Fate of domoic acid ingested by the copepod Acartia clausi

Marine Biology (2005) 148: 123–130 DOI 10.1007/s00227-005-0054-x R ES E AR C H A RT I C L E Isabel Maneiro Æ Paula Iglesias Æ Cástor Guisande Isabel Riveiro Æ Aldo Barreiro Æ Soultana Zervoudaki Edna Granéli Fate of domoic acid ingested by the copepod Acartia clausi Received: 19 January 2005 / Accepted: 30 May 2005 / Published online: 12 August 2005
Springer-Verlag 2005 Abstract Two important issues in the studies of harmful algae include ecological role of the toxic compounds and their fate through the food web. The aims of this study were to determine whether the production of domoic acid is a strategy evolved to avoid predation and the role of copepods in the fate of this toxic compound through the food web. The copepod Acartia clausi was fed with single and mixed cultures of the toxic diatom Pseudonitzschia multiseries and the non-toxic diatom Pseudonitzschia delicatissima. Ingestion rate as a function of diatom abundance was the same for the toxic and nontoxic Pseudo-nitzschia species, indicating no selective feeding behaviour against P. multiseries. The toxins ingested by the copepods did not affect mortality, feeding behaviour, egg production and egg hatching of the copepods. Copepods assimilated the 4.8% of the total domoic acid ingested. Although the amount of toxins daily detoxificated by the copepods was 63.6%, the copepods accumulated domoic acid in their tissues. We conclude that domoic acid is not toxic for copepods and, probably for this reason, this toxin does not act as feeding deterrent for copepods. However, even though the production of domoic acid has apparently not evolved to deter predation, copepods may play an Communicated by S. A. Poulet, Roscoff I. Maneiro (&) Æ P. Iglesias Æ C. Guisande Æ I. Riveiro A. Barreiro Facultad de Ciencias del Mar, Universidad de Vigo, Aptdo. 874, 36200 Vigo, Spain E-mail: [email protected] Tel.: +34-986814030 Fax: +34-986812556 S. Zervoudaki Hellenic Center for Marine Research, 46.7 km Athens Sounio, Mavro Lithari, 712-19013 Anavissos Attica, Greece E. Granéli Marine Sciences Department, University of Kalmar, 391-82 Kalmar, Sweden important role on the fate of this toxic compound through the marine food web. Introduction Harmful algae blooms are an important public health problem all over the world and their economic impact can produce losses of great magnitude in the affected areas. Diatoms were supposed to be non-toxic organisms for humans, but in 1987 Pseudo-nitzschia multiseries was identified as the causative species of a toxic event that caused five human deaths in Canada through the ingestion of contaminated shellfish (Quilliam and Wright 1989). Domoic acid (DA) was the toxin implicated in this event. Since 1987, DA was detected in other Pseudo-nitzschia species and poisoning events due to these harmful algae are spreading. In the interest of public health, there have been studies of trophic relations of shellfish and toxic Pseudonitzschia species (Blanco et al. 2002a, b; Vale and Sampayo 2002; Wekell et al. 2002; Campbell et al. 2003), but information on the presence of DA in other organisms of the marine food webs is scarce. Sierra et al. (1997) observed that mortality of pelicans in California in January of 1996 was related to the presence of DA in mackerel tissues. The same pathway of contamination, planktivorous fishes, was detected by Scholin et al. (2000) in the mortality of sea lions during a Pseudonitzschia bloom in California in 1998. In both these studies, the detection of DA in different levels of the food web confirmed the trophic transfer of this toxin. There is another potential entry point for DA in the food web other than fishes. Zooplankton are a key group in marine systems due to their intermediary position in the pelagic food web. The interaction of zooplankton–toxic phytoplankton is important for three reasons. First, toxins can act as a feeding deterrent to zooplankton. Several zooplanktonic species have been 124 observed to prey on toxic microalgae (see Turner and Tester 1997), but some species did not graze on toxic phytoplankton or exhibited a reduced feeding upon toxic phytoplankton due to either behavioural rejection of cells (Uye and Takamatsu 1990; Teegarden 1999) or physiological incapacitation after the ingestion of toxic cells (Ives 1985, 1987; Huntley et al. 1986). Bargu et al. (2003) found that Euphasia pacifica showed a discontinuous grazing rate over time when animals were fed with toxic P. multiseries. However, several studies indicate that DA does not function as feeding deterrent. Windust (1992) and Lincoln et al. (2001) observed that the feeding rates of copepods on toxic and non-toxic Pseudo-nitzschia species were the same and there was no selection against the toxic Pseudo-nitzschia species. Moreover, Shaw et al. (1997) observed that when the copepod Trigiopus californicus was fed with Thalassiosira pseudonana enriched with dissolved DA, the mortality of copepods increased, but toxins did not appear to act as a feeding deterrent. In some zooplankton species that ingest toxic algae have negative effects of toxins on egg production and hatching success have been reported (Miralto et al. 1999; Turner et al. 2001; Guisande et al. 2002). Lincoln et al. (2001) indicated that there is no effect of DA in copepod reproduction, but due to the low number of eggs produced by females, it remains unclear if DA had any effect on reproduction and offspring viability of the zooplankton. It has been reported that some zooplankton species can act as vectors for trophic transport of paralytic shellfish toxins (Turner et al. 2000; Guisande et al. 2002), okadaic acid (Maneiro et al. 2002) and DA (Lincoln et al. 2001; Tester et al. 2001; Bargu 2002) through the food web. Zooplankton species accumulate toxins in their tissues, via the ingestion of toxic phytoplankton (White 1981; Boyer et al. 1985; Turriff et al. 1995; Teegarden and Cembella1996; Frangópulos et al. 2000). Moreover, zooplankton also transfer toxins to their faecal pellets and/or eggs (Guisande et al. 2002; Maneiro et al 2002). Bargu (2002) reported that the levels of DA in euphausiid tissues corresponded to the abundance of toxic Pseudonitzschia species present in California waters during a bloom. Laboratory experiments (Bargu et al. 2003) confirmed that the euphausiids Euphasia pacifica and Thysanoessa spinifera ingested the toxic species P. multiseries. It has also been observed that several copepod species feed on toxic Pseudo-nitzschia species and accumulate toxins in their tissues (Windust 1992; Lincoln et al. 2001; Tester et al. 2001). However the assimilation rate of DA in zooplankton has not been quantified. However, other than these studies, there is little information about the role of copepods and the fate of DA through the food web. Since 1994, species of the toxic genus Pseudo-nitzschia have been identified as the toxic producers of DA outbreaks in northwest Spain (Rodriguez et al. 2001; Hasle 2002). However, DA has been detected only in P. multiseries and P. australis (Fraga et al. 1998; Rodriguez et al. 2001). These events caused toxicity in many shellfish species in the Galician Rias (Blanco et al. 2002a, b), but there is no information about the interaction between zooplankton and the species of the genus Pseudo-nitzschia in this area. The copepod Acartia clausi is one of the most abundant mesozooplanktonic species inside Galician Rı́as. The aims of this study were to determine the effect of DA on mortality, feeding behaviour and reproductive success of A. clausi, comparing copepods feeding on toxic and non-toxic Pseudo-nitzschia species together with the flagellate Tetraselmis suecica, and to determine whether copepods have an important role in the fate of DA in the pelagic community. Methods Phytoplankton cultures The non-axenic strains of the toxic Pseudo-nitzschia multiseries (strain from Kalmar University), the non-toxic Pseudo-nitzschia delicatissima (strain isolated in June 2003 by the team of University of Vigo in the Rı́a de Vigo, Spain), and Tetraselmis suecica used in this study, were cultured in K media at 18C and under a 12:12 h light:dark cycle. Cultures were used in stationary phase to enhance cell toxicity of the strain (see Bates 1998). The carbon and nitrogen contents of the phytoplankton species were determined from subsamples filtered on pre-combusted GF/F filters at low pressure, dried at 70C and combusted in a Fisons EA-1108. Sulphanilamide was used as the standard. The pg C and pg N contents (mean±SE) per cell for P. multiseries were 47.13±5.95 and 14.42±2.12, for P delicatissima 22.75±4.59 and 4.85±0.82, and for T. suecica 52.44±5.14 and 7.58±1.36, respectively. Zooplankton collection and acclimatization Zooplankton were collected by vertically integrated tows from a depth of 20 m to the surface, at a field station 39 m deep in Rı́a de Vigo, Spain (4213.3¢N, 847.7¢W). Samples were transported within 2 h of collection to the laboratory. Before the beginning of experiments, zooplankton community were kept in 20-l flasks with air bubbling and T. suecica as food. Experimental design of ingestion rate and copepod reproduction estimations For each experimental food concentration, 16 replicates of two females of A. clausi were sorted and transferred to 67 ml beakers containing food suspension. In all concentrations, T. suecica was offered as food in a constant concentration of approximately 0.5 lg C ml
1 to avoid starvation. There were one control concentration where T. suecica was the only food available to the 125 copepods, eight different concentrations contained T. suecica and P. multiseries with a maximum abundance of 0.16 lg C ml
1 (3,626 cell ml
1) and another four different concentrations contained T. suecica and P. delicatissima with a maximum abundance of 0.44 lg C ml
1 (19,659 cells ml
1). The maximum cell abundance observed in Rias of Galicia for toxic and nontoxic Pseudo-nitzschia species was around 1,600 cells ml
1 from the period between 1998 and 2003 (from weekly samples obtained by Centro de Control da Calidade do Medio Mariño-Xunta de Galicia). The carbon concentration in all experimental food concentrations was high enough to ensure that potential selection was not affected by food limitation, which might constrain grazers to consume less palatable prey (Teegarden 1999). The culture medium was prepared with aged natural seawater (salinity 33.6&) filtered through GF/F Whatman filters and autoclaved. In order to avoid sedimentation of diatoms, copepods were kept in a rotating wheel (1 rpm) at 18C under a 12:12 h light:dark cycle. Each day, the copepods were transferred to fresh phytoplankton suspensions at the experimental concentration and the mortality of copepods was checked. Ingestion rate (Frost 1972) and egg production were estimated after 2 days of acclimation (day 3) at all experimental food concentrations. The sample was not used if either of the two copepods have died. For each experimental concentration, the eggs produced by 4–8 copepods were pooled, so there were three replicates for each experimental food concentration for egg production data. From each of these replicates, 80–150 eggs produced by the females were incubated for a further 48 h period and the hatched nauplii and the remaining unhatched eggs were subsequently counted after fixation with formalin. Whatman filters and stored at
30C in ultracentrifuge plastic tubes and lyophilized 300 ll of methanol (80%) was added to the lyophilized material and the sample was homogenized using a pipette tip adapted to fit the shape of the vial. The sample was shaken followed by freezing twice. Finally, the extract was centrifuged twice at 4,000 rpm for 10 min, after which 100 ll of the supernatant was carefully collected with a Hamilton syringe and stored at
30C until liquid chromatography analysis. Liquid chromatography was performed on a Waters Alliance 2695 and a diode-array detector (Waters 996) following the method described by Vale and Sampayo (2001). Toxins were separated on a Merck Lichrospher 100 RP-18 (5 lm, 125·4 mm) column, protected by a guard-column Merck Lichrospher 100 RP-18 (5 lm, 125·4 mm). Detection wavelength was set at 242 nm with a 100 nm bandwidth. Column temperature was kept at 40C. The mobile phase consisted of acetonitrile:water (11:89, v/v), acidified with 5 ml 8.5% orthophosphoric acid. Flow rate was set at 0.45 ml min
1 and analysis time at 12 minutes. The concentration of DA in the P. multiseries strain used in this study (mean±SE) was 0.31±0.07 pg DA cell
1. To ensure that recently ingested toxins by copepods were excreted, 15–28 copepods were transferred from each experimental food concentration to 0.2 lm-filtered seawater and after 2 h the copepods were transferred to distilled water and immediately transferred to 50 ll distilled water, lyophilized and added to 100 ll methanol (80%). This experimental design allowed gut evacuation to measure the toxins accumulated in the copepod tissues. Results Experimental design of toxin accumulation and toxin detoxification estimations in the copepods To estimate copepod toxin accumulation, copepod females collected from the field were fed at the same temperature and light conditions described above for 4 days with a food concentration (mean±SE) of 0.306±0.029 lg C ml
1 of T. suecica and 0.107± 0.034 lg C ml
1 of P. multiseries. Copepod toxin content and grazing rate (Frost 1972) were estimated daily. To estimate copepod toxin detoxification, females were fed with a P. multiseries concentration of 0.09±0.02 lg C ml
1 (mean±SE) for two days and then transferred to a food medium of 0.5 lg C ml
1 of T. suecica; toxin content was analysed after 0, 12, 30 and 72 h. Toxin analysis To estimate the cell toxin content of P. multiseries, algal cells were collected on pre-combusted 13 mm GF/F Survival of copepods during experiments is showed in Fig. 1. An analysis of covariance (ANCOVA) taking days as covariable showed that there were no significant differences in copepod mortality between the control with only T. suecica and the treatments with T. suecica and P. delicatissima (F1,15 = 0.4, p = 0.515) and between the control and treatments with T. suecica and P. multiseries (F1,22 = 4.0, p = 0.056) in 3 days. From results obtained in copepod reproduction and toxin accumulation experiments, an ANCOVA, taking Pseudo-nitzschia spp. abundance as covariable and the ingestion rate of A. clausi on toxic and non-toxic Pseudo-nitzschia species as factor, showed that copepods ingested a higher amount of Pseudo-nitzschia cells as the diatom abundance increased (slope different from zero, F1,106 = 37, r2 = 0.36, p < 0.001), but there were no significant differences between Pseudo-nitzschia species (F1,106 = 0.3, p = 0.57) (Fig. 2), indicating no selective feeding behaviour against P. multiseries. The relationship between ingestion rate on Pseudo-nitzschia spp. (I, in lg C copepod
1 day
1) and Pseudo-nitzschia 126 Fig. 1 Survival (mean±SE) of A. clausi during both ingestion rate and accumulation experiments. T. suecica (grey symbols), P. delicatissima and T. suecica (white symbols) and P. multiseries and T. suecica (black symbols) abundance (A, in lg C ml
1) is described by the following equation: I ¼ 0:437 þ 5:375A ð1Þ Figure 3 shows egg production of A. clausi versus food availability on day 3 of the grazing experiment. An ANCOVA, taking food abundance as the covariable and the different food items as the factor, showed that egg production was higher as food abundance increased (slope different from zero, F1,31 = 19.4, r2 = 0.38, p < Fig. 2 Ingestion rates of A. clausi on Pseudo-nitzschia spp. (mean±SE) as a function of Pseudo-nitzschia spp. abundance (mean±SE). P. delicatissima (white circles) and P. multiseries (black circles) Fig. 3 Egg production of A. clausi (mean±SE) as a function of food abundance (the combined of T. suecica, P. delicatissima and P. multiseries, mean±SE). Symbols as in Fig. 1 0.001), but there were no significant differences between the control with only T. suecica and the treatments with T. suecica and P. delicatissima (F1,15 = 1.2, p = 0.29) and between the control and treatments with T. suecica and P. multiseries (F1,18 = 42.2, p = 0.147), indicating that the toxic P. multiseries does not affect egg production of A. clausi. Figure 4 also shows that P. multiseries does not affect egg hatching success of A. clausi, because a ANCOVA, taking Pseudo-nitzschia spp. abundance as covariable and egg hatching of A. clausi on toxic and non-toxic Pseudo-nitzschia species as factor, showed that egg Fig. 4 Egg hatching of A. clausi (mean±SE) as a function of Pseudo-nitzschia spp. abundance (mean±SE). Symbols as in Fig. 1 127 Fig. 5 Toxin content per copepod (mean±SE) during the detoxification experiment hatching did not change according to Pseudo-nitzschia spp. (F1,24 = 0.4, p = 0.492) and there were no significant differences between Pseudo-nitzschia species (F1,24 = 0.7, p = 0.391). The detoxification kinetic was described by the following equation (Fig. 5, r2 = 0.61, F1,6 = 8.1, p = 0.036): TCt ¼ TCt
1 e
1:01dt ð2Þ where TC is toxin concentration in copepods (in fmol copepod
1) and dt is the difference between t and t
1. The daily detoxification for DA toxins in A. clausi using this equation was 63.6% of the total toxin concentration in the copepod tissues. Following the methodology described by Guisande et al. (2002) we used all the equations and data obtained in this study to construct a model with the software STELLA (2001: High Performance Systems). Table 1 shows the specifications of the model. The model used a mean toxin content per ng cell carbon of 16.7 fmol ng C
1 (0.99 fmol cell
1). The assimilation efficiency of toxins ingested by the copepods that best reflects the results obtained in this study was 4.8%. Domoic acid concentration in copepods during accumulation experiments is showed in Fig. 6. Although Fig. 6 Toxin content per copepod (mean±SE) during the accumulation experiment (black circles) and predicted values by the model (white squares) copepods fed on toxic algae (Fig. 2), there were no differences in toxin concentration among days (analysis of variance, F3,4 = 0.78, p = 0.906) with a pooled mean±SD value of 1,116±110 fmol copepod
1. A paired t-test showed that there were no significant differences (df = 7, t = 0.2, p = 0.843) between the values of copepod toxin concentration predicted by the model and the real values (Fig. 6). Discussion One of the possible explanations of toxicity in phytoplankton is that, it has been evolved to prevent grazing and reduced interspecific competition. According to this hypothesis, those phytoplankton species that are toxic for the predators are rejected by them and consequently, predators redirect feeding pressure on nontoxic phytoplankton species and, hence indirectly, toxic phytoplankton species reduce also the interspecific competition. This selective feeding on toxic and nontoxic phytoplankton has been already observed in toxic dinoflagellates (Guisande et al. 2002; Teegarden et al. 2003). The rejection of phytoplankton species by Table 1 Specifications of the model Integration method Euler’s method Unit time days dt Toxin content carbon cell
1 Assimilation efficiency of toxins ingested Pseusonitzschia multiseries abundance lg C cell ml
1 Toxins assimilated (fmol copepod
1 d
1) days 1 day 16.7 fmol ng C
1 4.8% 0.105 Toxin content carbon cell
1 · 1000 · Equation 1 · Assimilation efficiency of toxins ingested fmol copepod
1 Eq. 2 (d
1) Toxins in copepods Detoxification 128 the predators implies that these species must be harmful for the predators. In this study, copepods do not select against Pseudo-nitzschia species and, in agreement with the previous studies on other copepod species about interaction with Pseudo-nitzschia species (Shaw et al. 1997; Lincoln et al. 2001; Tester et al. 2001), we did not observe any toxic effect of DA on A. clausi survival, egg production or egg hatching success. Therefore, it seems that DA is not toxic for copepods. It has been observed that copepod survival can be negatively affected by toxic marine phytoplankton, i.e. dinoflagellates (Carlsson et al. 1995; Bagøien et al. 1996). However, our results indicate that mortality of A. clausi adults did not increase when they fed toxic diatoms, as previously observed by Lincoln et al. (2001) for Temora longicornis and Acartia tonsa fed with toxic and non-toxic Pseudo-nitzschia genus. This fact does not mean that DA has no effect on copepod mortality. Windust (1992) found that high concentrations of domoic acid (several orders of magnitude higher than concentrations found at sea) can affect survival of copepods. Shaw et al. (1997) observed that dissolved domoic acid concentration of around 10 lM, produced a 50% of mortality in the copepod Tigriopus californicus after 24 h. However, this toxin concentration has been never reported in the field (diatom abundance higher than 40,000 cells ml
1), even in view of a very high cell toxin concentration of 68 pg DA cell
1 recorded by Bates (1998). In our study, although within the range observed for P. multiseries (Bates 1998; Lapworth et al. 2001), cell toxicity was only 0.31 pg DA cell
1. The effects of toxins can be also sublethal, including reduction in food intake, food assimilation or fecundity. It is known that grazing rates can be affected by size, motility, shape and nutritional value of the food (Frost 1972; Huntley et al. 1986). As the size of the algae used in these experiments ensures 100% filtering efficiency (Donaghay and Small 1979), it was expected that size of the Pseudo-nitzschia species used did not affect the feeding rate of A. clausi. However, the different toxic content of the Pseudo-nitzschia species could have influenced feeding rates of copepods. Toxin production in phytoplankton has been widely associated to feeding deterrence (see Turner et al. 1998) and it has been reported that toxins can affect feeding behaviour of predators (Teegarden 1999; Guisande et al. 2002; Colin and Dam 2003). Shaw et al. (1997) indicated that this was not the case for domoic acid that was toxic for copepods but had no effect on feeding rate. Our results showed that carbon ingestion rates on toxic and non-toxic Pseudo-nitzschia strains were the same, indicating that toxins did not affect feeding behaviour of copepods. This result is in agreement with the experiments carried out with other copepod species feeding on toxic and non-toxic Pseudonitzschia strains by Lincoln et al. (2001) and Tester et al. (2001). In both experiments, there were no differences on ingestion rates of copepods on toxic P. multiseries and non-toxic P. pungens. Thus, our findings and previous studies suggest that the role of the DA is not a feeding deterrent since neither feeding nor reproduction seem to be affected by the presence of toxic diatoms as food. Recent studies indicate that domoic acid binds iron and water in seawater, and therefore, a possible physiological role for DA in toxic Pseudo-nitzschia may include the acquisition (iron) or detoxification (copper) of trace metals (Rue and Bruland 2001). In agreement with this hypothesis, Maldonado et al. (2002) observed that DA production during exponential growth of two Pseudo-nitzschia species was directly induced by Fe-deficient or Cu stress. Although it seems clear that the production of DA is not a strategy evolved to avoid feeding pressure, copepods may play an important role on the transfer of DA through the food web. It has been observed that marine crustaceans can accumulate DA in their tissues (Lincoln et al. 2001; Tester et al. 2001). Falk et al. (1991) showed that domoic acid is water-soluble compound, and therefore, it is likewise not expected to be bioaccumulated in the food chain, unless a short food chain transferred toxins to higher trophic levels. Bargu et al. (2002) observed this kind of food chain in Monterey Bay. Krill accumulates domoic acid and seabird mortality due to DA has been reported (Sierra et al. 1997). Therefore, our findings and previous studies indicate DA is accumulated by zooplankton species, therefore, transferring the toxin toward higher trophic levels in marine food web. We observed an assimilation rate of 4.8% from toxins ingested. This rate is similar to the assimilation rate observed for PSP toxins in A. clausi (3.8%, Guisande et al. 2002). If we assume 4.5 lg dry wt per adult A. clausi (Guerrero and Rodriguez 1997), DA concentration in copepods during the experiment ranged between 40.84 and 97.47 lgDA g
1 dry wt. These levels exceed the regulatory limit of toxins in shellfish for human consumption (20 lg DA g
1 dry weight). The detoxification pattern observed in this study fits with the usual detoxification pattern observed in bivalves (Lassus et al. 2000) and copepods (Guisande et al. 2002). Domoic acid depuration time was shown to be species specific and have a wide variability that range from few days (Perna canalisculus, Mackenzie et al. 1993) to several months (Pecten maximus, Blanco et al. 2002a). The detoxification rate obtained (1.01 d
1) in this experiment is higher than that observed by Guisande et al. (2002) in this copepod species for PSP toxins. The faster detoxification of DA versus PSP toxins has been also observed in the mussels (Mytilus galloprovincialis) in waters of Galicia (Blanco et al. 1997; Blanco et al. 2002b). In summary, we conclude that DA production has not evolved to avoid predation, but some predators such as copepods and other zooplankton species may play an important role on the fate of this toxin through the marine food web. Acknowledgements We are very grateful to the crew of R/V ‘J.M. Navaz’ (I.E.O.) for technical support and helpful assistance and to 129 Jefferson Turner for English editing and constructive review. This research was supported by the project EVK3-CT2000-00055 and a FPU grant to A. Barreiro. 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