Cultured fish epithelial cells are a source of alarm substance.

In various species of fishes, the importance of visual cues in the determination of environmental threat and subsequent predator avoidance is clear. Chemical cues also play an essential role facilitating predator avoidance. Among fish in the superorder Ostariophysi, club cells in the epidermis produce an alarm substance. Damage to the skin during a predation event releases an alarm substance (AS), which diffuses through the water column and binds to olfactory receptors of conspecifics. Fish then engage in a number of anti-predator behaviors that may include darting, schooling, or hiding. Behavioral responses to AS and physiological mechanisms that underlie those responses is an active area of study. However, because the precise chemical composition of the alarm substance is unknown, AS is not commercially available. Thus, when fish are challenged alarm substance in various experiments and assays it is obtained from skin extracts or via perfusion of shallow cuts in the epidermis. Both procedures are effective but require the animal to be sacrificed. In this manuscript, we report: •A non-invasive primary cell culture protocol to obtain alarm substance and does not require the model organism to be killed.•The demonstration of anti-predatory behaviors in fish exposed to alarm substance collected by this method.