Predation is a strong driver for the evolution of prey behaviour. To properly assess the actual risk of predation, anuran tadpoles mostly rely on water-borne chemical cues, and their ability to evaluate environmental information is even more crucial when potential predators consist of unknown alien species. Behavioural plasticity, that is the capacity to express changes in behaviour in response to different environmental stimuli, is crucial to cope with predation risk. We explored the defensive behaviour of Italian agile frog (Rana latastei) tadpoles when exposed to the chemical cues of two predators’ species, one native (dragonfly larvae) and one alien (red swamp crayfish). Firstly, we observed if a sensible life history trait (i.e. hatching time) might be affected by native predatory cues. Secondly, we recorded a suite of behavioural responses (activity level, lateralization and sinuosity) to each cue. For assessing lateralization and sinuosity, we developed a C++ code for the automatic analysis of digitally recorded tadpole tracks. Hatching time seemed not to be affected by the potential risk of predation, while both predator species and diet affected tadpoles’ defensive behaviour. Tadpoles responded to predator threat by two main defensive strategies: freezing and “zig-zagging”. While the first behaviour had been previously reported, the analysis of individual trajectories pointed out that tadpoles can also increase path complexity, probably to prevent predators from anticipating their location. We also recorded a decrease in lateralization intensity, which suggested that under predation risk tadpoles tend to scrutinize the surrounding environment equally on both sides.

Two lateralization indices were calculated for tadpoles: LR = [(clockwise swimming time – anticlockwise swimming time) / (clockwise swimming time + anticlockwise swimming time)] × 100, and LA = |LR|, which assesses the intensity of lateralization. These procedures were applied to all 375 tadpoles, each recorded for 300 s before and after the stimulus for a total amount of about 15000 locations for each tadpole. See "Contextual behavioural plasticity in Italian agile frog (Rana latastei) tadpoles exposed to native and alien predator cues" (Gazzola et al.) for further details.