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.

1. Fly rearing and collection
<ol>
	<li>Maintain flies at 25 &#176;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.</li>
	<li>Transfer the flies every 2-3 days.</li>
	<li>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.</li>
	<li>Allow the flies to recover at 25 &#176;C for at least 24 h. 
	NOTE: By the time of the experiment, flies should be 1-4 days old. The frequency of collections described in step 1.3 can be set to narrow the age range of the flies.</li>
</ol>
2. Setup and assembly of respirometer chamber
<ol>
	<li>Turn on the water bath and set it to the desired temperature for the experiment. 
	NOTE: The experiments below were conducted at 25 &#176;C using 50 mL Schlenk tubes as chambers. Components are to be assembled as shown in Figures 1A, 1B, and 1C.</li>
	<li>Clean the ground glass joints of chambers and sensor plugs thoroughly by spraying 70% ethanol onto a laboratory wipe (not directly onto the joint) and wiping dust and old grease from the sensor plug (Figure 1A). Wipe off ethanol with a fresh laboratory wipe.</li>
	<li>Place 1 cm piece of cotton roll soaked in purified water into the bottom of the chamber to stabilize the humidity.
	<ol>
		<li>Add enough water (~0.5 mL) to form a small pool at the bottom of the cotton roll.</li>
	</ol>
	</li>
	<li>Wipe off any water that has spilled onto the joint of the chamber.</li>
	<li>Transfer the flies to labeled polypropylene tubes using a funnel.
	<ol>
		<li>Plug the tube with a cotton roll. 
		NOTE: Tubes consist of a 5 mL polypropylene test tube, trimmed to 5.5 cm in length and perforated with a hot knife to allow the free exchange of air with the experimental chamber. CO2 anesthesia is known to cause metabolic abnormalities, so flies are transferred without anesthesia which requires more care to avoid losing the flies.</li>
	</ol>
	</li>
	<li>Add one ventilated tube with flies into each respirometer chamber (on top of wet cotton).</li>
	<li>Fill soda lime cartridges (4-5 pellets per tube) and place them on the top of the tube containing flies inside the chamber. 
	NOTE: Soda lime cartridges consist of 800 &#181;L centrifuge tubes perforated 4-5 times with a power drill.</li>
	<li>Fill O2 generators with saturated copper sulfate (CuSO4) solution below level of vent holes 
	NOTE: O2 generators consist of screw-cap centrifuge tubes with 4 holes drilled below the threads. Platinum (Pt) and Copper (Cu) electrodes are soldered to two-pin connector, inserted into holes drilled in cap, and affixed with epoxy. Electrolysis of CuSO4 generates the O2 consumed by the experimental organism. CuSO4 is toxic to invertebrates, avoid spills or leakage and clean up immediately.</li>
	<li>Connect the filled O2 generator to two-pin connector on the sensor plug. 
	NOTE: The copper cathode must connect with the negative output of the controller and the platinum anode to the positive wire. Reversed connections will cause the failure of the experiment.</li>
	<li>Place two small dabs of clear silicone grease on opposite sides of the ground glass joint of the sensor plug.</li>
	<li>Insert the plug into the chamber and rotate the plug (or chamber) with moderate pressure to spread the grease in the joint.
	<ol>
		<li>Wipe off excess grease with a laboratory wipe.</li>
	</ol>
	</li>
	<li>Snap plastic Keck clamps onto joints to secure plugs in chambers. The assembled chamber should look like Figure 1C.</li>
	<li>Repeat the above steps for the number of chambers used for the day&#39;s experiment. 
	NOTE: The number of chambers that can be recorded is limited by the number of available chambers, controllers and USB inputs to the computer. For the present experiments, seven chambers were normally run in parallel. Experimental flies such as mutants should be matched with appropriate controls. A chamber set up identically but without flies should be included in each experiment as a control for environmental variation. Chambers containing different treatments (mutant, wildtype, no-fly) should be rotated between experiments.</li>
	<li>Place assembled chambers into a rack in the water bath with stopcocks open (Figure 1E). 
	NOTE: To avoid circadian variation, chambers were placed into the bath between 9:30 and 9:50 am for all experiments described here.</li>
	<li>Leave stopcocks open (Keep the handle parallel to the stopcock). 
	NOTE: Be careful not to allow water to enter the stopcocks.</li>
	<li>Allow the chambers to equilibrate with stopcocks open for about 30 min. 
	&#8203;NOTE: While the chamber is equilibrated, connect the electronics and set up data acquisition as described below.</li>
</ol>
3. Setting up controllers and computer
<ol>
	<li>Be sure that the switches supplying current to the O2 generators are in the OFF position (away from the connector; Figure 1D).</li>
	<li>Plug each controller box into an available Universal serial bus (USB) port. 
	NOTE: Construction and programming of controller units described elsewhere12.</li>
	<li>Connect controllers to respirometer chambers using 6-conductor cables.</li>
	<li>Check that the organic light emitting diode (OLED) displays of the controllers (Figure 1D) are displaying environmental parameters.</li>
	<li>Briefly turn on O2 generators using the switch on the controller (Figure 1D).
	<ol>
		<li>If the current value increases from zero to between 35 and 55 mA, the controller and chamber are ready for experiments.</li>
	</ol>
	</li>
	<li>Determine which COM ports are being used by the controllers, as described below.,
	<ol>
		<li>Click the Start Icon in Microsoft Windows.</li>
		<li>Click the Settings Icon.</li>
		<li>Click Bluetooth and Devices.</li>
		<li>Ensure that the controllers and their COM ports appear in the list of devices.</li>
	</ol>
	</li>
	<li>Open PuTTY on the desktop and set up a log file for each channel of the respirometer as described below. 
	NOTE: PuTTY is a free secure shell and telnet client that is used to transfer data to the computer via COM ports.
	<ol>
		<li>Select COM port for a controller by typing the number of the port in the &#34;Serial line&#34; box (Figure 2A).</li>
		<li>Click on Logging.</li>
		<li>Select Printable Output in &#34;Session logging&#34; (Figure 2B).</li>
		<li>Under Log File Name click Browse.</li>
		<li>In the folder of the choice, create a filename containing descriptive information (e.g., date, species, COM port number).</li>
		<li>Click Save.</li>
		<li>Click Open. A window will open showing comma-delimited data being logged (Figure 2C).</li>
		<li>Repeat for all other controllers in use for the experiment. Input to each COM port will appear as a separate window (Figure 2D).</li>
	</ol>
	</li>
</ol>
4. Running experiments
<ol>
	<li>Once chambers have equilibrated for 30 min, seal them by closing stopcocks.</li>
	<li>Cover the bath and chambers with a polystyrene foam box to maintain a stable environment.</li>
	<li>Allow to equilibrate for another hour.</li>
	<li>Turn on the current to the O2 generator of each chamber using the switch on the controller box.</li>
	<li>Once the O2 generators are activated, ensure that the pressure increases to pre-set OFF pressure. 
	NOTE: 1017 hPa, which is slightly above atmospheric pressure, was used as the &#34;OFF&#34; pressure in this series of experiments. Return to the ambient pressure will indicate leakage of gas from the chambers. Further, it allows the same pressure to be used across experiments regardless of ambient barometric pressure. The &#34;ON&#34; pressure was 1016 hPa, meaning that pressure only needed to drop 1 hPa before the O2 generator was activated. This provided adequate sensitivity to measure O2 consumption in Drosophila. Once a chamber is pressurized to the &#34;OFF&#34; setting, current should drop to zero.</li>
	<li>Let the experiment run for 3 or more h. 
	NOTE: Higher VO2 at&#160;elevated temperatures can allow for shorter experiment times. Monitor occasionally to ensure that equipment is functioning but avoid excessive activity near the chambers that may affect temperature stability.</li>
</ol>
5. Finishing experiment
<ol>
	<li>Turn off O2 generators on all controllers. 
	NOTE: Do first to avoid running the O2 generators while the chambers are open.</li>
	<li>Open the stopcocks to unseal the chambers.</li>
	<li>Leave the PuTTY windows open for another 5-15 min to provide a final baseline.</li>
	<li>Close the PuTTY window for each controller, ending recordings. 
	&#8203;NOTE: All experiments ended between 4:50 and 5:10 pm.</li>
	<li>Disconnect sensors from cables.</li>
	<li>Move chambers to dry rack.</li>
	<li>Remove sensor plugs one at a time from the chambers.</li>
	<li>Disconnect the O2 generators and place them in the tube rack.</li>
	<li>Wipe grease off the sensor plug and keep it in the rack.</li>
	<li>Clean grease from chamber joints and remove tubes with flies and soda lime.</li>
	<li>Anesthetize flies in each tube with CO2, tap onto a weight boat and weigh on a microbalance.
	<ol>
		<li>Log the weight and number of flies for each tube.</li>
	</ol>
	</li>
	<li>Discard flies or set them aside for additional procedures.</li>
	<li>Dump soda lime from cartridges into the waste container.</li>
	<li>Open the O2 generator and discard the CuSO4 solution into the waste container.
	<ol>
		<li>Rinse electrodes and tube with purified water.</li>
		<li>Place the tube racks for drying.</li>
	</ol>
	</li>
</ol>
6. Analysis of charge transfer data
<ol>
	<li>Import Data as comma-delimited text into a spreadsheet, with each record comprising a separate worksheet.</li>
	<li>Record the current and time data for each pulse of the O2 generator. Starting with the first pulse after the chamber was pressurized, record the start time and end time (as row numbers) of each current pulse. That is the row number when the current goes above zero (usually to about 45-50 mA) to the last row that is above zero.</li>
	<li>Make a table on the worksheet to record the following data:
	<ol>
		<li>The average current amplitude during the pulse: = AVERAGE([first row of pulse]:[last row of pulse]) for each pulse (from the current column).</li>
		<li>Pulse duration: ([Last row of pulse] - [first row of pulse[-one row]])/1000 for each pulse (from the time in milliseconds column).</li>
		<li>Total experiment time: [time at start of last pulse] - [time at end of first pulse after chamber pressurized] (from the time in minutes column).</li>
	</ol>
	</li>
	<li>Then calculate charge transfer (Q) for each pulse (average current X duration)</li>
	<li>Sum the charge from all pulses to calculate Total Charge (Qtot).</li>
</ol>
7. Analysis of O2 consumption
<ol>
	<li>Set up a new spreadsheet for all data and enter or calculate the following for each chamber:
	<ol>
		<li>Qtot (total charge)</li>
		<li>Moles (= Q &#247; 96485 &#215; 4)</li>
		<li>mL O2 (= moles &#215; 22413 mL/mol)</li>
		<li>Total time (from the data analysis above)</li>
		<li>mL min-1 (= ml O2 &#247; total time)</li>
		<li>Weight in grams (flies anesthetized and weighed measured after the experiment)</li>
		<li>mL min-1 g-1 (= mL min-1 &#247; weight in grams)</li>
		<li>mL/h/g (the above &#215; 60)</li>
		<li>mg/fly (= weight of flies &#247; number of flies)</li>
		<li>&#956;L fly-1 h-1 (= (mL min-1 &#215; 3600) &#247; number of flies).</li>
	</ol>
	</li>
	<li>Tabulate data for each treatment (genotype, e.g.)</li>
	<li>Compare treatments using ANOVA, t-test, or Mann-Whitney u-test 13.</li>
</ol>

Adapting Microrespirometry for &lt;em&gt;Drosophila&lt;/em&gt; - Insights into CASK Mutant Metabolism

<ol>
	<li>Arking, R., Buck, S., Wells, R. A., Pretzlaff, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+rates+in+genetically+based+long+lived+strains+of+Drosophila.">Metabolic rates in genetically based long lived strains of Drosophila.</a> Experimental Gerontology. 23 (1), 59-76 (1988).</li><li>Henry, Y., Overgaard, J., Colinet, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+nutrient+balance+shapes+phenotypic+traits+of+Drosophila+melanogaster+in+interaction+with+gut+microbiota.">Dietary nutrient balance shapes phenotypic traits of Drosophila melanogaster in interaction with gut microbiota.</a> Comparative Biochemistry and Physiology Part A: Molecular &amp; Integrative Physiology. 241, 110626 (2020).</li><li>Burggren, W., Souder, B. M., Ho, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+rate+and+hypoxia+tolerance+are+affected+by+group+interactions+and+sex+in+the+fruit+fly+(Drosophila+melanogaster):+new+data+and+a+literature+survey.">Metabolic rate and hypoxia tolerance are affected by group interactions and sex in the fruit fly (Drosophila melanogaster): new data and a literature survey.</a> Biology Open. 6, 471-480 (2017).</li><li>Lighton, J. R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Measuring Metabolic Rates. , (2019).</li><li>Fiorino, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallelized,+real-time,+metabolic-rate+measurements+from+individual+Drosophila.">Parallelized, real-time, metabolic-rate measurements from individual Drosophila.</a> Scientific Reports. 8 (1), 14452 (2018).</li><li>Berrigan, D., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+temperature+and+activity+on+the+rate+of+adult+Drosophila+melanogaster.">Influence of temperature and activity on the rate of adult Drosophila melanogaster.</a> Comparative Biochemistry and Physiology. 118 (4), 1301-1307 (1997).</li><li>Hulbert, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+rate+is+not+reduced+by+dietary-restriction+or+by+lowered+insulin/IGF-1+signalling+and+is+not+correlated+with+individual+lifespan+in+Drosophila+melanogaster.">Metabolic rate is not reduced by dietary-restriction or by lowered insulin/IGF-1 signalling and is not correlated with individual lifespan in Drosophila melanogaster.</a> Experimental Gerontology. 39 (8), 1137-1143 (2004).</li><li>Botero, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofibromin+regulates+metabolic+rate+via+neuronal+mechanisms+in+Drosophila.">Neurofibromin regulates metabolic rate via neuronal mechanisms in Drosophila.</a> Nature Communications. 12 (1), 4285 (2021).</li><li>Yatsenko, A. S., Marrone, A. K., Kucherenko, M. M., Shcherbata, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Metabolic+Rate+in+Drosophila+using+Respirometry.">Measurement of Metabolic Rate in Drosophila using Respirometry.</a> Journal of Visualized Experiments. (88), 51681 (2014).</li><li>Ross, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-specific+decrease+in+aerobic+efficiency+associated+with+increase+in+oxygen+free+radical+production+in+Drosophila+melanogaster.">Age-specific decrease in aerobic efficiency associated with increase in oxygen free radical production in Drosophila melanogaster.</a> Journal of Insect Physiology. 46 (11), 1477-1480 (2000).</li><li>Brown, E. B., Klok, J., Keene, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+metabolic+rate+in+single+flies+during+sleep+and+waking+states+via+indirect+calorimetry.">Measuring metabolic rate in single flies during sleep and waking states via indirect calorimetry.</a> Journal of Neuroscience Methods. 376, 109606 (2022).</li><li>Sandstrom, D. J., Offord, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+oxygen+consumption+in+Tenebrio+molitor+using+a+sensitive,+inexpensive,+sensor-based+coulometric+microrespirometer.">Measurement of oxygen consumption in Tenebrio molitor using a sensitive, inexpensive, sensor-based coulometric microrespirometer.</a> Journal of Experimental Biology. 225 (9), jeb243966 (2022).</li><li>Becker, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+dysfunction+in+CASK-related+neurodevelopmental+disorders.">Presynaptic dysfunction in CASK-related neurodevelopmental disorders.</a> Translational Psychiatry. 10 (1), 312 (2020).</li><li>Slawson, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+Regulation+of+Locomotor+Behavior+of+Drosophila+melanogaster+Depends+on+a+CASK+Isoform+Containing+CaMK-Like+and+L27+Domains.">Central Regulation of Locomotor Behavior of Drosophila melanogaster Depends on a CASK Isoform Containing CaMK-Like and L27 Domains.</a> Genetics. 187 (1), 171-184 (2011).</li><li>Slawson, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+dopamine+release+by+CASK-&#206;2+modulates+locomotor+initiation+in+Drosophila+melanogaster.">Regulation of dopamine release by CASK-&#206;2 modulates locomotor initiation in Drosophila melanogaster.</a> Frontiers in Behavioral Neuroscience. 8, (2014).</li><li>Andrew, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+motor-behavior+abnormalities+in+two+Drosophila+models+of+neurodevelopmental+disorders.">Spontaneous motor-behavior abnormalities in two Drosophila models of neurodevelopmental disorders.</a> Journal of Neurogenetics. 35 (1), 1-22 (2021).</li><li>Ueno, T., Tomita, J., Kume, S., Kume, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+Modulates+Metabolic+Rate+and+Temperature+Sensitivity+in+Drosophila+melanogaster.">Dopamine Modulates Metabolic Rate and Temperature Sensitivity in Drosophila melanogaster.</a> PLoS ONE. 7 (2), e31513 (2012).</li><li>Van Voorhies, W. A., Khazaeli, A. A., Curtsinger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+correlation+between+body+mass+and+metabolic+rate+in+Drosophila+melanogaster.">Lack of correlation between body mass and metabolic rate in Drosophila melanogaster.</a> Journal of Insect Physiology. 50 (5), 445-453 (2004).</li><li>Norton, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeation+of+gases+through+solids.">Permeation of gases through solids.</a> Journal of Applied Physics. 28 (1), 34-39 (1957).</li><li>Hoegh-Guldberg, O., Manahan, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coulometric+measurement+of+oxygen-consumption+during+development+of+marine+invertebrate+embryos+and+larvae.">Coulometric measurement of oxygen-consumption during development of marine invertebrate embryos and larvae.</a> Journal of Experimental Biology. 198 (1), 19-30 (1995).</li><li>Sohal, R. S., Agarwal, A., Agarwal, S., Orr, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+overexpression+of+copper-+and+zinc-containing+superoxide+dismutase+and+catalase+retards+age-related+oxidative+damage+and+increases+metabolic+potential+in+Drosophila+melanogaster.">Simultaneous overexpression of copper- and zinc-containing superoxide dismutase and catalase retards age-related oxidative damage and increases metabolic potential in Drosophila melanogaster.</a> Journal of Biological Chemistry. 270 (26), 15671-15674 (1995).</li><li>Van Voorhies, W. A., Melvin, R. G., Ballard, J. W. O., Williams, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+manometric+microrespirometers+for+measuring+oxygen+consumption+in+small+arthropods.">Validation of manometric microrespirometers for measuring oxygen consumption in small arthropods.</a> Journal of Insect Physiology. 54 (7), 1132-1137 (2008).</li><li>Herreid, C. F., Full, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cockroaches+on+a+treadmill:+Aerobic+running.">Cockroaches on a treadmill: Aerobic running.</a> Journal of Insect Physiology. 30 (5), 395-403 (1984).</li></ol>

The authors declare no conflicts of interest.

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...

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 of O2 consumption (VO2), CO2 production or both. Although direct calorimetry has been applied to small ectotherms, including Drosophila melanogaster5, respirometry is technically simpler and more commonly used.
Several forms of respirometry have been used successfully to measure metabolic rate in wildtype and mutant D. melanogaster and have provided insight into the metabolic effects of temperature6, social environment3, diet3,7,and neurodevelopmental disorders8. These fall into two classes, which vary considerably in cost and complexity. Manometry is the simplest and least expensive9,10, in which flies are placed into a sealed chamber that contains a CO2 absorbent and which is connected via a thin capillary to a fluid reservoir. As O2 is consumed and CO2 absorbed, pressure in the chamber decreases and fluid is drawn into the capillary. The fluid-filled volume of the capillary is therefore proportional to VO2. More elaborate versions, which compensate for the force exerted by the fluid in the capillary, have also been used on D. melanogaster1. Manometry has the advantages of being simple and inexpensive, but, because it is sensitive to pressure, requires constant environmental conditions. Further, because consumed O2 is not replaced, the partial pressure of O2 (PO2) gradually decreases inside the chambers.
Respirometry using gas analysis is also regularly used for D. melanogaster. In this case, gases are sampled at regular intervals from sealed chambers containing flies and sent to an infrared analyzer2,6,11. This type of apparatus has the advantages that it is available commercially, is less sensitive to environmental conditions, and gases are refreshed during sampling so that PO2 remains stable. However, the equipment can be expensive and complex to operate.
A recently developed coulometric microrespirometer12 provides an inexpensive, sensitive, and stable alternative to existing systems. In practice, an organism is placed into an airtight chamber where it consumes O2 and the exhaled CO2 is removed by an absorbent material, resulting in a net decrease in chamber pressure. When the internal pressure decreases to a pre-set threshold (ON-threshold), current is passed through an electrolytic O2 generator, returning pressure to a second threshold (OFF-threshold) stopping electrolysis. Charge transfer across the O2 generator is directly proportional to the amount of O2 required to re-pressurize the chamber and can therefore be used to measure the O2 consumed by the organism4. The method is highly sensitive, measures VO2 precisely, and the regular replacement of O2 can maintain PO2 at a nearly constant level for hours or days.
The coulometric microrespirometer used in this study employs a multi-modal (pressure, temperature, and humidity) electronic sensor. The sensor is operated by a microcontroller that detects small changes in pressure and activates O2 generation when a low pressure threshold is reached12. This apparatus is assembled from off the shelf parts, can be used with a wide variety of chambers and experimental environments, and has been employed successfully to examine the effects of body mass and temperature on the beetle Tenebrio molitor. In the present study, the microrespirometer has been adapted to measure O2 consumption in D. melanogaster, which has approximately 1% of the mass of T. molitor. Sensitivity of the apparatus has been increased by reducing the threshold for activating O2 generation, and environmental stability has been enhanced by conducting experiments in a temperature-controlled water bath and by maintaining humidity inside the chambers at or near 100%.
The CASK (Calmodulin-dependent Serine Protein Kinase) protein, part of the family of membrane-associated guanylate kinases (MAGUK), is a molecular scaffold in different multi-protein complexes, and mutations in CASK are associated with neurodevelopmental disorders in humans and in D. melanogaster13,14. A viable D. melanogaster mutant, CASK&#916;18, disrupts activity of dopaminergic neurons15 and reduces activity levels by more than 50% compared to congenic controls14,16. Because of the reduced activity levels of CASK mutants and the role of catecholamines in regulating metabolism17 we hypothesized that their standard metabolic rate, and therefore O2 consumption, would be dramatically reduced compared to controls.
O2 consumption was measured in CASK&#916;18 and their wildtype congeners, w(ex33). Groups of flies were placed into respirometry chambers, O2 consumption was measured, O2 consumption was calculated and expressed on both a mass-specific and per-fly basis. The apparatus recorded VO2 in wildtype flies that was consistent with previous studies, and it could differentiate between the per-fly O2 consumption of wildtype and CASK mutant flies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>19/22 Thermometer Adapter</td>
			<td>Wilmad-Labglass</td>
			<td>ML-280-702</td>
			<td>Sensor Plug</td>
		</tr>
		<tr>
			<td>2 ml Screwcap Tubes</td>
			<td>Fisher</td>
			<td>3464</td>
			<td>O2 generator</td>
		</tr>
		<tr>
			<td>2-Pin Connector</td>
			<td>Zyamy</td>
			<td>40PIN-RFB10</td>
			<td>O2 generator: cut to 2-pin</td>
		</tr>
		<tr>
			<td>4-Pin Female Connector</td>
			<td>TE Connectivity</td>
			<td>215299-4</td>
			<td>Sensor Plug</td>
		</tr>
		<tr>
			<td>5 ml Polypropylene Tube</td>
			<td>Falcon</td>
			<td>352063</td>
			<td>Cut to 5.5 cm and perforated&#160;</td>
		</tr>
		<tr>
			<td>50 ml Schlenk Tube 19/22 Joint</td>
			<td>Laboy</td>
			<td>HMF050804</td>
			<td>Chamber</td>
		</tr>
		<tr>
			<td>6-Conductor Cable</td>
			<td>Zenith</td>
			<td>6-Conductor 26 ga</td>
			<td>Cable</td>
		</tr>
		<tr>
			<td>6-Pin Female Bulkhead Connector</td>
			<td>Switchcraft</td>
			<td>17982-6SG-300</td>
			<td>Controller</td>
		</tr>
		<tr>
			<td>6-Pin Female Connector</td>
			<td>Switchcraft</td>
			<td>18982-6SG-522</td>
			<td>Sensor plug</td>
		</tr>
		<tr>
			<td>6-Pin Male Connector</td>
			<td>Switchcraft</td>
			<td>16982-6PG-522</td>
			<td>Cable</td>
		</tr>
		<tr>
			<td>800 ul centrifuge tube</td>
			<td>Fisher</td>
			<td>05-408-120</td>
			<td>Soda Lime Cartridge</td>
		</tr>
		<tr>
			<td>ABS Plastic Enclosure</td>
			<td>Bud Industries</td>
			<td>PS-11533-G</td>
			<td>Controller</td>
		</tr>
		<tr>
			<td>Arduino Nano Every</td>
			<td>Arduino LLC</td>
			<td>ABX00028</td>
			<td>Controller</td>
		</tr>
		<tr>
			<td>BME 280 Sensor</td>
			<td>DIYMall</td>
			<td>FZ1639-BME280</td>
			<td>Sensor Plug</td>
		</tr>
		<tr>
			<td>Circuit Board</td>
			<td>Lheng</td>
			<td>5 X 7 cm</td>
			<td>Controller</td>
		</tr>
		<tr>
			<td>Copper Sulfate</td>
			<td>BioPharm</td>
			<td>BC2045</td>
			<td>O2 Generator</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Azulle</td>
			<td>Byte4</td>
			<td>Data Acquisition</td>
		</tr>
		<tr>
			<td>Cotton Rolls</td>
			<td>Kajukajudo</td>
			<td>#2</td>
			<td>Cut in half to plug fly tubes 
			Cut in quarters for humidity</td>
		</tr>
		<tr>
			<td>Environmental Chamber</td>
			<td>Percival</td>
			<td>I30 VLC8</td>
			<td>Fly Care</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>JB Weld</td>
			<td>Plastic Bonder</td>
			<td>Secure Electrodes in O2 Generator</td>
		</tr>
		<tr>
			<td>Fly Food</td>
			<td>Lab Express</td>
			<td>Type R</td>
			<td>Fly Care</td>
		</tr>
		<tr>
			<td>Keck Clamps</td>
			<td>uxcell</td>
			<td>a20092300ux0418</td>
			<td>Secures glass joint of chamber to plug</td>
		</tr>
		<tr>
			<td>Low-Viscosity Epoxy</td>
			<td>Loctite</td>
			<td>E-30CL</td>
			<td>Sensor Plug</td>
		</tr>
		<tr>
			<td>OLED Display</td>
			<td>IZOKEE</td>
			<td>IZKE31-IIC-WH-3</td>
			<td>Controller</td>
		</tr>
		<tr>
			<td>Platinum Wire 24 ga</td>
			<td>uGems</td>
			<td>14349</td>
			<td>O2 generator</td>
		</tr>
		<tr>
			<td>Silicone grease</td>
			<td>Dow-Corning</td>
			<td>High Vacuum Grease</td>
			<td>Seals chamber-plug connection</td>
		</tr>
		<tr>
			<td>Soda Lime</td>
			<td>Jorvet</td>
			<td>JO553</td>
			<td>CO2 absorption</td>
		</tr>
		<tr>
			<td>Toggle Switch</td>
			<td>E-Switch</td>
			<td>100SP1T1B1M1QEH</td>
			<td>Controller</td>
		</tr>
		<tr>
			<td>USB Cable</td>
			<td>Sabrent</td>
			<td>CB-UM63</td>
			<td>Controller</td>
		</tr>
		<tr>
			<td>USB Hub</td>
			<td>Atolla</td>
			<td>Hub 3.0</td>
			<td>Connect controllers to computer</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Amersham</td>
			<td>56-1165-33</td>
			<td>Temperature Control</td>
		</tr>
		<tr>
			<td>Water Bath Tank</td>
			<td>Glass Cages</td>
			<td>15-liter rimless acrylic</td>
			<td>Bath for Respirometers</td>
		</tr>
	</tbody>
</table>

measuring o2 consumption in drosophila melanogaster using coulometric microrespirometry

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.

Author Spotlight: Influence of Temperature on Drosophila melanogaster and Desert-Adapted Beetles 

Measuring O2 Consumption in Drosophila melanogaster Using Coulometric Microrespirometry

Our research is focused on how temperature and activity affect insect metabolism. This is essential to understand how species have evolved to survive in their surroundings, and how they will continue to adapt in a changing world. Understanding the physiology and behavior of individual organisms is crucial to this endeavor.Metabolic rate can be measured directly by measuring heat production, or indirectly by measuring the consumption of oxygen or the production of carbon dioxide. Although it is technically possible to measure heat production by an organism as small as a fruit fly, indirect methods are used more commonly. The current challenges to measuring metabolism on this scale will include cost, environmental stability, and reliability.Commercially available systems are reliable, stable, and relatively easy to purchase, but they can be complex and expensive. DIY manometers are inexpensive, but can introduce error and require fabrication for each experiment. The coulometric respirometer is relatively inexpensive, easy to use, provides a convenient readout of environmental parameters, and all components are reusable.In addition, the regular replacement of oxygen maintains a stable environment for hours or even days. In the lab, we'll be studying the metabolic variation between established control strains of drosophila, and the effects of hyper and hypoactive mutants on oxygen consumption. In the field, we'll begin studying the metabolic rates and temperature tolerance of wild, desert-adapted beetles.

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...

Watch this Scientific Journal Video about Influence of Temperature on Drosophila melanogaster and Desert-Adapted Beetles at JoVE.com

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 ...

measuring-o2-consumption-drosophila-melanogaster-using-coulometric

Research

JoVE Journal

Neuroscience

Using a Respiratory Chamber to Measure Metabolic Rate in Drosophila melanogaster

Using a Respiratory Chamber to Measure Metabolic Rate in Drosophila melanogaster  

Begin by maintaining the flies at a temperature of 25 degrees Celsius, in narrow vials filled with standard drosophila food. Turn on the water bath and adjust the temperature as required. Use a laboratory wipe sprayed with 70%ethanol to clean the ground glass joints of the chambers and sensor plugs, and then use a fresh wipe to remove any remaining ethanol.Next, place a one centimeter piece of a cotton roll, soaked in purified water, at the bottom of the chamber to create a stable, humidity environment. Add sufficient water to form a modest pool at the base of the cotton roll. Ensure the joint of the chamber is dry by wiping any spilled water.Carefully transfer the flies into labeled polypropylene tubes using a funnel, and plug the tubes with a cotton roll. Now, add a ventilated tube containing flies into each respirometer chamber. Fill the soda lime cartridges and position them at the top of the tube that contains flies inside the chamber.Then fill the oxygen generators with a saturated solution of copper sulfate, ensuring that the level remains below the vent holes. Connect the filled oxygen generator to the two-pin connector located on the sensor plug. Place two small dabs of clear silicone grease on opposite sides of the ground glass joint of the sensor plug.Insert the plug into the chamber and gently rotate it with moderate pressure to evenly distribute the grease within the joint. Wipe off any excess grease using a laboratory wipe. Then secure the chamber plugs by snapping on the plastic keck clamps.Place the assembled chambers into a rack in the water bath, ensuring the stopcocks are open. Maintain stopcocks in the open position and let the chambers equilibrate for approximately 30 minutes. Ensure that the switches supplying current to the oxygen generators are off.Connect the controllers to the respirometer chambers using the six conductor cables, and verify that the organic light emitting diode displays on the controllers actively display environmental parameters. Now activate the oxygen generators using the switch on the controller. Once the current value rises from zero to between 35 and 55 milliamperes, the controller and chamber are ready for the experiments.After turning off the oxygen generators, identify the communication ports that the controllers are using. Open the PuTTY software on the desk desktop and set up a log file for each channel of the respirometer by selecting the communication port for the controller by entering the number of that port in the serial line box. Then click logging.Choose principal output in the session logging and under the log file name, click browse. In the chosen folder, create a file name that contains descriptive information. Then click save.Click open, and a window showing common delimited data being logged will open. Repeat this process for all other controllers being used. Each communication port input will be displayed in a separate window.After equilibrating the chambers for 30 minutes, seal them by closing the stopcocks. Cover the bath and the chambers with a polystyrene foam box to create a stable environment and equilibrate for an hour. Initiate the current to the oxygen generator of each chamber using the switch located on the controller box.Once the oxygen generators are activated, verify that the pressure increases to the predefined off pressure. After the experiment, turn off the oxygen generators on all controllers and open the stopcocks to unseal the chambers. Allow the PuTTY windows to remain open for five to 15 minutes to establish a final baseline, and then end the recordings by closing the PuTTY window for each controller.Disconnect the sensors from the cables and transport the chambers to the dry rack. One by one, remove the sensor plugs from the chambers. Disconnect the oxygen generators and carefully place them in the tube rack.Then wipe the grease off the sensor plug and place it back in the rack. Clean any grease from the chamber joints and remove the tubes containing flies and soda lime. Finally log the weight and the number of flies for each tube.Quantitative oxygen consumption data for wild type and cask delta 18 mutant flies showed that mass specific oxygen consumption was not significantly different among wild type controls and cask delta 18 mutants. Oxygen consumption analyzed on a per fly basis showed that the oxygen consumption was significantly reduced in cask delta 18 compared to wild type controls. However, the mean mass of cask delta 18 flies was lower than that of wild type controls.

606 Views.  University of Maryland, College Park, Universities at Shady Grove.  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.  

Adapting Microrespirometry for Drosophila - Insights into CASK Mutant Metabolism

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