Ontogenetic and Experience-Dependent Changes in Defensive Behavior in Captive-Bred Hawaiian Bobtail Squid, Euprymna scolopes

Cephalopod molluscs are known for their extensive behavioral repertoire and their impressive learning abilities. Their primary defensive behaviors, such as camouflage, have received detailed study, but knowledge is limited to intensive study of relatively few species. A considerable challenge facing cephalopod research is the need to establish new models that can be captive bred, are tractable for range of different experimental procedures, and that will address broad questions in biological research. The Hawaiian Bobtail Squid (Euprymna scolopes) is a small, tropical cephalopod that has a long history of research in the field of microbial symbiosis, but offers great promise as a novel behavioral and neurobiological model. It can be bred in the laboratory through multiple generations, one of the few species of cephalopod that can meet this requirement (which is incorporated in regulations such as EU directive 2010/63/EU). Additionally, laboratory culture makes E. scolopes an ideal model for studying ontogenyand experience-dependent behaviors. In this study, we show that captive bred juvenile and adult E. scolopes produce robust, repeatable defensive behaviors when placed in an exposed environment and presented with a visual threat. Further, adult and juvenile squid employ different innate defensive behaviors when presented with a size-matched model predator. When a 10-min training procedure was repeated over three consecutive days, defensive behaviors habituated in juvenile squid for at least 5 days after training, but memory did not appear to persist for 14 days. In contrast, adult squid did not show any evidence of long-term habituation memory. Thus we conclude that this species produces a range of quantifiable, modifiable behaviors even in a laboratory environment where ecologically-relevant, complex behavioral sequences may not reliably occur. We suggest that the lack of long-term memory in adult squid may be related to their less escalated initial response to the mimic, and thus indicates less motivation to retain memory and not necessary inability to form memory. This is the first demonstration of age-related differences in defensive behaviors in Euprymna, and the first record of habituation in this experimentally tractable genus of squid.