Introduction

Domoic acid (DA) is a potent neurotoxin produced by several species of the diatom genus Pseudo-nitzschia. DA has been demonstrated to elicit neuronal excitotoxicity and degeneration in mammals. Human exposure to sufficient quantities of this toxin through the ingestion of contaminated seafood causes amnesic shellfish poisoning (ASP) syndrome.

In addition to human ASP outbreaks, several marine bird and mammal mortality events have been associated with DA poisoning.
One factor common to such wildlife deaths is the transfer of toxin from algal producers to affected animals by one or more vector species. For example, DA-related bird (e.g., pelicans, cormorants) and sea lion (Zalophus californianus) casualties along the California, USA coast involved ingestion of contaminated planktivorous fish such as the northern anchovy, Engraulis mordax, that had fed directly on the toxic diatoms.

 Moreover, the natural krill populations that are consumed

by a wide range of predators, graze effectively on toxic Pseudo-nitzschia and accumulate DA , achieving concentrations more than twice the regulatory limit for human shellfish consumption (i.e., 20 g DA eq. g1). Zooplankton forms a critical component of many marine food webs. These grazers not only provide energy and nutrients assimilated from their algal diet to higher trophic levels, but can also serve as vectors for the transfer of algal biotoxins to their predators .

The ability of certain copepod species to graze on paralytic shellfish poisoning (PSP) toxin-producing dinoflagellates and accumulate these toxins has been reported from extensive laboratory and field studies.

In contrast, comparatively few Pseudo-nitzschia grazing or DA accumulation/retention experiments have been performed with copepods.

Overall, these few studies indicated that when copepods were exposed to either toxic or non-toxic Pseudo-nitzschia spp. there was no discernable difference in grazing rate.
Retention of DA by copepods following removal of the toxic Pseudonitzschia cells was also demonstrated. These albeit limited findings provide evidence suggesting that several copepod taxa can act as potential vectors for DA transfer in marine food webs.

Calanus finmarchicus is one of the most abundant copepod taxa in the N. Atlantic Ocean, with a geographical distribution ranging from the New England Shelf (40N) to the Greenland and Barents Seas (80N).
This species serves vital roles in pelagic food webs throughout this region as a phytoplankton grazer as well as prey for planktivorous fish and marine mammals, including the endangered N. Atlantic right whale (Eubalaena glacialis).

Grazing experiments and field work have shown that C. finmarchicus can ingest toxic Alexandrium spp. and accumulate PSP toxins, yet to date no similar investigations have been conducted with DA producing diatoms.

In the present study, C. finmarchicus was exposed, either simultaneously or independently, to toxic Pseudo-nitzschia multiseries and non-toxic P. pungens in both mixed- and monoculture experiments to address the following questions:
1) Can C. finmarchicus graze on DA-producing P. multiseries? 2) Does this copepod exhibit selective grazing when given a choice between both P. multiseries and P. pungens at varying initial cell concentrations? 3) Is this copepod capable of retaining DA after exposure to toxic P. multiseries, followed by depuration either in filtered seawater or in the presence of an alternate food source?

Evaluating the ability of C. finmarchicus to ingest DA producing diatoms and the extent to which this copepod retains the toxin will provide valuable insights into its role as a vector for DA transfer in marine food webs.

Materials and Methods

1. Algal culture maintenance
and zooplankton field sampling
Pseudo-nitzschia spp. P. multiseries non-toxic P. pungens C. finmarchicus
Cultures kept at 20C in f/2+Si medium on a 16 h:8 h light :dark cycle at a photon flux density of 100 mol m2s1, and acclimated to 10C for 4 h before each experiment.

2. Grazing experiment design

To assess whether copepods exhibit selective grazing when offered a choice between toxic P. multiseries and non-toxic P. pungens.
Five healthy adult female copepods were added to each experimental bottle using a wide mouth pipette and the bottles placed on a grazing wheel at 10C. Copepods were allowed to graze on the P.multiseries / P. pungens algal mixture for 12 hr.

4. Determination of grazing rates and

toxin ingestion
To discriminate between the morphologically similar P. multiseries and P. pungens for their enumeration in mixed algal culture samples, a fluorescence in-situ hybridization (FISH; i.e., whole cell hybridization) method was used. Total toxin ingested by C. finmarchicus during grazing experiments was estimated based on grazing rates and P. multiseries cellular DA content (cells ingested cop1 pg DA cell1). These values were used to calculate the percent of total ingested toxin present in copepods by the end of each grazing experiment.

3.Toxin accumulation/depuration
experiment design.
To determine whether C. finmarchicus is able to accumulate and assimilate DA into its tissues during exposure to P. multiseries. Depuration kinetics for DA following toxin accumulation by copepods (16 h, 38 h) and then removal of toxic P. multiseries cells were evaluated using GraphPad Prism (ver 4; Graphpad Software, San Diego, CA, USA)

Materials and Methods

5. DA extraction and analysis
DA was extracted from algal samples using 2.5 mL of 10% MeOH aq. added to the sample filters in glass extraction tubes. DA was quantified in algal (particulate and dissolved) and copepod extracts using a surface plasmon resonance (SPR) optical biosensor. Selected algal and copepod extracts were analyzed further by liquid chromatography coupled with mass spectrometry (LCMS/MS) to investigate the presence of a putative DA metabolite.

Results
1. Grazing Experiment
C. finmarchicus grazed on both toxic (P. multiseries) and non-toxic (P. pungens) diatom taxa in mixed-culture and mono-culture experiments, at all initial Pseudo-nitzschia cell concentrations tested. When offered a choice between P. pungens and P. multiseries, copepods did not significantly (two-way ANOVA; p-value >.05) ingest more of either food type regardless of total algal cell concentration.

Results
2. DA accumulation/depuration experiments

C. finmarchicus accumulated DA after being exposed to an initial P. multiseries cell concentration of 2200490 cells mL1 (mean SD; n=3), for either 16 h (Fig. 2) or 38 h (Fig. 3). By the end of each exposure, P. multiseries cell concentrations declined to 20099 (16 h) and 360140 (38 h) cells mL1 (meanSD; n=3). Maximum mean toxin levels accumulated by the copepods were 7.36 ng DA copepod1 and 2.99 ng DA copepod1 for the 16 h and 38 h exposures, respectively. During the depuration phase of each experiment, both starved and fed copepods showed an exponential decline in DA levels, with toxin still detectable in copepods at both the 24 h (Fig. 2) and 48 h (Fig. 3) depuration times.

Results
3. Detection of putative DA metabolite

LC-MS/MS analysis of copepod extracts before and after feeding P. multiseries indicated the possible presence of a methylated domoic acid metabolite after exposure to the culture (Fig. 4).

Discussion
The findings of this study confirm the ability of C. finmarchicus to ingest DA-producing Pseudo-nitzschia spp. and accumulate this neurotoxin in its tissues. Moreover, these results suggest that this ubiquitous N. Atlantic copepod can serve as a vector for DA transfer in marine food webs, potentially exposing planktivorous fish and certain marine mammals, including endangered N. Atlantic right whales (E. glacialis), to this toxin.

Discussion
In our grazing experiments, C. finmarchicus consumed toxic P.

multiseries cultures, regardless of the presence or absence of the morphologically similar, but non-toxic diatom, P. pungens.
When C. finmarchicus was exposed concurrently to equal

concentrations of P. multiseries and P. pungens, no significant difference or interaction was noted in grazing rates between the toxic and nontoxic species.

Discussion
These findings are consistent with several previous studies of

other copepod taxa and DA-producing Pseudo-nitzschia spp.

Several studies have shown that geographically distinct

populations of the marine copepod, Acartia hudsonica, cooccurring regularly with blooms of Alexandrium spp., feed more readily on these toxic algae than do nave populations from other regions.

Discussion
The amount of DA accumulated in C. finmarchicus by the end of

each grazing experiment was generally proportional to ingestion rates for P. multiseries as well as the amount of toxic food provided. Copepod toxin concen1trations ranged from 523 g DA g-1 cop,1 ,

occasionally exceeding the regulatory limit for human shellfish consumption (20 g DA g1). As a possible alternative explanation, the apparent loss of DA may be due to metabolism of the toxin in the copepod digestive system.

Conclusion
The present study is the first to establish the ability of the marine

copepod C. finmarchicus to ingest toxic P. multiseries cells and assimilate DA into its tissues.
C. finmarchicus did not discriminate between toxic vs. non-toxic

Pseudo-nitzschia spp. when exposed simultaneously to both; however, since blooms of DA-producing diatoms are generally not monospecific events, it will be important to perform similar experiments in the presence of other non-toxic algal prey.

Conclusion
Our findings also revealed that although C. finmarchicus

accumulated DA when exposed to toxic P. multiseries, the amount of toxin retained was generally less than 30% of that ingested still sufficient to facilitate trophic transfer of this toxin in natural systems.

Abstract
The marine algal biotoxin, domoic acid (DA), is produced by certain members of the diatom genus Pseudo-nitzschia. This neurotoxin has been responsible for several mass mortality events involving marine birds and mammals.

In all cases, the toxin was transferred from its algal producers through marine food webs by one or more intermediate vectors.

Abstract
The ability of some copepod taxa to serve as vectors for DA has been demonstrated; however, the role played in DA trophic transfer by Calanus finmarchicus, which often dominates N. Atlantic zooplankton assemblages and is a primary dietary component of the highly endangered N. Atlantic right whale (Eubalaena glacialis), has been uncertain.

Abstract
In the present study, we examined the ability of C. finmarchicus to consume DA-producing algae and retain the toxin. Results of grazing and toxin accumulation/depuration experiments showed that C. finmarchicus consumed DA-producing Pseudonitzschia multiseries regardless of the presence or absence of morphologically similar, but non-toxic P. pungens, across initial cell concentrations ranging from 1000-4000 cells ml-1 .

Abstract
Furthermore, C. Finmarchicus did not appear to preferentially consume or avoid either Pseudo-nitzchia species tested. After ingestion of P. multiseries, copepods accumulated DA and retained it for up to 48 h post-removal of the toxin source.

These findings provide evidence for the potential of C. finmarchicus to facilitate DA trophic transfer in marine food webs where toxic Pseudo-nitzschia is present.

Acknowledgments
Drs. S. Bates (Dept. of Fisheries & Oceans Canada, Moncton, NB, Canada) and R. Jones (Ocean Process Analysis Lab, Univ. New Hampshire, NH, USA), for providing the Pseudo-nitzschia cultures and Calanus finmarchicus used in this study. C. Mikulski for her assistance with fluorescence in-situ hybridization. Dr. C. Elliott, Queen's University Belfast, N. Ireland in giving DA antibody NOAA/NOS for providing the operationalfunds.

References
Bmstedt, U., 1985. Seasonal excretion rates of macrozooplankton from the Swedish west coast. Limnol. Oceanogr. 30, 607617. Bargu, S., Silver, M.W., 2003. Field evidence of krill grazing on the toxic diatom genus Pseudo-nitzschia in Monterey Bay, California. Bull. Mar. Sci. 72, 629638. Bargu, S., Powell, C.L., Coale, S.L., Busman, M., Doucette, G.J., Silver, M.W., 2002. Krill: a potential vector for domoic acid in marine food webs. Mar. Ecol. Prog. Ser. 237, 209216. Bargu, S., Marinovic, B., Mansergh, S., Silver, M.W., 2003. Feeding responses of krill to the toxin producing diatom Pseudo-nitzschia. J. Exp. Mar. Biol. Ecol. 284, 87104. Bates, S.S., 1998. Ecophysiology and metabolism of ASP toxin production. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. Springer-Verlag, Heidelberg, pp. 405426. Bates, S.S., Garrison, D.L., Horner, R.A., 1998. Bloom dynamics and physiology of domoicacid-producing Pseudo-nitzschia species. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. Springer-Verlag, Heidelberg, pp. 267292. Baumgartner, M.F., Mate, B.R., 2003. Summertime foraging ecology of North Atlantic right whales. Mar. Ecol. Prog. Ser. 264, 123135. Campbell, R.G., Wagner, M.M., Teegarden, G.J., Boudreau, C.A., Durbin, E.G., 2001. Growth and development rates of the copepod Calanus finmarchicus reared in the laboratory. Mar. Ecol. Prog. Ser. 221, 161183.

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