Drosophila Fecal Sampling

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Applied and Environmental Microbiology
Nov 2013



Fecal sampling is a non-invasive method which raises the possibility to study the development and the changes in the microbial community throughout different time points of a fly population or throughout different treatments. This method allows precise manipulation to trigger the fly’s physiology by nutritional interventions, bacterial infections or other stressors.

As in most other animals, the intestinal microbiota is essential for a healthy fly-life. Because Drosophila only harbors a relative simple bacterial community with a small variety of round about 8 to 10 different species, it is rather easy to build up the microbial community and to investigate microbial changes after treatment.

Another positive effect using the fly’s feces is that bacteria that are not part of the intestinal microbiome, for example Wolbachia, can be excluded directly from the analysis because they are not excreted.

Using this method, the generated datasets may reflect a good paradigm to study microbiome associated diseases in a simple fly model or furthermore, to test drugs in a high-throughput approach.

Keywords: Drosophila (果蝇), Intestine (肠道), Microbiome (微生物组), Fecal sampling (粪便取样), DNA isolation (DNA分离)


Until now, studies aiming to characterize the intestinal microbiome in the fruit fly Drosophila melanogaster used whole flies or dissected intestines as sources for bacterial DNA isolation. The main idea using feces sampling is to enable analyzing the dynamics of the intestinal microbiome in response to different treatments such as nutritional interventions or drug administration in the same cohort of flies. We were able to demonstrate that all general bacteria species, which are known to be important members of the Drosophila microbiome can be detected in fecal samples (Chandler et al., 2011; Wong et al., 2011; Matos and Leulier, 2014). Although whole fly samples are apparently the most convenient sources to generate microbiome data, several drawbacks are faced with this approach including contamination with surface bacteria or with intracellular bacteria such as Wolbachia spec (Saridaki and Bourtzis, 2010; Clark et al., 2005). Using feces as a source for microbiome data acquisition, these contaminations can be excluded almost completely, and, most importantly, the same cohorts of flies can be analyzed several times during the course of a complex experimental setup, thus unleashing the full potential of Drosophila genetics for microbiome studies.

Materials and Reagents

Note: For full name of the abbreviations in the text, please see Table S1 in Supplemental file.

  1. Filter tips, 0.5-10 µl, super slim, surface optimized (NERBE PLUS, catalog number: 07-613-8300 )
  2. Filter tips, 0-200 µl, super slim, surface optimized (NERBE PLUS, catalog number: 07-662-8300 )
  3. Filter tips, 0.5-10 µl, super slim, surface optimized (NERBE PLUS, catalog number: 07-695-8300 )
  4. Empty and clean Drosophila vials (NERBE PLUS, catalog number: 11-881-0052 )
  5. Cellulose plugs (NERBE PLUS, catalog number: 11-881-1010 )
  6. 0.22 μm sterilize filter filtropur (SARSTEDT, catalog number: 83.1826.001 )
  7. 1.5 ml reaction tubes (SARSTEDT, catalog number: 72.690.001 )
  8. 2 ml collecting tube
  9. Sterile spin swabs (Greiner Bio One International, catalog number: 420180 )
  10. X-tracta Tips (Biozym Scientific, catalog number: 615935 )
  11. Nitrogen (AIR LIQUIDE Deutschland, catalog number: I4001S10R2A001 )
  12. PowerSoil DNA isolation kit (NEW: Quiagen DNeasy PowerSoil Kit) (QIAGEN, catalog number: 12888 )
    Note: Attention this kit was from MOBIO in the past, also called Power Soil Kit!
  13. 70% ethanol diluted from 100% ethanol (Carl Roth, catalog number: 9065 )
  14. Proteinase K (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EO0491 )
  15. Phusion® Hot Start DNA polymerase (Thermo Fisher Scientific, catalog number: F540L )
  16. Crystal-Agarose (BIOLABPRODUCTS, catalog number: 16-005-805 )
  17. 100 bp DNA ladder (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15628019 )
  18. GeneRuler 50 bp DNA ladder (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: SM0371 )
  19. qPCR Bio SYBR-Hi-ROX (NIPPON Genetics, catalog number: PB20.12-20 )
  20. Deoxynucleotide triphosphates (dNTPs) (Promega, catalog number: U1330 )
  21. Oligo dT 7 primer (custom made, order from Thermo Fisher Scientific)
  22. Oligonucleotides for target genes for use in qPCR (custom made, order from Thermo Fisher Scientific, use [20 µM])
  23. RT-PCR grade water (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9935 )
  24. MinElute Gel Extraction Kit (QIAGEN, catalog number: 28604 )
  25. Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32851 )
  26. Agencourt AMPure XP (Beckman Coulter, catalog number: A63882 )
  27. Fly medium (see Recipes)
    1. Cornmeal (Mühle Schlingemann, catalog number: DAV-17080 )
    2. Brewer’s yeast (Leiber, catalog number: 17001636 )
    3. Glucose (Carl Roth, catalog number: 6780 )
    4. Agar-agar (Carl Roth, catalog number: 5210 )
    5. Sugar beat syrup (Kanne Brottrunk, catalog number: 2201 )
    6. Sugar molasses (Biohof Heidelicht, catalog number: 013aa )
  28. Preservatives for fly medium:
    1. Nipagin/Methyl 4-hydroxybenzoate (Sigma-Aldrich, catalog number: H5501 )
    2. Propionic acid (Carl Roth, catalog number: 6026 )
  29. 10x phosphate-buffered saline (PBS) (see Recipes)
    1. Sodium chloride (NaCl) (Carl Roth, catalog number: P029 )
    2. Potassium chloride (KCl) (Carl Roth, catalog number: 6781 )
    3. Potassium phosphate monobasic (KH2PO4) (Carl Roth, catalog number: P018 )
    4. Di-sodium hydrogen phosphate dihydrate (Carl Roth, catalog number: 4984 )
  30. 10x TBE (Tris-Borat-EDTA) buffer (see Recipes)
    1. Tris (Carl Roth, catalog number: 4855 )
    2. Borate acid (Carl Roth, catalog number: 6943 )
    3. Na2EDTA (Carl Roth, catalog number: X986 )


  1. Eppendorf Research® plus, single-channel, variable, 0.1-2.5 µl, dark grey (Eppendorf, catalog number: 3120000011 )
  2. Eppendorf Research® plus, single-channel, variable, 0.5-10 µl, light grey (Eppendorf, catalog number: 3120000020 )
  3. Eppendorf Research® plus, single-channel, variable, 2-20 µl, yellow (Eppendorf, catalog number: 3120000038 )
  4. Eppendorf Research® plus, single-channel, variable, 100-1,000 µl, blue (Eppendorf, catalog number: 3120000062 )
  5. Autoclave
  6. Sterile bench (NuAire, model: NU-437-400E )
  7. Anesthetizing pistol (Blowgun) (GENESE Scientific, model: 54-104 )
  8. Dumont forceps #5 (Fine Science Tools, catalog number: 11252-20 )
  9. Scissors (Bioform, model: B37e )
  10. Vortexer (Scientific Industries, model: Vortex Genie 2 , catalog number: SI-0256)
  11. Vortex adapter (MO BIO, catalog number: 13000-V1 )
  12. Thermomixer (Eppendorf, model: Thermomixer Comfort )
  13. Centrifuge (Eppendorf , model: 5424 )
  14. Qubit 3.0 Fluorometer (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q33216 )
  15. Sensoquest labcycler (BIOLABPRODUCTS, catalog number: 11-011-101-096 )
  16. NanoDrop 3300 Fluorometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 3300, catalog number: ND-3300 )
  17. 25 °C incubator (AQUALYTIC, model: TC 140 G , catalog number: 438210)
  18. StepOne qPCR cycler (Thermo Fisher Scientific, catalog number: 4376357 )
  19. Gel DocTM XR+ Gel Documentation System (Bio-Rad Laboratories, model: Molecular Imager® Gel DocTM XR+, catalog number: 1708195 )
  20. Wide Mini-Sub® Cell GT Horizontal Electrophoresis System, 15 x 10 cm tray, with PowerPacTM Basic Power Supply (Bio-Rad Laboratories, catalog number: 1640301 )
  21. Agilent Bioanalyzer (Agilent Technologies, model: 2100 Bioanalyzer , catalog number: G2939BA)
  22. Water bath
  23. 1.5 L beaker


  1. MOTHUR v1.23.1
  2. R statistics package v.2.13.1 (R_Development_Core_team, 2011)


  1. Medium preparation
    1. Cook desired sterile fly medium (see Recipes) by autoclaving the medium at 120 °C for 20 min.
    2. Decontaminate empty and clean Drosophila vials and plugs under a safety cabinet via UV light for at least 30 min (Figure 1A).
    3. Let the medium cool down to 65 °C and add preservative agents under sterile conditions, if necessary.
    4. Pour the sterile fly medium into the decontaminated Drosophila vials under sterile conditions (Figure 1B).
    5. Let the medium dry under the safety cabinet and plug the vials (Figure 1C).
    6. It is possible to store the sterile medium at 4 °C in a sterile plastic box.

      Figure 1. Preparation of sterile medium vials. A. Put empty (marked) vials and fresh plugs under the sterile bench and sterilize them using UV light for at least 30 min. B. Decant the fresh and sterile medium in the vials after decontamination under the bench and let the medium dry for about 45 to 60 min. C. Plug the vials. Vials can be kept at 4 °C in a sterile box.

  2. Feces collection
    1. Anesthetize the flies with nitrogen using the Blowgun (anesthetizing pistol). Transfer cohorts of 30 to 50 flies under sterile conditions to a sterile Drosophila vial and let them defecate for 24 h at 25 °C (Figure 2A).
    2. Then, transfer flies to a new vial under sterile conditions or discard them.
      Note: Figures 2B and 2C show how vials and fecal spots will look like after 24 h.

      Figure 2. Feces in the vial. Transfer 30 to 50 flies into a sterile prepared vial and let the flies defecate for about 24 h at 25 °C for standard wildtypes or crossings. B. After 24 h many fecal spots can be seen all over the vial (Figure 2C shows a magnification, red arrows mark only a few spots for better understanding).

    3. Place sterile spin swabs, sterile 1x PBS (see Recipes), sterile scissors and the Drosophila vial containing the feces under the safety cabinet (Figure 3A).
    4. For better control, filtrate the needed amount of 1x PBS using a sterile filtropour filter (Figure 3B).
    5. Soak sterile spin swab with sterile 1x PBS (Figure 3C; for preparation step 5-8, see Video 1).
    6. Open vial and carefully collect the feces with the soaked spin swab.
      Note: Don’t come in contact with the medium (Figures 3D and 3E).

      Figure 3. Feces collection. A. UV sterilization of all used materials, for example 1x PBS buffer, single packed swabs, sterile syringe, scissors and reaction tubes. B. Filter the needed amount of 1x PBS once again using a syringe filter. C. Soak the sterile swab with the fresh prepared and filtered 1x PBS. D. Start to collect/swab all fecal spots from the vial. ONLY from the walls, ignore spots on the plug AND take care NOT to touch the medium with the swab (E). F. Collected fecal spots from 40 flies after 24 h at 25 °C.

      Video 1. Feces collection

    7. Transfer the spin swab into a reaction tube and carefully cut off the stick with the sterile scissors (Figure 4C).
    8. Close the reaction tube.
    9. Prepare a negative control in the same way using an empty medium vial prepared at the same day as the used medium.
    10. Soak sterile spin swab with sterile 1x PBS (Figure 3C).
    11. Open vial and carefully collect the feces with the soaked spin swab (Figure 4A).
    12. After collection, the NTC (No Template Control) swab shows no feces (Figure 4B).
      Note: Don’t come in contact with the medium (Figures 3D and 3E).

      Figure 4. Negative control. A negative control (NTC), prepared at each time point of an isolation is necessary to check for a contamination of the vials and to have a baseline for later analysis. Start with the negative control always. A. Swab an empty medium vial (from the same charge as the treatment vials); B. Swab after ‘collection’; C. Transfer the swab into a Mobio Power Soil Tube.

  3. gDNA isolation (manufacturer’s data with some simple modifications)
    1. Add 60 µl solution C1 (Power Soil Kit) and 20 µl Proteinase K to the reaction tube. Mix by inverting the reaction tube.
      Note: If the solution is precipitated at room temperature, heat it up to 60 °C until it is homogenous again (Figures 5A-5C).
    2. Incubate the sample for 2 h at 50 °C and 750 rpm in the thermomixer (Figure 5D).
    3. Vortex (vortex adapter) the samples at highest speed for 10 min (Figure 5E).

      Figure 5. First steps of isolation using MOBIO Power Soil Kit. A. Add 60 µl of C1 (A, B) using sterile filter tips. C. Add 20 µl Proteinase K (STEP ADDED TO MANUFACTURERS PROTOCOL) and let the samples incubate in a thermos shaker (750 rpm) for 2 h at 50 °C (D, STEP ADDED TO MANUFACTURERS PROTOCOL) to induce Proteinase K activity. E. Transfer the vials into the MOBIO Vortex adapter and shake for 10 min at highest speed. F. The samples will look a lightly orange and may show a solid orange or red powder in the vial from the grinded silicate splints. This is absolutely normal. Go on by following the manufacturer’s protocol.

    4. Centrifuge the sample at 10,000 x g for 30 sec at 20 °C.
      Note: MAXIMUM 10,000 x g, tubes can break at higher rotations!
    5. Pipette up to 400 µl of the supernatant in a sterile 2 ml collecting tube.
    6. Add 250 µl solution C2 (Power Soil) to the supernatant and vortex for 5 sec.
    7. Incubate the sample for 5 min at 4 °C and centrifuge at 10,000 x g for 60 sec at 20 °C.
    8. Pipette up to 550 µl of the supernatant in a sterile 2 ml collecting tube.
    9. Add 200 µl solution C3 (Power Soil) to the supernatant and vortex shortly.
    10. Incubate the sample for 5 min at 4 °C and centrifuge at 10,000 x g for 60 sec at 20 °C.
    11. Pipette not more than 600 µl of the supernatant in a sterile 2 ml collecting tube, add 1,200 µl of C4 (shake solution 4 well before using!) and vortex 5 sec.
    12. Because the maximum spin column capacity is 675 µl, pipette only 600 µl of the mixture of step C11 onto the column, centrifuge at 10,000 x g for 60 sec at 20 °C. Discard the flow through and repeat this step twice.
    13. Afterwards, wash the column by pipetting 500 µl of C5 onto the column.
    14. Centrifuge at 10,000 x g for 30 sec.
    15. Discard flow through and dry the column by centrifugation at 10,000 x g for 60 sec at 20 °C.
    16. Put the spin-filter (column) into a new sterile collecting tube.
    17. Pipette 30 µl of TE onto the middle of the column and incubate for 1 min.
    18. Centrifuge the column at 10,000 x g for 30 sec at 20 °C.
    19. Dispose of the spin-filter.
    20. gDNA can be stored at -20 °C.
    Note: Keep a negative control for each isolation run!

  4. Further experiments
    After gDNA extraction, use a Qubit 3.0 Fluorometer to measure the gDNA concentration. This gDNA can be used for further analysis by qPCR or MiSeq (454 sequencing described in Fink et al., 2013).
    1. You need at least 5 ng/µl for metagenomics.
    2. Check all fly strains for a Wolbachia infection before performing your experiments. In some cases, Wolbachia OTUs (Operating Taxonimic Units) will dominate in your 454 data sets of whole fly samples or gut samples. This is important if feces samples are compared with whole tissue samples.
    1. PCR amplifications
      For PCR amplification we used 2.5 µl of gDNA as template in 25 µl reaction volume. Using 0.25 µl Phusion® Hot Start DNA polymerase, 0.5 µl of sense and antisense primer (10 mmol each); for PCR amplification, the Sensoquest labcycler was used. The general Wsp (Wolbachia specific) primers 81F (5’-TGGTCCAATAAGTGAAGAAAC-3’) and 691R (5’-AAAAATTAAACGCTACTCCA-3’) were used [10 mM] to amplify Wolbachia 16S RNA gene (Braig et al., 1994). To check the quality of the isolated DNA insect specific mitochondrial 12S ribosomal DNA primers were used, 12SAI (5’-AAACTAGGATTAGATACCCTATTAT-3’) and SBI (5’-AAGAGCGACGGGCGATGTGT-3’) (Simon et al., 1994). PCR conditions were: initial denaturation for 30 sec at 98 °C; 35 cycles of 9 sec at 98 °C, 30 sec at 55 °C, and 30 sec at 72 °C. The results are represented after gel electrophoresis in Figure 6 using 1.5 % Crystal-Agarose in 1x TBE (see Recipes).

      Figure 6. PCR analysis for Wolbachia contamination. A. PCR results using Wolbachia specific oligonucleotides (Wsp81F, Wsp691R) in lanes 1, 3 and 5. It is shown that fecal samples are free of Wolbachia contamination. To approve the success of DNA isolation gDNA specific 12S primers were used in lanes 2, 4 and 6 (12SAI and 12SBI) (Fink et al., unpublished data).

    2. qPCR amplifications
      PCR was performed in 10 µl reactions containing 2 µl of gDNA template in a total reaction volume of 10 µl using 5 µl of the qPCR Bio SYBR-Hi-ROX. The general V2 primers were used as general detection primer or control primer [8 pmol] when comparing to other species specific amplification. PCR conditions using the StepOne Cycler were: Holding stage for 10 min at 95 °C; cycling stage 40 cycles of 10 sec at 95 °C, 30 sec at 60 °C annealing as time point for data collection. Melting curve stage: 10 sec at 95 °C, 5 sec at 60 °C, starting of data collection every 0.3 °C until the temperature of 95 °C was reached for 10 sec.
    3. 454 procedures
      1. For PCR amplification of the 16sRNA gene a 311-nucleotide sequence flanking the hypervariable V1 and V2 region was used. For each sample, the 16S rRNA gene was amplified using the composite forward:
        (5’-CTATGCGCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3’) and reverse (5’-CGTATCGCCTCCCTCGCGCCATCAGXXXXXXXXXXCATGCTGCCTCCCGTAGGAGT-3’) primers. These primers include the 454 Life Sciences Adaptor A (for reverse primer) and B (for forward primer)–(labeled with italics). In addition, barcodes of 10 bp (designated as XXXXXXXXXX) were added to the reverse primers. The underlined sequences represent the broadly conserved bacterial primers 27F and 338R. A two-base linker sequence (TC/CA) and four-base key (TCAG) were added (see Supplemental file). The amplification mix contained the Phusion® Hot Start DNA polymerase which was used following the standard protocol (initial denaturation for 30 sec at 98 °C; 35 cycles of 9 sec at 98 °C, 30 sec at 55 °C, and 30 sec at 72 °C; final extension for 10 min at 72 °C). See results after amplification in Figure 7.
      2. All samples were done in duplicates. Duplicates were combined after PCR. PCR products were extracted with the QIAGEN MinElute Gel Extraction Kit and quantified with the Quant-iTTM dsDNA BR Assay Kit on a NanoDrop 3300 Fluorometer. Equimolar amounts of purified PCR product were pooled and further purified using Ampure Beads (Agencourt). A sample of each library was run on an Agilent Bioanalyzer prior to emulsion PCR (Rausch et al., 2011; SI) and sequencing as recommended by the manufacturer. Amplicon libraries were subsequently sequenced on a 454 GS-FLX using Titanium sequencing chemistry (Fink et al., 2013).

        Figure 7. Gel electrophoresis for PCR quality check and Gel extraction. Samples show an amplicon size about 400 bp. Negative controls (neg. Ktr.) are included for each amplification. Samples are cut off by using the Gel DocTM XR+ X-tracta Tips. All represented negative controls are free of contamination here. If negative controls (NTC) show any contamination, re-run the samples in the PCR.

Data analysis

Note: For statistical analysis, you should provide at least three independent biological replicates for each treatment, it’s suggested that it is better to prepare five.

  1. qPCR-data analysis
    We compared the Cycle threshold (Ct) from the PCR reaction, in which we used the primers specific to different bacterial species, to the Ct from the reactions with the general bacterial primers spanning the hypervariable regions V1 and V2 (V2 described above). By this relative comparison, we sought to control for the total amount of bacteria present in the sample. We averaged Cts between technical duplicates which resulted in a single Ct value per biological replicate using the following equation: ∆Ct = Ct (V2-primer set) - Ct Sample bacteria specific primer sets. The median ∆Ct of the three biological replicates was then used to transform this relative logarithmic measurement of bacterial abundance to a linear scale. We assumed doubling of the amount of PCR product with each cycle during the log-phase of the PCR reaction: x - fold = 2∆Ct (Fink et al., 2013).
  2. 454-data analysis
    1. 454 reads were sorted into groups according to their multiplex identifier (MID) tags using MOTHUR v1.23.1 (Schloss, 2009). During the process, tags and primer sequences were removed. Only sequences matching the MIDs and the bacterial primers perfectly were kept. The resulting sequences were quality filtered according to the following conditions: Minimum average quality of 35 in each 50 base pair window, minimum length of 260 bp, homopolymers no longer than 8 bp. Filtered sequences were aligned to the SILVA reference database (Pruesse et al., 2007) using the MOTHUR implemented kmer (all the possible substrings of length k that are contained in a string) algorithm with standard settings. Sequences not aligning in the expected region were removed.
    2. Passing sequences were filtered for sequencing errors using the MOTHUR pre.cluster command. Sequences were then searched for chimeras using Uchime (Edgar et al., 2011) as implemented in MOTHUR with standard settings. Sequences identified as chimeras were discarded. Sequences were classified into bacterial taxa with the classify.seqs command in MOTHUR using the SILVA reference database and taxonomy. Results were plotted using the R statistics package v.2.13.1 (R_Development_Core_team, 2011). Clustering sequences into OTUs was performed using MOTHUR with the average neighbor algorithm. (Fink et al., 2013). An example stack plot is represented in Figure 8.

      Figure 8. Stackplot of the relative abundance of OTUs detected after 454-sequencing. In the wildtype laboratory strain w1118 the Wolbachia infection/contamination could also be detected in the 454 data sets after sequencing (B, whole fly, gut samples). If there is a Wolbachia infection, most of the reads that are reflected by relative abundance (Y-axis) are reads from Wolbachia spec. This leads to the effect that it is not possible to find any other bacterial abundances in these samples. This will not affect fecal samples (Fink et al., unpublished data).


  1. Isolation is always reproducible. We could see in further analysis that there is no high variability between the samples of one treatment group. See Figure 8, feces samples: You nearly have the same abundance of all present bacteria. Sometimes, random sample dropouts may occur although the samples were extracted in the same run. Thus, we recommend to prepare always five samples of each treatment group.
  2. ALWAYS take care about a sterile environment, workspaces and tools. Any contamination could directly be seen in the further experiments.
  3. Take care NEVER touch the medium with the swab when doing feces collection (see Figure 3E).


  1. Fly medium (1.5 L beaker)
    62.5 g cornmeal
    62.5 g brewer’s yeast
    10 g agar-agar
    20 g glucose
    30 g molasses
    20 g sugar beat syrup
    Added in 1 L ddH2O
    Cooking the medium for 10-15 min in a water bath at 100 °C
    Autoclave for 15-20 min at 120 °C
    Cool down the medium to 60 °C before adding the preservatives
    30 ml Nipagin (10%)
    10 ml propionic acid (10% in EtOH)
    Prepare the vials under the clean bench. Add about 10 ml per vial
  2. 10x phosphate-buffered saline (PBS)
    80 g NaCl
    2 g KCl
    14.4 g Na2HPO4·2H2O
    2.4 g KH2PO4
    1 L ddH2O
    Autoclave for 15-20 min at 120 °C
    Adjust pH to 7.4
    Note: Dilute the 10x stock buffer by 1 to 10 with ddH2O freshly before usage.
  3. 10x TBE (Tris-Borat-EDTA) buffer
    108 g Tris
    55 g boric oxide
    40 ml of 0.5 M Na2EDTA
    1 L ddH2O
    Autoclave for 15-20 min at 120 °C
    Adjust pH to 8.0
    Note: Dilute the 10x stock buffer by 1 to 10 with ddH2O freshly before usage.


We thank Britta Laubenstein, Christiane Sandberg and Katja Cloppenborg-Schmidt for their excellent technical support. This work was supported by the Deutsche Forschungsgemeinschaft as part of the CRC 1182 (Project C2) and the Cluster of Excellence Inflammation@Interfaces.


  1. Braig, H. R., Guzman, H., Tesh, R. B. and O’Neill, S. L. (1994). Replacement of the natural Wolbachia symbiont of Drosophila simulans with a mosquito counterpart. Nature 367(6462): 453-455.
  2. Chandler, J. A., Lang, J. M., Bhatnagar, S., Eisen, J. A. and Kopp, A. (2011). Bacterial communities of diverse Drosophila species: ecological context of a host-microbe model system. PLoS Genet 7(9): e1002272.
  3. Clark, M. E., Anderson C. L., Cande J. and Karr, T. L. (2005). Widespread prevalence of wolbachia in laboratory stocks and the implications for Drosophila research. Genetics 170(4): 1667-75.
  4. Edgar, R. C., Haas, B. J., Clemente, J. C., Quince, C. and Knight, R. (2011). UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27(16): 2194-2200.
  5. Fink, C., Staubach, F., Kuenzel, S., Baines, J. F. and Roeder, T. (2013). Non-invasive analysis of microbiome dynamics in the fruit fly Drosophila melanogaster. Appl Environ Microbiol 79: 6984-6988.
  6. Huang, J. H., Jing, X. and Douglas, A. E. (2015). The multi-tasking gut epithelium of insects. Insect Biochem Mol Biol 67: 15-20.
  7. Matos, R. C. and Leulier, F. (2014). Lactobacilli-host mutualism: “learning on the fly”. Microb Cell Fact 13 Suppl 1: S6.
  8. Pruesse, E., Quast, C., Knittel, K., Fuchs, B. M., Ludwig, W., Peplies, J. and Glockner, F. O. (2007). SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 35(21): 7188-7196.
  9. Rausch, P., Rehmann, A., Keunzel, S., Haesler, R., Ott, S. J., Rosenstiel, P., Franke, A. and Baines, J. F. (2011). Colonic mucosa-associated microbiota is influenced by an interaction of Crohn’s disease and FUT2 (Secretor) genotype. Proc Natl Acad Sci U S A 108(47): 19030-5.
  10. Saridaki, A. and Bourtzis, K. (2010). Wolbachia: more than just a bug in insects genitals. Curr Opin Microbiol 13(1): 67-72.
  11. Schloss, P. D. (2009). A high-throughput DNA sequence aligner for microbial ecology studies. PLoS One 4(12): e8230.
  12. Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H. and Flook, P. (1994). Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am 87(6): 651-701.
  13. Wong, C. N., Ng, P. and Douglas, A. E. (2011). Low-diversity bacterial community in the gut of the fruitfly Drosophila melanogaster. Environ Microbiol 13(7): 1889-1900.



关键字:果蝇, 肠道, 微生物组, 粪便取样, DNA分离


注意:有关文本中缩写的全称,请参阅 补充文件中的表S1

  1. 过滤嘴,0.5-10μl,超薄,表面优化(NERBE PLUS,目录号:07-613-8300)
  2. 过滤嘴,0-200μl,超薄,表面优化(NERBE PLUS,目录号:07-662-8300)
  3. 过滤嘴,0.5-10μl,超薄,表面优化(NERBE PLUS,目录号:07-695-8300)
  4. 空的和干净的果汁小瓶(NERBE PLUS,目录号:11-881-0052)
  5. 纤维素塞(NERBE PLUS,目录号:11-881-1010)
  6. 0.22μm灭菌过滤器过滤(SARSTEDT,目录号:83.1826.001)
  7. 1.5ml反应管(SARSTEDT,目录号:72.690.001)
  8. 2 ml收集管
  9. 无菌旋转拭子(Greiner Bio One International,目录号:420180)
  10. X-tracta Tips(Biozym Scientific,目录号:615935)
  11. 氮(AIR LIQUIDE Deutschland,目录号:I4001S10R2A001)
  12. PowerSoil DNA分离试剂盒(新品:Quiagen DNeasy PowerSoil Kit)(QIAGEN,目录号:12888)
    注意:此套件的注意事项来自MOBIO,过去也称为Power Soil Kit!
  13. 从100%乙醇稀释的70%乙醇(Carl Roth,目录号:9065)
  14. 蛋白酶K(Thermo Fisher Scientific,Thermo Scientific TM,目录号:EO0491)
  15. Phusion ®热启动DNA聚合酶(Thermo Fisher Scientific,目录号:F540L)
  16. 水晶琼脂糖(BIOLABPRODUCTS,目录号:16-005-805)
  17. 100bp DNA梯(Thermo Fisher Scientific,Invitrogen TM,目录号:15628019)
  18. GeneRuler 50 bp DNA梯(Thermo Fisher Scientific,Thermo Scientific TM,目录号:SM0371)
  19. qPCR Bio SYBR-Hi-ROX(NIPPON Genetics,目录号:PB20.12-20)
  20. 脱氧核苷酸三磷酸(dNTP)(Promega,目录号:U1330)
  21. Oligo dT 7底漆(定制,Thermo Fisher Scientific订购)
  22. 用于qPCR的靶基因的寡核苷酸(定制,Thermo Fisher Scientific订购,使用[20μM])
  23. RT-PCR级水(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9935)
  24. MinElute凝胶提取试剂盒(QIAGEN,目录号:28604)
  25. Qubit dsDNA HS测定试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32851)
  26. Agencourt AMPure XP(Beckman Coulter,目录号:A63882)
  27. 飞行介质(参见食谱)
    1. 玉米粉(MühleSchlingemann,目录号:DAV-17080)
    2. 啤酒酵母(Leiber,目录号:17001636)
    3. 葡萄糖(Carl Roth,目录号:6780)
    4. 琼脂(Carl Roth,目录号:5210)
    5. 糖打糖浆(Kanne Brottrunk,目录号:2201)
    6. 糖蜜(Biohof Heidelicht,目录号:013aa)
  28. 防蝇剂:
    1. 尼泊金/ 4-羟基苯甲酸甲酯(Sigma-Aldrich,目录号:H5501)
    2. 丙酸(Carl Roth,目录号:6026)
  29. 10倍磷酸缓冲盐水(PBS)(参见食谱)
    1. 氯化钠(NaCl)(Carl Roth,目录号:P029)
    2. 氯化钾(KCl)(Carl Roth,目录号:6781)
    3. 磷酸二氢钾(KH 2 PO 4)(Carl Roth,目录号:P018)
    4. 二水合磷酸氢二钠(Carl Roth,目录号:4984)
  30. 10倍TBE(Tris-Borat-EDTA)缓冲液(参见食谱)
    1. Tris(Carl Roth,目录号:4855)
    2. 硼酸(Carl Roth,目录号:6943)
    3. Na 2 EDTA(Carl Roth,目录号:X986)


  1. Eppendorf Research ® plus,单通道,变量,0.1-2.5μl,深灰色(Eppendorf,目录号:3120000011)
  2. Eppendorf Research ® plus,单通道,变量,0.5-10μl,浅灰色(Eppendorf,目录号:3120000020)
  3. Eppendorf Research ® plus,单通道,变量,2-20μl,黄色(Eppendorf,目录号:3120000038)
  4. Eppendorf Research ® plus,单通道,变量,100-1,000μl,蓝色(Eppendorf,目录号:3120000062)
  5. 高压灭菌器
  6. 无菌台(NuAire,型号:NU-437-400E)
  7. 麻醉手枪(吹枪)(GENESE Scientific,型号:54-104)
  8. 杜邦镊子#5(精细科学工具,目录号:11252-20)
  9. 剪刀(Bioform,型号:B37e)
  10. Vortexer(Scientific Industries,型号:Vortex Genie 2,目录号:SI-0256)
  11. 涡旋适配器(MO BIO,目录号:13000-V1)
  12. Thermomixer(Eppendorf,型号:Thermomixer Comfort)
  13. 离心机(Eppendorf,型号:5424)
  14. Qubit 3.0荧光计(Thermo Fisher Scientific,Invitrogen TM,目录号:Q33216)
  15. Sensoquest labcycler(BIOLABPRODUCTS,目录号:11-011-101-096)
  16. NanoDrop 3300荧光计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM,目录号:ND-3300)
  17. 25℃培养箱(AQUALYTIC,型号:TC 140 G,目录号:438210)
  18. StepOne qPCR循环仪(Thermo Fisher Scientific,目录号:4376357)
  19. Gel Doc TM XR +凝胶文献系统(Bio-Rad Laboratories,型号:Molecular Imager Gel Doc TM XR +,目录号:1708195) br />
  20. 宽的Mini-Sub ® Cell GT卧式电泳系统,15 x 10厘米托盘,带PowerPac TM基本电源(Bio-Rad实验室,目录号:1640301) />
  21. 安捷伦生物分析仪(Agilent Technologies,型号:2100 Bioanalyzer,目录号:G2939BA)
  22. 水浴
  23. 1.5升烧杯


  1. MOTHUR v1.23.1
  2. R统计软件包v.2.13.1(R_Development_Core_team,2011)


  1. 中等制备
    1. 通过在120℃高压灭菌20分钟来烹制所需的无菌飞行培养基(参见食谱)。
    2. 将空的清洁果汁清洁干净的小瓶,并在安全柜下通过紫外线插入至少30分钟(图1A)。
    3. 让介质冷却至65°C,并在无菌条件下加入防腐剂(如有必要)
    4. 在无菌条件下将无菌飞行培养基倒入去污的果蝇小瓶中(图1B)。
    5. 让介质在安全柜下干燥并将小瓶塞住(图1C)
    6. 可以将无菌培养基在4℃下储存在无菌塑料盒中

      图1.无菌培养瓶的制备。 :一种。将空的(标记)小瓶和新鲜的塞子放在无菌台下,并使用紫外线对其进行灭菌至少30分钟。 B.在工作台上清洗后,将小瓶中的新鲜无菌培养基滗析,让培养基干燥约45至60分钟。 C.塞上小瓶。小瓶可以在无菌盒中保存在4°C。

  2. 粪便收集
    1. 使用吹枪(麻醉手枪)用氮气麻醉苍蝇。将无菌条件下将30至50只蝇的群体转移到无菌的果蝇小瓶中,并使其在25℃下排便24小时(图2A)。
    2. 然后,在无菌条件下转移到新的小瓶,或丢弃它们。

      图2.小瓶中的粪便。将30至50只苍蝇转移到无菌制备的小瓶中,并使苍蝇在25°C排便大约24小时,以进行标准野生型或交叉处理。 B. 24小时后,可以看到整个小瓶上有许多粪便斑点(图2C显示放大倍率,红色箭头仅标记几个点,以便更好地理解)。

    3. 将无菌旋转拭子,无菌1x PBS(见食谱),无菌剪刀和含有安全柜下面的粪便的果蝇小瓶(图3A)放置。
    4. 为了更好的控制,使用无菌过滤器过滤所需量的1x PBS(图3B)
    5. 用无菌1x PBS浸泡无菌旋转拭子(图3C;准备步骤5-8,见视频1)。
    6. 打开小瓶,并用浸泡的旋转拭子小心地收集粪便。

      图3.粪便收集。 :一种。所有使用的材料的紫外线灭菌,例如1x PBS缓冲液,单个棉签,无菌注射器,剪刀和反应管。 B.使用注射器过滤器再次过滤所需量的1x PBS。 C.用新鲜制备和过滤的1×PBS浸泡无菌拭子。 D.开始从小瓶收集/拭拭所有粪便点。只能从墙上忽略插头上的斑点,并注意不要用拭子(E)触摸介质。 F.在25℃下24小时后从40只蝇中收集粪便斑点。

      Video 1. Feces collection

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player

    7. 将旋转拭子转移到反应管中,并用无菌剪刀小心地切掉棒(图4C)
    8. 关闭反应管。
    9. 使用与使用的培养基相同的一天制备的空培养基小瓶,以相同的方式制备阴性对照。
    10. 用无菌1x PBS浸泡无菌旋转拭子(图3C)
    11. 打开小瓶,并用浸泡的旋转拭子小心地收集粪便(图4A)
    12. 收集后,NTC(无模板控制)拭子不显示粪便(图4B)。

      图4.阴性对照需要在每个隔离时间点准备的阴性对照(NTC),以检查小瓶的污染并具有用于后续分析的基准。始终以负面控制开始。 A.取一个空的培养瓶(与处理小瓶相同的电量); "收集"后的棉签C.将棉签转移到Mobio电力土壤管中

  3. gDNA分离(制造商的数据进行了一些简单的修改)
    1. 向反应管中加入60μl溶液C1(Power Soil Kit)和20μl蛋白酶K。混合反应管。
    2. 在温热混合器中,将样品在50℃和750rpm下孵育2小时(图5D)
    3. 涡旋(涡流适配器)将样品以最高速度进行10分钟(图5E)

      图5.使用MOBIO Power Soil Kit进行隔离的第一步。 :一种。加入60μl的C1(A,B)使用无菌过滤嘴。 C.加入20μl蛋白酶K(步骤加入制造商协议),并使样品在50℃的温度振荡器(750rpm)中孵育2小时(D,STEP ADDED TO MANUFACTURERS PROTOCOL)以诱导蛋白酶K活性。 E.将小瓶转移到MOBIO Vortex适配器中,以最高速度摇动10分钟。样品将看起来略带橙色,并且可以在磨碎的硅酸盐夹板上的小瓶中显示出一个坚实的橙色或红色粉末。这是绝对正常的。按照制造商的协议进行。

    4. 将样品以10,000 x g在20°C离心30秒。
      注意:最大10,000 x g,管子可以在更高的转速下断裂!
    5. 在无菌的2 ml收集管中吸取400μl上清液
    6. 向上清液中加入250μl溶液C2(Power Soil),旋转5秒
    7. 在4℃下孵育样品5分钟,并在20℃下以10,000×g离心60秒。
    8. 在无菌的2 ml收集管中吸取高达550μl的上清液
    9. 向上清液中加入200μl溶液C3(Power Soil),并迅速旋转
    10. 在4℃下孵育样品5分钟,并在20℃下以10,000×g离心60秒。
    11. 在无菌的2ml收集管中吸取不超过600μl的上清液,加入1,200μl的C4(摇匀溶液4次,然后使用!)并旋转5秒。
    12. 因为最大旋转柱容量为675μl,所以只将600μl步骤C11的混合物移液到柱上,在20℃下以10,000xg离心60秒。放弃流程并重复此步骤两次。
    13. 然后,通过将500μl的C5吸移到柱上来洗涤柱。
    14. 以10,000 x g离心30秒。
    15. 通过在20℃离心10分钟60分钟,在20℃下离心流经并干燥柱子。
    16. 将自动过滤器(色谱柱)放入新的无菌收集管中
    17. 将30μlTE移至色谱柱的中间并孵育1分钟
    18. 在20℃下以10,000×g离心柱30秒。
    19. 处理自动过滤器。
    20. gDNA可以保存在-20°C。

  4. 进一步实验
    gDNA提取后,使用Qubit 3.0荧光计测定gDNA浓度。该gDNA可用于通过qPCR或MiSeq进行进一步分析(Fink等人,2013年描述的454次测序)。

    1. 您需要至少5 ng /μl的元素
    2. 在进行实验之前,请检查所有感染Wolbachia感染的菌株。在某些情况下,Wolbachia OTUs(操作分类单位)将在您的454个全套样品或肠样品数据集中占主导地位。如果将粪便样本与全组织样本进行比较,这一点很重要。
    1. PCR扩增
      对于PCR扩增,我们在25μl反应体积中使用2.5μl的gDNA作为模板。使用0.25μlPhusion 热启动DNA聚合酶,0.5μl有义和反义引物(各10mmol);对于PCR扩增,使用Sensoquest labcycler。使用一般的Wsp (Wolbachia 特异性)引物81F(5'-TGGTCCAATAAGTGAAGAAAC-3')和691R(5'-AAAAATTAAACGCTACTCCA-3')[10mM]扩增Wolbachia 16S RNA基因(Braig et al。,1994)。为了检查分离的DNA昆虫特异性线粒体的质量,使用12S核糖体DNA引物,12SAI(5'-AAACTAGGATTAGATACCCTATTAT-3')和SBI(5'-AAGAGCGACGGGCGATGTGT-3')(Simon等人, >,1994)。 PCR条件为:98℃初始变性30秒; 98℃9秒,55℃30秒,72℃30秒35个循环。结果用图6中的凝胶电泳表示,使用1x TBE中的1.5%晶体琼脂糖(参见食谱)。

      图6. Wolbachia污染物的PCR分析。 :一种。在泳道1,3和5中使用Wolbachia特异性寡核苷酸(Wsp 81F,Wsp 691R)的PCR结果。显示粪便样品是免费的的Wolbachia 污染。批准DNA分离的成功gDNA特异性12S引物用于第2,4和6号(12SAI和12SBI)(Fink等人,未发表的数据)。

    2. qPCR扩增
      使用5μlqPCR Bio SYBR-Hi-ROX,在10μl总反应体积为10μl的10μl反应中进行PCR,所述反应含有2μl的gDNA模板。当与其他物种特异性扩增相比时,将一般V2引物用作一般检测引物或对照引物[8pmol]。使用StepOne Cycler的PCR条件为:在95℃下保持10分钟;在95℃下循环40个循环,10秒,60℃退火30秒,作为数据收集的时间点。熔融曲线阶段:在95℃下10秒,60℃下5秒,每0.3℃开始收集数据,直到95℃的温度达到10秒。
    3. 454程序
      1. 对于16sRNA基因的PCR扩增,使用侧翼于高变体V1和V2区域的311个核苷酸序列。对于每个样品,使用复合转发扩增16S rRNA基因:
        (5'-CTATGCGCCTTGCCAGCCCGC TCAGTCAGAGTTTGATCCTGGCTCAG-3')和反向(5'-CGTATCGCCTCCCTCGCGCCA TCAGXXXXXXXXXXCA TGCTGCCTCCCGTAGGAGT-3 ')引物。这些引物包括454生命科学适配器A(用于反向引物)和B(用于正向引物) - (用斜体标记)。此外,向反向引物加入10bp条形码(称为XXXXXXXXXX)。带下划线的序列代表广泛保守的细菌引物27F和338R。添加了两碱基连接序列(TC / CA)和四碱基键(TCAG)(参见补充文件)。扩增混合物含有按照标准方案使用的Phusion 热启动DNA聚合酶(98℃初始变性30秒; 98℃9秒,55℃30秒) ℃,72℃30秒; 72℃最终延伸10分钟)。参见图7中放大后的结果。
      2. 所有样品都重复完成。 PCR后结合重复。使用QIAGEN MinElute凝胶提取试剂盒提取PCR产物,并在NanoDrop 3300荧光计上用Quant-iT dsDNA BR测定试剂盒定量。合并等摩尔量的纯化的PCR产物,并使用Ampure Beads(Agencourt)进一步纯化。在乳液PCR(Rausch等人,2011; SI)和制造商推荐的测序之前,在Agilent Bioanalyzer上运行每个文库的样品。随后使用Titanium测序化学(Fink等人,2013)在454 GS-FLX上对Amplicon文库进行测序。

        图7.用于PCR质量检查和凝胶提取的凝胶电泳。 样品显示约400bp的扩增子大小。每个扩增包括阴性对照(负Ktr。)。通过使用Gel Doc TM XR + X-tracta Tips来切断样品。所有代表的阴性对照在这里都没有污染。如果阴性对照(NTC)显示任何污染,请在PCR中重新运行样品。



  1. qPCR数据分析
    我们将PCR反应的循环阈值(Ct)与来自与跨越高变区V1和V2(上述V2)的通用细菌引物的反应相比,将来自不同细菌种类的引物使用的引物与Ct进行比较。通过相对比较,我们试图控制样品中存在的细菌总量。我们在技术重复之间平均了Cts,其导致使用以下等式生成单个Ct值:子>。然后使用三个生物重复的中值ΔCt将细菌丰度的相对对数测量值转换为线性标度。在PCR反应的对数期间,我们假设每个循环的PCR产物的量加倍:x-fold =2ΔCt(Fink等人,2013年)
  2. 454数据分析
    1. 根据使用MOTHUR v1.23.1(Schloss,2009)的多路复用标识符(MID)标签将454个读取分组成组。在该过程中,去除了标签和引物序列。只保留了完全匹配MID和细菌引物的序列。所得序列根据以下条件进行质量过滤:每个50个碱基对窗口中的最小平均质量为35,最小长度为260bp,均聚物不超过8bp。使用标准设置的MOTHUR实现的kmer(包含在字符串中的长度为k的所有可能的子串)算法将筛选的序列与SILVA参考数据库(Pruesse等人,2007)进行比对。删除在预期区域不对齐的序列。
    2. 使用MOTHUR pre.cluster命令对传送序列进行过滤以进行测序。然后使用Uchime(Edgar等人,2011)在MOTHUR中使用标准设置来搜索序列。确定为嵌合体的序列被丢弃。使用SILVA参考数据库和分类法将序列分类为MOTHUR中的classify.seqs命令的细菌分类单元。使用R统计软件包v.2.13.1(R_Development_Core_team,2011)绘制结果。使用具有平均邻域算法的MOTHUR进行到OTU的聚类序列。 (Fink等人,2013)。示例堆叠图在图8中表示。

      图8. 454序列后检测到的OTU的相对丰度的堆积图。 在野生型实验室菌株 中,Wolbachia 感染/污染也可以在测序后的454个数据集中检测(B ,全飞,肠样品)。如果存在Wolbachia 感染,则相对丰度(Y轴)所反映的大部分读数是Wolbachia 规范中的读数。这导致在这些样品中不可能发现任何其他细菌丰度的效果。这不会影响粪便样品(Fink等人,未发表的数据)。


  1. 隔离总是可重复的。从进一步的分析可以看出,一个治疗组的样本之间没有高的变异性。见图8,粪便样本:您几乎拥有所有现有细菌的丰富度。有时,尽管在相同的运行中提取样品,但可能会发生随机抽样缺失。因此,我们建议每个治疗组总共准备五个样品。
  2. 始终关心无菌环境,工作空间和工具。在进一步的实验中可以直接看到任何污染物。
  3. 小心收集时不要用拭子触摸介质(见图3E)。


  1. 飞行介质(1.5L烧杯)
    10g琼脂 -
    20 g葡萄糖 30克糖蜜
    在1 L ddH 2 O
    中添加 在100°C水浴中烹调培养基10-15分钟 在120°C高压灭菌15-20分钟
    在加入防腐剂之前将介质冷却至60°C 30毫升尼泊金(10%)
  2. 10倍磷酸缓冲盐水(PBS)
    14.4g Na 2 HPO 4•2H 2 O
    2.4g KH PO 4
    1 L ddH 2 O
  3. 10倍TBE(Tris-Borat-EDTA)缓冲液
    40ml的0.5M Na 2 EDTA 1 L ddH 2 O
    注意:使用前,将10x储存缓冲液稀释1至10,ddH 2 O。


我们感谢Britta Laubenstein,Christiane Sandberg和Katja Cloppenborg-Schmidt的出色技术支持。作为CRC 1182(项目C2)和卓越炎症接口丛集的一部分,这项工作得到了德意志福神学院的支持。


  1. Braig,HR,Guzman,H.,Tesh,RB和O'Neill,SL(1994)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/ pobmed / 7906391"target ="_ blank">用蚊子对等体代替果蝇的自然Wolbachia共生体。 367(6462):453- 455.
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引用:Fink, C., von Frieling, J., Knop, M. and Roeder, T. (2017). Drosophila Fecal Sampling. Bio-protocol 7(18): e2547. DOI: 10.21769/BioProtoc.2547.