The document describes a study that developed an inquiry-based approach for undergraduate students to isolate and characterize natural Vibrio fischeri symbionts from the Hawaiian bobtail squid. Each semester, students isolated multiple strains directly from the light organ of a wild-caught squid. Phenotypic assays found variation in bioluminescence responses and motility rates among strains. Genetic sequencing revealed diversity in the luxIR intergenic region. Phylogenetic analysis classified strains into genetically distinct groups. Colonization experiments showed all strains could colonize juvenile squid, though colonization efficiency varied. This inquiry-based approach effectively characterized the phenotypic and genetic diversity of natural V. fischeri symbionts.

1. 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