HoTDAM! An easy-to-use automated assay expands the inducible thermotolerance phenotype in Drosophila melanogaster: Heat hardening reduces motility

Abstract

To quantify heat tolerance in insects, two manual observation measures are typically implemented: the time to physiological collapse at a static noxious temperature (time to knockdown; TKD) or the temperature at which collapse occurs as temperature increases (critical thermal maximum; CTmax). Both assay modalities focus on physiological collapse, neglecting the prior behavioral processes. In this study, the locomotion response of Drosophila melanogaster to relatively high temperature (39 and 40.5 °C) was quantified using the TriKinetics Drosophila Activity Monitor (DAM2 system). The absence of locomotion was defined as the state of physiological collapse resulting from extended exposure to high temperature. An easy-to-use executable application that allows the user to automatically extract individual TKD from the activity data was developed. For validation, manual TKD assays were performed in parallel to automated assays across multiple factors, including sex, hardening, recovery time after hardening, and assay temperature, which gave similar results. In terms of behavioral aspects, heat hardening consistently led to reduced activity during a subsequent heat stress, irrespective of assay temperature, sex, or recovery time after hardening. Our automated heat tolerance assay utilizing the DAM2 system is one way to expand the scope of the heat tolerance phenotype to include a behavioral component in conjunction with the traditional TKD measure.