Noxious Heat Avoidance in Drosophila

For adult avoidance of noxious heat, ∼20 four day old flies were placed into a behavioral chamber (35 mm×10 mm Petri dish; Nunclon) and the chamber was sealed with scotch tape. Flies were rested for at least 30 minutes in the dark. The chambers were then floated on a 46°C water bath for 4 minutes. The bottom of the chamber was heated to 46°C over 15 seconds by floating on a water bath while the sub-noxious zone was measured to be 31°C (inner top of chamber) and 33°C (middle edge of the chamber) at the end of the 4 minute experiment. Chamber temperature was monitored using an electronic thermometer (Testo 925, Germany) coated in heat sink gel (RS components, UK). Chambers were then removed from the water and immobilized flies were counted as “incapacitated”. In addition, total fly number was recorded. Percentage Avoidance was calculated by determining the percentage of flies that avoid the noxious temperature compared to the total number of flies in the chamber. All tests were performed under low red light. Of note, this assay is an absolute measurement where flies that avoid the bottom heated surface were considered capable of noxious thermal avoidance, independent of how far they move away from the 46°C surface, though the vast majority of flies avoiding the hot surface were found on the top of the chamber. For experiments involving UAS-Shibire^ts1, flies were transferred to the experimental chambers followed by a temperature shift to 30°C for 60 minutes. Larval pain assays were performed as described [7] (link). For assessing noxious temperature-induced paralysis, wild type flies were placed in 5 ml polystyrene round bottom tubes (BD Falcon, Germany) and exposed to temperatures ranging from 37–46°C with 1° increments) or only 46°C (for control vs TrpA1 flies) and the temperature at which 100% of flies were paralyzed was recorded. General coordination was assessed by tapping the test chamber on the bench and observing activity as flies move away from the site of impact [35] (link). This response was quantified using a geotactic repulsion assay where flies were knocked to the bottom of a 15 ml polystyrene tube and the geotactic response (number of flies climbing up the tube / total number of flies) was recorded.