Colonization of Euprymna scolopes Squid by Vibrio fischeri

Summary

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.

Abstract

Specific bacteria are found in association with animal tissue^1-5. 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 lifetime^6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities^8,9, 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 environment^11,12.

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

1. Preparation of Bacterial Inocula

1. Day 0
   Two days prior to squid inoculation, plate the relevant bacterial strains on LBS¹³ agar.
2. Incubate bacteria at 25-28°C overnight.
3. Day 1
   Inoculate 3 ml LBS medium in a glass culture tube with one colony of each V. fischeri strain for infection. Prepare duplicate tubes as backup.
4. Day 2
   (Coordinate bacterial steps 1.4-1.6 with squid steps 3.7-3.10)
   1 h prior to inoculation, subculture bacteria 1:80 (37.5 μl) into 3 ml LBS in a glass culture tube and grow for 1 h with aeratio....

Discussion

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 whi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glass Culture Tubes, 16 mm Diameter	VWR international	47729-580
Caps for Glass Culture Tubes	Fisher Scientific	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 Corp.	E5311	Equivalent to the Turner BioSystems 20/20n Luminometer. Includes the microcentrifuge tube holder.
GloMax 20/20 Light Standard	Promega Corp.	E5341	For luminometer calibration.
Refractometer, Handheld	Foster & Smith	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	CD-16881	Prepare at 35 ≥ in deionized water, using the refractometer, then filter through a 0.2 μm SFCA filter.
Filtration Unit	Nalge Nunc international	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 Scientific	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 international	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 Scientific	BP2818-100
Pestles	Kimble Chase	749521-1500
Plating Beads, 5 mm diameter	Kimble Chase	13500 5	Prepare 5 per tube and autoclave.