Preparing Drosophila larvae for feeding assays.

The food responses of Drosophila larvae offer an excellent opportunity to study the genetic and neural regulation of feeding behavior. Compared with fed larvae, hungry larvae are more likely to display aggressive foraging, rapid food intake, compensatory feeding, and stress-resistant food procurement. Behavioral assays have been developed to quantitatively assess particular aspects of the hunger-driven food response. In combination, these assays help define the specific role of signaling molecules or neurons in the regulation of feeding behavior in foraging larvae. Proper rearing of larvae is of paramount importance for all behavioral experiments. A time-tested procedure is described here to produce highly synchronized third-instar feeding larvae. To ensure synchronized growth of larvae, the rearing conditions must be optimized by controlling food quality, larval density, humidity, and temperature. Under such conditions, it takes ∼ 72 h for a wild-type or white egg to develop into a third-instar larva. One reliable way to judge the age of third-instar larvae is to determine the time of the second molting (into the third instar). A feeding step is included to ensure the selection of young third instars that are actively foraging to use in behavioral assays.