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Organisms have evolved the ability to detect, process, and respond to many different surrounding stimuli in order to successfully navigate their environments. Sensory experiences can also be stored and referenced in the form of memory. TheDrosophilalarva is a simple model organism that can store associative memories during classical conditioning, and is well-suited for studying learning and memory at a fundamental level. Much progress has been made in understanding larval learning behavior and the associated neural circuitry for olfactory conditioning, but other sensory systems are relatively unexplored. Here, we investigate memory formation in larvae treated with a temperature-based associative conditioning protocol, pairing normally neutral temperatures with appetitive (fructose, FRU) or aversive (salt, NaCl) stimuli. We test associative memory using thermal gradient geometries, and quantify navigation strength towards or away from conditioned temperatures. We find that larvae demonstrate short-term associative learning. They navigate towards warmer or cooler temperatures paired with FRU, and away from warmer or cooler temperatures paired with NaCl. These results, especially when combined with future investigations of thermal memory circuitry in larvae, should provide broader insight into how sensory stimuli are encoded and retrieved in insects and more complex systems. 

Detailed descriptions of behavior provide critical insight into the structure and function of nervous systems. InDrosophilalarvae and many other systems, short behavioral experiments have been successful in characterizing rapid responses to a range of stimuli at the population level. However, the lack of long-term continuous observation makes it difficult to dissect comprehensive behavioral dynamics of individual animals and how behavior (and therefore the nervous system) develops over time. To allow for long-term continuous observations in individual fly larvae, we have engineered a robotic instrument that automatically tracks and transports larvae throughout an arena. The flexibility and reliability of its design enables controlled stimulus delivery and continuous measurement over developmental time scales, yielding an unprecedented level of detailed locomotion data. We utilize the new system’s capabilities to perform continuous observation of exploratory search behavior over a duration of 6 hr with and without a thermal gradient present, and in a single larva for over 30 hr. Long-term free-roaming behavior and analogous short-term experiments show similar dynamics that take place at the beginning of each experiment. Finally, characterization of larval thermotaxis in individuals reveals a bimodal distribution in navigation efficiency, identifying distinct phenotypes that are obfuscated when only analyzing population averages.