Name: Using a Respiratory Chamber to Measure Metabolic Rate in Drosophila melanogaster
Uploaded: 2023-07-07T13:20:00
Description: Watch this Scientific Journal Video about Using a Respiratory Chamber to Measure Metabolic Rate in Drosophila melanogaster at JoVE.com

using a respiratory chamber to measure metabolic rate in drosophila melanogaster

Tilpasning av mikrorespirometri for Drosophila - Innsikt i CASK mutant metabolisme

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.

Forfatter Spotlight: Temperaturens innflytelse på Drosophila melanogaster og ørkentilpassede biller 

Måling avO2-forbruk i Drosophila melanogaster ved bruk av coulometrisk mikrorespirometri

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.

Watch this Scientific Journal Video about Using a Respiratory Chamber to Measure Metabolic Rate in Drosophila melanogaster at JoVE.com

Using a Respiratory Chamber to Measure Metabolic Rate in Drosophila melanogaster  

Bruk av et respiratorisk kammer for å måle metabolsk hastighet i Drosophila melanogaster

Begynn med å opprettholde fluene ved en temperatur på 25 grader Celsius, i smale hetteglass fylt med standard drosophila mat. Slå på vannbadet og juster temperaturen etter behov. Bruk en laboratorieserviett sprayet med 70 % etanol til å rengjøre de malte glassskjøtene i kamrene og sensorpluggene, og bruk deretter en ny klut for å fjerne eventuell gjenværende etanol.Deretter plasserer du et centimeter stykke bomullsrull, gjennomvåt i renset vann, i bunnen av kammeret for å skape et stabilt fuktighetsmiljø. Tilsett tilstrekkelig vann for å danne et beskjedent basseng ved foten av bomullsrullen. Sørg for at skjøten i kammeret er tørr ved å tørke av sølt vann.Overfør fluene forsiktig til merkede polypropylenrør ved hjelp av en trakt, og plugg rørene med en bomullsrull. Legg nå et ventilert rør med fluer inn i hvert respirometerkammer. Fyll brus kalkpatronene og plasser dem på toppen av røret som inneholder fluer inne i kammeret.Fyll deretter oksygengeneratorene med en mettet løsning av kobbersulfat, slik at nivået forblir under ventilasjonshullene. Koble den fylte oksygengeneratoren til topinnerskontakten på sensorpluggen. Plasser to små dabs med klart silikonfett på motsatte sider av jordglassskjøten på sensorpluggen.Sett pluggen inn i kammeret og roter den forsiktig med moderat trykk for å fordele fettet jevnt i skjøten. Tørk av overflødig fett med en laboratorieserviett. Fest deretter kammerpluggene ved å klikke på plastkeklemmene.Plasser de monterte kamrene i et stativ i vannbadet, og sørg for at stoppekranene er åpne. Hold stoppekranene i åpen stilling og la kamrene balansere i ca. 30 minutter. Forsikre deg om at bryterne som leverer strøm til oksygengeneratorene er slått av.Koble kontrollerne til respirometerkamrene ved hjelp av de seks lederkablene, og kontroller at de organiske lysemitterende diodedisplayene på kontrollerne aktivt viser miljøparametere. Aktiver nå oksygengeneratorene ved hjelp av bryteren på kontrolleren. Når den nåværende verdien stiger fra null til mellom 35 og 55 milliampere, er kontrolleren og kammeret klare for eksperimentene.Når du har slått av oksygengeneratorene, identifiserer du kommunikasjonsportene som kontrollerne bruker. Åpne PuTTY-programvaren på skrivebordet og sett opp en loggfil for hver kanal i respirometeret ved å velge kommunikasjonsporten for kontrolleren ved å skrive inn nummeret til den porten i serielinjeboksen. Klikk deretter logging.Velg hovedutdata i øktloggingen, og klikk bla gjennom under loggfilnavnet. Opprett et filnavn som inneholder beskrivende informasjon, i den valgte mappen. Klikk deretter lagre.Klikk åpne, og et vindu som viser vanlige data med skilletegn som logges, åpnes. Gjenta denne prosessen for alle andre kontrollere som brukes. Hver inngang for kommunikasjonsport vises i et eget vindu.Etter å ha balansert kamrene i 30 minutter, forsegl dem ved å lukke stoppekranene. Dekk badekaret og kamrene med en polystyrenskumboks for å skape et stabilt miljø og balansere i en time. Start strømmen til oksygengeneratoren i hvert kammer ved hjelp av bryteren på kontrollerboksen.Når oksygengeneratorene er aktivert, må du kontrollere at trykket øker til det forhåndsdefinerte trykket. Etter eksperimentet, slå av oksygengeneratorene på alle kontrollerne og åpne stoppekranene for å forsegle kamrene. La PuTTY-vinduene forbli åpne i fem til 15 minutter for å etablere en endelig grunnlinje, og avslutt deretter opptakene ved å lukke PuTTY-vinduet for hver kontroller.Koble sensorene fra kablene og transporter kamrene til tørrstativet. Fjern sensorpluggene fra kamrene én etter én. Koble fra oksygengeneratorene og plasser dem forsiktig i rørstativet.Tørk deretter fettet av sensorpluggen og legg det tilbake i stativet. Rengjør fett fra kammerleddene og fjern rørene som inneholder fluer og bruskalk. Logg til slutt vekten og antall fluer for hvert rør.Kvantitative oksygenforbruksdata for mutantfluer av villtype og fat delta 18 viste at massespesifikt oksygenforbruk ikke var signifikant forskjellig blant villtypekontroller og cask delta 18-mutanter. Oksygenforbruk analysert per fly viste at oksygenforbruket var signifikant redusert i fat delta 18 sammenlignet med villtype kontroller. Imidlertid var den gjennomsnittlige massen av cask delta 18 fluer lavere enn for villtypekontroller.

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JoVE Journal

Neuroscience

Using a Respiratory Chamber to Measure Metabolic Rate in Drosophila melanogaster

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 ...