Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

Published: July 30th, 2016

DOI:

Melinda L. Koyle¹, Madeline Veloz¹, Alec M. Judd¹, Adam C.-N. Wong^2,4, Peter D. Newell^2,5, Angela E. Douglas^2,3, John M. Chaston^1,2

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 …

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.

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 = OD₆₀₀of 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 = OD₆₀₀of 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).