Optimized criteria for locomotion-based healthspan evaluation in C. elegans using the WorMotel system

Abstract

Getting a grip on how we may age healthily is a central interest of biogerontological research. To this end, a number of academic teams developed platforms for life- and healthspan assessment in Caenorhabditis elegans. These are very appealing for medium- to high throughput screens, but a broader implementation is lacking due to many systems relying on custom scripts for data analysis that others struggle to adopt. Hence, user-friendly recommendations would help to translate raw data into interpretable results. The aim of this communication is to streamline the analysis of data obtained by the WorMotel, an economically and practically appealing screening platform, in order to facilitate the use of this system by interested researchers. We here detail recommendations for the stepwise conversion of raw image data into activity values and explain criteria for assessment of health in C. elegans based on locomotion. Our analysis protocol can easily be adopted by researchers, and all needed scripts and a tutorial are available in S1 and S2 Files.

Fig 1. Data collected after blue light stimulation are most suitable for quantification of lifespan.

Data are from one representative animal. Blue light stimulation (at “time 0” of the imaging interval) is crucial to ensure accurate lifespan determination, especially in older animals, which typically display little or no spontaneous movement within the 20 min imaging interval.

Fig 2. Overview of data analysis.

The activity of a single worm during one monitoring time (one day) can be summarized in different ways, relying on (A) the median, 99^th percentile (also: ‘maximal activity’), average of all values between the 95^th to 99^th percentiles (also: ‘peak activity’, red box) or integral (purple shading) values of the pixel difference data. (B) This process can be repeated for different days for the same worm, unveiling how activity (here: peak activity) changes over a lifetime. (C) This analysis is performed for all the worms of the population, based on which (D) the average survivor activity of worms belonging to the same population can be calculated. Worms showing an activity above the green threshold (panel C) are considered healthy—see main text for details.

Fig 3. Independent experiments comparing control, daf-2 and daf-16 RNAi-treated populations show inter-experiment differences yet adhere to expected relative survival changes.

To capture expected variation between possible end-users, four entirely independent experiments were performed, where many factors—including the robotic setup—could have contributed to differences in absolute effect size. Experiments I-IV shown in panels A-D.

Fig 4. All metrics used to produce an activity trace reflect the inherent day-to-day variation.

Traces of a representative daf-2 RNAi-treated worm, constructed based on (A) the median, (B) maximal activity, (C) peak activity or (D) integral—see main text.

Fig 5. Data based on peak or maximal values lead to lower overall variation than those based on median or integral values.

Data for activity values based on daily pixel differences of 100 second intervals (other intervals: S3 Fig) for control (EV, black), daf-2 RNAi (pink) and daf-16 RNAi (yellow) show similar trends. Box plots based on individual worm data from all worms of the same genotype across all experiments. Analysis on an individual experiment basis leads to the same conclusion (S4 Fig).

Fig 6. Choice of activity parameters does not affect variance of Z-score.

Data for activity values based on daily pixel differences of 100 second intervals (other intervals: S5 Fig) for control (EV, black), daf 2 RNAi (pink) and daf-16 RNAi (yellow) show similar trends. Box plots based on individual worm data from all worms of the same genotype across all experiments. Analysis on an individual experiment basis leads to the same conclusion (S6 Fig).

Fig 7. Average survivor activity is always higher for longer time intervals.

(A) Control populations display an activity decline in line with [7]. (B) The typical ‘twilight tail’ [13] or ‘gerospan’ [26] is observed in daf-2 RNAi-treated populations, where animals maintain low-level activity for the majority of their extended life. (C) In contrast, the activity of daf-16 RNAi-treated populations decreases slightly faster than that of controls.

Fig 8. Lifespan of the long-lived condition is most sensitive to the choice of time interval.

Population averages as calculated for control (black), daf-2 (pink) and daf-16 (yellow) RNAi-treated populations (error bars: standard error of the mean), when different time intervals are used for determination of lifespan. Time intervals of ≥ 60 seconds are advisable.

Fig 9. Decreasing pixel differences caused by worm movement correspond to decreasing locomotive health.

(A) Each dot in the figure represents the pixel difference (calculated by WorMotel analysis) for a single observation that was assigned to one of five qualitative categories (x-axis). (B) Above a pixel difference of 160, most animals are scored as healthy (~medium fast movement) by operators, whereas the majority of animals below this threshold are considered less healthy (~slow movement). Black line: fraction of animals in the 'slow' category with a pixel difference value < x-axis value; red line: fraction of animals in the 'medium fast' category with a pixel difference value >x-axis value.

Fig 10. Total days of health and healthspan quantifications are not sensitive to the choice of time interval within the 80–540 seconds range.

Population averages for control (black), daf-2 (pink) and daf 16 (yellow) RNAi-treated populations with accompanying standard error bars (reflecting standard error of the mean) when different time intervals are used for determination of (A) total days of health (TDH) and (B) healthspan (HS) are shown. Time intervals of >60 seconds are advisable.

Fig 11. Normalized IA helps distinguish treated populations from controls.

(A) The mean normalized lifespan (x-axis) vs total days of health (y-axis) and (B) normalized IA (x-axis) vs normalized HR (y-axis), are shown in relation to EV control populations (black, coordinates 1:1:1, all normalizations to internal controls). Average normalized values for populations treated with daf-2 (pink) or daf-16 (yellow) RNAi from Experiment I (‘◊’), Experiment II (‘Δ’), Experiment III(‘x’) or Experiment IV (‘o’) are used as coordinates.