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Food selectivity and grazing impact on toxic Dinophysis spp. by copepods feeding on natural plankton assemblages
Harmful Algae 5 (2006) 57–68 www.elsevier.com/locate/hal
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 Ru¨hl c, Edna Grane´li a a
Department of Biology and Environmental Science, University of Kalmar, Kalmar 39182, Sweden b Marine Biology, Lund University, Campus Helsingborg, Box 882, 25108 Helsingborg, Sweden c University 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
1. Introduction * Corresponding author. Present address: Departamento de Cieˆncias Naturais, Universidade Federal do Estado do Rio de Janeiro, UNIRIO, Av. Pasteur 458 Urca, 22290-040 Rio de Janeiro, Brazil. E-mail address: [email protected] (B. Kozlowsky-Suzuki).
Phytoplankton growth and loss rates are governed by several factors and in many cases, when growth control fails, algal blooms are established. Such blooms
1568-9883/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2005.05.002
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can be formed by different phytoplankton species and some of them have harmful impacts on the ecosystem. Thus, the understanding of bloom formation, maintenance and decay in nature is of major priority. Successful phytoplankton growth is primarily dependent on the maintenance of the reproductive stocks in suspension within the euphotic zone and on favourable nutrient conditions. Loss processes represent sedimentation 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 allelopathy (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 ranging 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). Parasitism 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 accumulate 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 (Diarrhetic 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 modulated 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 ¨ resund (Strait between Sweden and Denmark). In the O 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 ¨ land (568550 4000 N, outside the east coast of O 0 00 16853 10 E) on 7 June and 17 July 2001 and in the ¨ resund (558580 1200 N, 128400 2300 E) on 13 and 17 July O 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
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Table 1 Depth, salinity, temperature and dominant Dinophysis and copepod species at sampling depth in June–July 2001 (Baltic Proper) and in July 2002 ¨ resund) (O Site/date
Depth (m)
Baltic Proper 7 June 2001 17 July 2001
12 18
¨ resund O 13 July 2002 17 July 2002
14 10
Salinity (%)
Temperature (8C)
Dinophysis
Copepod species
6 6
12 12
D. acuminata D. norvegica
Acartia bifilosa Acartia bifilosa
18.7 15.6
16 18
D. acuta D. acuta
Acartia spp., T. longicornis 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 ¨ resund, 25–70, 70–100 and >100 mm. The conO 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 ¨ resund), which were used to prepare the food the O 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 collected 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 samples were done in micro-well plates or in 2 ml sedimentation chambers depending on the concentration 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:MilliQ 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. ¨ resund (also single Samples collected in the O 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 microcapillaries (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 (extraction, centrifugation and filtration as described above), the filtered extracts were analysed by liquid chromatography–electrospray ionisation mass spectrometry (LC–ESI-MS) (Hummert et al., 2000) using a PerkinElmer series 200 autosampler and pump, coupled to an Applied Biosystems API 165 mass spectrometer. Briefly, the extracts were separated on a reversed
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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 application 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 ¨ resund experiments (13 and typicus were used in the O 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 l1. In the ¨ resund experiments, similar Dinophysis spp. abunO 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 l1. In the Baltic Proper, calculated carbon concentrations in the food suspensions ranged from ca. 200 to 938 mg C l1, ¨ resund, they ranged from 19 to while in the O 1397 mg C l1. 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 ¨ resund) or 250-ml for 48 h in 550-ml plastic bottles (O
tissue culture flasks (Baltic Proper) with the designated 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 mm3 for phytoplankton and ciliates and 0.13 pg mm3 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 = m1 (where, m = number of food types available) and food items are fed upon in the same proportions as their availability, if a > m1 selection is positive; when a < m1 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: m¼
ln ðNt =N0 Þ t
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 perindividual copepod effect (Dr, grazing effect) on
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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.
the net growth rate of the different Dinophysis spp. was calculated using the metric described in Osenberg et al. (1997): Ntg ln N0g ln NN0ctc Dr ¼ mg mc ¼ 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 (MANOVA), 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
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 l1) was more abundant in the 40–70 mm size fraction than D. norvegica (42 cells l1), while the latter dominated in July (982 cells l1). 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 l1 to 20.11 and 38.51 ng l1, respectively) seemed con-
Table 2 Acartia bifilosa, Temora longicornis and Centropages typicus mean (S.D.) ingestion rate (ng C ind1 h1) on Dinophysis norvegica and D. acuta and total ingestion rate at each food concentration (total food concentration and Dinophysis spp. concentration) Copepod species
Food concentration (mg C l1)
Ingestion rate (ng C ind1 h1)
Total
D. norvegica
Total
D. norvegica
204 (13) 466 (28) 927 (62)
185 (11) 419 (17) 833 (60)
18 (13) 49 (16) 153 (31)
12 (13) 41 (13) 150 (29)
D. acuta
Total
D. acuta
Acartia bifilosa
Total Temora longicornis 83 194 970 1397
(4) (26) (127) (345)
3 7 36 70
(0.4) (2) (6) (12)
92 (5) 187 (72) 671 (118) 198 (183)
5 (0.3) 9 (5) 23 (17) 23 (7)
5 (1) 58 (9) 130 (41)
2 (0.6) 7 (2) 40 (20)
Centropages typicus 19 (2) 102 (13) 338 (39)
2 (0.6) 9 (2) 72 (12)
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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 cell1, respectively. ¨ resund, Dinophysis acuta (reaching In the O 1 712 cells l ) was the most abundant Dinophysis species, containing the highest OA cell quota (3.41 pg cell1). OA was also found in D. norvegica cells (1.70 pg cell1), but not detected in D. acuminata. OA concentration ranged from 0.27 0.01 (mean S.D.) on 13 July to 0.49 0.01 ng l1 (17 July) in the 25–70 mm fraction and from 0.02 0.00 (13 July) to 0.16 0.00 ng l1 (17 July) in the 70–100 mm fraction, but only found in the zooplankton fraction (>100 mm) on 17 July. At this occasion, Centropages typicus copepodites and adults (12 ind l1) were the most abundant animals in the zooplankton fraction, which contained less than 1% (0.005 ng l1) 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 ind1 h1 and from 12 to 150 ng C ind1 h1, respectively (Table 2). Both the total ingestion rate and the ingestion rate on D. norvegica were highest at the highest food concentration (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 nonselection 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. ¨ resund, Temora longicornis survival was In the O 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
Fig. 1. Selectivity coefficient (a) of Acartia bifilosa (mean S.D.) feeding at (a) 204 mg C l1, (b) 466 mg C l1 and (c) 938 mg C l1 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, Woronichinia; Dnor, Dinophysis norvegica.
on D. acuta ranged from 0.4 to 4 ml ind1 h1 and from 5 to 23 ng C ind1 h1, respectively. Maximum rates were calculated for T. longicornis feeding on C. furca (up to 5 ml ind1 h1 and 637 ng C ind1 h1). The total ingestion rate and ingestion rate on C. furca were highest at 970 mg C l1 (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 l1) than at 83 mg C l1 (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 concentration and was lower at 1397 than at 83 mg C l1 (one-way ANCOVA, Tukey HSD; p < 0.05). Even
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Fig. 2. Selectivity coefficient (a) of Temora longicornis (mean S.D.) feeding at (a) 83 mg C l1, (b) 194 mg C l1, (c) 970 mg C l1 and (d) ¨ resund. The dotted line shows the a-value (0.09). Ci, ciliate <25 mm; Da, Dactyliosolen sp.; Thal, Thalassiosira sp.; Cy, 1397 mg C l1 in the O 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, Pseudonitzschia sp., Chaetoceros sp. and ciliates <25 mm to T. longicornis diet increased at high food concentrations, 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 ind1 h1 and from 2 to 40 ng C ind1 h1, respectively. As observed for Temora longicornis, maximum ingestion rate was observed on Ceratium furca (up to 105 ng C ind1 h1). 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 (oneway ANOVA, Tukey HSD; p < 0.01).
3.3. Copepod grazing impact on Dinophysis spp. and toxin retention Acartia bifilosa, Temora longicornis and Centropages 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 population 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.
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However, only 5% (0.005 ng OA l1) of the calculated toxin content was detected in the zooplankton fraction. If we consider the total Centropages typicus population (12 ind l1 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 l1 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
Fig. 3. Selectivity coefficient (a) of Centropages typicus (mean S.D.) feeding at (a) 19 mg C l1, (b) 102 mg C l1 and ¨ resund. The dotted line shows the a-value (c) 338 mg C l1 in the O (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 l1) feeding on a natural concentration (ca. 700 cells l1) of D. acuta (both estimated at the Dinophysis depth), can be calculated by using Centropages typicus ingestion rate on D. acuta (14 cells ind1 day1 feeding at a D. acuta concentration similar to that observed in the Dinophysis depth and a toxin quota of 3.41 pg OA cell1) 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 day1, we would expect to find, at steady state, 0.087 (0.04) ng OA l1 in the zooplankton size-fraction (>100 mm) (assuming that C. typicus was the only copepod species feeding on D. acuta).
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,
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Table 3 Mean (S.D.) per-individual copepod effect (day1), and potential grazing impact (percentage of prey population removed daily) on the different ¨ resund) Dinophysis populations by the copepods Acartia bifilosa (Baltic Proper), Temora longicornis and Centropages typicus (O Copepod species
Dinophysis concentration (mg C l1)
Grazing effect (day1)
Grazing impact (%)
Acartia bifilosa
Dinophysis norvegica 185 (11) 419 (17) 833 (60)
0.008 (0.01) 0.011 (0.00) * 0.018 (0.01) *
0.009–0.15 0.01–0.18 0.02–0.29
Dinophysis acuta 3 (0.4) 7 (2) 36 (6) 70 (12)
0.17 0.20 0.04 0.02
Dinophysis acuta 2 (0.6) 9 (2) 72 (12)
0.08 (0.02)* 0.08 (0.02)* 0.06 (0.04)
Temora longicornis
Centropages typicus
* z
(0.06)* (0.07)* (0.03) (0.01)
1.12z 1.12z 0.50 0.30 7.01–25.85 6.88–25.40z 4.03–14.88
Significant effect ( p < 0.025). 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 suppression 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 lowest D. 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 l1 of C. typicus at the Dinophysis depth on 17 July, there was only 0.35 T. longicornis l1 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 l1) combined with a high D. norvegica density and net growth rates (0.15– 0.25 day1) led to an estimated low grazing impact despite the significant negative grazing effect. A high grazing impact could, however, be accomplished by
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the abundant Centropages typicus population (12 ind l1) on a less dense D. acuta population at sharp decline (0.13 to 0.79 day1). Low accumulation of OA (<1%) was observed in situ 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 kg1 tissue). By contributing to the diets of mysids (Viherluoto and Viitasalo, 2001), and fish, such as sprat and herring (Last, 1987; Mo¨llmann and Ko¨ster, 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; Mo¨llmann and Ko¨ster, 2002). However, considering the abundance of the sprat population (ca. 5.0 1010 age 1+ individuals in the same period; Mo¨llmann and Ko¨ster, 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 assemblages, 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. norvegica growing 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 conditions 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 following 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 ¨ resund experiments and Marja Koski used during the O 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-Environment Programme—Marine Ecosystems), through the FATE project (grant holder E. Grane´li, contract EVK3-2001-00050). [SS] References Bernays, E.A., Bright, K.L., Gonzalez, N., Angel, J., 1994. Dietary mixing in a generalist herbivore: test of two hypotheses. Ecology 75 (7), 1997–2006.
B. Kozlowsky-Suzuki et al. / Harmful Algae 5 (2006) 57–68 Calbet, A., Vaque´, D., Felipe, J., Vila, M., Sala, M.M., Alcaraz, M., Estrada, M., 2003. Relative grazing impact of microzooplankton and mesozooplankton on a bloom of the toxic dinoflagellates Alexandrium minutum. Mar. Ecol. Prog. Ser. 259, 303–309. Carlsson, P., Grane´li, E., Finenko, G., Maestrini, S.Y., 1995. Copepod grazing on a phytoplankton community containing the toxic dinoflagellate Dinophysis acuminata. J. Plankton Res. 17 (10), 1925–1938. Carpenter, E.J., Janson, S., Boje, R., Pollehne, F., Chang, J., 1995. The dinoflagellate Dinophysis norvegica: biological and ecological observations in the Baltic Sea. Eur. J. Phycol. 30, 1–9. Chesson, J., 1978. Measuring preference in selective predation. Ecology 59 (2), 211–215. DeMott, W.R., 1988. Discrimination between algae and artificial particles by freshwater and marine copepods. Limnol. Oceanogr. 33 (3), 397–408. DeMott, W.R., 1995. Optimal foraging by a suspension-feeding copepod: responses to short-term and seasonal variation in food resources. Oecologia 103, 230–240. Edler, L., 1979. Recommendations on Methods for Marine Biological Studies in the Baltic Sea. Phytoplankton and Chlorophyll. The Baltic Marine Biologists, Publications No. 5. Frost, B.W., 1972. Effects of size and concentration of food particles on the feeding behaviour of the marine planktonic copepod Calanus pacificus. Limnol. Oceanogr. 17 (6), 805–815. ˚ ., Carlsson, P., Grane´li, E., Pallon, J., 2002. DinoGisselsson, L-A physis blooms in the deep euphotic zone in the Baltic Sea: do they grow in the dark? Harmful Algae 1, 401–418. Goto, H., Igarashi, T., Watai, M., Yasumoto, T., Gomez, O.V., Valdivia, G.L., Noren, F., Gisselson, L.-A., Grane´li, E., 2000. Worldwide occurrence of pectenotoxins and yessotoxins in shellfish and phytoplankton. in: Ninth International Conference on Harmful Algal Blooms. p. 20 (Abstract). Goto, H., Igarashi, T., Yamamoto, M., Yasuda, M., Sekiguchi, R., Watai, M., Tanno, K., Yasumoto, T., 2001. Quantitative determination of marine toxins associated with diarrhetic shellfish poisoning by liquid chromatography coupled with mass spectrometry. J. Chromatogr. A. 907, 181–189. Guisande, C., Frango´pulos, M., Carotenuto, Y., Maneiro, I., Riveiro, I., Vergara, A.R., 2002. Fate of paralytic shellfish poisoning toxins ingested by the copepod Acartia clausi. Mar. Ecol. Prog. Ser. 240, 105–115. Hansen, B., BjØrnsen, P.K., Hansen, P.J., 1994. The size ratio between planktonic predators and their prey. Limnol. Oceanogr. 39 (2), 395–403. Hummert, C., Kastrup, S., Reinhardt, K., Reichelt, M., Luckas, B., 2000. Use of gel permeation chromatography for automatic and rapid extract clean-up for the determination of diarrhetic shellfish toxins (DSP) by liquid chromatography-mass spectrometry. Chromatographia 51 (7/8), 397–403. Huntley, M.R., 1981. Nonselective, nonsaturated feeding by three calanoid copepod species in the Labrador Sea. Limnol. Oceanogr. 26 (5), 831–842. ICES, 2001. Report of the Baltic Fisheries Assessment Working Group. ICES CM 2001/ACFM: 18. Irigoien, X., Head, R.N., Harris, R.P., Cummings, D., Harbour, D., Meyer-Harms, B., 2000. Feeding selectivity and egg production
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of Calanus helgolandicus in the English Channel. Limnol. Oceanogr. 45 (1), 44–54. Kleppel, G.S., 1993. On the diets of calanoid copepods. Mar. Ecol. Prog. Ser. 99, 183–195. Kozlowsky-Suzuki, B., Karjalainen, M., Lehtiniemi, M., Engstro¨m¨ st, J., Carlsson, P., 2003. Feeding, reproduction and toxin O accumulation by the copepods Acartia bifilosa and Eurytemora affinis in the presence of the toxic cyanobacterium Nodularia spumigena. Mar. Ecol. Prog. Ser. 249, 237–249. Last, J.M., 1987. The food of immature sprat (Sprattus sprattus (L.)) and herring (Clupea harengus L.) in coastal waters of the North Sea. J. Cons. Int. Explor. Mer. 44, 73–79. Maestrini, S.Y., 1998. Bloom dynamics and ecophysiology of Dinophysis spp. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. Springer-Verlag, Berlin, pp. 243–265. Maneiro, I., Frango´pulos, M., Guisande, C., Ferna´ndez, M., Reguera, B., Riveiro, I., 2000. Zooplankton as a potential vector of diarrhetic shellfish poisoning toxins through the food web. Mar. Ecol. Prog. Ser. 201, 155–163. Maneiro, I., Guisande, C., Frango´pulos, M., Riveiro, I., 2002. Importance of copepod faecal pellets to the fate of DSP toxins produced by Dinophysis spp. Harmful Algae 1, 333–341. Mo¨llmann, C., Ko¨ster, F.W., 2002. Population dynamics of calanoid copepods and the implications of their predation by clupeid fish in the Central Baltic Sea. J. Plankton Res. 24 (10), 959–977. Osenberg, C.W., Sarnelle, O., Cooper, S.D., 1997. Effect size in ecological experiments: the application of biological models in meta-analysis. Am. Nat. 150 (6), 798–812. Peres-Neto, P.R., 1999. How many statistical tests are too many? The problem of conducting multiple ecological inferences revisited. Mar. Ecol. Prog. Ser. 176, 303–306. Pimia¨, V., Kankaanpa¨a¨, H., Kononen, K., 1998. The first observation of okadaic acid in Mytilus edulis from the Gulf of Finland. Boreal Environ. Res. 2, 381–385. Reinikainen, M., Ketola, M., Walls, M., 1994. Effects of the concentrations of toxic Microcystis aeruginosa and alternative food on the survival of Daphnia pulex. Limnol. Oceanogr. 39 (2), 424–432. Reynolds, C.S., 1990. The Ecology of Freshwater Phytoplankton. Cambridge University Press. Salomon, P.S., Janson, S., Grane´li, E., 2003. Parasitism of Dinophysis norvegica by Amoebophrya sp. in the Baltic Sea. Aquat. Microb. Ecol. 33, 163–172. Sipia¨, V., Kankaanpa¨a¨, H., Meriluoto, J., 2000. The first observation of okadaic acid in flounder in the Baltic Sea. Sarsia 85, 471–475. Teegarden, G.J., Cembella, A.D., 1996. Grazing of toxic dinoflagellates, Alexandrium spp., by adult copepods of coastal Maine: implication for the fate of paralytic shellfish toxins in marine food webs. J. Exp. Mar. Biol. Ecol. 196, 145–176. Teegarden, G.J., Cembella, A.D., Capuano, C.L., Barron, S.H., Durbin, E.G., 2003. Phycotoxin accumulation in zooplankton feeding on Alexandrium fundyense—vector or sink? J. Plankton Res. 25 (4), 429–443. Turner, J.T., Anderson, D.M., 1983. Zooplankton grazing during dinoflagellate blooms in a Cape Cod embayment, with observations of predation upon tintinnids by copepods. Mar. Ecol. 4, 359–374.
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Turner, J.T., Tester, P.A., 1989. Zooplankton feeding ecology: nonselective grazing by the copepods Acartia tonsa Dana, Centropages velificatus De Oliveira, and Eucalanus pileatus Giesbrecht in the plume of the Mississipi River. J. Exp. Mar. Biol. Ecol. 126, 21–43. Turner, J.T., Ianora, A., Miralto, A., Laabir, M., Espo´sito, F., 2001. Decoupling of copepod grazing rates, fecundity and egg-hatching success on mixed and alternating diatom and dinoflagellate diets. Mar. Ecol. Prog. Ser. 220, 187–199. Uye, S., 1986. Impact of copepod grazing on the red-tide flagellate Chatonella antiqua. Mar. Biol. 92, 35–43.
Viherluoto, M., Viitasalo, M., 2001. Temporal variability in functional responses and prey selectivity of the pelagic mydis, Mysis mixta, in natural prey assemblages. Mar. Biol. 138, 575–583. Watras, C.J., Garcon, V.C., Olson, R.J., Chisholm, S.W., Anderson, D.M., 1985. The effect of zooplankton grazing on estuarine blooms of the toxic dinoflagellates Gonyaulax tamarensis. J. Plankton Res. 7, 891–908. Wexels Riser, C., Jansen, S., Bathmann, U., Wassmann, P., 2003. Grazing of Calanus helgolandicus on Dinophysis norvegica during bloom conditions in the North Sea: evidence from investigations on faecal pellets. Mar. Ecol. Prog. Ser. 256, 301–304.
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 Ru¨hl c, Edna Grane´li a a
Department of Biology and Environmental Science, University of Kalmar, Kalmar 39182, Sweden b Marine Biology, Lund University, Campus Helsingborg, Box 882, 25108 Helsingborg, Sweden c University 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
1. Introduction * Corresponding author. Present address: Departamento de Cieˆncias Naturais, Universidade Federal do Estado do Rio de Janeiro, UNIRIO, Av. Pasteur 458 Urca, 22290-040 Rio de Janeiro, Brazil. E-mail address: [email protected] (B. Kozlowsky-Suzuki).
Phytoplankton growth and loss rates are governed by several factors and in many cases, when growth control fails, algal blooms are established. Such blooms
1568-9883/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2005.05.002
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can be formed by different phytoplankton species and some of them have harmful impacts on the ecosystem. Thus, the understanding of bloom formation, maintenance and decay in nature is of major priority. Successful phytoplankton growth is primarily dependent on the maintenance of the reproductive stocks in suspension within the euphotic zone and on favourable nutrient conditions. Loss processes represent sedimentation 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 allelopathy (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 ranging 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). Parasitism 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 accumulate 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 (Diarrhetic 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 modulated 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 ¨ resund (Strait between Sweden and Denmark). In the O 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 ¨ land (568550 4000 N, outside the east coast of O 0 00 16853 10 E) on 7 June and 17 July 2001 and in the ¨ resund (558580 1200 N, 128400 2300 E) on 13 and 17 July O 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
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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 ¨ resund) (O Site/date
Depth (m)
Baltic Proper 7 June 2001 17 July 2001
12 18
¨ resund O 13 July 2002 17 July 2002
14 10
Salinity (%)
Temperature (8C)
Dinophysis
Copepod species
6 6
12 12
D. acuminata D. norvegica
Acartia bifilosa Acartia bifilosa
18.7 15.6
16 18
D. acuta D. acuta
Acartia spp., T. longicornis 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 ¨ resund, 25–70, 70–100 and >100 mm. The conO 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 ¨ resund), which were used to prepare the food the O 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 collected 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 samples were done in micro-well plates or in 2 ml sedimentation chambers depending on the concentration 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:MilliQ 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. ¨ resund (also single Samples collected in the O 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 microcapillaries (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 (extraction, centrifugation and filtration as described above), the filtered extracts were analysed by liquid chromatography–electrospray ionisation mass spectrometry (LC–ESI-MS) (Hummert et al., 2000) using a PerkinElmer series 200 autosampler and pump, coupled to an Applied Biosystems API 165 mass spectrometer. Briefly, the extracts were separated on a reversed
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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 application 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 ¨ resund experiments (13 and typicus were used in the O 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 l1. In the ¨ resund experiments, similar Dinophysis spp. abunO 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 l1. In the Baltic Proper, calculated carbon concentrations in the food suspensions ranged from ca. 200 to 938 mg C l1, ¨ resund, they ranged from 19 to while in the O 1397 mg C l1. 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 ¨ resund) or 250-ml for 48 h in 550-ml plastic bottles (O
tissue culture flasks (Baltic Proper) with the designated 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 mm3 for phytoplankton and ciliates and 0.13 pg mm3 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 = m1 (where, m = number of food types available) and food items are fed upon in the same proportions as their availability, if a > m1 selection is positive; when a < m1 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: m¼
ln ðNt =N0 Þ t
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 perindividual copepod effect (Dr, grazing effect) on
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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.
the net growth rate of the different Dinophysis spp. was calculated using the metric described in Osenberg et al. (1997): Ntg ln N0g ln NN0ctc Dr ¼ mg mc ¼ 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 (MANOVA), 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
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 l1) was more abundant in the 40–70 mm size fraction than D. norvegica (42 cells l1), while the latter dominated in July (982 cells l1). 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 l1 to 20.11 and 38.51 ng l1, respectively) seemed con-
Table 2 Acartia bifilosa, Temora longicornis and Centropages typicus mean (S.D.) ingestion rate (ng C ind1 h1) on Dinophysis norvegica and D. acuta and total ingestion rate at each food concentration (total food concentration and Dinophysis spp. concentration) Copepod species
Food concentration (mg C l1)
Ingestion rate (ng C ind1 h1)
Total
D. norvegica
Total
D. norvegica
204 (13) 466 (28) 927 (62)
185 (11) 419 (17) 833 (60)
18 (13) 49 (16) 153 (31)
12 (13) 41 (13) 150 (29)
D. acuta
Total
D. acuta
Acartia bifilosa
Total Temora longicornis 83 194 970 1397
(4) (26) (127) (345)
3 7 36 70
(0.4) (2) (6) (12)
92 (5) 187 (72) 671 (118) 198 (183)
5 (0.3) 9 (5) 23 (17) 23 (7)
5 (1) 58 (9) 130 (41)
2 (0.6) 7 (2) 40 (20)
Centropages typicus 19 (2) 102 (13) 338 (39)
2 (0.6) 9 (2) 72 (12)
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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 cell1, respectively. ¨ resund, Dinophysis acuta (reaching In the O 1 712 cells l ) was the most abundant Dinophysis species, containing the highest OA cell quota (3.41 pg cell1). OA was also found in D. norvegica cells (1.70 pg cell1), but not detected in D. acuminata. OA concentration ranged from 0.27 0.01 (mean S.D.) on 13 July to 0.49 0.01 ng l1 (17 July) in the 25–70 mm fraction and from 0.02 0.00 (13 July) to 0.16 0.00 ng l1 (17 July) in the 70–100 mm fraction, but only found in the zooplankton fraction (>100 mm) on 17 July. At this occasion, Centropages typicus copepodites and adults (12 ind l1) were the most abundant animals in the zooplankton fraction, which contained less than 1% (0.005 ng l1) 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 ind1 h1 and from 12 to 150 ng C ind1 h1, respectively (Table 2). Both the total ingestion rate and the ingestion rate on D. norvegica were highest at the highest food concentration (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 nonselection 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. ¨ resund, Temora longicornis survival was In the O 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
Fig. 1. Selectivity coefficient (a) of Acartia bifilosa (mean S.D.) feeding at (a) 204 mg C l1, (b) 466 mg C l1 and (c) 938 mg C l1 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, Woronichinia; Dnor, Dinophysis norvegica.
on D. acuta ranged from 0.4 to 4 ml ind1 h1 and from 5 to 23 ng C ind1 h1, respectively. Maximum rates were calculated for T. longicornis feeding on C. furca (up to 5 ml ind1 h1 and 637 ng C ind1 h1). The total ingestion rate and ingestion rate on C. furca were highest at 970 mg C l1 (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 l1) than at 83 mg C l1 (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 concentration and was lower at 1397 than at 83 mg C l1 (one-way ANCOVA, Tukey HSD; p < 0.05). Even
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Fig. 2. Selectivity coefficient (a) of Temora longicornis (mean S.D.) feeding at (a) 83 mg C l1, (b) 194 mg C l1, (c) 970 mg C l1 and (d) ¨ resund. The dotted line shows the a-value (0.09). Ci, ciliate <25 mm; Da, Dactyliosolen sp.; Thal, Thalassiosira sp.; Cy, 1397 mg C l1 in the O 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, Pseudonitzschia sp., Chaetoceros sp. and ciliates <25 mm to T. longicornis diet increased at high food concentrations, 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 ind1 h1 and from 2 to 40 ng C ind1 h1, respectively. As observed for Temora longicornis, maximum ingestion rate was observed on Ceratium furca (up to 105 ng C ind1 h1). 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 (oneway ANOVA, Tukey HSD; p < 0.01).
3.3. Copepod grazing impact on Dinophysis spp. and toxin retention Acartia bifilosa, Temora longicornis and Centropages 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 population 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.
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However, only 5% (0.005 ng OA l1) of the calculated toxin content was detected in the zooplankton fraction. If we consider the total Centropages typicus population (12 ind l1 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 l1 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
Fig. 3. Selectivity coefficient (a) of Centropages typicus (mean S.D.) feeding at (a) 19 mg C l1, (b) 102 mg C l1 and ¨ resund. The dotted line shows the a-value (c) 338 mg C l1 in the O (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 l1) feeding on a natural concentration (ca. 700 cells l1) of D. acuta (both estimated at the Dinophysis depth), can be calculated by using Centropages typicus ingestion rate on D. acuta (14 cells ind1 day1 feeding at a D. acuta concentration similar to that observed in the Dinophysis depth and a toxin quota of 3.41 pg OA cell1) 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 day1, we would expect to find, at steady state, 0.087 (0.04) ng OA l1 in the zooplankton size-fraction (>100 mm) (assuming that C. typicus was the only copepod species feeding on D. acuta).
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,
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Table 3 Mean (S.D.) per-individual copepod effect (day1), and potential grazing impact (percentage of prey population removed daily) on the different ¨ resund) Dinophysis populations by the copepods Acartia bifilosa (Baltic Proper), Temora longicornis and Centropages typicus (O Copepod species
Dinophysis concentration (mg C l1)
Grazing effect (day1)
Grazing impact (%)
Acartia bifilosa
Dinophysis norvegica 185 (11) 419 (17) 833 (60)
0.008 (0.01) 0.011 (0.00) * 0.018 (0.01) *
0.009–0.15 0.01–0.18 0.02–0.29
Dinophysis acuta 3 (0.4) 7 (2) 36 (6) 70 (12)
0.17 0.20 0.04 0.02
Dinophysis acuta 2 (0.6) 9 (2) 72 (12)
0.08 (0.02)* 0.08 (0.02)* 0.06 (0.04)
Temora longicornis
Centropages typicus
* z
(0.06)* (0.07)* (0.03) (0.01)
1.12z 1.12z 0.50 0.30 7.01–25.85 6.88–25.40z 4.03–14.88
Significant effect ( p < 0.025). 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 suppression 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 lowest D. 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 l1 of C. typicus at the Dinophysis depth on 17 July, there was only 0.35 T. longicornis l1 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 l1) combined with a high D. norvegica density and net growth rates (0.15– 0.25 day1) led to an estimated low grazing impact despite the significant negative grazing effect. A high grazing impact could, however, be accomplished by
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the abundant Centropages typicus population (12 ind l1) on a less dense D. acuta population at sharp decline (0.13 to 0.79 day1). Low accumulation of OA (<1%) was observed in situ 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 kg1 tissue). By contributing to the diets of mysids (Viherluoto and Viitasalo, 2001), and fish, such as sprat and herring (Last, 1987; Mo¨llmann and Ko¨ster, 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; Mo¨llmann and Ko¨ster, 2002). However, considering the abundance of the sprat population (ca. 5.0 1010 age 1+ individuals in the same period; Mo¨llmann and Ko¨ster, 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 assemblages, 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. norvegica growing 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 conditions 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 following 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 ¨ resund experiments and Marja Koski used during the O 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-Environment Programme—Marine Ecosystems), through the FATE project (grant holder E. Grane´li, contract EVK3-2001-00050). [SS] References Bernays, E.A., Bright, K.L., Gonzalez, N., Angel, J., 1994. Dietary mixing in a generalist herbivore: test of two hypotheses. Ecology 75 (7), 1997–2006.
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