Name: measuring exercise levels in drosophila melanogaster using the rotating exercise quantification system (reqs)
Uploaded: 2018-05-27T15:00:00
Description: Watch this Scientific Journal Video about Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS at JoVE.com

The work was supported by Award Number P30DK056336 from the National Institute Of Diabetes And Digestive And Kidney Diseases through a pilot grant from the Nutrition and Obesity Research Center at the University of Alabama at Birmingham to NCR.

The REQS consists of a Drosophila Activity Monitor unit (for source information, please see Table of Materials) mounted on a rotating arm that is controlled by a motor unit (Figure 1). The activity monitor determines how often in a given time span the array of laser beams dissecting the middle of the assay tube is disrupted. For detailed drawings and an in-depth characterization, see our previous publication9. While our system uses the LAM25H unit, th...

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As the representative results illustrate, the REQS is capable of accurately measuring the activity of exercising Drosophila. The REQS is flexible and allows researchers to address a variety of research questions related to exercise biology or exercise interventions. There are two critical steps in the protocol to highlight. First, it is essential to test the setup of the REQS to ensure that data transmission from the REQS to the DAMSystem308 is working properly. If not properly set up, the data cable can become tangled d...

Recently, researchers have begun to use the fruit fly Drosophila melanogaster to study exercise biology. D. melanogaster has been a genetic model system for over 100 years1,2. However, Drosophila research has made contribution to not just genetics, but also to a variety of other disciplines including neurobiology, behavioral biology, and physiology3. In 2009, the Power Tower, the first exercise machine for Drosophila was described4. The Power Tower takes advantage of the animals&#39; negative geotaxis response. When disturbed, Drosophila tend to move to the top of their enclosure. This response is well established and is the basis of the popular &#34;RING&#34; (Rapid Iterative Negative Geotaxis5) assay that is used to estimate climbing ability and/or physical fitness in Drosophila. The Power Tower uses a mechanical arm connected to a motor unit to repeatedly lift a set of animals within their enclosures by several inches and dropping them back to the ground to induce the negative geotaxis response (Tinkerhess et al. 20126 provide a video illustrating the use of the Power Tower). Prolonged treatment on the Power Tower thus increases the amount of physical activity (running or flying) the animals perform compared to untreated control animals and over time leads to improved performance in the RING assay for physical fitness4. Thus, this work demonstrated the feasibility of using Drosophila as a model for exercise biology.
To expand the repertoire of tools available for Drosophila exercise research, in 2016, Mendez and colleagues described a second Drosophila exercise machine, the Treadwheel7. Similar to the Power Tower, the Treadwheel exploits the negative geotaxis response of Drosophila. However, this response is induced by continued rotation of the animal enclosures, rather than by lifting and dropping them as in the Power Tower. This induction method is gentler and allows for a more endurance oriented exercise regime that avoids any physical trauma that might occur during exercise in the Power Tower (see Katzenberger, R. J. et al. 20138 for the impact of repeated physical trauma on Drosophila health). Similar to the Power Tower4, exercise treatment of animals on the Treadwheel leads to a variety of physiological responses, including changes in physical fitness, triglyceride levels, and body weight7. Thus, two complementary methods are available for Drosophila biologists studying exercise.
One limitation of both the Power Tower and the Treadwheel is the inability to measure the amount of activity induced by the exercise treatment. Analysis of video-recordings taken from the Treadwheel demonstrated that there were significant differences among the various Drosophila strains in how they respond to the exercise treatment7. Specifically, the strains studied differed in how much additional activity the animals performed when stimulated7. This observation prompted us to develop a third exercise system, the Rotating Exercise Quantification System (REQS), that allows us to measure animal activity levels during rotation-induced exercise9. The REQS utilizes a commercially available activity monitoring unit that is installed on a rotating arm to stimulate exercise through rotation as in the Treadwheel. Initial work with the REQS confirms that genetically different Drosophila strains &#8212; and sexes - can have significantly different responses to the rotational stimulation and thus the amount of exercise induced is not identical among different genotypes9. Thus, the REQS now enables Drosophila biologists to measure the amount of exercise induced by the treatment, opening a variety of new research avenues in the exercise field.
Here we describe in detail how to use the REQS for quantification of rotational exercise. The REQS induces rotational exercise and simultaneously measures the activity levels of the animals being treated. The REQS is able to accommodate a variety of exercise programs, ranging from the simple 2 h continuous exercise regime demonstrated here to more complex interval training methods as described by Mendez and colleagues7, and stimulation can be adjusted via rotation speed (between approximately 1-13 rotations per min). Depending on the activity monitor unit used to produce the REQS, this method is adaptable to the analysis of single flies or large populations of animals. Due to this versatility, the REQS provides Drosophila researchers with an array of opportunities to study, for example, different exercise regimes, diet interventions, or impact of population density.

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measuring exercise levels in drosophila melanogaster using the rotating exercise quantification system (reqs)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

The output from an individual run with the REQS is a data table produced by the DAMSystem308 software, which will be labeled &#34;Monitor1.txt&#34; by default (for an example see Supplemental File 1). An excerpt from such a table is shown in Table 1. Each column contains the data from an individual assay tube, while the rows contain the activity measured in each time interval from the beginning of the experiment (top) to the end (bottom). The first three ...

Watch this Scientific Journal Video about Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS at JoVE.com

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

This method can help answer key questions in the field of exercise biology, such as how does exercise response differ for animals of different ages and what genetic pathways control exercise response. The main advantage of this technique is that it allows researchers, for the first time, to measure activity in flies performing rotationally-induced exercise in real time. Demonstrating the procedure will be Louis Patrick Watanabe, a graduate student from my laboratory.Prior to the planned experiment, set up crosses of male and female flies so that each controlled progeny can be collected for the experiment. To begin, load eight glass specimen tubes with 10 anesthetized test flies per vial. A standard experimental group consists of 10 vials of 10 flies of each sex.To load the vials into the REQS, insert the vial into a slot of the activity monitor from the front. Then, put the second cap on the vial and center the vial, moving the O-ring as needed. House the REQS in an incubator for controlled environmental conditions.Allow the animals to recover from the anesthesia for an hour in this incubator before proceeding. For assays longer than eight hours, vials containing food that is very securely attached to one end of the tube may be used. Following the acclimatization period, power up the device to start the rotations.Adjust the speed to the desired speed, such as four RPM. No tube should make contact with the bottom surface if they are properly positioned. For a typical assay, simply initiate the software and record the activity for the desired time, such as for two hours when using vials without food.Upon initiation, the software automatically saves the data to a text file. In the software, a green connection light indicates the software is collecting data. The recording frequency can be adjusted under the Preferences tab.Typically, five minute intervals are used. During the assay, do not open the incubator, as the animals are very sensitive to any disturbance and are likely to respond with increased activity. At the end of the assay, terminate the data collection by closing the software, then turn off the REQS and inspect the tubes.Take note of any deaths that occurred during the exercise regime. Generally, four RPM for two hours is not lethal to healthy strains. Finally, unload the vials by anesthetizing the flies with carbon dioxide.The data file can be analyzed with any statistical package. If there are time points where an activity score of zero is recorded for all positions, then there was a cut out in the connection during these time points. If cut outs occur, stabilize the connections leading to the rotating phone jack using tape.Four genetically distinct drosophila lines were assayed with the REQS using the described protocol. Each group was composed of 100 flies split into 10 vials. The average activity was calculated in five minute bins over the two hour assay, conducted at four RPM.The mean exercise activity between all four genotypes was significantly different, however, no significant difference was found between virgin male and virgin female flies, so their data is presented together in this analysis. After its development, this technique paved the way for researchers in the field of exercise genetics to explore various exercise responses in drosophila melanogaster and have studied the impact of exercise on animal behavior and physiology. Following this procedure, other methods, such as body composition analysis, stress tests, and fitness tests can be used to answer additional questions regarding the impacts of exercise treatments on the animals.

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