Independent insulin signaling modulators govern hot avoidance under different feeding states

Thermosensation is critical for the survival of animals. However, mechanisms through which nutritional status modulates thermosensation remain unclear. Herein, we showed that hungry Drosophila exhibit a strong hot avoidance behavior (HAB) compared to food-sated flies. We identified that hot stimulus increases the activity of α⁰β⁰ mushroom body neurons (MBns), with weak activity in the sated state and strong activity in the hungry state. Furthermore, we showed that α⁰β⁰ MBn receives the same level of hot input from the mALT projection neurons via cholinergic transmission in sated and hungry states. Differences in α⁰β⁰ MBn activity between food-sated and hungry flies following heat stimuli are regulated by distinct Drosophila insulin-like peptides (Dilps). Dilp2 is secreted by insulin-producing cells (IPCs) and regulates HAB during satiety, whereas Dilp6 is secreted by the fat body and regulates HAB during the hungry state. We observed that Dilp2 induces PI3K/AKT signaling, whereas Dilp6 induces Ras/ERK signaling in α⁰β⁰ MBn to regulate HAB in different feeding conditions. Finally, we showed that the 2 α⁰β⁰-related MB output neurons (MBONs), MBONα⁰3 and MBON-β⁰1, are necessary for the output of integrated hot avoidance information from α⁰β⁰ MBn. Our results demonstrate the presence of dual insulin modulation pathways in α⁰β⁰ MBn, which are important for suitable behavioral responses in Drosophila during thermoregulation under different feeding states.