phenotypes. Over the last 30 years, the genetically controlled photoperiodic response of the pitcher-plant mosquito, Wyeomyia smithii, has shifted toward shorter, more southern daylengths as growing seasons have become longer. This shift is detectable over a time interval as short as 5 years. Faster evolutionary response has occurred in northern populations where selection is stronger and genetic variation is greater than in southern populations. W. smithii represents an example of actual genetic differentiation of a seasonality trait that is consistent with an adaptive evolutionary response to recent global warming.

We measured the egg size of six geographic populations of the pitcher-plant mosquito, Wyeomyia smithii, from Florida (30 degrees N) to Ontario (49 degrees N). Populations from northern latitudes produced larger eggs than populations from southern latitudes. Egg size increased with increasing latitude more rapidly when larvae were reared under low rather than high density. One southern (30 degrees N) and one northern (49 degrees N) population of W. smithii that persisted through 10 generations of selection for increased persistence under conditions of chronic thermal- and nutrient-limiting stress (conditions similar to southern rather than northern habitats) produced smaller eggs more rapidly than unselected control lines. However, there were no differences in lifetime fecundity or fertility between control and selected lines. Thus, laboratory evolution in an environment representative of extreme southern latitudes caused evolutionary changes consistent with geographic patterns of egg size. These results implicate temperature as a selective factor influencing the geographic variation of egg size in W. smithii, and demonstrate a novel trade-off in reproductive allocation between egg size and egg maturation time.

We determine the contribution of composite additive, dominance, and epistatic effects to the genetic divergence of photoperiodic response along latitudinal, altitudinal, and longitudinal gradients in the pitcher-plant mosquito, Wyeomyia smithii. Joint scaling tests of crosses between populations showed widespread epistasis as well as additive and dominance differences among populations. There were differences due to epistasis between an alpine population in North Carolina and populations in Florida, lowland North Carolina, and Maine. Longitudinal displacement resulted in differences due to epistasis between Florida and Alabama populations separated by 300 km but not between Maine and Wisconsin populations separated by 2000 km. Genetic differences between New Jersey and Ontario did not involve either dominance or epistasis and we estimated the minimum number of effective factors contributing to a difference in mean critical photoperiod of 5 SD between them as nE = 5. We propose that the genetic similarity of populations within a broad northern region is due to their more recent origin since recession of the Laurentide Ice Sheet and that the unique genetic architecture of each population is the result of both mutation and repeated migration-founder-flush episodes during the dispersal of W. smithii in North America. Our results suggest that differences in composite additive and dominance effects arise early in the genetic divergence of populations while differences due to epistasis accumulate after more prolonged isolation.

Adult longevity and lifetime fecundity, but not fertility, of northern (Maine, USA) Wyeomyia smithii (Coquillet) increase with female pupal weight. Mean egg size does not vary with pupal weight, but the standard deviation in egg size shows a marginally significant increase with pupal weight. Egg sizes are not skewed but are leptokurtic in their distribution; neither skewness nor kurtosis changes with female pupal weight. Mean egg size is not correlated with weight-specific adult longevity or with weight- and longevity-specific lifetime fecundity. Reproductive effort early in adult life does not affect longevity, reproductive effort late in life, or reproductive rate late in life. Finally, there is no significant correlation late in adult life between weight-specific rate of egg production and the size of eggs being produced. Egg size does vary within females but is not a variable entered into the physiological allocation of resources among survivorship, fecundity, or rate of egg production.

Parallel crosses between each of two southern (ancestral) and one northern (derived) population of the pitcher-plant mosquito, Wyeomyia smithii, were made to determine the genetic components of population divergence in critical photoperiod, a phenological trait that measures adaptation to seasonality along a climatic gradient. Joint scaling tests were used to analyze means and variances of first- and second-generation hybrids in order to determine whether nonadditive genetic variance, especially epistatic variance, contributed to divergence in critical photoperiod. In both crosses, digenic epistatic effects were highly significant, indicating that genetic divergence cannot have resulted solely from differences in additively acting loci. For one cross that could be tested directly for such effects, higher order epistasis and/or linkage did not contribute to the divergence of critical photoperiod between the constituent populations.

Third and fourth instars of Wyeomyia smithii were reared in Giemsa stain at 4 concentrations between 4 x 10(-7) and 10(-5) g/liter. The mosquitoes retained the blue mark as adults and remained marked throughout their laboratory life. Concentration of Giemsa significantly affected eclosion success but had no significant effect on mean days to pupation or days as a pupa, male or female adult longevity, per-capita female fecundity or fertility. Larval exposure to low concentrations (4 x 10(-7) or 10(-6) g/liter) of Giemsa stain provides an effective lifetime tag for otherwise indistinguishable laboratory populations.

1. Unchilled, diapausing larvae of Toxorhynchites rutilus rely on photoperiod for the maintenance of diapause. The photoperiodic clock is temperature-compensated between 16.5 degrees and 25 degrees C, maintaining both a similar set-joint and inherent accuracy over this range. The rates of development among larvae terminating diapause are dependent upon both temperature and photoperiod. 2. Chilling of dormant Toxorhynchites rutilus can promote response to progressively shorter daylengths, thus decreasing the critical photoperiod. Chilling can also accelerate response to long days, thereby decreasing the depth of diapause and, after prolonged exposure, can eventually terminate diapause directly, leaving subsequent morphogenesis independent of photoperiod. 3. The optimal temperature for these effects of chilling is above 4 degrees C, below 16.5 degrees C, and may lie around 7 degrees C. 4. Temperatures between 5 degrees and 15 degrees C are vernal and autumnal rather than hibernal. The interaction between chilling and photoperiod may then represent an adaptive compromise between selection due to long-term climatic trends and the vagaries of spring weather.

The National Ecological Observatory Network ( NEON) intends to monitor mosquito populations across its broad geographical range of sites because of their prevalence in food webs, sensitivity to abiotic factors, and relevance for human health. We describe the design of mosquito population sampling in the context of NEON's long-term continental scale monitoring program, emphasizing the sampling design schedule, priorities, and collection methods. Freely available NEON data and associated field and laboratory samples, will increase our understanding of how mosquito abundance, demography, diversity, and phenology are responding to land use and climate change. [ABSTRACT FROM AUTHOR]