FEMS Microbiology Ecology 52 (2005) 243–253
www.fems-microbiology.org
On-board flow cytometric observation of picoplankton
community structure in the East China Sea during the fall
of different years
L.A. Pan
a
c
a,*
, L.H. Zhang a, J. Zhang
a,b
, Josep M. Gasol c, M. Chao
a
State Key Laboratory of Estuarine and Coastal Research, East China Normal University, 3663 Zhongshan Road North, Shanghai 200062, PR China
b
College of Chemistry and Chemical Engineering, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China
Department de Biologia Marina i Oceanografia, Institut de Ciències del Mar, CSIC, Pg. Maritim de la Baceloneta, 37-49, E-08003 Barcelona, Spain
Received 1 June 2004; received in revised form 28 August 2004; accepted 16 November 2004
First published online 23 December 2004
Abstract
On-board flow cytometric determinations of picoplankton abundance (i.e. Synechococcus spp., Prochlorococcus spp., picoeukaryotes and also heterotrophic bacteria) were obtained in the East China Sea in fall of 2000 and 2003. The average abundances of Synechococcus, Prochlorococcus, picoeukaryotes and heterotrophic bacteria were 105, 105, 104 and 106 cells ml 1, respectively.
Synechococcus, picoeukaryotes and heterotrophic bacteria were abundant at all the stations and presented higher concentration
in the inner shelf where influences from the Changjiang effluent plumes and the coastal upwelling were evident, while Prochlorococcus was absent from the near-shore stations and became the dominant picophytoplankton population in offshore waters, where its
abundance was comparable to that for heterotrophic bacteria. All picoplankton groups showed a reduction in cell number with
depth, and a positive correlation with water temperature were observed, which reflected the importance of light and temperature
on picoplankton growth. A negative relationship with salinity was found for heterotrophic bacteria along two sections across the
East China Sea Shelf, and distribution of picoplankton was dominated by different water masses. The fixation could lead to loss
in Prochlorococcus cell numbers within one month, and all the picoplankton numbers decreased dramatically after three months.
2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: East China Sea; Flow cytometry; Synechococcus; Prochlorococcus; Picoeukaryotes; Heterotrophic bacteria
1. Introduction
Continental shelves represent <10% of the surface
area of the world ocean, but they are an important intermediate zone between the land and the open ocean,
where terrigenous and anthropogenic materials are
transported, deposited, and transformed. The continental shelf is characterized by important standing
stocks of organic carbon and high rates of primary
*
Corresponding author. Tel.: +86 21 62233420; fax: +86 21
62546441.
E-mail address: [email protected] (L.A. Pan).
and secondary productions, and is recognized as one
of the most important compartments of the global
biogeochemical cycles of carbon and other biogenic
materials [1].
The East China Sea is a marginal sea with one of the
most extensive continental shelves in the world, interacting with large rivers (e.g. Changjiang) and the Kuroshio
in its western and eastern boundaries, respectively. The
Kuroshio interacts actively with shelf waters through
frontal and upwelling processes (e.g. filaments and
eddies). Four water masses can be commonly differentiated within the East China Sea Shelf: the relatively fresh,
cool and nutrient-rich Changjiang Diluted Water, the
0168-6496/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsec.2004.11.019
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L.A. Pan et al. / FEMS Microbiology Ecology 52 (2005) 243–253
oligotrophic and highly saline Taiwan Current Warm
Water that reaches as far as the Changjiang (Yangtze
River) Estuary, the intrusion of cold and nutrient-rich
Kuroshio Subsurface Water, and the warm, saline and
nutrient-poor Kuroshio Surface Water (Fig. 1). The major contribution to the nutrients in the East China Sea
Shelf includes the Changjiang Diluted Water and Kuroshio Subsurface Water [2–4]. The nutrient supply via
these pathways sustains high primary production in
the East China Sea (e.g., 145 g C m 2 y 1) [5].
The operational concept of picoplankton (<2 lm) includes by definition the autotrophic cyanobacteria Synechococcus spp. [6] and Prochlorococcus spp. [7], small
eukaryotic algal groups, and heterotrophic bacteria [8],
which are important components of marine plankton
communities. Synechococcus is found ubiquitous in the
upper temperate and warm ocean [9], and Prochlorococcus has been found to be more abundant in oligotrophic
than in eutrophic waters [10,11]. These two phytoplankton groups, together with picoeukaryotes, have fast
growth rates matched by high mortality losses caused
by microzooplankton grazing, making them fundamental components of the biomass and primary production
of marine ecosystems, and hence participating in nutrient regeneration and cycling in the ocean [12]. The
widely distributed heterotrophic bacteria and their variability can parallel that of phytoplankton within ecosystems [8,13]. Heterotrophic activity contributes to the
conversion of dissolved organic matter (DOM) to bacte-
rial biomass that can be transferred to higher trophic
levels through the marine microbial food web with participation of flagellate and ciliate grazers [14]. The quantification of picoplanktonic organisms is therefore of
great importance for the characterization of marine ecosystems and for understanding the function of marine
food webs.
Picoplankton groups in the East China Sea have
been the subject of research in the past [3,15–25].
However, few data have been published on the distribution of picophytoplankton and bacterioplankton in
the shelf. Previous studies were mainly focused on
only one picoplankton group such as Synechococcus
or on heterotrophic bacteria rather than simultaneously observing the four picoplankton groups, and
most of the data in literature were obtained by epifluorescence microscopy, which was not as efficient,
sensitive and precise as flow cytometry (FCM) [26] except in the study by Jiao et al. [20]. It seems that none
of the analyses with flow cytometry was made on
board.
On-board simultaneous determination of these picoplankton groups was made during the fall of 2000 and
2003. The aim of this study was to better understand
the relationship between the spatial structure of hydrographic properties of different water masses and the variability of different picoplankton groups, and to discern
the factors controlling the distribution of picoplankton
in different parts of shelf ecosystems.
Fig. 1. Map of stations where both CTD data and picophytoplankton abundances were measured during 2000 (w, and station P12), and stations
where CTD data were collected (s), and where picoplankton abundances were measured (r) in 2003 in the East China Sea. The abundance of
heterotrophic bacteria was not determined in 2000. In 2003, the stations were sampled twice with a difference of 20 days; stations P4 and A6 were not
sampled during the first leg and station P3 was not sampled during the second leg. CDW, the Changjiang Diluted Water; TCWW, the Taiwan
Current Warm Water. The KSW (the Kuroshio Surface Water) and the KSSW (the Kuroshio Subsurface Water) are not shown in this figure.
L.A. Pan et al. / FEMS Microbiology Ecology 52 (2005) 243–253
2. Materials and methods
2.1. Station locations and sampling
Two cruises were conducted; cruise 2000 from October 20 to November 8, 2000, and cruise 2003 from September 4 to 26, 2003, on board R/V ‘‘Dong Fang Hong
2’’. Cruise 2000 had five stations located along the PN
section (named by Nagasaki Marine Observatory). On
cruise 2003, the stations were set on three different but
representative sections: PN section across the East China Sea shelf, AS section along the Okinawa Trough and
YT section from the Changjiang Estuary to the Tsushima Strait, and were sampled twice within 20 days
(Fig. 1). Stations P4 and A6 were not sampled in the first
leg, and station P3 was not sampled in the second leg because of a typhoon. In 2000, Synechococcus spp., Prochlorococcus
spp.,
and
picoeukaryotes
were
enumerated, and in 2003, heterotrophic bacteria were
counted as well. Temperature, salinity, turbidity, epifluorescence and dissolved oxygen were recorded in the
water column using a Sea-Bird 911 plus conductivitytemperature-depth (CTD)-Rosette assembly. Seawater
was sampled with Niskin bottles at different water
depths designed from the CTD profiles.
2.2. Analysis of picophytoplankton and heterotrophic
bacteria
Since fixation with added chemical reagents may result in loss of cells [27], samples were analyzed on a
FACScan flow cytometer (Becton Dickinson, San Jose,
CA, USA) equipped with an air-cooled argon laser
(488 nm, 15 mW), placed on-board so that after sample
collection the analyses could immediately be performed.
Forward light scatter (FSC), side light scatter (SSC),
green fluorescence (530 ± 15 nm, FL1), orange fluorescence (585 ± 21 nm, FL2) and red fluorescence
(>650 nm, FL3) were recorded for each particle in the
sample, and data obtained were processed with CELLQuest software (Becton Dickinson, San Jose, CA,
USA). Yellowish green fluorescent beads (1.002 lm)
(Polysciences Inc., catelogue # 18660) were added to calibrate cell fluorescence emissions and light scatter signals, which allows the comparison of fluorescence and
cell sizes among samples. The picophytoplankton
groups could be discriminated and enumerated according to their specific autofluorescence properties and light
scatter differences [28]. For the enumeration of heterotrophic bacteria, fresh water samples were stained with
1:10,000 (vol:vol) SYBR Green I (Molecular Probes,
Inc.), and incubated in the dark at room temperature
for 15 min before analysis [28]. Bacteria were measured
for their side light scatter and green fluorescence signals,
which were related to cell size and nucleic acid content,
respectively [28]. Triplicate measurements were made for
245
each sample with precision higher than 7.2% (relative
standard deviation).
To test the effects of preservation, duplicate samples
were fixed for 15 min with different fixatives including
paraformaldehyde (final concentration: 1%), glutaraldehyde (final concentration: 0.125%) and a mixture of
paraformaldehyde and glutaraldehyde (final concentration: 1% and 0.125%, respectively), then deep frozen in
liquid nitrogen [26,28] and analyzed after one and three
months, respectively. All the stock solutions except the
dye were pre-filtered through a 0.2 lm pore size filter before use to avoid contamination.
Statistical analysis of data was done with the SPSS
software (SPSS Inc.).
3. Results
3.1. Hydrographic conditions
In general, the coastal region was affected by fresh
water with low salinity discharged from the Changjiang
in fall 2000. In the surface water (0–30 m), hydrographic
parameters were vertically well mixed, with temperature
increasing from 21 to 25 C and salinity from 26.5 to
34.0, from off the Changjiang Estuary to the edge of
the shelf. There was vertical increase in salinity and decrease in temperature with depth, which was more pronounced in the outer shelf waters where a pycnocline at
a depth of about 40 m was formed. There was a salinity
gradient from the shore to the mid-shelf areas (i.e., salinity increasing from 26 to 33), which is quite distinctive in
summer when the Changjiang effluent plume spreads
over the continental shelf. In the bottom waters, an area
of uniform salinity (34.4–34.6) was formed but with a
strong vertical temperature gradient (15–22 C) in the
outer shelf. This could be an evidence of the intrusion
of the cool and saline Kuroshio Subsurface Water.
In 2003, the average surface water temperatures were
>25 C at all stations, and the surface salinity increased
from the inner shelf (23.0) to outer shelf (34.4) in early
fall. Both water temperature and salinity in the PN and
YT sections showed similar distributions (Fig. 2(a)),
which were comparable to that of 2000. However, an
upwelling of cold and saline water could be seen just outside the Changjiang Estuary at a depth of about 25 m in
2003 (Fig. 2(a)). As a result of the mixture of the Changjiang Diluted Water and the Kuroshio with strong surface heating by radiation, a pycnocline developed in
the PN section and became deeper towards the shelf
break (Fig. 2(a)). There was cold upwelling water at station T5 as well (Fig. 2(a)), which might indicate the shelf
mixing water. In the AS section, the water temperature
decreased steadily with depth until to <5 C in bottom,
while the salinity increased with depth (i.e. 33.3–34.9;
Fig. 2(a)). There was a high salinity area in the
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L.A. Pan et al. / FEMS Microbiology Ecology 52 (2005) 243–253
subsurface water near station A6, which seemed to be the
contribution of the Kuroshio Subsurface Water (Fig.
2(a)).
3.2. Distribution of picoplankton in two cruises
In most of the samples, three groups of picophytoplankton could easily be discriminated in unstained
samples according to their autofluorescence properties
and light scatter differences. Also, at least two groups
of heterotrophic bacteria, each with distinctive green fluorescences related to nucleic acid content, could be distinguished after DNA staining (Fig. 3). These results
were in agreement with studies in other world areas
[11,26,28,30].
On both cruises, the concentrations of Synechococcus
[(59.7 ± 98.0) · 103 cells ml 1] and Prochlorococcus
[(47.3 ± 101.8) · 103 cells ml 1] were comparable and
were one order of magnitude higher than that of
picoeukaryotes [(5.5 ± 7.7) · 103 cells ml 1] in the PN
section. In the YT section, the concentration of Synechococcus [(190.2 ± 527.7) · 103 cells ml 1] was one to
two orders of magnitude higher than those of Prochlorococcus [(39.1 ± 82.4) · 103 cells ml 1] and picoeukaryotes [(7.3 ± 14.2) · 103 cells ml 1]. Overall, the
distributions of three autotrophs were similar along
these sections, that is, the abundances of the picophytoplankton groups tended to increase in surface waters in
mid-shelf and decrease with depth, and were rarely detected beneath the euphotic zone (Fig. 4). Radiation
Fig. 2. Profiles of water temperature and salinity in 2003. (a) Profile of
temperature and salinity in different sections. Note the different scales
of depth above and below 200 m. (b) Relationship between temperature and salinity of seawater for all stations (two legs). Isopycnals are
shown. CDW, the Changjiang Diluted Water; TCWW, the Taiwan
Current Warm Water; KSW, the Kuroshio Surface Water; KSSW, the
Kuroshio Subsurface Water; KIW, the Kuroshio Intermediate Water;
SMW: the Shelf Mixed Water. The definitions of the water masses are
based on Gong et al. [3] and Ichikawa and Chaen [29].
Fig. 3. Flow cytometric analysis of marine samples from the surface
water at station P5 (a,b) and from a depth of 50 m at station A1 (c,d)
in the East China Sea in 2003. Samples were run without staining to
enumerate picophytoplanktons (a,b) and stained with SYBR Green I
to count bacteria (c,d). Syn, Synechococcus spp.; Proc, Prochlorococcus
spp.; Euk, picoeukaryotes; Bact, heterotrophic bacteria; Bact-I and
Bact-II, bacterial subpopulations; Beads: 1.002 lm yellowish green
fluorescent beads.
L.A. Pan et al. / FEMS Microbiology Ecology 52 (2005) 243–253
247
Station
P12
0
4
3
1 P12
2
P10
P8-1
P7
P3 P12 T9
P5
T7
T5
T3
S1 P3
A5
A1
A3
S1
-50
-100
<1
-150
-200
PN section, Cruise 2000
3
-1
Synechococcus (× 10 cells ml )
<1
<1
-400
YT section, Cruise 2003
3
-1
Synechococcus (× 10 cells ml )
-600
<1
-800
PN section, Cruise 2003
AS section, Cruise 2003
3
-1
Synechococcus (× 10 cells ml )
-1000 Synechococcus (× 103 cells ml -1)
0
-50
-100
-150
-200
<1
<1
PN section, Cruise 2000
3
-1
Prochlorococcus (× 10 cells ml )
<1
-400
YT section, Cruise 2003
3
-1
Prochlorococcus (× 10 cells ml )
-600
<1
Depth (m)
-800
-1000
PN section, Cruise 2003
3
-1
Prochlorococcus (× 10 cells ml )
AS section, Cruise 2003
3
-1
Prochlorococcus (× 10 cells ml )
0
-50
-100
<1
-150
<1
PN section, Cruise 2000
3
Picoeukaryotes (× 10 cells ml -1 )
<1
-200
-400
YT section, Cruise 2003
3
-1
Picoeukaryotes (× 10 cells ml )
-600
<1
-800
PN section, Cruise 2003
AS section, Cruise 2003
3
-1
Picoeukaryotes (× 10 cells ml )
-1000 Picoeukaryotes (× 103 cells ml -1 )
0
-50
-100
-150
< 200
< 200
-200
-400
YT section, Cruise 2003
-600
Heterotrophic bacteria
3
-1
(× 10 cells ml )
-800
PN section, Cruise 2003
Heterotrophic bacteria
-1000 ( 103 cells ml -1 )
×
< 100
AS section, Cruise 2003
Heterotrophic bacteria
3
-1
(× 10 cells ml )
Fig. 4. Distributions of Synechococcus spp., Prochlorococcus spp., picoeukaryotes and heterotrophic bacteria at different sections of the cruise 2000
and cruise 2003. Heterotrophic bacteria were not measured in 2000. Note the difference in scales of depth above and below 200 m. Refer to Fig. 1 for
station locations in the PN, YT and AS sections. The location of the PN section in 2000 was nearly the same as that of PN section in 2003.
was not limiting phytoplankton growth in surface water
offshore the turbid Changjiang effluent plume [5,15]. The
upwelling of the nutrient-rich Kuroshio subsurface
water, which can be found in the middle shelf of the East
China Sea [4] (Fig. 2(a)), stimulated growth of these
photosynthetic autotrophs at the mid-shelf along the
PN section in both years (Fig. 4). The supply of inorganic nutrients from the Changjiang runoff in combina-
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L.A. Pan et al. / FEMS Microbiology Ecology 52 (2005) 243–253
tion with and the coastal upwelling, induced the development of high picoeukaryote biomass at station P10
and high cell numbers of three autotrophs at station
T9 in 2003 (Fig. 2(a) and Fig. 4). It should be noted that
Prochlorococcus was almost absent near the Changjiang
Estuary (e.g. station P12 in Fig. 4). The Prochlorococcus
was abundant farther seawards than Synechococcus and
picoeukaryotes in the PN section in 2000 (Fig. 4). Also,
the high abundance site of Prochlorococcus was separated from those of Synechococcus and picoeukaryotes
in the PN and YT sections in 2003 (Fig. 4). Along the
AS section, high abundance sites of autotrophs appeared in high salinity area influenced by the Kuroshio
Subsurface Water (Fig. 2(a) and Fig. 4), where the abundance of Prochlorococcus was almost one to two orders
of magnitude higher than those of Synechococcus and
picoeukaryotes (Fig. 4). This was similar to the other reported results [11,20]. Furthermore, the numbers of
autotrophs remained relatively high and stable in surface mixing waters down to the pycnocline, and then decreased sharply with depth (Fig. 4).
Cell abundances of heterotrophic bacteria over the
East China Sea Shelf were often one to two orders of
magnitude higher than those of picophytoplankton.
The vertical distribution of bacteria decreased with
depth, similar to distributions of autotroph groups.
The presence of bacterioplankton tended to diminish
with offshore distance (Fig. 4). A high abundance site
formed also at station T5, where water was well mixed
because of the upwelling induced by the shelf mixing
water (Fig. 2(a) and Fig. 4).
4. Discussion
4.1. Effects of fixation
Fixation was reported to lead to cell loss [26,27]. In
this study, the difference between the different fixatives
was statistically insignificant, so only the data of samples fixed with paraformaldehyde are shown (Fig. 5).
The cell numbers and fluorescence parameters by
FCM were roughly unchanged during one month after
sampling, except in Prochlorococcus, shown by the variation of slopes of the simulation lines in Fig. 5. After
three months, the cell numbers of all picoplankton
groups dramatically declined and data scatter considerably, particularly for Prochlorococcus and picoeukaryotes (Fig. 5). If the samples are not fixed, the
abundances will decrease more dramatically and data
will scatter significantly. It can be concluded that
samples should be analyzed on board or kept in liquid
1000
50
Synechococcus
On board
1 m onth later
800
y=x
r2=1
3 months later
40
600
Cell numbers after fixation (x 103 cells ml-1)
Picoeukaryotes
y=x
r2=1
30
400
y = 0.92x + 0.09
r 2 = 0.99
200
y = 0.94x - 0.04
r 2 = 0.99
20
10
y = 0.39x + 3.56
r 2 = 0.95
y = 0.61x - 1.27
r 2 = 0.84
0
0
0
200
400
600
800
1000
0
10
20
30
40
50
4000
600
Prochlorococcus
Heterotrophic bacteria
500
y=x
r2=1
400
y=x
r2=1
3000
y = 1.20x - 22.11
r 2 = 0.99
2000
300
y = 0.84x - 1.35
r 2 = 0.98
200
1000
100
y = 0.79x - 70.32
r 2 = 0.96
y = 0.32x - 4.95
r 2 = 0.66
0
0
100
200
300
400
500
0
600
0
1000
2000
3000
4000
Cell numbers on board (x103 cells ml-1)
Fig. 5. Effects of paraformaldehyde (final concentration 1%) on cell numbers of picoplankton groups. Fixed samples were frozen in liquid nitrogen
until determination after one and three months. The results were compared with those of on-board measurements.
L.A. Pan et al. / FEMS Microbiology Ecology 52 (2005) 243–253
nitrogen before analysis (i.e. within one month) once
back to the laboratory.
4.2. Comparisons among different sections and cruises
Station P12 was always affected by the Changjiang diluted water, and stations in the mid-shelf at PN section
were mainly dominated by the Taiwan current warm
water [2] on both cruises (Fig. 2(b)). Nevertheless, the
distributions of water temperature and salinity were
quite different in the inner shelf between the two years.
Stratification was more evident, temperature was higher
in the inner shelf, and salinity was higher in the subsurface and bottom waters in the middle shelf in the year
2003 compared to 2000. This may explain the differences
in the locations of high abundance phytoplankton and
cell numbers between the two cruises (Fig. 4), as there
was a comparatively stronger effect from the Changjiang
Diluted Water in 2003 (Fig. 2(a)); the nutrients from the
Changjiang [2,3], together with a more favorable temperature, promoted the growth of autotrophs. It is possible that limited number of stations on the cruise in
2000 may have missed high biomass areas of varying
picophytoplankton populations. In 2003, the four picoplankton groups had similar locations of high abundances in two legs along the PN section, showing a
relative stability of cell abundances along with hydrographic features (Fig. 4).
The hydrographic properties in the YT section in
2003 are comparable to those in the PN section: affected
by the Changjiang Diluted Water in near-shore waters
249
and by the Taiwan Current Warm Water in offshore
waters (Fig. 2(b)), and showing a remarkable similarity
between the two legs. The exception is a lower salinity
(t-test, p < 0.01) above 10 m at stations in the near-shore
areas (e.g. station P12 to T8) for the first leg, which
could be seen in picophytoplankton abundances at stations T9 (Fig. 6). Similar high densities for Prochlorococcus and picoeukaryotes in the first leg at station T9
were not found in the second leg. The thermocline was
enhanced in the outer shelf at the YT section (i.e. station
T3, Fig. 6) of the second leg, with stronger stratification
preventing the supply of nutrients to the surface waters
[2]. The implication includes the lower concentrations of
picoplankton groups.
Overall, influences of the Changjiang Diluted Water
at the PN and the YT sections in the year 2003 are comparable (p < 0.01) with respect to the salinity data. The
temperature and salinity in the surface water at station
T5 (Fig. 2(a)) of the YT section were lower, indicating
upwelling that was probably induced by the exchange
of shelf mixing water with the Yellow Sea. The presence
of an upwelling that can facilitate vertical mixing and
bring up nutrients and DOM for Synechococcus and
heterotrophic bacteria, respectively, is a plausible explanation for the higher concentrations of those in the midshelf area of the YT section as compared to the PN
section (Fig. 4).
At the AS section, the hydrographic conditions were
more stable, and the stations were affected by the Kuroshio Surface and Subsurface Water (Fig. 2(b)). Cell numbers were rather low and stable, except for
Fig. 6. Typical vertical distributions of temperature, salinity and picoplankton cell numbers in different stations of cruise 2003 (n = 3). Syn,
Synechococcus spp.; Proc, Prochlorococcus spp.; Euk, picoeukaryotes; Bact, heterotrophic bacteria.
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L.A. Pan et al. / FEMS Microbiology Ecology 52 (2005) 243–253
Prochlorococcus that was more abundant in oligotrophic
waters [10,11]. Synechococcus and picoeukaryotes seem
to have a higher demand for nutrients and hence flourished in the Changjiang Diluted Water and in the upwelling rather than in the AS section. Heterotrophic bacterial
numbers were also lower in the AS section than in the
other two sections (Fig. 4), probably because the favorable conditions such as biodegradation of organic matters promote heterotrophic activity [14,17,25], and
those conditions originated presumably from the discharge from the Changjiang [31]. The high-salinity Kuroshio Subsurface Water incursion is a major nutrient
source for the East China Sea [2], sustaining picoplankton growth with high abundance at station A5 (Fig. 4).
Apparently, the uplift of the Kuroshio Subsurface Water
at station A5 was enhanced in the second leg, as was seen
from the difference in salinity (Fig. 6), with rather different distributions of Prochlorococcus and other picoplankton groups. Prochlorococcus cell numbers
decreased with depth in both legs, while the other groups
showed high numbers at depth of 60 m rather than at the
surface in the second leg (Fig. 6), indicating that the
Kuroshio Subsurface Water had an impact on other
picoplankton groups. This implies that Prochlorococcus
and the other photosynthetic groups in oligotrophic
Kuroshio waters have different demands for nutrients.
cell abundances are compared in this study, and the
biomass data from other studies are converted to
abundances with the relevant conversion factor for
each study.
The concentrations of different picoplankton groups
obtained in this study are roughly of the same order as
in the other studies conducted in the East China Sea
(Table 1). The abundance of heterotrophic bacteria in
this study is lower than that reported in literature
[3,19,24,25], which may be merely because of different
study areas, since high supply of DOM required for bacterial growth was reported at stations concentrated in
the inner shelf [17]. However, it was also reported that
Prochlorococcus could hardly be separated from bacterioplankton by means of epifluorescence microscopy,
and thereby might be mistaken for the latter [32]. Such
kind of bias can be significant especially in oligotrophic
waters, where Prochlorococcus can reach nearly 20–40%
of the total prokaryotes [10,30,32]. Furthermore, cell
loss caused by chemical fixation in other studies [26]
could have induced artifacts in previously published
data on picoplankton (cf. Fig. 5). Our on-board measurements using FCM provide more reliable information for the discrimination of picoplankton groups.
4.4. Relationship between picoplankton and hydrographic
properties
4.3. Comparison of picoplankton abundance
To avoid introducing additional biases related to
differences in biomass conversion factors [19,21,22],
Although radiation effects are weak in the fall [24],
there was a dramatic impact of temperature on cell numbers of all picophytoplankton groups in 2000 and 2003
Table 1
Abundances of picoplankton groupsa in the East China Sea from different studies
Location
Year
Month
Cell numbers (average ± standard deviation; 103 cells ml 1)
31–32N, 121–124E
1986
1986
1997
1998
1997
19941996
1996
1997, 1998
1998
1998
1997
1998
1998
2000
2001
2000
2003
January
July
February–March
July
February–March
May, November
March, April
May
December, March
June–July
October–November
October
May
July
October–November
March–April
October–November
September
0.1–1
0.1–200
0.04–24.5
0.6–19.7
Syn
27–32N, 122–130E
28–32N, 124–129E
24–27N, 120–124E
25–32N, 120–127E
31–32N, 122.5–124E
25–33N, 120–130E
25–32N, 120–128E
28–32N, 122–129E
a
b
c
Pro
Euk
Bact
[15]
[21]
1–56
110–2500
131
100–500
<100- >250
<700
<15
7.6–31.1
20.7–78.4
21.2 ± 44.9
97.3 ± 335.1
References
<120
<14
6.3 ± 15.0
52.5 ± 97.5
14.4 ± 15. 2
5.2 ± 10. 3
190–1765, 1000–3365
625–3425
1170 ± 1420b, 345 ± 190c
597 ± 268
197 ± 96
<540
690 ± 1780
733.3 ± 583.3
Syn, Synechococcus spp.; Pro, Prochlorococcus spp.; Euk, picoeukaryotes; Bact, heterotrophic bacteria.
Converted to abundances from bacterial biomass in the mesotrophic system with the conversion factor used by Chen et al. [25].
Converted to abundances from bacterial biomass in the oligotrophic system with the conversion factor used by Chen et al. [25].
[16]
[17]
[19]
[3,19,24]
[3,19,24,25]
[18]
[20]
[22,23]
[23]
This study
L.A. Pan et al. / FEMS Microbiology Ecology 52 (2005) 243–253
7
Heterotrophic bacterial
numbers (lg (cells ml -1))
6
5
4
3
2
r 2 = 0.66, n = 127, p < 0.01
1
10
15
20
25
30
Water temperature (˚C)
(a)
5
r 2 = 0.33, n = 76, p < 0.01
4
25
30
35
Het erotrophic bacterial
numbers (lg (cells ml -1))
6
5
r 2 = 0.69, n = 131, p < 0.01
4
600
5
10
15
900
Depth (m)
30
35
6
5
4
3
2
r 2 = 0.55, n = 127, p < 0.01
1
50
100
150
200
Depth (m)
7
6
5
r 2 = 0.56, n = 104, p < 0.01
4
1
(f)
25
7
0
1200
20
Water temperature (˚C)
(d)
7
300
4
40
Salinity
0
r 2 = 0.73, n = 131, p < 0.01
0
Picophytoplankton
numbers (lg (cells ml -1))
Heterotrophic bacterial
numbers (lg (cells ml -1))
6
20
5
(b)
7
(c)
6
35
Heterotrophic bacterial
numbers (lg (cells ml -1))
Picophytoplankton
numbers (lg (cells ml -1))
7
(e)
251
2
3
4
5
6
7
Picophytoplankton numbers
(lg (cells ml -1))
Fig. 7. Relationships between hydrographic conditions and picoplankton abundances and between total numbers of picophytoplankton and
heterotrophic bacteria for all sections of the cruise 2000 (·) and cruise 2003 (s). (a) Relationship between water temperature and picophytoplankton
abundance; (b) relationship between water temperature and heterotrophic abundance; (c) relationship between salinity and heterotrophic abundance;
(d) relationship between depth and total picophytoplankton abundance; (e) relationship between depth and heterotrophic bacterial abundance;
(f) relationship between total picophytoplankton abundance and heterotrophic abundance.
(Fig. 7(a)), and also of heterotrophic bacteria in 2003
(Fig. 7(b)).
The PN and YT sections covered broad range of
salinity in relation to the distance from the land; the
data of these sections were used to examine the relationship between picoplankton and salinity. The heterotrophic bacterial numbers decreased in the water column
with higher salinity (Fig. 7(c)), while no relationship
could be found between salinity and concentrations of
picophytoplankton, with Prochlorococcus disappearing
at salinity <29. This revealed that Prochlorococcus,
which is commonly more abundant in oligotrophic
waters [10,11], could be eliminated in the low-salinity
and turbid coastal waters, which is, however, opposite
to euryhaline groups such as Synechococcus and
picoeukaryotes.
Meanwhile, all picophytoplankton groups showed a
negative correlation between abundance and depth
(Fig. 7(d)). This may reflect the dependence of those
autotrophs upon light. Also, due to the stratification
that tends to constrain the biogenic DOM in the surface
water [31], the heterotrophic bacterial number displays a
negative correlation with depth as well (Fig. 7(e)).
Nutrient supply (e.g. N and P) is an important factor
in maintaining the picoplankton distribution [33]. Outside the Changjiang Estuary, we observed an upwelling
of cold and saline waters with high nutrients [34], similar
to the YT section. Other factors such as iron [35], grazing by ciliates [24] or other microzooplankton, viral
infections and co-sedimentation with organic particles
[36,37] may also affect the picoplankton distribution,
however, they were not tested in this study.
252
L.A. Pan et al. / FEMS Microbiology Ecology 52 (2005) 243–253
with depth were found for all four groups, and a negative correlation with salinity for heterotrophic bacteria
throughout the PN and YT sections was also seen.
Furthermore, picoplankton groups show different distributions in different water masses. The hydrographic
conditions to some extent confine the picoplankton
biomass.
Acknowledgements
Fig. 8. Distributions of four picoplankton groups in different water
masses in 2003. CDW, the Changjiang Diluted Water; TCWW, the
Taiwan Current Warm Water. Prochlorococcus cell number was lower
than 23 cells ml 1 in the CDW-controlled waters.
A positive correlation between heterotrophic bacteria
and the autotrophs (i.e. Synechococcus, Prochlorococcus
and picoeukaryotes) was found in this study (Fig. 7(f)),
suggesting the dependence of bacteria on the substrates
produced by the small primary producers.
Different water masses are found in the East China
Sea shelf (Fig. 2(b)). Synechococcus appears to be the
most abundant population in the areas affected by Taiwan current warm water, Prochlorococcus becomes the
dominant picophytoplankton population in the Kuroshio region. The concentrations of picoeukaryotes and
heterotrophic bacteria are comparable among different
water masses (Fig. 8). The abrupt change in picoplankton cell numbers within a short distance may be typical
of the marginal seas [20], where hydrographic conditions
are rather complicated.
5. Conclusion
Fixation can lead to loss in Prochlorococcus numbers within a month and there is also a notable decrease in abundances of all picoplankton groups after
three months, so the samples should be analyzed on
board or as soon as possible when back to the laboratory. In 2000 and 2003, the average numbers of Synechococcus, picoeukaryotes and heterotrophic bacteria
in the East China Sea were in the range of 105, 104
and 106 cells ml 1, respectively, with a relatively higher
abundance in the inner shelf, where nutrient levels are
higher due to Changjiang effluent plumes and the
coastal upwelling, compared to offshore oligotrophic
waters. Prochlorococcus was absent in the near-shore
zones partially because of high turbidity and low
salinity but became the dominant population among
picophytoplankton groups offshore. A positive correlation with water temperature and a negative correlation
This study was funded by the Ministry of Science and
Technology of PR China (Nos. G1999043705 and
2001CB711004) and by the Shanghai Priority Academic
Discipline Project. We thank Dr. D. Vaulot for the help
in population discrimination by FCM and Dr. H. Wei
for providing the hydrographic data and assistance in
field-work. Prof. R. Laanbroek and two anonymous
reviewers are acknowledged for their critical comments
to original manuscript.
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