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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

Measuring O2 Consumption in Drosophila melanogaster Using Coulometric Microrespirometry

Published: July 7th, 2023

DOI:

10.3791/65379

1Department of Biology, University of Maryland, College Park, Universities at Shady Grove
* These authors contributed equally

Coulometric respirometry is ideal for measuring the metabolic rate of small organisms. When adapted for Drosophila melanogaster in the present study, measured O2 consumption was within the range reported for wildtype D. melanogaster by previous studies. Per-fly O2 consumption by CASK mutants, which are smaller and less active, was significantly lower than the wildtype.

Coulometric microrespirometry is a straightforward, inexpensive method for measuring the O2 consumption of small organisms while maintaining a stable environment. A coulometric microrespirometer consists of an airtight chamber in which O2 is consumed and the CO2 produced by the organism is removed by an absorbent medium. The resulting pressure decrease triggers electrolytic O2 production, and the amount of O2 produced is measured by recording the amount of charge used to generate it. In the present study, the method has been adapted to Drosophila melanogaster tested in small groups, with the sensitivity of the apparatus and the environmental conditions optimized for high stability. The amount of O2 consumed by wildtype flies in this apparatus is consistent with that measured by previous studies. Mass-specific O2 consumption by CASK mutants, which are smaller and known to be less active, was not different from congenic controls. However, the small size of CASK mutants resulted in a significant reduction in O2 consumption on a per-fly basis. Therefore, the microrespirometer is capable of measuring O2 consumption in D. melanogaster, can distinguish modest differences between genotypes, and adds a versatile tool for measuring metabolic rates.

The ability to measure metabolic rate is crucial for a complete understanding of an organism in its environmental context. For example, it is necessary to measure metabolic rate in order to understand its role in lifespan1, the role of diet in metabolism2, or the threshold for hypoxic stress3.

There are two general approaches to measuring the metabolic rate4. Direct calorimetry measures energy expenditure directly by measuring heat production. Indirect calorimetry measures energy production through other means, often via respirometric measurement....

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1. Fly rearing and collection

  1. Maintain flies at 25 °C in narrow vials containing standard Drosophila food.
    NOTE: The sample size for each genotype should comprise at least nine replicates, each consisting of a single respirometer chamber containing 15-25 flies, set up as described below.
  2. Transfer the flies every 2-3 days.
  3. Anesthetize flies with CO2, collect groups of 15-25 males of each genotype, and place each group into fresh, unyeasted fo.......

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The pressure and current outputs of the respirometer controller are shown for one chamber in one experiment in Figure 3A. The first, long current pulse pressurized the chamber from ambient pressure (approximately 992 hPa) to the pre-set OFF threshold of 1017 hPa. As the flies consumed O2 and CO2 was absorbed, pressure decreased slowly until it reached the ON threshold of 1016 hPa, which activated current through the O2 generator. In the example shown, the ave.......

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The above procedure demonstrates measurement of O2 consumption in D. Melanogaster using an electronic coulometric microrespirometer. The resulting data for O2 consumption in wild-type D. melanogaster were within the ranges described in most previous publications using diverse methods (Table 1) although somewhat lower than that reported by others3,6.

Critical steps addressed the t.......

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We thank Dr. Linda Restifo at the University of Arizona for suggesting testing the O2 consumption of CASK mutants and for sending CASK mutants and their congenic controls. Publication fees were provided by the Departmental Reinvestment Fund from the Biology Department at the University of College Park. Space and some equipment were provided by the Universities at Shady Grove.

....

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Name Company Catalog Number Comments
19/22 Thermometer Adapter Wilmad-Labglass ML-280-702 Sensor Plug
2 ml Screwcap Tubes Fisher 3464 O2 generator
2-Pin Connector Zyamy 40PIN-RFB10 O2 generator: cut to 2-pin
4-Pin Female Connector TE Connectivity 215299-4 Sensor Plug
5 ml Polypropylene Tube Falcon 352063 Cut to 5.5 cm and perforated 
50 ml Schlenk Tube 19/22 Joint Laboy HMF050804 Chamber
6-Conductor Cable Zenith 6-Conductor 26 ga Cable
6-Pin Female Bulkhead Connector Switchcraft 17982-6SG-300 Controller
6-Pin Female Connector Switchcraft 18982-6SG-522 Sensor plug
6-Pin Male Connector Switchcraft 16982-6PG-522 Cable
800 ul centrifuge tube Fisher 05-408-120 Soda Lime Cartridge
ABS Plastic Enclosure Bud Industries PS-11533-G Controller
Arduino Nano Every Arduino LLC ABX00028 Controller
BME 280 Sensor DIYMall FZ1639-BME280 Sensor Plug
Circuit Board Lheng 5 X 7 cm Controller
Copper Sulfate BioPharm BC2045 O2 Generator
Computer Azulle Byte4 Data Acquisition
Cotton Rolls Kajukajudo #2 Cut in half to plug fly tubes
Cut in quarters for humidity
Environmental Chamber Percival I30 VLC8 Fly Care
Epoxy JB Weld Plastic Bonder Secure Electrodes in O2 Generator
Fly Food Lab Express Type R Fly Care
Keck Clamps uxcell a20092300ux0418 Secures glass joint of chamber to plug
Low-Viscosity Epoxy Loctite E-30CL Sensor Plug
OLED Display IZOKEE IZKE31-IIC-WH-3 Controller
Platinum Wire 24 ga uGems 14349 O2 generator
Silicone grease Dow-Corning High Vacuum Grease Seals chamber-plug connection
Soda Lime Jorvet JO553 CO2 absorption
Toggle Switch E-Switch 100SP1T1B1M1QEH Controller
USB Cable Sabrent CB-UM63 Controller
USB Hub Atolla Hub 3.0 Connect controllers to computer
Water bath Amersham 56-1165-33 Temperature Control
Water Bath Tank Glass Cages 15-liter rimless acrylic Bath for Respirometers

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