Name: rearing the fruit fly drosophila melanogaster under axenic and gnotobiotic conditions
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Description: Watch this Scientific Journal Video about Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions at JoVE.com

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.

1. Culture Bacteria (Start ~1 Week before Picking Eggs)
<ol>
	<li>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.</li>
	<li>Streak Acetobacter pomorum, A. tropicalis, Lactobacillus brevis, and L. plantarum on mMRS agar plates. Incubate Acetobacter overnight at 30 &#176;C. Incubate Lactobacillus anaer...

<ol>
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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+microbiome+modulates+host+developmental+and+metabolic+homeostasis+via+insulin+signaling.">Drosophila microbiome modulates host developmental and metabolic homeostasis via insulin signaling.</a> Science. 334 (6056), 670-674 (2011).</li><li>Storelli, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactobacillus+plantarum+promotes+Drosophila+systemic+growth+by+modulating+hormonal+signals+through+TOR-dependent+nutrient+sensing.">Lactobacillus plantarum promotes Drosophila systemic growth by modulating hormonal signals through TOR-dependent nutrient sensing.</a> Cell Metab. 14 (3), 403-414 (2011).</li><li>Wong, A. C., Chaston, J. M., Douglas, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inconstant+gut+microbiota+of+Drosophila+species+revealed+by+16S+rRNA+gene+analysis.">The inconstant gut microbiota of Drosophila species revealed by 16S rRNA gene analysis.</a> ISME J. 7 (10), 1922-1932 (2013).</li><li>Newell, P. D., Douglas, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecies+interactions+determine+the+impact+of+the+gut+microbiota+on+nutrient+allocation+in+Drosophila+melanogaster.">Interspecies interactions determine the impact of the gut microbiota on nutrient allocation in Drosophila melanogaster.</a> Appl Environ Microbiol. 80 (2), 788-796 (2014).</li><li>Broderick, N. 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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 ...

An erratum was issued for: <a href="https://www.jove.com/t/54219/rearing-fruit-fly-drosophila-melanogaster-under-axenic-gnotobiotic">Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions</a>. 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 (&#956;l), O = OD600 bacteria, B = OD600 blank media, D = fold-dilution, V = &#181;l 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 &#34;O&#34; in place of &#34;O-B&#34;. 
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 (&#956;l), O = OD600 bacteria, B = OD600 blank media, D = fold-dilution, V = &#181;l 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 &#34;O&#34; in place of &#34;O-B&#34;. 
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).

An erratum was issued for: <a href="https://www.jove.com/t/54219/rearing-fruit-fly-drosophila-melanogaster-under-axenic-gnotobiotic">Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions</a>. The Protocol was updated.

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

Most animals are intimately associated with bacteria (&#39;microbiota&#39;) from birth to death1. Comparisons of microorganism-free (&#39;axenic&#39;) and microorganism-associated (&#39;conventional&#39;) 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 individual (&#39;core&#39;), but Acetobacter and Lactobacillus species numerically dominate the microbiota of both laboratory-reared and wild-caught D. melanogaster. Other Acetobacteraceae (including Komagataeibacter and Gluconobacter), Firmicutes (such as Enterococcus and Leuconostoc), and Enterobacteriaceae are either frequently present in Drosophila individuals at low abundance, or irregularly present at high abundance7-12.
The microbiota of Drosophila and mammals is inconstant within and across generations14,19. Microbiota inconstancy can lead to phenotypic noise when measuring microbiota-dependent traits. For example, the Acetobacteraceae influence lipid (triglyceride) storage in Drosophila15-18. If Acetobacteraceae are more abundant in flies of one vial than in another19, isogenic flies can have different phenotypes20. A solution for the problem of microbiota inconstancy in mice14 has been in practice since the 1960&#39;s, by introducing a defined community of 8 dominant microbial species to mouse pups each new generation (altered Schaedler flora), ensuring that each pup is exposed to the same key members of the mouse microbiota. This practice controls for microbiota composition even when the microbiota is not the primary target of study32, and sets precedent to ensure the presence of key microbes in a variety of experimental conditions.
To define the influence of microbes on Drosophila nutrition, several protocols for deriving axenic fly lines have been developed, including hypochlorite dechorionation of embryos (either derived de novo each generation or maintained generationally by transfer to sterile diets) and antibiotic treatment13. There are benefits to different approaches, such as ease and rapidity for both of antibiotics treatment and serial transfer, versus greater control of confounding variables with de novo dechorionation (e.g., egg density, residual contaminating microbes, off-target antibiotic effects). Regardless of the method of preparation, introduction of specified microbial species to axenic embryos permits culture of Drosophila with defined (&#39;gnotobiotic&#39;) communities. Alternatively, mimicking the use of Schaedler flora, this community could be inoculated to conventionally-laid eggs (following steps 6-7 only) to ensure the presence of trait-influencing microbes in each vial and avoid complications of microbiota inconstancy. Here we describe the protocol for raising axenic and gnotobiotic Drosophila by de novo dechorionation of embryos, and for confirming the presence of introduced or contaminating microbial taxa.

<table border="0" cellpadding="0" cellspacing="0" width="851">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:259px;">Brewer&#39;s Yeast</td>
			<td style="width:291px;">MP Biomedicals, LLC.</td>
			<td style="width:108px;">903312</td>
			<td style="width:195px;">http://www.mpbio.com/product.php?pid=02903312</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma Aldrich</td>
			<td>158968-3KG</td>
			<td>http://www.sigmaaldrich.com/catalog/product/aldrich/158968?lang=en&#38;region=US</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>Fisher--Lab Scientific</td>
			<td>fly802010</td>
			<td>https://www.fishersci.com/shop/products/drosophila-agar-8-100mesh-10kg/nc9349177</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Welch&#39;s 100% Grape Juice Concentrate</td>
			<td>Walmart or other grocery store</td>
			<td>9116196</td>
			<td>http://www.walmart.com/ip/Welch-s-Frozen-100-Grape-Juice-Concentrate-11.5-oz/10804406</td>
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			<td height="20" style="height:20px;">Cage: 32 oz. Translucent Round Deli Container</td>
			<td>Webstaurant Store</td>
			<td>999L5032Y</td>
			<td>http://www.webstaurantstore.com/newspring-delitainer-sd5032y-32-oz-translucent-round-deli-container-24-pack/999L5032Y.html</td>
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			<td height="20" style="height:20px;">Translucent Round Deli Container Lid</td>
			<td>Webstaurant Store</td>
			<td>999YNL500</td>
			<td>http://www.webstaurantstore.com/newspring-delitainer-ynl500-translucent-round-deli-container-lid-60-pack/999YNL500.html</td>
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			<td height="20" style="height:20px;">Stock Bottles</td>
			<td>Genesee Scientific</td>
			<td style="width:108px;">32-130</td>
			<td>https://geneseesci.com/shop-online/product-details/?product=32-130</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Droso-Plugs</td>
			<td>Genesee Scientific</td>
			<td style="width:108px;">49-101</td>
			<td>https://geneseesci.com/shop-online/product-details/?product=49-101</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Nylon Mesh</td>
			<td>Genesee Scientific</td>
			<td>57-102&#160;</td>
			<td>https://geneseesci.com/shop-online/product-details/715/?product=57-102</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Plastic Bushing</td>
			<td>Home Depot</td>
			<td style="width:108px;">100343125</td>
			<td>http://www.homedepot.com/p/Halex-2-1-2-in-Rigid-Insulated-Plastic-Bushing-75225/100343125</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Specimen Cup</td>
			<td>MedSupply Partners</td>
			<td>K01-207067</td>
			<td>http://www.medsupplypartners.com/covidien-specimen-containers.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Repeater M4</td>
			<td>Eppendorf</td>
			<td>4982000322</td>
			<td>https://online-shop.eppendorf.us/US-en/Manual-Liquid-Handling-44563/Dispensers--Burettes-44566/Repeater-M4-PF-44619.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 ml Centrifuge Tubes</td>
			<td>Laboratory Product Sales</td>
			<td>TR2003</td>
			<td>https://www.lpsinc.com/Catalog4.asp?catalog_nu=TR2003</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Food Boxes</td>
			<td>USA Scientific</td>
			<td>2316-5001</td>
			<td>http://www.usascientific.com/search.aspx?find=2316-5001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lysing Matrix D Bulk</td>
			<td>MP Biomedicals, LLC.</td>
			<td>116540434</td>
			<td>http://www.mpbio.com/search.php?q=6540-434&#38;s=Search</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter Pipette Tips, 300&#956;l</td>
			<td>USA Scientific</td>
			<td>1120-9810</td>
			<td>http://www.usascientific.com/search.aspx?find=1120-9810</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri Dishes</td>
			<td>Laboratory Product Sales</td>
			<td>M089303</td>
			<td>https://www.lpsinc.com/Catalog4.asp?catalog_nu=M089303</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol</td>
			<td>Decon Laboratories, INC.</td>
			<td>2701</td>
			<td>http://www.deconlabs.com/products.php?ID=88</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paintbrush</td>
			<td>Walmart</td>
			<td>5133</td>
			<td>http://www.walmart.com/ip/Chenille-Kraft-5133-Acrylic-Handled-Brush-Set-Assorted-Sizes-colors-8-Brushes-set/41446005</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>Fisher</td>
			<td>08-882</td>
			<td>https://www.fishersci.com/shop/products/fisherbrand-medium-pointed-forceps-3/p-128693</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Household Bleach (6-8% Hypochlorite)</td>
			<td>Walmart</td>
			<td>550646751</td>
			<td>http://www.walmart.com/ip/Clorox-Concentrated-Regular-Bleach-121-fl-oz/21618295</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Universal Peptone</td>
			<td>Genesee Scientific</td>
			<td>20-260</td>
			<td>https://geneseesci.com/shop-online/product-details/?product=20-260</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yeast Extract</td>
			<td>&#160;Fisher Scientific</td>
			<td>BP1422-500</td>
			<td>https://www.fishersci.com/shop/products/fisher-bioreagents-microbiology-media-additives-yeast-extract-3/bp1422500?matchedCatNo=BP1422500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dipotassium Phosphate</td>
			<td>Sigma Aldrich</td>
			<td>P3786-1KG</td>
			<td>http://www.sigmaaldrich.com/catalog/search?term=P3786-1KG&#38;interface=All&#38;N=0&#38;mode=match%20partialmax&#38;lang=en&#38;region=US&#38;focus=product</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium Citrate</td>
			<td>Sigma Aldrich</td>
			<td>25102-500g</td>
			<td>http://www.sigmaaldrich.com/catalog/search?term=25102-500g&#38;interface=All&#38;N=0&#38;mode=match%20partialmax&#38;lang=en&#38;region=US&#38;focus=product</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Acetate</td>
			<td>VWR</td>
			<td>97061-994</td>
			<td>https://us.vwr.com/store/catalog/product.jsp?catalog_number=97061-994</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium Sulfate</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td>https://www.fishersci.com/shop/products/magnesium-sulfate-heptahydrate-crystalline-certified-acs-fisher-chemical-3/m63500?matchedCatNo=M63500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Manganese Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>10034-96-5</td>
			<td>http://www.sigmaaldrich.com/catalog/search?term=10034-96-5&#38;interface=CAS%20No.&#38;N=0&#38;mode=match%20partialmax&#38;lang=en&#38;region=US&#38;focus=product</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MRS Powder</td>
			<td>Sigma Aldrich</td>
			<td style="width:108px;">69966-500G</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/69966?lang=en&#38;region=US</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 Well Plate Reader</td>
			<td>BioTek (Epoch)&#160;</td>
			<td>NA</td>
			<td>http://www.biotek.com/products/microplate_detection/epoch_microplate_spectrophotometer.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.7 ml Centrifuge Tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td>http://www.usascientific.com/search.aspx?find=1615-5500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Filter Pipette Tips, 1000&#956;l</td>
			<td>USA Scientific</td>
			<td>1122-1830</td>
			<td>http://www.usascientific.com/search.aspx?find=1122-1830</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 Well Plates</td>
			<td>Greiner Bio-One</td>
			<td>655101</td>
			<td>https://shop.gbo.com/en/usa/articles/catalogue/article/0110_0040_0120_0010/13243/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ceramic Beads</td>
			<td>MP Biomedicals, LLC.</td>
			<td>6540-434</td>
			<td>http://www.mpbio.com/product.php?pid=116540434</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Homogenizer</td>
			<td>MP Biomedicals, LLC.</td>
			<td>116004500</td>
			<td>http://www.mpbio.com/product.php?pid=116004500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Class 1 BioSafety Cabinet</td>
			<td>Thermo Scientific&#160;</td>
			<td>Model 1395</td>
			<td>http://www.thermoscientific.com/en/product/1300-series-class-ii-type-a2-biological-safety-cabinet-packages.html</td>
		</tr>
	</tbody>
</table>

rearing the fruit fly drosophila melanogaster under axenic and gnotobiotic conditions

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.

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...

Watch this Scientific Journal Video about Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions at JoVE.com

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

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.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

Research

JoVE Journal

Developmental Biology

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...