title: Evolutionary divergence of core and post-translational circadian clock genes in the pitcher-plant mosquito, Wyeomyia smithii.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Ecology and Evolution, University of Oregon, Eugene, OR, USA.|Stowers Institute for Medical Research, Kansas City, MO, USA.
authors.forename: Duncan
authors.initials: D
authors.lastname: Tormey
authors.affiliation: Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN, USA.|School of Biosciences, University of Birmingham, Birmingham, UK.
authors.forename: John K
authors.initials: JK
authors.lastname: Colbourne
authors.affiliation: Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN, USA.|Pervasive Technology Institute, Indiana University, Bloomington, IN, USA.
authors.forename: Keithanne
authors.initials: K
authors.lastname: Mockaitis
authors.affiliation: Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN, USA.|GRU Cancer Center, Georgia Regents University, Augusta, GA, USA.
authors.forename: Jeong-Hyeon
authors.initials: JH
authors.lastname: Choi
authors.affiliation: Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN, USA.|Department of Biological Sciences, Notre Dame University, Notre Dame, IN, USA.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Lopez
authors.affiliation: Institute of Ecology and Evolution, University of Oregon, Eugene, OR, USA.|Burke E. Porter Machinery, Grand Rapids, MI, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: Burkhart
authors.affiliation: Institute of Ecology and Evolution, University of Oregon, Eugene, OR, USA. mosquito@uoregon.edu.
authors.forename: William
authors.initials: W
authors.lastname: Bradshaw
authors.affiliation: Institute of Ecology and Evolution, University of Oregon, Eugene, OR, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Holzapfel
mesh_terms: D000818:Animals; D057906:Circadian Clocks / Q000235:genetics*; D009033:Culicidae / Q000235:genetics*; D019143:Evolution, Molecular; D014644:Genetic Variation; D058977:Molecular Sequence Annotation; D017440:Photoperiod; D010802:Phylogeny; D012621:Seasons; D016415:Sequence Alignment; D059467:Transcriptome / Q000235:genetics*