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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Developmental Biology

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

Published: July 30th, 2016

DOI:

10.3791/54219

1Department of Plant and Wildlife Sciences, Brigham Young University, 2Department of Entomology, Cornell University, 3Department of Molecular Biology and Genetics, Cornell University, 4Division of Infectious Diseases, Boston Children's Hospital, Harvard Medical School, 5Biological Sciences, SUNY Oswego

ERRATUM NOTICE

Important: There has been an erratum issued for this article. Read more …

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Most animals are intimately associated with bacteria ('microbiota') from birth to death1. Comparisons of microorganism-free ('axenic') and microorganism-associated ('conventional') animals have shown microbes influence diverse aspects of animal health, including metabolic, nutritional, vascular, hepatic, respiratory, immunological, endocrine, and neurological function2. The fruit fly Drosophila melanogaster is a key model for understanding many of these processes in the presence of microbes3,4 and for studying microbiota influence on animal health5,6. No bacterial species is present in every ....

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1. Culture Bacteria (Start ~1 Week before Picking Eggs)

  1. Prepare modified MRS20 (mMRS) plates and broth tubes (Table 1). Pour 20 ml mMRS agar into each 100 mm Petri plate and allow to cool/dry overnight, or 5 ml mMRS broth into 18 mm test tubes.
  2. Streak Acetobacter pomorum, A. tropicalis, Lactobacillus brevis, and L. plantarum on mMRS agar plates. Incubate Acetobacter overnight at 30 °C. Incubate Lactobacillus anaer.......

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Successful rearing of axenic flies is confirmed by isolation of no CFUs from whole-body homogenizations of D. melanogaster adults (Figure 1). Alternatively, if the plated homogenate yields colonies, the vials are contaminated and should be discarded. For gnotobiotic flies, each of the four bacterial isolates were isolated from pools of 5 adult males, demonstrating differences in total viable CFUs associated with adult flies (Figure 1). Each bacte.......

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The method described here is one of several approaches for embryo dechorionation8,11,18,25,26,27, together with alternative methods of rearing axenic flies, including serial transfer of axenic adults18,27 or antibiotic treatment13,18. Other dechorionation methods include ethanol washes and reduce11,25,26 or extend8 hypochlorite treatment. Different wash steps may aid rearing different fly genotypes: in a previous study most of ~100 Drosophila genotypes were .......

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Some details of this protocol were optimized with the assistance of Dr. Adam Dobson, who also provided helpful comments on the manuscript. This work was supported by the Foundation for the National Institutes of Health (FNIH) grant number R01GM095372 (JMC, A(CN)W, AJD, and AED). FNIH grant number 1F32GM099374-01 (PDN), and Brigham Young University startup funds (JMC, MLK, MV). Publication costs were supported by the Brigham Young University College of Life Sciences and Department of Plant and Wildlife Sciences.

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Name Company Catalog Number Comments
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Nylon Mesh Genesee Scientific 57-102  https://geneseesci.com/shop-online/product-details/715/?product=57-102
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Specimen Cup MedSupply Partners K01-207067 http://www.medsupplypartners.com/covidien-specimen-containers.html
Repeater M4 Eppendorf 4982000322 https://online-shop.eppendorf.us/US-en/Manual-Liquid-Handling-44563/Dispensers--Burettes-44566/Repeater-M4-PF-44619.html
50 ml Centrifuge Tubes Laboratory Product Sales TR2003 https://www.lpsinc.com/Catalog4.asp?catalog_nu=TR2003
Food Boxes USA Scientific 2316-5001 http://www.usascientific.com/search.aspx?find=2316-5001
Lysing Matrix D Bulk MP Biomedicals, LLC. 116540434 http://www.mpbio.com/search.php?q=6540-434&s=Search
Filter Pipette Tips, 300μl USA Scientific 1120-9810 http://www.usascientific.com/search.aspx?find=1120-9810
Petri Dishes Laboratory Product Sales M089303 https://www.lpsinc.com/Catalog4.asp?catalog_nu=M089303
Ethanol Decon Laboratories, INC. 2701 http://www.deconlabs.com/products.php?ID=88
Paintbrush Walmart 5133 http://www.walmart.com/ip/Chenille-Kraft-5133-Acrylic-Handled-Brush-Set-Assorted-Sizes-colors-8-Brushes-set/41446005
Forceps Fisher 08-882 https://www.fishersci.com/shop/products/fisherbrand-medium-pointed-forceps-3/p-128693
Household Bleach (6-8% Hypochlorite) Walmart 550646751 http://www.walmart.com/ip/Clorox-Concentrated-Regular-Bleach-121-fl-oz/21618295
Universal Peptone Genesee Scientific 20-260 https://geneseesci.com/shop-online/product-details/?product=20-260
Yeast Extract  Fisher Scientific BP1422-500 https://www.fishersci.com/shop/products/fisher-bioreagents-microbiology-media-additives-yeast-extract-3/bp1422500?matchedCatNo=BP1422500
Dipotassium Phosphate Sigma Aldrich P3786-1KG http://www.sigmaaldrich.com/catalog/search?term=P3786-1KG&interface=All&N=0&mode=match%20partialmax&lang=en&region=US&focus=product
Ammonium Citrate Sigma Aldrich 25102-500g http://www.sigmaaldrich.com/catalog/search?term=25102-500g&interface=All&N=0&mode=match%20partialmax&lang=en&region=US&focus=product
Sodium Acetate VWR 97061-994 https://us.vwr.com/store/catalog/product.jsp?catalog_number=97061-994
Magnesium Sulfate Fisher Scientific M63-500 https://www.fishersci.com/shop/products/magnesium-sulfate-heptahydrate-crystalline-certified-acs-fisher-chemical-3/m63500?matchedCatNo=M63500
Manganese Sulfate Sigma Aldrich 10034-96-5 http://www.sigmaaldrich.com/catalog/search?term=10034-96-5&interface=CAS%20No.&N=0&mode=match%20partialmax&lang=en&region=US&focus=product
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96 Well Plate Reader BioTek (Epoch)  NA http://www.biotek.com/products/microplate_detection/epoch_microplate_spectrophotometer.html
1.7 ml Centrifuge Tubes USA Scientific 1615-5500 http://www.usascientific.com/search.aspx?find=1615-5500
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96 Well Plates Greiner Bio-One 655101 https://shop.gbo.com/en/usa/articles/catalogue/article/0110_0040_0120_0010/13243/
Ceramic Beads MP Biomedicals, LLC. 6540-434 http://www.mpbio.com/product.php?pid=116540434
Tissue Homogenizer MP Biomedicals, LLC. 116004500 http://www.mpbio.com/product.php?pid=116004500
Class 1 BioSafety Cabinet Thermo Scientific  Model 1395 http://www.thermoscientific.com/en/product/1300-series-class-ii-type-a2-biological-safety-cabinet-packages.html

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Erratum

Erratum: Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

An erratum was issued for: Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions. The Protocol was updated.

Step 6.1.4.2 of the Protocol was updated from:

If using the 4 species described here, normalize cells to equivalent colony forming unit (CFU)/ml densities (OD600 to CFU conversion determined previously20) using this equation:
E = ((O-B) x V x D)/C
where E = volume to resuspend pellet in (μl), O = OD600 bacteria, B = OD600 blank media, D = fold-dilution, V = µl bacterial culture prior to centrifugation, C = OD600 of predetermined constant. See Supplemental Code File for examples of calculations using these equations. For spectrophotometers that automatically blank, use "O" in place of "O-B".
Note: The predetermined constants (units OD600, normalized to 107 CFU ml-1, constants derived in20) are as follows: A. tropicalis (0.052), A. pomorum (0.038), L. brevis (0.056), L. plantarum (0.077).

to:

If using the 4 species described here, normalize cells to equivalent colony forming unit (CFU)/ml densities (OD600 to CFU conversion determined previously20) using this equation:
E = ((O-B) x V x D)/C
where E = volume to resuspend pellet in (μl), O = OD600 bacteria, B = OD600 blank media, D = fold-dilution, V = µl bacterial culture prior to centrifugation, C = OD600 of predetermined constant. See Supplemental Code File for examples of calculations using these equations. For spectrophotometers that automatically blank, use "O" in place of "O-B".
Note: The predetermined constants (units OD600, normalized to 107 CFU ml-1, constants derived in20) are as follows: A. tropicalis (0.053), A. pomorum (0.038), L. brevis (0.077), L. plantarum (0.056).

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