List of ANOVA results for Figure 3.

<p>The ANOVA table shows that at least one factor was significantly different for each sleep parameter. Results from individual Tukey pairwise comparisons are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037250#pone-0037250-g003" target="_blank">Figure 3</a>. All analyses were significant at DF_(3,115), <i>P</i><0.05, DAM Control (N = 30), DAM <i>CASK-β</i> (N = 30), Track Control (N = 29), Track <i>CASK-β</i> (N = 30).</p

List of ANOVA results for Figure 2.

<p>The ANOVA table shows that at there was a significant difference for each analysis type (DAM, Virtual Beam, 20%, 50%, and 100% FBL) at each time period (24, LP, or DP) except for Mean Sleep Episode Duration LP. Results from individual Tukey pairwise comparisons are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037250#pone-0037250-g002" target="_blank">Figure 2</a>. All analyses except Mean Sleep Episode Duration during LP were significant at DF_(4,30), <i>P</i><0.05, N = 7 for all analysis types.</p

Examples of descriptive statistics that can be gleaned from Tracker data that could not be determined from standard beam cross data (Track <i>CASK-β</i> N = 30, Track Control N = 29).

<p>Examples of descriptive statistics that can be gleaned from Tracker data that could not be determined from standard beam cross data (Track <i>CASK-β</i> N = 30, Track Control N = 29).</p

Location plots show that <i>CASK-β</i> mutant flies have altered place preference compared with controls.

<p>Location plots show the place preference of the wild type control and <i>CASK-β</i> mutant flies over the course of the experiment. Place preference was determined by recording the location of the flies within their tubes, and calculating the amount of time spent in each location as a function of time of day. Here, the fly locations were binned every minute of the experiment. (A) Female wild type control (Control, N = 29) flies have a periodic rhythmicity to place preference. (B) The female <i>CASK-β</i> mutants (<i>CASK-β</i>, N = 30) have altered place preference compared with control flies, and show longer durations spent in the center of the tube. Color intensity indicates longer dwell time for that location. Each individual vertical segment represents 1 min of the experiment. The tube was also divided into 10 equal sized sections (C). The food was always located just above section 10. White color in the time bar = daytime. Grey color in the time bar = night.</p

Sleep data capture comparison for DAM and Tracker sleep data.

<p>Sleep parameters from DAM and Tracker analysis for flies averaged for three days, DAM Control (N = 30), DAM <i>CASK-β</i> (N = 30), Track Control (N = 29), Track <i>CASK-β</i> (N = 30). Data from 3–5 day old female flies were collected for three consecutive days in a 25<b>°</b>C incubator with 12 hr LD cycle. Same letters indicate no significant difference between methods using Tukey HSD (<i>P</i><0.05). A separate Tukey test was run for each time comparison (24, LP, and DP). (A) Sleep profiles show higher sleep in the mutant line recorded by DAM. (B) Total sleep duration shows lower sleep at night and higher sleep during the night in the Tracker data as compared to control. (C) The number of sleep episodes was not significantly different in Tracker data. (D) Maximum sleep episode duration did not differ across methods, but was significantly different between genotypes. (E) DAM did not identify a difference in Mean sleep episode duration total. The Tracker program detected a difference at 24, LP, and DP. (F) DAM was not able to detect the locomotor deficiency in the <i>CASK-β</i> mutant. (G) Tracker data shows a greater latency difference between the genotypes in LP and DP. (H) The DAM and Tracker data show opposing results in mean wake duration depending on the time of day. DAM shows no difference in the LP, while Tracker data shows no difference in DP.</p

Control flies move much greater distances than <i>CASK-β</i> mutant flies and have a more dynamic locomotor pattern.

<p>Distance was averaged for each genotype and binned into minutes. (A) Female wild type control flies (Control, N = 29) had stereotypic locomotion across the day. (B) Female <i>CASK-β</i> mutant flies (<i>CASK-β</i>, N = 30) had a locomotor profile that was different from control. Nighttime movements were very low, with no morning light anticipation. (C) Speed plot for each genotype. (Green = control, Pink = <i>CASK-β</i>). The speed of the mutant fly followed closely with control only in the hours leading up to lights out. (D) All flies did not behave exactly the same all the time. Behavioral state was intrinsic to the individual. % Activity shows that control flies alternated their sleep/wake activity so that at least one fly was active all throughout the day. There was an overall sleep rhythm, but some individuals were awake while others were asleep. By contrast, there were long periods where all <i>CASK-β</i> mutant flies were not moving at night.</p

<i>CASK-β</i> mutant flies have different location preferences from controls when both asleep and awake.

<p>Location and Proportion of Occupancy plots show where the wild type control (Control, N = 29) and <i>CASK-β</i> mutant (<i>CASK-β</i>, N = 30) flies were when they were awake and asleep. (A, B) Female wild type control flies sleep near the food during siesta and at night and rarely venture to the half of the tube away from the food. (C) Wild type control flies show preference for being at the food when awake. The flies moved throughout the entire length of the tube, pausing at either end. This location preference coincides with peak activity times of day. (D) When awake, Control flies spent ∼24% of their time at the food. The rest of their waking time was dispersed throughout the tube. (E, F) Female <i>CASK-β</i> mutants sleep at different times and locations than the controls and do not have siesta. When the <i>CASK-β</i> mutants did sleep, they were dispersed throughout sections 4–9 of the tube. (G, H) When awake, the <i>CASK-β</i> mutants showed little location consolidation across the day as compared to control and spent the majority of their waking time at the food (∼22%). The rest of their time was spent dispersed throughout the rest of the tube, with some preference for the far end.</p

Data capture comparison for <i>CS</i> flies shows how restricting Tracker output conforms location data to DAM-like outputs.

<p>Sleep parameters from DAM and Tracker program analysis for <i>CS</i> wild type flies (N = 8). Data from 3–5 day old male flies were collected for three consecutive days in a 25<b>°</b>C incubator with 12-hr LD cycle. Same letters indicate no significant difference between methods using Tukey HSD (<i>P</i><0.05). NS = No Significant difference. Individual statistical tests were performed on each time comparison group (24, LP, DP). The order of letters represents the order of the analysis: DAM, Virtual Beam, Track 20%, Track 50%, Track 100% of the Fly Body Length (FBL). (A) Sleep profile for flies averaged for three days. (B, C, D, E, F) DAM data matches the Virtual Beam for all comparisons. Track 100% FBL matched DAM for all comparisons as well, but was also not significantly different from 50% FBL in many comparisons. 20% FBL was always significantly different from DAM. The 50% FBL showed significant differences from DAM in many comparisons, while still showing similarity, indicating that this resolution was most effective at capturing both DAM-insensitive and biologically meaningful locomotion.</p