Measuring O2 Consumption in Drosophila melanogaster Using Coulometric Microrespirometry

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 food vials.
   NOTE: Males were used to reduce variability due to reproductive status. The method applies to both sexes.
4. Allow the flies t....

Results

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.......

Discussion

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 others^3,6.

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

Materials

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