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 e…

Representative 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

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