Statistical tests for the significance of the power-law property in the distributions of rest and activity bouts according to the method of Clauset et al. [20].

<p>All the tests were separately conducted for the data obtained from individual flies. The mean statistical values for different flies are shown in the table. For each empirical distribution, we calculated the <i>p′-</i>value for the best power-law fit and likelihood ratios (LRs) of the power-law distribution to alternative distributions. We used the exponential distribution and the log-normal distribution as the alternative distributions. Positive values of the log likelihood ratio with p<0.05 indicate that the power-law distribution is statistically favored over the alternative distribution.</p

Impairment of cold avoidance in <i>dumb²</i> mutants.

<p>A. Distribution of flies along a thermal gradient after 25 min exposure to thermal gradient. B. Dynamics of temperature preference in control flies. C. Dynamics of temperature preference in <i>fmn</i> flies. D. Dynamics of temperature preference in <i>dumb²</i> flies.</p

Effects of circadian rhythm.

<p>a. Double logarithmic plots of the cumulative probability of the rest bout for the combined data of control male flies (n = 10). blue: subjective day. red: subjective night. b. Double logarithmic plots of the cumulative probability of the activity bout for the combined data of control male flies (n = 10). blue: subjective day. red: subjective night. c. The ratio of the total duration of the activity episodes. d. Power-law exponent of the fitted distribution of rest bouts. e. Mean activity bout length. Bars and error bars represent the mean and s.e.m., respectively. Asterisk indicates statistically significant difference (p<0.05 Student's <i>t</i> test).</p

dDAT expression in dopamine neurons rescues low-temperature preference phenotype of <i>fmn</i> mutants.

<p>A. Distribution of flies along a thermal gradient after 25 min exposure to thermal gradient. B. Preferred temperature of <i>fmn</i> mutants; <i>fmn</i> ; TH-GAL4 (<i>n</i> = 230), <i>fmn</i> UAS-dDAT ; TH-GAL4 (<i>n</i> = 242) and <i>fmn</i> UAS-dDAT (<i>n</i> = 245) after 25 min exposure to thermal gradient. In each box plot, the box encompasses the interquartile range, a line represents the median, and the error bars encompass the 10th and 90th percentiles. Asterisk indicates a statistically significant difference (Kruskal-Wallis test; <i>p</i><0.001) C. Dynamics of temperature preference in <i>fmn</i> ; TH-GAL4. D. Dynamics of temperature preference in <i>fmn</i> UAS-dDAT ; TH-GAL4. E. Dynamics of temperature preference in <i>fmn</i> UAS-dDAT.</p

<i>fmn</i> mutants showed increased cold tolerance due to enhanced dopamine signaling.

<p>A. Resistance to cold stress, which was tested by DAM assay with decreasing temperature. Solid line indicates flies fed with vehicle, dashed line indicates flies fed with 3IY. (n = 61–64) B. Resistance to cold stress with genetic rescue. (n = 39–45).</p

Dependence of the mean bout on the bin width.

<p>The degree of movement was summed up in each bin of the specified width. Then, we classified the bins of the specified width to rest or activity using the <i>k</i>-means clustering algorithm. The data analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032007#pone-0032007-g002" target="_blank">Figure 2a, b</a> were reexamined with different bin widths. a. Mean rest bout for different bin widths. Black: control. Red: <i>fmn</i>. b. Mean activity bout for different bin widths. Black: control. Red: <i>fmn</i>. Error bars represent s.e.m.</p

The dopamine biosynthesis inhibitor, 3-iodo-L-tyrosine, raised the preferred temperature both in control and <i>fmn</i> flies.

<p>A. Distribution of flies along a thermal gradient after 25 min exposure to thermal gradient. B. Preferred temperature of control flies fed with vehicle (<i>n</i> = 203), control flies fed with 3IY (<i>n</i> = 207), <i>fmn</i> flies fed with vehicle (<i>n</i> = 201) and <i>fmn</i> mutants fed with 3IY (<i>n</i> = 187) after 25 min exposure to thermal gradient. In each box plot, the box encompasses the interquartile range, a line represents the median, and the error bars encompass the 10th and 90th percentiles. Asterisk indicates a statistically significant difference (Kruskal-Wallis test; <i>p</i><0.001) C. Dynamics of temperature preference in control flies fed with vehicle. D. Dynamics of temperature preference in control flies fed with 3IY. E. Dynamics of temperature preference in <i>fmn</i> flies fed with vehicle. F. Dynamics of temperature preference in <i>fmn</i> flies fed with 3IY.</p

<i>fmn</i> mutants showed increased energy metabolism due to enhanced dopamine signaling.

<p>A. Metabolic rates as measured by CO₂ production (<i>n</i> = 12 for each genotypes). Data are represented as mean ± SEM. Asterisks indicate statistically significant differences (Student's <i>t</i>-test; <i>p</i><0.05). B. Metabolic rates as measured by CO₂ production without locomotion (<i>n</i> = 8 for each genotypes). Data are represented as mean ± SEM. Asterisks indicate statistically significant differences (Student's <i>t</i>-test; <i>p</i><0.05). C. Survival curves of control and <i>fmn</i> flies on starvation (<i>n</i> = 64 for each genotype). D. Mean survival time of control and <i>fmn</i> flies during starvation. Data are represented as mean ± SEM. Asterisks indicate statistically significant differences (Student's <i>t</i>-test; <i>p</i><0.05). E. Survival curves of <i>fmn</i>; TH-GAL4, <i>fmn</i> UAS-dDAT; TH-GAL4, and <i>fmn</i> UAS-dDAT flies on starvation (<i>n</i> = 64 for each genotype). F. Mean survival time of <i>fmn</i> ; TH-GAL4, <i>fmn</i> UAS-dDAT ; TH-GAL4, and <i>fmn</i> UAS-dDAT flies during starvation. Data are represented as mean ± SEM. Asterisks indicate statistically significant differences (one-way ANOVA followed by the Tukey-Kramer <i>post hoc</i> test; <i>p</i><0.05).</p

<i>fmn</i> mutant flies show a preference for lower temperatures than control flies.

<p>A. Temperature gradient of the apparatus. Actual temperature readout from thermocouples placed under the aluminum chamber indicating that a linear temperature gradient from 10 to 35°C was established. The temperatures were monitored simultaneously during the assay. B. Picture of a temperature preference assay after 25 min exposure to thermal gradient. Flies are reflected as white dots. Upper two lanes: control flies. Lower two lanes: <i>fmn</i> flies. Temperatures of horizontal positions are indicated. C. Quantified results showing the distributions of flies along the thermal gradient after 25 min exposure to thermal gradient. D. Preferred temperature calculated from the location of the control (<i>n</i> = 599) and <i>fmn</i> (<i>n</i> = 546) flies after 25 min exposure to thermal gradient. In each box plot, the box encompasses the interquartile range, a line represents the median, and the error bars encompass the 10th and 90th percentiles. Asterisk indicates a statistically significant difference (Mann–Whitney U test; <i>p</i><0.001). E. Dynamics of temperature preference in control flies. F. Dynamics of temperature preference in <i>fmn</i> flies.</p

Effect of transient dopamine neuron activation.

<p>a. Double logarithmic plots of the cumulative probability of the rest bout for the combined data of TH-GAL4/UAS-dTrpA1 flies(n = 10). blue: 22°C. red: 29°C. b. Double logarithmic plots of the cumulative probability of the activity bout for the combined data of TH-GAL4/UAS-dTrpA1 flies(n = 10). blue: 22°C. red: 29°C. c. The ratio of the total duration of the activity episodes. d. Power-law exponent of the fitted distribution of rest bouts. e. Mean activity bout length. Bars and error bars represent the mean and s.e.m., respectively. Asterisks indicate statistically significant difference (p<0.05 Student's <i>t</i> test).</p