Toxicology 199 (2004) 207–217

Isolation and characterisation of a cytotoxic polyunsaturatedaldehyde from the marine phytoplankterPhaeocystis pouchetii

(Hariot) Lagerheim

Espen Hansena,∗, Arild Ernstsenb, Hans Chr. Eilertsena

a Department of Aquatic Biosciences, NFH, University of Tromsø, N-9037 Tromsø, Norwayb Department of Biology, Faculty of Science, University of Tromsø, N-9037 Tromsø, Norway

Received 17 November 2003; received in revised form 14 January 2004; accepted 19 February 2004

Available online 10 April 2004

Abstract

Several investigators have documented that the marine phytoplankterPhaeocystis pouchetiiproduce and excrete some com-pound that has adverse effects on its surroundings, but the chemical composition and structure of the active agent has so farbeen unknown. In the present study we used mass spectrometry to investigate the structural properties of the putative toxin.Colonial cells ofP. pouchetiiwere collected along the coast of northern Norway and cultivated in the lab for a limited periodof time prior to harvesting by filtration. Harvested cells and culture filtrate were extracted separately with organic solvents, anda yeast cell bioassay was used to track the toxic fractions during extraction and purification with HPLC. We found the organicextract from the culture filtrate to be toxic, and after purification with RP-HPLC the cytotoxic activity was recovered as onefraction. When the toxic fractions were pooled and analysed by GC-MS we were able to identify 2-trans-4-trans-decadienalby comparing retention time and fragmentation pattern to a commercial standard. This is the first report of a polyunsaturatedaldehyde produced by a marine alga belonging to the class Haptophyceae, and this implies that production and release of thesereactive compounds are not limited to diatoms.© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Phaeocystis pouchetii; 2-trans-4-trans-decadienal; Polyunsaturated aldehydes

1. Introduction

Of all animal protein consumed by humans, 16%comes from marine organisms, and epidemiologicalstudies indicate lower risk for coronary heart dis-ease, hypertension and cancer among populations

∗ Corresponding author. Tel.:+47-77645577;fax: +47-77646020.

E-mail address:espenh@nfh.uit.no (E. Hansen).

of humans eating seafood (Simopoulos, 1997). Themajority of known marine toxins are produced byphytoplankton, and there are indications that bloomsof toxic algae are becoming more frequent (Smayda,1990; Van Dolah, 2000). Thus, in order to secure thesafety of seafood there are both toxicological andeconomical reasons why detection and identificationof marine toxins has attracted so much attention.Besides, as the toxins in question can affect growth,reproduction and survival of marine organisms from

0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.tox.2004.02.026

208 E. Hansen et al. / Toxicology 199 (2004) 207–217

all trophic levels, from microalgae to mammals(Turner and Tester, 1997), they are potential regula-tors of the channelling of energy up the marine foodchain.

Toxins produced by marine microalgae have beendivided into different classes based on the syndromesassociated with exposure to them, such as paralyticshellfish poisoning (PSP), diarrhetic shellfish poison-ing (DSP), neurotoxic shellfish poisoning (NSP) andciguatera fish poisoning (CFP) (reviewed byWhittleand Gallacher, 2000). Most of the toxins responsiblefor these syndromes have large and rather complexchemical structures with specific actions, e.g. block-age of voltage gated sodium channels (Leftley andHannah, 1998) and agonism of glutamate receptors(Bates et al., 1998). Intensive research over the lastyears has revealed a new class of phytotoxins pro-duced by diatoms with more subtle and less specificeffects, a discovery that has drawn a lot of attentionsince the diatoms have traditionally been regardeda key component of the food chain. Three closely-related polyunsaturated aldehydes (PUAs) have beenisolated from Thalassiosira rotula, SkeletonemacostatumandPseudo-nitzschia delicatissima, namely2-trans-4-cis-7-cis-decatrienal, 2-trans-4-trans-7-cis-decatrienal and 2-trans-4-trans-decadienal (Miraltoet al., 1999). In the same study these aldehydes werefound to inhibit cleavage of sea urchin embryos,reduce growth of Caco2 cells and hatching of cope-pod eggs. The structural element shared by thesecompounds, the�,�,�,∂-unsaturated aldehyde group(Fig. 1), is able to form adducts with nucleophiles andis thus capable of inducing reactions that are toxicto the cell (van Iersel et al., 1997; Comporti, 1998;Refsgaard et al., 2000). The harmful effects of PUAshave been demonstrated at the organism level as in-ducers of apoptosis in sea urchin embryos (Romanoet al., 2003), at the cell level as cytotoxicity in humancell lines (Nappez et al., 1996) and at the proteinlevel by deactivation of enzymes (van Iersel et al.,1997).

R O

Fig. 1. Structure of the reactive�,�,�,∂-unsaturated aldehydegroup. This Michael acceptor element can react with a wide rangeof nucleophiles.

The number of species belonging to the marinephytoplankton genusPhaeocystisis debated, but sofar five species have been described by ribosomalRNA gene sequencing (Medlin et al., 1994; Zingoneet al., 1999), and at least nine have been suggestedusing morphological characters (Sournia, 1988).Species belonging to the genusPhaeocystisare im-portant in all oceans (Kashkin, 1963), and Phaeo-cystis pouchetii (Hariot) Lagerheim (Lagerheim,1896) is an important component of the springbloom of phytoplankton in northern waters (Eilertsenet al., 1981b). Its life cycle is only partly resolvedbut is known to be polymorphic consisting of atleast two solitary and one colonial stage. Ribo-somal RNA from thePhaeocystisbloom-formingcells at the coast of northern Norway and fromthe Barents Sea have not so far been sequenced,but the morphology and physiological characteris-tics of the cells are similar to that ofP. pouchetii(Medlin et al., 1994) and it possesses the pen-tagonal thread-like material typical of this species(Eilertsen et al., 1989a; Pienaar, 1991; Baumann et al.,1994).

Already 70 years ago,P. pouchetiiwas suspectedto cause avoidance of herring (Savage, 1930). Laterit was demonstrated that copepods avoided gracingon healthyP. pouchetiicolonies (Estep et al., 1990),food intake and growth were reduced in sea cage cul-tivated salmon during the spring bloom ofP. pouchetii(Eilertsen and Raa, 1995) and water fromP. pouchetiicultures acted toxic towards cod larvae (Aanesen et al.,1998). Chemical extracts fromP. pouchetiiculturesturned out to be toxic to blowflies (Stabell et al.,1999), and this led us to test similar extracts in a seaurchin embryo development assay. We found that theorganic extracts blocked cell divisions, and key eventsin the cell cycle, such as microtubule assembly, pronu-clear migration and DNA replication, were hindered(Hansen et al., 2003).

Due to the relatively close phylogenetic relationshipbetween diatoms and Haptophyceans (Meeks, 1974),and the striking similarities between the toxic effectsof diatoms andP. pouchetiiextracts, we decided tosearch for PUAs in ourP. pouchetiiextracts. We useda yeast cell bioassay to track the toxic activity dur-ing extraction and sample purification, and HPLC andGC-MS were used for purification and structure elu-cidation.

E. Hansen et al. / Toxicology 199 (2004) 207–217 209

2. Materials and methods

2.1. Reagents

All chemicals were purchased from Sigma-Aldrichunless otherwise specified.

2.2. Collecting and cultivating algae

Field collections of the colonial form ofP. pouchetiiwere made from Balsfjorden and Krabbedjupet afterthe peak of the spring bloom in the end of April 2002.Both locations are situated at the coast of northernNorway in the vicinity of Tromsø (69◦40′N). Thealgae were sampled with a 180�m mesh size WP-2zooplankton net in vertical hauls from 50 m depth tothe surface. Further concentration was achieved using90�m mesh sieves. The concentrated algae were di-luted with GF/C-filtered (Whatman, Maidstone, UK)seawater in 10 l glass beakers to obtain cultures withdensities of approximately 106 cells l−1. The cul-tures were maintained at 5◦C and 18:6 h light:darkphotoperiod at a light intensity of approximately50�mol m2 s−1. The cultures were stirred daily, andevery 3rd day 2 l of the culture medium were replacedwith fresh GF/C-filtrated seawater. No extra nutrientswere added. After 1–2 weeks of growth the cultureswere thoroughly stirred and 2 l subsamples of algaesuspended in seawater were taken out and stored at−20◦C until use. The density of the cultures at thetime of sampling was about 107 cells l−1 and most ofthe algae were still in their colonial form.

2.3. Toxin extraction and purification

The frozen subsamples of theP. pouchetiicultureswere slowly thawed at 4◦C, and sequentially filteredwith Whatman No. 1 and GF/C filters. The pooled fil-ters were transferred to 250 ml Erlenmeyer flasks andsonicated on ice in 80% methanol for 2× 30 s priorto extraction at 4◦C in darkness for 12 h. The organicsolvent was then evaporated in a rotary evaporator atreduced pressure and low temperature (40◦C). Theresidue was dissolved in distilled water and partitionedthree times against equal volumes of ethyl acetate. Theorganic phases were combined and reduced to drynessat low temperature (40◦C) and reduced pressure.

The filtrate was partitioned three times againstequal volumes of ethyl acetate, and the organic phaseswere pooled and reduced to dryness (the pH of thiswater was 7.6, and thus labelled ‘neutral’). The pH ofthe remaining water phase was adjusted to 2.5 using0.1 M HCl and subsequently partitioned three timesagainst equal volumes of ethyl acetate (‘acidic’). Theorganic phases were pooled and reduced to dryness.The aqueous phase was adjusted to pH 8.0 using0.1 M KOH and partitioned three times against equalvolumes of ethyl acetate, and the pooled organicphase and the aqueous phase were reduced to dryness(‘basic’ and ‘aqueous’, respectively). All sampleswere stored at−20◦C until use. Each fraction wasdissolved in 1 ml 80% methanol and tested for toxicactivity in the yeast bioassay.

2.4. HPLC purification and analysis

Extracts from the filters and filtrates were furtherpurified by HPLC. The dried extracts were dissolvedin 100�l 80% methanol and injected into an XTerraRP18 HPLC-column (3.9 mm× 150 mm, 5�m parti-cle size) (Waters Corporation, Milford, MA, USA). Abinary mobile phase of methanol and 0.01% aqueoustrifluoroacetic acid was used. The column was elutedwith a gradient of 50–80% methanol over 25 min,and subsequently washed with 100% methanol for10 min. The temperature of the column was kept at30◦C. The Waters HPLC system consisted of a 2690separation module, 996 photodiode array detector andFraction Collector II. Waters Millenium32 chromatog-raphy software was used to control the HPLC system.One-minute fractions were collected and reduced todryness in a SpeedVac system (ThermoSavant, Hol-brook, NY, USA).

2.5. Yeast bioassay

Common bakers yeast (Saccharomyces cerevisiae)growing on a simple medium based on yeast ex-tract containing bacteriological peptone and glucose(YDP broth) was used for the yeast bioassay. Growthmedium (1% YPD broth, 136�l) was added to thewells of a sterile flat-bottomed 96-well Nunclon mi-crotitre plate (Nunc A/S, Roskilde, Denmark). Thesample to be tested was dissolved in 80% methanol,and 8�l of the test solution was added to each well.

210 E. Hansen et al. / Toxicology 199 (2004) 207–217

Bakers yeast was incubated in 1% YPD at 37◦C overnight, and diluted with 1% YPD to appropriate den-sities, and 16�l of the diluted yeast inoculum wastransferred to the microtitre plate wells. The plateswere sealed with SealPlate (Excel Scientific, Wright-wood, CA, USA) and incubated at 37◦C. Growth wasrecorded as increase in optical density atλ 560 nm asmeasured in a SpectraMax 190 microtitre plate readercontrolled by SoftMax Pro software (Molecular De-vices Corporation, Sunnyvale, CA, USA).

2.6. GC-MS analysis

For GC-MS analysis, samples were diluted in 100%methanol and 1�l was injected splitless into a CP-Sil 8 CB Low Bleed/MS column with 0.12�m filmthickness (25 m×0.25 m i.d., Varian, Middelburg, TheNetherlands). The GC-MS system consisted of a TraceDSQ single quadropole mass selective detector, TraceGC (ThermoFinnigan, Austin, TX, USA) and GC PALautoinjector (CTC Analytics AG, Zwingen, Switzer-land). XCalibur 1.3 software was used to control thesystem and to review data (ThermoFinnigan) and theNIST database was used for fragmentation spectrumidentification. The injection port was held at 220◦C,and helium grade 6.0 was used as carrier gas. Sepa-ration was performed by a temperature program that

Fig. 2. Growth ofS. cerevisiaemeasured as increase of absorbance after 6 h incubation with extracts fromP. pouchetii. Controls wereincubated in water containing similar amounts of methanol as the extracts. Values are means (±standard errors) of optical density in fivedifferent experiments.

started isothermally at 50◦C for 2 min and rampedwith 5◦C min−1 to 280◦C. Full spectra were recordedbetween 30 and 500 amu in the EI ionisation mode.A commercial standard of 2-trans-4-trans-decadienal(Acros Organics, Geel, Belgium) was injected to com-pare retention time and fragmentation pattern with theendogenous compound.

3. Results

3.1. Toxicity of raw extracts

The yeast cells grew well in the experimental setupused in the yeast bioassay, and the growth was notinfluenced by the addition of 5% methanol (data notshown). The growth medium had no significant ab-sorbance at 560 nm and did not influence the densitymeasurements of the yeast cells. The extracts of thecells contained components that to a certain degreeabsorbed light between 500 and 600 nm, so we usedincrease in absorbance from the beginning to the endof the incubation period (6 h) as response parameterin the bioassay.

In the control treatment, the average increase inabsorbance atλ 560 nm after 6 h was 0.423± 0.018absorbance units (AU) (Fig. 2). Yeast cells incubated

E. Hansen et al. / Toxicology 199 (2004) 207–217 211

Fig. 3. Growth ofS. cerevisiaemeasured as increase of absorbance after 6 h incubation with 1-min HPLC-fractions of neutral extracts offiltrate from P. pouchetiicultures. Controls (C) were incubated in water containing similar amounts of methanol as the HPLC-fractions.Values are means (±standard errors) of optical density in three different experiments.

in the methanol extracts of the sonicated filters con-taining P. pouchetiicells had an average increase inabsorbance of 0.415± 0.015 AU, i.e. not significantlydifferent from the control treatment. Yeast cells incu-bated in the organic extract of filtrated neutral water(‘neutral’) had a weak increase in optical density(0.056± 0.024 AU) compared to the control, indicat-ing that the growth of the yeast cells had been hinderedby the extract. The organic extracts of acidic and basicwater had no negative effect on the growth of yeastcells, and the average increases in absorbance forthese two treatments were 0.398± 0.013 and 0.432±0.019 AU, respectively, which are slightly lower butthough comparable to the control. The remaining wa-ter phase that had been extracted at three different pHlevels was also checked for remaining toxic properties,but the growth of yeast cells incubated in this samplewas normal (0.398±0.020 AU) relative to the control.

3.2. Purification with RP-HPLC

The extract found to be active in the yeast bioas-say (the organic extract from the neutral filtrate) wasredissolved in 80% methanol and purified with RP-HPLC. When the 1-min fractions were tested in theyeast bioassay they had an increase in optical density

at 560 nm ranging from 0.216± 0.007 AU to 0.279±0.023 AU, whereas the corresponding value for thecontrols was 0.252± 0.021 AU (Fig. 3). The excep-tions were fraction 11 and 12 in which the toxicitywas recovered, reflected by the low absorbance of0.094± 0.037 and 0.118± 0.025 AU, respectively.

3.3. Identification of PUA with GC-MS

After being pooled and reduced to dryness, frac-tions 11 and 12 were analysed by GC-MS. In the totalion current (TIC) chromatogram about 30 peaks weredetected (Fig. 4), but the majority of the observedpeaks contained fragments indicative of column andseptum bleed (m/z 73, 147, 207, 221, 281, 355, 429).Some peaks were identified as fatty acids in NISTlibrary searches, e.g. the spectrum of the peak at23.28 min matched with tetradecanoic acid and thespectrum of the peak at 24.49 min matched withhexadecanoic acid. When plotting ion chromatogramfor masses of fragments characteristic for PUAs pre-vious identified from marine microalgae, the peakthat eluted after 13.97 min gained our attention. Itsspectrum matched with 2-trans-4-trans-decadienal(Fig. 5) in the NIST library. The most important frag-ments observed (Fig. 6A) were M+ 152 (7), 95 (9),

212 E. Hansen et al. / Toxicology 199 (2004) 207–217

Fig. 4. Ion chromatogram (total ion current, 30–500 amu) of GC-MS analysis of the pooled fractions 11 and 12 from the HPLC-purificationof neutral extracts of filtrate fromP. pouchetiicultures.

83 (10), 82 (9), 81 (base peak), 67 (14), 55 (11) and41 (20). When compared to a commercial standard of2-trans-4-trans-decadienal, the retention time of theendogenous compound (13.97 min) was similar to thestandard (13.96 min) and the fragmentation patternswere equivalent (Fig. 6B).

4. Discussion

P. pouchetiiextracts from concentrated cells and al-gae culture seawater were tested for cytotoxic activityin a yeast cell bioassay. Extracts from the algal cells

O

Fig. 5. Structure of 2-trans-4-trans-decadienal.

had no effect on the growth of yeast cells, whereasorganic extracts from theP. pouchetiiculture waterreduced growth of yeast cells (Fig. 2). These findingsare in agreement with our studies on the effects ofP. pouchetiiextracts on sea urchin embryos, wherewe demonstrated that extracts from filtrated seawa-ter blocked cell divisions in sea urchin embryos andextracts from intact cells were only mildly cytotoxic(Hansen et al., 2003). When we purified this organicextract with RP-HPLC, we recovered the cytotoxicactivity in the two subsequent fractions 11 and 12(Fig. 3), and by using GC-MS we identified thePUA 2-trans-4-trans-decadienal in these two frac-tions (Figs. 4 and 6). We also searched for two otherclosely related compounds, namely 2-trans-4-cis-7-cis-decatrienal and 2-trans-4-trans-7-cis-decatrienal,since these two aldehydes have been isolated fromthe same microalgae as the decadienal (Miralto et al.,

E. Hansen et al. / Toxicology 199 (2004) 207–217 213

Fig. 6. Positive EI-MS background-subtracted spectra of (A) the peak eluting at 13.97 min in the pooled fractions 11 and 12 from theHPLC-purification of neutralP. pouchetiiextracts and (B) a commercial standard of 2-trans-4-trans-decadienal.

214 E. Hansen et al. / Toxicology 199 (2004) 207–217

1999) and they possess the same reactive Michaelelement (Comporti, 1998) (Fig. 1). We were thoughnot able to detect these two decatrienals in our sam-ples.P. pouchetiiis difficult to grow in culture, andas a consequence we had limited amounts of cellsavailable when making the extracts. Thus, we didnot have sufficient material for confirming the iden-tification of 2-trans-4-trans-decadienal by NMR.We cannot exclude that other PUAs may have beenpresent in the extracts but not in adequate amounts fordetection.

As the PUAs are reactive compounds they are alsounstable, and they are often derivatised prior to analy-sis in order to avoid decomposition (Vogel et al., 2000;d’Ippolito et al., 2002a). We decided not to derivatiseour samples because this could have complicated theinterpretation of the MS data. We found that the de-tection of 2-trans-4-trans-decadienal on the GC-MS,both as standard and endogenous compound, was verysensitive to the performance of the chromatographicsystem, especially the deactivation status of the injec-tion glass liner. Solid phase micro extraction (SPME)in combination with GC-MS have been used to anal-yse PUAs (Spiteller and Spiteller, 2000), but we didnot have any success with this technique. We believethat the PUAs were too diluted in the samples, andthat the SPME-fibre did not provide sufficiently strongconcentration of 2-trans-4-trans-decadienal or otherPUAs for detection on the GC-MS.

The fact that we found toxic activity in extractsfrom culture water and not from the algal cellsthemselves both in our experiments with sea urchinembryos (Hansen et al., 2003) and yeast cells, isfurther in support with our conclusion that 2-trans-4-trans-decadienal is responsible for the observed toxiceffects as the PUAs are not stored in the cells butreleased as a response to stress (Pohnert, 2000). Fattyacids released from membrane storage sites by phos-pholipases are transformed into lipid hydroperoxidesby lipoxygenases, which again are turned into PUAsby lyases (Pohnert, 2002; Pohnert and Boland, 2002).This enzymatic cascade is induced very fast, and thereactive PUAs are released only seconds after themechanical stress has occurred (Pohnert, 2000). Sev-eral investigators have used sonication as a mean toinduce production of PUAs in diatoms (Miralto et al.,1999; Caldwell et al., 2002; d’Ippolito et al., 2002b;Pohnert et al., 2002). We sonicated theP. pouchetii

cells after they had been concentrated on filters,but this did not induce any detectable production ofPUAs. The dehydration of the cells during the filtra-tion process might have inactivated the required en-zymes in the pathway leading to production of PUAs.On the other hand, the stress imposed on the cellsduring freezing and thawing and the concentrationwith sieves, the cultivation in dense cultures and thefollowing filtration seems to have been sufficient toinitiate the release of PUAs to the surrounding watermedia.

A wide array of bioassays have been used to screenfor PUAs from algal cultures and extracts rangingfrom copepods (Poulet et al., 1994; Ban et al., 1997),sea urchin embryos (Miralto et al., 1999; Pohnert,2002) and polychaetes (Caldwell et al., 2002) to hu-man cell lines (Berge et al., 1997; Miralto et al., 1999).Yeast cells have been used to screen for mycotox-ins (Whitehead and Flannigan, 1989), and these as-says can be conducted in 96-well plates using opticaldensity as measurement of cell density (Binder, 1999;Engler et al., 1999). The limitation of yeast cells asa toxicity-testing organism is that its permeability forlarger polar molecules can be restricted, but in the casewith PUAs this should be no problem. In our expe-rience yeast cells represents a rapid and easy methodfor screening biological extracts for PUAs.

P. pouchetii belongs to the class Haptophyceaewhich contains several other species associated withtoxin production, e.g.Chrysochromulina polylepisthatproduces and excretes glycolipids with haemolyticand ichtytoxic properties (Yasumoto et al., 1990).However, this is the first time a Haptophycean rep-resentative is reported to produce PUAs. Productionof PUAs by marine microalgae was first identified inthe diatomsT. rotula, S. costatumand P. delicatis-sima (Miralto et al., 1999), but recent studies haverevealed that the production of reactive aldehydesdoes not only differ from species to species, but alsowithin different strains of the same species (Pohnertet al., 2002). This makes it difficult to assess theecological significance of PUA-production by marinemicroalgae. This is especially true forPhaeocystis,a genus for which all members have not yet beendetermined by DNA sequencing and some taxonomicconfusion still remains (Sournia, 1988; Medlin et al.,1994; Zingone et al., 1999). Nevertheless, the PUA2-trans-4-trans-decadienal is released byP. pouchetii,

E. Hansen et al. / Toxicology 199 (2004) 207–217 215

and it is known to interfere with the proliferation ofdifferent cell types, both prokaryotic and eukaryotic(Miralto et al., 1999; Bisignano et al., 2001). Asmechanical stress is known to induce the release ofPUAs in other phytoplankton species (Pohnert, 2002;Pohnert and Boland, 2002), the release of 2-trans-4-trans-decadienal has been suggested to be a mean ofdeterring gracers, e.g. zooplankton or fish larvae.P.pouchetii is a common component of northern andtemperate spring blooms (Eilertsen et al., 1981a),and several publications reports thatP. pouchetii isgrazed by zooplankton at normal rates (Huntley et al.,1987; Eilertsen et al., 1989b) and that it also can bea diet preferable to diatoms (Connotec et al., 2001).On the other hand, it has been reported that copepodsmay avoid gracing on healthy colonies ofP. pouchetii(Estep et al., 1990). Thus, the production and excre-tion of PUAs seems to be depending on the state of thecells or on environmental factors, or both. When con-centrating and extracting the cells they were exposedto a rather mild mechanical stress (toxicity was notdetected in extracts from sonicated cells), which mayimply that the PUA can be released into the sea in theabsence of gracers. If this is the case, the PUA mayserve as an allelochemical (Legrand et al., 2003), i.e.a compound which givesP. pouchetiia competitiveadvantage over phytoplankton species blooming at thesame time by inhibiting their growth. It must thoughbe borne in mind that these ecological processes takesplace in a medium that experiences large horizontaland vertical displacements, and that dilution of re-leased substances will take place to varying degreesdepending on the prevailing mixing regimes. The lackof knowledge of the life cycle ofP. pouchetiiaddsfurther complexity to this picture. The colonies weworked with were from late in the spring bloom whenP. pouchetiioften dominates the phytoplankton stock.Future work on this issue should focus on quantifyingthe PUA in cultures ofP. pouchetiiat different devel-opmental stages, and in the ocean at different stages ofa bloom.

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