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Drosophila Fecal Sampling

果蝇粪便取样

CF Christine Fink
JF Jakob von Frieling
MK Mirjam Knop
TR Thomas Roeder

发布: 2017年09月20日第7卷第18期 DOI: 10.21769/BioProtoc.2547 浏览次数: 10575

评审: Jihyun KimAbhijit KaleAnonymous reviewer(s)

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Cover of Applied and Environmental Microbiology, featuring study using the protocol.

Nov 2013

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Abstract

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) search Intestine (肠道) search Microbiome (微生物组) search Fecal sampling (粪便取样) search DNA isolation (DNA分离) search

Background

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 )

Equipment

  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

Software

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

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Fink, C., von Frieling, J., Knop, M. and Roeder, T. (2017). Drosophila Fecal Sampling. Bio-protocol 7(18): e2547. DOI: 10.21769/BioProtoc.2547.

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分类

微生物学 > 微生物-宿主相互作用 > 细菌

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