Paralytic shellfish toxins or spirolides? The role of environmental and genetic factors in toxin production of the Alexandrium ostenfeldii complex

Harmful Algae 26 (2013) 52–59

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Paralytic shellfish toxins or spirolides? The role of environmental and genetic factors in toxin production of the Alexandrium ostenfeldii complex Sanna Suikkanen a,*, Anke Kremp a, Henna Hautala b, Bernd Krock c a b c

Finnish Environment Institute, Marine Research Centre, FI-00251 Helsinki, Finland Division of Biotechnology, Department of Biochemistry and Food Chemistry, University of Turku, FI-20520 Turku, Finland Alfred Wegener Institute for Polar and Marine Research, 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 30 November 2012 Received in revised form 4 April 2013 Accepted 4 April 2013

Dinoflagellates of the Alexandrium ostenfeldii complex (A. ostenfeldii, A. peruvianum) are capable of producing different types of neurotoxins: paralytic shellfish toxins (PSTs), spirolides and gymnodimines, depending on the strain and its geographic origin. While Atlantic and Mediterranean strains have been reported to produce spirolides, strains originating from the brackish Baltic Sea produce PSTs. Some North Sea, USA and New Zealand strains contain both toxins. Causes for such intraspecific variability in toxin production are unknown. We investigated whether salinity affects toxin production and growth rate of 5 A. ostenfeldii/peruvianum strains with brackish water (Baltic Sea) or oceanic (NE Atlantic) origin. The strains were grown until stationary phase at 7 salinities (6–35), and their growth and toxin production was monitored. Presence of saxitoxin (STX) genes (sxtA1 and sxtA4 motifs) in each strain was also analyzed. Salinity significantly affected both growth rate and toxicity of the individual strains but did not change their major toxin profile. The two Baltic Sea strains exhibited growth at salinities 6–25 and consistently produced gonyautoxin (GTX) 2, GTX3 and STX. The two North Sea strains grew at salinities 20–35 and produced mainly 20-methyl spirolide G (20mG), whereas the strain originating from the northern coast of Ireland was able to grow at salinities 15–35, only producing 13-desmethyl spirolide C (13dmC). The effects of salinity on total cellular toxin concentration and distribution of toxin analogs were strain-specific. Both saxitoxin gene motifs were present in the Baltic Sea strains, whereas the 2 North Sea strains lacked sxtA4, and the Irish strain lacked both motifs. Thus sxtA4 only seems to be specific for PST producing strains. The results show that toxin profiles of A. ostenfeldii/peruvianum strains are predetermined and the production of either spirolides or PSTs cannot be induced by salinity changes. However, changes in salinity may lead to changed growth rates, total cellular toxin concentrations as well as relative distribution of the different PST and spirolide analogs, thus affecting the actual toxicity of A. ostenfeldii/peruvianum populations. ß 2013 Elsevier B.V. All rights reserved.

Keywords: Alexandrium Dinoflagellate Paralytic shellfish toxin Spirolide Saxitoxin gene Toxin profile

1. Introduction Toxic marine dinoflagellates of the Alexandrium ostenfeldii complex (Balech, 1995) are widely distributed in coastal waters around the globe, occurring in the northern Atlantic as well as the Pacific Ocean, the Mediterranean and the brackish Baltic Sea. The two morphospecies A. ostenfeldii (Paulsen) Balech and Tangen and Alexandrium peruvianum (Balech and Mendiola) Balech and Tangen belonging to the complex are defined by subtle differences in plate structure, which do not conform to phylogenetic relationships. Hence, species boundaries have remained unclear. Both are

* Corresponding author. Tel.: +358 400 148866. E-mail address: sanna.suikkanen@ymparisto.fi (S. Suikkanen). 1568-9883/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.hal.2013.04.001

capable of producing different types of neurotoxins: paralytic shellfish toxins (PSTs), spirolides and/or gymnodimines. PSTs are a group of heat-stable, water-soluble alkaloids with close to 60 analogs, including saxitoxin, which is one of the most potent natural neurotoxins known (Wiese et al., 2010). In the aquatic environment, PSTs are mainly produced by marine eukaryotic dinoflagellates of the genera Alexandrium, Gymnodinium and Pyrodinium, and by at least 5 genera of freshwater prokaryotic cyanobacteria. Paralytic shellfish poisoning events with contaminated seafood and human intoxications are widespread and wellstudied (Anderson et al., 2012). Saxitoxin synthesis genes have recently been characterized in cyanobacteria (Kellmann et al., 2008) and dinoflagellates (Stu¨ken et al., 2011; Hackett et al., 2013). Spirolides and gymnodimines are fast-acting macrocyclic polyether toxins produced by Alexandrium ostenfeldii/peruvianum

S. Suikkanen et al. / Harmful Algae 26 (2013) 52–59

(Cembella et al., 2000; Franco et al., 2006; Van Wagoner et al., 2011), gymnodimines also by Karenia selliformis A. J. Haywood, K. A. Steidinger and L. MacKenzie (Kharrat et al., 2008). Although there are no reports of negative human health effects related to spirolides or gymnodimines, they have been found to accumulate in shellfish along the North Atlantic, Mediterranean and New Zealand coasts (Aasen et al., 2005; Villar Gonza´lez et al., 2006; Kharrat et al., 2008; Medhioub et al., 2011; Pistocchi et al., 2012). Biosynthesis genes for these toxins have not yet been confirmed. Individual strains of Alexandrium ostenfeldii/peruvianum usually produce either spirolides or PSTs. Causes for such variability in toxin profile are unknown, but salinity may play a role since toxin profiles of brackish water (salinity <30) strains clearly differ from those of strains isolated from oceanic salinities (>30). More specifically, A. ostenfeldii/peruvianum strains isolated from the northern Atlantic coast of Canada and USA (Cembella et al., 2000, 2001; Gribble et al., 2005), Ireland (Touzet et al., 2008) and the Mediterranean (Ciminiello et al., 2006; Franco et al., 2006) produce spirolides, whereas strains originating from Malaysia and the brackish water Baltic Sea produce PSTs (Lim et al., 2005; Kremp et al., 2009). Furthermore, strains originating from an intermediate salinity, i.e. the Danish Straits connecting the Baltic Sea to the North Sea, as well as a rivermouth in the eastern USA, contain both toxin types (Hansen et al., 1992; MacKinnon et al., 2004; Otero et al., 2010; Tomas et al., 2012). On the other hand, some A. ostenfeldii strains originating from oceanic salinities, i.e. from coastal New Zealand and Scotland, also produce both PSTs and spirolides (Mackenzie et al., 1996; Brown et al., 2010; Beuzenberg et al., 2012). In past experimental studies, it has been proposed that PST composition is a stable characteristic, fixed genetically for each clonal strain of Alexandrium spp., but that significant shifts may occur under changes in growth regime, e.g. inorganic nutrient limitation, temperature, irradiance, salinity or depending on growth phase (Boczar et al., 1988; Anderson et al., 1990; Hwang and Lu, 2000; Etheridge and Roesler, 2005; Anderson et al., 2012). Previous studies on effects of environmental factors on Alexandrium ostenfeldii/peruvianum toxin content and profile have mostly concentrated on spirolides, and it has been found that total toxin present in batch cultures can be affected by e.g. light, the amount of spirolides increasing with cell concentration but cell spirolide quota and profile remaining constant despite environmental changes (John et al., 2001; Maclean et al., 2003). On the other hand, in other studies (Otero et al., 2010; Medhioub et al., 2011; Tatters et al., 2012), salinity, culture media, photoperiod and nutrient limitation have been found to affect cellular spirolide toxicity and toxin profile of A. ostenfeldii/peruvianum, and it was suggested that even the production of either spirolides or PSP toxins by A. ostenfeldii might be determined by salinity, temperature or nutrients (Otero et al., 2010). In this study we investigated whether individual Alexandrium ostenfeldii/peruvianum strains are capable of producing both PSTs and spirolides, depending on salinity. We hypothesized that low salinity conditions will induce PSTs in oceanic spirolide producers and high salinities will lead to spirolide production in brackish water strains containing PSTs. To test this, batch culture

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experiments were performed with strains originating from the NE Atlantic and the Baltic Sea at salinities between 6 and 35, and growth rate, toxin production and composition was measured. To substantiate the genetic basis for PST production, strains were furthermore analyzed for presence of saxitoxin genes. 2. Material and methods 2.1. Batch culture experiments Growth and toxin production of five Alexandrium ostenfeldii and Alexandrium peruvianum strains originating from the Baltic Sea and NE Atlantic (Table 1) were monitored in batch cultures set up along a gradient of seven salinities (6, 10, 15, 20, 25, 30 and 35). Growth media with the different salinities were prepared by adding artificial sea salt (Tropic Marin1 Pro-Reef Sea Salt, Dr. Biener, Germany) to 0.45 mm filtered (Sartobran 300 sterile capsule filters, Sartorius Stedim Biotech, Germany), autoclaved Baltic Sea water (salinity 6), letting the salt dissolve for 2 h, and filtering the solution once more through 0.45 mm. Salinity of the media was measured using a conductivity meter (TetraCon 325, Cond 3210 SET 1, WTW, Germany). Finally, F/2 nutrients (excluding silicate) were added according to Guillard (1975). The Baltic Sea strains were initially grown in a salinity of 6, and the NE Atlantic strains in a salinity of 30, at 16 8C, in ca. 60 mmol photons m 2 s 1, and in a light:dark cycle of 12L:12D. The strains were stepwise acclimated (max. five salinity units at a time) to the different salinities: ca. 1000 exponentially growing cells mL 1 were inoculated to the next salinity and grown for ca. 4 weeks until exponential phase (>5000 cells mL 1). Then triplicate experimental cultures were inoculated at 500 cells mL 1 and their growth was monitored with fluorescence measurements (Varian Cary Eclipse fluorescence spectrophotometer, excitation 440 nm, emission 680 nm) every 2–3 days. The cultures were grown in 250 mL tissue culture flasks with an experimental volume of 200 mL, at 16 8C, 60 mmol photons m 2 s 1 and a 12:12 light:dark cycle. Samples for toxin analysis were taken both in exponential and stationary growth phases (V = 2  40 mL for spirolide and PST analyses, respectively). In addition, samples (V = 1.2 mL) were taken and preserved with a drop of acid Lugol solution for cell counts and length measurements on every toxin sampling occasion. Cell number was counted and mean cell length (n = 30/treatment) was measured in Sedgewick-Rafter cells (V = 1 mL) with an inverted microscope (Leica DMI 300B). The duration of the experiments varied between 50 and 78 days, depending on the growth rate of each strain. Growth rates, k, defined as doublings per day, were calculated based on the longest period of exponential growth, using the equation k = log2(Nt/N0)/Dt, where N = cell number and t = time (Wood et al., 2005). The interval of exponential growth was determined from growth curves established for each experimental culture replicate. For toxin analysis cells were concentrated by centrifugation, first 15 min at 4000 rpm (Heraeus Sepatech Megafuge 2.0), followed by 5 min at 10,000  g (IEC MicroCL 21R, Thermo Electron Corporation), and subsequent removal of the supernatant.

Table 1 Alexandrium ostenfeldii/peruvianum strains used in the study. Strain code

Morphotype

Geographic origin

Native salinity

Toxins produced

Isolated by

AOF 0927 AOVA 30 NCH 85 S06/013/01 LS A06

A. A. A. A. A.

Fo¨glo¨, A˚land (Baltic Sea) Gotland, Sweden (Baltic Sea) Skagerrak (North Sea, NE Atlantic) E Scotland (North Sea, NE Atlantic) N Ireland (Lough Swilly, NE Atlantic)

6 6 30 35 30

GTX2/3, STX GTX2/3, STX 20mG, 13dmC 20mG, 13dmC (and STX, NEO; Brown et al., 2010) 13 dmC

A. Kremp A. Kremp T. Alpermann L. Brown N. Touzet

ostenfeldii ostenfeldii ostenfeldii ostenfeldii peruvianum

54

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The samples were stored at 20 8C and freeze-dried (1 d, 60 8C; Edwards Pirani 501 Super Modulyo Freeze Drier, Thermo Electron Corporation) before measurement. 2.2. Extraction of toxins Cell pellets were suspended in 300 or 500 mL (depending on pellet size) 0.03 M acetic acid for PSP toxin analysis and with methanol for spirolide analysis, respectively. The samples were subsequently transferred into a FastPrep tube containing 0.9 g of lysing matrix D. The samples were homogenized by reciprocal shaking at maximum speed (6.5 m s 1) for 45 s in a Bio101 FastPrep instrument (Thermo Savant, Illkirch, France). After homogenization, samples were centrifuged (Eppendorf 5415 R, Hamburg, Germany) at 16,100  g at 4˚ C for 15 min. The supernatant (400 mL) was transferred to a spin-filter (pore-size 0.45 mm, Millipore Ultrafree, Eschborn, Germany) and centrifuged for 30 s at 800  g. The filtrates were transferred to HPLC vials and stored at 20˚ C until measurement. 2.3. Liquid chromatography with fluorescence detection (LC-FD) The aqueous extracts were analyzed by reverse-phase ion-pair liquid chromatography with fluorescence detection (LC-FD) and post-column derivatization following minor modifications of previously published methods (Diener et al., 2006; Krock et al., 2007). The LC-FD analysis was carried out on a LC1100 series liquid chromatography system consisting of a G1379A degasser, a G1311A quaternary pump, a G1229A autosampler, and a G1321A fluorescence detector (Agilent Technologies, Waldbronn, Germany), equipped with a Phenomenex Luna C18 reversed-phase column (250 mm  4.6 mm id, 5 mm pore size) (Phenomenex, Aschaffenburg, Germany) with a Phenomenex SecuriGuard precolumn. The column was coupled to a PCX 2500 post-column derivatization system (Pickering Laboratories, Mountain View, CA, USA). Eluent A contained 6 mM octanesulfonic acid, 6 mM heptanesulfonic acid, 40 mM ammonium phosphate, adjusted to pH 6.95 with dilute phosphoric acid, and 0.75% tetrahydrofurane. Eluent B contained 13 mM octanesulfonic acid, 50 mM phosphoric acid, adjusted to pH 6.9 with ammonium hydroxide, 15% acetonitrile and 1.5% tetrahydrofurane. The flow rate was 1 mL min 1 with the following gradient: 0–15 min isocratic A, 15–16 min switch to B, 16–35 min isocratic B, 35–36 min switch to A, 36–45 min isocratic A. The injection volume was 20 mL and the autosampler was cooled to 4 8C. The eluate from the column was oxidized with 10 mM periodic acid in 555 mM ammonium hydroxide before entering the 50 8C reaction coil, after which it was acidified with 0.75 M nitric acid. Both the oxidizing and acidifying reagents entered the system at a rate of 0.4 mL min 1. The toxins were detected by dual-monochromator fluorescence (lex 333 nm; lem 395 nm). The data were processed with Agilent Chemstation software and calibrated against external standards. Standard solutions of PSP toxins were purchased from the Certified Reference Material Programme of the Institute of Marine Biosciences, National Research Council, Halifax, NS, Canada. 2.4. Liquid chromatography with mass spectrometry (LC–MS) Mass spectral experiments were performed on an ABI-SCIEX4000 Q Trap (Applied Biosystems, Darmstadt, Germany), triple quadrupole mass spectrometer equipped with a TurboSpray1 interface coupled to an Agilent (Waldbronn, Germany) model 1100 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 mm  2 mm) packed with 3 mm Hypersil BDS 120 A˚ (Phenomenex, Aschaffenburg, Germany) and maintained at 25 8C. The flow rate was 0.2 mL min 1 and gradient elution was performed with two eluents, where eluent A was water and eluent B was methanol/ water (95:5, v/v), both containing 2.0 mM ammonium formate and 50 mM formic acid. Initial conditions were elution with 5% B, followed by a linear gradient to 100% B within 10 min and isocratic elution until 10 min with 100% B. The programme was then returned to initial conditions within 1 min followed by 9 min column equilibration (total run time: 30 min). Mass spectrometric parameters were as follows: curtain gas: 20 psi, CAD gas: medium, ion spray voltage: 5500 V, temperature: 650 8C, nebulizer gas: 40 psi, auxiliary gas: 70 psi, interface heater: on, declustering potential: 121 V, entrance potential: 10 V, exit potential: 22 V, collision energy: 57 V. Selected reaction monitoring (SRM) experiments were carried out in positive ion mode by selecting the following transitions (precursor ion > fragment ion): m/z 534 > 150, 536 > 150, 540 > 164, 552 > 150, 628 > 150, 640 > 164, 644 > 164, 650 > 164, 658 > 164, 674 > 164, 678 > 150, 678 > 164, 692 > 150, 692 > 164, 694 > 150, 694 > 164, 698 > 164, 706 > 164, 708 > 164, 710 > 150, 710 > 164, 720 > 164, 722 > 164, 766 > 164 and 784 > 164. Dwell times of 40 ms were used for each transition. 2.5. Detection of STX genes For the analysis of saxitoxin genes, cells were collected from exponentially growing cultures by centrifugation, 10 min, 4000 rpm (Centrifuge 5810 R, Eppendorf, Hamburg, Germany), and homogenized using a motor pestle and disposable pellet mixers (VWR, UK). Genomic DNA was extracted with the Plant DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The presence of sxtA1 and sxtA4 motifs was determined by PCR, using the primer pairs sxt001/ sxt002 for motif A1 and sxt007/sxt008 for A4 (Stu¨ken et al., 2011). The reactions were set up as follows: 1X Phire HotStart II buffer, 0.2 mM dNTPs, 0.4 mL Phire HotStart II polymerase (Finnzymes, Espoo, Finland), 0.5 mM forward and reverse primer (biomers.net), 1% v/v DMSO and 1 ng of template DNA. The reaction volume was brought up to 20 mL by sterile MQ-H2O. PCRs were conducted in a Peltier Thermal Cycler PTC-200 (MJ Research, Watertown, MA, USA), according to Stu¨ken et al. (2011) with slight modifications: 98˚ C 2 min 30 s, 5 cycles of (98˚ C 30 s, 68˚ C 30 s, 72˚ C 30 s), 5 cycles of (98˚ C 30 s, 66˚ C 30 s, 72˚ C 30 s), 30 cycles of (98˚ C 30 s, 64˚ C 30 s, 72˚ C 30 s) and 72˚ C 10 min. PCR products were visualized on a 2% (w/v) agarose gel stained with ethidium bromide (0.5 ng L 1). All approx. 550 bp (A1) and 750 bp (A4) products were excised, purified with the Qiaquick Gel Extraction kit (Qiagen) according to instructions and sequenced using the ABI PRISM dGTP BigDye1 Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) at the Turku Center for Biotechnology sequencing service. To confirm their identity, the obtained partial sequences (GenBank accession numbers KC835397-KC835402) were queried against the GenBank nr database using standard BLASTN 2.2.26+ (Zhang et al., 2000). 2.6. Statistical analysis Two-way ANOVA, followed by Tukey’s HSD post hoc comparisons, was used to analyze differences in growth rate between strains and salinities, and differences in strain-specific cell size, total toxin concentration and distribution of PST analogs (GTX2/3, STX) of total PST concentration between salinities and growth

S. Suikkanen et al. / Harmful Algae 26 (2013) 52–59

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0.20

phases (exponential vs. stationary). The toxin concentration data was log-transformed before analysis. Differences in the percentages of SPX analogs (13-desmethyl spirolide C, 13dmC; 20-methyl spirolide G, 20mG) of total SPX concentration between salinities was analyzed with one-way ANOVA, as both SPX analogs were only produced simultaneously in stationary growth phase. All analyses were conducted using the statistical package R 2.13.1 (R Development Core Team, 2011).

A

AOF 0927 AOVA 30

0.15

0.10

3. Results 3.1. Growth rate and cell size Growth rates of Alexandrium ostenfeldii/peruvianum differed significantly between the five strains and in the different salinities (two-way ANOVA, p < 0.001; Fig. 1). Overall, growth rates of all but two strains significantly differed from each other (Tukey’s HSD, p < 0.005), the exception being AOF 0927 and NCH 85: the strains LS A06 and AOVA 30 generally had the highest growth rates (mean  SD: 0.10  0.06 and 0.09  0.04 doublings day 1, respectively, n = 18), AOF 0927 and NCH 85 moderate (0.06  0.03 and 0.06  0.04 doublings day 1, respectively, n = 15), and S06/013/01 the lowest growth rates (0.05  0.02 doublings day 1, n = 9). Of the two Baltic Sea strains (with a native salinity of 6), AOF 0927 grew at salinities 6–25, with a significantly lowered growth rate at 25 (Tukey’s HSD, p < 0.001). AOVA 30 exhibited growth at salinities 6–30, but its growth rate was significantly lower at 25 and 30 compared to 6 (p < 0.001). The strain NCH 85, originating from Skagerrak, North Sea and salinity 30, grew at salinities 15–35, with a significantly lowered growth rate at 15 (p < 0.001). S06/ 013/01, isolated from the eastern coast of Scotland, North Sea and salinity 35, had a seemingly narrow salinity tolerance and was only able to grow at salinities 25–35. Its growth was significantly reduced at salinity 35 compared to 30 (p = 0.001). Finally, the strain LS A06 from coastal Northern Ireland, NE Atlantic grew at salinities ranging from 10 to 35, with the highest growth rate at salinity 15, but lowest in 10 (p < 0.001).

growth rate, k

0.05

0.00 0.20

B

NCH 85 S06/013/01 LS A06

0.15

0.10

0.05

0.00 6

10

15

20

25

30

35

Salinity Fig. 1. Maximum growth rates of the five A. ostenfeldii/peruvianum strains, originating from the Baltic Sea (A) and NE Atlantic (B), in the experimental salinities (mean  SD, n = 3).

Cells of all strains were significantly larger in stationary (mean length 30.01  4.67 mm, n = 720) than in exponential growth phase (26.76  4.44 mm, n = 540) (two-way ANOVA, p < 0.027 for all). Salinity had a small, but significant effect on cell size of LS A06 (twoway ANOVA, F5,20 = 12.64, p < 0.001): compared with the native salinity 30 (26.35  5.37 mm, n = 60), the cells were larger at 10 (32.47  4.32 mm, n = 30) and 35 (29.80  6.53 mm, n = 60) (Tukey’s HSD, p = 0.047 and 0.001, respectively).

Fig. 2. Cellular concentrations of PSP toxins in the two Baltic A. ostenfeldii strains in the experimental salinities in exponential (left) and stationary (right) growth phases (mean  SD, n = 3, n.d. = no data).

S. Suikkanen et al. / Harmful Algae 26 (2013) 52–59

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3.2. Toxin concentration and profile Total cellular PST concentrations in the Baltic strains AOF 0927 and AOVA 30 were generally on a similar level (up to 17.6 pg cell 1 in the stationary growth phase) and both STX and GTX2/3 were consistently produced by both strains in all salinities and both growth phases (Fig. 2). PSP toxins were not detected in any of the North Sea strains at any tested salinity. Spirolides were only found in the North Sea strains NCH 85 and S06/013/01 and the Irish LS A06 (Fig. 3), but never in the Baltic strains. NCH 85 and S06/013/01 only produced 20mG during exponential growth, but both 20mG and small amounts of 13dmC in the stationary phase (total spirolide concentration up to 0.97 pg cell 1). LS A06 exclusively produced 13dmC in both growth phases, and the concentrations were much higher than in the North Sea isolates (up to 27.0 pg cell 1). In 4 of the 5 strains investigated (AOF 0927, AOVA 30, NCH 85 and LS A06), total cellular toxin concentration was higher at stationary than exponential growth phase (two-way ANOVA, p < 0.002; Figs. 2 and 3). Also salinity significantly affected total toxin concentration in AOF 0927, AOVA 30 and S06/013/01 (two-way ANOVA, p < 0.05). In AOF 0927, total PST concentration was significantly higher at salinity 15 than at 6 (Tukey’s HSD, p = 0.039) or 20 (p < 0.001; Fig. 2). In AOVA 30, total PST concentration was significantly higher at 6 and 10 than at 20 (p = 0.003 and <0.001, respectively) and 25 (p = 0.002 and <0.001, respectively). In S06/013/01, total spirolide concentration was significantly higher at 25 than at 35 (p = 0.048; Fig. 3). In the Baltic strains, both growth phase and salinity significantly affected the percentages of GTX2/3 and STX of the total PST 1.0

Table 2 Results of sequencing analysis of presence of two motifs of sxt gene (A1 and A4) in the studied strains. + = present, = absent. Strain

A1

A4

AOF 0927 AOVA 30 NCH 85 S06/013/01 LS A06

+ + + +

+ +

concentration (two-way ANOVA, p < 0.001 for all). In AOF 0927, the ratio of STX was higher in the exponential than in the stationary phase, and it decreased significantly as salinity increased (Tukey’s HSD, p < 0.001 for all salinity pairs), from a mean of 49.5% at salinity 6 to 19.9% at salinity 25. In contrast, the percentage of STX in AOVA 30 was significantly higher in the stationary than in the exponential growth phase, being highest (mean 50.5%) at salinity 20 and lowest (44.2%) at salinity 10 (p < 0.030 for all). Distribution of spirolide analogs was also slightly affected by salinity in strain NCH 85 during stationary phase (one-way ANOVA, F3,8 = 6.93, p = 0.013): percentage of 20mG from total SPX concentration increased from 97.3% at salinity 20 to 99.4% at salinity 35 (Tukey’s HSD, p = 0.009). 3.3. Presence of saxitoxin genes Both sxt gene motifs, A1 and A4, were found to be present in the Baltic Sea strains AOF 0927 and AOVA 30 (Table 2). In the North Sea NCH 85 stat

NCH 85 exp 20mG

0.8

SPX total 13dmC 20mG

0.6 0.4 0.2 0.0 1.0

S06/013/01 exp

pg cell-1

S06/013/01 stat

20mG

0.8

SPX total 13dmC 20mG

0.6 0.4 0.2 0.0 30

LS A06 stat

LS A06 exp

13dmC

13dmC

20

10

n.d.

0 6

10

15

20

Salinity

25

30

35

6

10

15

20

25

30

35

Salinity

Fig. 3. Cellular concentrations of spirolides in the three NE Atlantic A. ostenfeldii/peruvianum strains in the experimental salinities in exponential (left) and stationary (right) growth phases (mean  SD, n = 3, n.d. = no data). Note different scaling of the y-axes.

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strains NCH 85 and S06/013/01 one of the motifs, sxtA1, was present, whereas the Irish strain LS A06 lacked both sxtA1 and sxtA4 motifs. 4. Discussion Salinity affected growth rate, toxin production and toxin profiles of the tested Alexandrium ostenfeldii and Alexandrium peruvianum strains, but did not change the suite toxins (i.e. PSTs or spirolides) produced by each strain. Salinity is likely to be an important physical factor for Alexandrium spp., which often form toxic blooms in estuarine and coastal environments (e.g. Anderson et al., 2012). It plays an especially important role in the ecology of the Baltic Sea, where a steep salinity gradient extends from near oceanic salinities in the south to close to freshwater conditions in the most northern and eastern areas. During the past decade, A. ostenfeldii has started to form recurrent toxic high-density blooms in shallow coastal embayments surrounding the Baltic Sea (Kremp et al., 2009, Hakanen et al., 2012). Investigations of the influence of salinity on A. ostenfeldii growth and toxicity may allow better characterization of risk areas for expansion of the blooms and their toxic effects. Globally, Alexandrium ostenfeldii and Alexandrium peruvianum seem to have a wide salinity tolerance. They occur in oceanic salinities in the North Atlantic, Arctic and Pacific Ocean and the Mediterranean Sea (Cembella et al., 2001; Gribble et al., 2005; Okolodkov, 2005; Ciminiello et al., 2006), but are often reported to prefer temperate coastal and estuarine areas with slightly decreased or fluctuating salinities (Lim and Ogata, 2005; Okolodkov, 2005; Gu, 2011; Tomas et al., 2012). The reported salinity ranges for A. ostenfeldii/peruvianum growth include 10–40 for Danish, 15–33 for Canadian, 10–35 for English, 10–30 for Malaysian and 7–27 for Chinese strains (Østergaard Jensen and Moestrup, 1997; Maclean et al., 2003; Percy et al., 2004; Lim and Ogata, 2005; Gu, 2011). In the Baltic Sea, A. ostenfeldii occurs in salinities down to ca. 5 (Kremp et al., 2009), however, several isolates from the Baltic Sea are able to grow at salinities as low as 2 (A. Kremp et al., unpublished). In the present study, effects of salinity on growth rate of Alexandrium ostenfeldii and Alexandrium peruvianum depended on the origin of the strain: the Baltic Sea strains were able to grow at salinities 6–25, even 30, although with reduced growth rates at salinities >20. The NE Atlantic strains originating from Skagerrak and Ireland had a wider salinity tolerance (15–35) than the strain from Scotland (25–35). This indicates that the strains are acclimated to their natural salinities. A. ostenfeldii has been reported from low salinity waters of the Baltic Sea for many decades (Nikolajew, 1953) and also recent molecular data indicate longstanding presence in the Baltic Sea (Tahvanainen et al., 2012), which supports the implication of the current data that local strains are well acclimated to the salinities prevailing in the Baltic Sea. A wide salinity range covering nearly the entire salinity gradient has been shown for other Baltic dinoflagellates (Kremp et al., 2005; Logares et al., 2007; Sundstro¨m et al., 2009). The strains from the Skagerrak and Ireland seem to be adapted to grow in environments with constant salinity fluctuations, but dominating oceanic influence. In fact, the Irish A. peruvianum strain LS A06 was isolated from a coastal inlet where salinities are usually lower than in the ocean. The Skagerrak where NCH 85 was isolated from is also influenced by periodic freshwater pulses from rivers and the Baltic Sea. The Scottish strain, in turn, clearly reflects oceanic origin with its restricted salinity tolerance range around marine conditions. In addition to growth, also toxin production of Alexandrium ostenfeldii and Alexandrium peruvianum was influenced by salinity. However, growth phase of a strain often had the most pronounced

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effect on total cellular toxin concentrations, which were usually significantly higher at stationary than exponential growth phase. Cells were also larger in the stationary growth phase, which might partly explain the higher toxin quotas; however, percentage differences in cellular toxin concentrations between the two growth phases were ca. 2–30 times as large as differences in cell length of each strain. Usually the highest cellular toxin quotas are found in the mid-exponential growth phase (e.g. Anderson et al., 1990), but in our study, the peak toxin concentrations are probably not represented, since the toxin samples were taken in the early exponential (ca. 5000 cells mL 1) and late stationary growth phases. Salinity had a significant effect on total cellular toxin concentrations of the Baltic Alexandrium ostenfeldii strains. Maximum cellular toxin concentrations were found at salinities 6, 10 (AOVA 30) and 15 (AOF 0927), i.e. in the optimum salinity range for growth of these strains. This effect cannot be explained by salinity-induced variations in cell size, since salinity did not have a significant effect on the cell size of the Baltic strains. Salinity only affected the cell size of the Irish Alexandrium peruvianum strain LS A06, however, there were no clear changes in total cellular spirolide concentration with variations in salinity in any of the Atlantic A. ostenfeldii and A. peruvianum strains. Total toxicity of a strain or a bloom is not only affected by the total concentration of toxins produced, but also by the toxin profile, i.e. the relative proportion of the individual toxins, as the different toxin variants usually vary in their toxicity. We hypothesized that salinity plays a role in determining whether a given strain of Alexandrium ostenfeldii or Alexandrium peruvianum produces spirolides or PS toxins, so that spirolides would mainly be produced in high salinities and PSTs in low salinities. This was not the case, i.e. the Baltic strains consistently produced PSTs (GTX2, GTX3 and STX) irrespective of the salinity, and no spirolides could be observed. Furthermore, all Atlantic strains produced spirolides (20mG and/or 13dmC) and no PSTs. This clearly indicates that the suite of toxins (PSTs/spirolides) produced by A. ostenfeldii/ peruvianum is not determined by salinity, but is either genetically fixed or governed by another environmental factor or a combination of several factors. There is a growing body of evidence that the PST composition of Alexandrium isolates is a stable phenotypic trait (Anderson et al., 2012 and references therein), and it has been suggested that geographic isolation has led to the development of intraspecific indigenous populations in different regions (Mackenzie et al., 1996; Cembella et al., 2001). The marked and relatively stable differences in toxin profile confirm that the strains used in the present study represent three distinct populations: the Baltic Sea (AOF 0927, AOVA 30), the open North Sea (NCH 85, S06/013/01) and the coastal inlets of Ireland (LS A06). In fact, Alexandrium ostenfeldii and Alexandrium peruvianum form a genetically structured species complex with genetic clades reflecting biogeographic distribution or habitat types. The strains used here represent 3 of the 6 major different phylogenetic clades constituting the A. ostenfeldii complex. Baltic strains belong to a clade comprising mostly brackish estuarine strains most of which produce PSTs. Scottish and Skagerrak strains in turn, fall into a clade of spirolide producing strains originating from the NE Atlantic. Irish LS A06 belongs to yet a third clade (A. Kremp et al., unpublished), containing a mixture of estuarine spirolide producing A. ostenfeldii and A. peruvianum strains. Interestingly, the close relatives of Baltic strains from the estuaries of the US East coast produce both PSP toxins and spirolides, as well as a third toxin form, gymnodimines which are structurally similar to spirolides (Tomas et al., 2012). Gymnodimine-like compounds were also detected in a Baltic strain (B. Krock, unpublished), which could be an

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indication for the presence of a pathway for spirolide synthesis in PST producing Baltic strains. Although the profile of major toxin groups (PST/spirolide) did not change due to salinity or growth phase, changes in profiles of the PST and spirolide variants were observed. In the northern Baltic strain, AOF 0927, the percentage of STX of total PST concentration decreased from exponential to stationary phase, and with increasing salinity (from ca. 50 to 20% with salinity increasing from 6 to 25). Decreased percentages of STX in Alexandrium spp. PST profile with increasing culture age (i.e. extent of nutrient limitation), as well as increased percentages of GTX2/3 in high salinity medium have also been reported in other studies (Boczar et al., 1988; Anderson et al., 1990; Hwang and Lu, 2000). As the toxicity of GTX2 and GTX3 is only 36 and 64% of that of STX, respectively (Luckas et al., 2003), more toxic blooms of A. ostenfeldii may develop if summertime salinity keeps decreasing in the northern Baltic Sea in the future (Suikkanen et al., 2007). Similarly, the percentage of STX of total PST concentration in several A. ostenfeldii isolates with the same origin generally increased with increasing temperatures and pCO2 concentrations, although the intraspecific variation was high (Kremp et al., 2012). The current study used only two strains from the Baltic Sea, and thus clear conclusions can hardly be drawn on the effect of salinity on A. ostenfeldii toxicity, especially as in the other strain originating from the central Baltic (AOVA 30), the trend of increasing percentage of STX with decreasing salinity was not confirmed, instead the highest percentage of STX (51%) was reached at salinity 20 and lowest (44%) at 10. Distribution of spirolide analogs was only slightly affected by salinity in strain NCH 85 during stationary phase. Instead, growth phase induced the greatest change in spirolide profiles, as the two North Sea strains only produced 20mG during exponential growth, but also minor amounts of 13dmC in the stationary phase. The Irish A. peruvianum strain, on the other hand, exclusively produced 13dmC throughout the salinities and in both growth phases. Of the spirolide analogs, 13dmC seems to dominate in NW Atlantic (coastal Canada and USA, but also Spain, France, England, Ireland, Denmark; Cembella et al., 2000; MacKinnon et al., 2004; Percy et al., 2004; Villar Gonza´lez et al., 2006; Amzil et al., 2007; Touzet et al., 2008; Van Wagoner et al., 2011) and Mediterranean (Ciminiello et al., 2006), while 20mG dominates in North Sea and adjacent waters (Norway, Scotland; Aasen et al., 2005; Krock et al., 2008; Brown et al., 2010). Our findings confirm this. Of the strains used in the present study, S06/013/01 has previously been found to produce only 20mG and not 13dmC, but it was only analyzed in exponential phase (Brown et al., 2010). In the present study, presence of a putative STX gene (sxtA4 motif) was found to reliably predict STX production. Both sxt gene motifs, A1 and A4 (Stu¨ken et al., 2011), were found to be present in the Baltic Sea strains that produced PSTs. In the 20mG and 13dmC spirolide-producing North Sea strains one of the motifs, sxtA1, was present, whereas the 13dmC spirolide-producing Irish strain lacked both sxtA1 and sxtA4 motifs. This indicates that only sxtA4 is necessary for STX production, since no PST production could be observed in the North Sea strains. Stu¨ken et al. (2011) found both sxtA1 and sxtA4 genomic sequences to be present in all STXproducing dinoflagellate strains analyzed, however, both fragments were also amplified from A. tamarense (Lebour) Balech strains for which no STX-production was detected. This was hypothesized to be due to (1) other genes of the STX pathway missing in these strains, (2) different post-transcriptional mechanisms between STX-producing and non-producing strains, or (3) production of lower amounts of STX by these strains than the detection limit of the toxin determination methods used. In the present study, the role of the sxtA1 motif in the North Sea strains remains unclear. One alternative is that the strains did produce

PSTs, but the concentrations were lower than the detection limit of the toxin analysis, since production of low PST concentrations has previously been reported for strain S06/013/01 (Brown et al., 2010). It is, however, also possible that the analyzed S06/013/01 clone has lost its ability to produce PSTs in culture (and thus the relevant A4 gene sequences). This kind of phenomenon has been documented in Alexandrium at least once (Martins et al., 2004). On the other hand, also Hackett et al. (2013) found that the N-terminal portion of sxtA (corresponding to the sxtA1 motif in Stu¨ken et al., 2011) is not specific to STX-producing dinoflagellates, whereas the C-terminal portion (sxtA4 motif) is. As a conclusion, whether isolates of Alexandrium ostenfeldii/ peruvianum produce PSTs, spirolides or both is not determined by salinity, but is probably a genetically fixed character reflecting their phylogeny and biogeographic origin. Changes in salinity may, however, affect the actual toxicity of A. ostenfeldii/peruvianum populations via changed growth rates, total cellular toxin concentrations and composition of the toxin analogs. The PSTproducing A. ostenfeldii seems to thrive in the salinity range of the Baltic Sea, whereas the spirolide-producing populations are more typical for the ocean, but both groups have potential to grow in intermediate salinities, such as ocean coasts and estuaries. Acknowledgements We are grateful to T. Alpermann, E. Bresnan and N. Touzet for providing A. ostenfeldii/peruvianum strains, as well as to J. Oja for assistance in the lab. We also thank A. Mu¨ller for sample extraction and PST analysis. The study was financed by the Academy of Finland, grant 128833 to SS and AK, the Finnish Agency for Technology and Innovation (TEKES) grant 955/31/09 and the Finnish Doctoral Programme in Environmental Science and Technology (EnSTe) to HH.[SS] References Aasen, J., MacKinnon, S.L., LeBlanc, P., Walter, J.A., Hovgaard, P., Aune, T., Quilliam, M.A., 2005. Detection and identification of spirolides in Norwegian shellfish and plankton. Chemical Research in Toxicology 18, 509–515. Amzil, Z., Sibat, M., Royer, F., Masson, N., Abadie, E., 2007. Report on the first detection of pectenotoxin-2, spirolide-A and their derivatives in French shellfish. Marine Drugs 5, 168–179. Anderson, D.M., Kulis, D.M., Sullivan, J.J., Hall, S., Lee, C., 1990. Dynamics and physiology of saxitoxin production by the dinoflagellates Alexandrium spp. Marine Biology 104, 511–524. Anderson, D.M., Alpermann, T.J., Cembella, A.D., Collos, Y., Masseret, E., Montresor, M., 2012. The globally distributed genus Alexandrium: multifaceted roles in marine ecosystems and impacts on human health. Harmful Algae 14, 10–35. Balech, E., 1995. The Genus Alexandrium Halim (Dinoflagellata). Sherkin Island Marine Station Publication, Sherkin Island, Co. Cork, Ireland. Beuzenberg, V., Mountfort, D., Holland, P., Shi, F., MacKenzie, L., 2012. Optimization of growth and production of toxins by three dinoflagellates in photobioreactor cultures. Journal of Applied Phycology 24, 1023–1033. Boczar, B.A., Beitler, M.K., Liston, J., Sullivan, J.J., Cattolico, R.A., 1988. Paralytic shellfish toxins in Protogonyaulax tamarensis and Protogonyaulax catenella in axenic culture. Plant Physiology 88, 1285–1290. Brown, L., Bresnan, E., Graham, J., Lacaze, J.-P., Turrell, E., Collins, C., 2010. Distribution, diversity and toxin composition of the genus Alexandrium (Dinophyceae) in Scottish waters. European Journal of Phycology 45, 375–393. Cembella, A.D., Lewis, N.I., Quilliam, M.A., 2000. The marine dinoflagellate Alexandrium ostenfeldii (Dinophyceae) as the causative organism of spirolide shellfish toxins. Phycologia 39, 67–74. Cembella, A.D., Bauder, A.G., Lewis, N.I., Quilliam, M.A., 2001. Association of the gonyaulacoid dinoflagellate Alexandrium ostenfeldii with spirolide toxins in size-fractionated plankton. Journal of Plankton Research 23, 1413–1419. Ciminiello, P., Dell’Aversano, C., Fattorusso, E., Magno, S., Tartaglione, L., Cangini, M., Pompei, M., Guerrini, F., Boni, L., Pistocchi, R., 2006. Toxin profile of Alexandrium ostenfeldii (Dinophyceae) from the Northern Adriatic Sea revealed by liquid chromatography-mass spectrometry. Toxicon 47, 597–604. Diener, M., Erler, K., Hiller, S., Christian, B., Luckas, B., 2006. Determination of Paralytic Shellfish Poisoning (PSP) toxins in dietary supplements by application of a new HPLC/FD method. European Food Research and Technology 224, 147–151. Etheridge, S.M., Roesler, C.S., 2005. Effects of temperature, irradiance, and salinity on photosynthesis, growth rates, total toxicity, and toxin composition for

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