Food selectivity and grazing impact on toxic Dinophysis spp.

by copepods feeding on natural plankton assemblages

Betina Kozlowsky-Suzuki a,b,*, Per Carlsson a,b, Alexander Ruhl c, Edna Graneli a

aDepartment of Biology and Environmental Science, University of Kalmar, Kalmar 39182, SwedenbMarine Biology, Lund University, Campus Helsingborg, Box 882, 25108 Helsingborg, SwedencUniversity of Jena, Department of Food Chemistry, Dornburger Str. 24, 07743 Jena, Germany

Received 25 February 2005; received in revised form 25 April 2005; accepted 9 May 2005

Abstract

Food selectivity and grazing impact by Acartia bifilosa, Temora longicornis and Centropages typicus on Dinophysis spp.

plankton assemblages were experimentally investigated in the Baltic Sea. Toxin analyses were carried out on phyto- and

zooplankton-dominated size fractions from field-collected samples to assess if toxins produced by Dinophysis spp. would end up

in the zooplankton. All copepod species fed actively on toxic Dinophysis spp. (Dinophysis acuta and Dinophysis norvegica).

Despite the non-selective feeding behaviour by T. longicornis and C. typicus, selectivity coefficients on D. acuta progressively

decreased as food availability increased. Similar response was not observed for A. bifilosa, which displayed an even less selective

behaviour. A. bifilosa had no significant negative effect on the net growth of D. norvegica at the lowest food concentration

offered, whereas T. longicornis and C. typicus had significant negative effects on the net growth of D. acuta at low

concentrations, similar to those observed in situ. Both species could potentially contribute as a substantial loss factor for

Dinophysis spp. provided they are abundant at the onset of the blooms. The estimated grazing impact by the copepod populations

was only considerable when C. typicus abundance was high and D. acuta population in sharp decline. Our results suggest that

when high abundance of grazers and poor growth condition of prey populations prevail, grazing impact by copepods can

contribute considerably to prevent Dinophysis spp. populations to grow or to cause the populations to decline. Okadaic acid was

detected in the zooplankton size fraction at one occasion, but the concentration was far lower than the one expected from the

ingested toxins. Thus, even if copepods may act as vectors of DSP-toxins to higher trophic levels, the amount of these toxins

transported in the food web by copepods seems limited.

# 2005 Elsevier B.V. All rights reserved.

Keywords: Baltic Sea; Dinophysis; Food selectivity; Grazing impact; Toxin retention

www.elsevier.com/locate/hal

Harmful Algae 5 (2006) 57–68

* Corresponding author. Present address: Departamento de Cien-

cias Naturais, Universidade Federal do Estado do Rio de Janeiro,

UNIRIO, Av. Pasteur 458 Urca, 22290-040 Rio de Janeiro, Brazil.

E-mail address: betinaksuzuki@unirio.br

(B. Kozlowsky-Suzuki).

1568-9883/$ – see front matter # 2005 Elsevier B.V. All rights reserved

doi:10.1016/j.hal.2005.05.002

1. Introduction

Phytoplankton growth and loss rates are governed

by several factors and in many cases, when growth

control fails, algal blooms are established. Such blooms

.

B. Kozlowsky-Suzuki et al. / Harmful Algae 5 (2006) 57–6858

can be formed by different phytoplankton species and

some of them have harmful impacts on the ecosystem.

Thus, the understanding of bloom formation, main-

tenance and decay in nature is of major priority.

Successful phytoplankton growth is primarily depen-

dent on the maintenance of the reproductive stocks in

suspension within the euphotic zone and on favourable

nutrient conditions. Loss processes represent sedimen-

tation of the cells to the bottom, or mortality caused by

other abiotic factor(s) as well as by negative biotic

interactions, such as grazing, parasitism and allelo-

pathy (Reynolds, 1990).

Dinoflagellates of the genus Dinophysis do not

usually attain high cell numbers, which are sufficient

to discolour the water. Therefore, the term bloom for

these dinoflagellates represents concentrations ran-

ging from several hundred to thousands of cells per

liter, which is very low compared to other blooming

phytoplankton (Maestrini, 1998). The most common

feature of Dinophysis spp. blooms seems to be the

formation of dense patch populations (Maestrini,

1998). Patchy distribution of Dinophysis norvegica,

usually concentrated in the thermocline, could

possibly be adopted to sequester inorganic nutrients,

but most likely to meet growth demands through

heterotrophic means (Gisselsson et al., 2002). Para-

sitism seems to contribute with minor loss rates for D.

norvegica in the Baltic Sea (Gisselsson et al., 2002;

Salomon et al., 2003). Although it has been suggested

that low grazing pressure may be one of the major

factors allowing D. norvegica populations to accu-

mulate in this system (Carpenter et al., 1995), this

hypothesis has yet not been tested. Grazing impact by

zooplankton on harmful algal blooms has been

reported (Turner and Anderson, 1983; Watras et al.,

1985; Uye, 1986), however, the importance of

copepods on the suppression of toxic blooms seems

limited (Calbet et al., 2003; Wexels Riser et al., 2003).

Scientific interest on the dinoflagellate genus

Dinophysis derives from the worldwide DSP (Diar-

rhetic Shellfish Poisoning) outbreaks. The occurrence

of DSP toxins in field samples has often been reported

and the relationship with shellfish contamination

thoroughly investigated. In the Baltic Sea,D. norvegica

can produce okadaic acid (OA), pectenotoxin 2 (PTX2)

and seco acid (PTX2SA) (Goto et al., 2000). OA has

also been found in mussels (Pimia et al., 1998) and

flounder (Sipia et al., 2000) in the Baltic Sea. The fate of

these toxins in the environment and their effects on

zooplankton are however relatively unknown (Carlsson

et al., 1995; Maneiro et al., 2000, 2002). Both

avoidance and consumption of toxic Dinophysis spp.

by copepods have been reported (Turner and Anderson,

1983; Carlsson et al., 1995; Maneiro et al., 2000, 2002;

Wexels Riser et al., 2003) and the importance of

copepod faecal pellets to the fate of DSP toxins

evaluated (Maneiro et al., 2002).

In this study, we aimed to investigate to what extent

Dinophysis species are consumed by different

copepod species and how food selectivity is modu-

lated under increasing food concentrations. According

to optimal foraging theory, consumers should switch

to better quality food types as food availability

increases (e.g. DeMott, 1988, 1995). In this sense,

toxic food particles would be progressively selected

against when food is abundant. We also wanted to

assess whether the grazing impact by copepods on

Dinophysis spp. accounts for a substantial loss factor

for these toxin-producing dinoflagellates in the Baltic

Sea. Experiments were conducted in the brackish

Baltic Proper and in the more marine environment of

the Oresund (Strait between Sweden and Denmark). In

addition, toxin analyses on different size fractions and

single cells of the different Dinophysis species from

field-collected samples were conducted to observe if

these dinoflagellates produced toxins and whether the

toxins would be detectable in the zooplankton under

natural conditions.

2. Material and methods

2.1. Sampling

Water samples were collected in the Baltic Proper

outside the east coast of Oland (5685504000N,

1685301000E) on 7 June and 17 July 2001 and in the

Oresund (5585801200N, 1284002300E) on 13 and 17 July

2002. At each sampling occasion, several hundred

liters of water were pumped from the depth (between

10 and 18 m) (Table 1), where Dinophysis spp.

(hereafter termed Dinophysis depth) concentrations

were highest (checked with a portable microscope),

and filtered on a 25 mm net, partly submersed in water.

These concentrated samples (>25 mm) were diluted

with 25 mm-screened in situ water and transported to

B. Kozlowsky-Suzuki et al. / Harmful Algae 5 (2006) 57–68 59

Table 1

Depth, salinity, temperature and dominant Dinophysis and copepod species at sampling depth in June–July 2001 (Baltic Proper) and in July 2002

(Oresund)

Site/date Depth (m) Salinity (%) Temperature (8C) Dinophysis Copepod species

Baltic Proper

7 June 2001 12 6 12 D. acuminata Acartia bifilosa

17 July 2001 18 6 12 D. norvegica Acartia bifilosa

Oresund

13 July 2002 14 18.7 16 D. acuta Acartia spp., T. longicornis

17 July 2002 10 15.6 18 D. acuta Centropages typicus

the laboratory. In the laboratory, they were filtered

through nets of decreasing mesh size and concentrated

in different size fractions. In the Baltic Proper, those

were 40–70, 70–150 and >150 mm, while in the

Oresund, 25–70, 70–100 and >100 mm. The con-

centrated fractions were divided in aliquots for the

enumeration of the dominant phyto- and zooplankton

and for toxin analysis.

Dinophysis spp.-dominated size fractions (40–

70 mm from the Baltic Proper and 25–70 mm from

the Oresund), which were used to prepare the food

suspensions for the feeding experiments were

obtained at the Dinophysis depth applying the same

procedure described above. At the laboratory, these

samples were further diluted with 25 mm-filtered

seawater and placed in a cold room with similar

temperature to that recorded at the sampling depth.

Copepods for the feeding experiments were collec-

ted with a 100 mm net by vertical hauls (20 m to

surface).

2.2. Cell counts and toxin analysis from the

Dinophysis depth

Phytoplankton counts from the fractionated sam-

ples were done in micro-well plates or in 2 ml

sedimentation chambers depending on the concentra-

tion of the cells. At least 200 Dinophysis spp. cells,

and more than 600 cells in total were counted per

sample. In most cases, entire sub-samples were

counted, but occasionally dense taxa were enumerated

in diagonals. Zooplankton in the concentrated

zooplankton-size fractions (>100 or >150 mm) was

counted in 2 ml sedimentation chambers.

Samples from the Baltic Proper (40–70 mm size

fraction and initial food suspensions for the feeding

experiments) were analysed for OA, dinophysistoxin-

1 (DTX1), pectenotoxin-1 (PTX1), pectenotoxin-6

(PTX6), PTX2 and PTX2SA according to Goto et al.

(2001). Filters were extracted in 80% methanol:Milli-

Q water in a sonicator bath (Bandelin, Sonorex TK 52)

for 15 min and centrifuged (5417 C, Eppendorf) at

14,000 rpm for 10 min. The supernatant was filtered

through a 0.2 mm PTFE membrane and 0.5 ml was

dried with gaseous N2.

Samples collected in the Oresund (also single

cells of Dinophysis acuminata, D. acuta and D.

norvegica), and the larger size fractions from the

Baltic Proper were only investigated for the presence

of OA. Single Dinophysis spp. cells were obtained by

fractionating plankton cells using 25 and 70 mm

nylon nets (small circular nets glued onto 10 cm high

plastic cylinders). The cells were back-flushed from

the 25 mm net with cold filtered seawater into 1 ml

Sedgewick-Rafter counting chambers, which hade

been kept cold on ice packs. Single cells of D.

acuminata, D. acuta and D. norvegica were then

manually picked at 100� magnification in an

inverted microscope using microcaps and micro-

capillaries (100 ml) that had been melted in a flame

and drawn to become very thin in the end. The cells

were then washed in filtered sewater three times in

SR-counting cells, placed in scintillation vials

containing 10 ml GF/F-filtered seawater. The cells

were then filtered onto GF/F-filters and frozen

(�20 8C) until analysis.

After treatment of the plankton samples (extrac-

tion, centrifugation and filtration as described above),

the filtered extracts were analysed by liquid chroma-

tography–electrospray ionisation mass spectrometry

(LC–ESI-MS) (Hummert et al., 2000) using a Perkin-

Elmer series 200 autosampler and pump, coupled to an

Applied Biosystems API 165 mass spectrometer.

Briefly, the extracts were separated on a reversed

B. Kozlowsky-Suzuki et al. / Harmful Algae 5 (2006) 57–6860

phase column (Luna, 3 mm C18, 150 mm � 4.60 mm,

i.d. with a 30 mm � 4.60 mm, i.d. guard column, both

from Phenomenex, Torrance, CA, USA) by applica-

tion of acetonitrile, methanol and 0.1 M acetic acid as

eluents (gradient mode). The mass spectrometer was

operated in selective ion monitoring (SIM) mode

using negative ionisation of OA, detected as [M � H]�

ion at m/z 803.4. Quantification was performed using

peak areas.

2.3. Feeding experiments

We tested the effect of the incubation time (24 or

48 h) on Acartia bifilosa and Temora longicornis

feeding rates and on the net growth of Dinophysis spp.

on June 2001 in the Baltic Proper. Since significant

feeding was only detected after 48 h and this

incubation time did not affect Dinophysis spp. net

growth rates compared to a shorter incubation time,

we decided to use 48 h incubations for all other

experiments.

The dominant copepod species at the Dinophysis

depth (Table 1) were used in the feeding experiments.

Acartia bifilosa was used in the Baltic Proper (July

2001), while Temora longicornis and Centropages

typicus were used in the Oresund experiments (13 and

17 July 2002, respectively). Dinophysis spp. densities

in the food suspensions offered to the copepods in the

Baltic Proper were high but realistic (e.g. Carpenter

et al., 1995), and ranged from 26,733 � 1547

(mean � S.D.) to 120,171 � 8692 cells l�1. In the

Oresund experiments, similar Dinophysis spp. abun-

dances as those recorded in situ (determined by

counting phytoplankton samples collected with a 2.5 l

Niskin bottle at the same depth as the sample taken

with the pump) were provided. They ranged from

170 � 49 to 5867 � 995 cells l�1. In the Baltic

Proper, calculated carbon concentrations in the food

suspensions ranged from ca. 200 to 938 mg C l�1,

while in the Oresund, they ranged from 19 to

1397 mg C l�1.

Copepod adult females were picked up individually

and kept in filtered seawater (Whatman GF/C)

overnight at the same temperature used during the

incubations. After the starvation period, ca. 10

(Temora longicornis and Centropages typicus) or 15

(Acartia bifilosa) females per bottle were incubated

for 48 h in 550-ml plastic bottles (Oresund) or 250-ml

tissue culture flasks (Baltic Proper) with the desig-

nated food suspension. Bottles with copepods were

usually run in triplicates for each food concentration,

except in two cases where four bottles were used.

Control bottles were also mostly run in triplicates,

except in two cases where two bottles were used. All

bottles were placed on a plankton wheel at a

temperature similar to that observed in situ

(Table 1) and dim light in a 12-h light:12-h dark

cycle.

Phytoplankton and ciliate counts were done from

samples (50–250 ml) taken at 0 and 48 h of the feeding

experiment and preserved in acid Lugol’s solution.

Entire samples were counted in sedimentation

chambers of different volumes depending on the

density of the cells, except for very dense that taxa

were enumerated in diagonals. At least 20 cells of the

initial samples were measured to estimate the cell

volumes.

Clearance and ingestion rates were estimated

according to Frost (1972), where the disappearance

of food particles over the incubation time in the

copepod bottles is compared with the controls.

Ingestion rates of the different food types were

converted to carbon by employing a conversion factor

of 0.11 pg mm�3 for phytoplankton and ciliates and

0.13 pg mm�3 for armoured dinoflagellates (Edler,

1979). Food selectivity was determined by the

selectivity coefficient a relating the ingestion rates

on the different food types to their availability

(Chesson, 1978). No selection occurs when a = m�1

(where, m = number of food types available) and food

items are fed upon in the same proportions as their

availability, if a > m�1 selection is positive; when

a < m�1 selection is negative.

To assess the effect of copepod grazing on the net

growth of Dinophysis spp. under our experimental

conditions, we estimated the grazing effect. For this,

Dinophysis spp. net growth rates were estimated by

cell counts and calculated as:

m ¼ ln ðNt=N0Þt

where m is the net growth rate of the different

Dinophysis spp. populations, Nt and N0 the densities

of Dinophysis spp. at time t and 0 h, respectively and

t is the incubation time (2 days). Then, the per-

individual copepod effect (Dr, grazing effect) on

B. Kozlowsky-Suzuki et al. / Harmful Algae 5 (2006) 57–68 61

the net growth rate of the different Dinophysis spp.

was calculated using the metric described in Osen-

berg et al. (1997):

Dr ¼ mg � mc ¼

�ln

�Ntg

N0g

�� ln

�Ntc

N0c

��

tG

where mg and mc are the net growth rates of the

different Dinophysis spp. populations, Ntg and N0g

(and Ntc and N0c) are the densities of Dinophysis

spp. in the grazers (g) and control (c) bottles at time

t and 0 h, respectively, t the incubation time (2 days)

and G is the number of grazers per bottle. The

potential grazing impact by copepods on Dinophysis

spp. in situ was estimated by multiplying the natural

(at Dinophysis depth) copepod abundance by their

ingestion rates on Dinophysis species at each prey

concentration offered in the incubations.

All data were tested for homogeneity of variances

and normality. If those assumptions were not met,

the data were log- or square root-transformed.

Clearance and ingestion rates were tested with

one-way multivariate analysis of variance (MAN-

OVA), followed by one-way analysis of variance

(ANOVA) and the Tukey HSD a posteriori test if

responses were significant. In order to test if the

selectivity coefficients for Dinophysis spp. decreased

with increasing food availability, one-way analysis

Table 2

Acartia bifilosa, Temora longicornis and Centropages typicus mean (S.D.) i

and total ingestion rate at each food concentration (total food concentrat

Copepod species Food concentration (mg C l�1)

Total D. norv

Acartia bifilosa

204 (13) 185 (11

466 (28) 419 (17

927 (62) 833 (60

Total D. acut

Temora longicornis

83 (4) 3 (0.

194 (26) 7 (2)

970 (127) 36 (6)

1397 (345) 70 (12

Centropages typicus

19 (2) 2 (0.

102 (13) 9 (2)

338 (39) 72 (12

of covariance (ANCOVA) was used with food

concentration as a covariable. t-Tests, followed by

the sequential Bonferroni method applied to adjust

the alpha values (Peres-Neto, 1999), were used to

assess whether the selectivity coefficients for the

different food types were significantly different from

the non-selection value and if the grazing effect on

Dinophysis spp. by the different copepods were

significantly different from zero.

3. Results

3.1. Dinophysis spp. cell concentrations and toxin

profile in the phyto- and zooplankton size fractions

at Dinophysis depth

In the Baltic Proper, Dinophysis acuminata and D.

norvegica were the dominant species. D. rotundata

occurred at lower numbers. In June, D. acuminata

(208 cells l�1) was more abundant in the 40–70 mm

size fraction than D. norvegica (42 cells l�1), while the

latter dominated in July (982 cells l�1). OA, DTX1,

PTX1 and PTX6 were not detected in any of the samples

and size fractions. PTX2 and PTX2SAwere detected in

the 40–70 mm size fraction and the increase in their

concentrations in July (from 0.69 and 0.14 ng l�1 to

20.11 and 38.51 ng l�1, respectively) seemed con-

ngestion rate (ng C ind�1 h�1) on Dinophysis norvegica and D. acuta

ion and Dinophysis spp. concentration)

Ingestion rate (ng C ind�1 h�1)

egica Total D. norvegica

) 18 (13) 12 (13)

) 49 (16) 41 (13)

) 153 (31) 150 (29)

a Total D. acuta

4) 92 (5) 5 (0.3)

187 (72) 9 (5)

671 (118) 23 (17)

) 198 (183) 23 (7)

6) 5 (1) 2 (0.6)

58 (9) 7 (2)

) 130 (41) 40 (20)

B. Kozlowsky-Suzuki et al. / Harmful Algae 5 (2006) 57–6862

Fig. 1. Selectivity coefficient (a) of Acartia bifilosa (mean � S.D.)

feeding at (a) 204 mg C l�1, (b) 466 mg C l�1 and (c) 938 mg C l�1

in the Baltic Proper. The dotted line shows the a-value (0.17) where

no selection occurs. Meso, Mesodinium sp.; Diat, centric diatom;

Pse, Pseudoanabaena sp.; Aph, Aphanizomenon sp.; Wor, Woroni-

chinia; Dnor, Dinophysis norvegica.

nected to an increase in the abundance of D. norvegica.

Accordingly, PTX2 and PTX2SA were found in the D.

norvegica-dominated food suspensions of the feeding

experiment, yielding a toxin quota of 43.78 and

8.16 pg cell�1, respectively.

In the Oresund, Dinophysis acuta (reaching

712 cells l�1) was the most abundant Dinophysis

species, containing the highest OA cell quota

(3.41 pg cell�1). OA was also found in D. norvegica

cells (1.70 pg cell�1), but not detected inD. acuminata.

OA concentration ranged from 0.27 � 0.01 (mean �S.D.) on 13 July to 0.49 � 0.01 ng l�1 (17 July) in the

25–70 mm fraction and from 0.02 � 0.00 (13 July) to

0.16 � 0.00 ng l�1 (17 July) in the 70–100 mm frac-

tion, but only found in the zooplankton fraction

(>100 mm) on 17 July. At this occasion, Centropages

typicus copepodites and adults (12 ind l�1) were the

most abundant animals in the zooplankton fraction,

which contained less than 1% (0.005 ng l�1) of the

toxin content in the phytoplankton size fractions.

3.2. Feeding rates on Dinophysis spp. and food

selectivity with increasing food availability

In the Baltic Proper, Acartia bifilosa survival

during the experiment was high (96 � 4%,

mean � S.D.). D. norvegica was the most abundant

food type comprising up to 90% of the total available

carbon. Clearance and ingestion rates on D. norvegica

ranged from 0.08 to 0.2 ml ind�1 h�1 and from 12 to

150 ng C ind�1 h�1, respectively (Table 2). Both the

total ingestion rate and the ingestion rate on D.

norvegica were highest at the highest food concentra-

tion (one-way ANOVA, Tukey HSD; p < 0.01). The

contribution of D. norvegica to the total ingested

carbon by A. bifilosa ranged from 83% to 99%.

However, the selectivity coefficient for the toxic

dinoflagellate was neither different from the non-

selection value (Fig. 1) nor affected by food

availability (one-way ANCOVA; p > 0.05). Although

Mesodinium sp. was readily included in the diet of A.

bifilosa, selectivity for this ciliate was not significantly

different from the non-selection value.

In the Oresund, Temora longicornis survival was

88 � 15% (mean � S.D.). Ceratium furca was the

dominant food type contributing to at least 70% of the

total available carbon followed by D. acuta (4% of the

total food availability). Clearance and ingestion rates

on D. acuta ranged from 0.4 to 4 ml ind�1 h�1 and

from 5 to 23 ng C ind�1 h�1, respectively. Maximum

rates were calculated for T. longicornis feeding on C.

furca (up to 5 ml ind�1 h�1 and 637 ng C ind�1 h�1).

The total ingestion rate and ingestion rate on C. furca

were highest at 970 mg C l�1 (one-way ANOVA,

Tukey HSD; p < 0.05), whereas the ingestion rate on

D. acuta was higher at the two highest food

concentrations (970 and 1397 mg C l�1) than at

83 mg C l�1 (one-way ANOVA; p < 0.05). Although

not different (t-test; p > 0.0045) from the non-selection

value (Fig. 2), the selectivity coefficient for D. acuta

progressively decreased with increasing food con-

centration and was lower at 1397 than at 83 mg C l�1

(one-way ANCOVA, Tukey HSD; p < 0.05). Even

B. Kozlowsky-Suzuki et al. / Harmful Algae 5 (2006) 57–68 63

Fig. 2. Selectivity coefficient (a) of Temora longicornis (mean � S.D.) feeding at (a) 83 mg C l�1, (b) 194 mg C l�1, (c) 970 mg C l�1 and (d)

1397 mg C l�1 in the Oresund. The dotted line shows the a-value (0.09). Ci, ciliate <25 mm; Da, Dactyliosolen sp.; Thal, Thalassiosira sp.; Cy,

cyanobacteria; Ch, Chaetoceros sp.; Tha, Thalassionema sp.; Ps, Pseudo-nitzschia sp.; Din, dinoflagellates (cf. cysts); Cf, Ceratium furca; Ske,

Skeletonema costatum; Dacuta, Dinophysis acuta.

though the contribution of S. costatum, Pseudo-

nitzschia sp., Chaetoceros sp. and ciliates <25 mm to

T. longicornis diet increased at high food concentra-

tions, the selectivity coefficients for these taxa were not

different from the non-selection value (t-test;

p > 0.0045).

Survival by Centropages typicus was 79 � 14%

(mean � S.D.). Clearance and ingestion rates on D.

acuta ranged from 1.2 to 1.8 ml ind�1 h�1 and from

2 to 40 ng C ind�1 h�1, respectively. As observed for

Temora longicornis, maximum ingestion rate was

observed on Ceratium furca (up to 105 ng C ind�1

h�1). Ingestion rate on D. acuta and total ingestion

rate increased with increasing food availability

(Table 2), and were highest at the highest food

concentration (one-way ANOVA; p < 0.001, Tukey

HSD; p < 0.02). Peridinales >30 mm was the only

food type selected for (Fig. 3). Although not

different (t-test; p > 0.016) from the non-selection

value (Fig. 3), the selectivity coefficient for D. acuta

was lowest at the highest food concentration (one-

way ANOVA, Tukey HSD; p < 0.01).

3.3. Copepod grazing impact on Dinophysis spp.

and toxin retention

Acartia bifilosa, Temora longicornis and Centro-

pages typicus affected negatively the net growth of

Dinophysis spp. (negative grazing effect in Table 3).

However, whereas the grazing effect by T. longicornis

and C. typicus on D. acuta was significant at the lower

food concentrations, the effect by A. bifilosa on D.

norvegica was significant at the highest food

concentrations. We would expect a considerable

grazing impact in situ (as a percentage of prey

standing stock removed daily) on D. acuta by the

C. typicus population (up to 25%), whereas the grazing

impact by A. bifilosa and T. longicornis on Dinophysis

spp. in situ would be insignificant (0.009–1.12%).

Toxin retention in the Centropages typicus popula-

tion can be calculated by comparing the amount of

OA found in the animals collected in the Dinophysis

depth to the amount estimated taking into account

their feeding rates and egestion. The estimated toxin

content in the C. typicus female population (ca.

B. Kozlowsky-Suzuki et al. / Harmful Algae 5 (2006) 57–6864

Fig. 3. Selectivity coefficient (a) of Centropages typicus

(mean � S.D.) feeding at (a) 19 mg C l�1, (b) 102 mg C l�1 and

(c) 338 mg C l�1 in the Oresund. The dotted line shows the a-value

(0.33). Cf, Ceratium furca; Peri, peridinales >30 mm; Dacuta,

Dinophysis acuta. (*) a-Value significantly higher than no-selection

value at p < 0.016.

3 ind l�1) feeding on a natural concentration (ca.

700 cells l�1) of D. acuta (both estimated at the

Dinophysis depth), can be calculated by using

Centropages typicus ingestion rate on D. acuta

(14 cells ind�1 day�1 feeding at a D. acuta concen-

tration similar to that observed in the Dinophysis depth

and a toxin quota of 3.41 pg OA cell�1) and Eq. (2) in

Maneiro et al. (2002), which estimates the number of

Dinophysis spp. cells excreted daily with pellets.

Thus, assuming that one adult female C. typicus

ingests 0.047 (0.016) ng OA daily and egests 0.020

(0.002) ng OA day�1, we would expect to find, at

steady state, 0.087 (0.04) ng OA l�1 in the zooplank-

ton size-fraction (>100 mm) (assuming that C. typicus

was the only copepod species feeding on D. acuta).

However, only 5% (0.005 ng OA l�1) of the calculated

toxin content was detected in the zooplankton fraction.

If we consider the total Centropages typicus popula-

tion (12 ind l�1 including males and copepodites) the

toxin retention would be even lower (1.5%). Similar

calculation can be made for Temora longicornis

feeding on D. acuta. Then, 0.005 ng OA l�1 should

have been found in the zooplankton size fraction,

instead no toxin was detected on 13 July 2002.

4. Discussion

4.1. Feeding rates on Dinophysis spp. and food

selectivity

All the copepod species used in the experiments fed

on Dinophysis spp. and had high survival. In general,

feeding rates on Dinophysis spp. incresead as they

became more abundant, though none of the copepod

species selected for these dinoflagellates. Instead, the

selectivity coefficients of Temora longicornis and

Centropages typicus on D. acuta progressively

decreased with increasing food concentrations.

Despite their non-selective feeding behaviour towards

the toxic dinoflagellate, these results suggest some

avoidance tendency as food availability increases. T.

longicornis avoidance towards Dinophysis spp. with

increased food availability has been previously

reported (Maneiro et al., 2000). However, we cannot

rule out the effect of size shaping food selectivity as

other food types (e.g. S. costatum, Chaetoceros sp. and

ciliates <25 mm in the diet of T. longicornis, and

peridinales >30 mm for C. typicus), which fall within

optimum size particles for both copepods (e.g. Hansen

et al., 1994), became more abundant at higher food

concentrations. Such response was not observed for

Acartia bifilosa feeding on high concentrations of D.

norvegica indicating that this species was even less

selective than T. longicornis and C. typicus. Low or

non-selective feeding behaviour under natural food

conditions (e.g. Huntley, 1981; Turner and Tester,

1989; Irigoien et al., 2000), with the inclusion of a

variety of food items in the diet, should be

advantageous (Kleppel, 1993). Although this feeding

behaviour diminishes the probability of optimal

foraging in nature, it increases the chances of

obtaining a nutritionally complete ration (Kleppel,

B. Kozlowsky-Suzuki et al. / Harmful Algae 5 (2006) 57–68 65

Table 3

Mean (S.D.) per-individual copepod effect (day�1), and potential grazing impact (percentage of prey population removed daily) on the different

Dinophysis populations by the copepods Acartia bifilosa (Baltic Proper), Temora longicornis and Centropages typicus (Oresund)

Copepod species Dinophysis concentration (mg C l�1) Grazing effect (day�1) Grazing impact (%)

Acartia bifilosa Dinophysis norvegica

185 (11) �0.008 (0.01) 0.009–0.15

419 (17) �0.011 (0.00)* 0.01–0.18

833 (60) �0.018 (0.01)* 0.02–0.29

Temora longicornis Dinophysis acuta

3 (0.4) �0.17 (0.06)* 1.12z

7 (2) �0.20 (0.07)* 1.12z

36 (6) �0.04 (0.03) 0.50

70 (12) �0.02 (0.01) 0.30

Centropages typicus Dinophysis acuta

2 (0.6) �0.08 (0.02)* 7.01–25.85

9 (2) �0.08 (0.02)* 6.88–25.40z

72 (12) �0.06 (0.04) 4.03–14.88* Significant effect ( p < 0.025).z Corresponding to in situ prey concentration at the time of the experiments.

1993). In addition to this nutritional advantage, diet

mixing or the availability of other food types can, for

instance, benefit herbivores by diluting the effect of

chemical defenses in particular foods (e.g. Bernays

et al., 1994), improving not only survival but also

reproductive success in zooplankton (e.g. Reinikainen

et al., 1994; Turner et al., 2001).

4.2. Copepod grazing impact on Dinophysis spp.:

importance to bloom decline and to the fate of

toxins

The grazing impact of zooplankton on the suppres-

sion of harmful algal blooms may, at certain times, be

important (Turner and Anderson, 1983; Watras et al.,

1985; Uye, 1986). However, even when the feeding

activity by copepods on toxic dinoflagellates under

natural circumstances can be representative, their

grazing impact as a loss factor for these algal blooms

may be unimportant (Calbet et al., 2003; Wexels Riser

et al., 2003). It has, though, been suggested that grazing

by copepods could exert considerable impact at the

onset of bloom formation (Uye, 1986). This author

estimated a high removal (up to 30%) of the red tide

Chatonella antiqua by the copepod assemblage at low

densities of the flagellate, whereas lower removal (up to

4%) at high densities of the algae (Uye, 1986).

Similarly, we found that at the lowestD. acuta densities

(similar to those found at the in situ Dinophysis depth),

the Centropages typicus population would be able to

remove up to 25% of the prey population.

Despite the significant negative effect on the net

growth of D. acuta at the lowest prey concentrations

by both Centropages typicus and Temora longicornis,

the estimated low grazing impact by T. longicornis

could be explained by its low abundance in situ. While

there were 12 ind l�1 of C. typicus at the Dinophysis

depth on 17 July, there was only 0.35 T. long-

icornis l�1 on 13 July. This suggests that these two

copepod species could potentially contribute as a

substantial loss factor for Dinophysis spp., provided

they are abundant at the onset of the blooms. Acartia

bifilosa had, however, no significant negative effect on

the net growth of D. norvegica at the lowest food

concentration. We would then expect an even lower

effect by this copepod at lower D. norvegica densities,

which are commonly observed in the Baltic Sea (e.g.

Salomon et al., 2003).

The effectiveness of grazing as a loss factor for

Dinophysis spp. populations will depend on the set of

conditions regarding copepod abundance and feeding

behaviour, and prey abundance and growth conditions.

In our study, for instance, the low Acartia bifilosa in

situ abundance (0.6 copepod l�1) combined with a

high D. norvegica density and net growth rates (0.15–

0.25 day�1) led to an estimated low grazing impact

despite the significant negative grazing effect. A high

grazing impact could, however, be accomplished by

B. Kozlowsky-Suzuki et al. / Harmful Algae 5 (2006) 57–6866

the abundant Centropages typicus population

(12 ind l�1) on a less dense D. acuta population at

sharp decline (�0.13 to �0.79 day�1).

LowaccumulationofOA(<1%)wasobserved insitu

and low retention (�5%) of the toxin by Centropages

typicus was roughly estimated. Low retention of

ingested toxins has been reported for copepods feeding

on toxic Alexandrium spp. (Teegarden and Cembella,

1996; Guisande et al., 2002; Teegarden et al., 2003) and

Nodularia spumigena (Kozlowsky-Suzuki et al., 2003).

In spite of low toxin retention, higher toxin burdens than

the commonly accepted regulatory limit for safe

consumption of shellfish containing Paralytic Shellfish

Poisoning toxins can be found for copepods feeding on

Alexandrium spp. (Guisande et al., 2002; Teegarden

et al., 2003). In our study, considering 5% retention of

ingested toxins, 1 kg of Centropages typicus and

Temora longicornis (assuming individual wet weights

of 100 and 55 mg, respectively) would contain at the

most 136 and 114 mg OA, respectively. Those values

are, however, lower than the regulatory limit for safe

human consumption of shellfish contaminated with

DSP toxins (200 mg kg�1 tissue).

By contributing to the diets of mysids (Viherluoto

and Viitasalo, 2001), and fish, such as sprat and

herring (Last, 1987; Mollmann and Koster, 2002),

copepods could still act as vectors to higher trophic

levels. Temora longicornis could potentially transport

0.16 kg OA daily to the sprat population in the Central

Baltic Sea (assuming a daily population consumption

of up to 1400 tonnes of T. longicornis wet weight

during the summer of 1995; Mollmann and Koster,

2002). However, considering the abundance of the

sprat population (ca. 5.0 � 1010 age 1+ individuals in

the same period; Mollmann and Koster, 2002) with an

individual average weight ranging from 8.1 to 12.1 g

(ICES, 2001), the toxin amount per kg of sprat would

be 0.34 mg OA. This value is even lower than the

calculated toxin amount per kg of copepod

(136 � 114 mg), and indicates that toxin losses take

place at each step up in the food chain.

5. Conclusions

Here we provide evidence that copepods feed on

toxic Dinophysis spp. in natural plankton assem-

blages, even when the availability of other food types

increases. Despite their non-selective behaviour,

Temora longicornis and Centropages typicus, tended

to avoid D. acuta at high food concentrations, thus

presenting a significant grazing effect only at low D.

acuta densities. This suggests that these copepod

species could potentially contribute as a substantial

loss factor for Dinophysis spp. provided they are

abundant at the onset of the blooms. Despite the

significant grazing effect by Acartia bifilosa on D.

norvegica, the low observed copepod density in situ

would not impose any grazing impact to a dense D.

norvegicagrowing at high rates. Our results suggest that

the effectiveness of grazing as a loss factor for

Dinophysis spp. populations depends on the abundance

of copepods in situ and their feeding behaviour (e.g.

avoidance), and on the abundance and growth condi-

tions of the prey populations. Our calculations indicate

that copepods retain low amounts of okadaic acid and

that their relative importance as vectors of toxins to

higher trophic levels should be limited and appears to

not pose any harm to human consumption. Here, we

simulated Dinophysis spp. blooms by providing

increasing prey concentrations, further studies follow-

ing different stages of Dinophysis spp. blooms (with

several ranges of prey concentrations and growth

conditions) should give new insights of the impact of

copepod grazing on the dynamics of these blooms.

Acknowledgements

We would like to thank Prof. Takeshi Yasumoto for

running part of the toxin analysis and reviewing the

manuscript, Per Juel Hansen for the plankton wheel

used during the Oresund experiments and Marja Koski

for comments on the manuscript. This study was

supported by CNPq (The Brazilian National Council

for Research), MISTRA (Swedish Foundation for

Strategic Environmental Research) and the European

Commission (Research Directorate General-Environ-

ment Programme—Marine Ecosystems), through the

FATE project (grant holder E. Graneli, contract

EVK3-2001-00050). [SS]

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