We would like to thank Max-Planck Society for funding our research.

1. Preparation of Respirometers
<ol>
	<li>Cut the 1,000 &#956;l pipette tip with a razor blade to allow insertion of the 50 &#956;l capillary micropipette, try to get the pipette tip as straight as possible.</li>
	<li>Place a piece of foam into the pipette and push it down in the pipette tip.</li>
	<li>Add a small amount of CO2 absorbent and contain it by a second piece of foam.</li>
	<li>Apply glue at the place where the micropipette is inserted into the pipette tip.</li>
	<li>Leave the respirometer overnight to allow the glue to dry. 
	A schematic of a respirometer is shown in Figure 1A.</li>
</ol>
2. Preparation of the Measurement Chamber
<ol>
	<li>Prepare the chamber solution by mixing water with eosin in a ratio that will result in visible colorization.</li>
	<li>Pour the eosin/water solution into the chamber.</li>
	<li>Label one of the sides of the chamber with a centimeter scale.</li>
</ol>
3. Placing Flies into Respirometers
<ol>
	<li>Label the individual respirometers with a marker.</li>
	<li>Anesthetize flies using an alternative method to CO2 and place 3-5 flies of the desired genotype inside each respirometer.</li>
	<li>Seal the respirometers tightly at the top using plasticine putty.</li>
	<li>Allow flies to recover from anesthetization for approximately 15 min.</li>
	<li>Prepare one respirometer without flies, which will be used as the atmospheric control.</li>
</ol>
4. Performing the Experiment
<ol>
	<li>Hang the respirometers in the chamber by attaching a 1.5 ml Eppendorf tube holder that is open on the top and the bottom at the top of the chamber.</li>
	<li>Insert respirometers with the micropipette tip down into the chamber allowing the tip to submerge into the colored solution.</li>
	<li>Add Vaseline between the lid cover and the chamber to provide stronger isolation from temperature and pressure fluctuations.</li>
	<li>Close the lid and allow the system to equilibrate for 15 min.</li>
	<li>Take a photograph of the chamber making sure that the level of liquid within each micropipette is visible and so is the scale (see example shown in Figure 1B).</li>
	<li>After 1-2 hr, take another picture.</li>
	<li>When experiment is finished, remove the flies from respirometers and weigh if desired or transfer them back to the vial if needed further.</li>
</ol>
5. Analysis of Results
<ol>
	<li>Open acquired images using ImageJ software11.</li>
	<li>Using the scale in each picture, set the pixel scaling in the software.</li>
	<li>Measure the distance (&#916;d) that the liquid traveled from a determined reference spot in images taken at the beginning (d1) and end of experiment (d2). A schematic example is shown in Figure 1C.</li>
	<li>Calculate the amount of produced CO2 (&#181;l/hr/fly) with the formula:</li>
</ol>
<img alt="Figure 1" fo:content-width="2in" src="/files/ftp_upload/51681/51681eq1.jpg" />
R= radius of micropipette tube in centimeters 
&#916;d= distance the liquid has moved up in the micropipette of test samples measured in centimeters 
&#916;c= distance the liquid has moved up in the micropipette of the negative control sample (without flies) 
n = number of flies used 
h= hours

<ol>
	<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=Testing+the+&amp;#34;rate+of+living&amp;#34;+model:+further+evidence+that+longevity+and+metabolic+rate+are+not+inversely+correlated+in+Drosophila+melanogaster.">Testing the &amp;#34;rate of living&amp;#34; model: further evidence that longevity and metabolic rate are not inversely correlated in Drosophila melanogaster.</a> J Appl Physiol. 97, 1915-1922 (2004).</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, 1477-1480 (2000).</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=Selected+contribution:+long-lived+Drosophila+melanogaster.+lines+exhibit+normal+metabolic+rates.">Selected contribution: long-lived Drosophila melanogaster. lines exhibit normal metabolic rates.</a> J Appl Physiol. 95, 2605-2613 (2003).</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, 1137-1143 (2004).</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, (2012).</li><li>Takeuchi, K., 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=Changes+in+temperature+preferences+and+energy+homeostasis+in+dystroglycan+mutants.">Changes in temperature preferences and energy homeostasis in dystroglycan mutants.</a> Science. 323, 1740-1743 (2009).</li><li>Kucherenko, M. M., Marrone, A. K., Rishko, V. M., Magliarelli Hde, F., 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=Stress+and+muscular+dystrophy:+a+genetic+screen+for+dystroglycan+and+dystrophin+interactors+in+Drosophila.+identifies+cellular+stress+response+components.">Stress and muscular dystrophy: a genetic screen for dystroglycan and dystrophin interactors in Drosophila. identifies cellular stress response components.</a> Developmental Biology. 352, 228-242 (2011).</li><li>Marrone, A. K., Kucherenko, M. M., Wiek, R., Gopfert, M. C., Shcherbata, H. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+and+metabolism+in+Drosophila+melanogaster:+genetic+correlations+between+life+span+and+age-specific+metabolic+rate+in+populations+artificially+selected+for+long+life.">Longevity and metabolism in Drosophila melanogaster: genetic correlations between life span and age-specific metabolic rate in populations artificially selected for long life.</a> Genetics. 169, 231-242 (2005).</li><li>Elia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+equivalents+of+CO2+and+their+importance+in+assessing+energy+expenditure+when+using+tracer+techniques.">Energy equivalents of CO2 and their importance in assessing energy expenditure when using tracer techniques.</a> The American Journal of Physiology. 260, (1991).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9, 676-682 (2012).</li><li>Bharucha, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epicurean+fly:+using+Drosophila+melanogaster.+to+study+metabolism.">The epicurean fly: using Drosophila melanogaster. to study metabolism.</a> Pediatric Research. 65, 132-137 (2009).</li><li>Rajan, A., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+flies+and+men:+insights+on+organismal+metabolism+from+fruit+flies.">Of flies and men: insights on organismal metabolism from fruit flies.</a> BMC Biology. 11, 38 (2013).</li></ol>

We have nothing to disclose.

In this protocol, we describe an inexpensive and reliable method for measuring CO2 production in flies. We found that this experiment is easy, quick to conduct and generates reproducible data that is in agreement with other studies1,6,9. The protocol outlined here can be easily modified to fit any laboratory&#39;s budget and available materials. The construction of each individual respirometer can be adapted as long as the chamber remains airtight. However...

The biochemical Kreb's cycle generates ATP through the oxidation of acetate derived from carbohydrates, fats, and proteins producing CO2. In Drosophila, O2 input is directly correlated with CO2 output and reflects the level of metabolism1. Thus, measurement of CO2 output has successfully been used in studies related to aging and metabolism2-5. Here our laboratory has modified previously designed experimental setups, allowing measurement of CO2 production in up to eighteen samples without requiring any specialized equipment. Others and we have previously used this method to show differences in metabolic rates in flies that are deficient in the muscular dystrophy associated protein, Dystroglycan (Dg)6-8.
O2 used for oxidative metabolism is converted into CO2, which is expelled as respiratory waste. The construction of hand-made respirometers is described that allows for the determination of the rate of O2 consumed. Flies are placed in a sealed container with a substance that absorbs expelled CO2, efficiently eliminating it from the gaseous phase. The change in gas volume (decreased pressure) is measured by the displacement of fluid in a glass capillary attached to the closed respirometer.
The main advantage of this technique over others is the cost. Previous studies have measured CO2 production by Drosophila using gas analyzers and technically advanced respirometry systems1,9. Despite the more complex equipment, the sensitivity of the method described here is similar to reported values (Table 1). Additionally, several other groups have used variations of this technique to determine relative metabolic rates in Drosophila4-6. Therefore, this assay can be used to generate reliable, reproducible data relevant to Drosophila metabolism without the purchase of specialized equipment which can be setup in any lab and can be used for educational purposes.
In general, the accepted techniques to determine the metabolism of an organism is to measure the CO2 produced, the O2 consumed, or both3,4,9. Though, it can be assumed that one equivalent of O2 generates one equivalent of CO2, the precise ratio of CO2 generated is dependent on the metabolic substrate utilized10. Thus, to accurately determine the metabolic rate in energy units it is necessary to measure both O2 consumed and CO2 produced. Due to this, the method described here is specifically relevant to comparing differences in CO2 production between animals and not the absolute value. Our technique integrates multiple animal CO2 production over a period of time (1-2 hr) and therefore returns an average of the animals&#39; activity. If there is reason to believe that the experimental animals are less active than the control animals the measurement could reflect different levels of activity and not necessarily metabolism.

<table><tbody><tr><td>BlauBrand IntraMark 50 &amp;micro;l micropipettes</td><td>VWR</td><td>612-1413</td><td></td></tr><tr><td>Soda Lime</td><td>Wako</td><td>CDN6847</td><td></td></tr><tr><td>Eosine&amp;nbsp;</td><td>Sigma</td><td>031M4359</td><td>Any dye that can create visible colorization of liquid can be used</td></tr><tr><td>Thin Layer Chromatorgaphy (TLC) Developing Chamber</td><td>VWR</td><td>21432-761</td><td>Any transparent glass chamber that can be closed with the lid</td></tr><tr><td>Anesthetizer, Lull-A-Fly Kit</td><td>Flinn</td><td>FB1438</td><td></td></tr><tr><td>Power Gel Glue</td><td>Pritt</td><td></td><td></td></tr><tr><td>1 ml pipett tips</td><td>Any</td><td></td><td></td></tr><tr><td>Foam</td><td>Any</td><td></td><td></td></tr><tr><td>Plaesticine Putty</td><td>Any</td><td></td><td></td></tr><tr><td>Scalpel</td><td>Any</td><td></td><td></td></tr><tr><td>Tweezers</td><td>Any</td><td></td><td></td></tr></tbody></table>

measurement of metabolic rate in drosophila using respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

In order to show that the method is sensitive we measured CO2 production from wild type (Oregon R) male flies at 18, 25, and 29 &#176;C and flies mutant for Dg. Flies were raised at 25 &#176;C and then shifted to the experimental temperature for 5 days prior to measurement. As expected for this ectothermic species, the amount of CO2 produced increased with temperature (Figure 2). We have in the past shown that a sugar free diet reduces the metabolic rate of both wi...

Watch this Scientific Journal Video about Measurement of Metabolic Rate in Drosophila using Respirometry at JoVE.com

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

measurement-of-metabolic-rate-in-drosophila-using-respirometry

Research

JoVE Journal

Biology

19.5K Views.  Max Planck Institute for Biophysical Chemistry.  Metabole stoornissen behoren tot een van de meest voorkomende ziekten bij mensen. De genetisch handelbaar modelorganisme D. melanogaster kan worden gebruikt om nieuwe genen te identificeren die metabolisme regelen. Dit artikel beschrijft een relatief eenvoudige methode die het mogelijk maakt het bestuderen van de stofwisseling in vliegen door het meten van hun CO 2-productie. 

Video: Measurement of Metabolic Rate in Drosophila using Respirometry

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

Biochemistry

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Neuroscience

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Adapting Microrespirometry for Drosophila - Insights into CASK Mutant Metabolism

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 

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 ...

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human disease in this organism. Sensitive methods for measurements of parameters such as metabolic rates are needed to drive the understanding of physiology and disease in small animals such as the fruit fly. The method described here assesses fuel oxidation in small numbers of adult flies fed food containing trace amounts of 14C-labeled substrates such as glucose or fatty acid. After the feeding period and any additional experimental manipulations, flies are transferred to short tubes capped with mesh, which are then placed in glass vials containing KOH-saturated filter paper that traps exhaled, radiolabeled CO2 generated from oxidation of radiolabeled substrates as potassium bicarbonate, KHCO3. This radiolabeled bicarbonate is measured by scintillation counting. This is a quantitative, reproducible, and simple approach for the study of fuel oxidation. The use of radiolabeled glucose, fatty acids, or amino acids allows determination of the contribution of these different fuel sources to energy metabolism under different conditions such as feeding and fasting and in different genetic backgrounds. This complements other approaches used to measure in vivo energy metabolism and should further the understanding of metabolic regulation.

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

This paper describes a method for the measurement of fuel oxidation in Drosophila melanogaster in which trace amounts of specific radiolabeled metabolic substrates are fed to flies. The exhaled radiolabeled CO2 that is a produced from fuel oxidation is collected and measured.

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human ...

Measurement of Metabolic Rate in Drosophila using Respirometry

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