Project description: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.

Project description:Gene expression of two populations was compared at a daylength intermediate to the two population critical photoperiods after 0 and 6 days. Genes with interaction terms are candidates for involvement in long-day response. 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 covert transcriptional events involved in the termination of diapause following exposure to long-days. We incorporate a novel approach that compares two populations (DB - southern, DR - northern) that differentially respond to a single day length. After six long days, 50% of individuals of population DB have terminated diapause and are irrevocably committed to development, though no phenotyping differences are observed. We can compare gene expression between these two populations after six long days and zero long days. The zero long day treatment will control for evolved differences between the populations and the 6 long day treatment allows for the identification of genes that are differentially expressed due to different responses to a single daylength. We identify 30 transcripts associated with differential response to day length. All of the corresponding genes with a previously annotated function are consistent with a role in the termination of diapause, with downstream developmental events, or with the transition from potentially oxygen-poor to oxygen-rich environments; none appears to be specifically part of the photoperiodic switch mechanism itself. However, among 10 unannotated genes, a gene homologous to Drosophila melanogaster CG13043 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 (prg1). Prg1 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. Three independent forward genetic approaches identify prg1 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.

Project description:Gene expression of two populations was compared at a daylength intermediate to the two population critical photoperiods after 0 and 6 days. Genes with interaction terms are candidates for involvement in long-day response. 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 covert transcriptional events involved in the termination of diapause following exposure to long-days. We incorporate a novel approach that compares two populations (DB - southern, DR - northern) that differentially respond to a single day length. After six long days, 50% of individuals of population DB have terminated diapause and are irrevocably committed to development, though no phenotyping differences are observed. We can compare gene expression between these two populations after six long days and zero long days. The zero long day treatment will control for evolved differences between the populations and the 6 long day treatment allows for the identification of genes that are differentially expressed due to different responses to a single daylength. We identify 30 transcripts associated with differential response to day length. All of the corresponding genes with a previously annotated function are consistent with a role in the termination of diapause, with downstream developmental events, or with the transition from potentially oxygen-poor to oxygen-rich environments; none appears to be specifically part of the photoperiodic switch mechanism itself. However, among 10 unannotated genes, a gene homologous to Drosophila melanogaster CG13043 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 (prg1). Prg1 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. Three independent forward genetic approaches identify prg1 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. 4 treatments, (Population DR, Day 0; Population DB, Day 0; Population DR, Day 6, Population DB, Day6), one dye swap (treatment Cy3 or Cy5, each replicated three times for a total of 24 arrays

Dataset Information

Quantitative trait loci associated with photoperiodic response and stage of diapause in the pitcher-plant mosquito, Wyeomyia smithii.

Publications

Quantitative trait loci associated with photoperiodic response and stage of diapause in the pitcher-plant mosquito, Wyeomyia smithii.

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