Calanus Finmarchicus-A Journal Report

7/28/2019 Calanus Finmarchicus-A Journal Report

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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 anddegeneration in mammals.

Human exposure to sufficient quantities of this toxin through theingestion of contaminated seafood causes amnesic shellfishpoisoning (ASP) syndrome.

Introduction


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In addition to human ASP outbreaks, several marine bird andmammal mortality events have been associated with DA poisoning.

One factor common to such wildlife deaths is the transfer of toxinfrom algal producers to affected animals by one or more vectorspecies.

For example, DA-related bird (e.g., pelicans, cormorants) and sea

lion (Zalophus californianus) casualties along the California, USAcoast involved ingestion of contaminated planktivorous fish such asthe northern anchovy, Engraulis mordax, that had fed directly onthe toxic diatoms.


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Moreover, the natural krill populations that are consumed by awide range of predators, graze effectively on toxic Pseudo-nitzschiaand 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 foodwebs.

These grazers not only provide energy and nutrients assimilatedfrom their algal diet to higher trophic levels, but can also serve as

vectors for the transfer of algal biotoxins to their predators .


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The ability of certain copepod species to graze on paralyticshellfish poisoning (PSP) toxin-producing dinoflagellates andaccumulate these toxins has been reported from extensive

laboratory and field studies.

In contrast, comparatively few Pseudo-nitzschia grazing or DAaccumulation/retention experiments have been performed withcopepods.


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Overall, these few studies indicated that when copepods wereexposed to either toxic or non-toxic Pseudo-nitzschia spp. there wasno discernable difference in grazing rate.

Retention of DA by copepods following removal of the toxicPseudonitzschia cells was also demonstrated.

These albeit limited findings provide evidence suggesting thatseveral copepod taxa can act as potential vectors for DA transfer inmarine food webs.


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Calanus finmarchicusis one of the most abundant copepod taxa inthe N. Atlantic Ocean, with a geographical distribution rangingfrom the New England Shelf (40N) to the Greenland and Barents

Seas (80N).

This species serves vital roles in pelagic food webs throughout thisregion as a phytoplankton grazer as well as prey for planktivorousfish and marine mammals, including the endangered N. Atlanticright whale (Eubalaena glacialis).


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Grazing experiments and field work have shown that C.finmarchicus can ingest toxicAlexandrium spp. and accumulate PSPtoxins, yet to date no similar investigations have been conducted

with DA producing diatoms.


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In the present study, C. finmarchicuswas exposed, eithersimultaneously or independently, to toxic Pseudo-nitzschiamultiseries and non-toxicP. pungens in both mixed- and mono-

culture 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 choicebetween both P. multiseries and P. pungens at varying initial cellconcentrations?3) Is this copepod capable of retaining DA after exposure to toxicP. multiseries, followed by depuration either in filtered seawater orin the presence of an alternate food source?


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Evaluating the ability ofC. finmarchicusto ingest DA producingdiatoms and the extent to which this copepod retains the toxin willprovide valuable insights into its role as a vector for DA transfer inmarine food webs.


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Materials and Methods

3.Toxin accumulation/depurationexperiment design.

Pseudo-nitzschia spp.P. multiseriesnon-toxic P. pungensC. finmarchicus Cultures kept at 20C in f/2+Si medium on a 16 h:8h light :dark cycle at a photon flux density of 100mol m2s1, and acclimated to 10C for 4 h before

each experiment.

4. Determination of grazing rates andtoxin ingestion

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

To determine whether C. finmarchicus is able to accumulateand assimilate DA into its tissues during exposure to P.

multiseries.Depuration kinetics for DA following toxin accumulation bycopepods (16 h, 38 h) and then removal of toxic P. multiseriescells were evaluated using GraphPad Prism (ver 4; GraphpadSoftware, San Diego, CA, USA)

To discriminate between the morphologically similar P.

multiseries and P. pungens for their enumeration in mixedalgal culture samples, a fluorescence in-situ hybridization(FISH; i.e., whole cell hybridization) method was used. Total toxin ingested by C. finmarchicus during grazingexperiments was estimated based on grazing rates and P.multiseries cellular DA content (cells ingested cop1 pg DAcell1). These values were used to calculate the percent of totalingested toxin present in copepods by the end of eachgrazing experiment.

1. Algal culture maintenance

and zooplankton field sampling

2. Grazing experiment design


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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 extractiontubes.DA was quantified in algal (particulate and dissolved) andcopepod extracts using a surface plasmon resonance (SPR)

optical biosensor.Selected algal and copepod extracts were analyzed further byliquid chromatography coupled with mass spectrometry(LCMS/MS) to investigate the presence of a putative DAmetabolite.


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Results

1. Grazing Experiment

C. finmarchicus grazed on both toxic (P.multiseries) and non-toxic (P. pungens) diatomtaxa in mixed-culture and mono-cultureexperiments, at all initial Pseudo-nitzschia cell

concentrations tested. When offered a choice between P. pungensand P. multiseries, copepods did notsignificantly (two-way ANOVA; p-value >.05)ingest more of either food type regardless oftotal algal cell concentration.


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Results2. DA accumulation/depuration experiments

C. finmarchicus accumulated DA after being exposed to an initial P. multiseries cell concentration of2200490 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 DAcopepod1 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.


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Results

3. Detection of putative DA metabolite

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


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Discussion

The findings of this study confirm the ability ofC. finmarchicus toingest DA-producing Pseudo-nitzschia spp. and accumulate thisneurotoxin 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 marinemammals, including endangered N. Atlantic right whales(E. glacialis), to this toxin.


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In our grazing experiments, C. finmarchicus consumed toxic P.multiseries cultures, regardless of the presence or absence of themorphologically similar, but non-toxic diatom, P. pungens.

When C. finmarchicuswas exposed concurrently to equalconcentrations ofP. multiseries and P. pungens, no significantdifference or interaction was noted in grazing rates between thetoxic and nontoxic species.

Discussion


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These findings are consistent with several previous studies ofother copepod taxa and DA-producing Pseudo-nitzschia spp.

Several studies have shown that geographically distinct

populations of the marine copepod,Acartia hudsonica, co-occurring regularly with blooms of Alexandrium spp., feed morereadily on these toxic algae than do nave populations from otherregions.

Discussion
http://www.zooplankton-online.net/gallery_Ahudsonicawhole.htm


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The amount of DA accumulated in C. finmarchicus by the end ofeach grazing experiment was generally proportional to ingestionrates for P. multiseries as well as the amount of toxic foodprovided.

Copepod toxin concen1trations ranged from 523 g DA g-1 cop,1 ,

occasionally exceeding the regulatory limit for humanshellfish consumption (20 g DA g1).

As a possible alternative explanation, the apparent loss ofDA may be due to metabolism of the toxin in the copepod

digestive system.

Discussion


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The present study is the first to establish the ability of the marinecopepod C. finmarchicus to ingest toxic P. multiseries cells andassimilate DA into its tissues.

C. finmarchicus did not discriminate between toxic vs. non-toxicPseudo-nitzschia spp. when exposed simultaneously to both;however, since blooms of DA-producing diatoms are generally notmonospecific events, it will be important to perform similarexperiments in the presence of other non-toxic algal prey.

Conclusion


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Our findings also revealed that although C. finmarchicusaccumulated DA when exposed to toxic P. multiseries, the amountof toxin retained was generally less than 30% of that ingested stillsufficient to facilitate trophic transfer of this toxin in naturalsystems.

Conclusion


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Abstract

The marine algal biotoxin, domoic acid (DA), is produced by certainmembers of the diatom genus Pseudo-nitzschia.

This neurotoxin has been responsible for several mass mortalityevents involving marine birds and mammals.

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


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Abstract

The ability of some copepod taxa to serve as vectors for DA has been

demonstrated;

however, the role played in DA trophic transfer byCalanusfinmarchicus, which often dominates N. Atlantic zooplanktonassemblages and is a primary dietary component of the highlyendangered N. Atlantic right whale (Eubalaena glacialis), has beenuncertain.


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Abstract

In the present study, we examined the ability ofC. finmarchicus toconsume DA-producing algae and retain the toxin.

Results of grazing and toxin accumulation/depuration experiments

showed that C. finmarchicusconsumed DA-producing Pseudo-nitzschia multiseries regardless of the presence or absence ofmorphologically similar, but non-toxic P. pungens, across initial cellconcentrations ranging from 1000-4000 cells ml-1 .


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Abstract

Furthermore, C. Finmarchicus did not appear to preferentiallyconsume or avoid either Pseudo-nitzchia species tested.

After ingestion ofP. multiseries, copepods accumulated DA andretained it for up to 48 h post-removal of the toxin source.

These findings provide evidence for the potential ofC.finmarchicus to facilitate DA trophic transfer in marine food webswhere toxic Pseudo-nitzschia is present.


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Acknowledgments

Drs. S. Bates (Dept. of Fisheries & Oceans Canada, Moncton, NB,Canada) and R. Jones (Ocean Process Analysis Lab, Univ. NewHampshire, NH, USA), for providing the Pseudo-nitzschia culturesand 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 DAantibodyNOAA/NOS for providing the operationalfunds.


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ReferencesBmstedt, 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-nitzschiain 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 vectorfor 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.), PhysiologicalEcology 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 anddevelopment rates of the copepod Calanus finmarchicus reared in the laboratory. Mar. Ecol. Prog. Ser.

221, 161183.


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BACKGROUND OF CRISENCIO M. PANER

Ph.D. in Biological Science (Candidate), USTM.S. in Microbiology, UST

B.S. Biochemistry, UST

Italian Government ScholarCollege Scholar

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Calanus Finmarchicus-A Journal Report

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