The Molecular Clockwork of the Fire Ant Solenopsis invicta

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Schematic models for Clockwork Orange (CWO) proteins from various insects.

<p>The depicted models are for the fire ant <i>Solenopsis invicta</i> (siCWO), the honey bee <i>Apis mellifera</i> (amCWO), the fruit fly <i>Drosophila melanogaster</i> (dmCWO), Red Flour Beetle <i>Tribolium castaneum</i> (tcCWO). Also shown are related proteins from the house mouse <i>Mus musculus</i> (mmDEC2) and zebrafish <i>Danio rerio</i> (drDEC2). Highlighted areas on diagrams represent putative functional domains and motifs. For more details see legend to Fig. 3. Inset shows a CLUSTALW multiple sequence alignment of a new conserved domain discovered on the CWO protein sequence that we termed 'Clockwork Orange C-tail Domain' (CWOCD) the CLUSTALW alignment includes several additional CWO proteins from drosophilid and ant species (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045715#pone.0045715.s005" target="_blank">Table S3</a>). Asterisks in the bottom of alignment indicate amino acids conserved between insects and vertebrates. Alignments were generated with CLUSTALW and colored with JalView according to the default CLUSTALX convention. • bHLH - Basic-helix-loop-helix. Proteins containing this domain are typically dimeric transcription factors, each with one helix containing basic amino acid residues that facilitate DNA binding to an E-box. (Pfam domain accession number PF00010.). • CWOCD - Clockwork orange C-tail domain. • Hairy Orange - The Orange domain is found in the <i>Drosophila</i> proteins Hesr-1, Hairy, and Enhancer of Split. The Orange domain is proposed to mediate specific protein-protein interaction that confers specificity among members of the Hairy/E(SPL) family. • PCRs - regions of targeted PCR for confirmation of mRNA sequence.</p

Summary representation of oscillation in mRNA for five core clock genes in fire ant brains under LD illumination.

<p>Shown are four genes (<i>SiPer, SiCry, SiCwo,</i> and <i>SiCyc)</i> that show significant oscillations and have significant correlations to the cosine model with R²adj≥0.5, and one gene, <i>SiClk</i>, which does not oscillate. Lines represent schematic cartoons of the actual oscillations. <i>SiPer</i> and <i>SiCry</i> cycle in the same phase and peak during the night and <i>SiCwo</i> and <i>SiCyc</i> cycle antiphase to <i>SiPer</i> and peak during the day. Not shown on the figure are the two transcription factors, <i>SiVri</i> and <i>SiPdp1</i>; <i>SiTim</i> was excluded because the expression of this gene was not consistent across nests.</p

Parsimony tree for <i>clockwork orange</i> orthologs.

<p>Shown is a consensus tree with bootstrap values from 250 replicates. Insect CWO orthologs clearly separate from mammalian orthologs <i>Dec1</i> & <i>Dec2</i>.</p

Schematic models for CRY proteins from various animals.

<p>The protein models depicted are from the fire ant <i>Solenopsis invicta</i> (siCRY), the Western honey bee <i>Apis mellifera</i> (amCRY-m), the fruit fly <i>Drosophila melanogaster</i> (dmCRY), the Monarch butterfly <i>Danaus plexippus</i> (dpCRY1) the jewel wasp <i>Nasonia vitripennis</i> (nvCRY) and the domestic mouse <i>Mus musculus</i> (mmCRY1). Highlighted areas on the diagrams represent putative functional domains and motifs. The numbers below the domains indicate percents of identity/similarity to corresponding sequences on the protein of the fire ant. The numbers at the end of each diagram indicate the predicted protein size (number of amino acid residues). The protein domains on the CRY sequence are:. • FAD binding. Proteins containing this domain are photolyases (DNA repair enzymes) or function as blue light photoreceptors (Pfam domain accession number: PF03441). • DNA photolyase. This domain is an evolutionary conserved protein domain from bacteria to mammals. It binds to UV-damaged DNA containing pyrimidine dimers and, upon absorbing a near-UV photon 300 to 500 nm, breaks the cyclobutane ring joining the two pyrimidines of the dimer (Pfam domain accession number: PF00875). • ICAT - Inhibition CLOCK-ARNTL Transcription. A domain required for the inhibition of CLOCK-ARNTL-mediated transcription (Swiss-Prot record of mmCRY1 accession number: P97784). • RD-2b – A domain defined by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045715#pone.0045715-Hirayama1" target="_blank">[58]</a> based on studies with the clock proteins of the zebrafish. The domain is necessary for nuclear localization and the repression of CLOCK: BMAL-mediated transcription. • NLS - Nuclear localization signal in the RD-2b region, following <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045715#pone.0045715-Hirayama1" target="_blank">[58]</a>. • EST - Expressed sequence tags.</p

Relative gene expression patterns of 8 principle clock genes across colonies.

<p>Expression values for each data point (±SE) are plotted as the average relative expression level (ΔΔCt) across colonies (n = 3 colonies). Relative expression is calculated as the number of standard deviations above and below the mean value for all data points (across time). Standard error bars are calculated from variation across three colonies. The open stripe in the horizontal bar at base of the plot represents the daylight phase (12 hrs) and the solid stripe represents the dark phase (12 hrs) during the night. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045715#pone-0045715-t001" target="_blank">Table 1</a> for additional details on clock gene expression.</p

Summary of gene expression data.

*<p>Values not consistent across nests.</p>†<p>p-values in <b>bold</b> are significant at α = 0.05.</p>§<p>Amplitudes calculated from peak/trough values of cosine model.</p