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

Summary

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.

Abstract

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.

Introduction

Drosophila melanogaster was introduced to the scientific community as a potentially powerful model organism more than 100 years ago. That potential has been firmly validated in several areas of the biological and biomedical sciences, such as genetics, evolution, developmental biology, neurobiology, and molecular and cell biology. As a result, six Nobel Prizes in Medicine or Physiology have been awarded to ten Drosophila researchers who have substantially contributed to our understanding of heredity, mutagenesis, innate immunity, circadian rhythms, olfaction and development¹. Perhaps more importantly, D. melanogaster has not ceased to provide us with new models of human biology and diseases, as a quick search on PubMed reveals almost 600 publications in the last 5 years, using the search term "drosophila model" (², as of February, 2021). In the US, where Drosophila is a wide spread model organism in the biomedical community, about 2.2% of all R01 research awards granted by the NIH in 2015 were allocated to Drosophila researchers³. In Brazil, on the other hand, a search for currently funded projects on the website of the Sao Paulo Research Foundation (FAPESP), the most important funding agency for research in all scientific areas in the state of Sao Paulo, showed only 24 grants and fellowships with Drosophila as the main subject of study⁴. Considering all 13205 projects currently funded by FAPESP (⁵, as of February, 2021), those 24 Drosophila projects represent a ratio of less than 0.2% of the total projects, which is nearly 12 fold lower than that of the NIH. If we remove the funded projects that aim at studying Drosophila from an ecological and/or evolutionary point of view, and assume that the remaining projects use this organism as a model for understanding human biological processes in health and disease, that ratio decreases to a shocking ~0.1%.

In fact, a proper investigation is warranted to reveal the reasons why Drosophila research in Brazil/Sao Paulo does not appear to be as significant in number of funded projects. Culturing Drosophila is not expensive^6,7,8 and is relatively simple, as unlike vertebrates, no permission from a bioethical committee is necessary for experimentation^9,10. An approval to work with genetically modified fly lines is, however, required in Brazil¹¹, adding a layer of bureaucracy inherent to all work involving genetically modified organisms. However, this would likely not prevent interested researchers from initiating a flylab. We speculate that misinformation about the power of the model, and about the expected high costs associated with setting up a flylab and performing meaningful experiments are important factors in this decision. As for most science equipment and supplies, the appropriate apparatuses to perform general fly maintenance and behavioral analyses must be imported into Brazil from North America, Europe and/or elsewhere, which is an expensive and extremely time consuming process^12,13.

Recently, an alternative to importing specialized apparatuses has emerged as 3D printers have become more affordable and accessible to any person, including Drosophila researchers in developing countries. The 3D-printing technology has been widely used in the last 10 years by members of the "maker culture", which is based on the idea of self-sufficiency over exclusively relying on company manufactured products¹⁴. Such an idea has always been present in academic research laboratories around the globe, so it is not surprising that 3D printers have become standard lab equipment in many places^15,16. For a number of years, we have been 3D-printing fly vial racks, mating arenas, climbing apparatus, among other devices, for a fraction of the cost of brand-named equivalents. The reduced costs of printing and assembling homemade lab equipment is classically represented by the FlyPi, which can be built for less than €100.00 and serves as a light and fluorescence microscope able to use sophisticated opto- and thermogenetic stimulation of the genetically tractable zebrafish, Drosophila and nematodes¹⁵. Here, we provide a series of protocols for anyone interested in becoming a Drosophila researcher (or in expanding his/her own existing flylab) to 3D-print many of the necessary material. By investing time and developing a little expertise, the reader will even be able to optimize the protocols presented here to print apparatuses better adapted to his/her own research needs.

However, a flylab is not a place for "cheap" equipment only, especially when one intends to associate behavioral analyses with underlying metabolic phenomena. We have also been interested in the roles of mitochondria in the modulation of Drosophila behavioral patterns, as these organelles are responsible for the bulk production of ATP in most tissues through several metabolic pathways whose products converge to oxidative phosphorylation (OXPHOS). Analyzing mitochondrial oxygen consumption as a way to understand mitochondrial metabolism does require an oxygraph, which is a more sophisticated piece of equipment that unfortunately cannot yet be 3D-printed. Because OXPHOS impacts practically all cellular processes since it depends on a series of exergonic redox reactions that occur in the cell^17,18, oxygen consumption rates based on the oxidizable substrate provided to mitochondria may help reveal whether the organelle´s functioning is cause or consequence of a particular behavior. Therefore, we also provide here a protocol for measuring mitochondrial oxygen consumption in larva samples, as we realize the vast majority of published protocols are focused on analyzing adult samples. We show that changes in mitochondrial respiration, induced by the transgenic expression of the Ciona intestinalis alternative oxidase (AOX), leads to increased larval mobility under cold stress. This is most likely due to thermogenesis, since AOX is a non-proton pumping terminal oxidase that can bypass the activity of OXPHOS complexes III and IV (CIII and CIV), without contributing to the mitochondrial membrane potential (ΔΨm) and ATP production^19,20,21. No insect, including Drosophila, or vertebrate naturally possesses AOX^21,22,23, but its expression in a myriad of model systems^{24,25,26,27,28,29} has been successful to show its therapeutic potential for conditions of general mitochondrial respiratory stress, especially when caused by CIII and/or CIV overload. AOX confers resistance to toxic levels of antimycin A²⁴ and cyanide^24,25, and mitigates diverse phenotypes related to mitochondrial disfunction^{24,25,30,31,32}. The fact that AOX expression changes larval behavior and mitochondrial function justifies more in-depth studies of this enzyme's roles in the metabolism and physiology of metazoan cells and tissues^33,34.

We hope that with this article we can help raise awareness within the scientific community of developing countries such as Brazil that using the excellent genetic toolset that D. melanogaster presents, in combination with efficient and affordable homemade apparatuses for behavioral analyses, can generate relatively fast basic research data on interesting biological processes with significant translational impact, supporting future therapeutic studies in clinical research. Developing such communal ideals would greatly benefit Drosophilists, medical researchers, and the biological and biomedical sciences. Most importantly, it would benefit society in general, as public funding could be applied more translationally to understand and treat human diseases.

The protocols we provide here for 3D printing the apparatuses for a flylab were designed for use with the RepRap 3D printer, based on the Prusa I3 DIY model available at³⁵. We use the 1.75-mm white polylactic acid (PLA) filament (SUNLU) as raw material for printing, the Tinkercad platform³⁶ for model design, and the Repetier-Host software³⁷ for STL to G-Code conversion, a necessary step to provide coordinates to the printer. Further optimization of the protocols is required should the reader want to use alternative equipment, materials and software.

Protocol

1. 3D model design

NOTE: The workflow for 3D printing has three basic steps: (1) 3D modeling; (2) importing the model into the slicing software; and (3) selecting the correct filament, configuring the printer, and finally, printing. A basic protocol for modeling a small fly vial rack/tray is shown below; this rack is to be used with standard fly vials, which have approximately 2.5 cm in diameter and 9.8 cm in height. For new model designs, the tools provided by the Tinkercad software allow the easy handling of three-dimensional structures, by creating pieces of different shapes, sizes and thickness, according to one's own needs. For Drosophilists venturing for the first time into the realm of 3D printing, following the protocols below, even with all their details, may still be challenging, so we strongly recommend becoming acquainted with the software for best results.

1. Sign in to Tinkercad online³⁸ (Figure 1A). Prior registration with personal information is required to access the platform, free of charge.
2. Click on Create a new Project to begin a new design, and rename the Project accordingly on the upper right corner of the window. Press Enter to be directed to the project´s workplane (Figure 1B).
3. Verify if the workplane has the correct dimensions of 200 mm x 200 mm, by clicking with the mouse´s left button on Edit Grid at the lower right corner (red square in Figure 1B). In the popup window (Figure 1C), make sure that the "Units" are millimeters, and the "Presets" are default. Enter 200.00 in the "Width" and in the "Length" fields, and click on Update Grid to save the changes.
4. Verify if the Snap Grid is set to 1.0 mm (red square in Figure 1B). If it is not, click on the dropdown menu and select 1.0 mm.
5. Under the Basic Shapes menu on the right (blue square 2 in Figure 1B), select a solid box and drag it to the center of the workplane.
6. Click anywhere on the box in the workplane with the mouse´s left button to see its edges and vertices. Click on any vertex (which will then turn red) to show the box dimensions (red squares in Figure 1D). Click with the mouse´s left button on each dimension and type 130 mm for length (L), 130 mm for width (W) and 40 mm for height (H). Recenter the box by dragging it to the middle of the workplane.
7. Click on the tool Ruler on the upper right corner of the screen (red square 1 in Figure 1E). Immediately click on the lower left vertex of the box, as indicated in Figure 1E (red square 2), to set the initial point (x = 0, y = 0, z = 0) of a tridimensional Cartesian coordinate system. Note that the distance between the selected vertex and the coordinate initial point will now show (red squares in Figure 1F), where "A", "B" and "C" represent the distance to the x, y and z axes (which should be zero in this case), respectively.
8. Next, select an empty (hole) box from the Basic Shapes menu on the right (blue square 2 in Figure 1B) and drag it to the workplane. Set its dimensions to 30 (L) x 30 (W) x 40 (H) mm, and elevate it 2 mm from the workplane, by typing "2.00" in the textbox next to the green arrowhead on the lower right corner of the empty box (red square in Figure 1G). Position the empty box inside the solid box 2 mm away from the x,y coordinate initial point, by typing "2.00" in the textboxes next to the green arrows on the lower left corner of the box (red squares in Figure 1H; compare with Figure 1G).
9. With the empty box still selected, press the CtrL+D keys on the keyboard to deploy the "duplicate" command and create a new empty box of the exact same dimensions. Position the new empty box inside the solid box, next to the first empty box, by typing "34.00" in the textbox next to the green arrow along the y axis, and "2.00" in the textboxes next to the two remaining green arrows (red squares in Figure 1I).
10. Repeat this step, adjusting for the correct distances from the coordinate initial point, until the entire solid box is filled with empty boxes spaced 2 mm apart from each other (Figure 1J).
11. Select all the boxes (solid and empty ones) by clicking with the mouse´s left button and dragging to the entire area. Press the CtrL+G keys on the keyboard to deploy the "group" command and create a single box with 16 empty spaces for fly vials (Figure 1K). This is the final design of the vial rack.
12. Click on Export on the upper right corner of the Tinkercad window. On the window box displayed (Figure 1L), select Everything in the design next to Include, and .STL under For 3D Print as the file type. Choose a proper name for the design file and save it in an appropriate place in the computer.

2. 3D printing

NOTE: In this section, we provide instructions on how to use the STL file created in Step 1 and convert it to the G-Code file containing the printing instructions to the 3D printer. This is the slicing process, for which we use the Repetier-Host software.

1. Download Supplemental File 1 and save it on an appropriate place in the computer. This is a .rcp file containing the printer configurations to be used below. To get more information on the .rcp file type, please visit³⁹.
2. Open the Repetier-Host software, which should already be installed the computer, following instructions from³⁷. Press the CtrL+O keys on the keyboard to open the STL file on the computer created in Protocol 1.
3. Once opened, click on the designed vial rack and press the R key on the keyboard to open the editing menu on the right side of the screen (red square 1 in Figure 2A). Centralize the object on the printing table by clicking on the Center Object button, indicated with red square 2 in Figure 2A, on the Object Placement tab.
4. Click on the Slicer tab (red square 1 in Figure 2B) next to Object Placement tab, and then click on the Configuration button (red square 2 in Figure 2B) below. Note that a new window on the left will open where the printer parameters such as velocity, layer thickness and holders can be defined (see more details in the Discussion below).
5. Click on the Import button (red square 3 in Figure 2E), select Supplemental File 1 from the files, and press Enter. Note that this .rcp file (downloaded in step 2.1) provides the parameters for the automatic configuration of the printer we have optimized for this vial rack.
6. To finish configuring the printing parameters, select None for Support Type on the menu on the right (red square 4 in Figure 2E), as printing of this piece does not require a support to prevent bending or other deformities. In Infill Density (red square 5 in Figure 2E), choose 20% to create a solid structure (see more details about these parameters in the Discussion below).
7. Click on Slice with CuraEngine on the upper right corner of the screen to run the slicing program and generate the G-Code, which has the information necessary for the printer to print the piece. Note that under the Print Preview tab on the menu on the right (next to the Slicer tab), information about the time and amount of material required for the printing job to be completed will then show (red square 1 in Figure 2F).
8. Click on Save for SD Print to save the G-Code file in a SD card (red square 2 in Figure 2F). Note that the G-Code contains the 3D coordinates of the designed piece, sliced into layers, for proper function of the printer.
9. Insert the SD card in the RepRap 3D printer, and then follow the information displayed on the printer screen to select printing from the SD card.
10. Select the G-Code file of the vial rack. Note that the printer will automatically warm up and start printing the designed piece, which should take several hours. The rack (Figure 3A) should be ready for use immediately after the printing job is completed.

3. Behavioral analysis apparatuses

NOTE: The steps described in Protocols 1 and 2 can be repeated with appropriate adjustments to print several of the pieces of lab equipment needed. However, we realize that designing new pieces may be challenging and time-consuming for beginner users of Tinkercad, so instead of providing step-by-step protocols on how to design all models, we are making available for download several design models we created as STL files (see Supplemental Files 2-11).

1. Download Supplemental File 2 for a model of a small funnel (Figure 3B), routinely used in flylabs to help transfer adult flies to new vials or bottles by avoiding flies crawling up the inside walls of these containers to escape.
2. Download Supplemental File 3 for a tapping mat support (Figure 3C), which can hold an ethylene-vinyl acetate foam or a thick cotton mat onto which glass vials or bottles can be tapped when one is tipping flies into new containers with freshly made food.
3. Download Supplemental File 4 and Supplemental File 5 for the model of a camera stand that we named Stalker (Figure 3D).
   NOTE: The apparatus allows any camera (professional, webcams, cellphones, etc.) to be positioned on top of a base where a Petri dish containing larvae or adult flies can be imaged or video recorded. Stalker allows the imaging of animal behavior that takes place horizontally, always at the same distance from the Petri dish, which avoids introducing variability into the experimental measurements if the recordings must be made on different days, for example, or if the camera needs to be used for another purpose in between recordings. The apparatus is conveniently modular and can be easily assembled after printing the base and the top from Supplemental File 4, and the sides from Supplemental File 5. The 1 cm² squares at the base, which can be highlighted using a permanent marker, help track distance travelled by individual animals. Print the apparatus (at least the base) using white filament, so that there is enough contrast between the background and the animals for the tracking software to identify each fly.
4. Download Supplemental File 6 for the printable design of the Fly Motel (Figure 3E), which has ten courtship and mating arenas (rooms) organized in a manner to facilitate video recordings of ten individual mating pairs at a time. Note that the Fly Motel is based on the device published in⁴⁰, where detailed explanation for its use in behavioral studies is found. In addition to the 3D-printed parts, the apparatus requires 12 screws (3 x 8 mm) for fixing the upper part to the lower one to stabilize the assembled device, an acrylic plate (60 x 60 x 3 mm), and a zip tight. Because the structure of the Fly Motel is more complex, we also provide an instructional video (Supplemental File 7) of how to assemble it correctly, given that all required pieces and a screwdriver are available.
5. Download Supplemental File 8 for the model of a T-Maze (Figure 3F), which is used for memory assays using adult flies. A detailed explanation of how the T-Maze is used to make flies associate repulsive odor stimuli with their phototropic behavior is found in the original publication⁴¹. The apparatus also makes use of two translucent 15 mL conical tubes, which are commonly found in any lab and are attached to the 2 cm-wide circular openings in each side of the central piece. Note that the printing of the T-Maze requires a support (see more details in the legend to Figure 2). Select "Everywhere" for Support Type (red square 4 in Figure 2E) after following steps 2.1-2.6 above.
6. Download Supplemental File 9 and Supplemental File 10 for the design of the printable parts of our version of the apparatus for rapid iterative negative geotaxis (RING) assays (Figure 3G), used to perform climbing assays with adult flies of several genotypes or environmental conditions simultaneously, generating results in a more standardized and higher-throughput fashion⁴². The RING apparatus is also modular, and in addition to the 3D-printed parts, it requires other pieces that can be easily purchased online or in a hardware store at low cost: two Φ8 x 300 mm rectified shafts, four Φ8 mm linear bearings, rubber bands (or pieces of a string), a 240 x 60 x 20 mm piece of wood for the base, and eight wood screws (8 mm) to fix the printed parts to the wood base. Download Supplemental File 11 for instructions on how to assemble the device once all parts are printed or purchased. A detailed explanation of how to use the RING apparatus is found in the original publication⁴².

4. Larval mobility assay

NOTE: We have optimized this protocol, originally based on Nichols et al.⁴², to study the effects of AOX expression on Drosophila development under cold stress. The lines 3xtubAOX²⁵ and w¹¹¹⁸, used as examples of AOX-expressing and control larvae, respectively, were cultured on standard diet²⁴ at 12 °C, according to Saari et al.³⁴. We recommend this protocol to analyze the mobility of larval samples of any genetic condition, cultured under any environmental condition of interest.

1. Prepare 2% agar plates by boiling the equivalent amount of agar in deionized water, pouring into Φ90 X 15 mm Petri dishes, and allowing them to solidify at room temperature. For plates with a final volume of 20 mL each, use 0.4 g of agar.
2. Carefully collect wandering L3 larvae from the side of the culture vials/flasks using a pair of round tip forceps or a brush, and place the individuals in a Φ90 x 15 mm Petri dish with deionized water for less than 10 seconds to rinse food particles attached to their body.
3. Transfer individual larva to dishes with agarose and wait 5 minutes for the animals to acclimate.
4. Position the dishes containing the individual larva on top of a graph paper (0.2 cm² grid), and count the number of lines crossed by the animal for 1 minute, as it moves on top of the agar. Each line crossed represent a distance of 2 mm. Repeat the procedure with the same individual 10-15 times to obtain technical replicates.
5. Repeat step 4.4 with at least 8-10 individuals from different tubes/flasks, cultured at different times to obtain biological replicates of the same line.
6. Repeat steps 4.4 and 4.5 to obtain data for the other fly line (or for as many lines as one intends to analyze).
7. The same individual larvae used in steps 4.4-4.6 may be used to obtain additional data on body movement by placing the agar dishes with an individual larva under a stereomicroscope and counting the number of peristaltic contractions of the body wall for 1 minute. Obtain technical and biological replicates for an estimate of average mobility among the fly lines analyzed.
8. Apply the statistical test to calculate the probability of the values of these larval mobility parameters being distinct among the lines of interest. Because we are comparing here data from only two lines, a Student's t test may be applied.

5. Mitochondrial respirometry using larval homogenates

NOTE: The following protocol was optimized to measure mitochondrial oxygen consumption from larval homogenates of the AOX-expressing line 3xtubAOX and the control w¹¹¹⁸, cultured at 12°C, but we also recommend it to be used for larval samples of any genetic and environmental conditions. We realize that conducting such experiments should not be included as an "affordable" goal for a "home-made" flylab, unlike all other protocols we provide in this article, as a considerable initial investment must be made for a lab to acquire a high resolution oxygraph. The protocol is to be used with the Oxygraph-2k (O2k) and the DatLab software from Oroboros Instruments, so further optimization is required should the reader want to use an alternative equipment.

1. Turn on the O2k and start the DatLab software in the computer connected to the oxygraph. The magnetic bars inside the assay chambers should start stirring automatically. Remove the ethanol storage solution from the chambers.
2. Wash the chambers at least 3 times with 100% ethanol and 3 times with ultrapure water.
3. In DatLab, the window O2k Control will open automatically. In Block temperature [°C], enter 12 °C (or the preferred temperature in the range of 2-47 °C) and press OK.
4. A second window, Edit Experiment, will then open automatically. Enter sample names in the Sample fields, according to what will be added in chambers A and B, and press Save.
5. Add 1800 µL of the assay buffer (120 mM KCl, 5 mM KH₂PO₄, 3 mM Hepes, 1 mM EGTA, 1 mM MgCl₂, 2% BSA, pH 7.4) in each chamber and close them partially, still allowing oxygen exchange with the outside air. Allow the oxygen concentration and oxygen flux signals to stabilize, showing minimal fluctuations for at least 10 min. This step will provide the calibration of the equipment with the outside air on the day of the experiment (see steps 6.3 and 6.4 below for further details on experimental calibrations).
6. During the air calibration step, initiate sample preparations by carefully collecting from the culture tubes/flasks 20 wandering larvae of the appropriate genotype, using a pair of forceps or a small paintbrush.
7. Rinse each larva quickly but thoroughly with deionized water or 1x PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄ and 2 mM KH₂PO₄) and transfer them to a 1 mL glass homogenizer on ice, containing 500 µL of ice-cold isolation buffer (250 mM sucrose, 5 mM Tris HCl, 2 mM EGTA, pH 7.4)
8. Homogenize the whole larvae with 5 strokes and pour the homogenate into a 1.5 mL microcentrifuge tube on ice.
9. Add 300 µL of isolation buffer to the glass homogenizer, macerate the remaining larval tissues further with 3 strokes, and pour the homogenate again into the same microcentrifuge tube on ice.
10. Add 200 µL of isolation buffer to the glass homogenizer, macerate the residual larval tissues further with 2 strokes, and pour the homogenate again into the same microcentrifuge tube on ice.
11. Mix the final homogenate (~1 mL) by inverting the tube twice gently and keep it on ice until the second sample is processed.
12. Repeat steps 5.6-5.11 to obtain the larval homogenate of the other genotype.
13. Open the oxygraph chambers A and B, and transfer 200 µL of each homogenate to the assay buffer in each chamber, which must be exactly at 12 °C at this point. Close the chambers completely and allow the oxygen concentration and oxygen flux signals to stabilize for approximately 10 min.
14. Initiate oxygen consumption measurements by adding to each chamber 5 µL of a solution of 2 M pyruvate, 2 M proline (final concentration in the chambers = 5 mM each) and 7.5 µL of a solution of 0.4 M malate (1.5 mM). Allow at least 5 minutes for signal stabilization. Note that at this point, the mitochondria will be charged with oxidizable substrates to initiate the tricarboxylic acid (TCA) cycle reactions. Any increase in the oxygen consumption signal may be due to the functions of uncoupling proteins (or due to other uncoupling phenomena), and can be used to calculate general uncoupled respiration (also referred to as Leak respiration in the absence of adenylates, L_N- see Representative Results for details).
15. Add to each chamber 4 µL of a solution of 0.5 M ADP (1 mM) and allow at least 5 minutes for signal stabilization. A significant increase in the oxygen consumption signal is usually immediately observed, representing the oxidative phosphorylative (OXPHOS) respiration. The great majority of this OXPHOS respiration is driven by complex I (CI).
16. Add to each chamber 2 µL of a solution of 0.05 M antimycin A (0.05 mM) to inhibit CIII, and allow at least 5 minutes for signal stabilization. A decrease in the oxygen consumption signal down to the basal levels seen before the addition of pyruvate, proline and malate should be observed for the w¹¹¹⁸ control sample. Mitochondrial respiration from AOX-expressing larvae will be partially resistant to antimycin-A, as the electrons can now be directed to AOX. About 40% of the total OXPHOS respiration should remain after CIII inhibition, which should be all supported solely by AOX.
17. Add to each chamber 4 µL of a solution of 0.1 M propyl-gallate (0.2 mM) to inhibit AOX, and allow at least 15 minutes for signal stabilization. The oxygen consumption levels in the AOX-expressing larval samples should now decrease to the basal levels seen before the addition of pyruvate, proline and malate. This decrease proves that the antimycin-A resistant respiration observed is due to AOX function.
18. Add to each chamber 1 µL 0.01 M rotenone (0.005 mM) to inhibit CI, and allow at least 5 minutes for signal stabilization to obtain the oxygen consumption signal of the sample independent of mitochondrial respiration. This signal is usually as low as that observed before the addition of pyruvate, proline and malate.
19. Save the experiment and close the DatLab software.

6. Mitochondrial respirometry data processing

NOTE: Oxygen consumption values are obtained as an average of the oxygen flux signals in a determined period of time and are expressed as pmol O₂ consumed per second per mg total protein in the sample. The values are first referenced against the maximal oxygen concentration available in the assay buffer on the day of the experiment, based on the experimental temperature (referred to as air saturation), and the minimal oxygen concentration, which is determined previously in each chamber by the addition of Na₂S₂O₄ to the assay buffer (see ⁴³ for the manufacturer's guidelines to obtain zero oxygen calibration). The values are also normalized by the amount of total protein in the larval homogenates added to the assay buffer of each chamber.

1. Determine total protein concentration of each sample using the Bradford method⁴⁴. Reopen the saved experiment on the DatLab software, press Experiment at the top menu, and then Edit. In the opened window, select mg in the Unit section and in the Amount section enter the protein amount contained in the 200 µL of the sample added in each chamber. The concentration will be automatically calculated by the software, considering the total chamber volume of 2 mL. Press the Save button.
2. Press Graph in the top menu, and then Select plots. In the opened window, select Flux per mass for both graphs (1 and 2, which refer to chambers A and B, respectively). Press Ok. The experimental outcome is now normalized by the protein concentration of the samples (pmol O₂/s/mg total protein).
3. On the upper right corner of each graph (1 and 2) of the main experimental outcome page, click on O₂ Concentration. Along the x axis, identify a time range prior to addition of the samples in the chambers, in which the oxygen concentration and flux signals are very stable.
   1. Press and hold the Shift key on the computer's keyboard, click with the left mouse button on the initial time selected, drag the cursor along the time axis to select the desired region, and release the mouse button. Do this procedure for each of the graphs individually.
   2. Double-click on the blue bars of the selected areas at the bottom of the graphs, and type "air" to indicate that these are the selected regions used to calculate the oxygen air saturation.
4. Click on Calibration on the top menu bar, and select A: Oxygen, O₂ to calibrate chamber A. In the opened window, verify that O₂ Calib is selected (yellow color).
   1. For the Zero calibration, click on Copy from file and choose the file with the previously performed zero oxygen calibration (see ⁴³ for the manufacturer’s guidelines).
   2. For Air calibration, select air in the Select Mark column. Click on Calibrate and copy to clipboard.
5. Click on Calibration on the top menu bar, select B: Oxygen, O₂ and repeat step 6.4 to calibrate chamber B.
6. On the upper right corner of each graph (1 and 2) of the main experimental outcome page, click on O₂ flux per mass. Select desired stable regions of the oxygen consumption signal by holding the Shift key on the keyboard, clicking with the left mouse button, and dragging the cursor along the time axis. The stable regions selected between additions of pyruvate/proline/malate and ADP represents the L_N; between ADP and antimycin A, OXPHOS; between antimycin A and propyl gallate (upon CIII inhibition), antimycin A-resistant respiration; between propyl gallate and rotenone (upon CIII+AOX inhibition), residual respiration; after rotenone (upon CI+CIII+AOX inhibition), residual non-mitochondrial respiration. Double-click on the red bars of the selected areas at the bottom of the graphs, and enter appropriate labels.
7. Click on Marks on the top menu bar, and then Statistics. In the Show tab of the opened window, uncheck all options except O₂ flux per mass. In the Select tab of the same window, select chamber A to obtain the respiration data for the first sample. Click on Copy to clipboard and paste the data into a spreadsheet. Repeat the procedure to obtain the data for the second sample in chamber B.
8. In a spreadsheet, subtract all respiration values by the residual non-mitochondrial respiration (the data after rotenone addition). Calculate averages from multiple experimental replicates and plot the data as preferred.

Results

By following the steps in Protocols 1 and 2, one should be able to design a simple fly vial rack, and run the model STL file through the slicing program to generate coordinates for the 3D printer. Figure 3A shows a printed unit of the model next to its design. We also hope step 1 can provide the basic skills for one to use the basic shapes available in the Tinkercad platform to create useful apparatuses for the lab. Developing these skills, however, may require constant practice and frequent...

Discussion

The 3D-printing protocols and STL files provided here are intended to facilitate the setup of a new flylab or to increase the repertoire of apparatuses in an existing Drosophila behavioral facility, using "homemade" equipment. The 3D-printing strategy may be particularly useful in developing countries such as Brazil, where research using Drosophila as a model organism for studying human biology appears to be underrepresented, and specialized equipment is costly. Our protocols provide instruction...

Materials

Name	Company	Catalog Number	Comments
3D Printer RapRep			A popular 3D-printer based on the Prusa I3 DIY mode, instructions available in https://www.instructables.com/Building-a-Prusa-I3-3D-Printer-Revisited/
3xtubAOX fly line	Howy Jacobs´s lab, Tampere University		Drosophila line expressing the AOX gene from C. intestinalis under the control of the constitutive α-tubulin promoter. 5 and 6 copies of this construct are present in males and females in homo/hemizigosity, respectively, one in each of the chromosomes X, 2 and 3.
Acrylic plate			60 x 60 x 3 mm
ADP	Sigma-Aldrich	A2754	Adenosine 5′-diphosphate sodium sal (CAS number 20398-34-9); ≥95%; molecular weight = 427.20 g/mol; solubility in water at 50 mg/ml
Antimycin-A	Sigma-Aldrich	A8674	Antimycin A from Streptomyces sp. (CAS number 1397-94-0); molecular weight ~ 548.63 g/mol; solubility in 95% ethanol at 50 mg/mL
Agar	Kasv	K25-611001	For bacteriologal use; powder; solidifying agent (12-20 g/L)
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A7030	Heat shock fraction, protease free, fatty acid free, essentially globulin free (CAS number 9048-46-8);pH 7; ≥98%; solubility in water 40g/ml
Deionized water
EGTA	Sigma-Aldrich	E4378	Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (CAS number 67-42-5); ≥97%; molecular weight = 380.35g/mol
Ethanol 99.5%
Ethylene-vinyl acetate foam			Can be replaced with thick pieces of cotton
Graph paper			0.2 cm2 grid
Hepes	Sigma-Aldrich	H4034	4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (CAS number 7365-45-9), BioPerformance Certified; ≥99,5% (titration), cell cultured tested; molecular weight =238.30g/mol
Homogenizer	Sartorius		Hand glass homogenizer (S), 1 mL; composed of a cylinder made of borosilicate glass plus plunger S; often used for simple sample preparation, e.g. crushing of tissue samples.
KCl	Amresco	0395-2	Potassium chloride (CAS number 7447-40-7); ≥99,0%; molecular weight = 74.55g/mol
KH2PO4	Sigma-Aldrich	P5379	Potassium phophate monobasic (CAS number 7778-77-0); ReagentPlus; molecular weight = 136.09g/mol
Linear bearings (LM8UU)			8 mm, any brand
Malate	Sigma-Aldrich	M1000	L-(-)-Malic acid (CAS number 97-67-6); ≥95-100%; molecular weight = 134.09 g/mol), solubility in water: 100 mg/mL. A solution is pH adjusted to approximately 7.0.
MgCl2	Amresco	0288-1KG	Magnesium chloride, hexahydrate (CAS number 7791-18-6); 99%-102%; molecular weight = 203.3g/mol
Microcentrifuge tubes			1.5mL; Graduated every 100µL, autoclavable
Na2HPO4	Amresco	0348-1KG	Sodium phosphate, dibasic, heptahydrate (CAS number 7782-85-6); 98-102%; molecular weight = 268.07 g/mol
NaCl	Honeywell	31434-1KG	Sodium chloride (CAS number 7647-14-5); ≥99,5%; molecular weight 58,44g/mol. For laboratory use only.
Oxigraph-O2k	Oroboros	10000-02	Series D-G; O2k-Core: includes O2k-Main Unit with stainless steel housing, O2k-Assembly Kit, two OroboPOS (polarographic oxygen sensors) and OroboPOS-Service Kit, DatLab software, the ISS-Integrated Suction System and the O2k-Titration Set.
Permanent marker			Preferably black
Petri dishes			90 X 15 mm dishes; commonly used for bacteriological culture
PLA 3D Printing Filament	Quantum3D Printing	http://quantum3dprinting.com/	High quality polylatic acid filament (PLA), strongly recomended, (1.0 kg Roll), any brand
Proline	Sigma-Aldrich	P0380	L-Proline (CAS number 147-85-3); powder; 99%; molecular weight = 115.13 g/mol
Propyl gallate	Sigma-Aldrich	P3130	Propyl gallate (CAS number 121-79-9); powder; ≥98%; molecular weight = 212.2 0g/mol; solubility in ethanol at 50 mg/ml
Pyruvate	Sigma-Aldrich	P2256	Sodium pyruvate (CAS number 113-24-6), ≥99%; molecular weight = 110.04 g/mol; solubility in water at 100 mg/mL
Rectified shafts			8 x 300 mm, any brand
Rotenone	Sigma-Aldrich	R8875	Rotetone (CAS number 83-79-4); ≥95%, molecular weight 394.42 g/mol
Rubber bands			Can be replaced with pieces of a string
Screwdriver			To assemble some of the 3D-printed apparatuses
Screews			M3 x 8 mm
SD Card			At least 32Mb in size; usually provided with 3D printers
Software Repetier Host	Hot-World GmbH & Co. KG	https://www.repetier.com/	Excellent slicing software, available free of cost
Software Tinkercad	Autodesk	https://www.tinkercad.com	3D model design software, available free of cost
Stereomicroscope	Leica	M-80	Stereomicroscope, zoom 7.5-60X + Leica cls 150 led light source
Sucrose	Merck	107,651,000	Sucrose for microbiology use (CAS number 57-50-1);
Tris	Amersham Biosciences	17-1321-01	Tris (hydroxymethyl)-aminomethane (CAS number 77-86-1); 99,8-100.1%; molecular weight 121.14 g/mol
Tweezer/forceps	Stark	ST08710	Histological tweezer, straight, round tip, 12 cm, AISI-410 stainless steel
w1118 fly line	Howy Jacobs´s lab, Tampere University		Drosophila line used as genetic background control for 3XtubAOX
Wood plate			240 x 60 x 20 mm
Zip tights			2 x 210 mm, any brand