Abstract
To study the behavior of Drosophila, it is often necessary to restrain and mount individual flies. This requires removal from food, additional handling, anesthesia, and physical restraint. We find a strong positive correlation between the length of time flies are mounted and their subsequent reflexive feeding response, where one hour of mounting is the approximate motivational equivalent to ten hours of fasting. In an attempt to explain this correlation, we rule out anesthesia side-effects, handling, additional fasting, and desiccation. We use respirometric and metabolic techniques coupled with behavioral video scoring to assess energy expenditure in mounted and free flies. We isolate a specific behavior capable of exerting large amounts of energy in mounted flies and identify it as an attempt to escape from restraint. We present a model where physical restraint leads to elevated activity and subsequent faster nutrient storage depletion among mounted flies. This ultimately further accelerates starvation and thus increases reflexive feeding response. In addition, we show that the consequences of the physical restraint profoundly alter aerobic activity, energy depletion, taste, and feeding behavior, and suggest that careful consideration is given to the time-sensitive nature of these highly significant effects when conducting behavioral, physiological or imaging experiments that require immobilization.