CeleST reveals novel information on aging phenotypes.

<p><b>A–J</b>, Age-associated changes in swimming parameters in wild-type adults. <b>A</b>, Wave initiation rate; <b>B</b>, Body wave number; <b>C</b>, Asymmetry; <b>D</b>, Stretch; <b>E</b>, Attenuation; <b>F</b>, Reverse swimming; <b>G</b>, Curling; <b>H</b>, Travel speed; <b>I</b>, Brush stroke; and <b>J</b>, Activity index. from 9 independent trials, for each age day 4 and day 11. Data for ages ranging from day 4 to day 20 are presented in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s004" target="_blank">Figure S4</a>. <b>K–M</b>, CeleST reports great differences in graceful agers vs. poor agers for measures that change with age. We selected animals that appeared to have robust crawling capacity (Class A, graceful agers) and those that had decrepit crawling capacity (Class C, poor agers) at day 11 and compared swim behavior. <b>K</b>, Activity index; <b>L</b>, Asymmetry; <b>M</b>, Attenuation. from 3 independent trials, for each class. Data for all ten measures in this series are shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s005" target="_blank">Figure S5</a>. <b>N</b>, Locomotory changes under life-extending and progeric insulin signaling pathway manipulation suggest complex influences of signaling over the lifetime. Activity index, WT: blue line (middle dashed line); long lived <i>age-1(hx546)</i>: green (top line); short-lived <i>daf-16(mgDf50)</i>: red (bottom line). Note that the <i>age-1</i> mutant has a higher activity index in young adult life as compared to WT, which suggests differences in swim performance are not limited to aging. Also, at day 15, WT and <i>age-1</i> scores appear increased, which we suggest reflects the preferential death of the poorest swimmers, rather than an actual increase in average swimming of individuals. in each data point from 4 independent trials. Data on all measures are presented in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s006" target="_blank">Figure S6</a>. Error bars show SEM, *** , **** .</p

Summary of CeleST components and usage.

<p>Input files are videos of multiple swimming <i>C. elegans</i>. Files are stored in a database that records identifying features (strain, date, etc.) to permit easy selection of animals to be compared by analysis. After selection of animals to be compared, swimmers are automatically tracked from videos, and computation from curvature data or posture is used to score ten swim measures in 30 second swim trials (see description in text). Measures from the scored animals are compiled and can be exported in several alternative data analysis formats, including dot plots, line graphs, histograms and two dimensional comparison (ellipses indicate the principal directions and the standard deviations of the data). Statistical analysis is automated. A dynamic demonstration of CeleST tracking and computing of measures can be found in Video S1 1–4 on <a href="http://celest.mbb.rutgers.edu" target="_blank">http://celest.mbb.rutgers.edu</a>.</p

Examples of ten CeleST measure outputs for an individual <i>C. elegans</i> swim trial.

<p>All measures reflect analysis of a 30/sec, with <b>C–G</b> calculated from analysis of radius of curvature over 12 body segments (curvature plot vs. time example is in <b>B</b>). For <b>C–G</b> and <b>M–O</b>, instantaneous values are plotted in black; the median value for each swim is drawn in red, and the 10–90 percentile range of values is shown in gray; median and range over the 30 s trial are listed on the right. Note that although this panel demonstrates analysis of measurements of a single animal swimming, the CeleST program score multiple animals simultaneously and can compile data from thousands of individual swim trials (examples in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi-1003702-g003" target="_blank">Figure 3</a>). <b>A</b>, Scored animal at three indicated time points in the video, with the curvature measure superimposed on the body; color key shown in <b>B</b>. <b>B</b>, Curvature heat map of an individual swim trial. Map is of curvature at a given body point (Y axis) as a function of time (X axis), with head curvature score at the top and tail curvature at the bottom on the Y axis, deep bend in one direction dark blue, and in the other direction dark red. Lines indicate posture of the animal depicted at that time point in panel <b>A</b>. Note that the posture of an individual at any point in time could be reconstructed from the measure of curvature over body position. Further details on each parameter measurement <b>C–O</b> are given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s009" target="_blank">Text S1</a>. Informative videos (Videos S2 01–10) that feature extreme examples for each measure can be found on <a href="http://celest.mbb.rutgers.edu" target="_blank">http://celest.mbb.rutgers.edu</a>.</p

CeleST analysis reveals features of <i>C. elegans</i> swimming considerable individuality, gait preference, and reverse swimming.

<p><b>A–C</b>, <i>C. elegans</i> exhibit diverse swimming abilities, despite genetic and environmental homogeneity. CeleST can plot scores for two parameters against each other, for example: <b>A</b>, Travel speed vs. Asymmetry; <b>B</b>, Body wave number vs. Activity index; <b>C</b>, Brush stroke vs. Stretch. Data for WT 4-day old animals from 9 independent trials are plotted. <b>D</b>, <i>C. elegans</i> swim at specific wave initiation rates. We plotted in the form of line histograms the distribution of median Wave initiation rates (WIR) in wild-type animals as occurs over a 30 second interval. WIR values are binned to integers and the plot line delineates the contour of the bins in the histogram. X axis is median WIR, Y axis is the number of individuals exhibiting the indicated WIR. Data in this panel are combined to represent 3,372 animals ranging from 4 to 20 days old from 9 independent trials to emphasize the peaked distribution. Although animals in this large population do swim over the range of possible <u>median</u> WIRs (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s001" target="_blank">Figure S1F</a> for example), CeleST analysis reveals an unexpected bias for particular “gaits” in a subset of the population (about 14% total appear in favored WIRs). Older animals swim at lower median WIRs than young adult animals, but the preferred WIRs remain. WIR distributions for specific individual ages are depicted in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s001" target="_blank">Figure S1</a>. Note that mean WIR rates do not exhibit a distribution bias (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s001" target="_blank">Figure S1F</a>), so this study emphasizes the value of also considering median scores in swim behavioral analysis. <b>E–G</b>, For brief periods, swimming animals reverse, with the tail initiating the body wave. Reverse swimming is illustrated in Videos S3 and S4 on <a href="http://celest.mbb.rutgers.edu" target="_blank">http://celest.mbb.rutgers.edu</a>. In 4-day old animals, the <i>glr-1(ky176)</i> mutant, lacking a neuronal glutamate receptor, reversal frequency is increased relative to WT (<b>E</b>), although the trend to increased time spent in reverse is not statistically significant () (<b>F</b>). Unexpectedly, <i>glr-1</i> mutants swim more symmetrically than WT at day 4 (<b>G</b>). from 3 independent trials for each strain. Data for all 10 measures, young and old age are shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s002" target="_blank">Figure S2</a>. Error bars show SEM, **** .</p