Heat Tolerance Assays Using the Drosophila Activity Monitor System: A Guide to an Executable Application for Data Analysis

In grad school, I became very interested in heat tolerance, especially its behavioral components, and ever since I've been very interested in behavioral thermo regulation and the mechanisms that endotherms employ to regulate their body temperature. Heat tolerance has been studied for decades with researcher observation of organisms. More recently, there has been an advent of video-based and automated methods to measure aspects of heat tolerance.

And I think that it'll be really cool to see where these new technologies will take us in terms of exploring some of the intricacies of behavior and activity as it relates to thermal regulation. One of the challenges in the field is that although we have a way now to monitor and collect large amounts of activity data, the interpretation of what the activity data means in terms of its biological relevancy for real-life applications remains a question that needs further research. One of the advantages of having the automated program is that you can deal with large datasets in a much more easily accessible way.

Previously, one had to manually go through large Excel files to analyze and interpret the results. The automated program makes data analysis much easier. To begin, load the flies into the DAM2 monitor tubes and cap both ends of the tubes with cotton.

Aspirate individual flies from the holding vials into the monitor tubes. Insert the assay tubes into the activity monitors and carefully document the slot numbers corresponding to each group. Load the monitors into the assay incubator.

Start the acquisition software and ensure preferences are set before beginning data acquisition for the experiment. View the monitor data and index number in real time through the DAMSystem interface. Set the noxious temperature on the incubator after the defined number of indexes have passed, marking the end of the acclimation period.

Monitor activity counts in real time using the DAMSystem display in the acquisition software. Alternatively, it can be easier to monitor activity data directly in the DAMSystem files. Copy the text files and open the copies instead of the originals to prevent interference with live data recording.

After observing no movement in any of the flies for several minutes, stop the acquisition software. Once the data has been acquired, scan the text files for errors using the referenced software. Based on the implemented acclimation interval prior to the induction of the noxious temperature, select specific start and stop points for binning the activity data before saving the scanned file.

Now, open the HoTDAM! analysis software and click File and load monitor data to import the scanned monitor data files. Add group designations to indicate the treatment groups corresponding to each cell within the DAM2 monitors.

Click on Start Multi-Group Definition to open the dialogue box for adding a group designation. Then, click on the respective cells to apply the group designation and click Stop Multi-Group Designation. To export the TKD for each fly as a CSV file, click File, Export Knockdown Data, and Export All Monitors or Export Selected Monitors.

The output will organize the TKD for each fly by group designation. Now click File, Export Activity Data, and Export All Monitors or Export Selected Monitors to export the activity data for each fly as a CSV file, retaining only the timestamp and count data to simplify the data file and assigning designated group labels for each fly. Hardened W1118 females exhibited a significantly lower probability of knockdown during heat stress compared to control females.

While no significant difference was observed in males, hardened TRPA1 males exhibited a significantly higher probability of knockdown compared to control males during heat stress, whereas no significant difference was found between hardened and control females. Average activity during heat tolerance varied distinctly between hardened and control groups for W1118 stocks, showing a peak in activity counts around 30 minutes for both males and females. Activity for TRPA1 stocks showed a sharp spike in initial activity, followed by a rapid decline for all groups.