The authors would like to thank Drs. Roger Cone and Chao Zhang for their contributions to validating the application of this assay as previously published. This work was supported by NIH 1F32DK082167-01 (BJR).

Note: This protocol follows the guidelines of the University of Arizona Office for the Responsible Conduct of Research and has been approved by the Institutional Animal Care and Use Committee
1. Zebrafish Breeding and Embryo Maintenance
<ol>
	<li>Prepare E3 embryo medium8. To Make 60x stock solution, dissolve 34.8 g NaCl, 1.6 g&#160;KCl, 5.8g CaCl2&#9679;2H2O, and 9.78 g MgCl2&#9679;6H2O in 1.95 L ddiH2O. Adjust pH to 7.2 using NaOH and HCl Adjust volume to 2 L using ddiH2O.</li>
	<li>To prepare 1x E3 embryo medium, dilute 16.5 ml 60x stock in 1 L ddiH20. Add 100 &#181;l 1% methylene blue.</li>
	<li>Breeding
	<ol>
		<li>House Male and Female brood stock (7 - 18 months of age) separately to maximize fecundity.</li>
		<li>Turn the lights off 1 - 2 hr before on the day before eggs are desired. Set up mating (1 male and 1 - 2 females) in crossing cages as previously described9. Crossing cages allow for the eggs to escape predation as they pass through the perforated base of the tank insert in which the adult fish are held.</li>
		<li>Optional: For timed breeding, (as would be needed for studies involving morpholino injection), separate the male and female fish by a clear plastic divider., Remove this in the morning to result in timed laying of the eggs and fertilization. For morpholino studies, perform power calculations to assess the number of embryonic fish required per treatment. Without an estimate of the magnitude of effect or variation, a preliminary trial can be run with an n = 8.</li>
	</ol>
	</li>
	<li>Pour the eggs from the crossing cage into a petri dish (100 - 150 embryos/10 cm petri dish). Allow the eggs to settle to the bottom of the dish and pour off the water. Remove any remaining water using a fine tip disposable transfer pipette. Add back E3 embryo medium made in step 1.1.</li>
	<li>Make a flame polished disposable pipette for future egg and fish transfer. Using scissors, cut a graduated disposable transfer pipette at the 0.25 ml graduation. To remove rough edges, flame polish the cut tip over a Bunsen burner.</li>
	<li>Maintain embryonic fish to 4 days post-fertilization (dpf) in 10 cm petri dishes in zebrafish E3 embryo medium at 28.5 &#9675;C. Twice each day, use a flame polished graduated disposable transfer pipette to remove any unfertilized eggs or dead embryos (opaque white appearance). Once each day, replace E3 embryo medium. Pour off E3 embryo medium, remove remaining liquid using a fine tip disposable transfer pipette, and add back pre-warmed (28.5 &#9675;C) E3 embryo medium.</li>
</ol>
2. Prepare Assay Solution
<ol>
	<li>Prepare the assay solution according to Table 1. Sterile filter assay solution using a 0.22 &#181;M filter. Prior to assay initiation, warm the sterile assay solution in a 28.5 &#9675;C water bath.</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Ingredient</td>
			<td>% of Total</td>
			<td>Volume (&#181;l/10 ml)</td>
		</tr>
		<tr>
			<td>60x E3 Embryo Medium</td>
			<td>1.667</td>
			<td>166.7</td>
		</tr>
		<tr>
			<td>Double deionized (ddi) H20</td>
			<td>96.233</td>
			<td>9623.3</td>
		</tr>
		<tr>
			<td>Alamar Blue</td>
			<td>1</td>
			<td>100</td>
		</tr>
		<tr>
			<td>400 mM NaOH</td>
			<td>1</td>
			<td>100</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>0.1</td>
			<td>10</td>
		</tr>
	</tbody>
</table>
Table 1. Assay Medium
3. Perform Assay
<ol>
	<li>Rinse zebrafish embryos 3 times with sterile filtered E3 embryo medium. Combine all viable zebrafish embryos from the clutch into a beaker. Sterile filter 75 ml E3 embryo medium. Using a fine tip disposable transfer pipette, remove as much of the E3 embryo medium from the zebrafish embryos as possible. Add back 20 ml sterile filtered E3 embryo medium. Remove and replace E3 embryo medium 3 times.</li>
	<li>Plate 1 fish into each of 93 wells of a 96-well black sided clear bottom plate. Transfer the embryonic fish to the plate by individually capturing an embryo within a flame polished graduated plastic disposable transfer pipette (step 1.5) and gently ejecting into the well. Transfer E3 embryo medium taken from the beaker containing rinsed fish into the 3 blank wells. This ensures that blank wells have been exposed to the exact same water as the wells that contain fish. Both wells that contain fish and blank wells should be at least &#189; full (200 &#181;l) at the conclusion of this step.</li>
	<li>Replace E3 embryo medium with assay solution in all 96 wells, including blank wells. This step should be performed 1 column at a time for each of the 12 columns in a 96 well plate. Using a fine tip disposable transfer pipette, remove E3 embryo medium from every well in one column of the plate (8 wells). Take care to not touch the zebrafish embryo. Add 300 &#181;l&#160;of assay solution to each well from which water was removed. Repeat the removal of E3 embryo medium and replacement with assay solution for all 12 columns of the plate.</li>
	<li>Optional: To test the metabolic rate change in response to a compound, make a solution that is 100 times (100X) the desired treatment concentration. Of note, ensure that the 100x solution does not exceed 10% DMSO to limit the final DMSO concentration to no more than 0.1%. Add 3 &#181;l of the 100x solution to each of the treatment wells. 
	NOTE: Perform power calculations to assess the number of treatment wells. However, without an estimate of the magnitude of response or variation, a preliminary trial can be run in 8 compound treated and 8 vehicle treated control wells that contain fish.
	<ol>
		<li>Add 3 &#181;l of vehicle control to control (no drug treatment) wells that contain fish. To ensure that the fluorescent response to compound is a result of actions within the fish, apply drug and vehicle treatments to 3 blank wells in a 96 well plate.</li>
	</ol>
	</li>
	<li>Read fish filled plate on fluorescent plate reader with excitation wavelength at 530 nM and emission wavelength at 590 nM.</li>
	<li>Place plate into humidified incubator set to 28.5 &#9675;C (maintain plate in darkness throughout assay to avoid photobleaching of assay solution). The assay can be run for durations of time that range from 1 hr to 24 hr. The duration of assay depends on the objective of the study. For testing the response to a short acting non-stable compound a short duration may be most suitable. Yet, for stable compound tests or tests of genetic effects, maximizing assay duration is preferable to exploit the advantages associated with the cumulative signal.</li>
	<li>At a pre-determined time point (see step 3.6 to help determine timing) read fluorescence of fish-filled plate again with the same settings as step 3.5.</li>
</ol>
4. Assess the Relative Change in Fluorescence Associated with Treatment
<ol>
	<li>Calculate change in fluorescence of each well by subtracting the fluorescence at time 0 from the fluorescence at the conclusion of the study. Wells without fish (blanks) should have values near 0, far below values from wells that contain fish. 
	&#160;&#160;&#160;&#160;&#160;&#160;&#160; Change in Fluorescence = Fluorescence at conclusion - Fluorescence at time 0</li>
	<li>To ascertain the relative change in fluorescence, calculate the average change in fluorescence for control wells then divide the change in fluorescence of each well by the average change in fluorescence of the control wells. Of note, controls could be either vehicle controls or genetic controls. 
	&#160;&#160;&#160;&#160;&#160;&#160;&#160; <img alt="Equation 1" src="/files/ftp_upload/53297/53297eq1.jpg" /></li>
</ol>

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	<li>Jao, L. E., Wente, S. R., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+multiplex+biallelic+zebrafish+genome+editing+using+a+CRISPR+nuclease+system.">Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.</a> PNAS. 110, 13904-13909 (2013).</li><li>Kimura, Y., Hisano, Y., Kawahara, A., Higashijima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+knock-in+transgenic+zebrafish+carrying+reporter/driver+genes+by+CRISPR/Cas9-mediated+genome+engineering.">Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.</a> Sci. Rep. 4, 6545 (2014).</li><li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat. Rev. Genet. 8, 353-367 (2007).</li><li>Brand, M. D., Couture, P., Else, P. L., Withers, K. W., Hulbert, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+energy+metabolism.+Proton+permeability+of+the+inner+membrane+of+liver+mitochondria+is+greater+in+a+mammal+than+in+a+reptile.">Evolution of energy metabolism. Proton permeability of the inner membrane of liver mitochondria is greater in a mammal than in a reptile.</a> Biochem. J. 275 (Pt 1), 81-86 (1991).</li><li>Renquist, B. J., Zhang, C., Williams, S. Y., Cone, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+assay+for+high-throughput+energy+expenditure+monitoring+in+the+zebrafish.">Development of an assay for high-throughput energy expenditure monitoring in the zebrafish.</a> Zebrafish. 10, 343-352 (2013).</li><li>Livingston, 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=A+volumetric+respirometer+for+long-term+studies+of+small+aquatic+animals.">A volumetric respirometer for long-term studies of small aquatic animals.</a> J.Mar. Biol. Assoc. U.K. 48, 485-497 (1968).</li><li>Makky, K., Duvnjak, P., Pramanik, K., Ramchandran, R., Mayer, A. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+a+genetic+model+of+obesity+in+a+teleost.">Creation of a genetic model of obesity in a teleost.</a> FASEB J. 21, 2042-2049 (2007).</li><li>Maddison, L. A., Joest, K. E., Kammeyer, R. M., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+insulin+resistance+in+zebrafish+induces+alterations+in+beta-cell+number+and+glucose+tolerance+in+an+age+and+diet+dependent+manner.">Skeletal muscle insulin resistance in zebrafish induces alterations in beta-cell number and glucose tolerance in an age and diet dependent manner.</a> Am. J. physiol. Endocrinol. , (2015).</li><li>Flynn, E. J., Trent , C. M., Rawls, J. 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The authors have no competing financial interests to disclose.

Contamination with bacteria or fungi will greatly limit application of this assay. The methylene blue in the E3 embryo medium limits the possibility of fungal contamination. Throughout embryo rearing it is critical that care is taken to remove dead embryos twice each day. Additionally, it is essential to thoroughly rinse the embryos with sterile E3 embryo medium on the day of assay initiation. These 2 steps limit the potential for bacterial contamination of the embryos. The inclusion of blank wells described in step 3.2 allows for detection of any bacterial contamination. Bacterial contamination that yields a measurable signal is rare. However, if the blank wells do show a robust signal, the assay should be repeated with care taken to maintain embryo cleanliness. If the user expects that contamination is arising prior to embryo collection, the embryos may be bleached as previously described (p. 22) 10. Alternatively, if the experimenter suspects that contamination is occurring after collection the user may use E2 with penicillin and streptomycin to rear the embryos to 4 dpf (p. 23)10.
Although this assay is valuable for application in embryos, we have not tested for validity in adult zebrafish. We suspect that functionality of this assay would be limited in adult fish by poor tissue penetration of AlamarBlue. Moreover, in feeding fish the gut microbiota may produce a profound signal, not indicative of the fish metabolism. Thus, this assay is restricted to use in yolk feeding embryonic fish. The relative inactivity of embryonic fish provides a unique advantage, as this limits the variability that would be associated with activity induced energy expenditure 11. Thus this assay is a unique tool to screen the metabolic rate response to genetic or pharmacological manipulation in a whole animal system.
The zebrafish is rapidly becoming a leading model for the metabolic perturbations that accompany obesity. In fact, there are published models of both transgenic and diet-induced zebrafish obesity 12,13. Similar to mouse models, diet induced obesity in the zebrafish increases serum triglycerides and hepatic lipid accumulation12. Furthermore, overfed zebrafish have elevated fasting glucose, suggesting glucose intolerance 14. Using the assay described here we have shown that 24 hr of pretreatment with Metformin, an insulin sensitizer, increases the metabolic rate response to insulin treatment 5. Thus, the zebrafish may prove to be an ideal whole animal model to discover drugs that enhance insulin sensitivity (embryo) and to establish their effectiveness in alleviating obesity related insulin resistance (adult).
The development of genetic and diet induced obese zebrafish models that mimic the phenotypes observed in obese mammals creates the impetus for initiating zebrafish studies focused on metabolic disease. High throughput assays are essential for fully appreciating the value of the zebrafish model to studies of energy homeostasis. Innovative tools designed to assess phagic drive will allow for screens focused on energy intake, while high throughput lipid storage assays will lead to studies on whole body lipid homeostasis 15-17. The assay we describe here provides a high throughput tool to assess the role of genes, compounds or their interactions on whole body energy expenditure.

The mouse is currently the predominant model for obesity research. The short generation interval and genetic tools available in the mouse have been unmatched to date. However, the zebrafish also has a short generation interval (3 - 4 months) and surpasses even the mouse in ease of genetic manipulation 1,2. The zebrafish maintains nearly 90% of mammalian genes, while far exceeding the mouse in number of offspring and potential for use in genetic and drug screens 3.
To tap the potential of the zebrafish model for studies in obesity, assays must be developed to investigate factors that influence body weight regulation, including energy expenditure. As metabolites are processed through &#946;-oxidation and the tricarboxylic acid cycle oxygen is consumed and NADH2 is produced. Thus, NADH2 is a direct indicator of the flux of metabolites through metabolic pathways (metabolite oxidation). In poikilotherms, H+ leak through the inner mitochondrial membrane, which uncouples NADH2 oxidation from ATP synthesis, is 4 - 5 fold lower than in homeotherms 4. Accordingly, in zebrafish NADH2 is very tightly linked to ATP production through oxidative phosphorylation. Herein, we describe an assay that measures NADH2 production in larval zebrafish as a proxy for energy expenditure5.
Oxygen consumption is the gold standard for measuring energy expenditure. Yet, to best take advantage of the high throughput potential of the zebrafish, assays of energy expenditure must be amenable to high-throughput. Oxygen consumption systems that depend on a closed chamber circulating system are limited in throughput by the number of chambers available6. Open air O2 consumption/CO2 production assays have also been applied in the zebrafish5,7. These open air 96-well plate based systems are amenable to high throughput. Unfortunately, gas exchange with the environment limits sensitivity of these assays. We recently published the application of an assay that monitors NADH2 using the redox indicator alamarBlue5. This assay overcomes the limitations in throughput and sensitivity common to analyses of oxygen consumption in the zebrafish.
The zebrafish is becoming an increasingly important model for studies of whole body energy homeostasis. In part, because zebrafish are amenable to use in forward genetic screens and drug screens. Moreover, targeted genetic manipulation, including knockdown and knock-in, can be quickly applied. We have previously shown that this assay can be combined with bath drug application and genetic knockdown or knockout to identify compounds and genes that alter metabolic rate5. Moreover, this assay is designed to exploit the high throughput advantages inherent to the zebrafish.

<table><tbody><tr><td>AlamarBlue</td><td>Fisher Scientific</td><td>10-230-103</td><td>Manufactured by Thermo Scientific</td></tr><tr><td>Fine Tip DisposableTransfer Pipette</td><td>Fisher Scientific</td><td>13-711-26</td><td>Manufactured by Thermo Scientific</td></tr><tr><td>Fisherbrand Graduated Disposable Transfer Pipette</td><td>Fisher Scientific</td><td>13-771-9AM</td><td>Manufactured by Thermo Scientific</td></tr><tr><td>Black sided clear bottom 96-well plates</td><td>Fisher Scientific</td><td>50-320-785</td><td>Manufactured by E&amp;amp;K Scientific Products Inc.</td></tr></tbody></table>

high throughput danio rerio energy expenditure assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

Assay solution does not increase fluorescence in the absence of embryonic fish (Figure 1). However, the assay is highly sensitive to small changes in metabolic rate within the well. A single fish within a well creates a signal significantly different from blank wells within 1 hr (P&#60;0.0001). Figure 2 provides a visual representation of signals generated by embryonic fish after 24 hr of incubation. For the purpose of this picture clear sided wells were used. Yet, black sided wells are preferred for assay performance to prevent leaching of the fluorescent signal from nearby wells.
Because the NADH2 induced reduction of alamarBlue is non-reversible, the signal accumulates with time. This allows for small changes to be amplified. To show that differences accumulate and thus increase with time, we monitored the relative change in fluorescence induced by 1 to 5 fish at 1, 2, and 4 hr (Figure 3). At each number of fish/well the relative change in fluorescence significantly increased with time (P&#60;0.001). More importantly, because signal accumulates with time, the magnitude of differences in fluorescence change that result from manipulating the number of fish/well is more robust at 4 hr than at 1 hr of incubation. Thus, by increasing the duration of exposure we can enhance the ability to detect differences associated with a given treatment.
<img alt="Figure 1" src="/files/ftp_upload/53297/53297fig1.jpg" /> 
Figure 1. Comparison of Noise to Signal. A detectable change in fluorescence is generated by 1 zebrafish in a 96 well plate at 1 hr. Wells that do not contain fish (blanks) generate little signal. Error bars represent SEM (n = 5 - 8). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53297/53297fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53297/53297fig2.jpg" /> 
Figure 2. Visual Representation of Assay Plate at 24 hr. Representative picture of results obtained using this assay. Pink wells are indicative of fish with a high metabolic rate, purple wells a moderate metabolic rate, and blue wells a low metabolic rate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53297/53297fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53297/53297fig3.jpg" /> 
Figure 3. Signal Differences Amplify With Time. The difference in fluorescent signal generated by varying the number of fish/well increases with time. Because signal accumulates with time, the magnitude of fluorescence change diverges with time betweensamples that differ in rate of signal generation. Error bars represent SEM (n = 7).

Watch this Scientific Journal Video about High Throughput Danio Rerio Energy Expenditure Assay at JoVE.com

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

high-throughput-danio-rerio-energy-expenditure-assay

Nanyang Technological University

Research

JoVE Journal

Biology

12.6K Views.  Vanderbilt University Medical Center. Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery. 

Video: High Throughput Danio Rerio Energy Expenditure Assay

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Genetics

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, Drosophila research lacks popularity in developing countries, possibly due to the misinformed idea that establishing a lab and performing relevant experiments with such tiny insects is difficult and requires expensive, specialized apparatuses. Here, we describe how to build an affordable flylab to quantitatively analyze a myriad of behavioral parameters in D. melanogaster, by 3D-printing many of the necessary pieces of equipment. We provide protocols to build in-house vial racks, courtship arenas, apparatuses for locomotor assays, etc., to be used for general fly maintenance and to perform behavioral experiments using adult flies and larvae. We also provide protocols on how to use more sophisticated systems, such as a high resolution oxygraph, to measure mitochondrial oxygen consumption in larval samples, and show its association with behavioral changes in the larvae upon the xenotopic expression of the mitochondrial alternative oxidase (AOX). AOX increases larval activity and mitochondrial leak respiration, and accelerates development at low temperatures, which is consistent with a thermogenic role for the enzyme. We hope these protocols will inspire researchers, especially from developing countries, to use Drosophila to easily combine behavior and mitochondrial metabolism data, which may lead to information on genes and/or environmental conditions that may also regulate human physiology and disease states.

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

We provide protocols for anyone with a "maker culture" mind to start building a flylab for quantitative analysis of a myriad of behavioral parameters in Drosophila melanogaster, by 3D-printing many of the necessary pieces of equipment. We also describe a high resolution respirometry protocol using larvae to combine behavioral and mitochondrial metabolism data.

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, ...

Behavior

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental factors, and the toxicological and pharmacological effects of numerous substances. Few methods currently implemented are amenable to high throughput measurement. The Microplate Feeder Assay (MFA) was developed for quantifying the consumption of liquid food for individual flies using absorbance. In this assay, flies consume liquid food medium from select wells of a 1536-well microplate. By incorporating a dilute tracer dye into the liquid food medium and loading a known volume into each well, absorbance measurements of the well acquired before and after consumption reflect the resulting change in volume (i.e., volume consumed). To enable high throughput analysis with this method, a 3D-printed coupler was designed that allows flies to be sorted individually into 96-well microplates. This device precisely orients 96- and 1536-well microplates to give each fly access to up to 4 wells for consumption, thus enabling food preference quantification in addition to regular consumption. Furthermore, the device has barrier strips that toggle between open and closed positions to allow for controlled containment and release of a column of samples at a time. This method enables high throughput measurements of consumption of aqueous solutions by many flies simultaneously. It also has the potential to be adapted to other insects and to screen consumption of nutrients, toxins, or pharmaceuticals.

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

The microplate feeder assay offers an economical, high throughput method for quantifying liquid food consumption in Drosophila. A 3D-printed device connects a 96-well microplate in which flies are housed to a 1536-well microplate from which flies consume a feeding solution with a tracer dye. The solution volume decline is measured spectrophotometrically.

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

Neuroscience

Mitochondrial Functional Assessment in Brush Lizard Liver Mitochondria

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of metabolic pathways. Therefore, experimentally assessing mitochondrial functionality can provide insight into variations among different populations or disease states. Additionally, it is valuable to assess whether isolated mitochondria are healthy enough to proceed with experiments. One characteristic often used to compare mitochondrial function in different samples is the rate of oxygen consumption. Oxygen consumption and subsequent calculation of the respiratory control ratio in either intact cells or mitochondria isolated from tissue can serve all three purposes. Using mitochondria isolated from the livers of brush lizards in conjunction with a phosphorescent probe that is sensitive to the fluctuations in oxygen concentration of a solution, we measured oxygen consumption using a fluorescent plate reader. This method is not only quick and efficient but also can be conducted with a small amount of mitochondria and without the need for specialized equipment. The step-by-step protocol described here increases the accessibility of mitochondrial functional assessment to researchers.

Assessment of oxygen consumption provides integral information about mitochondrial function. Using a phosphorescent probe with a fluorescent plate reader, accurate and reproducible data can be obtained easily without specialized equipment. This assay enables any lab to measure the oxygen consumption of isolated mitochondria and calculate respiratory control ratios.

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of ...

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 ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. ...

High-Throughput Assays of Critical Thermal Limits in Insects

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are essential for predicting responses to climate change. This work describes two high-throughput protocols for measuring insect thermal limits: one for assessing critical thermal minima (CTmin), and the other for assessing heat knock down time (KDT) in response to a static heat stressor. In the CTmin assay, individuals are placed in an acrylic-jacketed column, subjected to a decreasing temperature ramp, and counted as they fall from their perches using an infrared sensor. In the heat KDT assay, individuals are contained in a 96 well plate, placed in an incubator set to a stressful, hot temperature, and video recorded to determine the time at which they can no longer remain upright and move. These protocols offer advantages over commonly used techniques. Both assays are low cost and can be completed relatively quickly (~2 h). The CTmin assay reduces experimenter error and can measure a large number of individuals at once. The heat KDT protocol generates a video record of each assay and thus removes experimenter bias and the need to continuously monitor individuals in real time.

Thermal limits can predict the environments organisms tolerate, which is valuable information in the face of rapid climate change. Described here are high-throughput protocols to assess critical thermal minima and heat knockdown time in insects. Both protocols maximize the throughput and minimize the cost of the assays.

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

High Throughput Danio Rerio Energy Expenditure Assay

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