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 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'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.
Childhood and adult obesity are growing epidemics in cultures that consume high caloric diets and remain idle for extended periods of time, which can lead to severe long-term consequences, including insulin resistance, chronic inflammation, and osteoarthritis1,2,3,4. The prevalence of these disorders continues to rise due to the growing imbalance in caloric intake and expenditure attributed to elevated consumption of fats and sugars and a primarily sedentary lifestyle5. Correspondingly, this energy imbalance has led to an increase in cases of Type 2 Diabetes Mellitus (T2DM) and Cardiovascular Disease (CVD)5. Individuals are at a higher risk of developing both disorders if they have been diagnosed with the disorder Metabolic Syndrome (MetS), whose symptoms include abdominal obesity and dyslipidemia2. MetS is shaped by the complex interactions between genotype and various environmental factors, such as diet and exercise6. Thus, to gain a full understanding of the underlying mechanisms of this complex disease, all of these factors should be considered.
When it comes to combating MetS, doctors first recommend undergoing lifestyle changes that include eating a healthy, well-balanced diet and physical activity2,7,8. Since effective medications are limited and gastric bypass surgery is costly and requires lifelong medical monitoring, pharmacological and surgical intervention is only recommended for severe cases and only in combination with these lifestyle changes3,7,8. While lifestyle interventions, such as exercise and dieting, can produce and maintain long-term weight loss goals, whether or not these corrective measures can fully ameliorate the negative effects associated with MetS needs further study7,8.
Mouse models have been used to examine the effects of exercise on metabolic diseases for years; however, the introduction of exercise to fly research on MetS is a relatively recent endeavor9,10,11,12. Flies provide the perfect vehicle to study exercise in a controlled laboratory setting, since they are easily manipulated, have a short lifespan, are inexpensive to maintain, and energy-related metabolic pathways are highly conserved between Drosophila and humans13. D. melanogaster genomes are well characterized, and there is a wealth of genetics tools available for use in Drosophila that can provide insight into various genotypes and genotype-by-environment interactions that could modulate the effect of exercise on organismal health14.
Current methods of exercising Drosophila invoke the fly's innate, negative geotaxis tendency, the behavioral instinct to climb upwards, to stimulate climbing in adults within their enclosures11,12,15. The Power Tower, one method to stimulate exercise in flies, systematically raises the fly enclosures vertically and then drops them back to the bench surface, effectively knocking the flies to the bottom of vial, thus inducing their instinctual negative geotaxis12,16. Experiments conducted using this machine showed that exercise is a powerful protective factor against many age-related diseases, including CVD and T2DM, and promotes healthy aging12,17,18. Specifically, they demonstrated that exercise can reduce the onset of age-related mobility decline in flies and improve multiple age-related symptoms, such as cardiac performance and stress response17,18. However, control flies never placed on the tower showed higher climbing scores than those experiencing the machine, suggesting the force of the repetitive drops may be injuring the flies and affecting mobility12. This suggests that an alternative method of inducing exercise that is less forceful and avoids causing physical trauma would be a useful, complementary method to the Power Tower protocol16.
To gently induce exercise in Drosophila, we developed an exercise machine called the TreadWheel (Figure 1). The TreadWheel's (henceforth abbreviated as TW) rotational motion triggers the flies' innate, negative geotaxis tendency by constantly redefining the gravitational top of the vials, which, in turn, stimulates climbing in the flies. Unlike other methods, the rotational motion of the TW is inherently gentle, which minimizes the number of additional stressors that may arise and impact the results.Thus, the machine provides the means to induce exercise in large numbers of flies without inducing stress, which will enable researchers to study the effects of exercise on metabolic health (Figure 2), aging, sleep, and many other topics11.
Our method follows an inverse pyramidal, interval training protocol, which largely incorporates aspects of aerobic interval training (AIT) with some endurance exercise training. The standard AIT regime is altered in this protocol to gradually increase the duration of each interval over a five-day period to promote endurance. AIT has been particularly useful in preventing MetS relative to other intervention methods and was more effective in reversing Metabolic Syndrome's common risk factors than continuous moderate exercise19,20. However, a disadvantage of the TW relative to the Power Tower is that flies more quickly habituate to the rotating motion, thus variation in innate tendency to habituate among the flies can complicate the interpretation of the benefits of exercise.11 An elegant solution to this limitation is described by Watanabe and Riddle15 and their complementary article21.
1. TreadWheel Setup and Operation
NOTE: See Supplemental Figure 1 for schematics on TW exercise machine construction. The parts required are listed in the Table of Materials. The phrases "TW", "TreadWheel", "exercise machine", and "machine" are used interchangeably throughout the protocol.
2. Fly Collection and Maintenance
NOTE: All flies are maintained in a 25 °C incubator with 50% humidity and a 12 h light/dark cycle between benchtop manipulations described below. Flies are fed a standard cornmeal-molasses lab diet unless otherwise noted.
3. Exercise Protocol
4. Climbing Assessment
5. Triglyceride Storage Assay
NOTE: The samples, standards, glycerol standard solution, and triglycerol working solution used throughout the assay should be kept on ice for the duration of the protocol and should be stored in the refrigerator when not in use.
We are especially interested in identifying the factors that contribute to the overall metabolic health of an individual. It was previously found that genotype-by-diet interactions contribute substantially to population level variation in metabolic traits14. This means that each genotype responds to environmental differences in a unique and complex manner. To extend our work on genotype-by-environment effects to include physical exercise, we developed the TreadWheel, which is capable to exposing large numbers of genotypes to aerobic interval training (AIT) in a high-throughput manner.
To establish whether exercise on the TW influenced metabolic traits, we measured triglyceride storage in Oregon-R (OreR) and y1w1 flies, common wildtype flies (Figure 2B–C), and normalized those values against the flies' protein concentration, as originally reported in Mendez et al.11. We analyzed the data by multivariate analysis of variance (MANOVA) accounting for genotype, sex, exercise treatment (and their interactions), and experimental block effects such as time replicates and food vial and found that there was a significant genotype-by-exercise interaction (p = 0.0017) affecting triglyceride storage. There was a significant sexual dimorphic effect between males and females, with males storing more triglycerides than females (p < 0.0001). We saw that in females, exercised flies had significantly lower triglyceride levels than their unexercised counterparts (Figure 2B, p <0.0001). In the males, while the decrease in triglyceride storage observed in Oregon-R exercised flies (as compared to controls) was not statistically significant, a significant difference in triglyceride storage was observed between the two separate lines (Figure 2C, p <0.0001). Note that while standardizing triglyceride concentration against the protein concentration provides insight on overall fly body composition ratios, comparison of direct triglyceride or protein concentration among different groups of flies can also provide specific information on the effect of exercise on these phenotypes individually.
Since variables like adult exercise, sex, and genotype are shown to affect triglyceride storage, it was expected that these factors also impact other phenotypes and interact with diet. We raised larvae from a representative wildtype Drosophila Genetic Reference Panel line (DGRP 153)25 on either a high fat or normal diet and induced exercise in adult flies for one week (Figure 2A). Subsequently, we performed a RING-like negative geotaxis assay to measure climbing ability. The climbing assay used differed from a standard RING assay; instead of a RING apparatus, vials with paraffin film covering the openings were used to house flies during the assay. Other aspects of the original RING assay, such as time between tapping the flies to the bottom of the vial and taking the photo, were retained22.
All treatments were repeated three separate times with a minimum of 59 individual flies per time replicate and treatment. Data was analyzed by MANOVA accounting for diet, genotype, sex, exercise treatment (and their interactions), as well as experimental block effects of time replicate, assay vial, and vial assay replicate. We found that the exercised females climbed significantly higher (p <0.005) when reared on the high fat diet than any of the other female treatments (Figure 2D). For the males, it was seen that exercise only improved climbing when males were raised on the normal diet, and those raised on the high fat diet showed no significant change (Figure 2E). We also found significant sexually dimorphic effects (p <0.0001) in climbing with males climbing higher than females. The surprising result of a decrease in climbing performance following exercise for females from the DGRP 153 line (Figure 2D) for females consuming a normal diet (p <0.0001) is an example of how this type of exercise may not be a uniformly positive intervention for all genotypes and could be contingent on other environmental factors. In Mendez et al.11, females from four other genetic lines tested for their climbing performance after having been raised on a normal diet all showed enhanced climbing ability with exercise training. This suggests that the response observed in DGRP 153 is genotype-specific and not a general property of the TW exercise treatment. The variability in response across sex, diet, and exercise treatment groups indicates that there are significant sex-by-diet-by-exercise interactions that affects the climbing ability of the line (p <0.0001).
Taken together, the results indicate that the impact of exercise on an adult fly's metabolic health can be a function of its sex, genotype, and larval diet. The phenotypic variation observed in response to genotype, environment variables (e.g., diet and exercise), and sex has also been observed in other studies11,12,14,15. Thus, the TW and Drosophila can be a powerful strategy to elucidate the genetic and environmental factors shaping metabolic health.
Figure 1: The TreadWheel exercise machine. (A) The machine holds 48 vials and has an adjustable speed feature. Here experiments were performed at 4 rpm. (B) Individual food vials containing the experimental flies were snapped into brackets attached to a rotating axle. (C) The distance between vial plug and food was 6 cm for exercise vials and 1 cm for control vials. The vials were then placed on the machine for exercise. (D) The 5 day inverse pyramid exercise regime was used to exercise adult flies. Each day, an additional five minutes was added to one of the exercise bouts to ramp up exercise intensity incrementally, which models endurance, interval training. This figure has been modified from Mendez et al.11. Please click here to view a larger version of this figure.
Figure 2: Visual methods and representative results. (A) Methods for testing larval diet and adult exercise interaction effects. Larvae were raised on high fat or normal laboratory diet and switched to normal food upon eclosion. Adult flies were separated by sex, placed into experimental groups and exercised for five consecutive days. Afterwards, a RING-like negative geotaxis climbing assay was conducted, and flies were frozen for triglyceride measurements. Representative triglyceride data are shown for two genetic lines, Oregon R and y1w1 for (B) females and (C) males. All flies were reared on a normal diet and exercised in adulthood. Levels with different letters are significantly different (p <0.05) using a post hoc Student's t-test. This figure contains a subset of data reported in Mendez et al.11. Representative climbing data are shown for line DGRP 153 for (D) females and (E) males. Each point represents the averaged climbing performance of 232 or more individuals across three independent time points. Error bars indicate one standard error. Please click here to view a larger version of this figure.
Supplemental Figure 1: A detailed schematic of the TreadWheel. (A) Diagram of the dual vial clamp structure used to attach vials to the machine. (B) Interior view of the machine's rotational drive system. (C) Parallel projection of the machine's front face. Please click here to view a larger version of this figure.
Supplemental Code File: TreadWheel.skp Please click here to download this file.
The exercise protocol detailed here has been shown to successfully stimulate gentle exercise in Drosophila and can be used to simulate endurance exercise in a controlled laboratory setting11. It should be noted that when initially developing the TreadWheel concept, we considered commercial products that might be modified to perform similarly (e.g., a laboratory rotisserie). However, we ultimately rejected this approach, choosing a custom-built design because the commercial equipment did not have a sufficiently low rotational speed (4 rpm) and lacked sufficient vial capacity for high-throughput sample generation.
The TW protocol itself can be adjusted to encompass a wide range of research topics. For example, adjusting the frequency and duration of the flies' exercise regimes can alter the intensities of the work-outs they receive. By extending the protocol longer than one week or to other age groups, it would be possible to study the effects of exercise on aging and various age-related diseases. We recommend that if an extended exercise approach is implemented, then at least one rest day per week should be added to allow for recovery. Performance and improvement was shown to be greater in regimes that included a recovery day than those regimes that used consecutive daily exercise12. Also, we would caution against extending exercise bout lengths past 30 min since habituation to the rotational motion can occur after long periods of continuous exercise11. We have also observed genetic variations in flies' motivation to maintain their activity levels on the TW11. Thus, if longer bouts are desired, consider monitoring the flies' activity levels, as presented by Watanabe and Riddle15,21 describing their modification of the TW concept.Other factors that could be varied include temperature and sleep-wake patterns, which are shown to also affect fly movements26,27.
There is a plethora of available analyses during and after protocol completion to address the effects of exercise at the physiological, behavioral, and molecular level, as well as the potential to unravel epigenetic effects of exercise. Additional metabolic assays (e.g., glucose, glycogen, and protein), cardiac performance, and inflammatory response measures can be used to further explore the effect exercise has on fly physiology and body composition12,14,24,28. Various behavioral changes associated with exercise induction, such as changes in feeding behavior, locomotor activity, and sleep, can also be measured using tools like the CAFE assay29 or activity monitoring devices12,15,30. Changes in gene expression and cellular respiration attributed to exercise can also be quantified using methods like qRT-PCR11 and respirometry31. Finally, Drosophila have valuable genetic resources available, such as the Drosophila Genetics Reference Panel 2 and the Drosophila Synthetic Population Resource that provide researchers the platform to perform quantitative genetic studies25,32. These tools enable mapping experiments, such as Genome-Wide Association Studies and Quantitative Trait Loci mapping, to identify candidate loci associated with exercise and diet.
Studies completed using the TreadWheel demonstrated that, on average, exercise decreases body weight, total triglyceride storage, and glycogen, while increasing protein content and climbing performance11. Additionally, there were variable responses to exercise across sexes and genotypes for body weight, as well as triglycerides, protein, glycogen, glucose, and activity levels11,15. While the variability of response to exercise and diet across genotypes and sex may be challenging to interpret and, at times, counterintuitive, it reflects the sources of real biological variation observed in natural populations. As we strive to understand the diversity of underlying factors contributing to the high rates of MetS, tools that facilitate disentangling the relative role of various contributing factors by testing these factors in model organisms will be critical to our ability to develop personalized prevention and treatment interventions. To fully assess the effectiveness of exercise, each of these factors and how they interact must be considered when conducting experiments and formulating conclusions.
The TW, like most other fly exercise machines, is limited in its ability to quantify fly motion. Recently, Watanabe and Riddle developed the Rotating Exercise Quantification System (REQS), a TW backbone modified with an activity monitoring unit (LAM25H) from the Drosophila Monitoring System series15,21. Like the TW, this system uses rotary motion to gently induce exercise while using the LAM25H arm to track and quantify fly activity, but it holds a smaller number of vials (32 vials) than the TW15. For high-throughput exercise studies, if activity quantification is not necessary or desired, the TW allows for a greater sample number. The TW design could also be modified from its current form to accommodate a greater number of vials. This system, along with other existing exercise methods, helped to establish that exercise is not only achievable in Drosophila but can also be studied to determine the effect of physical activity on a variety of physiological and molecular responses11,12,15. Thus, the TW, as a proven method of inducing gentle exercise in flies, can be used to probe a large variety of biological questions.
The authors have nothing to disclose.
We would like to thank Julie Jarnigan, Meredith Owens, Rachel Hill, Brandon Moye, Laura Mafla, Olivia Fish, and the rest of the Reed Lab for their help with fly husbandry and image processing. Sean Mendez built the original TreadWheel with assistance from the UAB machine shop. Funding for this study was provided by NIH-R01 GM 098856 to LKR and the Undergraduate Creativity and Research Academy at the University of Alabama to KEL and BJW.
Materials for TreadWheel Construction: | |||
Heavy Duty Vibration-Damping leveling Mount | McMaster-Carr | 60855K71 | Quantity: 4 |
Stainless Steel Ball Bearing | McMaster-Carr | 57155K306 | Quantity: 8 |
Plug-in Voltage Transformer (500MA, 120VAC input, 24 VAC output) | McMaster-Carr | 70235K16 | Quantity: 1 |
Compact Square-Face DC Gear motor | McMaster-Carr | 6409K23 | Quantity: 1 |
Tool Holder (clamps) | McMaster-Carr | 1723A22 | Quantity: 5 (10x) |
12L14 Carbon Steel Tight-Tolerance Rod | McMaster-Carr | 5227T24 | Quantity: 1 |
Set Screw Shaft Collar | McMaster-Carr | 6432K13 | Quantity: 8 |
Round-Belt Pulley | McMaster-Carr | 6284K51 | Quantity: 5 |
Dart Controls – 25 Max RPM, Electric AC DC Motor | McMaster-Carr | 13DV 1A | Quantity: 1 |
Materials for Fly Maintenace and Husbandry | |||
6 oz Square Bottom Bottles (polypropylene) | Genesee Scientific | 32-130 | Quantity: 1 |
35x10mm Petri Dishes | VWR | 82050-536 | Quantity: 1 |
Narrow Drosophila vials | Genesee Scientific | 32-116 | Quantity: 1 |
Flystuff Flypad | Genesee Scientific | 59-114 | Quantity: 1 |
Blowgun, Mini | Genesee Scientific | 54-104 | Quantity: 1 |
Materials for RING-like Assay: | |||
ImageJ software | NIH | https://imagej.nih.gov/ij/ | Quantity: 1 |
1 cM graph paper or drawn grid (at least 20 cM by 30 cM) | various | Quantity: 1 | |
digital camera with timer or smart phone with camera timer app | various | Quantity: 1 | |
Materials for Triglyceride Assay: | |||
Dewar Flask | VWR | 14200-960 | Quantity: 1 |
Serum Triglyceride Determination Kit | Sigma Aldrich | TRO100 | Quantity: 1 |
Cordless Pestle Motor | VWR | 47747-370 | Quantity: 1 |
Pestles | VWR | 47747-358 | Quantity: 1 |