FLEX: Flight Exercise Training Protocol for the Fruit Fly Drosophila

Summary

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.

Abstract

Drosophila has recently become a popular model for studying obesity and related disorders. While it's relatively easy to induce metabolic disorders in Drosophila using specific diets, creating an exercise model for the flies is more challenging. Existing methods, such as the Power Tower and Treadwheel, utilize shaking or rotating vials to induce the flies to climb, leveraging their negative geotaxis response. However, continuous climbing for long periods is unnatural for the flies and can lead to exercise-related injuries and mortalities. This paper introduces a new exercise protocol that leverages the fly's natural ability to fly, offering a more natural, physiological, and injury-free exercise approach. The experiments show that a 5-day flight exercise regimen increases the fly's bioenergetic capacity and counteracts the adverse effects of a Western diet (WD), improving climbing behavior and reducing WD-associated mortality. This protocol enables the simultaneous training of a large number of flies to study the interaction between exercise and various factors, including diet, drugs, aging, and genetics. Together, this method can complement existing methods for studying the effects of exercise on fly physiology and behavior.

Introduction

Physical activity and exercise are the focus of intense research for their immediate and long-term health benefits¹. Due to its powerful systemic effect on metabolism, exercise is increasingly used to prevent and treat metabolic, cardiovascular, and neurological disorders^2,3,4. Despite the increasing evidence of the numerous health benefits associated with regular exercise, the exact molecular mechanisms underpinning those benefits remain poorly understood⁵. To understand these mechanisms, the use of innovative and high-throughput animal models is essential. While rodent models are commonly used to investigate the impact of exercise on metabolic diseases, such studies require significant costs, regulatory oversight, and specialized equipment^6,7.

Recently, Drosophila melanogaster has emerged as a new model for researching exercise biology (reviewed^8,9,10). Equipped with a wide array of potent genetic tools, a short life cycle, extensive progeny, and economical maintenance requirements, the fruit fly presents a valuable resource for investigating genetic, epigenetic, and transgenerational mechanisms. When modeling exercise in flies, current methods typically rely on negative geotaxis, the behavioral reaction to climb upwards^11,12.

One of the first automated exercise protocols utilized a special exercise apparatus named the Power Tower¹¹. The Power Tower uses a motor unit that repeatedly lifts vials with animals and then drops them down to induce the negative geotaxis response¹¹. Prolonged exercise in the Power Tower results in a significant reduction in age-related decline in mobility and cardiac performance¹³. Additionally, the Power Tower regimen can be stressful for flies, and excessive training intensity has been reported to cause injury or mortality¹¹.

To address this issue, another exercise paradigm called Treadwheel was developed¹⁴. The Treadwheel also leverages the negative geotaxis response of Drosophila, but instead of lifting and dropping flies as in the Power Tower, it inverts vials with animals. Unlike the Power Tower, this method is gentler on flies and prevents exercise-related injuries and mortality⁹. However, the flies in the Treadwheel could habituate to the rotating motion, which might complicate the interpretation of the results¹². To address this issue, a Locomotor Activity Monitor (LAM25H, Trikinetics) was added to measure activity during exercise, to quantify Drosophila activity, and account for habituation. Subsequently, the name of the system was updated to the Rotational Exercise Quantification System (REQS)¹².

Interestingly, very few studies took advantage of the natural flight abilities of Drosophila to model exercise. The predominant focus of flight research has been on the aerodynamics and biomechanical aspects of hovering and maneuvering in the air^15,16,17,18. Several studies have delved into the physiological and cellular mechanisms related to flight^19,20. In these studies, flight ability was assessed via the flight assay^21,22, where individual flies were released into a clear acrylic box and their ability to maintain steady elevation and flight was observed²³.

In one particular study, researchers examined the effects of induced flight exercise by placing flies in bottles attached to a vortex-type shaker. The shaker gently shook the bottles at random intervals between 5 min and 10 min, using a programmable timer¹⁹. This study reported that induced flight behavior accelerated senescence and the age-related loss of antioxidant capacity in flight muscles¹⁹. Building on the previous research, our laboratory has developed a new exercise protocol called FLEX (flight exercise) by integrating an automated negative geotaxis system with housing flies in large containers²⁴. For the FLEX training, flies were placed in 1-gallon clear plastic drum fish bowls strapped to a horizontal platform attached to a motor. The motor was controlled by a timer, which initiated motor revolutions every 5 min. Each revolution elevated the platform and then dropped it down, triggering the flies into flight. The FLEX was performed daily for 7 h for 5 days. No mortalities or injuries associated with exercise were observed²⁴. Moreover, a 5-day FLEX regimen in flies on the Western Diet (WD) markedly negated the adverse effects of the WD, improving survival, climbing behavior, and mitochondrial respiration²⁴. Together, this paper presents a novel exercise paradigm based on natural flight activity that does not cause injury. This is a high-throughput procedure that allows a large number of flies to be trained simultaneously to study the interaction between exercise, diet, drugs, aging, environment, and/or genetics. The protocol can be used to complement existing exercise methods to study the effects of physical activity on fly physiology and behavior.

Protocol

1. Exercise machine setup and operation

1. Build a machine at the University Machine Shop. Use a 35 mm x 35 mm x 1.4 mm rectangular aluminum base with an AC/DC motor and a 35 mm x 35 mm x 0.5 mm horizontal aluminum platform (Figure 1A).
2. For the conversion of rotary motion to linear motion, build a custom-made rotating round cam 15 cm in diameter mounted on the motor (Figure 1B). Attach a follower with a 2 cm roller to the bottom of the horizontal platform so that the platform is lifted by the follower and then dropped back to its original position once the follower roller clears the cam.
3. Adjust the speed of the motor with a rotary potentiometer such that a full rotational period is about 5,360 ms long.
   NOTE: Exact motor specifications, dimensions of follower and roller, as well as cam geometry, are not critical. Any motorized configuration that reliably lifts and drops the platform to dislodge the flies and permits user control over the drop frequency is acceptable.
4. Secure 1-gallon clear plastic drum fish bowls to the platform by bungee cords that are hooked onto screw eyes (Figure 1C).

2. FLEX protocol

1. Keep flies in a 24 °C incubator or room with 60% humidity and a 12 h light/dark cycle.
2. Collect age-matched flies within 3-4 days of hatching. After collection, randomly assign flies to exercise and sedentary groups. Use an equal number of flies for both the exercise and sedentary groups.
   NOTE: Flies may be housed at densities ranging from 30 to 400 per bowl, as this does not interfere with flight exercise. However, experimental and control groups should always be matched for number, sex, and age to ensure comparability.
3. Provide sedentary and controlled flies with 7 mL of formulation food. Secure food vials in the bowls with tape.
4. Transfer flies into the bowls using light and brief CO₂ anesthesia a day before starting the exercise regimen. Cover bowls' openings with a mesh secured with rubber bands.
   NOTE: Flies should be anesthetized briefly under light CO₂ on a pad. The exposure must remain short to avoid lasting behavioral effects; therefore, anesthesia should be performed 1 day before the exercise experiment.
5. Secure the bowls to the platform with bungee cords. Conduct the exercise training in a room with a temperature of 24 °C and a humidity of 60%. If the humidity in the room is not controlled, provide flies with access to water by placing vials of wet cotton into the fish bowls and securing vials to the bowl's wall with tape.
6. For control experiments (sham exercise), place an equal number of flies in a bowl positioned next to the exercise machine but not attached to the platform.
7. Exercise flies for 5 consecutive days.
   1. Control the motor with two timers (Figure 1D). The first timer turns the machine on and off at a specific time of the day. Begin exercise at 8 am and finish at 3 pm.
   2. Use the second timer to initiate a series of three motor revolutions, with each sequence occurring every 5 min. Each revolution will elevate the platform and then drop it down, triggering the flies into flight.
   3. Once the exercise treatment for a given day is completed, return the bowls to their incubators. This protocol can be easily modified to suit a particular experiment.
      NOTE: A humidity below 60% may cause an increase in mortality. A small number of flies may not initiate flight after the first drop, but typically begin flying after the second or third drop. While it is not feasible to track flight in every individual, the majority of flies engage in flight, and it is assumed that all flies eventually participate in exercise. In addition, the large container provides space for voluntary flight between drops.
8. At the end of the FLEX regimen, immobilize flies by placing the bowls in the cold room for 5 min. Then transfer flies to new food vials for further analyses of phenotypic characteristics or to microcentrifuge tubes for molecular and/or biochemical assay of interest.
   NOTE: Examples of further analyses include behavioral and biochemical assay and/or gene expression. Assays involving living flies will need to be conducted promptly in accordance with the specific goals of the phenotypic assay. The protocol can be paused here with samples for biochemistry/molecular biology frozen and stored appropriately, depending on the assessments to be performed. A summary of the protocol is provided as Table 1.

Results

The FLEX protocol was previously validated in a published work from our laboratory. In a study published, we showed that flight exercise mitigates the negative metabolic effects of a Western Diet (WD)²⁴. This diet was made on the basis of the standard Nutri-Fly Bloomington diet with the following additions of 15% Nutiva USDA Certified Organic, non-GMO, Red Palm Oil, 15% Sucrose, and 0.1 M NaCl.

Three-to-four-day old adult male fruit flies were randomly assigned to each ...

Discussion

The FLEX protocol provides a novel and physiologically relevant method for inducing aerobic exercise in Drosophila melanogaster through flight. Unlike traditional paradigms such as the Power Tower and TreadWheel, which rely on negative geotaxis and mechanical agitation, FLEX harnesses the fly's innate flight behavior. This approach minimizes exercise-induced injuries and stress responses, making it a safer and more biologically appropriate model for exercise biology.

Insects rely ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
17 x 100 mm 14 mL polypropylene culture tube	VWR	60818-689
1-gallon clear plastic drum fish bowls	Petco	N/A	Available from Petco
8 Outlet Surge Protector with 7-Day Digital Timer	BN-LINK	EGPH020028	Available from Amazon
AC/DC motor	Dayton	Model 12W368 with torque 80 IN-LBS
Bloomington Formulation diet	Nutri-Fly	66-112	Available from Genesee Scientific Inc., San Diego, CA
Kim-wipes	Fisher Scientific	06-666	Kimberly-Clark Professional 34120
Outlet Multifunctional Programmable Timer with Countdown and Infinite Short Cycle	NEARPOW	SPM10188153502	Available from Amazon