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

Summary

This report describes a method for measuring adult Drosophila melanogaster time to knockdown using a Drosophila Activity Monitor (DAM2) in response to an air conduction heat stressor within an incubator chamber. The DAM2 measures activity by recording individual fly movements as they cross an infrared beam. Data analysis is facilitated by a novel executable file created by the authors.

Abstract

The study of heat tolerance in Drosophila melanogaster has been of particular interest to researchers for decades, with a common approach to assessing heat tolerance being to monitor the time to knockdown (TKD) after exposure to an elevated temperature. Classically, flies are housed in individual vials and placed inside a heated water bath. TKD is then monitored manually by researchers. While very well-established, there remain problems of subjectivity and consistent application of a tangible definition of cessation of all movement, including muscular spasms, when implementing these manual assays. We have developed a high-throughput method for automating heat tolerance assays using the TriKinetics Drosophila Activity Monitors (DAM2). To accompany the DAM2 system, we have written a program and created an easy-use executable to automatically read the last time of movement from the activity data generated. This script then writes to a .csv file the time to heat paralysis (TKD) for each fly. Our data show that this automated DAM2 method is consistent and reliable. Meanwhile, activity profiles created from the activity count data are of interest. These activity profiles can be compiled and have the potential to expand heat tolerance assays to include the relatively unstudied behavioral components of heat tolerance. This protocol will describe in detail how to use the DAM2 system and the HoTDAM! software to estimate heat tolerance in D. melanogaster.

Introduction

Ectotherms typically respond to heat stress with increased locomotor activity. This phenomenon has been apparent to researchers for decades, with the characteristic behavioral response described by Cowles and Bogert in 1944¹. They described how an organism under heat stress will first show increased locomotor movement. As the heat stress builds, short bursts of activity are interspersed with periods of inactivity. The temperature at which the organism can no longer show coordinated movement is the critical thermal maximum (CTmax). Muscular spams follow, and ultimately the organism collapses^1,....

Protocol

1. Fly husbandry

1. Choose the appropriate fly stock/line for the investigation.
   NOTE: To illustrate the assay, we used the transient receptor potential TRPA1 knockout stock and its genetic control, the w¹¹¹⁸ stock.
2. Maintain the fly stocks under consistent conditions as appropriate. To follow this protocol, house stocks at 25 ˚C under a 12:12 diurnal cycle on standard food (cornmeal, molasses, and torula yeast).
3. Separate males and fema.......

Discussion

The heat tolerance assay method we describe here is versatile and scalable. We have previously published a validation study where we compared the HoTDAM! method to a classic, observation-based TKD assay and found the automated assay to give show the same general trend across several factors⁴ (Figure 3). In other words, in the same way as the classic manual TKD assay, the DAM2 automated assay was able to differentiate organisms by sex, assay temperature, hardening pret.......

Materials

Name	Company	Catalog Number	Comments
14 mL polystyrene test tubes	Falcon	352057
30 gallon fish tank	Wal-mart
8 oz bottles	Genesee	32-129F
Constant Climate Chamber	Memmert	HPP750eco
cornmeal	Lab Scientific	FLY801010
DAM2  Drosophila Activity Monitor	TriKinetics	DAM2	(DAMSystem3 Data Sheet) https://www.trikinetics.com/Downloads/DAMSystem%20Price%20List%202024.7.pdf
DAMSystem data acquisition software	TriKinetics		free download
Drosophila agar	Lab Scientific	FLY80201
ethanol	Fisher Scientific	BP82011
Ether	Fisher Scientific	E134-4
FileScan software	TriKinetics		for scanning for text errors, binning data, and output
FlyStuff Flugs for bottles	Genesee	49-100
FlyStuff Flugs for vials	Genesee	49-102
FlyStuff vials	Genesee	32-113RL
HoTDAM software	Github or Trikinetics		https://github.com/MatthewR47/HoTDAM
Immersion circulating heater	PolyScience	MX-CA11B
molasses	Lab Scientific	FLY80084
propionic acid	Fisher Scientific	A258-500
Pyrex Glass tubes 5 x 65 mm for DAM2	TriKinetics	PGT 5x65	https://www.trikinetics.com/Downloads/DAMSystem%20Price%20List%202024.7.pdf
small paint brush	Wal-mart
SPSS Statistics	IBM
tegosept	Lab Scientific	FLY55015
torula yeast	MP Biomedicals	290308505
TRPA1 mutant stock	Bloomington Stock center	26504	w[1118]; TI{w[+mW.hs]=TI}TrpA1[1]
w1118 stock	Bloomington Stock center	3605