Introduction The Hawaiian bobtail squid harbors populations of Vibrio

fischeri within a dedicated structure called the light organ

(Fig. 1). Previous work has shown that the V. fischeri

infections within wild-caught squid are polyclonal, i.e., each

animal hosts multiple strains of V. fischeri (1). The assembly

process and biological function of such polyclonal infections

in the squid-Vibrio symbiosis remain unclear, particularly

because the symbiotic strains currently available

underrepresent the phenotypic and genetic diversity. We

recently reported experiments that enable undergraduate

researchers to study natural V. fischeri isolates (2)

Methods Media and growth conditions In this study, V. fischeri

strains were grown at 28°C in LBS medium (1% tryptone,

0.5% yeast extract, 2% NaCl, 50 mM Tris-HCl [pH 7.5]). Bioluminescence assay Luminescence was measured for

cultures grown in LBS ± 120 nM 3-oxo-C6 HSL. Motility assay 10-μl sample of culture at OD600 ~0.2 was

injected into 0.25% agar plates with TB-IO medium at 28°C.

Diameter of motility ring was measured over time. Sequencing The luxIR intergenic region was amplified by

PCR and cloned into pCR-blunt. Sanger sequencing was

performed by PSU sequencing core facility. Alignment was

performed using MEGA 6 software. Phylogenetic analysis PCR amplification and analysis was

performed as previously described (2). Squid colonization Newly hatched squid were exposed to

~5,000 CFU/ml inoculum for 18 h. At 48 h post-inoculation

(p.i.), animal luminescence was measured.

Figure 1 The squid-Vibrio symbiosis.

A. Juvenile squid. Dotted box highlights light organ. Bar = 1 mm.

B. Juvenile light organ colonized by GFP-labeled V. fischeri (green).

Bar = 100 µm.

C. Crypt highlighted in B showing V. fischeri population. Host actin is

stained with phalloidin (blue). Bar = 10 µm.

A B C

Goal: To implement inquiry-based modules for

undergraduates to examine V. fischeri symbiont diversity

within a semester.

Results Each semester-long study involved four undergraduate

students isolating strains directly from the light organ of a

wild-caught E. scolopes adult animal. For each strain, the

bioluminescence response to 120 nM 3-oxo-C6 HSL, which

induces bioluminescence in ES114, and the motility rate

through soft-agar were determined (Fig. 2). The

bioluminescence response of ES114 was remarkably

consistent across trials, showing approximately 100-fold

enhanced luminescence levels (Fig. 2B). In contrast,

variability was observed for motility rates (Fig. 2D), which

may be due to different motility-plate preparations among

individuals. Together, these assays demonstrate striking

phenotypic diversity among the strains isolated from

individual animals, as previously reported (1).

Figure 2 Bioluminescence and motility of natural isolates.

A. Bioluminescence assay performed for one isolate, with positive and

negative controls for V. fischeri response to 3-oxo-C6 HSL.

B. Bioluminescence assay for ES114 by different students.

C. Bioluminescence response of natural isolates to 3-oxo-C6 HSL.

D. Motility rates of ES114 performed by different students.

E. Motility rates of natural isolates (normalized by rate of ES114).

F. Scatter plot showing bioluminescence (C) and motility rates (E) for

natural isolates. Colors indicate strains co-isolated from the same

animal. ES114 is represented black circle.

Finally, we examined the ability of V. fischeri strains to

colonize the light organ of juvenile squid, which hatch un-

colonized. Based on animal luminescence, all strains were

able to function in symbiosis, although the efficiency of

colonization varied among the strains (Fig. 5). Based on

CFU plating, co-colonization assays showed strains

CHS319, ECT001, NAD004, ABM004, and EMG003 were

out-competed by ES114 and strain ZJH004 competed

equally with ES114 (data not shown).

Sequencing of the luxIR intergenic region, which is

hypervariable among V. fischeri strains (3), revealed

variation among the isolates (Fig. 3), which suggest a genetic

basis for the different luminescence responses observed

among the strains.

Figure 3 Alignment of luxIR intergenic region among natural isolates.

Strain names are labeled according to isolated animal. Known binding

sites for transcription factor are indicated.

Phylogenetic analysis was performed using four genetic loci

(recA, mdh, katA, and pyrC). Each undergraduate group

isolated between 2-3 genetically distinct strains (Fig. 4).

Figure 5 Squid colonization assay.

A. Colonization assay for one natural isolate, with positive (ES114)

and negative (apo-symbiotic) controls for animal luminescence. Lum+

animals are defined as animals with > 500 RLU at 48 h p.i.

B. Colonization efficiency for natural isolates as determined by

luminescence.

Conclusions These results demonstrate that an inquiry-based approach is

sufficient to isolate and characterize natural V. fischeri

symbionts with phenotypic and genetic diversity.

Furthermore, our studies suggest that the bioluminescence/

motility profile of the type strain ES114 is unique relative to

those of other isolates obtained thus far.

References 1. Wollenberg, M.S. and Ruby, E.G. (2009) Population structure of Vibrio fischeri

within the light organs of Euprymna scolopes squid from Two Oahu (Hawaii)

populations. Appl Environ Microbiol 75: 193-202.

2. Sun, Y., LaSota, E.D., Cecere, A.G., LaPenna, K.B., Larios-Valencia, J.,

Wollenberg, M.S. and Miyashiro, T. (2016) Intraspecific Competition Impacts Vibrio

fischeri Strain Diversity during Initial Colonization of the Squid Light Organ. Appl

Environ Microbiol 82: 3082-91.

3. Bose, J.L., Wollenberg, M.S., Colton, D.M., Mandel, M.J., Septer, A.N., Dunn,

A.K. and Stabb, E.V. (2011) Contribution of rapid evolution of the luxR-luxI

intergenic region to the diverse bioluminescence outputs of Vibrio fischeri strains

isolated from different environments. Appl Environ Microbiol 77: 2445-57.

4. Wollenberg, M.S. and Ruby, E.G. (2012) Phylogeny and fitness of Vibrio fischeri

from the light organs of Euprymna scolopes in two Oahu, Hawaii populations. ISME

J 6: 352-62.

Acknowledgements We thank members of the Miyashiro lab for valuable advice during this study. This

work was supported by the Eberly College of Science.

For further information Please e-mail Tim Miyashiro at tim14@psu.edu.

Future directions 1. This inquiry-based approach will be used as a training

program for undergraduate researchers who join the lab.

Following completion of this program, students will

propose independent projects to pursue in the lab.

2. To what extent do co-occurring strains segregate within

the light organ?

3. How do different strains interact via quorum signaling?

4. What strain types are transmissible?

Figure 4 Phylogenetic analysis was performed

with concatenated sequences of recA, mdh, katA,

and pyrC. Names of strains isolated for this study

are colored according animal. Strains in stippled

box are competitively dominant strains (4)

Development of an inquiry-based approach to investigate strain diversity

within the squid-Vibrio symbiosis A. G. Cecere1, E. Grandinette1, Z. J. Houston1, A. Mouchref1, A. N. Murtha1, N. C. Ortega1, E. B.

Schwendeman1, C. H. Steingard1, E. C. Tatsumi1, M. S. Wollenberg2, and Tim Miyashiro1

1Department of Biochemistry and Molecular Microbiology, The Pennsylvania State University, University Park, PA 2Department of Biology, Kalamazoo College, Kalamazoo, MI