Harmful Algae 23 (2013) 3445

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Harmful Algae
journal homepage: www.elsevier.com/locate/hal

Production and excretion of okadaic acid, pectenotoxin-2 and a novel dinophysistoxin from the DSP-causing marine dinoagellate Dinophysis acuta Effects of light, food availability and growth phase
Lasse Tor Nielsen a,*, Bernd Krock b, Per Juel Hansen a
a b

Marine Biological Section, University of Copenhagen, Strandpromenaden 5, DK-3000 Helsingr, Denmark Alfred-Wegener-Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany

A R T I C L E I N F O

A B S T R A C T

Article history: Received 20 September 2012 Received in revised form 21 December 2012 Accepted 25 December 2012 Available online 23 January 2013 Keywords: Diarrhetic shellsh poisoning (DSP) Dinophysis acuta Food availability Light Mixotrophy Okadaic acid (OA)

Diarrhetic shellsh poisoning (DSP) toxins constitute a severe economic threat to shellsh industries and a major food safety issue for shellsh consumers. The prime producers of the DSP toxins that end up in lter feeding shellsh are species of the marine mixotrophic dinoagellate genus Dinophysis. Intraspecic toxin contents of Dinophysis spp. vary a lot, but the regulating factors of toxin content are still poorly understood. Dinophysis spp. have been shown to sequester and use chloroplasts from their ciliate prey, and with this rare mode of nutrition, irradiance and food availability could play a key role in the regulation of toxins contents and production. We investigated toxin contents, production and excretion of a Dinophysis acuta culture under different irradiances, food availabilities and growth phases. The newly isolated strain of D. acuta contained okadaic acid (OA), pectenotoxins-2 (PTX-2) and a novel dinophysistoxin (DTX) that we tentatively describe as DTX-1b isomer. We found that all three toxins were excreted to the surrounding seawater, and for OA and DTX-1b as much as 90% could be found in extracellular toxin pools. For PTX-2 somewhat less was excreted, but often >50% was found extracellularly. This was the case both in steady-state exponential growth and in food limited, stationary growth, and we emphasize the need to include extracellular toxins in future studies of DSP toxins. Cellular toxin contents were largely unaffected by irradiance, but toxins accumulated both intra- and extracellularly when starvation reduced growth rates of D. acuta. Toxin production rates were highest during exponential growth, but continued at decreased rates when cell division ceased, indicating that toxin production is not directly associated with ingestion of prey. Finally, we explore the potential of these new discoveries to shed light on the ecological role of DSP toxins. 2012 Elsevier B.V. All rights reserved.

1. Introduction Diarrhetic shellsh poisoning (DSP) toxins pose a serious threat to both human health and shellsh industries in many areas of the world (Reguera et al., 2012). Although rst described from two marine sponges, the primary producers of DSP toxins are species from the two marine dinoagellate genera Dinophysis and Prorocentrum (Yasumoto et al., 1980; Tachibana et al., 1981; Murakami et al., 1982). In terms of shellsh poisoning and human health issues, Dinophysis is considered the key player, since DSP producing Prorocentrum species are benthic, and usually not readily available for suspension feeding mussels. The genus Dinophysis contains more than 100 mixotrophic species and representatives can be found in most oceans and mez, 2005). Dinophysis spp. marine environments of the world (Go

* Corresponding author. Tel.: +45 35321992; fax: +45 35321951. E-mail addresses: ltnielsen@bio.ku.dk, ltnielsen@gmail.com (L.T. Nielsen). 1568-9883/$ see front matter 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.hal.2012.12.004

have long been considered obligate mixotrophs (Jacobson and Andersen, 1994), but Park et al. (2006) were the rst to successfully grow a Dinophysis species in laboratory culture, by feeding it the marine ciliate Mesodinium rubrum (= Myrionecta rubra). Prior to that, all studies on Dinophysis spp. were limited to in situ populations and single cells picked from natural populations (e.g. Draisci et al., 1996; Miles et al., 2004; Setala et al., 2005). The mixotrophic nature of Dinophysis spp. extends beyond regular mixotrophy, since the genus has recently been shown to sequester and utilize the chloroplasts of its ciliate prey, M. rubrum (Park et al., 2007; Wisecaver and Hackett, 2010; Kim et al., 2012). Therefore, high photosynthetic activity of Dinophysis spp. relies on continuous food uptake. Starved cells of mixotrophic Dinophysis species will remain photosynthetically active, although at reduced rates. This allows mixotrophic Dinophysis spp. to survive without food for several months, as long as light is available (Kim et al., 2008; Riisgaard and Hansen, 2009; Nielsen et al., 2012). Dinophysis spp. produce two groups of DSP toxins: (1) okadaic acid (OA) and the structurally similar dinophysistoxins (DTXs) and

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(2) pectenotoxins (PTXs) (Yasumoto et al., 1985). OA and DTXs are free polyether acids that inhibit serine/threonine phosphatase, and affect the secretion and gene transcription of nerve growth factor (Pshenichkin and Wise, 1995; Garcia et al., 2003). PTXs are polyether lactones, but the actual toxicity, and their status as DSP toxins, is currently under debate. For now, however, the toxin group remains included in the 160 mg kg1 regulatory threshold set for all commercial shellsh (EC, 2004; Miles et al., 2004; Reguera et al., 2012). Currently, two DTXs (DTX-1 and DTX-2) have been described with full molecular structures from species of Dinophysis. In addition, other DTXs with yet undisclosed exact molecular structures have been reported from Irish waters, and the list of known DTXs could grow within the next years (James et al., 1997; Draisci et al., 1998). PTX-2 is the primary PTX produced by Dinophysis spp. but PTX can be found in a variety of forms, with at least 15 different derivatives presently identied (Miles, 2007; Anonymous, 2009). Most of these are believed to occur only as metabolites in shellsh, however (Suzuki et al., 1999). Dinophysis acuta normally contains OA and PTX-2 as well as either DTX-1 or DTX-2, but the cellular content of each toxin can vary a lot (Lee et al., 1989; James et al., 1999; Lindahl et al., 2007; Pizarro et al., 2008, 2009; Fux et al., 2010). The ecological role and signicance of DSP toxins are currently largely unknown (Reguera et al., 2012). Several functions seem plausible, including food capture, grazer defense, allelopathy and anti-bacterial deterrent, and some of these have already been visited (Nagai et al., 1990; Carlsson et al., 1995; Gross, 2003). The theory of DSP toxins as a defense against grazing is supported by the nding that some copepods seem to discriminate against D. acuminata as a food source, whereas another non-discriminating copepod species experienced reduced survival rates (Carlsson et al., 1995). The theory of allelopathic effects has also received backing (Windust et al., 1996), but both ideas remain unproven, and the ecological role of DSP toxins is still undisclosed. Many harmful microalgae are mixotrophic (i.e. use particulate food for growth; e.g. Prymnesium parvum, Alexandrium spp., Karenia spp., and Karlodinium spp.). In fact, only the non-motile algal groups like diatoms (e.g. Pseudo-nitzschia spp.) and cyanobacteria (e.g. Nodularia spumigena) can be considered autotrophs or auxotrophs (Flynn et al., 2013). Dinophysis spp. are among the very few toxic microalgae that rely on chloroplasts sequestered from their prey. Psteria spp. may be another group with similar abilities, but data on rates of photosynthesis are still lacking. The available data on Dinophysis spp. suggest they are obligate mixotrophs. Hence they 1) cannot live in the long run without food and 2) cannot live in complete darkness even when supplied excess amounts of food (Park et al., 2008; Nagai et al., 2008; Nishitani et al., 2008; Kim et al., 2008; Riisgaard and Hansen, 2009). This raises questions about the role of food uptake and light for toxin production. It also raises questions about the possible excretion of DSP toxins and ultimately about the ecological role of DSP toxins. Here, we investigate the dependence of toxin contents and production of D. acuta upon irradiance, food availability and growth phase. We measure both intra- and extracellular levels of DSP toxins in order to quantify toxin excretion under various conditions. The aim is to understand Dinophysis spp. toxicity better and ultimately to unravel the ecological function of DSP toxins. 2. Materials and methods 2.1. Cultures and culturing conditions Cultures of the cryptophyte Teleaulax amphioxeia (K-0434 (SCCAP)) and the ciliate M. rubrum (MBL-DK2009) were established from water samples from Helsingr Harbor in 2009. Cultures

of M. rubrum were fed T. amphioxeia at a predator:prey ratio of 1:10 twice a week. During a scientic cruise in the North Atlantic ca. 100 km south of the Faroe Islands (608240 N; 68580 W), a nonclonal culture (DANA-2010) of the DSP producing dinoagellate D. acuta was established in June 2010, by picking and washing several cells. M. rubrum was added as prey organism twice per week at a predator:prey ratio of 1:10 to allow mixotrophic growth. All three species were grown in f/2 medium based on autoclaved seawater, and with a salinity of 32 1, a dissolved inorganic carbon (DIC) concentration of 2.3 0.1 mmol L1 and a pH of 8.0 0.05 (Guillard and Ryther, 1962). They were grown at 15.0 1.0 8C in a temperature controlled room, at an irradiance of 130 mmol photons m2 s1 (PAR), controlled by a timer to a light:dark cycle of 16:8 h. All cultures were non-axenic. DSP toxins of a D. acuta stock culture were sampled by transferring 0.5 ml subsamples to spin lters (pore size = 0.45 mm, VWR, Denmark), and centrifuging these at 400 g for 2 min. Filtrates were removed, and spin lters were stored at 18 8C until extraction and analysis. 2.2. Experiment 1 effects of irradiance on growth, photosynthesis and toxin production D. acuta was kept well-fed for a minimum of 14 days at four different irradiances to evaluate the effects of light on photosynthesis, growth rate and toxin content. The four irradiances were 7, 15, 30 and 130 mmol photons m2 s1 (PAR), henceforth termed I7, I15, I30 and I130 respectively. All treatments were setup in the same room, in front of the same light source consisting of Osram cool white 58 W/640 uorescent tubes, but with different combinations of neutral density lters in front of I7, I15 and I30 in order to achieve the designated irradiances. All four treatments were run in triplicate 65 ml polystyrene bottles lled to capacity. The ciliate, M. rubrum, was cultured at I130, but was light acclimated for a week at the appropriate irradiances before being used as prey for D. acuta. The same was done with the cryptophyte T. amphioxeia. Initial cell concentrations at the experimental setup were 200 and 2500 cells ml1 of D. acuta and M. rubrum, respectively. Every 24 days, 3 ml subsamples were removed from each ask for enumerations of D. acuta, M. rubrum and T. amphioxeia. 1 ml SedgewickRafter sedimentation chambers were used, and cells were counted on an Olympus CK2 inverted microscope at 40 400. A minimum of 200 cells were counted, unless cell concentrations were below 200 ml1 at which point a maximum 1 ml was inspected. After each sampling, cell concentrations of D. acuta and M. rubrum were adjusted to 200 and 2000 cells ml1 respectively, by adding f/2 medium and light acclimated, well fed M. rubrum. On the nal day, 1 ml subsamples were removed from each triplicate bottle for determination of photosynthetic activity. 80 D. acuta cells were picked from each subsample under a stereo microscope, and photosynthetic activities were determined exactly like presented earlier, including 14C addition (as HCO3) to both light and dark samples, 3 h incubations and checks of specic activity (Nielsen et al., 2012). Toxin samples were also taken from each ask on the nal day of the rst experiment. Subsamples of 0.5 ml were transferred to spin lters, and these were centrifuged at 400 g for 2 min. Filtrates were removed, and spin lters stored at 18 8C until extraction and analysis. This toxin extraction method has previously been shown not to affect intracellular toxins quotas of Dinophysis acuminata at centrifugal forces up to 12,800 g (Nielsen et al., 2012). For growth rates and toxin contents, averages of the last three values were dened as the well-fed, light acclimated values (hereafter termed steady-state), and these were used for comparisons between irradiances.

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2.3. Experiment 2 toxin content, production and excretion as a function of growth phase Based on results from the rst experiment, the two irradiances I15 and I130 were selected for the set of subsequent experiments. Triplicate 65 ml polystyrene bottles of each of the two irradiances were set up with 200 D. acuta cells ml1 and 2500 M. rubrum cells ml1 initially. Under both treatments, D. acuta was kept well fed for the rst 14 days to ensure light acclimation. This was done by sampling, enumerating and diluting every few days, parallel to the rst experiment. After the rst 14 days, food additions were stopped, and bottles were instead diluted with f/2 medium only, in order to ensure optimal conditions of nutrients, pH, etc. Dilutions with f/2 were stopped once division rates of D. acuta declined due to starvation. In this set of experiments, DSP toxins were sampled in three different ways. Firstly, standard spin lter samples of 0.5 ml were taken on all samplings following the method described for the rst experiment. Secondly, on four occasions during the experiments, 100 D. acuta cells from each bottle were picked and washed with micropipettes under the stereoscope. After washing, cells were picked and transferred to spin lters that were subsequently treated like the regular spin lter samples. Thirdly, on ve occasions during the experiments, a 10 ml subsample from each bottle was transferred to a glass scintillation vial, which was then stored at 18 8C until further analysis. Since D. acuta cells were not removed from these samples prior to freezing, these samples were a measure of total toxin (intra- and extracellular). As in the rst experiment, averages of the last three values from the well-fed period were dened as steady-state values representing well-fed and light acclimated conditions, and these values were used for comparisons. 2.4. Toxin extraction and analysis For extraction of spin lter samples, 100 ml methanol (100%) was added, and after 1 h incubation, these were centrifuged at 800 g for 2 min. The extract was then transferred to a 2 ml glass HPLC vial with a 250 ml glass insert. Extractions from the 10 ml water samples taken in the second experiment were achieved with solid phase extraction (SPE). 1 ml LC-18 SPE cartridges (Sigma Aldrich1, Germany) were pre-conditioned with 100% methanol, and then ushed with distilled water to remove excess methanol. All 10 ml of each sample were then slowly (1 ml min1) passed through an SPE cartridge. The cartridge was washed three times with 1 ml distilled water, after which DSP toxins were eluded with 1 ml 100% methanol directly into a 2 ml glass HPLC vial. Toxin samples from the rst experiment were only analyzed in the positive ionization mode, whereas all toxin samples from the second experiment were analyzed in duplicates: once in positive ionization mode to detect PTXs, and once in negative ionization mode to detect OA and DTXs. Separation of toxins was achieved on an Agilent (Waldbronn, Germany) model 1100 liquid chromatograph (LC). The LC equipment included a solvent reservoir, in-line degasser (G1379A), binary pump (G1311A), refrigerated autosampler (G1329A/G1330B), and temperature-controlled column oven (G1316A). After injection of 5 ml of sample, separation of lipophilic toxins was performed by reverse-phase chromatography on a C8 column (50 2 mm) packed with 3 mm Hypersil BDS 120 A (Phenomenex, Aschaffenburg, Germany) and maintained at 20 8C. The ow rate was 0.2 mL min1 and gradient elution was performed with two eluants, where eluant A was water and eluent B was acetonitrile/water (95:5, v/v), both containing 2.0 mmol l1 ammonium formate and 50 mmol l1 formic acid. Initial conditions were 12 min column equilibration with 5% B, followed by a

linear gradient to 100% B within 10 min and isocratic elution until 15 min with 100% B. The programme was then returned to initial conditions for 18 min (total run time: 30 min). For okadaic acid (OA) and dinophysistoxins (DTXs) large volume injection of 50 ml was used. After injection the sample was rst ushed for 1 min by the initial eluent composition via a six-port valve onto a cartridge (Oasis HLB 5 mm, 2.1 20 mm; Water, Eschborn, Germany) for removal of sample solvent, and the analytes were then backushed by valve switch onto the analytical column and chromatographed as described above. Detection of toxins was performed on an ABI-SCIEX-4000 Q Trap (Applied Biosystems, Darmstadt, Germany), triple quadrupole mass spectrometer equipped with a TurboSpray1 interface. PTX-2 was detected in the positive ionization mode by selected reaction monitoring (SRM) experiments with the transition m/z 876 > 213 using the following parameters: curtain gas: 10 psi, CAD gas: medium, ion spray voltage: 5500 V, temperature: ambient, nebulizer gas: 10 psi, auxiliary gas: off, interface heater: on, declustering potential: 50 V, entrance potential: 10 V, collision energy: 55 V, exit potential: 15 V. OA and DTXs were detected in the negative ionization mode with the transitions m/z 803 > 255 and m/z 803 > 113 for OA and DTX-2 and m/z 817 > 255 and m/z 817 > 113 for DTX-1 using the following parameters: curtain gas: 10 psi, CAD gas: high, ion spray voltage: 4500 V, temperature: 500 8C, nebulizer gas: 30 psi, auxiliary gas: 30 psi interface heater: off, declustering potential: 120 V, entrance potential: 10 V, collision energy: 60 V, exit potential: 2 V. Dwell times of 100 200 ms were used for each transition. Due to the nding of a novel DTX-1 isomer, collision induced dissociation (CID) spectra of DTX-1 and the new compound were recorded in the enhanced product ion (EPI) mode of m/z 836 (mass range m/z 100830) in the positive and m/z 803 (mass range m/z 100820) in the negative mode. Mass spectrometric parameters were as in the respective SRM experiments. 2.5. Calculations and statistical analysis In both sets of experiments, toxin production rates, Rtox were calculated as: Rtox T2 T1 t 2 t 1 N

where T1 and T2 are toxin contents per ml culture at two is the lnsubsequent samplings taken on days t1 and t2, and N average of the cell concentration per ml calculated as: N N2 N1 lnN 2 =N 1

Total toxin values from SPE samples were used for this in the second experiment. However, calculations of toxin production rates require two consecutive measurements, and since only one SPE measurement was taken during the well-fed period, rates from this period could only be obtained by assuming that the steadystate condition gave similar toxin contents at day 11 and 14. Thus, we set the toxin contents (per cell) on day 11 to mirror those found in the only steady-state measurement from day 14. This assumption seems justied, since D. acuta had grown well for several samplings up until this point. At the same time, carry-over effects from the stock culture were minimal, since dilutions had left only 4 and 2% of the original stock culture in the two treatments I15 and I130, respectively. One-way ANOVA was used for all statistical comparisons, with a predened a of 0.05 and a Tukey test for pairwise comparisons. Analyses were done using the software SigmaStat 3.5.

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3. Results 3.1. Dinophysis acuta toxin prole The only pectenotoxin (PTX) produced by the D. acuta strain DANA-2010 was PTX-2. The strain also produced okadaic acid (OA) and a yet undescribed isomer of DTX-1, in the following named compound 1. Compound 1 had the same molecular mass and the same two identical fragments after collision induced fragmentation (CID), recorded by the mass transitions (m/z 817 > 255 and 817 > 113), as DTX-1 in the SRM. However, compound 1 eluted under our experimental conditions at 9.13 min, which was between OA (9.0 min) and DTX-1 (9.87 min), almost co-eluting with OA (data not shown). The CID spectrum of DTX-1 in the positive mode was characterized by an intense pseudo-molecular ion cluster consisting of seven subsequent water losses from the pseudomolecular ion m/z 836 [M+NH4]+ (Fig. 1A). In addition there was a fragment m/z 447 consisting of the fragment C1C22, followed by two water losses (m/z 429 and 411) (Fig. 1A inset), which originated from the cleavage of the common bond of ring D and E, i.e. between C22 and C23 of the molecule (Fux et al., 2011) (Fig. 3A). By an almost identical cleavage, a fragment m/z 337 consisting of the other part of the original molecule C23C35 was formed, followed by two water losses (m/z 319 and 301). Compound 1 showed the same fragments, but the fragment consisting of molecular part C1C22 had an m/z value of 461, which is 14 Da higher than the corresponding fragment of DTX-1 with m/z 447 (Fig. 1B). The opposite was observed for the other fragment consisting of C23C35, which, with m/z 305, was 14 Da lighter than the corresponding fragment of DTX-1 (m/z 319) (Fig. 1, insets). In our experiment, a comparison in the negative mode, showed the same phenomenon as that observed in the positive mode, namely a 14 Da reduction of fragment C23C35 and a 14 Da enlargement of fragment C1C22 of compound 1 compared to DTX-1. This indicates that compound 1 is not only isobaric, but also structurally closely related to DTX-1 (see Section 4). Therefore,

255

DTX-1

817

151 191
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335 307
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563

m/z

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255

817

compound 1

151 577 191 321 293

300

200

m/z
400

400

500

200

300

m/z 500

600

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800

Fig. 2. CID spectra in the negative ionization mode of (A) DTX-1 and (B) compound 1; insets: enlargements of the mass range 150580 m/z.

compound 1 is termed DTX-1b in the subsequent experiments concerning D. acuta toxicity. 3.2. Experiment 1 effects of irradiance on growth, photosynthesis and toxin production In the rst set of experiments, D. acuta concentrations were kept below 600 cells ml1 by diluting every 24 days depending on observed growth rates (Fig. 4A, not all data shown). In I7 and I15 M. rubrum decreased in abundance between samplings, whereas it generally increased in I30 and I130 due to increased growth rates. This resulted in average cell concentrations of M. rubrum of 1601 159, 1748 31, 2241 288 and 2128 125 cells ml1 in the four treatments I7, I15, I30 and I130 respectively (Fig. 4B, not all data shown). Exponential growth rates of well-fed D. acuta under the four irradiance treatments I7, I15, I30 and I130 were 0.06 0.02, 0.21 0.06, 0.23 0.04 and 0.34 0.03 day1, respectively (Fig. 5A). All treatments except I15 vs. I30 were statistically signicantly different (p < 0.001, one-way ANOVA, n = 3). Similarly, rates of photosynthetic activity were 30.8 0.5, 56.5 6.6, 68.3 2.5 and 139.7 14.5 pg C cell1 h1 (Fig. 5B), and the same treatment combinations were statistically signicantly different (p < 0.001, one-way ANOVA, n = 3). Light acclimated PTX-2 contents of the four treatments I7, I15, I30 and I130 were 81.4 13.5, 99.6 11.7, 77.3 8.5 and 59.2 7.2 pg cell1, respectively (Fig. 5C), and only the I15 and the I130 treatments were statistically signicantly different (p = 0.01, oneway ANOVA, n = 3). In addition to PTX-2, most samples also contained okadaic acid (OA) and DTX-1b. However, the levels were close to the detection limit, and data were not t for quantitative analysis and presentation. Calculated rates of light acclimated cellular PTX-2 production of the four treatments I7, I15, I30 and I130 were 6.34 0.35, 12.30 3.65, 14.24 0.95 and 18.19 0.30 pg cell1 d1. The I30

301 319

DTX-1
429 447 747

783

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801
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287 305

compound 1

443 461 747 783

300

350

m/z

400

450

801
200 300 400

m/z 500

600

700

800

Fig. 1. CID spectra in the positive ionization mode of (A) DTX-1 and (B) compound 1; insets: enlargements of the mass range 280470 m/z.

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-H O
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O
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O OH O OH
12

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31

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35

O OH

29

Fig. 3. Fragmentation scheme of DTX-1 in (A) the positive mode and (B) the negative mode. Fragments of DTX-1/compound 1, respectively, observed in the spectra are printed in bold.

treatment was not statistically signicantly different from I15 and I130, but all other treatment combinations were (p < 0.02, one-way ANOVA, n = 3). 3.3. Experiment 2 toxin content, production and excretion as a function of growth phase 3.3.1. Growth characteristics When well-fed, D. acuta concentrations increased between samplings from averages of 159 20 and 167 4 to 262 33 and 359 8 cells ml1 in the two treatments I15 and I130 respectively.

Comparably, M. rubrum concentrations decreased from averages of 2306 100 and 2313 65 cells ml1 in the two treatments to 695 244 and 913 81 cells ml1 between samplings (Fig. 6). After the nal feeding at day 11, the two treatments developed somewhat differently. In I15, M. rubrum decreased to 231 220 cells ml1 on day 17, and was all but gone four days later. Simultaneously, D. acuta grew unaffectedly until day 21, where after growth continued at a decreasing rate until day 55 (maximum 690 103 cells ml1). In the I130 treatment, M. rubrum decreased comparably, but D. acuta only grew unaffectedly for six days (until day 17), and only showed positive growth until day 28. 3.3.2. DSP toxins The three DSP toxins PTX-2, okadaic acid (OA) and DTX-1b found in experiment 1 also dominated the toxin prole of D. acuta during experiment 2. 3.3.2.1. Spin lters. Based on spin lter samples, steady-state (well-fed, light acclimated) contents of PTX-2, OA and DTX-1b were 58.8 12.4, 3.0 0.4 and 7.6 1.4 pg cell1 in the I15 treatment, and 52.6 1.1, 2.7 0.2 and 6.5 0.4 pg cell1 in the I130 treatment respectively (Fig. 7). All three toxins accumulated under both irradiances when D. acuta was starved, and corresponding values on the nal day of the experiments were 97.2 10.6, 15.4 4.3 and 33.2 10.2 pg cell1 for I15 and 130.5 17.1, 12.9 3.0 and 29.5 8.5 pg cell1 for I130. 3.3.2.2. Intracellular toxin from picked cells. From samples of picked cells, intracellular contents of PTX-2, OA and DTX-1b in the steadystate phase were 43.7 7.5, 1.7 0.5 and 4.0 1.3 pg cell1 under I15 and 32.8 1.4, 2.8 0.3 and 6.0 0.9 pg cell1 under I130, respectively (Fig. 7). When cultures were starved, intracellular toxin values increased in a similar way as with the spin lter samples. On the nal day of the experiment, values of PTX-2, OA and DTX-1b had thus increased to 67.6 0.6, 7.2 1.0 and 16.0 2.3 pg cell1 under I15 and 84.2 3.1, 8.2 0.3 and 17.7 0.8 pg cell1 under I130, respectively.

800 600

A
D. acuta M. rubrum

Cell concentration (cells ml-1)

400 200 0 4000 3000 2000 1000 0 0

10

12

14

16

Day
Fig. 4. Steady-state growth of Dinophysis acuta (A) and Mesodinium rubrum (B) in the rst experiment. Example of experimental design and dilution scheme at the irradiance treatment I30. Symbols and error bars represent means SD (n = 3).

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0,40 0,35 0,30 0,25

0,20 0,15 0,10 0,05 0,00

3.3.2.3. Total toxin via SPE. By comparison, the total amounts of PTX-2, OA and DTX-1b found in the steady-state phase were 125.2 30.0, 16.3 3.0 and 37.9 8.0 pg cell1 respectively under I15, and the corresponding values for I130 were 93.9 7.4, 10.1 0.5 and 22.6 1.1 pg cell1 (Fig. 7). As with the other two types of toxin measurements, most values of total toxin also increased when cells were starved. The only exception was the PTX-2 value at I15, which did not increase noticeably during starvation. Thus, on the nal day of the experiment, values of PTX-2, OA and DTX-1b were 134.1 14.9, 33.6 5.2 and 78.0 11.6 pg cell1, respectively in the I15 treatment, and 182.0 31.1, 51.8 14.3 and 115.4 33.0 pg cell1 under I130. 3.3.3. Intracellular vs. total toxin content When in steady-state, intracellular PTX-2 from picked cells of I15 and I130 each accounted for 35.7 7.0 and 35.0 1.0% of the total amount of PTX-2 found in the corresponding whole-water SPE samples (Fig. 8C and D). When starved, the intracellular proportions initially increased markedly to 72.9 8.0 and 74.3 14.8% respectively, but these later decreased again somewhat, and ended at 53.2 7.7 and 43.0 8.3% on the nal day of the experiment. In terms of intracellular versus extracellular toxin pools, OA and DTX-1b almost perfectly mirrored each other at both irradiances (Fig. 8C and D). Initially, when well-fed, 10.7 2.6 and 10.4 2.3% of OA and DTX-1b respectively were found intracellularly at I15. Thus, 90% of both OA and DTX-1b were found extracellularly in the growth medium. With corresponding values of 27.3 2.6 and 26.5 3.9%, statistically signicantly more was found intracellularly at I130 (OA: p = 0.001, DTX-1b: p = 0.003, one-way ANOVA, n = 3). At I15, the intracellular proportion increased a little during starvation, especially toward the end of the experiment, where intracellular proportions of OA and DTX-1b were 21.6 2.4 and 20.6 1.5%, respectively. At I130, the intracellular proportions of OA and DTX-1b were unaffected for the rst 38 days of starvation. At the last measurement, on day 66, the intracellular proportions decreased somewhat to 16.8 5.0 and 16.4 5.5% for OA and DTX-1b, respectively. 3.3.4. Toxin ratios Average ratios of PTX-2: OA from the steady-state period were 20.9 5.5 and 20.4 1.0 for I15 and I130, respectively (Fig. 8E and F). Corresponding values for PTX-2: DTX-1b were 8.1 2.2 and 8.2 0.3, and for DTX-1b: OA they were 2.6 0.1 and 2.5 0.1. The DTX-1b: OA ratios were remarkably stable throughout the experiment, regardless of irradiance and starvation. The ratios of PTX-2 to OA and to DTX-1b decreased, on the other hand, under both irradiances when D. acuta was starved. Thus, PTX-2: OA ratios ended at 6.6 1.5 and 10.3 1.5 at I15 and I130 respectively, and the corresponding ratios of PTX-2: DTX-1b were 3.1 0.7 and 4.6 0.8. 3.3.5. Rates of toxin production Total toxin production rates in steady-state were 21.8 6.4 pg PTX-2 cell1 d1, 2.9 1.0 pg OA cell1 d1 and 6.7 2.1 pg DTX1b cell1 d1 for I15 and 28.9 0.9 pg PTX-2 cell1 d1, 3.1 0.2 pg OA cell1 d1 and 7.0 0.4 pg DTX-1b cell1 d1 for I130 (Fig. 9). All these rates declined markedly when growth rates declined, and the corresponding values on the nal day were 1.5 0.9, 0.2 0.2 and 0.4 0.5 pg PTX-2/OA/DTX-1b cell1 d1 for I15 and 3.0 0.5, 0.1 0.3 and 0.3 0.8 pg PTX-2/OA/DTX-1b cell1 d1 for I130. 4. Discussion 4.1. Structure of compound 1 and the toxin prole of Dinophysis acuta Under collision induced dissociation (CID) conditions in the positive and negative ionization mode, compound 1 showed the same fragmentation patterns as DTX-1, except for a 14 Da

(d-1)

180

Photosynthesis (pg C cell-1 h-1)

160 140 120 100 80 60 40 20 0

140 120 100 80 60 40 20 0

PTX-2 content (pg cell-1)

25

PTX-2 production (pg cell-1 d-1)

D
20

15

10

0 0 20 40 60 80 100
-2

120
-1

140

PAR (mol photons m s )

Fig. 5. Steady-state values of growth rate (A), photosynthetic activity (B), PTX-2 content (C) and daily PTX-2 production rate (D) in Dinophysis acuta as functions of treatment irradiance. For A, C and D, mean values of the last three measurements during steady-state growth were used. Only one measurement was made for B. A, B and D were statistically signicantly affected by irradiance, whereas C was not. Symbols and error bars represent means SD (n = 3).

40
800

L.T. Nielsen et al. / Harmful Algae 23 (2013) 3445

800

15 mol
600

130 mol
600

Cell concentrations (cells ml-1)

400

400 D. acuta M. rubrum

200

200

3000

3000

2000

2000

1000

1000

0 0 10 20 30 40 50 60 70

0
0 10 20 30 40 50 60 70

Time (d)
Fig. 6. Cell concentrations of Dinophysis acuta (top) and its prey Mesodinium rubrum (bottom) at the two irradiance treatments I15 and I130 used in the second experiment. Flasks were diluted with f/2 growth medium every 24 days until Day 21. Mesodinium rubrum was added every 24 days until day 14. Symbols and error bars represent means SD (n = 3).

enlarged C1C22 fragment and a 14 Da smaller C23C35 fragment (Figs. 1 and 2). This is strong evidence that in comparison to DTX-1, compound 1 carries an additional methyl (or methylene) group at C1C22, and a methyl (or methylene) group less in the part C23C35. Carey et al. (2012) established a fragmentation pattern of DTX-1 for the negative ionization mode, which basically gave the same fragments as in the positive mode (Fig. 3B). The fact that DTX-1 and compound 1 showed the exact same differences in the positive and the negative ionization mode strengthen our argument that compound 1 is closely related to DTX-1. Interestingly, both DTX-1 and compound 1 formed the fragments m/z 151, 191 and 255, the latter originating from a cleavage between C11 and C12 (Fig. 3B). This is strong evidence that both molecules share an identical structure from C1 to C11. Taking into account that OA, which was also produced by this strain of D. acuta, does not possess a methyl group at C35, the information at hand strongly suggest the structure of compound 1 to be 1222-methyl OA. However, the exact locations of the missing methyl (or methylene) group between C26 and C35 and the additional methyl (or methylene) group between C12 and C22 cannot be unambiguously assigned by mass spectrometry alone. For an unambiguous structural elucidation of compound 1, approximately 500 mg of pure substance is required for nuclear magnetic resonance (NMR) spectroscopy, and this was unfortunately not possible to achieve within this work. Even though the complete structure of compound 1 remains unresolved, we demonstrate that there is a yet undescribed dinophysistoxins in this strain of D. acuta, and that the molecular structure seems to be that of a DTX-1 isomer. The high similarity of the spectra and the partly identical mass fragments may have caused compound 1 to be misidentied as DTX-1 in the past. D. acuta normally contains only OA, PTX-2 and either DTX-1 or DTX-2; only on a single occasion has it been found with both DTX-1 and DTX-2 (Johansen, 2008). Two novel DTX-2 isomers have previously been reported in D. acuta, but with precise chemical structures yet to be determined (James et al., 1997; Draisci et al., 1998). Together with our report on a new DTX-1 isomer, this demonstrates that DTXs D. acuta are not limited to the standard DTX-1 and DTX-2 molecules. Future work should be meticulous

with the identication of DTXs, and more information on novel toxins is required, not least on their toxicity. 4.2. Effects of light on growth, photosynthesis and PTX-2 content and production Irradiance signicantly affected the growth rates of D. acuta, even when prey was supplied in excess. The growth rate at the I7 treatment was reduced to 18% of that obtained at I130. Irradiance also affected the photosynthetic rate of D. acuta, and the rate of photosynthesis at I7 was reduced to 22% of that obtained at I130. Similar effects of irradiance on growth and photosynthesis have been found in other Dinophysis species, indicating that they are functionally identical (Kim et al., 2008; Riisgaard and Hansen, 2009; Nielsen et al., 2012). The fact that the growth rate was so tightly coupled to the rate of photosynthesis indicates that food ingestion cannot substitute for phototrophically derived carbon. Irradiance had only marginal effects on the cellular contents of PTX-2 in D. acuta. If anything, the cellular PTX-2 content decreased with irradiance. On the other hand, with growth rates signicantly affected by irradiance and PTX-2 contents more or less stable, cellular PTX-2 production rates were obviously higher at increased irradiances. This was also the case for D. acuminata, although this species showed slightly increased PTX-2 contents at higher irradiances (Nielsen et al., 2012). Contrary, Tong et al. (2011) found that irradiance had no effect on rates of toxin production in D. acuminata, but they applied only irradiances from 65 to 300 mmol photons m2 s1 that were all growth saturating. Together, these result suggest, that PTX-2 production is linked somehow to growth rate. In fact, in the current dataset, there was an almost perfect linear relationship between growth rates and PTX-2 production rates of the four well-fed irradiance treatments, I7I130 (r2 = 0.99). The inuence of irradiance on toxin production seems, thus, to owe to the inuence held via effects on growth rates. Beyond that, PTX-2 production appears unaffected by irradiance. 4.3. Effects of growth phase on toxin content and production Production of all three DSP toxins continued as growth rates declined due to starvation, resulting in signicant accumulation of

L.T. Nielsen et al. / Harmful Algae 23 (2013) 3445

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15 mol
0,5 0,4 0,3 0,2 0,1 0,0

130 mol
B

(d-1)

250

C
200

PTX-2 (pg cell-1)

150

100

50

0 70 60 50

E
SPE (total) Spin filters Picked cells

OA (pg cell-1)

40 30 20 10 0 160 140

DTX-1b (pg cell-1)

120 100 80 60 40 20 0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70

Time (d)

Time (d)

Fig. 7. Toxin contents of Dinophysis acuta at irradiance treatments I15 (left) and I130 (right) during the second experiment. PTX-2 (C and D), okadaic acid (E and F) and the novel toxin DTX-1b (G and H) in the three different sample types total toxin (dashed), spin lters (solid) and picked cells (dotted). Growth rates of D. acuta (A and B) for references. Dashed, vertical lines indicate when growth rates of D. acuta started to decline. Symbols and error bars represent means SD (n = 3).

these toxins in D. acuta (Figs. 7 and 9). This is parallel to what has been observed before, and it is now evident that senescent populations of Dinophysis spp. contain more toxin than those in exponential growth (Tong et al., 2011; Nagai et al., 2011; Nielsen et al., 2012). This has also been observed in eld populations of

D. acuta (Pizarro et al., 2008, 2009). The fact that toxin production is not coupled directly to the growth rate when growth rates decline due to starvation seems in direct contrast to the conclusion reached in Section 4.2, and the correlation appears to only hold true under food replete conditions. The obvious interpretation

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L.T. Nielsen et al. / Harmful Algae 23 (2013) 3445

15 mol
0,5 0,4 0,3 0,2 0,1 0,0

130 mol
B

(d-1)

1,0

0,8

PTX-2 OA DTX-1b

Cellular : total

0,6

0,4

0,2

0,0

40

F
PTX-2:OA PTX-2:DTX-1b DTX-1b:OA

30

Toxin ratios

20

10

0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70

Time (d)

Time (d)

Fig. 8. Toxin contents of Dinophysis acuta at irradiance treatments I15 (left) and I130 (right) during the second experiment. Ratios of cellular: total toxin (C and D) and ratios between the three toxins PTX-2, okadaic acid (OA) and the novel toxin DTX-1b (E and F). Growth rates of D. acuta (A and B) for references. Dashed, vertical lines indicate when growth rates of D. acuta started to decline. Symbols and error bars represent means SD (n = 3).

30

Toxin production (pg cell-1 d-1)

25 20 15 10 5 0 -5 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
PTX-2 OA DTX-1b

Time (d)

Time (d)

Fig. 9. Rates of PTX-2, OA and DTX-1b production in Dinophysis acuta at irradiance treatments I15 (left) and I130 (right) during the second experiment. Dashed, vertical lines indicate when growth rates of D. acuta started to decline. Production rates were calculated from measures of total toxin (SPE samples). Symbols and error bars represent means SD (n = 3).

L.T. Nielsen et al. / Harmful Algae 23 (2013) 3445

43

would be that both growth and toxin production are limited by irradiance, but that only growth, and not toxin production, is limited by heterotrophic intake of ciliate prey. 4.4. Intra- and extracellular toxin pools The spin lters used for the standard toxin analysis in both experiments had a pore diameter of 0.45 mm, and thus sampled particles of roughly this size and larger. However, this could potentially encompass several toxin pools including intracellular toxin from D. acuta and from bacteria as well as extracellular toxins bound to organic matter particles, e.g. cell debris. In addition, spin lters omitted toxin from the <0.45 mm fraction, neglecting any excreted toxins. From the toxin samples of picked and washed cells, and by comparing whole-water toxin samples with measures of intracellular toxin and with spin lter samples, DSP toxins could be divided into the three pools (1) intracellularly in D. acuta, (2) the particulate organic matter (POM) fraction (>0.45 mm) and (3) the dissolved organic matter (DOM) fraction (<0.45 mm). 4.4.1. Intracellular toxins The toxin contents found in picked cells followed the same patterns as those observed from the spin lter samples, but the values were almost always lower especially in the stationary growth phase. No differences were found in intracellular toxin contents of the two irradiances in experiment 2 when D. acuta was in exponential, steady-state growth. We can thus conclude that irradiance did not affect the intracellular toxin contents. When growth rates of D. acuta declined, intracellular toxin contents of all three toxins increased, albeit the values were always lower than those found in POM samples. There is no doubt, however, that toxins accumulated intracellularly when subjected to starvation and suppressed growth. This matches the conclusions of earlier studies on other Dinophysis species, and intracellular accumulation of different DSP toxins seems ubiquitous when Dinophysis spp. exhibit reduced growth rates due to starvation (Nagai et al., 2011; Nielsen et al., 2012). 4.4.2. Extracellular toxins in POM and DOM When D. acuta was well-fed and in exponential growth, 65% of the PTX-2 and 7390% of the OA and DTX-1b was found outside D. acuta cells (Fig. 8C and D). Thus, we demonstrate that the majority of the DSP toxin produced by D. acuta was found extracellularly, and that it is imperative to include extracellular toxins when studying Dinophysis spp. toxicity. Extracellular DSP toxins have been reported before in eld studies (Pizarro et al., 2009; Fux et al., 2009, 2011), but only one study has previously documented quantitatively the importance of intra- and extracellular DSP toxins in Dinophysis spp. cultures (Nagai et al., 2011). Interestingly, they also found that extracellular proportions of OA and DTX-1 comprised the majority (up to 80%) of the total toxin in D. acuminata and D. fortii, and that PTX-2 was mostly found intracellularly. However, compared to our results, toxin excretion seemed to occur mostly when cells were in the stationary growth phase. For OA and DTX-1b, the majority of the extracellular toxin was found in the DOM pool. Thus, these two toxins were to a large extent excreted to the surrounding seawater, and leakage continued even long after D. acuta had ceased growing. The contribution of the POM pool (>0.45 mm) was much smaller, generally at the same level at that found intracellularly in D. acuta. In steady-state, exponential growth, the DOM pool also contained the larger part of the extracellular PTX-2. This changed when cell growth ceased, and here the POM pool was often as large as the DOM pool. The awareness that all DSP toxins are excreted, but to completely different levels, and that growth phase affects the

excretion of the different toxins differently could lead to a better understanding of the biological role of the different DSP toxins. For OA and DTX-1b, starvation caused both intra- and extracellular levels to increase, resulting in fairly stable ratios between intra- and extracellular pools of toxin. PTX-2, on the other hand, only accumulated intracellularly, and the proportion of toxin found extracellularly thus decreased as growth ceased. At the same time, the ratios of PTX-2 to OA and PTX-2 to DTX-1b continuously decreased during the stationary growth phase (Fig. 8E and F). PTX2 to OA/DTX-1 ratios also decreased markedly during stationary growth in the experiment by (Tong et al., 2011). Hence, it would seem that PTX-2 production and excretion are regulated differently than OA and DTX-1b, and this may relate to the function of these different toxins. 4.5. Potential functions of DSP toxins The results presented here could help reveal the ecological role of DSP toxins. With the nding that the majority of OA and DTX-1b was found outside D. acuta cells, the idea that these compounds play an extracellular role seems increasingly likely. 4.5.1. Allelopathy Observations of swimming and feeding behavior in mixed M. rubrum and Dinophysis spp. cultures have previously led to the suggestion that some form of allelopathic interaction occurs (Nagai et al., 2008; Nishitani et al., 2008). Also, free OA has been shown to have allelopathic effects on other, potentially competing, microalgae (Windust et al., 1996). However, concentrations of free OA 1 mmol l1 were needed to induce a 10% inhibition of growth in the competing organisms. Even by optimistically using the highest possible toxin contents achieved in this experiment (150 pg OA + DTX-1b cell1), this corresponds to >5 million cells l1 a concentration far beyond those found in natural blooms. It is worth mentioning that this could potentially be different for the other OA producing genus, Prorocentrum: its benthic habitat could facilitate local hotspots of high OA concentrations in the vicinity of Prorocentrum spp. populations, due to stagnant water and high local cell densities. Besides, competing microalgae, such as those studied by (Windust et al., 1996), may not even be the intended target organisms of allelopathic effects of OA and DTXs. Okadaic acid has been shown to affect fungi, and these, or even a third group of organisms, could be the primary intended targets (Nagai et al., 1990). For now, OA and DTXs as allelochemicals remains an unproven, largely unexplored, but still plausible theory. 4.5.2. Grazer defense DSP toxins have also been suggested to work as grazer repellents (Carlsson et al., 1995), and some copepod species have been shown to select against Dinophysis spp. in mixed phytoplankton assemblages (Carlsson et al., 1995; Kozlowsky-Suzuki et al., 2006; Setala et al., 2009). But food selectivity is common in copepods, and may result from sizes or food quality rather than toxicity. This was exemplied in a study involving Dinophysis norvegica by (Jansen et al., 2006), who showed that only one out of three copepods grazed on D. norvegica, but that the same copepod species was also the only one to graze Ceratium furca, a similarsized non-DSP containing dinoagellate. The study by Carlsson et al. (1995) is, to our knowledge, so far the only study to show direct negative effects of Dinophysis spp. consumption on the copepod grazer. Negative effects on e.g. survival or egg production, or behavioral changes such as those found when copepods are fed other toxic algae (Huntley et al., 1986; Sykes and Huntley, 1987), needs to be demonstrated if the theory of DSP toxins as grazer repellents is to gain favor.

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L.T. Nielsen et al. / Harmful Algae 23 (2013) 3445 Fux, E., Bire, R., Hess, P., 2009. Comparative accumulation and composition of lipophilic marine biotoxins in passive samplers and in mussels (M. edulis) on the West Coast of Ireland. Harmful Algae 8, 523537. Fux, E., Gonzalez-Gil, S., Lunven, M., Gentien, P., Hess, P., 2010. Production of diarrhetic shellsh poisoning toxins and pectenotoxins at depths within and below the euphotic zone. Toxicon 56, 14871496. Fux, E., Smith, J.L., Tong, M.M., Guzman, L., Anderson, D.M., 2011. Toxin proles of ve geographical isolates of Dinophysis spp. from North and South America. Toxicon 57, 275287. Garcia, A., Cayla, X., Guergnon, J., Dessauge, F., Hospital, V., Rebollo, M.P., Fleischer, A., Rebollo, A., 2003. Serine/threonine protein phosphatases PP1 and PP2A are key players in apoptosis. Biochimie 85, 721726. mez, F., 2005. A list of free-living dinoagellate species in the worlds oceans. Go Acta Botanica Croatica 64, 129212. Gonzalez-Gil, S., Pizarro, G., Paz, B., Velo-Suarez, L., Reguera, B., 2011. Considerations on the toxigenic nature and prey sources of Phalacroma rotundatum. Aquatic Microbial Ecology 64, 197203. Gross, E.M., 2003. Allelopathy of aquatic autotrophs. Critical Reviews in Plant Sciences 22, 313339. Guillard, R.R., Ryther, J.H., 1962. Studies of marine planktonic diatoms. 1. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Canadian Journal of Microbiology 8, 229. Huntley, M., Sykes, P., Rohan, S., Marin, V., 1986. Chemically-mediated rejection of dinoagellate prey by the copepods Calanus pacicus and Paracalanus parvus mechanism, occurrence and signicance. Marine Ecology-Progress Series 28, 105120. Jacobson, D.M., Andersen, R.A., 1994. The discovery of mixotrophy in photosynthetic species of dinophysis (Dinophyceae) light and electron-microscopic observations of food vacuoles in Dinophysis acuminata, D. Norvegica and 2 heterotrophic Dinophysoid dinoagellates. Phycologia 33, 97110. James, K.J., Bishop, A.G., Healy, B.M., Roden, C., Sherlock, I.R., Twohig, M., Draisci, R., Giannetti, L., Lucentini, L., 1999. Efcient isolation of the rare diarrhoeic shellsh toxin, dinophysistoxin-2, from marine phytoplankton. Toxicon 37, 343357. James, K.J., Carmody, E.P., Gillman, M., Kelly, S.S., Draisci, R., Lucentini, L., Giannetti, L., 1997. Identication of a new diarrhoetic toxin in shellsh using liquid chromatography with uorimetric and mass spectrometric detection. Toxicon 35, 973978. Jansen, S., Riser, C.W., Wassmann, P., Bathmann, U., 2006. Copepod feeding behaviour and egg production during a dinoagellate bloom in the North Sea. Harmful Algae 5, 102112. Johansen, M., 2008. On Dinophysis occurrence and toxin content. Ph.D. Thesis. teborg. Department of Marine Ecology, University of Go Kim, M., Nam, S.W., Shin, W., Coats, D.W., Park, M.G., 2012. Dinophysis caudata (Dinophyceae) sequesters and retains plastids from the mixotrophic ciliate prey Mesodinium rubrum. Journal of Phycology 48, 569579. Kim, S., Kang, Y.G., Kim, H.S., Yih, W., Coats, D.W., Park, M.G., 2008. Growth and grazing responses of the mixotrophic dinoagellate Dinophysis acuminata as functions of light intensity and prey concentration. Aquatic Microbial Ecology 51, 301310. Kozlowsky-Suzuki, B., Carlsson, P., Ruhl, A., Graneli, E., 2006. Food selectivity and grazing impact on toxic Dinophysis spp. by copepods feeding on natural plankton assemblages. Harmful Algae 5, 5768. Lee, J.S., Igarashi, T., Fraga, S., Dahl, E., Hovgaard, P., Yasumoto, T., 1989. Determination of diarrhetic shellsh toxins in various dinoagellate species. Journal of Applied Phycology 1, 147152. Lindahl, O., Lundve, B., Johansen, M., 2007. Toxicity of Dinophysis spp. in relation to population density and environmental conditions on the Swedish west coast. Harmful Algae 6, 218231. Miles, C.O., Wilkins, A.L., Munday, R., Dines, M.H., Hawkes, A.D., Briggs, L.R., Sandvik, M., Jensen, D.J., Cooney, J.M., Holland, P.T., Quilliam, M.A., MacKenzie, A.L., Beuzenberg, V., Towers, N.R., 2004. Isolation of pectenotoxin-2 from Dinophysis acuta and its conversion to pectenotoxin-2 seco acid, and preliminary assessment of their acute toxicities. Toxicon 43, 19. Miles, C.O., 2007. Pectenotoxins, chapter 9. In: Botana, L. (Ed.), Phycotoxins Chemistry and Biochemistry. Blackwell Publishing, Iowa, pp. 159186. Murakami, Y., Oshima, Y., Yasumoto, T., 1982. Identication of okadaic acid as a toxic component of a marine dinoagellate prorocentrum-Lima. Bulletin of the Japanese Society of Scientic Fisheries 48, 6972. Nagai, H., Satake, M., Yasumoto, T., 1990. Antimicrobial activities of polyether compounds of dinoagellate origins. Journal of Applied Phycology 2, 305 308. Nagai, S., Nitshitani, G., Tomaru, Y., Sakiyama, S., Kamiyama, T., 2008. Predation by the toxic dinoagellate Dinophysis fortii on the ciliate Myrionecta rubra and observation of sequestration of ciliate chloroplasts. Journal of Phycology 44, 909922. Nagai, S., Suzuki, T., Nishikawa, T., Kamiyama, T., 2011. Differences in the production and excretion kinetics of okadaic acid, dinophysistoxin-1, and pectenotoxin-2 between cultures of dinophysis acuminata and Dinophysis fortii isolated from Western Japan. Journal of Phycology 47, 13261337. Nielsen, L.T., Krock, B., Hansen, P.J., 2012. Effects of light and food availability on toxin production, growth and photosynthesis in Dinophysis acuminata. Marine Ecology-Progress Series 471, 3750. Nishitani, G., Nagai, S., Takano, Y., Sakiyama, S., Baba, K., Kamiyama, T., 2008. Growth characteristics and phylogenetic analysis of the marine dinoagellate Dinophysis infundibulus (Dinophyceae). Aquatic Microbial Ecology 52, 209221.

4.5.3. Food capture Given the mixotrophic nature of Dinophysis spp., the potential for DSP toxins to play a role in food capture is a logic suggestion. Nevertheless, the presently available information seems to contradict both an intra- and an extracellular role of DSP toxins in food capture. Firstly, the high amounts of extracellular toxin suggest an extracellular effect such as immobilization of prey items. This, however, has never been reported and was certainly not the case in our experiments. Secondly, Dinophysis spp. with only PTXs or OA/DTXs has been found (Blanco et al., 2005, 2007; Suzuki et al., 2009). This would hardly have been possible if one of the toxin groups played a major role in its obligate food capture. Thirdly, the closely related Phalacroma rotundatum catch prey the same way as Dinophysis spp., but seems not to produce PTXs, OA or DTXs (Gonzalez-Gil et al., 2011). Thus, the idea that DSP toxins play a key role in food capture (either intra- or extracellularly) seems unlikely at present. 5. Conclusions We found a novel DTX in Dinophysis spp. for the rst time in nearly 15 years, but we concluded that cellular contents of PTX-2, OA and the new DTX-1 isomer were not affected signicantly by irradiance. We also argued that production of DSP toxins follows growth rate when D. acuta has ample food supplies. On the other hand, when growth of D. acuta is limited by food availability, toxin production continues (albeit at decreased rates) and toxins are accumulated both intra- and extracellularly. Finally, we have demonstrated that extracellular toxin pools of all three DSP toxins were often larger than amounts found intracellularly also when D. acuta was in exponential growth. Future studies on DSP toxins should always include extracellular toxin pools. Acknowledgements We thank Wolfgang Drebing for technical assistance with toxin extractions and measurements. The work was supported nancially from project no. 2101-07-0084 from The Danish Counsel for Strategic Research.[SS] References
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