饲养果蝇<em>果蝇</em>在无菌和悉生条件
Summary
提出了一种用于无菌和限菌条件下饲养果蝇的方法。飞胚dechorionated在次氯酸钠，无菌转移到无菌的饮食，并在密闭容器中饲养。接种饮食和胚胎与细菌导致限菌协会，和细菌的存在是通过电镀全身果蝇匀浆证实。
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
大多数动物密切与细菌（'微生物'）从出生到死亡1有关。免费微生物（'无菌'）和微生物相关的（“传统”）的动物的比较表明微生物影响动物健康的各个方面，包括代谢，营养，血管，肝脏，呼吸系统，免疫，内分泌和神经功能2。果蝇果蝇是用于理解许多微生物3,4的存在下这些方法的并为研究对动物健康5,6-菌群影响的关键模式。没有细菌种类存在于每一个个体（“核心”），但醋酸和乳酸杆菌数字都占据主导地位实验室饲养和野生捕捞D的微生物果蝇 。其他Acetobacteraceae（包括Komagataeibacter和葡萄糖酸 ）， 菲尔米cutes（如肠球菌和明串珠菌 ）和肠杆菌科细菌无论是在低丰度经常出现在果蝇中的个人，或不定期出现在高丰度7-12。
果蝇和哺乳动物的微生物是内和跨世代14,19见异思迁。测量微生物相关的特征时，菌群世态炎凉会导致表型的噪音。例如，Acetobacteraceae影响脂质（甘油三酯）存储在果蝇 15-18。如果Acetobacteraceae是比另外19一小瓶的苍蝇更丰富，同基因苍蝇可以有不同的表型20。自1960年代以来，一种用于小鼠14菌群世态炎凉的问题的解决方案已经在实践中，通过引入定义8优势微生物物种社会的乳鼠每一代新产品（改变Schaedler菌群）确保每个小狗暴露于小鼠菌群的同一主要成员。即使当菌群不研究32的主要目标，并设置先例，以确保密钥的微生物在各种实验条件下的存在于微生物群组成这种做法控制。
上定义果蝇营养微生物的影响下，用于导出无菌飞线几个协议已被开发，包括胚胎次氯酸钠dechorionation（无论是衍生从头每一代或通过转移到无菌的饮食的世代保持）和抗生素治疗13。有益处的不同方法，如方便与快捷为抗生素治疗和串行传输，相对于与从头 dechorionation混杂变量（ 例如 ，卵密度，残留污染微生物，脱靶抗生素效果）的更大的控制。不管该方法的准备，出台规定微生物物种对无菌胚胎允许果蝇的文化与自定义（“悉生'）社区。可替代地，模仿使用Schaedler菌群，该社区可以接种到常规的鸡蛋（以下步骤6-7只），以确保在每个小瓶特质影响微生物的存在，并避免菌群反复无常的并发症。在这里，我们描述了协议用 于通过胚胎从头 dechorionation提高无菌和限菌果蝇 ，并确认导入或污染微生物类群的存在。
Protocol
1.细菌培养（开始采摘〜鸡蛋前1周） 制备改良的MRS 20（MMRS）板和肉汤管（ 表1）。倾20毫升MMRS琼脂到每个百毫米培养皿并冷却/干燥过夜，和5ml MMRS肉汤分为18 mm试管。 条纹醋pomorum，A. 热带 ， 短乳杆菌 ，和L.植物上MMRS琼脂平板上。孵育醋杆菌在30℃下过夜。通过在密封前用二氧化碳将所述板在密封容器中，并驱孵育?…
Representative Results
无菌蝇饲养成功是没有从D的全身homogenizations CFU的隔离证实果蝇成虫（ 图1）。或者，如果镀覆匀浆得到的菌落，小瓶被污染并应该被丢弃。为限菌苍蝇，四个细菌菌株是从5成年男性池隔离，以表明其与成蝇（ 图1）相关联的总活菌的CFU的差异。每个细菌物种具有不同的形态，并可以在视觉上加以区分（ 图2）。如果没有?…
Discussion
此处所描述的方法是几种方法用于胚胎dechorionation 8,11,18,25,26,27之一，饲养无菌蝇，包括无菌的成年人18,27的串行传输或抗生素治疗13,18的替代方法在一起。其他dechorionation方法包括乙醇洗涤，减少11,25,26或延长8次氯酸盐治疗。不同的洗涤步骤可以帮助饲养不同蝇基因型：在先前的研究中最〜的饲养时如这里概述（无乙醇洗涤）100 果蝇基因型无菌?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
该协议的一些细节与亚当博士布森，谁也对稿件提供了有益的意见的协助下进行了优化。这项工作是由该基金会为美国国立卫生研究院的支持（FNIH）授权号R01GM095372（JMC，A（CN）W，AJD和AED）。 FNIH授权号码1F32GM099374-01（PDN），和杨百翰大学的启动资金（江铃汽车，MLK，MV）。出版费用由生命科学杨百翰大学学院和植物和野生动物科学系的支持。
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®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 |
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Citer Cet Article
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).