RESEARCH ARTICLE
Association of colony morphotypes with virulence, growth and
resistance against protozoan predation in the fish pathogen
Flavobacterium columnare
Ji Zhang1,2, Jouni Laakso1,2, Johanna Mappes1, Elina Laanto1,3, Tarmo Ketola1, Jaana
K.H.Bamford1,3, Heidi Kunttu1 & Lotta-Riina Sundberg1
Centre of Excellence in Biological Interactions, Department of Biological and Environmental Science, University of Jyv€
askyl€
a, Jyv€
askyl€
a, Finland;
Department of Biological and Environmental Science, University of Helsinki, Helsinki, Finland; and 3Department of Biological and Environmental
Science and Nanoscience Centre, University of Jyv€askyl€a, Jyv€askyl€
a, Finland
1
2
Correspondence: Lotta-Riina Sundberg,
Centre of Excellence in Biological Interactions,
Department of Biological and Environmental
Science, University of Jyv€askyl€a, P.O. Box 35,
Jyv€
askyl€
a 40014, Finland.
Tel.: +358 408 053 931; fax: +358 14 617
239; e-mail: [email protected]
Received 3 February 2014; revised 8 April
2014; accepted 13 May 2014. Final version
published online 6 June 2014.
MICROBIOLOGY ECOLOGY
DOI: 10.1111/1574-6941.12356
Editor: Patricia Sobecky
Keywords
colony morphotypes; columnaris disease;
phage; predation; protozoa; trade-off.
Abstract
Many opportunistic pathogens can alternate between inside- and outside-host
environments during their life cycle. The opportunistic fish pathogen Flavobacterium columnare is an inhabitant of the natural microbial community and
causes significant yearly losses in aquaculture worldwide. The bacterium grows
in varying colony morphotypes that are associated with either virulence (rhizoid type) or resistance to starvation and phages (rough type). Rough type
strains can arise spontaneously or can be induced by phage infection. To identify the determinants of morphotype fitness, we measured virulence, growth
parameters, biofilm-forming ability and resistance to amoeba and ciliate predation of both morphotypes in thirteen F. columnare strains. The (phage-sensitive) rhizoid type had a fitness advantage over the rough type in virulence,
growth rate and maximum population size. Phage-induced rough type was
found to be significantly weakest in resisting both ciliate and amoeba predation, and produced more biofilm in the presence of amoebae, whereas the
spontaneous rough types did not differ from rhizoid in biofilm production. In
co-culture experiment, the ciliate population sizes were higher when co-cultured with rough type than with rhizoid type. Our results thus suggest that the
resistance to phages and starvation of the F. columnare rough type may have
strong a trade-off, as the performance of the ancestral rhizoid type is better
under environmental conditions.
Introduction
While obligate pathogens live mainly in the within-host
environment, opportunistic bacterial pathogens are usually host generalists and alternate between inside- and
outside-host phases in their life cycles (Brown et al.,
2012). Therefore, the ability to cope with fluctuations in
environmental conditions is especially important for
opportunists. Having the ability to switch phenotypes
according to available growth strategies allows environmentally transmitting pathogens to respond to environmental threats, increase their survival in the outside-host
environment, and the ability to invade and infect a host
FEMS Microbiol Ecol 89 (2014) 553–562
population (Thattai & van Oudenaarden, 2004; Beaumont
et al., 2009; Brown et al., 2012). Understanding the
mechanisms that enable the switch between the insideand outside-host phases could be useful in the prevention
of diseases, but requires detailed knowledge of the tradeoffs in the life cycle of the pathogen.
Life outside the host exposes pathogens to selective
pressures and trade-offs that are not present in the
inside-host environment. Although fluctuations in nutrients and the surrounding microbial community affect
natural bacterial population dynamics (e.g. Eiler & Bertilsson, 2007; Foster & Bell, 2012), parasitism and predation are the main biotic factors that maintain phenotypic
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
J. Zhang et al.
554
variation in free-living bacterial populations (J€
urgens &
Matz, 2002). For example, phage parasitism (Buckling &
Rainey, 2002; Holmfeldt et al., 2007) and protozoan predation (Matz et al., 2004; Matz, 2005) can diversify bacterial characteristics that can correlate either positively or
negatively with bacterial virulence (J€
urgens & Matz, 2002;
Greub & Raoult, 2004; Meltz Steinberg & Levin, 2007;
Friman et al., 2009; Adiba et al., 2010). Yet, information
on the factors that select against bacterial virulence under
natural conditions in environmentally transmitting bacterial pathogens is scarce, but needed for understanding
disease dynamics.
Flavobacterium columnare (Bacteroidetes) is an environmentally growing opportunistic fish pathogen that can
cause disease outbreaks and large economic losses in
freshwater fish farming worldwide (Wagner et al., 2002;
Pulkkinen et al., 2010; Declercq et al., 2013). When isolated from disease outbreaks, the bacteria grow as the rhizoid (Rz) morphotype that is highly virulent in
comparison with the other known morphotypes (rough
and soft) (Kunttu et al., 2009a; Laanto et al., 2012)
(Fig. 1). F. columnare benefits from high virulence
because it increases fish mortality and promotes the bacterium’s saprotrophic growth in the dead fish, which in
turn acts as an efficient transmission strategy (Kunttu
et al., 2009b). The rough (R) phenotype of F. columnare
appears either after the bacterium has gained resistance
against a phage (Laanto et al., 2012) or spontaneously as
a result of starvation or serial culture (Kunttu et al.,
2009a, 2012; Sundberg et al., 2014). This phenotype
should therefore be superior when lytic phages are present, or under starvation in the outside-host environment.
It is therefore possible that different morphotypes are
beneficial in various parts of the life cycle. Indeed, in
some cases, the R phenotype has been isolated from fish
experimentally infected with solely the Rz type (Kunttu
et al., 2009a). However, to date, rough types tolerating
starvation and phage infection have not been isolated
(a)
from nature, even though in boreal regions the outsidehost period of F. columnare is long: columnaris disease
outbreaks occur only during the warmest time of the
year, and the lytic phages are present in the rearing units
(Laanto et al., 2011). This suggests that being virulent
could outweigh the benefits of being tolerant to starvation
or antagonistic species interactions. Alternatively, the
nonvirulent R phenotype could be rare because they are
selected against in the outside-host environment, for
example via protozoan predation, and are thus not
detectable.
Understanding the biotic factors that influence bacterial
performance in the environment is crucial to understand
disease dynamics. To map the factors selecting for the
presence of the virulent rhizoid type in environment and
in disease outbreaks, we measured virulence, growth rate,
maximum population size, biofilm-forming ability, and
resistance to protozoan predation (surface-feeding
amoeba Acanthamoeba castellanii and particle-feeding ciliate Tetrahymena thermophila) of 13 F. columnare strains.
These bacterial strains were isolated from disease outbreaks at fish farms and their upstream water bodies.
Each strain comprised of two colony morphotypes: ancestral Rz and R that was triggered by either laboratory starvation or exposure to lytic phage. The virulence of all
morphotypes was measured using zebrafish (Danio rerio)
as an infection model. By measuring these traits, we
aimed to identify fitness benefits that could explain the
frequent occurrence of the rhizoid type instead of the
rough type under environmental pressures.
Materials and methods
Bacterial strains, phages, and predators
We used 13 different strains of F. columnare in this study,
and all strains included both colony morphotypes, Rz and
R, although the method to induce the R type differed
(b)
Fig. 1. The ancestral rhizoid colonies of
Flavobacterium columnare strain B245 (Rz, a)
are flat and spreading. The starved, derivative
rough morphotype (R, a) resembles the Rz
type, but is smaller and more adherent to
agar. After exposure to a lytic phage, the
phage-resistant R type (b, strain B392) has lost
all root-like protrusions and has round and
solid edges.
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
FEMS Microbiol Ecol 89 (2014) 553–562
Flavobacterium columnare and protozoan predation
555
Table 1. Flavobacterium columnare strains used in the study, and their ARISA genotypes (N.A., not analysed)
Strain code
Isolation source
Isolation year
ARISA group
Rough type induced by
Reference
B392_P
B067
B185
B245
B398
B401
B392_S
B394
B402
B425
B408
H
B355
Environment
Farm L
Farm L
Farm V
Environment
Environment
Environment
Environment
Farm V
Farm V
Environment
Farm V
Environment
2010
2007
2008
2009
2010
2010
2010
2010
2010
2007
2010
2003
2009
A
A
G
C
A
G
A
A
C
N.A.
C
H
N.A.
FCL-1 phage
FCL-1 phage
FCL-2 phage
FCV-1 phage
FCL-1 phage
FCL-2 phage
Starvation
Starvation
Starvation
Starvation
Starvation
Starvation
Starvation
Kunttu et al. (2012)
Laanto et al. (2011, 2012)
Laanto et al. (2011, 2012)
Laanto et al. (2011, 2012)
Kunttu et al. (2012)
Kunttu et al. (2012)
Kunttu et al. (2012)
Kunttu et al. (2012)
Kunttu et al. (2012)
N.A.
Kunttu et al. (2012)
Suomalainen et al. (2006)
N.A.
Each strain included the ancestral rhizoid and its derivative rough morphotypes triggered by either exposure to phages or to starvation. For more
information on the phages see Laanto et al. (2011).
between strains (Table 1). The strains originated from
fish farms or natural freshwater bodies (Table 1). They
were originally isolated using standard culture methods
on Shieh medium (Shieh, 1980) or in Shieh medium supplemented with tobramycin (Decostere et al., 1997) and
were stored frozen in 80 °C with 10% glycerol and 10%
foetal calf serum. When reviving from the stocks, the bacterial strains were grown in Shieh medium at room temperature (RT, c. 24 °C) under constant shaking
(110 r.p.m.).
We infected F. columnare with three previously isolated
lytic phages to induce phage resistance in six of the original Rz type strains; the treatment changed the morphotype to the phage-resistant R type (Table 1). The phages
originated from two different fish farms in Central Finland and were previously characterised (Laanto et al.,
2011). The bacteria were exposed to phages following
standard protocol (Laanto et al., 2012). Shortly, the
phages were enriched and isolated by adding 5 mL of
Shieh medium (Shieh, 1980) on top of an agar plate with
confluent lysis. The plate was shaken at 8 °C, 95 r.p.m.
for 24 h. Phage lysates were filtered through a 0.45 lm
Supor Membrane (PALL Corporation) and stored in
4 °C. Phage lysate was pipetted on Shieh agar plate, and
fresh F. columnare culture was inoculated on top of the
phage lysate. After 48 h, the R type colonies growing in
the presence of the phage were isolated and purified for
further use. The starvation-triggered R type (seven strains;
Table 1) was obtained from our previous study where
F. columnare cultures were inoculated and maintained
in starvation for 2 months in sterile distilled water (see
Kunttu et al., 2012).
We tested the bacterial morphotypes’ resistance against
protozoan predation by co-culturing them with a ciliate
and an amoeba (see below). The predatory particle-feeding
FEMS Microbiol Ecol 89 (2014) 553–562
ciliate T. thermophila strain ATCC 30008, which has a
minimum generation time of c. 2 h (Kiy & Tiedtke,
1992), was obtained from American Type Culture Collection and is routinely maintained in PPY medium (Proteose Peptone and Yeast medium) (Friman et al., 2008) at
25 °C. Free-living amoeba A. castellanii strain CCAP
1501/10, which has a generation time of c. 7 h (Kennedy
et al., 2012), was obtained from Culture Collection of
Algae and Protozoa and routinely maintained in PPG
medium (Proteose Peptone Glucose medium) (Page,
1976) at 25 °C.
Bacterial growth measurements
We measured the maximum growth rate and population
size in the absence of predators from both morphotypes,
Rz and R of all strains individually. First, the master cultures of the bacteria were prepared by inoculating single
colonies of either R or Rz to 5 mL of modified Shieh
medium, where the concentration of nutrients was
reduced to 10% of the original medium (0.05 g L 1 yeast
extract and 0.5 g L 1 peptone). After cultivation at 25 °C
for 48 h on a shaker (120 r.p.m.), 10 lL of the master
culture was applied onto 100-well Bioscreen C plates
containing 400 lL of fresh modified Shieh liquid medium. Bacterial biomass was then measured as optical density (OD) at 460–580 nm wavelength (broad band) using
Bioscreen C spectrophotometer (Oy Growth Curves Ab
Ltd) at 25 °C. The bacteria were cultured without shaking, and the OD measurements were repeated at 5-min
intervals for c. 50 h. To find maximum growth rate (OD
460–580 nm h 1) and maximum population size (yield),
we fit linear regressions to the ln-transformed population
growth data consisting of 30 datapoint’s sliding time
window. The time window with maximum slope was
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
J. Zhang et al.
556
determined as the maximal growth rate. Maximum population size (yield) was determined as the maximal arithmetic mean OD value in the sliding window data. The
MATLAB code to perform these analyses was provided by
Ketola et al. (2013).
Resistance to protozoan predation
To measure the resistance of the F. columnare morphotypes to protozoan predation, we co-cultured both Rz
and R type bacteria with either T. thermophila (ciliate) or
A. castellanii (amoeba) and compared bacterial growth in
these co-cultures to cultures without predators.
The amoebas and ciliates were washed twice in 40 mL
of modified Shieh liquid medium and pelleted with centrifugation at 1200 g for 15 min, after which they were
suspended in the modified Shieh medium and adjusted to
a final concentration of 10–20 cells per lL. After c. 50 h
of static culture of bacteria on Bioscreen C plates, 20 lL
of amoeba or ciliate suspension was added to the bacterial culture (20 lL fresh modified Shieh liquid medium
was added to predator-free wells). The plates were incubated in Bioscreen C spectrophotometer to monitor
bacterial growth with the method described above. Each
F. columnare morphotype (Rz or R) had at least three
replicates of every treatment (ciliate, amoeba or no predators) on the plates.
Resistance to predation was then measured by subtracting the minimum population size (OD) of the bacterial
morphotypes co-cultured with protozoan predators
(T. thermophila or A. castellanii) from their minimum
population size without the predators. As in the growth
measurements, the OD data were smoothed to remove
measurement noise. The small population sized of predators in the experiments (maximum of 50 cells per lL)
did not hamper the OD measurements. High values indicate high protozoan predator resistance, zero indicates
that the predator has no effect on the gross bacterial biomass, and positive values indicate that the predator can
increase the gross biomass.
Biofilm formation
To measure the amount of the biofilm the F. columnare
morphotypes formed on the Bioscreen plate wells during
the protozoan predation experiment, after 50 h culture,
we removed all culture medium from the wells and
stained the wells with 100 lL of 1% crystal violet solution
(Sigma-Aldrich). After 10 min of staining, the plates were
rinsed three times with distilled water. Next, the crystal
violet was dissolved from the wells with 450 lL of 96%
ethanol for 24 h (O’Toole & Kolter, 1998). The amount
of formed biofilm was quantified from the OD of the
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dissolved crystal violet at 460–580 nm with a Bioscreen
C spectrophotometer (Friman et al., 2011).
Virulence test
We tested the virulence of the Rz and R morphotypes
using a zebra fish bath immersion infection model. Unsexed adult disease-free wild type zebrafish were obtained
from Core Facilities (COFA) and the research services of
the University of Tampere, Finland. The fish were
infected with either the Rz or the R type F. columnare.
We utilised the infection method and bacterial dose that
were developed and adjusted in preliminary experiments
(Laanto et al., 2012): the fish were individually challenged
in 50 mL of ground water with 1 9 107 colony-forming
units (CFU) mL 1 of overnight grown bacteria for
30 min at 25–26.5 °C. For each bacterial strain, we
infected six fish individually with Rz and six with R type,
and six individuals exposed to sterile Shieh medium
served as the negative control (totalling 168 fishes, when
strain B392 had both R types, see Table 1). After the
challenge, the fish were transferred into separate 750-mL
(11.5 9 8 9 9 cm) plastic aquaria (one fish per aquarium) with 500 mL of ground water and monitored for
2 days for disease symptoms every 12 h. The light regime
during the experiment was 12:12, and fish were not fed
during the experiment. Morbid fish that had lost their
natural swimming buoyancy and that which did not
respond to external stimuli were considered dead and
were removed from the experiment. They were euthanatized to meet the ethical end point of the experiment and
to avoid suffering of the fish. After the experiment, the
fish that had survived were further monitored for another
2 days. To confirm that the cause of death was indeed
the columnaris disease, a bacterial culture sample from all
dead and moribund fish were taken on Shieh agar. Virulence of the morphotypes was measured as the number of
survived fish per bacterial strain 48 h post-infection.
Palatability of F. columnare to T. thermophila
and A. castellanii
To find out how well the protozoan predators are able to
exploit the R and Rz morphotypes of F. columnare as a
nutrient source, we measured the population size of the
predators whilst they were co-cultured with the bacteria.
First, the bacteria (only strains B245 and B67 and their
two morphotypes, ancestral Rz, and the R type induced
by phages, see Table 1) were cultured for 48 h in 20 mL
of modified Shieh medium. The cultures were kept in
Sarstedt 25 cm2 flasks with filter caps at 25 °C without
shaking. Next, 1 mL of washed ciliates or amoebae (10–
20 cells per lL) were added to the flasks (three replicates
FEMS Microbiol Ecol 89 (2014) 553–562
Flavobacterium columnare and protozoan predation
557
for each treatment). For the negative control, the same
volume of ciliate or amoeba cells were added to 20 mL of
fresh modified Shieh liquid medium.
The population size of the ciliate and amoeba predators was measured daily for 7 days. To measure the
amoeba population size, the flasks were first chilled in ice
for 15 min and shaken vigorously to detach the cells from
the flask wall. A 240-lL sample of the culture was taken
out and stained with 10 lL of Lugol’s solution and
placed into a 250-lL glass cuvette. For each sample, eight
randomly placed images (depth 2.34 mm, total area
18 mm2) were digitized with an Olympus SZX microscope (329 magnification). The cell numbers in each
image were counted with an IMAGE PRO PLUS script (Laakso
et al., 2003). Ciliate population size was measured similarly but without chilling.
Statistical analyses
We analysed the effects of morphotype and morphotype
triggering method and the effect of strain identity using
restricted maximum likelihood mixed models implemented in SPSS. In this statistical model, we fitted the
effect of treatment (untreated Rz, starved R, and phageinduced R) as a fixed factor. Isolate identity was fit as a
random effect because the same isolates are found in at
least two treatments groups, and three measurement replicates cause the observations to be nonindependent. In
addition to this, we also tested whether inclusion of origin of isolate (fish farm, or wild) or interaction with
treatment and origin could explain the data better. However, by Akaike information criterion, we deduced that
the best model for all of the traits is a simple model that
contained only treatment and strain identity. In our
analysis, we considered maximal growth rate, maximum
population size, biofilm-forming ability (in the absence of
predators), resistance against amoeba and ciliate predators, and biofilm-forming ability in the presence of the
amoeba and the ciliate, as dependent factors.
(a)
(b)
The palatability of the bacteria to protozoans was
tested with repeated MANOVA. Virulence of the bacterial
morphotypes (the number of morbid fish per bacterial
strain and morphotype) was analysed by Kruskall–Wallis
test. All the analyses were performed with SPSS v. 20 software (IBM).
Results
Rhizoid morphotypes (Rz) grew faster than either of the
rough (R) types (P < 0.001). For both pairwise comparisons, maximum growth rates per h were 0.096 0.005,
0.076 0.005, and 0.076 0.006 for Rz, spontaneous R,
and phage-induced R, respectively, measured as mean OD
at 460–580 nm SEM (see ‘Materials and methods’ for
calculating growth rate). Growth rate of the two R types
were comparable to each other (P > 0.9, Fig. 2). The
overall statistics for comparison between morphotypes
and their triggering methods are given in Table 2.
Maximum population size was larger in Rz type
(0.181 0.008, mean SEM) compared with R types
(spontaneous
0.140 0.010,
phage-induced
0.120 0.012, pairwise tests P < 0.001 in both; see Fig. 2
and Table 2 for overall statistics).
Biofilm-forming ability was highest in phage-induced R
type (0.293 0.019), followed by Rz (0.270 0.017)
and R triggered by starvation (0.215 0.019). The pairwise tests indicated that biofilm-forming ability of the Rz
and the phage-induced R were similar (P = 0.206), but
all the rest combinations differed (P < 0.001, Table 2,
Fig. 2).
The lowest resistance against the ciliate T. thermophila
was found in phage-induced R types ( 0.089 0.011,
measured as OD difference between individual and
co-culture), followed by Rz ( 0.070 0.010) and spontaneous R ( 0.065 0.011). The phage-induced R types
had the lowest resistance to ciliate predation (phageinduced R vs. Rz P = 0.004, phage-induced R vs. starved R
P = 0.019, spontaneous R vs. Rz P < 0.9, Table 2, Fig. 3).
(c)
Fig. 2. (a) Maximum growth rates (h 1, mean OD 460–580 nm SEM), (b) Maximum population size (OD SEM), and (c) Biofilm-forming
ability (OD SEM) of the Flavobacterium columnare strains (n = 13) measured in modified Shieh medium. The changes from rhizoid to rough
type appeared spontaneously in the culture in response to starvation (n = 7) or were triggered by exposure to phages (n = 6).
FEMS Microbiol Ecol 89 (2014) 553–562
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Published by John Wiley & Sons Ltd. All rights reserved
J. Zhang et al.
558
Table 2. Overall statistics of the effect of morphotype (ancestral Rhizoid, Rough type triggered by starvation or phage) and identity of the strain
on Flavobacterium columnare growth and defensive traits
Morphotype
F
Trait
Maximum growth rate
Maximum population size
Biofilm-forming ability
Resistance against:
Ciliates
Amoebae
Biofilm-forming ability with:
Ciliates
Amoebae
Strain ID
d.f.1, d.f.2
P
Estimate
12.119
19.092
11.954
2, 59.58
2, 58.17
2, 69.90
< 0.001
< 0.001
< 0.001
1.39 9 10
3.77 9 10
3.21 9 10
4
5.864
19.606
2, 86.04
2, 91.17
0.004
< 0.001
1.25 9 10
7.30 9 10
3
2.257
3.905
2, 70.57
2, 70.49
0.112
0.025
3.09 9 10
3.35 9 10
6
SE
4
3
4
3
9.20 9 10
2.72 9 10
1.45 9 10
5
5.35 9 10
3.24 9 10
4
1.73 9 10
1.52 9 10
6
4
3
4
3
Wald Z
P
1.510
1.387
2.223
0.131
0.165
0.026
2.341
2.253
0.019
0.024
1.785
2.205
0.074
0.027
Results of pairwise comparisons between morphotypes are given in text.
0.04
0.04
(a)
0.33
(b)
(c)
Ciliate resistance
–0.04
Biofilm with amoebae
0.31
0.00
0.00
–0.04
–0.08
–0.08
–0.12
–0.12
0.29
0.27
0.25
0.23
0.21
Rhizoid
Rough
(starv.)
Rough
(phage)
Rhizoid
Rough
(starv.)
Rough
(phage)
0.19
Rhizoid
Rough
(starv.)
Rough
(phage)
Fig. 3. (a) shows resistance of Flavobacterium columnare against amoebae (mean SEM), (b) resistance to ciliates (mean SEM), and (c) the
biofilm-forming ability in the presence of amoebae (mean SEM). The changes from rhizoid to rough type appeared spontaneously in the
culture in response to starvation (n = 7) or were triggered by exposure to phages (n = 6).
Also the amoeba A. castellanii predation resistance was
weakest in phage-induced R types ( 0.012 0.009), in
comparison with Rz (P < 0.001, 0.023 0.008, Table 2,
Fig. 3) and starved R (P < 0.001, 0.026 0.009). Rz and
starved R did not deviate from each other (P < 0.9).
In the presence of ciliates, biofilm-forming ability was
comparable between the different morphotypes (Rz
0.268 0.018, spontaneous R 0.245 0.020, phageinduced R 0.298 0.020). However, in the presence of
amoebae, the phage-induced R produced more biofilm
than spontaneous R (P = 0.020). Other pairwise comparisons were nonsignificant (Rz vs. phage-induced R:
P = 0.103, Rz vs. starved R P = 0.293, Table 2, Fig. 3).
The ciliate population size increased when co-cultured
with both B245 and B67 bacterial strains (population size:
F4,10 = 92.036, P < 0.001; Fig. 4b), but feeding on the
phage-induced R type increased it more than feeding on
the Rz type (Fisher’s LSD: B245 R vs. B245 Rz,
P < 0.001; B67 R vs. B67 Rz, P = 0.015). Interestingly,
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Published by John Wiley & Sons Ltd. All rights reserved
however, both B245 and B67 inhibited the growth of the
amoeba (population size: F4,10 = 23.271, P < 0.001;
Fig. 4a), and there was no morphotype effect (Fisher’s
LSD: B245 R vs. B245 Rz, P = 0.488; B67 R vs. B67 Rz,
P = 0.052).
The Rz type bacteria were more virulent than the R
type, causing higher mortality in the zebra fish
(v2 = 79.636, d.f. = 2, P < 0.01). Flavobacterium columnare was isolated from all the individuals that died during
the virulence test, except for those that died after exposure to the R type of strain B394. The fish that remained
alive after the 48 h of infection did not show signs of columnaris disease during the extra 2 days of monitoring.
Discussion
The fish pathogen F. columnare is variable in colony morphology (Kunttu et al., 2009a; Laanto et al., 2012). In line
with previous findings using rainbow trout (Oncorhynchus
FEMS Microbiol Ecol 89 (2014) 553–562
Flavobacterium columnare and protozoan predation
(a)
559
(b)
Fig. 4. Growth curves (cells lL 1) for the amoeba Acanthamoeba castellanii (a) and ciliate Tetrahymena thermophila (b) populations feeding
separately on the rhizoid and rough morphotypes of the strain B67 or B245 of Flavobacterium columnare. Population size of the predators
reflects palatability of the bacteria.
mykiss, Walbaum) (Kunttu et al., 2009a) and zebra fish
(Laanto et al., 2012), our study confirmed that the Rz
(rhizoid) morphotype is highly virulent and should have
a higher fitness during the columnaris disease outbreak
season. Indeed, research does show that mainly this
morphotype is isolated from fish farms and environmental water bodies during the warmest summer months
(Kunttu et al., 2012). However, as the infection period is
short, it is likely that due to their environmental origin,
the bacteria live outside their fish host for most of the
year. This would suggest a trade-off between virulence
and resistance to environmental threats like presence of
phages or starvation. However, even under these conditions, the Rz type dominates, and the starvation-tolerant
and phage-resistant R phenotypes are rare, suggesting that
there could be additional costs for displaying the nonvirulent R morphotype in the outside-host environment.
Here, we show that indeed the virulent Rz type bacteria
have both higher growth rate and population size compared with both R types. This result is consistent with other
findings showing a positive correlation between virulence
and growth rate in bacteria (Chesbro et al., 1969; West &
Buckling, 2003; Pulkkinen et al., 2010) (but see Sturm
et al., 2011; Ketola et al., 2013 for opposite results). In
F. columnare, the biofilm formation was higher in phageinduced R and Rz morphotypes when compared to starved
R type. The majority of bacteria live in biofilms, especially
in aquatic communities, and the biofilms protect bacteria
from disturbances such as predation (Beveridge et al.,
1997; Dunne, 2002). Although beneficial for bacteria, biofilms are problematic for pathogen eradication as bacteria
living in biofilms often persist on surfaces and are resistant
FEMS Microbiol Ecol 89 (2014) 553–562
to treatments (Dunne, 2002). In addition to environmental
transmission via natural waters, biofilm formation may be
one reason why F. columnare causes frequent outbreaks in
rearing units. In natural conditions, bacteria living in biofilms are expected to be especially vulnerable to amoeba
predation, whereas ciliate predators prey mostly upon
planktonic bacteria (Rodrıguez-Zaragoza, 1994; Molmeret
et al., 2005). In our study, the phage-resistant R type
formed significantly more biofilm when exposed to amoeba
predation than the other colony types, but in the presence
of ciliates biofilm-forming ability between the morphotypes
did not differ.
When measured in free water, the phage-induced R
type had significantly lower resistance against protozoan
predation than the Rz. This was confirmed in the co-culture test for strains B245 and B67, where ciliates could
more efficiently exploit the phage-resistant R type of
F. columnare as a nutrient source than the Rz type
(Fig. 4). In exponential phase of the bacterial growth, the
population decrease by predation can be compensated by
rapid bacterial growth, and it could be speculated if the
faster growth of the Rz type gives an overestimation
of protozoan resistance. In our experiment, however,
the predators were added to the bacterial culture after the
exponential phase (c. 50 h after inoculation), when the
majority of nutrients have already been converted to
bacterial biomass. In this phase, difference in growth rate
would have little effect on the estimation of predation
resistance because of nutrient depletion of the medium
prevents rapid compensatory growth.
Our results indicate that protozoan predation may be
an important factor selecting against the phage-induced
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
J. Zhang et al.
560
rough type in aquatic food webs. Even though F. columnare and its phages co-occur in aquaculture (Laanto et al.,
2011), the trade-off between phage susceptibility and virulence would suggest strong selection also for the presence of the phage-resistant R type. If, however, predators
preferably feed on the phage-resistant type, the benefits of
being rhizoid could be higher. Ciliates are generally found
in aquatic systems (Beaver & Crisman, 1989; Finlay &
Esteban, 1998). Farming units in freshwater aquaculture
can maintain high numbers of ciliate protozoans, many
of which feed mainly on bacteria and other organic particles in the water (e.g. fish ectoparasitic trichonomids,
Apiosoma sp., and Epistylis sp.; (Buchmann et al., 1995;
Rintamaki-Kinnunen & Valtonen, 1997), possibly selecting against the R type. Interactions between predatory
protozoans and F. columnare have not, to our knowledge,
been previously studied, and the effect of ciliates on the
fish farming environment can only be speculated. Furthermore, it is possible that the R phenotype in nature
enters into a viable but nonculturable state explaining (in
addition to the benefits of Rz type growth) why the R
type is rarely isolated. However, this seems unlikely, as a
nonculturable state has not been yet demonstrated in
F. columnare (Arias et al., 2012), and in our previous
study, both the ancestral Rz and its derivative starved R
type remained culturable under prolonged starvation
(Sundberg et al., 2014).
Based on our results, it seems that the Rz type has a
higher fitness due to higher virulence, higher outside-host
growth rate and yield, and higher resistance to protozoan
predation. This suggests that the R morphotypes are
selected only in extreme conditions, and the potential for
morphotype changing could be linked to very rare occasions, such as prolonged starvation or high virus load. If
the maintenance of ability to change morphotype is cheap
in evolutionary currency, the ability to express the R
morphotype can persist even with the strong positive
selection for the Rz morphotype. In general, it seems that
across species, the costs of this kind of morphological
changes, for example in the case of phenotypic plasticity,
are very rarely shown or negligible (reviewed in DeWitt &
Schneider, 2004). If this is the case also in F. columnare,
the persistence of the rare R morphotype becomes far less
paradoxical.
In F. columnare, the mechanism behind the change
from Rz to R is not known, but both phase variation and
bet-hedging have been suggested (Kunttu et al., 2009a;
Laanto et al., 2012; Sundberg et al., 2014). In general,
bacterial phenotypic variation may arise through different
mechanisms that are based on different strategies to cope
with alternating environments. Bacteria may induce phenotypic variation by sensing environmental cues and
changing their gene expression accordingly (L
opez et al.,
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
2009). In contrast to such inducible variation, bacteria
can hedge their bets by stochastic gene expression (Thattai & van Oudenaarden, 2004). Bet-hedging can also
involve several other genetic mechanisms such as on/off
switches and epigenetic regulation (Veening et al., 2008;
Be’er et al., 2011). Regardless of the underlying mechanisms, phenotypic variation is visible even in homogenous laboratory environments where genetically similar
individuals can differentiate into phenotypically distinct
populations (Elowitz et al., 2002; Dubnau & Losick, 2006;
Davidson & Surette, 2008). In future, the mechanisms
underlying phenotypic change in F. columnare may be
revealed by genetic studies.
To summarise, it seems that the virulent rhizoid morphotype of F. columnare has a higher fitness in both
inside-host and outside-host environment, although it is
susceptible for phage infection. Antagonistic interactions
between bacteria, phages and protozoa are likely to be
even more complex under natural conditions, but our
study is the first step to understand the association
between F. columnare and its protozoan predators.
Acknowledgements
This work was supported by the Finnish Centre of Excellence Program of the Academy of Finland; the CoE in
Biological Interactions 2012–2017 (#252411), by grants
#272995 (L.-R.S.) and #1255572 (J.L.), from the Academy
of Finland, grant from the Finnish Cultural Foundation
(J.Z.) as well as a grant from the Maj and Tor Nessling
Foundation (J.K.H.B. and H.K.). We would like to thank
Prof. Angus Buckling and Drs Anna-Liisa Laine and
Swanne Gordon for comments, Dr Katja Pulkkinen, Kaisa
Suisto, Irene Helkala and Petri Papponen for assistance in
laboratory. The authors declare no conflict of interest.
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