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 individual ('core'), 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'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 ('gnotobiotic') 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.
1. Culture Bacteria (Start ~1 Week before Picking Eggs)
2. Prepare Sterile Diet
3. Prepare Egg-laying Cages
4. Collect Eggs
5. Dechorionate Eggs and Transfer to Sterile Diet
6. Make Gnotobiotic Flies Using 4 Bacterial Species
7. Measure CFU Load/Test for Sterility
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 bacterial species has a distinct morphology and can be distinguished visually (Figure 2). If one or more colony types are not detected there may have been errors in preparing the bacterial inoculum (e.g., washing, normalizing, mixing) or the corresponding species may not be compatible with culture conditions (e.g., Drosophila density23 or genotype24). To rule out technical errors we recommend plating a portion of the bacterial mixture immediately after inoculating fly vials (step 6.2).
Figure 1: Colony Forming Units Found in Axenic and Gnotobiotic Drosophila. The number of colony forming units (CFU) per fly in whole-body homogenates of 4-species gnotobiotic and axenic D. melanogaster. Lack of colonies in the axenic homogenates confirms D. melanogaster sterility. Values are presented as mean ±SEM of 24 replicate values. Please click here to view a larger version of this figure.
Figure 2: Differentiating Bacterial Colonies. CFUs from a sample homogenization showing different morphologies for each of the 4-species in the gnotobiotic D. melanogaster. Acetobacter colonies are a clear, light brown color and come in varying sizes depending on the species. ~24-36 hr after plating, A. tropicalis colonies are opaque, whereas A. pomorum is transparent, though over time the difference becomes less pronounced. L. brevis colonies are small and white and L. plantarum colonies are large and yellow. If necessary, growth from the homogenate can be compared to the original bacteria plates to help distinguish each type of colony20. Please click here to view a larger version of this figure.
mMRS Recipe | ||
Amount in g | Notes | |
Distilled Water | 1,000 | |
Universal Peptone | 12.5 | |
Yeast Extract | 7.5 | |
Glucose | 20 | |
Dipotassium Phosphate | 2 | |
Ammonium Citrate | 2 | |
Sodium Acetate | 5 | |
Magnesium Sulfate | 0.1 | |
Manganous Sulfate | 0.05 | |
Agar | 12 | Do not add to broth |
Table 1: mMRS recipe.
Yeast-glucose diet recipe | |
Amount in g | |
Distilled water | 500 |
Brewer's Yeast | 50 |
Glucose | 50 |
Agar | 6 |
Table 2: Yeast-glucose diet recipe.
Supplemental Code File: Sample Calculations. Please click here to download this file.
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 axenic when reared as outlined here (without ethanol washes), but some lines were contaminated and discarded24. Perhaps some contaminated lines would have been axenic if reared with ethanol washes or longer hypochlorite treatment. If the method outlined here does not lead to isolation of axenic flies for particular host genotypes we recommend ethanol rinses or longer hypochlorite treatment to remedy the problem.
When serial transfers of Drosophila are used, a parental axenic generation is made axenic by egg dechorionation as described here, and subsequent generations are maintained by aseptic transfer in a biosafety cabinet, with or without antibiotics18,27. Serial transfer is faster than deriving axenic flies anew each generation, and transferred flies can remain axenic for multiple generations. One complication of serial transfers is maintaining matched densities of conventional/gnotobiotic and axenic flies since axenic flies tend to lay fewer eggs over a comparable time interval (data not shown). Since Drosophila density influences multiple traits, including bacterial composition in fly diet23,28,29, transferring eggs anew each generation may be a superior approach for fly density-sensitive traits. De novo dechorionation also avoids the possibility of bacterial contamination during transfers. Thus, while serial transfers can save time, dechorionation allows more control of complicating variables.
Antibiotics can also be used to create axenic flies, although in contrast to dechorionation, antibiotic treatment is usually insufficient to completely remove colonizing microbes13. Additionally, antibiotics may influence the host directly. For example, raising flies on a diet with antibiotics decreases their fecundity and protein content, but these effects were not observed in flies raised from dechorionated embryos13. We note that antibiotics are necessary to eliminate endosymbiotic bacteria that are transmitted vertically and are not affected by surface sterilization, such as Wolbachia33.
Several steps are critical to the success of preparing dechorionated axenic or gnotobiotic flies. First, it is crucial to shake the fly diet as in steps 2.3. If the racks are not shaken by hand for about 15 sec each before and after an exact 45 min interval on the shaker, the yeast and agar will settle, reducing fly access to yeast and making the agar surface too soft for fly culture. Second, the thickness of yeast paste and the agar concentration of grape juice plates influences egg removal (step 3.3). A thin layer produced by a 1:15 yeast:water dilution will prevent eggs from embedding in the agar when rinsing and removing from the food plates (step 4.4). Third, if grape juice plates are too soft and break up during egg collection, firmness of plates can be increased by adding less grape juice concentrate to the next batch of plates. Fourth, egg yields are higher if flies have 24 hr to acclimatize to the cage environment: this can be addressed by placing flies in the cage ~40 hours in advance of collection, including transfer to a new collection cage with a fresh agar plate <20 hr before the desired collection date. Fifth, if the eggs do not adhere to the side of the bushing during dechorionation (step 5.7), the rinsing period should be extended by 15-30 sec (Caution: extending the rinse time too long may kill the eggs). Also, egg loss into the liquid rinses can be prevented by checking the mesh-bushing seal for complete closure and verify that eggs do not spill outside of the sieve during re-suspension. If the bushing is well sealed, spilled eggs can be recovered by pouring the wash through the sieve as the wash is discarded. Sixth, after flies have hatched, a presumptive test to determine if the flies are axenic is to examine the color of the diet. If the flies are axenic, the top layer of diet will be a dark-brown coffee color and no air-bubbles will be present throughout the diet. Air bubbles or tan color of the top diet layer indicate bacterial presence. Bacterial presence should still be confirmed by homogenization. Alternatively, PCR amplification of the 16S rRNA gene can also be performed to detect unculturable microbes (e.g., strict anaerobes)30. Finally, after homogenization, ceramic beads can reused by rocking in a solution of 2% hypochlorite + 0.05 M potassium hydroxide for 30 min, rinsing generously (10 times or more) in H2O, washing once with 100% ethanol (to facilitate drying), and drying at 65 °C. If all steps are followed carefully, axenic flies should be isolated every time the protocol is performed.
This work outlines a method for re-associating sterile Drosophila embryos with a 4-species microbiota that is representative of the bacterial communities of laboratory flies raised on a yeast-glucose diet. Previous work has used a 5-species community including the 4-species here and Lactobacillus fructivorans, which has been numerically abundant in several fly surveys9,19. We recommend omitting L. fructivorans for several reasons, including that phenotypes in D. melanogaster monoassociated with L. fructivorans are largely congruent with axenic phenotypes 31 and L. fructivorans is more fastidious than other fly isolates. If L. fructivorans is included, it should be cultured as described for L. plantarum and L. brevis: static liquid culture; and in an airtight container flooded with CO2 for solid culture (the latter step reduces ambient oxygen levels to support Lactobacillus growth).
The approaches outlined here can be readily varied to raise Drosophila under diverse gnotobiotic conditions. For example, monoassociated flies can be reared by inoculating sterile Drosophila embryos with one microbial species at a time15,25,31. Multi-species associations are formed by inoculating multiple species in equivalent ratios20,24. Although we provided CFU/OD600 constants for each of the 4-species to facilitate normalization, it may not be necessary to derive a constant for most species mixtures. It was previously shown that the abundance of microbes in 5-7 d.p.e. adults was not significantly different in 2-species associations when the starting density of bacteria was varied over three orders of magnitude 20. Also, the Drosophila gut is highly permissive, and many species that are readily cultured on fly diet can associate with Drosophila at high densities in monoassociation (e.g., E. coli, B. subtilis25) enabling genetic dissection of microbial influence by microbes with extensive genetic and genomic resources. Finally, studies of Drosophila phenotypes that are influenced by the microbiota could adapt the approach of Altered Schaedler Flora in mice by inoculating naturally laid conventional eggs with a defined microbial community to ensure each vial has access to a specific set of microbes.
The authors have nothing to disclose.
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.
Brewer's Yeast | MP Biomedicals, LLC. | 903312 | http://www.mpbio.com/product.php?pid=02903312 |
Glucose | Sigma Aldrich | 158968-3KG | http://www.sigmaaldrich.com/catalog/product/aldrich/158968?lang=en®ion=US |
Agar | Fisher–Lab Scientific | fly802010 | https://www.fishersci.com/shop/products/drosophila-agar-8-100mesh-10kg/nc9349177 |
Welch's 100% Grape Juice Concentrate | Walmart or other grocery store | 9116196 | http://www.walmart.com/ip/Welch-s-Frozen-100-Grape-Juice-Concentrate-11.5-oz/10804406 |
Cage: 32 oz. Translucent Round Deli Container | Webstaurant Store | 999L5032Y | http://www.webstaurantstore.com/newspring-delitainer-sd5032y-32-oz-translucent-round-deli-container-24-pack/999L5032Y.html |
Translucent Round Deli Container Lid | Webstaurant Store | 999YNL500 | http://www.webstaurantstore.com/newspring-delitainer-ynl500-translucent-round-deli-container-lid-60-pack/999YNL500.html |
Stock Bottles | Genesee Scientific | 32-130 | https://geneseesci.com/shop-online/product-details/?product=32-130 |
Droso-Plugs | Genesee Scientific | 49-101 | https://geneseesci.com/shop-online/product-details/?product=49-101 |
Nylon Mesh | Genesee Scientific | 57-102 | https://geneseesci.com/shop-online/product-details/715/?product=57-102 |
Plastic Bushing | Home Depot | 100343125 | http://www.homedepot.com/p/Halex-2-1-2-in-Rigid-Insulated-Plastic-Bushing-75225/100343125 |
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®ion=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®ion=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®ion=US&focus=product |
MRS Powder | Sigma Aldrich | 69966-500G | http://www.sigmaaldrich.com/catalog/product/sial/69966?lang=en®ion=US |
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 |
Filter Pipette Tips, 1000μl | USA Scientific | 1122-1830 | http://www.usascientific.com/search.aspx?find=1122-1830 |
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 |