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

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

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

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

Gonadotropic and Physiological Functions of Juvenile Hormone in Bumblebee (<i>Bombus terrestris</i>) Workers

<div><p>The evolution of advanced sociality in bees is associated with apparent modifications in juvenile hormone (JH) signaling. By contrast to most insects in which JH is a gonadotropin regulating female fertility, in the highly eusocial honey bee (<i>Apis mellifera</i>) JH has lost its gonadotrophic function in adult females, and instead regulates age-related division of labor among worker bees. In order to shed light on the evolution of JH signaling in bees we performed allatectomy and replacement therapies to manipulate JH levels in workers of the "primitively eusocial" bumblebee <i>Bombus terrestris</i>. Allatectomized worker bees showed remarkable reduction in ovarian development, egg laying, <i>Vitellogenin</i> and <i>Krüppel homolog 1</i> fat body transcript levels, hemolymph Vitellogenin protein abundance, wax secretion, and egg-cell construction. These effects were reverted, at least partially, by treating allatectomized bees with JH-III, the natural JH of bees. Allatectomy also affected the amount of ester component in Dufour's gland secretion, which is thought to convey a social signal relating to worker fertility. These findings provide a strong support for the hypothesis that in contrast to honey bees, JH is a gonadotropin in bumblebees and lend credence to the hypothesis that the evolution of advanced eusociality in honey bees was associated with major modifications in JH signaling.</p></div

The influence of allatectomy on hemolymph JH titers.

<p>Bumblebee workers receiving the same treatment were housed in groups of three in small cages without a queen. JH titers were determined at the age of 7 days with a JH-III specific radioimmunoassay. Shown are mean ± SE, and sample size within or above the bars. The p-value summarizes the results of one-way ANOVA; groups with different letters differ significantly in a LSD post-hoc test (p<0.05). CA-  =  allatectomized bees; Sham  =  sham operated bees, Control  =  control bees.</p

The influence of JH on vitellogenin levels.

<p><b>A.</b> Vitellogenin (<i>Vg</i>) transcript abundance in the fat body (mean ± SE; sample size within bars). The p-value summarizes the results of one-way ANOVA; groups with different letters differ significantly in a LSD post-hoc test (p<0.05). <b>B</b>. VG protein levels in the hemolymph. The upper blot shows immunostaining with an antibody directed against the <i>Apis mellifera</i> VG protein. The lower panel shows non-specific protein staining of the same blot with Ponceau S. Each column contains data from a single bee. For additional details see legends to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100650#pone-0100650-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100650#pone-0100650-g002" target="_blank">2</a>.</p

The influence of JH on fat body <i>Kr-h1</i> mRNA levels.

<p>For plot details see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100650#pone-0100650-g003" target="_blank">Fig. 3A</a>.</p

The influence of allatectomy on ester amounts in the Dufour's gland secretion.

<p><b>A.</b> Total amount of secretion. <b>B</b>. The relative amounts of esters out of the Dufour's gland secretion. The Dufour's gland secretion was analyzed at the age of 7 days using gas chromatography/mass spectrometry (GC/MS). Shown are the mean ± SE, sample size within bars. The p-value summarizes the results of one-way ANOVA; groups with different letters differ significantly in a LSD post-hoc test (p<0.05).</p