Contribution of auto- and heterotrophic

protozoa to the diet of copepods in the
Ulleung Basin, East Sea/Japan Sea
EUN JIN YANG
1
*, HYUNG-KU KANG
2
, SINJAE YOO
2
AND JUNG-HO HYUN
3
1
DEEP-SEA AND MARINE GEORESOURCES RESEARCH DEPARTMENT, KORDI, ANSAN PO BOX 29, SEOUL 425-600, KOREA,
2
MARINE LIVING RESOURCE RESEARCH
DEPARTMENT, KORDI, ANSAN PO BOX 29, SEOUL 425-600, KOREA AND
3
DEPARTMENT OF ENVIRONMENTAL AND MARINE SCIENCE, HANYANG UNIVERSITY,
ANSAN 426-791, KOREA
*CORRESPONDING AUTHOR: ejyang@kordi.re.kr
Received October 17, 2008; accepted in principle February 5, 2009; accepted for publication February 6, 2009; published online 4 March, 2009
Corresponding editor: John Dolan
We estimated the ingestion rates and prey selectivity of two calanoid copepods, Calanus sinicus and
Neocalanus plumchrus, on auto- and heterotrophic protozoa in the Ulleung Basin in the East
Sea/Japan Sea. Both copepod species preferentially ingested auto- and heterotrophic protozoa. The
heterotrophic protozoa comprised between 42.9 and 86.7% of the total carbon ration ingested by
the copepods, although they comprised on average 21.5% of the total available prey carbon. In
particular, ciliates comprised the major dietary component for the copepods in the study area. The
copepod consumption of ciliates was on average 38.2% of the total daily carbon ration of auto-
and heterotrophic protozoa. Auto- and heterotrophic protozoa in the 20–50 mm size were the frac-
tion that was most actively consumed by copepods and they comprised on average 45.1% of the
total carbon ration ingested. In all experiments, the copepods selected the ciliate and heterotrophic
dinoflagellate (HDF) groups over the other protozoa. Our results indicate that the copepod feeding
regime was influenced by the composition and size of the potential food and that, in this study
area, feeding behavior of copepods may control the populations of ciliates and HDFs that are
larger than 10 mm in size. Thus, the selective feeding patterns and higher grazing pressure of
copepods on heterotrophic protozoa, compared with autotrophic protozoa, indicate a trophic coupling
between copepods and the microbial food web in the Ulleung Basin.
I NTRODUCTI ON
Traditionally, mesozooplankton such as copepods have
been considered as herbivorous feeding on phytoplank-
ton, channeling energy from primary producers
through to higher trophic level. Recently, copepod diets
have been shown to be much more diverse than pre-
viously reported based on the classical food chain
(Calbet and Landry, 1999; Broglio et al., 2004; Liu et al.,
2005; Castellani et al., 2008). Thus, special research
interest is now focused on understanding the role of het-
erotrophic protozoa, specifically ciliates and dinoflagel-
lates, in the copepod diet. The importance of
heterotrophic protozoa in the diet of copepods has been
quantified in a number of studies in very different
trophic areas, ranging from spring bloom situations
(Leising et al., 2005b; Liu et al., 2005; Fileman et al.,
2007) to oligotrophic (Atkinson, 1996; Pe´rez et al., 1997;
Zeldis et al., 2002; Broglio et al., 2004). Copepod grazing
on heterotrophic protozoa and selection of specific prey
can directly affect the protozoan community compo-
sition and impact the biomass and structure of popu-
lations at lower trophic levels via trophic cascades
(Leising et al., 2005b; Liu et al., 2005; Olson et al., 2006;
Vargas et al., 2008). Therefore, understanding the
feeding selectivity of a copepod species when presented
with multiple food types is critical to understanding the
carbon flow in pelagic ecosystem. However, previous
studies on copepod diets have focused on certain het-
erotrophic protozoa and/or phytoplankton groups and
doi:10.1093/plankt/fbp014, available online at www.plankt.oxfordjournals.org
# The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
JOURNAL OF PLANKTON RESEARCH j VOLUME 31 j NUMBER 6 j PAGES 647–659 j 2009

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microplankton (Gifford and Dagg, 1991; Levinsen et al.,
2000; Vincent and Hartmann, 2001; Calbet et al., 2002;
Broglio et al., 2004; Olson et al., 2006; Castellani et al.,
2008). There is little information on the relative import-
ance of multiple food items including all groups of
auto- and heterotropic protozoa within the natural diet
of copepods (Liu et al., 2005; Castellani et al., 2008;
Vargas et al., 2008). Without including the dietary con-
tribution of both heterotrophic and autotrophic prey
composition and size, the total grazing rate, prey selec-
tivity and egg production of copepods cannot be pre-
dicted or causally related to measurements of food
availability.
The Ulleung Basin is a deep oceanic depression
located in the southwestern part of the East Sea/Japan
Sea, surrounded by Korea and Japan. In this study area,
most zooplankton research has focused on copepod
abundance and composition related to the physical prop-
erties of the water column (Park and Choi, 1997; Kang
et al., 2004) in the Uleung Basin and transition regions of
the North Korean Cold Current/East Korean Warm
Current. The calanoid copepods Calanus sinicus and
Neocalanus plumchrus are the major large species of meso-
zooplankton, together with the other large copepod
Metridia pacifica and the small copepods Paracalanus parvus
and Clausocalanus sp. (Lee, 2004; Ashjian et al., 2005).
They are also an important prey of fishes such as Pacific
saury and mackerel (Odate, 1994; Uye et al., 1999).
However, studies on feeding activity of these copepods in
the Ulleung Basin have not been made previously.
Therefore, this paper represents the first study toward
improving our understanding of the protozoa–copepod
trophic link in a pelagic ecosystem of the Ullueng
Basin. Our study objective was to elucidate the relative
contribution of auto- and heterotrophic protozoa to the
diet of copepods residing in the Ulleung Basin. The
impact of two calanoid copepods, namely C. sinicus and
N. plumchrus, on auto- and heterotrophic protozoan
assemblages was investigated, and the copepod ingestion
rates and selective predation preferences were evaluated
in terms of the prey carbon biomass and size spectra.
Furthermore, as ingestion rates and selective predation
can vary according to copepod species, trophic status
and prey items, we conducted experiments at different
stations during two different seasons to compare results.
METHOD
Grazing experiments
To analyze data under various food conditions, exper-
iments were conducted at two or three stations during
summer (August 2005 and 2007) and spring (April
2006) in the Ulleung Basin (Fig. 1; Table I). Water
samples were collected from the depth of the 70% light
level and surface using Niskin bottles and were gently
transferred to a 20-L carboy. Live zooplankton were col-
lected with a conical-type net (mouth diameter 60 cm;
mesh 200 mm) towed vertically from a depth of 150 m
to the surface. For the grazing experiments, copepods
larger than 2 mm in body length, which included either
a mixture of C. sinicus adults and copepodites, and
N. plumchrus copepodites (hereafter referred to as
C. sinicus and N. plumchrus), were selected using a wide-
mouthed pipette. C. sinicus, which was dominant during
the summer, was collected for the experiments in
August 2005 and August 2007. N. plumchrus, which was
dominant during the spring was collected for the exper-
iments in April 2006. As difficulties in shipboard identi-
fication precluded the rapid separation of copepodites,
they were sorted without consideration of gender or
stage. Approximately 50% of these were later verified as
C. sinicus adults, and the remainder was almost entirely
their late-stage copepopodites. Owing to the absence of
adult N. plumchrus in April at this study site, we selected
copepodite stages of N. plumchrus for the experiment in
April. The sorted copepods were transferred to 2.3-L
polycarbonate bottles, two to four individuals of cope-
pods per bottle, with water prescreened by gentle
reverse filtration through a 200-mm mesh to remove
Fig. 1. Map showing the location of the sampling stations where
water and copepods for the grazing experiments were collected in the
Ulleung Basin. See Table I for additional information on the station
characteristics.
JOURNAL OF PLANKTON RESEARCH j VOLUME 31 j NUMBER 6 j PAGES 647–659 j 2009
648

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other copepods and large grazers. Each experiment was
prepared as three sets of replicate treatment bottles,
including initial samples and control bottles without
copepods. To override any nutrient enrichment effects
from copepod excretion in the grazing bottles, the
experimental water was enriched with a nutrient
mixture of 5 mM NH
4
Cl and 1 mM Na
2
HPO
4
. The
experimental bottles were incubated in a flow system
incubator, maintained at a temperature within +0.58C
of the ambient field temperature, and manually rotated
every 3–4 h during incubations. The experimental
incubators were illuminated with light at an intensity of
70% of the ambient light, and ambient light for
Experiments 2A and 2C. At the beginning and end of
each incubation period, we collected subsamples for
assessing the microbial abundance and composition
and chlorophyll (Chl a).
Sample analysis and calculations
We fractionated the Chl a pigments into two size cat-
egories, total and pico Chl a (,3 mm), by passing water
samples through 3-mm polycarbonate membrane filters.
We filtered 500 mL of seawater through Whatman GF/F
filters. The Chl a concentrations in each sample
were determined using a Turner Designs fluorometer
(TD-700) following extraction with 90% acetone
(Parson et al., 1984). To determine the abundance of
picoplankton and nanoflagellates, 100 mL of water was
preserved with glutaraldehyde (1% final concentration),
and subsamples of 70–100 mL were filtered through
black Nuclepore filters ( pore size 0.45 mm), stained with
4
00
-6-diamidino-2-phenylindole (DAPI; 5 mg mL
21
final
concentration) and proflavin (0.33%), and examined
under an epifluorescence microscope (Nikon type 104)
at a magnification of Â600–1000. We counted the
autotrophic and heterotrophic flagellates under both
UV and blue excitation wavelengths to distinguish the
autofluorescence of chlorophyll and other pigments. We
counted the cells in at least 50 fields (n 100). To deter-
mine the abundance of microplankton we preserved
250 mL of water with acid Lugol’s iodine (5% final con-
centration) and formalin (2% final concentration),
respectively. The Lugol-preserved samples were stored
in the dark, and the formalin-preserved samples were
stored at 48C in the dark until analysis. To determine
the abundance of the ciliates and diatoms, 100 mL
samples preserved in Lugol’s solution were concentrated
in sedimentation chambers for 48 h and counted
under an inverted microscope (Olympus IX 70) at mag-
nifications of Â200. To determine the abundance of
the dinoflagellates, 100 mL samples preserved in forma-
lin were concentrated in sedimentation chamber for
48 h in a refrigerator (48C), stained with DAPI (5%
final concentration), and then counted under an
inverted epifluorescence microscope at a magnification
of Â200. To estimate the carbon biomass of all organ-
isms, we calculated the cell volume by measuring the
cell dimensions using an ocular micrometer (Edler,
1979). The factors and equations used to convert cell
volume to carbon biomass were as follows:
220 fgC mm
23
for nanoflagellates and picoplankton
(Børsheim and Bratbak, 1987); 0.19 mgC mm
23
for
naked ciliates (Putt and Stoecker, 1989); carbon ( pg) ¼
44.5 þ 0.053 Â lorica volume (mm
3
) for loricate ciliates
(Verity and Langdon, 1984); and carbon ( pg) ¼
0.216 Â (volume, mm
3
)
0.939
for dinoflagellates and
diatoms (Menden-Deuer and Lessard, 2000).
In the laboratory, we identified the copepods under a
stereomicroscope (Stemi 2000C; Zeiss), and their
prosome lengths were measured using an ocular
micrometer. The body carbon weights of the copepods
were estimated from the prosome lengths, using
the body length–weight relationship established for
C. sinicus by Uye (Uye, 1988) and for N. plumchrus by Liu
and Hopcroft (Liu and Hopcroft, 2006). The mean
body carbon content was 46.2 mgC copepod
21
for
C. sinicus and 183.1 mgC copepod
21
for N. plumchrus.
Table I: Initial conditions for copepod grazing experiments conducted with C. sinicus adults and
copepodites and N. plumchrus copepodites in the study area
Experiment
Number Date
Station (sample
depth, m) Temperature (88888C) Predator
Number
per bottle
Incubation
time (h) Chl a a (mg L
21
)
Pico-Chl
a a (%)
1A 5 August D2 (10) 18.8 C. sinicus (adult and stage V) 3 24 0.40 70.0
1B 5 August D4 (10) 20.1 C. sinicus (adult and stage V) 4 20 0.21 52.4
2A 7 August D2 (0) 22.1 C. sinicus (adult and stage V) 4 24 2.90 15.5
2B 7 August D2 (20) 19.5 C. sinicus (adult and stage V) 4 24 0.79 43.0
2C 7 August D4 (0) 22.5 C. sinicus (adult and stage V) 4 24 0.15 66.7
2D 7 August D4 (30) 15.4 C. sinicus (adult and stage V) 4 24 0.95 61.1
3A 6 April D1 (30) 12.5 N. plumchrus (stage V) 4 22 2.00 34.9
3B 6 April D2 (30) 11.1 N. plumchrus (stage V) 4 24 1.28 68.8
3C 6 April D4 (10) 10.3 N. plumchrus (stage V) 4 24 1.45 68.7
E. J. YANG ET AL. j CONTRIBUTION OF PROTOZOA TO THE DIET OF COPEPODS
649

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Ingestion rate and clearance rate of copepods on
auto- and heterotrophic protozoa were calculated using
Frost’s equation (1972). In experiments, the results from
all replicates were averaged. Ingestion rate was calcu-
lated only when the difference in prey concentration
between control and experimental bottles proved signifi-
cant (t-test, P ,0.05). Copepod prey selectivity was
determined using Chesson’s index of selectivity (a),
which relates ingestion rates of the different food types
to their availability (Chesson, 1983). The parameter a
indicates capture probability on the basis of the prob-
ability of prey encounter: a ¼ (r
i
/p
i
)/
P
(r
i
/p
i
), where r
i
is
the proportion of the prey i in the diet, p
i
is the pro-
portion of the prey i in the environment and
P
a ¼ 1.
If the total number of prey species is n, then a . 1/n,
selective copepod predation may have occurred.
Alternatively, if a , 1/n, prey avoidance may have
occurred. This index is density independent and
permits the determination of whether prey items were
ingested in higher or lower proportion relative to what
would be expected owing to their relative biomass in
the field (Vargas et al., 2008). Using these data, we
examined whether certain size ranges or groups were
selected preferentially by the copepods.
RESULTS
Initial grazing condition, prey biomass
and composition
The water temperatures ranged from 15.4 to 22.58C in
August and from 10.3 to 12.58C in April (Table I). In
summer 2007, there was a seasonal thermocline with
temperature difference between surface and mixed
layer. Initial Chl a concentrations ranged between 0.15
and 2.90 mg L
21
and were relatively high in April
during the early spring bloom except for Experiment
2A. The Chl a concentration was higher at the coastal
sites (St. 1 for spring and St. 2. for summer) than in the
more oceanic site (St. 4). In particular, high Chl a con-
centration was observed at St. 2 of 2007 summer
(Experiment 2A) and St. 1 of spring (Experiment 3A).
The pico-size fractionated Chl a comprised the greater
percentage of the total Chl a in all experiments, with
the exceptions of Experiments 2A and 3A. Individual
auto- and heterotrophic protozoan cells were character-
ized according to their size and composition. The auto-
trophic protozoa were classified as diatoms, autotrophic
dinoflagellates (ADFs), autotrophic nanoflagellates
(ANFs) and autotrophic picoplankton (APPs), including
cyanobacteria (CYAN) and autotrophic picoeukaryotes
(APEs). The carbon biomass of the autotrophic
protozoa ranged from 12.4 to 55.2 mgC L
21
and was
highest in Experiment 2A because of the high abun-
dance of ADFs (Fig. 2). High abundance of ADFs
observed in Experiment 2A probably resulted from
coastal upwelling that often occurs off the west coast of
the East Sea/Japan Sea during summer. With the
exceptions of Experiments 2A and 3A, the APP com-
prised .50% of the total autotrophic carbon biomass.
Additionally, the diatom biomass, dominated by
Thalassiosira spp. (20–30 mm), was highest in
Experiment 3A. The heterotrophic protozoa were classi-
fied as ciliates, heterotrophic dinoflagellates (HDFs) and
heterotrophic nanoflagellates (HNFs). The carbon
biomass of the heterotrophic protozoa ranged from 3.3
to 13.0 mgC L
21
and exhibited a pattern similar to that
of the APP. The ciliate biomass comprised on average
42.2% of the heterotrophic protozoan biomass, thus
constituting the largest portion of heterotrophic proto-
zoa. The biomass of the naked ciliates accounted for
89.0% of the total ciliate biomass. The HDF biomass
comprised on average 39.1% of the heterotrophic pro-
tozoan population. The size-fractionated autotrophic
protozoa showed that the pico-sized fraction was the
most dominant of the fractions, except in Experiments
2A and 2B, and accounted for 46.7% of the total auto-
trophic protozoan biomass. Most of the heterotrophic
protozoan biomass comprised cells with sizes of 3–10,
10–20 and 20–50 mm on average, and these rep-
resented 24.4, 20.7 and 20.9% of the total biomass,
respectively. The heterotrophic protozoa comprised on
average 21.8% of the total protozoan biomass.
Copepod grazing on auto- and
heterotrophic protozoa
The carbon ingestion by the copepods was based on the
auto- and heterotrophic protozoan groups, which dif-
fered according to prey type and size (Figs 3 and 4). The
ADFs were ingested at high rates, with the exception of
those in Experiment 3A, in which the autotrophic proto-
zoa were dominated by diatoms. The maximum ADFs
ingestion rate of 5.45 mgC copepod
21
day
21
was
measured for C. sinicus in Experiment 2A (Fig. 3). The
ADFs comprised 18.4% of the total autotrophic carbon
available and 47.5% of the carbon consumed by the
copepods. The ingestion rate on diatoms was highest, at
3.6 mgC copepod
21
day
21
, in Experiment 3A. The
CYAN and APEs were ingested at undetectable or low
rates, despite the fact that they were the dominant
species in the autotrophic protozoan biomass. Of the
heterotrophic protozoa, the ciliates were ingested at high
rates by both copepods. Of the ciliate groups, the naked
ciliates were ingested at a maximum rate of
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3.4 mgC copepod
21
day
21
by C. sinicus. The naked cili-
ates comprised 40.1% of the total heterotrophic carbon
available and 49.3% of the carbon consumed by the
copepods. The copepods ingested the athecate HDFs at
an average rate of 1.3 mgC copepod
21
day
21
, with the
highest rate observed in Experiment 2A. The loricate
ciliates and the thecate HDFs did not contribute signifi-
cantly to the copepod dietary intake of heterotrophic
protozoa. Of total protozoa, the ciliates were the most
important prey item in the copepod diet (Fig. 5),
accounting for roughly 38.2% of the total daily summed
carbon rations of the auto- and heterotrophic protozoa.
The auto- and heterotrophic protozoa in the 20- to
50-mm size class were consumed preferentially during
all experiments and were ingested at rates of 1.7 and
1.3 mgC copepod
21
day
21
, respectively (Figs 4 and 5).
The copepods did not ingest, or ingested at a low
rate, the auto- and heterotrophic protozoa in the pico-
sized fraction. Overall, all of the auto- and heterotrophic
protozoa groups were ingested by the copepods at
rates of 0.3–8.6 mgC copepod
21
day
21
and 1.9–
6.5 mgC copepod
21
day
21
, respectively (Table II).
Clearance rates of copepods on auto- and heterotrophic
protozoa also ranged from 80.2 to 575.9 mL copepod
21
day
21
and from 229.8 to 705.3 mL copepod
21
day
21
,
respectively (Table II). Of the auto- and heterotrophic
groups, the heterotrophic protozoa comprised between
42.9 and 86.7% of the total carbon ration ingested,
although they comprised on average 21.5% of the total
available prey carbon (Figs 2 and 5; Table II). The daily
ingestion of auto- and heterotrophic protozoa was 4.3–
32.6% of body carbon for C. sinicus and 3.0–5.5% of
body carbon for N. plumchrus (Table II).
Prey selection
Within the auto- and heterotrophic categories, a selec-
tivity index showed an obvious difference in the auto-
and heterotrophic assemblages as well as size-dependent
patterns of prey selection (Fig. 6). The feeding patterns
exhibited by the two copepod species showed positive
selection for the ciliates and/or HDFs. The two
copepod species showed a strong preference for hetero-
trophic over autotrophic protozoa. The size-dependent
selective feeding strategy exhibited by the copepods
showed a trend toward positive selection for
Fig. 2. Initial carbon biomass (A and B) and size biomass (C and D) of auto- and heterotrophic protozoa in all experiments. APP, autotrophic
picoeukaryote; CYAN, cyanobacteria; ANF, autotrophic nanoflagellate; ADF, autotrophic dinoflagellate; HNF, heterotrophic nanoflagellate; HDF,
heterotrophic dinoflagellate.
E. J. YANG ET AL. j CONTRIBUTION OF PROTOZOA TO THE DIET OF COPEPODS
651

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heterotrophic protozoa in the 20- to 50-mm and 50- to
100-mm size categories. The auto- and heterotrophic
protozoa in the small range (,10 mm) were not selected
by the copepods. The heterotrophic protozoa in the
10- to 20-mm size range were positively selected,
whereas the autotrophic protozoa in the same size
range were negatively selected.
DI SCUSSI ON
Our results on protozoa–copepod prey/feeding inter-
actions in the Ulleung Basin in the East Sea/Japan Sea
demonstrated that: (i) C. sinicus and N. plumchrus prefer-
entially ingested heterotrophic over autotrophic proto-
zoa and at high rates, (ii) the auto- and heterotrophic
Fig. 3. Mean (+SD) ingestion rates of the copepods C. sinicus adults and copepodites and N. plumchrus copepodites on autotrophic protozoa (A)
and heterotrophic protozoa (B) based on protozoan compositions. Ingestion rates were significant between control and experimental prey
concentration (t-test, P , 0.05). NS, not significant.
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protozoa in the 20- to 50-mm size fraction were con-
sumed in the greatest abundance and preferentially by
the copepods in all experiments, and (iii) the copepods
positively selected the ciliates and HDFs from among
the protozoan populations.
Calanoid copepods are considered to be largely
omnivorous and capable of feeding on a wide range of
food items (Vincent and Hartmann, 2001; Zeldis et al.,
2002; Liu et al., 2005; Fileman et al., 2007). In our exper-
iments, the copepods grazed on both auto- and hetero-
trophic protozoa. However, both copepod species
consumed heterotrophic protozoa from among
the natural plankton assemblages at higher rates than
the autotrophic protozoa, although the autotrophic
Fig. 4. Mean (+SD) ingestion rates of the copepods C. sinicus adults and copepodites and N. plumchrus copepodites on autotrophic protozoa (A)
and heterotrophic protozoa (B) based on size class. Ingestion rates were significant between control and experimental prey concentration (t-test,
P , 0.05). NS, not significant.
E. J. YANG ET AL. j CONTRIBUTION OF PROTOZOA TO THE DIET OF COPEPODS
653

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protozoan biomass was consistently higher than that of
the heterotrophic protozoa in all experiments (Fig. 3;
Table II). The heterotrophic protozoan population in
the Ulleung Basin comprised 22.5% of the available
carbon and 62.1% of the carbon consumed by
C. sinicus, and 21.1% of the available carbon and
58.5% of the carbon consumed by N. plumchrus (Figs 3
and 5). Even though the two copepod species have
different body sizes, grazing activity on auto- and het-
erotrophic protozoa showed a similar pattern. The per-
centage of the heterotrophic protozoa in the diet was
significant when phytoplankton biomass was low and
dominated by pico-sized cell. The contribution of het-
erotrophic protozoa in the diet decreased linearly with
Chl a concentration and increased with the percent of
pico-size fractionated Chl a (Fig. 7). This result was not
related to predator size and sampling season (i.e. temp-
erature) (r
2
¼ 0.02, P .0.05). The result of our study is
in agreement with the results of Liu et al. (Liu et al.,
2005) and Fileman et al. (Fileman et al., 2007), whereas
our result contrasts with those of other studies showing
that Calanus pacificus ingested heterotrophic protozoa
preferentially and at high rates, even when diatom and
ADFs biomass far exceeded that of heterotrophic
protozoa (Leising et al., 2005a; Olson et al., 2006). The
difference in results may come from the appearance of
toxic aldehyde producing diatom and ADF on which
C. pacificus avoid feeding in Leising et al. (2005a) and
Olson et al. (2006) studies. Therefore, our study suggests
that both copepods were omnivorous in all experiments,
and the contribution of the heterotrophic protozoa to
the diet may be varied with the trophic status, prey size
and composition. The importance of the heterotrophic
protozoa in the diet of N. plumchrus has been recognized
in the subarctic North Pacific (Gifford and Dagg, 1991;
Gifford, 1993). In one study, Gifford and Dagg (Gifford
and Dagg, 1991) showed that heterotrophic prey (e.g.
ciliates and HDFs) was ingested at an average rate of
1.53 mgC copepod
21
day
21
and was 1.6–3.7 times as
important as autotrophic prey in supporting the dietary
Fig. 5. Relative significance of each protozoan compositions (A) and size class (B) to the total diets of copepods. Semi-vertical line indicates a
boundary line between autotrophic protozoa and heterotrophic protozoa.
JOURNAL OF PLANKTON RESEARCH j VOLUME 31 j NUMBER 6 j PAGES 647–659 j 2009
654

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needs of the large N. plumchrus. The feeding of C. sinicus
on heterotrophic protozoa has been studied in the
Yellow Sea and in the Japanese Inland Sea (Uye and
Murase, 1997; Huo et al., 2008). In the Yellow Sea, C.
sinicus preferentially ingested ciliates over other com-
ponents of the microplankton biomass (Huo et al.,
2008). In contrast, Uye and Murase (1997) reported that
the microzooplankton population was not an important
food source for female C. sinicus in spring in the inland
Sea of Japan.
The grazing patterns of C. sinicus and N. plumchrus on
heterotrophic protozoan communities appeared to be
very similar in all experiments (Fig. 3). Among the auto-
and heterotrophic protozoa, ciliates were ingested at sig-
nificantly higher rates by both copepod species, except
when the autotrophic protozoan biomass was domi-
nated by ADFs and diatoms (Experiments 2A and 3A).
Although the autotrophic protozoa represented a higher
biomass constituent than the ciliates, the copepod con-
sumption of ciliates comprised 60% of the summed
ingested heterotrophic protozoa and accounted for an
average of 38.3% of the total daily carbon ration of
auto- and heterotrophic protozoa (Fig. 5). Thus, the cili-
ates were identified as an important dietary item for
copepods, and the relative importance of ciliate con-
sumption clearly depends on the trophic state of the
ecosystem (Calbet and Saiz, 2005). Our results were
consistent with those reported by Calbet and Saiz
(Calbet and Saiz, 2005), who have shown that the con-
tribution of ciliates to the copepod diet can be expected
to range, on average, from 49% under oligotrophic con-
dition (i.e. phytoplankton ,50 mg L
21
) to 25% in pro-
ductive environments (i.e. phytoplankton .50 mg L
21
).
The result from this study showed that in stations where
phytoplankton carbon biomass is low (,40 mgC L
21
),
ciliates and phytoplankton contribute equally to
copepod diets (average 47.2%), whereas at coastal
stations where phytoplankton biomass is .40 mgC L
21
(Experiments 2A and 3A), the contribution of ciliate
carbon to the copepod diet decreased (average 27.4%).
Despite the low abundance of ciliates in nature, high
consumption rates of ciliates by copepods have been
reported in previous studies. The high food quality of
ciliates, encounter rates and optimal cell size of prey
have been proposed as the most likely reasons for ciliate
consumption (Wickham, 1995; Levinsen et al., 2000:
Castellani et al., 2005; Liu et al., 2005). Here, we report
that the HDFs ranked as the second most important
prey item for the copepods in all of the experiments,
comprising on average 22.3% of the total daily carbon
ration from auto- and heterotrophic protozoa (Fig. 5).
Previous studies have shown that HDFs may also play a
significant role in copepod trophodynamics and may
T
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E. J. YANG ET AL. j CONTRIBUTION OF PROTOZOA TO THE DIET OF COPEPODS
655

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represent an important link between primary producers
and higher trophic levels in certain ecosystems at
various time periods (Suzuki et al., 1999; Calbet et al.,
2002). Calbet and Saiz (Calbet and Saiz, 2005) also
reported that copepods cleared the HDFs at rates com-
parable with those at which they cleared ciliates (P .
0.05). However, despite the fact that the initial ciliate
and HDF biomasses measured by Calbet and Saiz
(Calbet and Saiz, 2005) were similar to those measured
in our experiments, we found that ciliates were ingested
at twice the rate of the HDFs. One possible explanation
for this difference in ingestion rate is that the behavior
of the faster swimming ciliates influenced the magnitude
of ingestion by enhancing the prey–predator encounter
rates (Levinsen et al., 2000). Alternatively, this difference
may be attributable to the differences that we deter-
mined between the dominant cell sizes in both prey
groups. The ciliates were dominated by cells of 20–
30 mm, whereas the HDFs were ,20 mm in size in this
study area (data not shown). Thus, the larger ciliates
encountered by the copepods in our study area might
have been more suitable as prey. Conversely, the
diatoms and ADFs were consumed preferentially by the
copepods in Experiments 2A and 3A, respectively
(Fig. 3), both of which were dominated by the 20- to
30-mm size range. This result indicates that the two
dominating trophic characteristics in our study area
include a suitable prey size and a relatively high abun-
dance of diatoms and ADFs, which may be important
for copepod feeding selectivity. Interestingly, we found
that APPs contributed little to the copepod diet, despite
their high abundance in the water column. The negli-
gible copepod grazing activity on APPs that we
observed may be due to the negative feeding selectivity
Fig. 6. Chesson’s index values calculated for selectivity patterns of the copepods with respect to protozoan composition (A) and size-class of
protozoa (B). For protozoan composition, value above 0.14 (¼1/n classes, n ¼ 7, n is number of prey classes) indicates positive selection for that
prey, while value below 0.14 indicates negative selection for that prey. For size-class of protozoa, value above 0.08 (n ¼ 12) indicates positive
selection for that prey, while value below 0.08 indicates negative selection for that prey.
JOURNAL OF PLANKTON RESEARCH j VOLUME 31 j NUMBER 6 j PAGES 647–659 j 2009
656

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on certain taxa and/or prey sizes. In particular, the pre-
ferential grazing of the copepods on APEs versus
CYAN indicates that C. sinicus and N. plumchrus had less
of a preference for CYAN.
Prey-size dependency is a well-known feature of
copepods grazing on phytoplankton (e.g. Frost, 1972);
however, little is known about size-dependent predation
on heterotrophic protozoan prey (Atkinson, 1996; Liu
et al., 2005; Castellani et al., 2008; Vargas et al., 2008).
Our results showed that the protozoan ingestion rates
were higher for prey sizes .20 mm, a size that corre-
sponds to the ciliates, dinoflagellates and diatoms. Both
the copepods also ingested heterotrophic protozoa at
higher rates than similarly sized autotrophic protozoa.
This result is consistent with the result of Atkinson
(Atkinson, 1996), who reported that ciliates and dinofla-
gellates were cleared higher than centric diatoms of
similar overall dimensions. Furthermore, the maximum
ingestion rates in all experiments occurred with prey
sizes in the range of 20–50 mm (Fig. 4). In arctic ecosys-
tems, the maximum food capture efficiency for Calanus
spp. was in the size range of 30–40 mm equivalent
spherical diameters (Levinsen et al., 2000). Calanus
finmarchicus also fed preferentially on ciliates that were
.20 mm in size (Nejstgaard et al., 1997; Liu et al.,
2005). As mentioned earlier, the ingestion rates by the
copepods on the pico-sized fraction (,3 mm) of auto-
and heterotrophic protozoa were negligible in our
experiments (Fig. 5). Interestingly, APPs contributed
little to the copepod diet, despite their high abundance
in the water column. The negligible copepod grazing
on APPs that we observed may be due to their negative
feeding selectivity on certain taxa and/or prey sizes. In
particular, the preferential grazing of the copepods on
APEs versus CYAN indicates that both copepods had
less of a preference for CYAN. This result agrees with
those of previous studies that have demonstrated that
copepods did not feed efficiently on prey ,5 mm in size
(Uye and Murase, 1997; Calbet et al., 2002; Liu et al.,
2005). Hua et al. (Hua et al., 2006) also reported that
C. sinicus avoided small-sized food items when presented
with a mixture of large- and small-sized algae. These
findings indicate that the pico-sized fraction was near or
below the threshold size at which both copepod species
can perceive and capture individual cells. In our exper-
iments, picoplankton (,3 mm) frequently increased in
cell number in the experimental bottles relative to the
control level. This increase might have been a result of
a potential trophic cascade, whereby the grazing mor-
tality of the ciliates and HDFs released the picoplankton
from any grazing pressure within the experimental
bottles. The simplest scenario to explain our result is
that both copepod species fed on a diverse assemblage
of heterotrophic protozoa (i.e. larger ciliates and HDFs),
and the larger heterotrophic protozoa fed mostly on the
picoplankton and small flagellates. As a result, a large
part of the primary production capacity might have
indirectly reached the copepods through their consump-
tion of ciliates and HDFs. Thus, the ciliates and HDFs
may provide a direct trophic linkage between the
primary producers and copepods in the study area.
Most predators exhibit prey selectivity as a strategy to
maximize their survival and reproduction capacity.
Selective feeding patterns depend on factors such as the
energetics (nutritional value) of prey, encounter rate
(search time), specific composition of the predators
(Calbet et al., 2002; Castellani et al., 2008) and life stage
of predator (Castellani et al., 2008). Our study showed
clearly that the copepods positively selected ciliates and
HDFs as food from the protozoan populations, and
specifically selected heterotrophic protozoa .10 mm
(Fig. 6) in all experiments. This selective feeding beha-
vior exhibited by the both copepods was independent of
the concentration of autotrophic protozoa. Previous
Fig. 7. Relationship between the percentage of heterotrophic
protozoa in the diet of copepods and initial autotrophic protozoan
carbon biomass (A), and the percentage of heterotrophic protozoa in
the diet of copepods and percentage of initial pico-size fractionated
Chl a (B).
E. J. YANG ET AL. j CONTRIBUTION OF PROTOZOA TO THE DIET OF COPEPODS
657

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studies reported that copepod prey selection can depend
on copepod species and life stage of copepod (Fileman
et al., 2007; Castellani et al., 2008). Although we cannot
distinguish copepod stage, selective feeding from our
results was also independent of copepod species (i.e.
C. sinicus and N. plumchrus). The effects of selective
feeding on the composition and size of food particles
indicate that the larger ciliates and HDFs may experi-
ence a stronger top-down regulation than smaller
species. Therefore, selective feeding behavior by cope-
pods, both negative and positive, may control the com-
position and size structure of natural plankton
communities in this study area, particularly those of the
heterotrophic protozoa.
In conclusion, our results indicate that both copepods
ingested heterotrophic protozoa in amounts that were
sufficient to achieve copepod nutrition and to impact
heterotrophic protozoan populations. More significantly,
both copepods did not indiscriminately ingest all sizes
of particles but instead primarily impacted the large
particles and thereby modified the size composition and
structure of the microplankton community, indirectly
altering the carbon flow of the pelagic ecosystem by a
trophic cascade. These observations emphasize that
grazing impact, copepod prey selection and potential
trophic cascades depend not only on the trophic status
but also on the composition and size of the food par-
ticles. Although C. sinicus and N. plumchrus cannot rep-
resent all the copepod community in the study areas,
and our experiments did not also cover a wide range of
food environments, the selective feeding behavior and
higher grazing pressure on the heterotrophic versus
autotrophic protozoa indicate a strong coupling
between copepods and the microbial food web in this
study area. Such studies of trophic coupling interactions
between copepods and protozoa in the Ulleung Basin
of East Sea/Japan Sea have not been reported pre-
viously. This link requires further investigation if the
East Sea/Japan Sea ecosystem dynamics are to be
better understood. Our initial study reported herein
should serve as a basis for determining feeding prefer-
ences and life stages of other copepod species to eluci-
date this potential trophic interaction.
ACKNOWLEDGEMENTS
We are grateful to Dr Roger Harris, Dr John Dolan and
two anonymous reviewers for their comments, which
improved an earlier version of the manuscript. We also
thank captain and crew of the R/V Eardo and all the
members of “Carbon Cycle in the East Sea” project for
their professional assistance during the cruise.
FUNDI NG
This work was funded by the Korea Research
Foundation Grant from the Korean Government
(MOEHRD) (KRF-2006-532-C00012) and by a grant
from the Basic Research Program of KORDI (PE
98312).
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