Expression profiles of <i>Td'HR3</i> and <i>Td'E75</i> mRNA and effects of their dsRNAs on their mRNA levels and the survival rate in firebrats.

<p>Both <i>Td'HR3</i> (A, C) and <i>Td'E75</i> (B, D) were rhythmically expressed in LD (A, B) and DD (C, D), peaking late (subjective) day to early (subjective) night (black symbols). Ds<i>HR3#1,2</i> (pink triangles and squares for #1 and #2, respectively) and ds<i>E75#1,2</i> (green triangles and squares for #1 and #2, respectively) downregulated mRNA levels and disrupted the rhythm of their respective genes (A and B). Treatment with ds<i>DsRed2</i> as a negative control did not disrupt the rhythmic expression in both genes (A and B, blue symbols), but caused a significant decrease in <i>Td'HR3</i> mRNA levels at ZT 2 and ZT 18 (+ <i>P</i><0.05, <i>t</i> test vs intact). White, black, and gray bars above each graph indicate day, night/subjective night, and subjective day, respectively. Total RNA was extracted from firebrats collected at 4-h intervals starting 2 h after lights on or 2 h into subjective day (ZT2 or CT2, respectively). The data collected from 3–4 independent experiments were averaged and plotted as the mean ± SEM values relative to the value of <i>rp49</i> mRNA used as the reference. Values with different letters significantly differ from each other within the same treatment groups (<i>P</i><0.05, ANOVA with Tukey test, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114899#pone.0114899.s001" target="_blank">S1 Table</a>). *<i>P</i><0.05, **<i>P</i><0.01, <i>t</i> test vs ds<i>DsRed2</i> (exact <i>P</i> values are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114899#pone.0114899.s002" target="_blank">S2 Table</a>). (E) Survival rate after injection of 10.23–13.95 µg/g ds<i>HR3</i>, ds<i>E75</i>, or ds<i>DsRed2</i> into the abdomen. Ds<i>HR3</i> and ds<i>E75</i> significantly reduced the survival rate. For further explanation, see text.