Download Diel cell cycle analysis of Heterosigma akashiwo (Raphidophyceae

GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012
Diel cell cycle analysis of Heterosigma akashiwo
(Raphidophyceae) with special reference to
vertical migration behavior
Juyun Lee1, Myung-Soo Han2* and Man Chang1*
1
Marine Ecosystem Research Division, Korea Institute of Ocean Science and Technology (KIOST), 787, Haeanro, Ansan 426-744,
Korea
2
Department of Life Science, Hanyang University, 133-791 Seoul, Korea
*
Corresponding author
E-mail: [email protected]
Tel: +82-31-400-6468; Fax: +82-31-400-6495
been widely applied in the eco-physiological study of
algal plankton (Li 2002; Marie et al. 1997; Olson et al.
1985; Olson et al. 2003). One useful application of
flow cytometry is for diel cell cycle analysis (Marie et
al. 1997) including changes that occur during a full
light-dark cycle. Cells of Amphidinium operculatum
divide at night before the transition from dark to light
(Leighfield and Van Dolah 2001), while Karenia brevis
cells start to divide 2 h before the dark-light transition,
and continue 6 h after the transition (Van Dolah et al.
2008). Kohata and Watanabe (1986) investigated the
changes in H. akashiwo cell size, which indirectly
equates to cell division. Based on that information,
they hypothesized that the cell division occurred near
the dark-light transition in the laboratory. They also
found that the decrease in cell size and upward
migration occurred simultaneously in the morning,
suggesting synchronous division with the vertical
migration.
Generally, the cell cycle is composed of 4
phases with respect to the DNA content of each cell.
This phase is preceded by G1-phase which is the first
gap stage before the DNA synthesis which named Sphase. After S-phase, The G2-phase is the second gap,
between the S-phase and mitosis which named Mphase of cell cycle (for review see Chisholm 1981).
Regardless of whether diel cell cycle is synchronous or
not, Smith and Dendy (1962) showed that the specific
growth rate can be calculated from the fraction and the
duration of the M-phased population. And McDuff and
Chisholm (1982) established an equation for estimating
the daily mean growth rate in phytoplankton. Diel cell
cycle analysis has also been applied to measure the
growth rate of oceanic picophytoplankton (Campbell
and Carpenter 1986). Since this analysis does not
require incubation, it is appropriate for the
measurement of microalgae growth when vertical
1.
INTRODUCTION
The
marine
alga,
Heterosigma
akashiwo(Raphidophyceae), is known to frequently
form blooms in temperate coastal waters throughout
the world (Horner et al. 1997; Smayda 1998; Taylor
and Haigh 1993; Taylor and Horner 1994). H.
akashiwo populations usually develop rapidly in early
summer (Imai 2000; Shikata et al. 2007), forming
dense blooms in the upper water column at or near the
surface (Handy et al. 2005; Honjo 1993; Yamochi and
Abe 1984). Its blooms can cause massive fish kills
through the production of chemicals such as
neurotoxins, cardiotoxins, and reactive oxygen (Endo
et al. 1992; Khan et al. 1997). In addition to the
significant impacts to fish populations, algal blooms
can cause a decrease in plankton biodiversity and are
sometimes accompanied by low oxygen in deeper
water (Tsutsumi 2006) which may kill shellfish and
other invertebrates. Given these potential impacts, a
better understanding of the conditions leading to an
algal bloom, and the bloom process itself, are
important in helping to protect coastal ecosystems.
The accumulation of algae on the surface
water is caused by biological processes such as growth
and vertical migration (Bearon et al. 2006) and/or
physical processes such as wind-driven advection (Hill
1991). These events should be considered separately in
the analysis of the bloom-development process. Since
cells of H. akashiwo can accumulate in “nutrientdepleted” surface waters during the daytime and move
downward to “nutrient-rich” bottom waters at night
(Watanabe et al. 1983; Watanabe et al. 1988), growth
rate measurement for this species is quite difficult
when employing in situ incubation experiments using
bottles.
To study the biological processes of a H.
akashiwo bloom, we used flow cytometry which has
DOI: 10.5176/2251-3140_2.1.19
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© 2012 GSTF
GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012
migration is a factor. Recently, this technique was also
applied to the dinoflagellate Karenia brevis (formerly
Gymnodinium breve), which is the cause of toxic red
tides (Van Dolah and Leighfield 1999; Van Dolah et al.
2008). Van Dolah et al. (2008) investigated the growth
rate of K. brevis in natural environments over five
years and found that the growth rate varied from 0.17
to 0.55 day-1; however, the timing of cell division was
apparently not dependent on the growth rate. For H.
akshiwo, this cell cycle analysis has not yet been
applied to natural populations.
The cell membrane of H. akashiwo is
generally not well preserved by fixation probably due
to osmotic changes. Moreover, the cells sometimes
form aggregates after fixation. Therefore, it is hard to
apply flow cytometry for H. akashiwo. Recently, we
found that a fixative using sodium cacodylate works
well with H. akashiwo cells, and have used it (Lee et al.
2012), in the present study to investigate the diel cell
cycle with flow cytometry. This study was thus carried
out to clarify the cell cycle and pattern of vertical
migration to enhance the bloom formation and
furthermore the relation between diel vertical migration
and the cell cycle.
analysis was performed on a FACS Calibur (BD
Biosciences, San Jose, CA) equipped with an aircooled argon laser (488 nm, 150 mW). Histograms of
the relative DNA amount were analyzed using the
Modfit program (Verity Software House, Inc., Palo
Alto, CA, USA) to quantify the percentage of cells in
each of the growth stages (G1, S, G2+M). At least
10,000 cells were analyzed.
Using the sum of the S- and G2+M-phase
fractions, the daily mean specific growth rate, µS+G2M,
was calculated according to the following equation
from Chang and Carpenter (1991);
Where (tS)i is the sampling interval of sample i,
measured in hours. We used the combined S, G2, and
M-phases as the terminal event, and measured the
duration term (Tx) by monitoring the diel change of the
S- and G2+M-phase fractions [fs(t) and fG2M(t)].
2.4. Counting
H. akashiwo cells were fixed with Lugol’s
solution for counting. An aliquot of the preserved
sample was placed in a Sedgewick-Rafter counting
chamber and cells were enumerated under a light
microscope (Axioplan, ZEISS, Germany).
2. METHODS
2.1. Cultures
A cyst of H. akashiwo was isolated with a
capillary pipette on June 2005 from Masan Bay,
located in the southern part of Korea. The cyst was
incubated in F/2 media (Guillard and Ryther 1962) at
20oC under a 12:12 h light:dark cycle. A unialgal
culture established from the germinated cyst was
tentatively named as strain HYM06HA (Ki and Han
2007). The culture was maintained under the same
conditions described above. The light intensity under
cool white bulbs was 100 µmol photons m-2 s-1. All
experimental cultures were sampled during the
logarithmic phase.
2.5. Laboratory experiment
We conducted laboratory studies to clarify the
relationship between the diel cell cycle and vertical
migration of H. akashiwo. Synchronized cells were
inoculated to flasks for Experiment 1 or cylinders for
Experiment 2, each containing 2 L of F/2 media at a
final density of 1 x 104 cells mL-1. These cells were
incubated at 20oC under 100 µmol photons m-2 s-1 with
a 12:12 h light:dark cycle. Experiments were initiated
after 24 h of stabilization.
Experiment 1: Diel phasing of the cell cycle
progression
The amount of DNA in cells was monitored
with a flow cytometer every 3 h for 48 h. To confirm
that cell division was occurring, cell density was also
monitored as described above. Culture flasks
containing H. akashiwo were mixed gently and 25 ml
were removed for cell cycle analysis and 1 ml for
measurement of cell density. Since culture flasks were
mixed, we could not evaluate vertical migration in this
experiment; however, we analyzed the diel cell cycle
timing and cell division. The experiment was
conducted in triplicate using three flasks.
2.2. Cell fixation
Cells of H. akashiwo were fixed with
paraformaldehyde (PFA) at a final concentration of
0.1%. The fixative contains sodium cacodylate (pH 7.4,
0.2 M, final concentration) and sucrose (0.1 M, final
concentration) and F-68 (0.1%, final concentration).
2.3. Diel cell cycle analysis and the calculation of the
growth rate
Fixed cells were washed with deionized water
(DW) and ethanol, and suspended with phosphatebuffered saline (PBS, pH 7.5). We confirmed that
ethanol treatment removed chlorophyll, which
interferes with propidium iodide (PI) fluorescence used
in the flow cytometric analysis. Cells treated with
RNase (final concentration 20 mg mL-1, Sigma, USA)
were incubated at 35oC for 30 min then were stained
with 20 µg mL-1 PI (Sigma, USA). Diel cell cycle
Experiment 2: Diel phasing of cell cycle progression
and vertical migration
H. akashiwo cells were incubated using 2 L
cylinders (10 cm in diameter 40 cm in height) under
the same conditions as Experiment 1. In the
preliminary experiment, we found that cells migrated
to the surface from CT0 (circadian time, 0 h) at least
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GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012
until CT9, and to the bottom from CT12 until CT21.
We collected samples every 6 h from CT21 without
mixing. For the diel cell cycle analysis, samples were
taken from the surface during the day and from the
bottom during the night. To confirm the diel vertical
migration, each surface and bottom sample was
measured for in vivo chlorophyll fluorescence using a
10-AU fluorometer (Turner Designs, Sunnyvale, CA,
USA). The experiment was completed in triplicate
using three cylinders after 24 h of stabilization.
day as occurring from CT21 to the following day
CT21. The cell density of H. akashiwo increased from
1.6 x 104 cells mL-1 to 4.8 x 104 cells mL-1 after 48 h of
incubation (Figure 1. A). The cell density increased
from CT21 to CT0 on the first day and from CT18 on
the first day to CT6 on the second day. On the second
day, cell density increased again from CT18.
DNA histograms of H. akashiwo cells are
shown in Figure 1. B. A distinct G2/M peak was found
in the later dark period (CT18, CT21). These peaks
diminished in the light period, indicating cell division.
Coefficient of variation (CV) values for these
histograms ranged between 6.5% and 17.3% with a
mean of 10.8%. G1-phase cells increased from CT15
on the first day, reaching a peak (90%) at CT3 on the
second day (Figure 1. C). DNA synthesis began in the
early light period; the percentage of cells in the Sphase increased from CT0 on the first day and from
CT3 on the second day. These values reached their
daily maximum at CT15 and CT18 on the first and
second days, respectively. As the percentage of S-phase
cells dropped, the percentage of G2+M-phase cells
increased. The G2+M-phase cells reached a peak at
CT21 on all days. G2+M-phase cells shifted to the G1phase from CT21 to CT6, which coincided with cell
division (Figures, 1. A and C).
The growth rate based on the diel cell cycle
analysis was 0.34 d-1 for the first day and 0.23 d-1 for
the second day. The reduced growth rate observed
during the second day was probably due to a lower
fraction of cells in the S-phase (Figure 1. C).
To clarify the relation between vertical
migration pattern and cell division in the laboratory, we
observed the concentration of chlorophyll fluorescence
and the diel cell cycle. High chlorophyll fluorescence
from H. akashiwo cells was observed at the surface
during the light period (CT3, CT9) and at the bottom
during the dark period (CT15, CT21, Figure 2. A).
Percentages of fluorescence in the bottom layers
increased from 13.4% at CT3 to 20.1% at CT9 on the
first day and from 17.9% to 19.7% on the second day,
suggesting that a downward migration began during
the late light period.
The diel cell cycle pattern followed by
vertical migration was quite similar to those observed
in the previous experiment (Figure 2. B). When the
percentage of G1 cells was maximized, the cells
formed a patch at the surface (CT3, CT9, Figures, 2. A
and B), and the percentage of the G1-phase cells
decreased from CT3 (Figure 2. B). The percentage of
S-phase cells began to rise from CT9, reaching a peak
at CT15 on both days. When the cells started to migrate
downward (Figure 2. A), the percentage of S-phase
cells also started to increase (Figure 2. B). The
percentage of S-phase cells increased from 5.9% at
CT3 to 19.2% at CT9 during the first day and from
4.6% at CT3 to 12.1% at CT9 on the second day. When
the cells migrated to the bottom, the percentage of S-
Experiment 3, Microscopic observation of dividing
cells
We sampled H. akashiwo cells from the surface
at CT3 when they formed a patch on the surface water.
This method was used for Experiment 2. The cells
were stained with 4’-6-diamidino-2-phenylindole
(DAPI) at a final concentration of 1 µg mL-1. Stained
cells were observed under UV excitation with an
epifluorescence microscope (Axioplan, ZEISS,
Germany) at 400Х magnification. Micrographs were
also taken with a CCD camera (Qincom fast,
Qimaging, Canada).
2.6. Field observations
Field observations were performed in Masan
Bay (35o 12’ 12.33’’, 128o 35’ 03.95’’), when H.
akashiwo formed a dense bloom (ca. 2.4 x 104 cells
mL-1) from 20 to 21 June 2007. Vertical water
temperature measurements were made with a portable
(Model U-10, HORIBA, Japan) at 0, 1, 2, 3, and 4 m.
Photosynthetically available radiation was measured
vertically with a quantum sensor (Li-250, Li-Cor,
Lincoln, NE, USA). Water samples were taken every 4
h from 1900 h on 20 June to 2300 h on 21 June from 0,
1.5 and 3 m with a 3-L Van-Dorn sampler. The water
samples were fixed immediately with the fixative
mixture as described above. All samples were packed
on dry ice and transported to the laboratory, where they
were stored in a freezer (-70oC) until the analysis. For
the enumeration of H. akashiwo cells, the sample was
fixed with Lugol’s solution at a final concentration of
0.5%. To determine the concentration of chlorophyll a
(Chl. a) and nutrients, water samples were collected at
1900 h on 20 June and 1500 h on 21 June. A portion
(250 ml) of the water sample was filtered with a GF/F
filter (Whatman, 47 mm diameter) and the Chl. a
concentrations were determined after extraction with
90% acetone (APHA 1995). Each of the nutrient
samples (NH4-N, NO3-N+NO2-N and PO4-P) was
prepared from 50 ml of filtered water through GF/F
filters; the nutrient concentrations were determined
following standard methods (APHA 1995).
3.RESULTS
3.1. Laboratory experiments
In this study, all laboratory experiments
were started at CT21. Therefore, we regarded the first
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GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012
phase cells maximized and, when the cells remained at
the bottom, the percentage of G2+M-phase cells
increased from CT15. The percentage of G2+M-phase
cells reached a maximum at CT21, and decreased until
CT9. This shift of G2+M-phase cells into G1-phase
occurred simultaneously with upward migration.
To summarize, in the upward migration and
shift from M-phase to G1-phase, we observed the
nucleus in the cells which were sampled from the
surface. Epifluorescence-microscopic observation of
cells at CT3 revealed that many cells had a binary
nucleus in one cell (Figure 7). It clearly demonstrates
that these cells were in the M-phase.
values ranged between 13.1% and 16.3% with a mean
of 14.5%. The percentage of G1-phase cells decreased
from 1900 h on 20 June to 0300 h on 21 June and from
1500 h to 2300 h on 21 June (Figure 5. B). Then, it
increased until 1500 h on 21 June. The percentage of
the G1-phase cells reached its peak at 1500 h on 21
June. The percentage of S-phase cells increased from
1900 h to 2300 h on 20 June and from 1500 h to 23:00
on 21 June. The percentage of G2+M-phase cells
increased from 1900 h on 20 June to 0300 h on 21 June
and decreased until 1500 h on 21 June. The percentage
of G2+M-phase cells reached a maximum of 14.7% at
2300 h on 20 June.
The growth rate based on the diel cell cycle
analysis was 0.34 d-1 during the investigated period.
This value was higher than the growth rates in the
laboratory experiment, probably due to a higher
number of S-phase cells (Figure 5. B) in the natural sea
water.
3.2. Field observation
The concentrations of NH4-N were higher at
the surface (3.1 and 6.7 µM) than those at the bottom
(1.5 and 2.2 µM, Table 1). The highest concentration of
NO3-N +NO2-N was 2.5 µM and the lowest
concentration was 2.2 µM (Table 1). There was no
trend in its vertical distribution. The concentrations of
PO4-P, ranging between 0.6 and 3.1 µM, also showed a
similar trend to those of NH4-N.
High chl. a concentrations were detected at
the surface (306.9 and 466.8 µg L-1, Table 1).
Microscopic observation revealed that more than 95%
of phytoplankton cells were H. akashiwo. Other
phytoplankton taxa present at less than 5% of the total
phytoplankton cell concentrations were Cosinodiscus
sp. and Thalassiosira decipiens (Bacillariophyceae),
Eutreptiella
gymnastica
(Euglenophyceae),
Gymnodinium sp. and Prorocentrum triestinum
(Dinophyceae).
The temperature at the surface ranged
between 22.5 and 25.5oC during the sample period
(Figure 4. A). Dawn and dusk occurred, respectively, at
0511 h and 1943 h on 21 June. Light intensity rapidly
attenuated with depth from 685.4 µmol photons m-2 s-1
at the surface to 6.8 µmol photons m-2 s-1 at 1m (Figure
4. B) at 1100 h. At that time, the attenuation coefficient
was 3.08 m-1, and Z1%, the depth where light was
attenuated to 1% of the surface, was 1.50 m.
The cell density of H. akashiwo ranged from
0.3 to 13.3 x 104 cells mL-1 (Figure 4. C). H. akashiwo
cells dispersed vertically from 1900 h on June 20 when
sampling was started. The cells migrated to the surface
from 0300 h before dawn and formed a dense patch at
the surface during the day to a maximum of 13.3 x 104
cells mL-1. At the same time, the lowest values were
recorded at 4 m depth. Most cells were distributed
above the Z1% during daytime (Figures, 4. B and C).
These cells started to descend around 1500 h, before
the sunset. At 1900 h, a patch moving downward was
observed at 3 m depth.
The DNA histograms of the natural H.
akashiwo population are shown in Figure 5. A. Overall,
peaks were broad compared to those observed in the
strain in the laboratory experiment (Figure 1. B). CV
4.DISCUSSION
The rapid appearance of H. akashiwo blooms
in coastal environments (Bearon et al. 2006; Handy et
al. 2005; Hard et al. 2000; Honjo 1993; Odebrecht and
Abreu 1995; Rensel et al. 1989; Smayda 1998; Taylor
and Haigh 1993; Yamochi 1987) is due to physical and
biological processes. Among them, it is widely
accepted that the higher growth rate of H. akashiwo
compared to other raphidophytes and dinoflagellates is
the reason for the rapid appearances. In addition to
rapid growth of H. akashiwo, wind sometimes
concentrates H. akashiwo cells near the coast. Thus, in
situ growth should be separately evaluated from such
wind driven processes in analyzing the bloom
development.
The cell density of H. akashiwo drastically
increased from 0300 h (13.1 x 103 cells mL-1) to 1100 h
(133.0 x 103 cells mL-1) (Figure 4. C). From 0700 h
(118.0 x 103) to 1100 h (133.0 x 103 cells mL-1), cell
density at the surface increased again (Figure 4. C). At
the same time, percentage of G2/M-phase decreased
from 11.3% to 2.6% (Figure 5. B). G2/M-phase cell
numbers at 0700 h and 1100 h were, respectively, 13.3
x 103 and 3.5 x 103 cells mL-1. Therefore, the increase
in cell density by cell division between these two times
accounted for 9.8 x 103 cells mL-1, while apparent
increase was 15.0 x 103 cells mL-1. This means that
65.3% of the apparent increase in cell concentrations
was explained by cell division. In contrast, from 0300
h to 0700 h the change in percentage of G2/M-phase
was less obvious (14.3% to 11.3%), while increase in
cell density was more significant from 13.1 x 103 to
118.0 x 103 cells mL-1. Thus, the cause for the apparent
increase in cell density at the surface shifted from
vertical migration to the cell division.
High cell density of H. akashiwo (Figure 4.
C) and chlorophyll concentration (Table 1) in Masan
Bay indicated that our investigation was carried out
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GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012
around the time of at the peak of the bloom. Generally,
the growth rate of microalgae becomes low in late
exponential and stationary phases, as described by Pan
and Cembella (1998). However, we detected positive
growth (0.34 day-1) of H. akashiwo in the dense patch,
although the rate did not reach a maximum rate
previously recorded (1.14 day-1, Yamatogi et al. 2006).
This positive growth at the peak of the bloom suggests
that losses to grazing and cell dispersion to the adjacent
waters may be substantial. Recently, Jeong et al.
(2005) pointed out the significance of heterotrophic
dinoflagellates as the grazer for H. akashiwo. In
analyzing population dynamics, the loss process
requires more attention.
In the laboratory experiment, G1-phase cells
started to increase when they migrated upward
(Figures. 2. A and B). Cells undergoing mitosis were
found at the surface at CT3 (Figure 3). Moreover, the
cell density increased between CT18 and CT6 (Figure
1. A). Indeed, in Masan Bay, H. akashiwo cells
migrated upward when G2+M-phased cell decreased
before the sunrise (Figures. 4. C and 5. B). In a
previous laboratory study, enlarged H. akashiwo cells
migrated downward and newly-divided cells started to
migrate upwards with cell division and upward
migration at CT21 (Kohata and Watanabe 1986). Thus,
it is clear that cell division occurs simultaneously with
upward migration in H. akashiwo.
The overlapping of vertical migration and
cell division has been reported for studies of other
dinoflagellate species (Table 2). For example, cell
division of Prorocentrum micans started at CT2 (Pan
and Cembella 1998), while upward migration of this
species started at CT22 (Kamykowski 1981). In
Lingulodinium polyedrum, cell division started 2 h
before dawn in the laboratory (Homma and Hasting
1989). Upward migration was also found to have
started at dawn in Baja California (Blasco 1978). In the
case of Karenia brevis, cell division appeared to be
completed before dawn (Van Dolah and Leighfield
1999; Van Dolah et al. 2008), and upward migration
also started before dawn (Van Dolah et al. 2008). It has
been demonstrated that the diel cell cycle progression
and vertical migration behavior are under the control of
circadian rhythm (Ault 2000; Brunelle et al. 2007; Van
Dolah and Leighfield 1999; Weiler and Karl 1979).
The variation in these timings can be a factor to
determine phytoplankton succession.
Although rhythms in vertical migration and
diel cell cycle seem to be synchronized, it is not known
whether there is interaction between the two. There
was no difference in the percentage of different cell
phase in K. brevis between depths (Van Dolah and
Leighfield 1999; Van Dolah et al. 2008). Thus, Van
Dolah et al. (2008) concluded that cell cycles of K.
brevis occur independently of vertical migration
behavior (Van Dolah et al. 2008). However, vertical
positioning of this dinoflagellate species was affected
by the internal cellular condition (Kamykowski et al.
1998). For H. akashiwo, we observed that vertical
migration became less obvious when the culture
entered into a stationary phase (data not shown). In the
present study, most cells of H. akashiwo were
concentrated at the surface; hence, we could not
analyze cells in the sub-surface layer. It should be
further determined in laboratory experiment.
The height of the cylinder used for our
vertical migration experiment was no more than 40 cm,
while of > 1 m height mesocosms were employed
previously to study vertical migration behavior (Dublin
et al. 2006; Kamykowski et al. 1998; Kohata and
Watanabe 1986; Watanabe et al. 1988). In the vertical
migration experiment, most cells accumulated at the
surface during the light period, and the thickness of the
H. akashiwo cell patches was less than 5 (data not
shown). During the dark period, cells concentrated on
the bottom of the cylinders. Thus, we believe that
vertical migration of H .akashiwo observed in the
present study well presented their original behavior.
H. akashiwo grow more rapidly (1.14 day-1)
than the dinoflagellates such as Karenia brevis (0.69
day-1). Honjo (1993) observed that a cell of H.
akashiwo divided up to three times during one night.
We showed that the cell cycle progression of H.
akashiwo seemed to be synchronized. However, both
the laboratory experiments and the field study did not
show more than one division per day. High initial cell
density (1 x 104 cells mL-1) in the laboratory
experiments is one possible explanation for the lower
growth rate (< one division per day). It is of interest
whether cells can divide more than one time during a
night without accumulation of carbon by
photosynthesis. This is a topic requiring further
investigation.
One of the potentially important ecological
advantages of vertical migration behavior is nutrient
uptake from deeper layers during the night. However,
our data did not support this idea; concentrations of
NO3-N and PO4-P were higher in the surface layer than
the bottom (Table 1). This leads to a tentative
conclusion that in the bay, vertical migration behavior
does not give such an advantage for H. akashiwo over
other competitive phytoplankton species. Another
explanation for the lower nutrient concentrations at the
bottom of the bay is a result of nutrient uptake by H.
akashiwo cells during night time. Since our
investigation was carried out at the peak of the bloom,
it is possible that these nutrients had been already
depleted. Unfortunately, we do not have conclusive
date to decide this issue. It is certain, however, that
nutrient dynamics is one of the important
environmental factors for the development of H.
akashiwo population in the bay.
In conclusion, we demonstrated a diel cell
cycle of H. akashiwo for both a cultured strain and
natural populations. Upward migration and cell
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GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012
division appeared to occur simultaneously. The
increase in cell density at the surface in Masan Bay
was partly due to cell division, although bloom had
already developed. To more accurately analyze the
bloom developmental process, the present study
emphasizes that population growth should be
separately evaluated from horizontal advection and
vertical migration. Since the growth rate of this
harmful alga is high compared to that of other
dinoflagellates, the cell cycle behavior of H. akashiwo
may differ from that of dinoflagellates. Our study
shows that diel cell cycle analysis is a useful tool to
clarify the bloom development process, and the method
will provides us with important new insights.
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Acknowledgements
This work was supported by the Korea
Institute of Ocean Science and Technology Research
Fund (#PE98747). Last, but not the least, the authors
thank the two anonymous referees for their helpful
critical comments.
Horner RA, Garrison DL and Plumley FG 1997 Harmful algal
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GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012
Figure legends
Figure 1 (A) The growth curve of H. akashiwo strain (HYM06HA) based on the cell counting. (B) Cell cycle
progression of H. akashiwo strain HYM06HA. Histograms of DNA fluorescence of cell determined by flow
cytometry. Experiment was carried out in triplicate, and results from one of the representative flask are
shown. (C) Ratio of cell phase (%) which was analyzed of each cell phase from the laboratory experiment. All
experiments were conducted over 48 h with 3 h intervals. ■: G1, ●: G2+M, ▲: S, CT: circadian time.
Figure 2 (A) The daily fluctuation of percentages of cell migration based on the Chl. a concentration. Each sample was
collected at the top and bottom over 48 h with 6 h intervals. (B) Ratio of cell phase (%) from the laboratory
experiment over 48 h. The cells of H. akashiwo collected from the patch were analyzed for their diel cell
cycle at 6 h intervals. ······ : G1, ―: G2+M, ---: S, □: Dark period, ●: Light period, CT: circadian time.
Figure 3 Binary nucleus in one cell of H. akashiwo strain HYM06HA, which is stained with DAPI. Chlorophyll showed
red auto-fluorescence with the DAPI-specific filter set. These cells in two figures were collected at CT2 from
the top of the cylinder.
Figure 4 Changes in the vertical profiles of water temperature (oC, panel A), light intensity (µmol photons m-2 s-1, panel
C) and vertical distribution of H. akashiwo cells (x 103 cells mL-1) in Masan Bay from 20 to 21 Jun 2007.
Figure 5 (A) Cell cycle progression of H. akashiwo population in Masan Bay. Histograms of DNA fluorescence of cell
determined by flow cytometry are shown. (B) Ratio of cell phase (%) from the field experiment over 28 h. The
daily fluctuation of each cell phase was analyzed at 4 h intervals. ■: G1, ○: S, ▲: G2+M.
Figure 1
37
© 2012 GSTF
GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012
Figure
Figure 3
38
© 2012 GSTF
GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012
Figure 4
39
© 2012 GSTF
GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012
Figure 5
Table 1. The vertical concentrations of nutrients (NH4, NO3+NO2, PO4) (µM) and Chl. a (µg L-1) in Masan Bay on 20 and 21 June
2007.
Date
20 June
2007
Time Depth
19:00
0m
1.5m
3m
21 June
2007
15:00
0m
1.5m
3m
NH4
(µM)
6.7
NO3+NO2
(µM)
2.5
3.1
1.5
2.3
2.4
3.1
2.4
0.0
2.2
2.2
2.3
40
PO4
(µM)
Chl.a
(µg L-1)
1.8
466.8
1.3
1.0
68.9
111.8
3.1
306.9
0.6
0.9
18.9
59.1
© 2012 GSTF
GSTF International Journal of BioSciences (JBio) Vol.2 No.1, December 2012
Table 2. Beginning times (circadian time, CT or local time) of cell division (CD) and upward migration (VM) for various species of
flagellates.
Species
Location
L:D
Beginning times
cycle
CD
VM
Method
Reference
Dinophyceae
Karenia brevis
Lingulodinium polyedrum
Laboratory
16:08
CT16CT22
Gulf of
16:00-
Mexico
21:00
Laboratory
12:12
Laboratory
12:12
Prorocentrum minimum
1999
Van Dolah et al. 2008
FL
Komykowski et al. 1998
FCM
Homma and Hasting 1989
LM
Blasco1978
FCM
Pan and Cembella 1998
FL
Kamykowski 1981
FCM
Pan and Cembella 1998
CT2
LM
Watanabe et al. 1982
CT22
CT0
California
Van Dolah and Leighfield
FCM
CT2
Baja
Prorocentrum micans
FCM
Laboratory
12:12
CT2
Laboratory
12:12
Laboratory
12:12
Laboratory
12:12
Laboratory
12:12
CT21
CT21
LM
Kohata and Watanabe 1986
Laboratory
12:12
CT21
CT21
FCM
This study
Masan Bay
14:10
CT21
CT21
FCM
This study
CT22
CT4
Raphidopyceae
Heterosigma akasniwo
*FCM: flow cytometry
*FL: fluorometry (in vivo
fluorescence)
*LM: light microscopy
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© 2012 GSTF