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Contribution of auto- and heterotrophicprotozoa to the diet of copepods in the

Ulleung Basin, East Sea/Japan SeaEUN JIN YANG1*, HYUNG-KU KANG2, SINJAE YOO2 AND JUNG-HO HYUN3

1DEEP-SEA AND MARINE GEORESOURCES RESEARCH DEPARTMENT, KORDI, ANSAN PO BOX 29, SEOUL 425-600, KOREA, 2MARINE LIVING RESOURCE RESEARCH

DEPARTMENT, KORDI, ANSAN PO BOX 29, SEOUL425-600, KOREA AND 3DEPARTMENT OF ENVIRONMENTAL AND MARINE SCIENCE, HANYANG UNIVERSITY,

ANSAN426-791, KOREA

*CORRESPONDING AUTHOR: [email protected]

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 2050 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 N T R O D U C T I O N

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; Perezet al., 1997;

Zeldis et al., 2002; Broglioet 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

(Leisinget 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: [email protected]

JOURNAL OF PLANKTON RESEARCH j VOLUME31 j NUMBER6 j PAGES647659 j 2009


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microplankton (Gifford and Dagg, 1991; Levinsen et al.,

2000; Vincent and Hartmann, 2001; Calbetet 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 protozoacopepodtrophic 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.

M E T H O D

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 mto 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

adultN. 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 wherewater and copepods for the grazing experiments were collected in theUlleung Basin. See Table I for additional information on the stationcharacteristics.


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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 NH4Cl and 1 mM Na2HPO4. 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 throughblack Nuclepore filters (pore size 0.45 mm), stained with

400-6-diamidino-2-phenylindole (DAPI; 5 mg mL21 final

concentration) and proflavin (0.33%), and examined

under an epifluorescence microscope (Nikon type 104)

at a magnification of 600 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 Lugols 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 Lugols solution were concentrated

in sedimentation chambers for 48 h and counted

under an inverted microscope (Olympus IX 70) at mag-

nifications of 200. 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 200. 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 mm23 for nanoflagellates and picoplankton

(Brsheim and Bratbak, 1987); 0.19 mgC mm23 for

naked ciliates (Putt and Stoecker, 1989); carbon (pg)

44.5 0.053 lorica volume (mm3) for loricate ciliates

(Verity and Langdon, 1984); and carbon (pg)

0.216 (volume, mm3)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 theirprosome lengths were measured using an ocular

micrometer. The body carbon weights of the copepods

were estimated from the prosome lengths, using

the body lengthweight 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 copepod21 for

C. sinicusand 183.1 mgC copepod21 forN. plumchrus.

Table I: Initial conditions for copepod grazing experiments conducted with C. sinicus adults andcopepodites and N. plumchrus copepodites in the study area

Experiment

Number Date

Station (sample

d ep th , m ) Te mp eratu re (88888C) Predator

Number

per bottle

Incubation

time (h) Chl aa(mg L21)

Pico-Chl

aa(%)

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

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Ingestion rate and clearance rate of copepods on

auto- and heterotrophic protozoa were calculated using

Frosts 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 wasdetermined using Chessons 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 (ri/pi)/P

(ri/pi), whereriis

the proportion of the prey i in the diet, pi is the pro-

portion of the prey i in the environment andP

a 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.

R E S U L T S

Initial grazing condition, prey biomassand composition

The water temperatures ranged from 15.4 to 22.58C inAugust 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 L21 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, withthe 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 L21 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. (2030 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 L21 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 310,

1020 and 2050 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- andheterotrophic 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 copepod21 day21 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 thecopepods. The ingestion rate on diatoms was highest, at

3.6 mgC copepod21 day21, 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 copepod21 day21 byC. sinicus. The naked cili-

ates comprised 40.1% of the total heterotrophic carbonavailable and 49.3% of the carbon consumed by the

copepods. The copepods ingested the athecate HDFs at

an average rate of 1.3 mgC copepod21 day21, 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 and1.3 mgC copepod21 day21, 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 0.38.6 mgC copepod21 day21 and 1.9

6.5 mgC copepod21 day21, respectively (Table II).

Clearance rates of copepods on auto- and heterotrophic

protozoa also ranged from 80.2 to 575.9 mL copepod21

day21

and from 229.8 to 705.3 mL copepod21

day21

,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 3.05.5% of

body carbon forN.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, autotrophicpicoeukaryote; CYAN, cyanobacteria; ANF, autotrophic nanoflagellate; ADF, autotrophic dinoflagellate; HNF, heterotrophic nanoflagellate; HDF,heterotrophic dinoflagellate.


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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.

D I S C U S S I O N

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 preyconcentration (t-test, P, 0.05). NS, not significant.


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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 anddominated 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) (r2 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

Olsonet 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 sizeand composition. The importance of the heterotrophic

protozoa in the diet ofN. 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 copepod21 day21 and was 1.63.7 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 aboundary line between autotrophic protozoa and heterotrophic protozoa.


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needs of the large N. plumchrus. The feeding ofC. 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 e t al .,

2008). In contrast, Uye and Murase (1997) reported thatthe microzooplankton population was not an important

food source for female C. sinicus in spring in the inland

Sea of Japan.

The grazing patterns ofC. 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 L21

) to 25% in pro-ductive environments (i.e. phytoplankton .50 mg L21).

The result from this study showed that in stations where

phytoplankton carbon biomass is low (,40 mgC L21),

ciliates and phytoplankton contribute equally to

copepod diets (average 47.2%), whereas at coastal

stations where phytoplankton biomass is .40 mgC L21

(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 ciliateconsumption (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

TableII:Cleara

ncerates(+SD)andingestionrates

(+SD)ofthecopepodsC

.sinicusadultandcopepoditesandN

.plumchruscopepoditeson

auto-andheterotrophicprotozoa

Experiment

num

ber

Autotrop

hicprotoz

oa

(mLcopepo

d2

1

da

y2

1

)

Heterotrop

hicprotozoa

(mLcopepo

d2

1

day2

1

)

Autotrop

hicprotozoa

(mg

Ccopepo

d2

1

day2

1

)

Heterotrop

hicprotozoa

(mg

Ccopepo

d2

1

day2

1)

Autotrop

hic

protozoa

(%bo

dy

Cday2

1

)

Heterotrop

hic

protozoa

(%bo

dy

C

day2

1

)

Ingestion

(%heterotorp

hic

protozoa

)

1A

112

.3(10

.3)

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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 measuredin 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 preypredator 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 twodominating 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. Chessons index values calculated for selectivity patterns of the copepods with respect to protozoan composition (A) and size-class ofprotozoa (B). For protozoan composition, value above 0.14 (1/n classes, n 7,n is number of prey classes) indicates positive selection for thatprey, while value below 0.14 indicates negative selection for that prey. For size-class of protozoa, value above 0.08 (n 12) indicates positiveselection for that prey, while value below 0.08 indicates negative selection for that prey.


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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 thatC. 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 athigher 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 2050 mm (Fig. 4). In arctic ecosys-

tems, the maximum food capture efficiency for Calanus

spp. was in the size range of 3040 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 ourexperiments (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 assemblageof 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(Calbetet 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 heterotrophicprotozoa in the diet of copepods and initial autotrophic protozoancarbon biomass (A), and the percentage of heterotrophic protozoa inthe diet of copepods and percentage of initial pico-size fractionatedChla (B).


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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 particlesindicate 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 versusautotrophic 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.

A C K N O W L E D G E M E N T S

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.

F U N D I N G

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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