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Biologia do Desenvolvimento

Opdræt bananfluen<em> Drosophila melanogaster</em> Under Axeniske og gnotobiotiske Betingelser

Published: July 30, 2016
doi:
10.3791/54219
, , , , , ,
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

Summary

En metode til opdræt Drosophila melanogaster under Axeniske og gnotobiotiske betingelser præsenteres. Fly embryoner dechorionated i natriumhypochlorit, overføres aseptisk til sterilt kost, og opdrættet i lukkede beholdere. Pode kost og embryoner med bakterier fører til gnotobiotiske foreninger, og bakteriel tilstedeværelse bekræftes ved plating hele kroppen Drosophila homogenater.

Abstract

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.

Introduction

De fleste dyr er intimt forbundet med bakterier (mikrobiota «) fra fødsel til død 1. Sammenligninger af mikroorganismer-fri ( 'axenisk «) og mikroorganisme-associerede (' konventionelle ') dyr har vist mikrober påvirke forskellige aspekter af dyrs sundhed, herunder metaboliske, ernæringsmæssige, vaskulær, lever-, respiratorisk, immunologiske, endokrine og neurologiske funktion 2. Frugten flyve Drosophila melanogaster er en vigtig model til forståelse mange af disse processer i tilstedeværelse af mikrober 3,4 og for at studere mikrobiota indflydelse på dyresundhed 5,6. Ingen bakteriearter er til stede i hver enkelt ( "kerne"), men Acetobacter og Lactobacillus arter numerisk dominere mikrobiota af både laboratorie-opdrættet og vild-fanget D. melanogaster. Andre Acetobacteraceae (herunder Komagataeibacter og Gluconobacter), Firmicutes (såsom Enterococcus og Leuconostoc), og enterobakterier er enten ofte til stede i Drosophila enkeltpersoner på lav tæthed, eller uregelmæssigt til stede ved høj overflod 7-12.

Den mikrobiota af Drosophila og pattedyr er ustadig inden for og på tværs af generationer 14,19. Mikrobiota ustadighed kan føre til fænotypisk støj ved måling mikrobiota-afhængige egenskaber. For eksempel Acetobacteraceae indflydelse lipid (triglycerid) opbevaring i Drosophila 15-18. Hvis Acetobacteraceae er mere rigelige i fluer med et hætteglas end i et andet 19, kan isogene fluer har forskellige fænotyper 20. En løsning på problemet med mikrobiota ustadighed i mus 14 har været i praksis siden 1960'erne, ved at indføre et defineret fællesskab af 8 dominerende mikrobielle arter til muse unger hver ny generation (ændret Schaedler flora),sikre, at hver hvalp er udsat for de samme centrale medlemmer af musen mikrobiota. Denne praksis kontrollerer for mikrobiota sammensætning, selv når mikrobiota er ikke det primære mål for studiet 32, og satte præcedens for at sikre tilstedeværelsen af centrale mikrober i forskellige eksperimentelle betingelser.

At definere indflydelse mikrober på Drosophila ernæring, har flere protokoller til at udlede Axeniske flueliner blevet udviklet, herunder hypochlorit dechorionation af embryoner (enten afledt de novo hver generation eller vedligeholdes generationally ved overførsel til sterile diæter) og antibiotisk behandling 13. Der er fordele for forskellige tilgange, såsom lethed og hurtighed for både af antibiotika behandling og seriel overførsel, versus større kontrol med forstyrrende variabler med de novo dechorionation (fx æg tæthed, resterende kontaminerende mikrober, off-target antibiotiske virkninger). Uanset fremgangsmåden tilforberedelse, indførelse af bestemte mikrobielle arter Axeniske embryoner tillader kultur af Drosophila med definerede ( 'gnotobiotiske «) samfund. Alternativt efterligner brugen af ​​Schaedler flora, dette samfund kunne podes til konventionelt lagt æg (følgende trin 6-7 kun) for at sikre tilstedeværelsen af ​​egenskab-påvirke mikrober i hvert hætteglas og undgå komplikationer af mikrobiota ustadighed. Her beskriver vi protokollen for at hæve axenisk og gnotobiotiske Drosophila ved de novo dechorionation af embryoner, og til bekræftelse af tilstedeværelsen af indførte eller kontaminerende mikrobiel taxa.

Protocol

1. Kultur Bakterier (Start ~ 1 uge før Picking æg) Forbered modificeret MRS 20 (mMRS) plader og bouillon rør (tabel 1). Hæld 20 ml mMRS agar i hver 100 mm Petri plade og lad den køle / tørre natten, eller 5 ml mMRS bouillon i 18 mm reagensglas. Streak Acetobacter pomorum, A. tropicalis, Lactobacillus brevis, og L. plantarum på mMRS agarplader. Inkuber Acetobacter natten over ved 30 ° C. Inkuber Lactobacillus an…

Representative Results

Vellykket opdræt af Axeniske fluer bekræftes af isolering af nogen CFU'erne fra hele kroppen homogenizations af D. melanogaster voksne (figur 1). Alternativt, hvis belagte homogenatet giver kolonier, er hætteglassene kontamineret og skal kasseres. For gnotobiotiske fluer, blev hver af de fire bakterielle isolater isoleret fra puljer af 5 voksne mænd, hvilket viser forskelle i de samlede levedygtige CFU'er forbundet med voksne fluer (figur 1)….

Discussion

Den her beskrevne metode er en af flere metoder til embryo dechorionation 8,11,18,25,26,27, sammen med alternative metoder til opdræt Axeniske fluer, herunder seriel overførsel af Axeniske voksne 18,27 eller antibiotikabehandling 13,18. Andre dechorionation metoder omfatter ethanol vaske og reducere 11,25,26 eller forlænge 8 hypochlorit behandling. Forskellige vasketrin kan støtte opdræt forskellige flyve genotyper: i en tidligere undersøgelse meste af ~…

Declarações

The authors have nothing to disclose.

Acknowledgements

Nogle oplysninger om denne protokol blev optimeret med hjælp fra Dr. Adam Dobson, der også nyttige bemærkninger til manuskriptet. Dette arbejde blev støttet af Fonden for National Institutes of Health (FNIH) tilskud nummer R01GM095372 (JMC, A (CN) W, AJD, og ​​AED). FNIH tilskud nummer 1F32GM099374-01 (PDN), og Brigham Young University startup midler (JMC, MLK, MV). Offentliggørelse omkostninger blev støttet af Brigham Young University College of Life Sciences og Institut for Plantebiologi og Wildlife Sciences.

Materials

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&region=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&region=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&region=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&region=US&focus=product
MRS Powder Sigma Aldrich 69966-500G http://www.sigmaaldrich.com/catalog/product/sial/69966?lang=en&region=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
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Drosophila MelanogasterAxenicGnotobioticMicrobiotaSterile DietMRS AgarLactobacillusAcetobacterGrape Juice AgarYeast PasteEgg Collection
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Koyle, M. L., Veloz, M., Judd, A. M., Wong, A. C., Newell, P. D., Douglas, A. E., Chaston, J. M. Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions. J. Vis. Exp. (113), e54219, doi:10.3791/54219 (2016).
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