The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.
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.
1. Preparation of Bacterial Inocula
2. Preparation of Agar Plates for Enumeration of the Inocula
3. Collection of Squid Juveniles
4. Squid Colonization
5. Determination of Colonization Levels
6. Data Analysis
7. Representative Results
Results from a sample colonization assay are shown in Figure 4. Two strains of V. fischeri that exhibit different relative levels of luminescence were each inoculated into six squid, along with six squid that served as aposymbiotic (uncolonized) controls. The E. scolopes symbiont, ES11414, and the brighter Sepiola robusta symbiont, SR515,16. Similar inoculum levels (Fig. 4A) lead to 100% colonization within 3 h. At 48 h, the luminescence levels (Fig. 4B) and CFU counts (Fig. 4C) were determined to assess the colonization proficiency of the strain. Determination of the specific luminescence (Fig. 4D; per bacterium) allows for determination of the brightness of each bacterial strain during the symbiosis.
Figure 1. Flow chart of the colonization procedure. Bacteria and squid are harvested separately, then mixed at the specified inoculum. Squid are washed, then transferred to new water at 3 h, 24 h, and 48 h post inoculation. At 48 h the luminescence is measured and the animals are frozen, which serves to surface-sterilize the animals. Light-organ colonized bacteria remain viable at -80°C through one thaw (no additional freeze-thaw cycles).
Figure 2. Flow chart illustrating homogenization and dilution plating of the bacteria. Serial 20-fold dilutions provide the appropriate dynamic range for enumeration of colonized bacteria.
Figure 3. Transfer pipettes with an appropriately narrow shaft taper to a narrow bore (A) that would damage juvenile squid. Pretreatment by cutting off the narrowest section with scissors or a razor blade yields an appropriate tool (B) for transferring juvenile squid.
Figure 4. Sample data for a colonization assay. (A) Levels of the bacteria in the inoculum bowls. (B) Luminescence of individual squid. (C) Colony counts of individual squid. (D) Specific luminescence of individual squid. Apo, Aposymbiotic (uncolonized negative control).
The colonization assay described allows for analysis of a natural symbiotic process in a controlled laboratory environment. As such, it can be used to assess colonization by mutant strains, by different natural isolates, and under different chemical regimes. Variations on the experiments described are commonly used to assess different aspects of the symbiosis. The kinetics of colonization can be measured by examining luminescence during the first 24 h, which can be detected automatically in a scintillation counter in which the coincidence detector has been removed. Furthermore, the relative colonization ability of one strain relative to another can be measured by a competitive colonization assay, in which the output ratio of the two strains in a set of animals are normalized to the input ratio (differential detection by distinct antibiotic resistance, fluorescence, or chromogenic [LacZ/Xgal] markers). Finally, colonization can be imaged directly by confocal microscopy.
It is critical to use healthy juvenile squid for the experiments. Behavioral indicators of poor squid health include swimming in circles (euthanize the animal), or animals that remain white and do not alter their chromatophores to turn brown on a dark surface (use with care). As there exists variation in the host populations, a greater number of replicate experiments with smaller numbers of animals is often more valuable than a smaller number of replicate experiments with large sample sizes.
The authors have nothing to disclose.
The authors thank Mattias Gyllborg for squid facility support and for comments on this manuscript, Michael Hadfield and the Kewalo Marine Laboratory for assistance during field collection, and members of the Ruby and McFall-Ngai Laboratory for contributions to this protocol. Work in the Mandel Laboratory is supported by NSF IOS-0843633.
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. |