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, the REQS can be modified to accommodate other Drosophila Activity Monitor units as well.
1. Testing the Setup of the REQS
<ol>
	<li>Load test flies into glass specimen tubes by anesthetizing the animals with CO2 or other chosen method for handling, moving them into the empty tubes, and capping the tubes.</li>
	<li>Insert capped glass specimen tubes into the slots of the activity monitor unit. Secure the tubes using rubber O-rings (17-18 gauge) and/or rubber bands on both sides of the unit to ensure the tubes do not move during rotation:
	<ol>
		<li>Slip one O-ring over the specimen tube and position the O-ring close to the midpoint of the tube. Insert the specimen tube into a slot of the activity monitor from the front and center it.</li>
		<li>Move the O-ring as needed and ensure that once the specimen tube is centered, the O-ring is slid as close to the activity monitor unit as possible. Fix the specimen tube in place by slipping a second O-ring over the glass tube from the back, again moving the O-ring as close to the activity monitor unit as possible.</li>
	</ol>
	</li>
	<li>Initiate rotation of the REQS by flipping the power switch on the front of the unit. Adjust the rotation speed with the dial on the front of the unit to the desired speed (4 rpm (rotations per min) in this example). 
	NOTE: The rotational speed in this example was chosen to match a previously published study7. As different rotational speeds will result in different animal activity levels, when initiating a new study, optimization of rotational speed for the specific experimental set-up (genotype, length of exercise bout, etc.) may be necessary. 
	NOTE: It is important to ensure that the tubes are properly positioned so that they do not drag along the bottom of the REQS unit during rotation.</li>
	<li>Test the connection to the data collection system provided with the Drosophila Activity Monitor unit by opening the DAMSystem308 software and ensuring that the connection light remains green for several rotations. The software will begin recording data into a text file &#34;monitorX&#34; (X being the unit number if multiple monitors are in use) immediately upon initiation. Recording frequency can be adjusted in the &#34;Preferences&#34; tab; we typically record in 5 min intervals.</li>
	<li>Investigate the text file generated by the DAMSystem308 software to ensure that the data connection is functioning properly. Problems with the connection result in cutouts, time points where 0 activity is recorded for all positions in the REQS (see Representative Results and Table 2). If cutouts occur, adjust data connections leading to the rotating phone jack, as during rotation, this connection can become loose or twisted. We find that stabilizing the connection with tape helps to prevent this problem.</li>
</ol>
2. Preparation of Animals
NOTE: All animals were raised and tested under standard conditions in an incubator (25 &#176;C, 60-70% humidity, 12 h light/dark cycle) on molasses/cornmeal media10.
<ol>
	<li>Two weeks prior to the planned experiment, set up vials with controlled animal density to collect the experimental flies from; we typically set up vials with 7 males and 10 females. With a healthy stock, from a single vial, approximately 15 virgin males and 15 females can be collected over a 4 day time period. Adjust the number of vials set up based on the number of animals needed for the assay; a typical assay in our laboratory includes 10 sets of 10 virgin males and females per genotype (100 males and 100 females). 
	NOTE: Non-virgin flies can be used depending on the specific experimental question. If conducting longer-term experiments with non-virgin animals, crawling larvae may interfere with accurate activity monitoring.</li>
	<li>Remove the parent flies one week after setting up the vials.</li>
	<li>Starting on day 10 after setting up the vials, collect virgin flies from the vials and store them separated by sex.</li>
	<li>Collect sufficient animals for the experiment and age them as needed, 3 days in this example. 
	NOTE: We typically anesthetize the animals with CO2 for handling. CO2 is known to impact animal activity levels for extended periods of time after treatment (for example, see Bartholomew et al. 201511). If the CO2 effect interferes with planned downstream assays or analyses, use a different anesthesia method such as ice anesthetization.</li>
</ol>
3. Data Collection with the REQS
<ol>
	<li>Anesthetize the animals to be used for the exercise quantification study with CO2 or another anesthesia method. Divide them into groups as needed and load the groups of animals into the empty glass tubes of the activity monitor; in this example, 10 animals of the same age are loaded per glass tube, with 10 replicates for each animal type (sex/genotype). Make sure to note which animal type is loaded in each tube. 
	NOTE: For longer experiments, food can be included in the specimen tubes. In this case, it is essential that the food is secure and does not become dislodged during rotation.</li>
	<li>Load the glass assay tubes into the REQS unit and secure them with the rubber O-rings. If working with more than one sex/genotype, using a randomized block design or randomization of the position of the animals in the REQS will eliminate any potential position effects. Randomization can be achieved by assigning each vial a random number, for example from a web-based random number generator (e.g., https://www.randomizer.org/ ) or in a spreadsheet, and then ordering the vials based on the random number.</li>
	<li>Place the REQS into the incubator to ensure constant temperature, humidity, and light conditions. Ensure that both the data and power cables are properly connected.</li>
	<li>Allow the animals to recover from anesthesia and to acclimatize to the new environment for 1 h.</li>
	<li>Begin the experiment by initiating rotation of the REQS at the desired speed. 
	NOTE: Another option is to first collect baseline activity data from the animals before the initiation of rotation, followed by recording activity levels in response to rotational stimulation.</li>
	<li>Initiate data collection by opening the DAMsystem308 software (simply opening the software initiates data collection). Data are written into a text file at the set interval (here, 5 min); if needed, change interval settings in the &#34;Preferences&#34; tab (see step 1.4).</li>
	<li>To ensure that the data are successfully transmitted to the computer, open the text file generated by the DAMsystem308 software after one or two time intervals have passed and confirm that data are being written to the file. Close and re-open this file to see data from any additional time points. Collect data for the desired amount of time; in this example, 2 h. 
	NOTE: Do not open the incubator during the assay period, as the animals are very sensitive to any disturbance and will likely respond with increased activity.</li>
	<li>At the end of the exercise assay, terminate data collection by closing the DAMsystem308 software, then turn off the REQS. Remove the animals from the glass assay tubes and clean the tubes. The animals can be moved back into food vials if repeated measures at different ages are needed. 
	NOTE: If deaths occur during the exercise regime, it should be recorded, as the presence of dead flies can impact activity counts. In our experience with the DGRP2 fly lines12,13, exercise at 4 rpm for 2 h did not result in any deaths, but weaker strains might respond differently.</li>
</ol>
4. Data Analysis
<ol>
	<li>Open the .txt file labeled &#34;Monitor1&#34; produced by the DAMSystem308 software.</li>
	<li>Inspect the data file for any problems that might have occurred during the data acquisition (missed time points, etc.; Table 1). If necessary, censor the file by removing data points at the beginning and end of the recording.</li>
	<li>Using the statistical software of your choice (e.g., R), analyze the data collected. Generate descriptive statistics and carry out analysis of variance (ANOVA) to investigate effects such as sex and genotype, if the data are normally distributed. If the data are not normally distributed, use non-parametric methods, such as Kruskal-Wallis tests, to compare groups. The specific analyses required will depend on the specific scientific question and the experimental design.</li>
</ol>

<ol>
	<li>Rieder, L. E., Larschan, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wisdom+from+the+fly.">Wisdom from the fly.</a> Trends Genet. 30 (11), 479-481 (2014).</li><li>Ugur, B., Chen, K., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+tools+and+assays+for+the+study+of+human+diseases.">Drosophila tools and assays for the study of human diseases.</a> Dis Model Mech. 9 (3), 235-244 (2016).</li><li>Hales, K. G., Korey, C. A., Larracuente, A. M., Roberts, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+on+the+fly:+A+primer+on+the+Drosophila+Model+System.">Genetics on the fly: A primer on the Drosophila Model System.</a> Genetics. 201 (3), 815-842 (2015).</li><li>Piazza, N., Gosangi, B., Devilla, S., Arking, R., Wessells, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise-training+in+young+Drosophila+melanogaster+reduces+age-related+decline+in+mobility+and+cardiac+performance.">Exercise-training in young Drosophila melanogaster reduces age-related decline in mobility and cardiac performance.</a> PLoS One. 4 (6), e5886 (2009).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp Gerontol. 40 (5), 386-395 (2005).</li><li>Tinkerhess, M. J., Ginzberg, S., Piazza, N., Wessells, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endurance+training+protocol+and+longitudinal+performance+assays+for+Drosophila+melanogaster.">Endurance training protocol and longitudinal performance assays for Drosophila melanogaster.</a> J Vis Exp. (61), (2012).</li><li>Mendez, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TreadWheel:+A+novel+apparatus+to+measure+genetic+variation+in+response+to+gently+induced+exercise+for+Drosophila.">The TreadWheel: A novel apparatus to measure genetic variation in response to gently induced exercise for Drosophila.</a> PLoS One. 11 (10), e0164706 (2016).</li><li>Katzenberger, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+of+closed+head+traumatic+brain+injury.">A Drosophila model of closed head traumatic brain injury.</a> Proc Natl Acad Sci U S A. 110 (44), E4152-E4159 (2013).</li><li>Watanabe, L. P., Riddle, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Rotating+Exercise+Quantification+System+(REQS),+a+novel+Drosophila+exercise+quantification+apparatus.">Characterization of the Rotating Exercise Quantification System (REQS), a novel Drosophila exercise quantification apparatus.</a> PLoS One. 12 (10), e0185090 (2017).</li><li>Reed, L. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype-by-diet+interactions+drive+metabolic+phenotype+variation+in+Drosophila+melanogaster.">Genotype-by-diet interactions drive metabolic phenotype variation in Drosophila melanogaster.</a> Genetics. 185 (3), 1009-1019 (2010).</li><li>Bartholomew, N. R., Burdett, J. M., VandenBrooks, J. M., Quinlan, M. C., Call, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+climbing+and+flight+behaviour+in+Drosophila+melanogaster+following+carbon+dioxide+anaesthesia.">Impaired climbing and flight behaviour in Drosophila melanogaster following carbon dioxide anaesthesia.</a> Sci Rep. 5, 15298 (2015).</li><li>Huang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+variation+in+genome+architecture+among+205+Drosophila+melanogaster+Genetic+Reference+Panel+lines.">Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines.</a> Genome Res. 24 (7), 1193-1208 (2014).</li><li>Mackay, T. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+melanogaster+Genetic+Reference+Panel.">The Drosophila melanogaster Genetic Reference Panel.</a> Nature. 482 (7384), 173-178 (2012).</li><li>Berlandi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swing+Boat:+Inducing+and+recording+locomotor+activity+in+a+Drosophila+melanogaster+model+of+Alzheimer's+disease.">Swing Boat: Inducing and recording locomotor activity in a Drosophila melanogaster model of Alzheimer's disease.</a> Front Behav Neurosci. 11, 159 (2017).</li><li>Faville, R., Kottler, B., Goodhill, G. J., Shaw, P. 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The authors have nothing to disclose.

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.

<table><tbody><tr><td>Drosophila Activity Monitor&amp;nbsp;</td><td>Trikinetics</td><td>LAM25H</td><td>REQS component</td></tr><tr><td>Telephone Cord Detangler</td><td>Uvital</td><td>uv20170719</td><td>REQS component</td></tr><tr><td>Vial closures (flugs)</td><td>Genesee Scientific</td><td>49-102</td><td>Drosophila culture supplies</td></tr><tr><td>Vials&amp;nbsp;</td><td>Genesee Scientific</td><td>32-120</td><td>Drosophila culture supplies</td></tr><tr><td>Drosophila culture netting</td><td>Carolina Biological Supply</td><td>173090</td><td>Drosophila culture supplies</td></tr><tr><td>Cornmeal</td><td>Pepsico</td><td>43375</td><td>Drosophila media</td></tr><tr><td>Molasses</td><td>Golden Barrel</td><td>BLA-GAL</td><td>Drosophila media</td></tr><tr><td>Agar</td><td>Apex Bioresearch</td><td>66-103</td><td>Drosophila media</td></tr><tr><td>Inactive Dry Yeast</td><td>Genesee Scientific</td><td>62-106</td><td>Drosophila media</td></tr><tr><td>Tegosept</td><td>Apex Bioresearch</td><td>20-258</td><td>Drosophila media</td></tr><tr><td>Propionic acid</td><td>Genesee Scientific</td><td>20-271</td><td>Drosophila media</td></tr></tbody></table>

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 ...

measuring-exercise-levels-drosophila-melanogaster-using-rotating

Universidad San Sebastian

Research

JoVE Journal

Genetics

7.4K Views.  University of Alabama at Birmingham. 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.

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

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

Neuroscience

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly&#39;s innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies&#39; innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health.

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

Behavior

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders such as diabetes and cardiovascular disease 1-3. An important topic of research into these associated health problems involves the role of endurance exercise as a beneficial intervention.
Exercise training is an inexpensive, non-invasive intervention with several beneficial results, including reduction in excess body fat 4, increased insulin sensitivity in skeletal muscle 5, increased anti-inflammatory and antioxidative responses 6, and improved contractile capacity in cardiomyocytes 7. Low intensity exercise is known to increase mitochondrial activity and biogenesis in humans 8 and mice, with the transcriptional coactivator PGC1-&alpha; as an important intermediate 9,10. 
Despite the importance of exercise as a tool for combating several important age-related diseases, extensive longitudinal genetic studies have been impeded by the lack of an endurance training protocol for a short-lived genetic model species. The variety of genetic tools available for use with Drosophila, together with its short lifespan and inexpensive maintenance, make it an appealing model for further study of these genetic mechanisms. With this in mind we have developed a novel apparatus, known as the Power Tower, for large scale exercise-training in Drosophila melanogaster 11. The Power Tower utilizes the flies' instinctive negative geotaxis behavior to repetitively induce rapid climbing. Each time the machine lifts, then drops, the platform of flies, the flies are induced to climb. Flies continue to respond as long as the machine is in operation or until they become too fatigued to respond. Thus, the researcher can use this machine to provide simultaneous training to large numbers of age-matched and genetically identical flies. Additionally, we describe associated assays useful to track longitudinal progress of fly cohorts during training.

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders ...

Biology

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Tracking Freely Walking Flies: A Method to Assess Locomotion in Drosophila

Source: Sun, M. and Chen, L. L. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/55602/" target="_blank">A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila</a>. J. Vis. Exp. (2017).
This video describes how to track freely walking flies to assess their general locomotion ability. The featured protocol demonstrates the assay&#39;s setup and data analysis using video tracking software.

Source: Sun, M. and Chen, L. L. A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila. J. Vis. Exp. (2017).
This video describes how to ...

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

Summary

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Chapters in this video

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

A Simple Technique to Assay Locomotor Activity in Drosophila

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Measurement of Metabolic Rate in Drosophila using Respirometry

Measurement of Larval Activity in the Drosophila Activity Monitor

Tracking Freely Walking Flies: A Method to Assess Locomotion in Drosophila