Name: Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster (Video) | JoVE Journal
Uploaded: 2012-03-26T13:00:00.000+00:00
Duration: 9 min 49 s
Description: Learn an endurance training protocol for Drosophila melanogaster using an automated power tower device. This method enables longitudinal assessment of climbing ability and mobility improvements through behavioral assays.

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This work was supported by a grant from the NHLBI to RW.

1. Power Tower Setup and Operation
<ol>
 <li>The Power Tower is placed on a rectangular piece of plywood that is clamped to a table. A 4.5 rpm gear motor from Grainger rests on top of four 2"x6" boards and one ¾ inch plywood board. The motor, an AC/DC speed control, a fuse, and an on/off switch are wired together into a juncture box.</li>
 <li>To cool the motor, a hole 3 inches in diameter is cut into a vertical ½ inch piece of plywood. On one side, a 3-inch fan is attached to the plywood, and on the other, a 3-inch diameter metal cylinder is attached.</li>
 <li>A custom-made rotating arm with a roller mounted on the end is connected to the motor. The motor and attached arm rotate in a clockwise direction, pushing down one side of a bent ¾ inch square tube stock that is attached to the plywood with a door hinge. A wheel is attached to the top side of the tube stock below the platform to allow it to roll against the platform that is directly above it.</li>
 <li>The arm pushes down on one end of the tube stock and lifts the opposite end, raising a two-level platform (Figure 1D). When the roller on the arm clears the tube stock it allows the arm to return to the starting position, dropping the platform back down (Figure 1F).</li>
 <li>The platforms consist of racks that are placed on top of plywood and secured by bungee cords that are hooked onto screw eyes. The platforms slide up and down along drawer sliders that are attached to the plywood with brackets.</li>
 <li>Vials are placed into the racks and secured by a square grid screen that is held in place with bungee cords.</li>
 <li>A Styrofoam cushion is placed below the platform to dampen the shock of the drop.</li>
 <li>The power of the motor is adjusted such that a full rotational period is 15 seconds long.</li>
</ol>
2. Exercise Protocol
<ol>
 <li>Flies are housed in a 25 °C incubator with 50% humidity and a 12-hour light/dark cycle. Both exercise training and assessment of exercise capacity are conducted in a 25 °C temperature-controlled room.</li>
 <li>Flies are collected and age-matched within 1-2 days of each other. A minimum of 240 flies is required for longitudinal monitoring of endurance and negative geotaxis (climbing) ability, two physiological indicators that reflect the effects of exercise training. Longitudinal monitoring allows isolation of induced change relative to starting values for each cohort, without confounding effects from variation between cohorts. Flies for additional experiments should be collected as needed.</li>
 <li>After collection, flies are divided into 120 experimental flies, which will be subjected to the exercise regimen, and 120 control flies, which will not be exercised. The flies are stored in vials of 20 flies each. The vials of experimental flies are plugged with firm cellulose acetate Flugs and the vials of control (unexercised) flies are plugged with soft sponge stoppers, which can be pushed down to immobilize the flies while on the Power Tower. Sponge is used for immobilization because its adaptive size allows easy placement near the bottom of the vial. The softer sponge material also reduces injuries caused by the more solid Flugs.</li>
 <li>Flies on the Power Tower are stored and exercised in vials that contain 5 mL of food, which provides them with a softer landing than flies that are exercised in empty vials. The food is typically composed of 10% sucrose, 10% yeast, and 2% agar in water, although we do not observe flies to eat often during the course of a training session. If empty vials are used during training, the percentage of flies that suffer injuries is dramatically increased.</li>
 <li>The Power Tower is operated in a temperature-controlled room that is maintained at 25 °C. Experimental flies are placed on the Power Tower and made to climb. Control flies are also placed on the Power Tower, but the sponge stopper is pushed down into the vial approximately 3 mm above the flies in order to limit movement. This group serves as a control for any effects of the Power Tower that are unrelated to exercise. Although some room still exists for these flies to maneuver, we have repeatedly observed that flies under these conditions run very little and do not demonstrate a physiological response to exercise. It may also be desirable to include an additional control group that is not placed on the Power Tower at all.</li>
 <li>Flies are exercised five consecutive days each week, utilizing a ramped schedule (Figure 2). During week one the flies are exercised for 2 hours per session, week two for 2.5 hours per session, and week three for 3 hours per session. Although other regimens have been tested, this protocol produces consistent results across genotypes and under divergent conditions. This protocol is, however, subject to a wide array of possible variations that might suit a particular experiment.</li>
</ol>
3. Exercise and Locomotor Fatigue
<ol>
 <li>Time to fatigue during endurance exercise is a useful physiological indicator to confirm and/or quantitate exercise capacity of a given cohort. Fatigue assays can be performed longitudinally on cohorts that will be used in training experiments. Assays may be conducted either prior to training, after the completion of the training protocol, or both. Other intermediate time points can be added if charting incremental progress during training is desired. The fatigue assay can also be used to measure exercise endurance as a response to treatments other than the training protocol, such as diet alteration (Figure 3B).</li>
 <li>Fatigue assays should be conducted on a day when training is not taking place. Both the experimental and control flies are placed on the Power Tower in vials of 20 each and made to climb until fatigued. Fatigue is recognized as the failure to respond to a negative geotaxis stimulus with climbing behavior. Fatigue behavior is scored by visual observation.</li>
 <li>A vial of flies is considered "fatigued" when 5 or fewer flies are able to climb higher than 2 inches for four consecutive drops. Fatigued vials are removed from the Power Tower while it is still operating, and the time of removal is recorded. Times of removal can be plotted as a nested histogram, or as a "time-to-failure" plot. An example of data plotted as a "time-to-failure" plot is shown in Figure 3B.</li>
 <li>Vials are monitored at 10 minute intervals for a maximum of 10 hours (Figure 3B).</li>
</ol>
4. Exercise, Age, and Locomotor Ability
<ol>
 <li>Since this training protocol relies on induced running behavior, a primary assay for quantitating effects of training is to longitudinally measure change in average running speed during training. During the course of training, 120-fly cohorts of exercised and unexercised flies are tested daily for their response to a negative geotaxis stimulus. The RING technique described in Gargano et al. (2005) is used to measure the flies' climbing ability 12. Briefly, this technique measures average height climbed by a vial of flies during a defined time after induction of negative geotaxis. The height climbed during a defined time is equivalent to climbing speed. Measurements are performed before placing flies on the machine for daily training so as to avoid complications from fatigue after the day's run.</li>
 <li>In our standard protocol, flies are exercised for a total of three weeks and climbing ability is examined for a total of five weeks: three weeks during exercise-training and two weeks after cessation of exercise (Figure 2). This allows the experimenter to chart whether effects of exercise on mobility persist after cessation of the program.</li>
 <li>Four photographs of each cohort's daily RING performance are used for the analysis of climbing ability. Six vials are included in each photograph. Negative geotaxis is induced, and using a timed camera, a photograph is taken after a defined number of seconds. We typically use two seconds, although this can be varied depending on experimental conditions. For every photograph, each vial is divided into four quadrants of equal height, and image processing software is used to assign each fly a score based on the quadrant it reached within the allotted time period. Flies that climb to the topmost quadrant receive a score of 4, flies in the next highest quadrant receive a 3, flies in the second highest quadrant receive a 2, and flies in the lowest quadrant receive a 1. Flies that do not climb off of the bottom at all are given a score of 0.</li>
 <li>For each vial in a given photograph, a "climbing index" is generated by averaging the scores of all the flies in that vial. Each vial's indices from the four photographs are then averaged to give a final climbing index for that day.</li>
 <li>All negative geotaxis experiments are conducted longitudinally. Results are normalized to the initial "pre-exercise" score in order to reduce the effect of cohort variation in climbing ability and highlight the relative longitudinal change in a cohort's climbing capacity as the result of exercise training. The initial score is determined by the average of the first three days' climbing indices and is assigned a value of 1. Subsequent trials are then expressed as a percentage of the initial index.</li>
</ol>
5. Representative Results
Wild-type flies respond to an endurance protocol with a diminished age-related decrease in climbing ability that persists following the end of training, as reflected in longitudinal RING assays across five weeks of age (Figure 3A). This delayed decline in negative geotaxis is a standard phenotypic response that can serve as a positive control to ensure that wild-type exercise response is occurring normally. This data set is presented as an example of how the induction of exercise by the Power Tower program can be used as a behavioral input. The ability of various genetic or environmental factors to modulate this effect can then be assessed.
Conversely, the Power Tower can also be used as an output in various experimental designs. For example, genotype, diet, or other conditions can be varied. Then, the effect of these variations on exercise physiology can be tested using the Power Tower. Here, we show an example of this approach. When flies with a varying percentage of sucrose in their diets were tested for time to fatigue, increased sucrose content correlated with increased endurance capacity (Figure 3B).
<img alt="Static equilibrium experiment setup; torque apparatus; six configurations; mechanical physics study." src="/files/ftp_upload/3786/3786fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Operation of the Power Tower. (A-C) The motorized bent arm with attached roller rotates clockwise until it comes into contact with the bent square tube. (D,E) The arm pushes down on the bent square tube, causing the platform that is laden with vials of flies to lift. (F) As the arm clears the tube the platform is allowed to fall back down, forcing the flies to return to the bottom of the vial.
<img alt="Fly experiment diagram; exercise impact on fatigue; treatment and assay schedule." src="/files/ftp_upload/3786/3786fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Suggested Exercise Protocol. Flies subjected to the training protocol are made to exercise for five days each week under a three-week long ramped regimen that progressively increases the duration of exercise from the initial 2 hours by 30 minutes each week. Standard analyses include fatigue assays before and following the exercise program and RING assays from week 1 through week 5. All assays are performed in duplicate on an equal number of unexercised flies as a negative control. Other various physiological or biochemical tests can be conducted as determined by the researcher.
<img alt="Exercise impact graph; climbing index vs. day, percent failure vs. time; data analysis chart." src="/files/ftp_upload/3786/3786fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Endurance exercise alters multiple aspects of mobility. (A) RING assays performed longitudinally across ages in a single pair of male Y1W67C23 cohorts. Age-matched, genetically identical exercised and unexercised control flies were measured daily for average climbing speed. Results are expressed in terms of a climbing index that is normalized to the average climbing height across the first three days of measurement. Exercise-trained flies displayed a diminished age-related decline in negative geotaxis ability compared to age-matched unexercised siblings across ages (2-way ANOVA, p &lt; 0.005). (B) Fatigue assays conducted for 8 hours on age-matched female Canton S flies show a significant effect of dietary sucrose content on time to fatigue (Log-rank, p &lt; 0.0001). Prior to experiment, flies were fed a yeast/sucrose/agar diet, with 10% weight/volume yeast concentration, and a varying percentage of dietary sucrose. Five vials of 20 flies each were tested for each diet. Graph displays how many vials still have five or more flies running at a given time point. These results can be treated statistically and graphically as a survival (or time to failure) curve, with "failure" for a vial being defined as a point in time when less than five flies continue to respond to negative geotaxis stimulus. Note that many other possible study designs and statistical treatments are possible, and data treatment and measurement should be tailored to fit individual purposes.

<ol><li>Wild, S., Roglic, G., Green, A., Sicree, R., King, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+Prevalence+of+Diabetes%3A+Estimates+for+the+year+2000+and+projections+for+2030%5BTitle%5D)+AND+%22Diabetes+Care%22%5BJournal%5D)">Global Prevalence of Diabetes: Estimates for the year 2000 and projections for 2030.</a> Diabetes Care. 25, 1047-1053 (2004).</li>
<li>Flegal, K. M., Carroll, M. D., Ogden, L. C., Johnson, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevalence+and+Trends+in+Obesity+Among+US+Adults%5BTitle%5D)+AND+%22JAMA%22%5BJournal%5D)">Prevalence and Trends in Obesity Among US Adults.</a> JAMA. 288, 1723-1727 (1999).</li>
<li>Hubert, H. B., Feinleib, M., McNamar, P. M., Castelli, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Obesity+as+an+Independent+Risk+Factor+for+Cardiovascular+Disease%3A+A+26-year+Follow-up+of+Participants+in+the+Framingham+Heart+Study%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Obesity as an Independent Risk Factor for Cardiovascular Disease: A 26-year Follow-up of Participants in the Framingham Heart Study.</a> Circulation. 67, 698-977 (1983).</li>
<li>Ross, R., Dagnone, D., Jones, P. J. H., Smith, H., Paddags, A., Hudson, R., Janssen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduction+in+obesity+and+related+comorbid+conditions+after+diet-induced+weight+loss+or+exercise-induced+weight+loss+in+med+%C3%A2%E2%82%AC%E2%80%9C+A+randomized%2C+controlled+trial%5BTitle%5D)+AND+%22Annals+of+Internal+Medicine%22%5BJournal%5D)">Reduction in obesity and related comorbid conditions after diet-induced weight loss or exercise-induced weight loss in med â€“ A randomized, controlled trial.</a> Annals of Internal Medicine. 133, 92-103 (2000).</li>
<li>Goodyea, L. J., Kahn, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exercise+Glucose+Transport%2C+and+Insulin+Sensitivity%5BTitle%5D)+AND+%22Annual+Review+of+Medicine%22%5BJournal%5D)">Exercise Glucose Transport, and Insulin Sensitivity.</a> Annual Review of Medicine. 49, 235-261 (1998).</li>
<li>Linke, A., Adams, V., Schulze, P. D., Erbs, S., Gielen, S., Fiehn, E., Mobius-Winkler, S., Schubert, A., Schuler, G., Hambrecht, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antioxidative+Effects+of+Exercise+Training+in+Patients+With+Chronic+Heart+Failure%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Antioxidative Effects of Exercise Training in Patients With Chronic Heart Failure.</a> Circulation. 111, 1763-1763 (2005).</li>
<li>Kemi, O. J., Ellingsen, O., Smith, G. L., Wisloff, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exercise-induced+changes+in+calcium+handling+in+left+ventricular+cardiomyocytes%5BTitle%5D)+AND+%22Frontiers+in+Bioscience%22%5BJournal%5D)">Exercise-induced changes in calcium handling in left ventricular cardiomyocytes.</a> Frontiers in Bioscience. 13, 356-368 (2008).</li>
<li>Wang, H., Hiatt, W. R., Barstow, T. J., Brass, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relationships+between+muscle+mitochondrial+enzyme+activity+and+oxidative+capacity+in+man%3A+alterations+with+disease%5BTitle%5D)+AND+%22Eur.+J.+Appl.+Physiol%22%5BJournal%5D)">Relationships between muscle mitochondrial enzyme activity and oxidative capacity in man: alterations with disease.</a> Eur. J. Appl. Physiol. 80, 22-27 (1999).</li>
<li>Wu, Z., Puigserver, P., Andersson, U., Zhang, C., Adelmant, G., Mootha, V., Troy, A., Cinti, S., Lowell, B., Scarpulla, R. C., Spiegelman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+Controlling+Mitochondrial+Biogenesis+and+Respiration+through+the+Thermogenic+Coactivator+PGC-1%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Mechanisms Controlling Mitochondrial Biogenesis and Respiration through the Thermogenic Coactivator PGC-1.</a> Cell. 98, 115-124 (1999).</li>
<li>Goto, M., Terada, S., Kato, M., Katoh, M., Yokozeki, T., Tabata, I., Shimokawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((cDNA+Cloning+and+mRNA+Analysis+of+PGC-1+in+Epirtrochlearis+Muscle+in+Swimming-Exercised+Rats%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">cDNA Cloning and mRNA Analysis of PGC-1 in Epirtrochlearis Muscle in Swimming-Exercised Rats.</a> Biochemical and Biophysical Research Communications. , 274-350 (2000).</li>
<li>Piazza, N., Gosangi, B., Devilla, S., Arking, R., Wessells, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exercise-Training+in+Young+Drosophila+melanogaster+Reduces+Age-Related+Decline+in+Mobility+and+Cardiac+Performance%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Exercise-Training in Young Drosophila melanogaster Reduces Age-Related Decline in Mobility and Cardiac Performance.</a> PLoS ONE. 4, e5886-e5886 (2009).</li>
<li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+iterative+negative+geotaxis+%28RING%29%3A+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila%5BTitle%5D)+AND+%22Exp+Gerontol%22%5BJournal%5D)">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp Gerontol. 40, 386-395 (2005).</li>
</ol>

We have nothing to disclose.

The general protocol presented here has been successful in documenting physiological effects following training. However, several areas in this protocol are subject to modification to fit particular experimental needs. For example, the length of training and number of bouts could potentially be varied to make the program more or less challenging, as desired. The height of the container in which negative geotaxis ability is measured could be altered to increase the available area for improvement to be documented. Various methods of automating quantitation of climbing speed may also be applicable. In principle, any software program capable of distinguishing a fly from background can be used to speed the data gathering process.
Some aspects of the protocol should be modified only with great caution, however. For example, preliminary experiments strongly indicate that at least one day of rest per week tends to facilitate greater improvement than relentless daily exercise. Additionally, circadian rhythms and temperature are known to affect the movement of cold-blooded animals. The time of day that training takes place can be varied, but should always be consistent within particular groups under comparison, in order to avoid the possibility of confounding effects of circadian rhythms. Temperature control is also essential, and we recommend a dedicated room at constant temperature to house exercise equipment. Lastly, males and females must be reared and measured separately, in order to avoid the potential of confounding effects of fertility and sex differences in exercise capacity.
Potential applications of this methodology are limited only by the imagination of the researcher. In preliminary work, we have utilized this methodology in three broad applications:
<ol>
	<li>To measure the effect of endurance exercise on various aspects of wild type biology across ages.</li>
 <li>To measure the effect of endurance exercise on specific mutant phenotypes.</li>
 <li>To screen for genetic factors that are necessary to execute the benefits of exercise.</li>
</ol>
Each of these applications encompasses a wide variety of specific possibilities. Based on our preliminary experience, mutant phenotypes tend to vary with exercise level as much as they vary with diet. The use of invertebrate models to better understand the relationship between diet, exercise, and aging physiology is perhaps the most important general application of this protocol.

<table><tbody><tr><td>Dayton Gearmotor</td><td>Grainger</td><td>1LRA6A</td><td></td></tr><tr><td>Raco Electrical Box</td><td>Grainger</td><td>5A052</td><td></td></tr><tr><td>Raco Cover</td><td>Grainger</td><td>5A053</td><td></td></tr><tr><td>Cooper Bussmann Fuse</td><td>Grainger</td><td>6F043</td><td></td></tr><tr><td>Cooper Bussmann Fuse Holder</td><td>Grainger</td><td>1DD33</td><td></td></tr><tr><td>Carling Technologies Switch</td><td>Grainger</td><td>2X464</td><td></td></tr><tr><td>Dayton Control, AC/DC Speed</td><td>Grainger</td><td>4X796</td><td></td></tr><tr><td>Flugs for Narrow Plastic Vials</td><td>Genesee Scientific</td><td>49-102</td><td></td></tr></tbody></table>

endurance training protocol and longitudinal performance assays for drosophila melanogaster

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.

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 ...

The goal of the following experiment is to train Dsof and Melanogaster using a novel endurance exercise program and to assess the effects of exercise training. This is achieved by using an automated device that lifts and drops several vials of flies repetitively activating the fly's instinctive climbing response, and thus giving the flies a workout session. The flies are trained daily on a ramped schedule from two to three hours over a three week long training period.Next flies are subjected to various behavioral assays in order to measure the effects of exercise. Results show that exercise flies climb faster as measured by the rapidly induced negative geo axis or ring assay. The main advantage of this technique over existing methods like endurance training other animals is the extensive collection of tools available for manipulation of drosophila genetics.In the study of endurance exercise, We first had the idea for this method when we were conducting many single negative geo taxis assays. We wondered how long flies could keep climbing and if we could endurance train them in this way. The power tower is built on a rectangular piece of plywood that is clamped to a table.A 4.5 rotation per minute. Gear motor rests on several boards and is wired to a speed control, a fuse, and a power switch. In a juncture box, a three inch fan held in a metal tube is directed at the motor for cooling.Connected to the motor is a rotating arm with a roller. On the end, the motor and arm rotate pushing down one side of a bent rod that is attached to the plywood. With a door hinge on the other side of the rod, a wheel is attached so it can roll against a platform.It pushes up and down as the motor spins, the power of the motor is adjusted, such that a full rotational period is 15 seconds long On the platform, fly vial racks are attached by bungee cords, and the vials placed into these racks are secured with a screen that is also attached to the platform. By bungee cords, the platform is able to slide up and down along standard draw sliders and a styrofoam cushion dampens the shock of its drop. The power tower is operated in a temperature controlled room to match the conditions the animals are reared under.To run an exercise experiment, a minimum of 240 age matched male flies are required for longitudinal monitoring of endurance and negative geo axis or climbing ability. Maintain the flies in populations of 20 per vial. Divide the vials into two equally sized groups, one of which will be exercised, that group's vials should be plugged with flugs, whereas the control group should be plugged with sponge stoppers.A sponge is used for immobilization because its adaptive size allows for easy placement near the bottom of the vial. The softer sponge material also reduces injuries caused by the more solid flugs. The food vials should be replaced every other day before testing.During training, the flies remain in the food vials because if empty vials are used, many more flies suffer. Training related injuries. When loading the vials of control flies on the power tower, push down their sponge stoppers to about three millimeters above the food level, hence, limiting the flies.Movement for consistent results. Test both groups of flies five consecutive days per week, utilizing a ramped schedule that increases the time per session from two to three hours a day over a three week period. Of course, any regime could be employed.Consistency in treatment, time of day, and assay methods over a three week time span is essential. Careful scheduling and practice procedures reduce the possibility for error Fatigue. Assays can be performed longitudinally on flies before, after, or during the training regime.It can also be used to ass safer response to an environmental variable. Run the fatigue assay on a day in which training is not taking place. At the beginning of a day load both groups of flies on the power tower.Activate the apparatus and observe each vial once every 10 minutes. If on four consecutive drops, five or fewer flies in a vial climb higher than two inches, then that cohort of flies is considered fatigued. Remove the fatigued flies from the power tower while it is still operating and record the time it is removed.Observe all the vials for a maximum of 10 hours and compile the results. One assay for quantitating. The effects of training is a daily measurement of running speed, which can be performed throughout the training regime and beyond.To measure the long-term effect of training. To begin the climbing speed or ring assay first transfer flies into new empty vials and place a row of six vials into a vial rack. Rolled pieces of tape are used to secure the vials to the rack.Then set the rack in front of a light source, like an x-ray view box that will provide even illumination of the vials. Now set up a camera to photograph the rack so the position of each fly will be visible in each vial. Then tap the six vials to knock down all the flies to the bottom of the vials and take a timed photograph.Two seconds after the flies are all knocked down. If the camera's flash is used, it should be used consistently. Repeat this process four times for each set of vials before proceeding with the power tower.To analyze the data, score each photo individually using software to find the upper and lower boundaries of the vial. Then measure the climbing height for each fly. Then copy the climbing heights into excel.Each fly in each vial is given a score from zero to four corresponding to the height of the quadrant. They climb to four for the highest quadrant and zero for not climbing at all. For each vial, calculate the score for each of the 20 flies and take the average score of the cohort.This is called the climbing index. The average of each cohort's four climbing indices on that day is used for data analysis. If image processing software is not available, quadrant lines can be drawn onto the empty vials, and flies can be manually assigned Quadrant values while type flies responded to an endurance protocol with a diminished age-related decline in climbing ability.That persisted following the end of training as reflected in longitudinal ring assays across five weeks of age. Thus, induction of exercise by the power tower program can be used as a behavioral input and the ability of genetic or environmental factors to modulate this effect can be assessed. Conversely, the power tower can also be used as an output.For example, when flies with a varying percentage of sucrose in their diet, were tested for time to fatigue. Increased sucrose content correlated with increased endurance capacity. While attempting this procedure, it's important to remember to make sure that all fly cohorts are age matched, and that methods are performed consistently throughout the course of the experiment.After watching this video, you should have a good understanding of how to endurance, train, and assess flies of any genotype.

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Research

JoVE Journal

Biology

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement.  This article demonstrates how to do this using a high-resolution tracking system.

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

This video describes a simple method to quantify fly locomotion and shows the climbing assay performed.  The fly climbing assay is a general test of motor function which relies on a negative geotaxis response.

Fly Climbing Assay: A Method to Test General Locomotion in Drosophila melanogaster

Scientists use the Drosophila lifespan protocol to measure how different experimental conditions affect flies' longevity and generate survival curves in the process to quantify the experimental outcomes. The sample protocol features the assay being applied to study the effects on longevity from exposure to varying concentrations of endocrine disruptor chemicals, or EDCs, such as 17-α-ethinylestradiol (EE2).

Lifespan Protocol for Drosophila melanogaster: Generating Survival Curves to Identify Differences in Fly Longevity

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

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)

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies' 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 TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

Methods to Test Endocrine Disruption in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

The goal of this protocol is to provide flight exercise for the fruit fly Drosophila. Representative results show that flight exercise training results in an increased aerobic capacity and resistance to a high-fat diet.

FLEX: Flight Exercise Training Protocol for the Fruit Fly Drosophila

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Learn an endurance training protocol for Drosophila melanogaster using an automated power tower device. This method enables longitudinal assessment of climbing ability and mobility improvements through behavioral assays.

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

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