Bloom formation process of Noctiluca scintillans
in the coastal waters of Sagami Bay, Japan
相模湾沿岸域における Noctiluca scintillans の大増殖を引き起こすメカニズムについて
04D5504
宮口 英夫
指導教員 戸田龍樹
SYNOPSIS
相模湾北西部に位置する真鶴半島沿岸域において、1997 年 1 月から 2006 年 9 月までの間に、従属栄養性渦鞭毛藻 Noctiluca
scintillans(夜光虫)の大増殖についての調査を行った。周年変化については、3 月から増加し始め、4 月下旬から 5 月にかけて細
胞密度のピークを示した。また、N. scintillans の細胞密度は年ごとに大きく変動した。調査期間を通して細胞密度が最も高かった
のは、2000 年の 5 月下旬で約 7.5×105cells/m3 に達した。1997 年と 2000 年には、他の年に比べて細胞密度高く、1998 年は細胞
密度が低かった。また重回帰分析の結果、N. scintillans の細胞密度：D＝－0.14 R－0.20 Wd＋0.04 Chl＋0.12 B－0.70 (R：降雨
量、Wd：風向、Chl：Chl. a 濃度、B：ネット動物プランクトン生物量)の重回帰式 (P<0.01, 寄与率: 53%)が得られた。よって、 N.
scintillans の細胞密度の変動には、増殖や競争関係をもたらす水質・生物要因よりも、降雨量・風向といった気象要因が最も強く
影響を及ぼしていることが明らかになった。さらに、調査期間中の月ごとの平均値からの残差分析から、1997 年と 2000 年は、調
査期間を通して降雨量が少なく、春季の初めに南東寄りの風が頻繁に観測された年であった。一方、1998 年は、1 年を通して降
雨量が多く、春季には南西からの風が観測された。すなわち海表面付近に多く分布する N. scintillans は、降雨による海表面の撹
乱が起こらず、湾口から真鶴半島に向かう南東の風が吹くことにより、相模湾真鶴半島沿岸域において、集積されることが示唆さ
れた。実験室における培養実験から、細胞密度が高濃度条件下になると、遊走子が栄養細胞に無性的な 2 分裂を誘発・継続さ
せ、大増殖を引き起こすことが考えられている (Sato et al., 1998)。そこで、実際の現場海域で、遊走子の大増殖に対する貢献を
評価するために、N. scintillans の遊走子の定量法の確立・細胞密度の変動の追跡を行った。その結果、N. scintillans の大増殖
期の 4 月下旬から 5 月にかけて、N. scintillans の遊走子は 5.2 ×107 cells/m3 の最大細胞密度を示し、現場海水中には春から
夏にかけて遊走子が豊富に存在していることが明らかとなった。以上より、N. scintillans が大増殖引き起こすには、本研究によっ
て初めて提案された、以下に示す 3 段階の過程を経るメカニズムがあると考えられる。まず、第一段階として、夜光虫の成長を制
限している、環境要因が好適になり、夜光虫の成長速度が上昇し、細胞密度の増加が見られる。次に、第二段階 として、風、安
定した海表面などにより、海水の集積作用が起こり、夜光虫細胞が高濃度に集積される。次に、第三段階として、高濃度状態に
なった、夜光虫の栄養細胞は遊走子に刺激されることで、遊走子母細胞になることなく、二分裂を続けて行い、素早く増殖するこ
とが可能になる。現場海域において、夜光虫の大増殖に対する、海水の集積作用と遊走子の果たす役割が大きいことが示唆さ
れた。
Keywords: Noctiluca scintillans, Bloom, Accumulation, Rainfall, Wind, Zoospore
(Schaumann et al., 1988). In the same area, the southeasterly
winds from the German littoral towards the North Sea were
known to be dominant from April to July, and the accumulation
leading to the bloom of N. scintillans seemed not to occur
during these periods (Uhlig and Sahling, 1990). The
accumulation trigger is thought to be influenced by wind
direction and topography at each region including the condition
of surface seawater change by meteorological factors such as
rain, wind velocity, wind direction, tide and ocean current
(Smayda, 1997; Dela-Cruz et al., 2003). Therefore, not only are
the relationship between N. scintillans and hydrographic and
biological factors significant, but also meteorological factors
influencing the accumulation of the cells are crucial factors in
bloom formation. Blooms of N. scintillans have been reported
to correlate with environmental factors but the initial trigger of
the large-scale bloom formations have not specifically been
attributed to the any particular condition. The cause of the
large-scale bloom formation in coastal embayments is still
controversial.
The vegetative cell of N. scintillans is extremely large and
ranges between 100 and 2000 μm (Elbrächter and Qi, 1998). N.
scintillans has been known to reproduce by binary fission and
the formation of flagellate zoospore (Zingmark, 1970). The
process of binary fission starts with a reorganization of cell
organelles. During the formation of flagellate zoospores, the
nucleus in the vegetative cell migrates to the surface of the
zoospore-mother cell. The zoospore divide several times
INTRODUCTION
The large heterotrophic dinoflagellate Noctiluca scintillans is
one of the most common “red tide” organisms. N. scintillans is
a widespread organism and its blooms occur from spring to
summer in temperate and tropical neritic waters all over the
world (Elbrächter and Qi, 1998). N. scintillans is considered to
be a species harmful to fish since it has been responsible for
large-scale deaths of caged fish by clogging the gill (Smayda,
1997). N. scintillans produces a simple ichthyotoxin (NH3) and
causes O2 depletion of surface waters during bloom decay
(Smayda, 1997). The seasonality of N. scintillans bloom
formations has previously been proposed to be regulated by a
variety of environmental factors such as hydrographical and
biological factors.
Accumulation of buoyant cells caused by convergence of
surface seawater is suggested as one of the factors leading to N.
scintillans blooms (Elbrächter and Qi, 1998). At Tai Tam Bay
on the southern coast of Hong Kong, the southerly winds
following typhoons were responsible for concentrating the red
tide on beaches in July (Morton and Twentyman, 1971). In
Mirs Bay and Port Shelter around the northern region of Hong
Kong, the prevailing northeast monsoon winds promoted the
formation of a bloom of N. scintillans (Yin, 2003). In the
German Bight when the bloom of N. scintillans was observed
in August 1984, the northwesterly winds from the North Sea
towards the German littoral were considered important
1
without cytoplasmic fission and eventually up to 1000-2000
zoospores are formed from the one zoospore-mother cell in less
the 24 h (Uhlig and Sahling, 1982). Zingmark (1970) proposed
that the zoospores result from meiosis in a gametocyte mother
cell and that they represent isogametes which fuse. Zingmark
(1970) also suggested that the zoospore is haploid while the
vegetative cell is diploid. The resulting zygote was suggested to
give rise directly to a vegetative cell, but this was not
convincingly proven. Schnepf and Drebes (1993) proposed that
the zoospore is a microgamete whereas the macrogamete is
identical or very similar to a vegetative cell. Thus, the
reproduction of N. scintillans is still controversial and the
resting stages as with most dinoflagellate are so far not well
known. Recently, a new reproductive mechanism of N.
scintillans was proposed suggesting that the vegetative cell of
N.
scintillans
is
programmed
to
become
the
zoospore-mother-cell after a defined number of binary fissions
(Sato et al., 1998). Moreover, if the released zoospores from the
zoospore mother cells contact with and stimulate the vegetative
cell, the program is reset and the vegetative cells continue to
conduct binary fissions. This suggests that an intensive
multiplication of N. scintillans occurs in high-density
conditions of the cells, as the encounter rate between vegetative
cells and zoospore is high. In order to demonstrate the role of
zoospores in the life cycle of N. scintillans and to estimate
accurately the contribution of the zoospores to bloom
formations of N. scintillans, the study is firstly required to
establish a quantitative method in determining of the number of
N. scintillans zoospore in both laboratory experiments and field
studies.
Contrary to the vegetative cell of N. scintillans, the zoospore
is 10-15 μm in size and morphologically indistinguishable from
other algae and protozoa. Light or electron microscopy cell
counts are therefore not possible on environmental samples
because the cells cannot be positively identified. These
identification methods are not appropriate for long-term
monitoring and are time-consuming because microscopy
analysis needs a great deal of taxonomic experience to identify
the species. Because of the genetic diversity of different genera
and species, molecular techniques such as fluorescence in situ
hybridization (FISH) and polymerase chain reaction (PCR)
assays that use a genetic markers as criterion for identification
allow for the detection and quantification of microorganisms
including harmful algae (Bowers et al. 2000; Galluzzi et al.
2004; Sako et al. 2004; Hosoi-Tanabe and Sako, 2005). FISH
has known to be a rapid, precise and quantitative method that
might prove to be a useful tool (Miller and Scholin, 1996).
However, when the cells of targeted organism do not have a
hard cell wall like Alexandrium spp. and are broken easily by
chemical reagents or centrifugation, this FISH method might be
less useful because of the difficulty of obtaining intact cells. In
the case of fragile cells, the PCR assay may be better (Sako et
al. 2004). PCR assay is an important technique in several
studies. Real-time PCR offers all the advantages of
conventional PCR, such as high sensitivity and specificity, and
also allows for the quantification of PCR amplicon formation
during the exponential phase of the reaction. Even if real-time
PCR was originally developed for microbial ecology, it has
recently been applied to detect small-subunit rRNA in natural
harmful algae and for rapid detection in culture and
environmental water samples (Popel et al., 2003; Galluzzi et al.
2004; Hosoi-Tanabe and Sako, 2005). The purpose of this study
was to examine the relationship between the blooms of N.
scintillans and environmental factors, and to establish rapid
detection and quantification methods of N. scintillans zoospore
in order to resolve the complex processes that initiate and
sustain bloom formations. Our goal is to examine the trigger
mechanism of the large-scale bloom formation N. scintillans in
Sagami Bay.
MATERIALS AND METHODS
Field monitoring
Studies were conducted at three coastal stations, St. A, St. 40,
St.70, St. M, and St. G, near Manazuru Peninsula, Sagami Bay,
Japan. Sts. A, 40, 70 and M were selected to examine the
seasonal and annual variation of plankton including N.
scintillans as long-term stations for cooperative research of
Soka University and Yokohama National University. St. A lain
to the mouth of Manazuru Port (35゜09’49”N, 139゜10’33”E and
depth 5m) was chosen as a representative station of the coastal
area. Sts 40 (35゜09’30”N, 139゜09’25”E and depth 40m) and 70
(35゜09’30”N, 139゜09’25”E and depth 70m) were located in the
middle stations between coastal area and offshore region. St. M
was situated about 2km north off Manazuru Peninsula (35゜
09’30”N, 139゜09’25”E and depth 120m) and was located in the
off-shore environment. St. G was chosen to observe the
accumulation process of N. scintillans to the bloom forming. St.
G was located about 1km north off Manazuru Peninsula (35゜
09’30”N, 139゜09’25”E and depth 20m).
Water samples for analysis of temperature, salinity and
chlorophyll a and zooplankton samples for study of N.
scintillans were collected at all five stations. The cell
abundance of N. scintillans in zooplankton sample was counted
and the cell diameter of 100 randomly chosen cells was
determined under a stereo microscope. The cell volume was
calculated on the assumption that N. scintillans cells are
spherical (Hillebrand et al., 1999).
Meteorological data, rainfall (mm), wind velocity (m/s),
wind direction and daylight hours (h) were obtained by the
Japan Meteorological Business Support Center at the Odawara
Office, Kanagawa, Japan (35゜15’01”N, 139゜09’03”E).
Real-Time PCR standard curve construction
Matured zoospore-mother cells of N. scintillans were
transferred to the laboratory to a dish containing 0.22 μm
filtered seawater and washed three times to eliminate other
organisms. The matured zoospore-mother cells of were washed
three times by using 0.22 μm filtered seawater and were sorted
at 1, 5, 10, 20, 50, and 100 cells, respectively. The cells were
transferred to 1.5 mL Eppendorf tube containing 0.22
μm-filtered seawater and were incubated at 20 oC for 24 h. The
waters samples including released zoospores were fixed in 5 %
borate-buffered formaldehyde and in 5 % Lugol iodine,
respectively. The serial dilutions of the zoospore were prepared
as, the formaldehyde fixation series; F-(i): 10000 cells/mL,
F-(ii): 5000 cells/mL, F-(iii): 2500 cells/mL, F-(iv): 1250
cells/mL, F-(v): 625 cells/mL and F-(vi): 312 cells/mL, and
Lugol fixation series; L-(i): 12000 cells/mL, L-(ii): 6000
cells/mL, L-(iii): 3000 cells/mL, L-(iv): 1500 cells/mL, L-(v):
750 cells/mL and L-(vi): 350 cells/mL. For each of the series,
2
the 200 μL of samples were transferred to 1.5 mL Eppendorf
tube. Then 1 μL of 10 % TritonX-100 was added in the tubes,
and the tubes were boiled at 75 oC for 5 min. After
centrifugation (15000 rpm. for 3 min), supernatant was placed
into a new tube and was filtered through a Sephacryl S-400H
(Amersham Biosciences) column. The samples as genomic
DNA of N. scintillans zoospores were preserved at -30 oC the
until real-time PCR assay. Real-time PCR assays were
conducted in 20 μL volumes containing 2 μL of the genomic
DNA, 2 μL of primer sets i) Ns63F - Ns260R and 10 μL of Full
VelocityTM SYBR® Green QPCR Master Mix (STRATAGENE)
on Mx 3000PTM Real-Time PCR System (STRATAGENE). The
following cycling protocol was used: 95 oC for 2.5 min,
followed by 50 cycles at 95 oC for 10 sec and 60 oC for 30 sec.
At the end of each run, a dissociation protocol: 95 oC for 1 min,
60 oC for 30 sec and 95 oC for 30 sec was performed to ensure
that only the specific PCR amplicon was present. The standard
curves for the primer sets were constructed with the serial
dilutions of N. scintillans zoospores containing the targeted 18S
rDNA sequence. All the experiments for real-time PCR assays
were carried out with triplicates.
Quantification of N. scintillans zoospores in the field samples
The real-time PCR assays with Ns63F and Ns260R primers
were also applied for the sea water samples. Field sampling of
net-samples and seawater have been continuously conducted in
the costal waters of Sagami Bay since spring 2005. Surface
seawater was collected by bucket at St. A and prescreened
through a 180 μm mesh to eliminate large zooplankton
including the vegetative cells and zoospore mother cells of N.
scintillans. Water samples were gently rinsed on a 35 µm mesh
sieve to remove large size phytoplankton and debris. Then
duplicate 1 L of samples were filtered through a 5 μm pore-size
Millipore hydrophilic Durapore filter in twice. One filter was
then placed into a 15 mL corning tube containing 3 mL of 5 %
borate-buffered formaldehyde in 0.22 μm filtered seawater, and
the other filter was transferred to 15 mL corning tube
containing 3 mL of 5 % Lugol iodine in 0.22 μm filtered
seawater. The corning tubes were agitated by a vortex mixer for
1 min and the 200 μL of suspension in the tubes were then
placed into 1.5 mL Eppendorf tube, respectively. Next, 1 μL of
10 % TritonX-100 was added in the tubes, and the tubes were
boiled at 75 oC for 5 min. After centrifugation (15000 rpm. for
3 min), supernatant was placed into a new tube and was filtered
through a Sephacryl S-400H (Amersham Biosciences) column.
In order to quantify the number of the N. scintillans zoospores
in an unknown sample, a standard curve for the primer sets was
constructed with the serial dilutions of N. scintillans zoospores
containing the targeted 18S rDNA sequence for each real-time
PCR assay. The numbers of the cells in the unknown sample
were calculated by plotting the Ct value against the serial
dilutions on the standard curve.
RESULTS AND DISCUSSIONS
the abundance declined abruptly and remained low from June
to December. Abundance of N. scintillans showed variation
from year to year in Sagami Bay. The maximum abundance
through the sampling period reached 7.5×105cells/m3 in late
May. The abundance in 1997 and 2000 was relatively high
compared with other years. While the abundance in 1998 was
relatively low. In 2000, the abundance was about ten times
larger than in other years. The seasonal variation in the
abundance of N. scintillans was statistically more significant
with relation to meteorological factors than hydrographic and
biological factors. The multiple linear regression analysis
showed that the most significant meteorological factors relating
to the abundance of N. scintillans were wind direction and
rainfall. These meteorological factors seemed to be directly
related to the variation in the abundance of N. scintillans.
During the period of high abundance of N. scintillans in
1997 and 2000, the rainfall residuals were lower than average
and average wind velocity was high (Fig. 2). Moreover, the
wind blowing from the south-southeast was frequently
observed in 1997 and 2000. During the bloom period of N.
scintillans in 1997 and 2000, the winds from south-southeast
and calm surface layers thereafter without any ecological
turbulence such as heavy rain, may have forced the cells of N.
scintillans to accumulate in the surface layer. The calm surface
layer condition may be a key factor in forming and maintaining
the high density conditions of N. scintillans.
(a)
(b)
100
20
80
15
60
40
5
N. D.
0
20
80
1998
15
60
10
40
Volume of Noctiluca (×107 μm3)
Abundance of Noctiluca ( ×103 cells m-3)
20
0
100
1997
10
20
0
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
100
5
0
20
1999
15
10
5
0
20
2000
15
10
5
0
20
2001
15
10
5
0
40
2002
80
60
20
40
20
0
100
0
20
80
10
40
20
5
0
100
0
20
80
2004
15
60
Relationship between the bloom and environmental factors
In Sagami Bay, seasonal variation in abundance of N.
scintillans showed the same pattern every year (Fig. 1).
Abundance of N. scintillans started to increase from March and
it reached the maximum abundance in spring between April and
May throughout the study periods. After maximum abundance,
2003
15
60
10
40
5
20
0
0
J F MAMJ J A S O N D
J F MAMJ J A S O N D
Month
Fig. 1. Abundance (a) and the cell volume (b) of Noctiluca
scintillans from 1997 to 2004.
3
4
10
(a)
-2
-6
4
6
4
2
-4
40
Stability
0.5
0.4
(c)
0.3
20
0
-0.2
-0.4
-0.8
200
(e)
0.2
0
0.1
0.2
-0.6
Wind direction
-8
Net - zooplankton
biomass
-4
-6
(g)
0.4
Wind velocity
0
300
200
100
0
-100
-200
0.6
-2
-2
(f)
-300
0
(b)
2
Salinity
Rainfall
0
-4
Residuals
600
500
400
(d)
8
Chlorophyll a
Temperature
2
0
-0.1
(h)
100
0
-100
-0.2
-0.3
1997 1998 1999 2000 2001 2002 2003 2004
-20
-200
1997 1998 1999 2000 2001 2002 2003 2004
Month
1997 1998 1999 2000 2001 2002 2003 2004
Month
Month
Fig. 2. Deviation of environmental factors from the monthly averages of study period, temperature (a), salinity (b), water stability (c),
chlorophyll a concentration (d), other zooplankton biomass (e), rainfall (f), wind velocity (g) and wind direction normalized at
south-southeast (h). Vertical dark bars denote the bloom period of Noctiluca scintillans in 1997 and 2000.
Quantification of N. scintillans zoospores in the field samples
The standard curves were constructed with the serial
dilutions of N. scintillans zoospores containing the targeted 18S
rDNA sequence (Fig. 3). The high linearity of curves for each
assay in the present study indicates it might be possible to
apply the real-time PCR assay to field samples and achieve
accurate quantification. Variation in the concentration of the
cells could be detected at St. A in Manazuru Port of Sagami
Bay, Japan. The highest peak of N. scintillans zoospores, which
was about 5.2 x104 cells/L, was observed in the end of April
with a small peak detected in the end of June in 2005 (Fig. 4).
The peaks of the zoospore were followed by the high
abundance of the vegetative cells.
Our results suggest that bloom formation can be separated into
a three-step process 1) initial increase in the abundance of N.
scintillans is attributed to an increase in optimum hydrographic
and biological factors (temperature, salinity, water stability and
chlorophyll a), 2) N. scintillans are then accumulated by
convergence of seawater by the factors of low rainfall and wind
and 3) swarmer-effects suggested by Sato et al. (1998) enhance
bloom formation. Accumulation is considered to be a key
trigger in this process of the formation of large-scale blooms.
Previous studies concerning the bloom formation of N.
scintillans have been focused on analyzing the individual
factors controlling the variation in the abundance. Based on the
findings of the present study, large-scale bloom occur once
follow to the linkage and combination of environmental factors
in the three steps of the bloom formation. Thus, the
environmental factors regulating the population growth are
different between the phases of the bloom formation process.
The concept of bloom-phase formation mechanisms was
applied to other blooms of N. scintillans observed in other areas.
N. scintillans possibly has also unknown stages resting in a
bottom sediment as other dinoflagellate. More data from field
samples are necessary to confirm the potential. Real-time PCR
assay will support to estimate not only the contribution of the
zoospore to the bloom formation of N. scintillans in the field
Cell density of N. scintillans swarmers cells (cells/mL)
studies, but also the function of zoospore in the life cycle of N.
scintillans in laboratory experiments.
16
Formaldehyde
12
8
ΔRn (×10000)
4
0
16
Lugol
12
8
4
0
0
10
20
30
40
100000
R= 0.85
10000
1000
100
10
1
100000
R= 0.99
10000
1000
100
10
1
20
50
30
40
50
Ct
Cycle number
Fig. 3. Amplification and standard curves using primer set (i)
Ns63F-Ns260R for real-time PCR assay of Noctiluca
scintillans 18S rDNA sequence with the zoospores fixed by
formaldehyde and lugol.
2.0
6.0
Zoospore
1.5
3.0
1.0
1.5
0.5
Vegetative cell
(×105 cells m-3)
Zoospore
(×107 cells m-3)
Vegetative cell
4.5
0
0
Apr
Maｙ
Jun
Jul
Fig. 4. Variation in the cell density of zoospore and vegetative
cells of Noctiluca scintillans at Manazuru Port of Sagami Bay
from April to July 2005.
4