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Method Article
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, 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.
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 expensive6,7,8 and is relatively simple, as unlike vertebrates, no permission from a bioethical committee is necessary for experimentation9,10. 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 process12,13.
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 places15,16. 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 cell17,18, 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 production19,20,21. No insect, including Drosophila, or vertebrate naturally possesses AOX21,22,23, but its expression in a myriad of model systems24,25,26,27,28,29 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 cyanide24,25, and mitigates diverse phenotypes related to mitochondrial disfunction24,25,30,31,32. 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 tissues33,34.
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.
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.
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.
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).
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.
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.
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.
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...
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...
The authors declare no conflict of interest.
We would like to thank Emily A. McKinney for English editing of the manuscript. G.S.G. was supported by a fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number 141001/2019-4). M.T.O. would like to acknowledge funding from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant numbers 2014/02253-6 and 2017/04372-0), and the CNPq (grant numbers 424562/2018-9 and 306974/2017-7). C.A.C.-L. would like to acknowledge internal financial support from the Universidade do Oeste Paulista. The work with genetically modified Drosophila lines was authorized by the Local Biosafety Committee (CIBio) of the Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, under the protocols 001/2014 and 006/2014, and by the National Technical Committee on Biosafety (CTNBio), under the protocols 36343/2017/SEI-MCTIC, 01200.706019/2016-45, and 5488/2017.
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 |
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