की उपनिवेशण Euprymna scolopes द्वारा व्यंग्य विब्रियो fischeri</em
Summary
विधि है जिसके द्वारा हवाई कट दुम व्यंग्य प्रक्रिया की रूपरेखा,<em> Euprymna scolopes</em> और इसके जीवाणु symbiont<em> विब्रियो fischeri</em> अलग और फिर उठाया बैक्टीरिया द्वारा विद्रूप प्रकाश अंग के विशिष्ट बसाना के लिए अनुमति देने के लिए शुरू कर रहे हैं. Bacterially व्युत्पन्न luminescence के द्वारा और प्रत्यक्ष कॉलोनी गिनती द्वारा औपनिवेशीकरण का पता लगाने में वर्णित हैं.
Abstract
Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.
Protocol
Discussion
बसाना वर्णित परख एक नियंत्रित पर्यावरण प्रयोगशाला में विश्लेषण के लिए एक प्राकृतिक सहजीवी प्रक्रिया की अनुमति देता है. जैसे, यह विभिन्न प्राकृतिक आइसोलेट्स द्वारा उत्परिवर्ती उपभेदों, और विभिन्न र?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखकों व्यंग्य सुविधा समर्थन के लिए और इस पांडुलिपि पर टिप्पणी, माइकल Hadfield और क्षेत्र संग्रह के दौरान सहायता के लिए Kewalo समुद्री प्रयोगशाला, और रूबी और McFall-Ngai इस प्रोटोकॉल के लिए योगदान के लिए प्रयोगशाला के सदस्यों के लिए Gyllborg Mattias धन्यवाद. मेंडल प्रयोगशाला में काम NSF के IOS 0843633 द्वारा समर्थित है.
Materials
| Name of reagent | Company | Catalogue Number | Comments |
| Glass Culture Tubes, 16 mm Diameter | VWR | 47729-580 | |
| Caps for Glass Culture Tubes | Fisher | NC9807998 | |
| Visible Spectrophotometer for Determination of OD600 | Biowave | CO8000 | Any spectrophotometer capable of measuring OD600 will work. This unit can measure the OD600 of liquid directly in the glass culture tubes. Some adjustment of the inoculum calculation may be necessary depending on the instrument used. |
| GloMax 20/20 Single-Tube Luminometer | Promega | E5311 | Equivalent to the Turner BioSystems 20/20n Luminometer. Includes the microcentrifuge tube holder. |
| GloMax 20/20 Light Standard | Promega | E5341 | For luminometer calibration. |
| Refractometer, Handheld | Foster and Smith Aquatics | CD-14035 | Calibrate before each use with deionized water. Rinse after every use with deionized water to prevent salt build-up. |
| Instant Ocean (artificial seawater concentrate) | Foster & Smith Aquatics | CD-16881 | Prepare at 35 ‰ in deionized water, using the refractometer, then filter through a 0.2 μm SFCA filter. |
| Filtration Unit | Nalgene | 158-0020 | Surfactant-free cellulose acetate (SFCA) membrane, 0.2 μm. We have observed variable results with some surfactant-containing PES filters. |
| Transfer Pipettes | Fisher | 13-711-9AM | Using scissors or razor blade, cut the tip cleanly above the first ridge to increase the diameter of the pipette tip and avoid squeezing the squid hatchlings. |
| Disposable Sample Bowls (plastic tumblers) | Comet | T9S (9 oz.) | Bowls for inoculation, with upper diameter 3 ¼”, lower diameter 2 ¼”, height 3″. Bowls create a homogenous environment as they have no bottom rim, in which squid can get trapped in a low-oxygen niche. The size is optimized for 40-ml inoculum. Available at webstaurantstore.com, #619PI9. |
| Drosophila Vials | VWR | 89092-720 | Vial diameter matches the opening on the luminometer PMT. |
| 1.5 ml Microcentrifuge Tubes | ISC Bioexpress | C-3217-1CS | Tubes must fit the shape of the pestles. |
| Ethanol, 200 Proof | Fisher | BP2818-100 | |
| Pestles | Kimble Chase/Kontes | 749521-1500 | |
| Plating Beads, 5 mm diameter | Kimble Chase | 13500 5 | Prepare 5 per tube and autoclave. |
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