Name: rearing the fruit fly drosophila melanogaster under axenic and gnotobiotic conditions
Uploaded: 2016-07-30T14:00:00
Description: Watch this Scientific Journal Video about Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions at JoVE.com

Vissa detaljer i detta protokoll har optimerats med hjälp av Dr Adam Dobson, som också värdefulla kommentarer på manuskriptet. Detta arbete stöddes av Stiftelsen för National Institutes of Health (FNIH) licensnummer R01GM095372 (JMC, A (CN) W, AJD och AED). FNIH licensnummer 1F32GM099374-01 (PDN), och Brigham Young University startmedel (JMC, MLK, MV). Kostnader för publicering stöddes av Brigham Young University College of Life Sciences och Institutionen för växt- och djurliv Sciences.

1. Kultur Bakterier (Start ~ 1 vecka innan plocka ägg) <ol><li> Förbered modifierad MRS 20 (mMRS) plattor och buljong rör (Tabell 1). Häll 20 ml mMRS agar i varje 100 mm petriplatta och låt svalna / torka över natten, eller 5 ml mMRS buljong i 18 mm provrör. </li><li> Strimma Acetobacter pomorum, A. tropicalis, Lactobacillus brevis, och L. plantarum på mMRS agarplattor. Inkubera Acetobacter natten vid 30 ° C. Inkubera Lactobacillus</...

<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. A., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut-associated+microbes+of+Drosophila+melanogaster.">Gut-associated microbes of Drosophila melanogaster.</a> Gut Microbes. 3 (4), 307-321 (2012).</li><li>Ren, C., Webster, P., Finkel, S. E., Tower, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+internal+and+external+bacterial+load+during+Drosophila+aging+without+life-span+trade-off.">Increased internal and external bacterial load during Drosophila aging without life-span trade-off.</a> Cell Metab. 6 (2), 144-152 (2007).</li><li>Wong, A. 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=The+Host+as+the+Driver+of+the+Microbiota+in+the+Gut+and+External+Environment+of+Drosophila+melanogaster.">The Host as the Driver of the Microbiota in the Gut and External Environment of Drosophila melanogaster.</a> Appl Environ Microbiol. 81 (18), 6232-6240 (2015).</li><li>Dobson, A. J., 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=Host+genetic+determinants+of+microbiota-dependent+nutrition+revealed+by+genome-wide+analysis+of+Drosophila+melanogaster.">Host genetic determinants of microbiota-dependent nutrition revealed by genome-wide analysis of Drosophila melanogaster.</a> Nat Commun. 6, 6312 (2015).</li><li>Bakula, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+persistence+of+a+microbial+flora+during+postembryogenesis+of+Drosophila+melanogaster.">The persistence of a microbial flora during postembryogenesis of Drosophila melanogaster.</a> J Invertebr Pathol. 14 (3), 365-374 (1969).</li><li>Ryu, J. H., 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=Innate+immune+homeostasis+by+the+homeobox+gene+caudal+and+commensal-gut+mutualism+in+Drosophila.">Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila.</a> Science. 319 (5864), 777-782 (2008).</li><li>Blum, J. E., Fischer, C. N., Miles, J., Handelsman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequent+replenishment+sustains+the+beneficial+microbiome+of+Drosophila+melanogaster.">Frequent replenishment sustains the beneficial microbiome of Drosophila melanogaster.</a> MBio. 4 (6), 00860 (2013).</li><li>Bitner-Mathe, B. C., Klaczko, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+Drosophila+melanogaster+wing+morphology:+effects+of+sex,+temperature+and+density.">Plasticity of Drosophila melanogaster wing morphology: effects of sex, temperature and density.</a> Genetica. 105 (2), 203-210 (1999).</li><li>Edward, D. A., Chapman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-specific+effects+of+developmental+environment+on+reproductive+trait+expression+in+Drosophila+melanogaster.">Sex-specific effects of developmental environment on reproductive trait expression in Drosophila melanogaster.</a> Ecol Evol. 2 (7), 1362-1370 (2012).</li><li>Ridley, E. V., Wong, A. C., Westmiller, S., Douglas, A. 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<html> Den metod som beskrivs här är en av flera metoder för embryo dechorionation 8,11,18,25,26,27, tillsammans med alternativa metoder för uppfödning axenisk flugor, inklusive seriell överföring av axenisk vuxna 18,27 eller antibiotikabehandling 13,18. Andra dechorionation metoder inkluderar etanol tvättar och minska 11,25,26 eller förlänga 8 hypoklorit behandling. Olika tvättsteg kan underlätta uppfödning olika flyga genotyper: i en tidigare studie fl...

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

De flesta djur är intimt förknippade med bakterier (mikroorganismer) från födseln till döden 1. Jämförelser av mikroorganismer fria (axenisk &quot;) och mikroorganismassocierade (&quot; konventionella &quot;) djur har visat mikrober påverkar olika aspekter av djurhälsa, inklusive metaboliska, näringsmässiga, vaskulär, lever-, respiratoriska, immunologiska, endokrina och neurologisk funktion 2. Bananflugan Drosophila melanogaster är en viktig modell för att förstå många av dessa processer i närvaro av mikrober 3,4 och för att studera mikrobiota inverkan på djurhälsa 5,6. Inga bakteriearter finns i varje individ ( &quot;kärna&quot;), men Acetobacter och Lactobacillus arter dominerar numeriskt mikrobiota av både laboratorie uppfödda och vildfångad D. melanogaster. Andra Acetobacteraceae (inklusive Komagataeibacter och Gluconobacter), Firmicutes (t.ex. Enterococcus och Leuconostoc), och Enterobacteriaceae är antingen ofta förekommer i Drosophila individer med låg förekomst, eller oregelbundet närvarande vid hög förekomst 7-12. Mikrobiota av Drosophila och däggdjur obeständig inom och mellan generationer 14,19. Mikrobiota förgänglighet kan leda till fenotypisk buller vid mätning mikrobiota beroende egenskaper. Till exempel Acetobacteraceae inflytande lipid (triglycerider) lagring i Drosophila 15-18. Om Acetobacteraceae är mer rikligt i flugor i en flaska än i en annan 19, kan isogena flugor ha olika fenotyper 20. En lösning på problemet med mikrobiota förgänglighet i möss 14 har i praktiken sedan 1960-talet, genom att införa en definierad gemenskap av 8 dominerande mikrobiella arter mus valpar varje ny generation (förändrad Schaedler flora),se till att varje valp utsätts för samma nyckelpersoner i musen mikroorganismer. Denna praxis kontroller för mikrobiota sammansättningen även när mikrobiota är inte det primära målet för studien 32, och sätter prejudikat för att säkerställa förekomsten av viktiga mikrober i en mängd olika experimentella betingelser. För att definiera påverkan av mikrober på Drosophila näring har flera protokoll för att härleda axenisk fluglinor utvecklats, bland annat hypoklorit dechorionation av embryon (antingen härrör de novo varje generation eller underhålls generationally genom överföring till sterila dieter) och antibiotikabehandling 13. Det finns fördelar för olika metoder, såsom lätthet och snabbhet för både av antibiotika behandling och seriell överföring, jämfört med större kontroll över störande variabler med de novo dechorionation (t.ex. ägg densitet, restkontaminerande mikrober, off-target antibiotika effekter). Oberoende av metoden förberedning, införandet av specificerade mikrobiella arter axenisk embryon tillåter odling av Drosophila med definierade ( &quot;gnotobiotiska) samhällen. Alternativt imitera användningen av Schaedler flora, denna gemenskap kan ympas till konventionellt som ägg (efter steg 6-7) för att säkerställa närvaron av egenskapspåverkande mikrober i varje flaska och undvika komplikationer av mikrobiota förgänglighet. Här beskriver vi protokoll för att höja axenisk och gnotobiotisk Drosophila av de novo dechorionation av embryon, och för att bekräfta närvaron av infört eller kontaminerande mikrobiella 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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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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		<tr height="20">
			<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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		<tr height="20">
			<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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			<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>
		</tr>
		<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.

Framgångsrik uppfödning av axenisk flugor bekräftas genom isolering av någon CFU från hela kroppen homogenizations av D. melanogaster vuxna (Figur 1). Alternativt, om det pläterade homogenatet ger kolonier, är flaskorna förorenade och måste kasseras. För gnotobiotiska flugor, var och en av de fyra bakterieisolat isolerades från pooler av 5 vuxna hannar, vilket visar skillnader i totala viabla CFUs associerade med vuxna flugor (Figur 1).</stron...

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

En metod för uppfödning Drosophila melanogaster enligt axenisk och gnotobiotiska villkor presenteras. Fly embryon dechorionated natriumhypoklorit, överfördes aseptiskt till sterila diet, och föds upp i slutna behållare. Ympning kost och embryon med bakterier leder till gnotobiotiska föreningar, och bakteriell närvaro bekräftas genom plätering hela kroppen Drosophila homogen.

Uppfödning bananfluga<em&gt; Drosophila melanogaster</em&gt; Under axenisk och gnotobiotiska Villkor

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

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Analysera blodcellpopulationer av<em&gt; Drosophila melanogaster</em&gt; 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

Produktion och administrering av terapeutiska mesenkymala stam / Stromacells- (MSC) sfäroiderna Primed i 3-D kulturer Under Xeno fria förhållanden

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

Protokoll för Visualisering steroidogena organ och deras interaktiva organ med immunfärgning i fruktflugan<em&gt; Drosophila melanogaster</em

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

Dissektion och Immunofluorescerande färgning av svamp kropp och ljusmätare nervceller i vuxen Drosophila melanogaster hjärnor

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

Induktion av Endothelial differentiering i hjärt stamceller låga Serum villkor

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

Upptining, odling och bevarandet Drosophila cellinjer

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

Metoder för att testa endokrina störningar i 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

Analys för blod-hjärnbarriären integritet i 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

Effektiv differentiering av postganglitoniska sympatiska nervceller med hjälp av mänskliga pluripotenta stamceller under Feeder-fria och kemiskt definierade kulturförhållanden

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