Measuring Anxiety-like Behavior in Crayfish by Using a Sub Aquatic Dark-light Plus Maze

(Abstract) Crayfish are omnivorous freshwater arthropods that naturally explore their environment during day and night, but also frequently hide under a shelter or in a hole in case of danger. They may be submitted to various stressors, including predation, social interactions or changes in environmental parameters (temperature, water quality, oxygen, etc.). It has been recently demonstrated that, as a consequence of stress, crayfish is able to adapt its exploratory behavior by restricting movements to protective areas, a response similar to the anxiety-like behavior (ALB) observed in rodents. To reveal such a behavior in an aquatic species, we designed a plus-shaped sub aquatic maze divided in two protective dark arms and two more aversive illuminated arms. The aim of this paradigm was to place crayfish in a conflicting situation between its innate curiosity for novel environment and its aversion for light (Leo, 2014; Pellow et al., 1985). Unstressed crayfish generally explore the whole maze, including illuminated arms. By contrast stressed crayfish remain preferentially in the dark arms (Fossat et al., 2014). Stressed crayfish injected with anxiolitics (chlordiazepoxide-CDZ), behave as unstressed animals. Several parameters, related to the light arms can be easily measured from video records by commercial software This protocol could be suitable for analyzing the effects of any stressful situation on ALB in crayfish, as well as in many other aquatic species.