Material and methods
Tardigrades of the species M. tardigradum Doyère, 1840 (Eutardigrada, Apochela) were cultured in the laboratory in plastic culture dishes with a thin layer of agar, covered with VolvicTM water (Danone Waters Deutschland, Wiesbaden, Germany). The animals were fed rotifers of the species Philodina citrina Ehrenberg, 1832 which were raised on the green algae Chlorogonium elongatum (Dangeard, 1897). Young tardigrades were additionally fed green algae C. elongatum. We used animals from a parthenogenetic population, which was previous collected in Tübingen, Germany, and cultured over several years in an environmental chamber at 20°C using an artificial light source with a 12 h light/12 h dark cycle. The age of the tested tardigrades was approximately 40 days and the animals had a mean body size of about 1 mm.
For the behavioural experiments and the observation of the specimens a simple ‘arena’ construction was developed (Fig. 1). For this purpose 36 openings were drilled through a plexiglas plate to form the arena grid, with each individual ‘arena’ having a diameter of 7 mm. Preliminary tests showed that the optimal adhesive material for fixing the ‘arena’ grid was hot paraffin wax. The surface of paraffin is rough (not slippery as normal Petri dishes), so this material seemed to be suitable for the movement of tardigrades using their clawed limbs without further adaptation.
The behavioural experiments were carried out in a room with constant climate conditions controlled by a climate control system and using a video tracking system. For the experiments the Petri dish with arena grid was positioned on a custom-made infrared light table and filled with VolvicTM water (Fig. 1). Thereafter, well fed animals were placed individually into each ‘arena’, and the behaviour of tardigrades was recorded using an infrared sensitive camcorder (Sony DCR–HC23E). For the duration of the experiments the infrared light table was left ON to produce the optimal illumination for the infrared camcorder. The only visible light (daytime) was emitted by a standard halogen lamp of type ‘Philips TLD 58 W’ with the colour temperature of 4000 K. The intensity of illumination in the experimental area was measured at 200 lux. The room lightning was controlled by an automated relay. The working mode of visible light was 12 h ON (light regime), 12 h OFF (dark regime). For capturing and saving of video information the freely available software VirtualDub (http://www.virtualdub.org) was used. The video was recorded directly to the hard drive of a computer system at 25 frames per second, and because of the small size of the animals (≤ 1 mm) it was possible to record two arenas simultaneously by one camcorder.
Video analysis and statistics
The sampled videos were analysed using the custom made video tracking software "BioMotionTrack D.S.", programmed by D. Shcherbakov. Analysis of animal movements by our video tracking program was based on an automated body contours recognition algorithm. The software calculated the location coordinates of the animals, their movement speed and length of the tracks, portion of time in different areas of the arenas, and angle of movement direction. Due to the very slow movement and speed of the animals it was sufficient to analyse 1 frame per 10 second of the original video source. A video file with the animal tracks was created as a result of this calculation (see Fig. 1, embedded supplementary video) and calculated data were saved as ‘.asc’-text. These results were imported into Access (Microsoft) database for data management and analysis. The calculation of statistical significance was carried out by Wilcoxon matched pairs test (Statistica 6.1 – StatSoft, Inc). Significance levels were P≥0.05 (not significant), 0.05>P≥0.01 (weakly significant *), 0.01>P≥0.001 (significant **), and P<0.001 (highly significant ***).