This work was supported in part by a scholarship from the Behavioural and Cognitive Neuroscience Program of the University of Groningen and a graduate scholarship from the Consejo Nacional de Ciencia y Tecnolog&#237;a (CONACyT) from Mexico, granted to Andrea Soto-Padilla, and a grant from the John Templeton Foundation for the study of time awarded to Hedderik van Rijn and Jean-Christophe Billeter. We are also thankful to Peter Gerrit Bosma for his participation in developing the FlySteps tracker.
Scripts TemperaturePhases,FlySteps, and FlyStepAnalysis can be found as supplementary information and in the following temporary and publicly available link: 
https://dataverse.nl/privateurl.xhtml?token=c70159ad-4d92-443d-8946-974140d2cb78

1. Preparation of Fly Food Medium
<ol>
	<li>Pour 1 L of tap water into a 2 L glass beaker and add a magnetic stir bar. Put the beaker on a magnetic hot plate at 300 &#176;C until boiling temperature is reached.</li>
	<li>Stir at 500 rounds/min and add the following: 10 g of agar, 30 g of glucose, 15 g of sucrose, 15 g of cornmeal, 10 g of wheat germ, 10 g of soy flour, 30 g of molasses, and 35 g of active dry yeast.</li>
	<li>When the mix foams vigorously, turn down the hot plate temperature to 120 &#176;C while contin...

<ol>
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The authors declare that they have no competing financial interests.

Here we have presented an automated temperature-controlled arena (Figure 1) that produces precise temperature changes in time and space. This method allows exposure of individual Drosophila not only to pre-programmed gradual increases of temperature (Figure 2 and Figure 3), but also to dynamic temperature challenges in which each tile of the fly arena was heated independently to a different temperature (<strong class="xfig"...

Temperature is a constant environmental factor that affects how organisms function and behave1. Differences in latitude and altitude lead to differences in the type of climates organism are exposed to, which results in evolutionary selection for their responses to temperature2,3. Organisms respond to different temperatures through morphological, physiological, and behavioral adaptations that maximize performance under their particular environments4. For instance, in the fruit fly Drosophila melanogaster, populations from different regions have different temperature preferences, body sizes, developmental times, longevity, fecundity, and walking performance at different temperatures2,5,6,7. The diversity observed between flies of different origins is explained in part by genetic variation and plastic gene expression8,9. Similarly, Drosophila species from different areas distribute differently among temperature gradients and show differences in resistance to extreme heat and cold tests10,11,12.
Drosophila has also recently become the model of choice to understand the genetic and neural basis of temperature perception13,14,15,16,17. Broadly, adult flies perceive temperature through cold and hot peripheral temperature sensors in the antennae and through temperature sensors in the brain13,14,15,16,17,18,19,20. The periphery receptors for hot temperatures express Gr28b.d16 or Pyrexia21, while the periphery cold receptors are characterized by Brivido14. In the brain, temperature is processed by neurons expressing TrpA115. Behavioral studies on mutants of these pathways are improving our understanding of how temperature is processed and give insights into mechanisms that vary among populations of Drosophila from different regions.
Here we describe a temperature-controlled arena that produces fast and precise temperature changes. Investigators can pre-program these changes, which allows for standardized and repeatable temperature manipulations without human intervention. Flies are recorded and tracked with specialized software to determine their position and speed at different phases of an experiment. The main measurement presented in this protocol is the walking speed at different temperatures, because it is an ecologically relevant index of physiological performance that can identify individual thermal adaptability5. Together with temperature receptor mutants, this technique can help reveal the mechanisms of thermal adaptation at cellular and biochemical levels.

<table border="0" cellpadding="0" cellspacing="0" width="965">
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			<td height="21" style="height:21px;width:332px;">Arduino Due</td>
			<td style="width:223px;">Arduino</td>
			<td style="width:189px;">A000062</td>
			<td style="width:221px;">Software RUG</td>
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			<td>Fisher Technik</td>
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			<td>Decoupled for vibration</td>
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			<td height="21" style="height:21px;">Temperature sensors</td>
			<td>Measurement Specialties</td>
			<td>MCD_10K3MCD1</td>
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			<td height="21" style="height:21px;">Tesa 4104 white tape 25 x 66 mm</td>
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</table>

an automated method to determine the performance of drosophila in response to temperature changes in space and time

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

The temperature-controlled arena (Figure 1A) consists of three copper tiles whose temperature can be individually controlled through a programmable circuit. Each copper tile possesses a temperature sensor that gives feedback to the programmable circuit. The circuit activates a power supply to increase the temperature of each tile. Passive thermoelectric elements act as constant heating elements to maintain the desired temperature, while a heat sink cooled by ...

Watch this Scientific Journal Video about An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time at JoVE.com

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Here we present a protocol to automatically determine the locomotor performance of Drosophila at changing temperatures using a programmable temperature-controlled arena that produces fast and accurate temperature changes in time and space.

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

This method can help us answer key questions about flies'response to temperature changes, such as differences between genotypes, the interaction with other sensory cues, or the function of different temperature receptors. The main advantage of this technique is that it allows for multiple fast and precise temperature changes controlling time and space and in an automated fashion. Begin by placing 20 male and 20 female flies in a rearing bottle containing 45 milliliters of fly food medium, and placing the bottle inside a 25 degree celsius incubator under 12 hour light, 12 hour dark cycles.After 10 days, anesthetize the newly eclosed flies on carbon dioxide pads for a maximum of four minutes. And use a paintbrush to collect virgin flies in 2.5 by 9.5 centimeter fly rearing vials containing 6.5 milliliters of fresh fly food medium, separated by sex into groups of 20 flies per vial. Then return the vials to the incubator for five to seven days.To set up a temperature-controlled arena, turn on the arena and open the temperature phases script in the control computer. Verify that the temperature sequence is properly set and check that the duration of each experimental phase is set to 60 seconds. Under the start experimental block section, confirm these settings:the desired number of phases, iterative on/off setup of the indicative red light emitting diodes, two degrees celsius temperature increase per phase, and 16 degrees celsius as the starting temperature.Next, run the temperature phases script. The software will initialize for five seconds then stop. For temperature behavioral experiments, place a strand of white conductive tape on the top of the copper tiles of the arena, ensuring that all the edges are covered.Place a heated aluminum ring around the copper tiles and use a clean tissue to wipe the glass cover. Place the cover on the top of the aluminum ring, leaving a gap through which a fly can be blown in and tap an acclimated fly vial two times to force the flies to the bottom of the vial. Using a mouth aspirator, trap one fly and close the vial before putting it back into the incubator.Blow the fly into the arena through the gap between the glass cover and the aluminum ring and immediately push the glass clover to close the gap. Place a frame of lights around the arena to ensure symmetric illumination and start recording with the video recording program. Then press the space bar of the control computer to begin running the experimental phases.To track the videos, open the fly steps tracking software and open the configuration file inside the fly tracker folder. Set the location of the videos in video folder, and the names of the videos in video files. Specify the borders of the fly arena in arena settings based on the x/y pixel coordinates of multiple points at the edge of the arena.Specific the location of the inactive red LEDs in LED settings, based on the x/y pixel coordinates of the location of the center of the LEDs. To check the location of the borders of the fly arena, set debug to true in the arena settings, click save, and run the script in the terminal. A screen capture of the video will appear with a blue square formed by the coordinates inputted in arena settings.Then change debug in arena settings to false. Click save, and run the screen in the terminal again to begin the tracking process. In a typical temperature behavioral experiment, single flies exposed to 16, 20, or 24 degrees celsius exhibit a higher locomotion at the beginning of the experiment, than after five minutes.The temperature-controlled arena can also be used to compare fly behavioral responses from different genetic backgrounds to dynamic temperature changes. For example, in this experiment, the speed of all of the tested species increased according to their own response curves as the temperature increased, until reaching a point of maximum performance, after which their speeds decayed and the flies perished. When individual flies were exposed to 40 degrees celsius in the middle and one side tile, with the other side tile at a comfortable 22 degrees celsius the the Wild-type flies quickly stopped moving along the arena and remain in the comfortable location.In contrast, the classic memory mutant Dunce flies keep exploring the arena and spend less time than controls in the comfortable location. Further, testing combinations of temperature and location are also useful in understanding the function of different temperature receptors during dynamic temperature changes. As illustrated in this experiment in which individual d melanogaster mutants were exposed to increasing temperatures while a shifting comfortable location at 22 degrees celsius was also provided.While performing these procedure, remember to perform the steps in order and quickly so you can capture as much of flies behavior as possible. Next to this procedure other methods like negative geotaxis or heat choke assays can be used to answer additional questions such as where do flies move normally or have a normal heat resistance. The implications of this technique extend to us understanding neurological disorders or mutations in which temperature perception or pain play a role.Generally, individuals new to this method will struggle because each step is simple by itself but they're required to be perfectly coordinated so the technique needs to be practiced.

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JoVE Journal

Behavior

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