Behavioral adaptations play an important role in predator-prey interactions as they reduce predation risk. Prey organisms have therefore evolved a tremendous variability in behavioral adaptations. In case of small crustaceans of the genus Daphnia, which are common and important herbivores transferring energy from primary producers to higher trophic levels, such as predatory fish, and insects, altered migration patterns, swarming, or adaptive swimming speeds may increase survival rates. However, hitherto it has been difficult to analyze predator-induced behavioral adaptations as the small body size, as well as the low contrast between the transparent animals and their environment, most often impede behavioral movement analysis of individual animals. Therefore, we worked with a newly established technique providing higher contrast. We tagged daphniids with fluorescent nanoparticles and used a three-dimensional movement analysis system. We analyzed behavioral defense strategies of Daphnia clones from three species against different types of predators by measuring their behavior in presence and absence of predator cues. We analyzed swimming speed, depth selection, and motion patterns of Daphnia, as well as swarming behavior. We observed differences in the general swimming behavior in all analyzed aspects and show that daphniids change their behavioral strategies in the presence of predator cues, e.g., decrease their swimming speed as well as their vertical position or increase their nearest neighbor distance. Based on the observed changes in behavioral patterns, we conclude that the swimming behavior of daphniids may play an important role as inducible defense strategy that has the potential to improve prey survival chances.

Analyzing movement is essential for understanding complex behavioral interactions. Up to date, there are different movement analysis methods. Previous studies working with 2D movement analysis systems provided first insights into this field. 2D systems can capture only two of three spatial dimensions and thus allow analyses of movements on surfaces. 3D systems include all three spatial dimensions. Thus, 3D movement analysis is essential for analyzing movements in air or in water. Especially during the last years, 3D movement analysis has progressed a lot thanks to technological advances. Unfortunately, these technological advanced systems are often very expensive and handling flexibility is limited. Therefore, we implemented a customizable 3D movement analysis system for aquatic organisms. The system is composed of an experimental arena, backgrounds with different contrasts, three visible light diode spots, one infrared diode spot for analysis in the dark, and two Raspberry Pi 3 Model B V 1.2 equipped with cameras without infrared filters. We assembled a user-friendly system that is controlled directly via a graphical user interface (GUI). It not only delivers raw data but also runs detailed analyses of common behavioral parameters. The accuracy of those analyses can be enhanced by the integrated manual error-correction method that allows to interrupt the automatic track analysis and to correct detection errors by hand. In our case study, we show the spectrum of opportunities of the system by determining the swimming behavior of the freshwater crustacean Daphnia magna. We successfully recorded its swimming behavior under visible light and infrared light (simulating darkness for the daphniids) conditions and analyzed swimming velocity, motion patterns, depth selection, and tendency of clustering. In our case study, we show that using the integrated manual error-correction method leads to a clear accuracy improvement. Our movement analysis system is not restricted to aquaria but can be adopted to movements in cages and even open areas of different sizes both in water or air, which emphasizes its potential. Together with a user manual, the opensource MATLAB algorithm and the recommended hardware components, the here presented system enables semi-automatic 3D movement analysis, which can be adapted to the individual needs.