Morphology, toxin composition and molecular analysis of Dinophysis ovum Schütt, a dinoflagellate of the “Dinophysis acuminata complex”

Harmful Algae 7 (2008) 839–848

Contents lists available at ScienceDirect

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

Morphology, toxin composition and molecular analysis of Dinophysis ovum Schu¨tt, a dinoflagellate of the ‘‘Dinophysis acuminata complex’’ Nicola´s Raho a,1, Gemita Pizarro b,1, Laura Escalera b, Beatriz Reguera b, Irma Marı´n a,* a b

Centro de Biologı´a Molecular, Universidad Auto´noma de Madrid, Cantoblanco, 28049 Madrid, Spain Instituto Espan˜ol de Oceanografı´a, Centro Oceanogra´fico de Vigo, Aptdo. 1552, E-36280 Vigo, Spain

A R T I C L E I N F O

A B S T R A C T

Article history: Received 24 January 2008 Received in revised form 24 April 2008 Accepted 24 April 2008

This work provides a morphological, toxinological and molecular description of Dinophysis ovum Schu¨tt, a species included in the Dinophysis acuminata complex, and often misidentified as D. acuminata Clapare`de and Lachmann. A dinoflagellate bloom occurred in the Galician Rı´as Baixas (NW Spain) in June 2006, where D. ovum was the overwhelmingly dominant (97%) Dinophysis species, accompanied by small numbers of D. acuminata, and other Dinophysis spp., Protoperidinium spp. and Ceratium spp. D. ovum was discriminated from D. acuminata at the light microscope on the basis of their different cell contours; it had only okadaic acid (OA) (7.1 pg cell1) as a detectable lipophilic toxin component. Molecular analyses of the ITS1-5,8S-ITS2 rDNA region showed that D. ovum was grouped in a common clade of small-sized species of Dinophysis, close to D. sacculus. Analyses of mitochondrial genes (Cytochrome c oxydase 1) of D. ovum and D. acuminata showed this region exhibited a much higher variability 25 bp difference between the 2 species under study and was therefore more appropriate than rDNA genes for phylogenetic analyses of Dinophysis spp. This is the first report of lipophilic shellfish toxins in D. ovum, and of analyses of mitochondrial genes to discriminate between different species of Dinophysis. ß 2008 Elsevier B.V. All rights reserved.

Keywords: Dinophysis acuminata Dinophysis ovum DSP toxins PCR ITS rDNA Cytochrome c

1. Introduction Different dinoflagellate species of the genus Dinophysis have been reported as causative agents of Diarrhoeic Shellfish Poisoning (DSP) outbreaks throughout the world (Lee et al., 1989). Up to date, 11 species of Dinophysis have been confirmed, by chromatographic analyses on single cell isolates, to contain lipophilic toxins (Moestrup, 2004), but this number is very likely to increase when new species of the genus are tested with advanced chromatographic systems coupled to mass spectrometers (LC/MS) that require a very small number of cells per sample. Proliferations of some species of Dinophysis are extremely persistent throughout the year and may lead to lengthy shellfish-harvesting closures. A precise taxonomic and toxinological characterization of the species responsible for these outbreaks is essential, because species with very close morphology may show large differences in their toxin profile and content.

* Corresponding author at: Centro de Biologı´a Molecular ‘‘Severo Ochoa’’, C\ Nicola´s Cabrera n8 1. Lab 104, Campus de Cantoblanco, 28049 Madrid, Spain. Tel.: +34 91 4978078; fax: +34 91 4978300. E-mail address: [email protected] (I. Marı´n). 1 These authors contributed equally to this work. 1568-9883/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2008.04.006

Dinophysis acuminata Clapare`de et Lachmann and D. acuta Ehrenberg have been reported as the main causative agents of Lipophilic Shellfish Toxins (LST) events – presence of okadaiates and/or pectenotoxins in shellfish above regulatory levels – in New Zealand, Chile and in the Atlantic coasts of Europe (van Egmond et al., 1993; Reguera and Pizarro, 2007). The taxonomic identification of Dinophysis spp. is largely based on the cell contour – size and shape of their large hypothecal plates – and the shape of the left sulcal lists and their ribs (Larsen and Moestrup, 1992). Nevertheless, each species of Dinophysis, on each locality, may exhibit a continuum of shapes between large vegetative specimens and small gamete-like cells as a result of their polymorphic life cycle (Reguera and Gonza´lez-Gil, 2001). This morphological variability causes uncertainty in the identification and quantification of phytoplankton samples, especially in those where two close species of Dinophysis, such as the pair D. acuminata and D. sacculus Stein co-occur. The term ‘‘D. acuminata complex’’ was coined by several authors to label a group of co-occurring species, difficult to discriminate with conventional light-microscopy methods (Lassus and Bardouil, 1991; Bravo et al., 1995; Koukaras and Nikolaidis, 2004). A large array of morphologically distinct morphotypes of Dinophysis have been labeled either as D. acuminata or as D. cf acuminata provided their large hypothecal plates were dorsally convex and with a more or less oval/suboval shape.

840

N. Raho et al. / Harmful Algae 7 (2008) 839–848

Molecular biology may provide powerful tools to improve identification of organisms in the laboratory, as well as in field samples. Due to the difficulties to establish species of Dinophysis in culture, very sensitive techniques for single-cell DNA amplification were developed (Marı´n et al., 2001a,b; Edvardsen et al., 2003; Hart et al., 2007; Kai et al., 2006). In the last years, these techniques have generated sequences for a rank of species of Dinophysis (RehnstamHolm et al., 2002; Edvardsen et al., 2003; Hart et al., 2007) and provided an important dataset for phylogenetic studies. The nuclear rDNA operon has been extensively used to infer taxonomic relationships in a large array of organisms, including microalgae. Its large (LSU, 28S rRNA) and small (SSU, 18S rRNA) subunits have highly conserved regions that are widely used in phylogeny studies (Bhattacharya and Medlin, 1995; Puel et al., 1998; Guillou et al., 2002). However, the SSU has shown a low resolution power to understand relationships within dinoflagellates (Saunders et al., 1997; Saldarriaga et al., 2001). In the case of Dinophysis, sequence comparisons of the D1–D2 LSU regions of different species have identified two major clades, and little variation between D acuminata, D. acuta and D. norvegica Clapare`de et Lachmann (Edvardsen et al., 2003). Further, phylogeographical analysis based on LSU sequences was not successful to establish intra-specific differences between Dinophysis isolates from different locations (Hart et al., 2007). The internal transcribed spacers, ITS1 and ITS2, of the nuclear rDNA operon have less conserved regions and are more useful at close phylogenetic levels, such as those observed between species and their populations. Nevertheless, these sequences were identical in the species D. acuminata and D. norvergica (Edvardsen et al., 2003), and 99% identical in D. acuminata and D. sacculus (Marı´n et al., 2001a). Mitochondrial DNA is a useful candidate as a molecular marker in closer phylogenetic reconstructions because their genes are generally conserved, but they are subject to more variations than nuclear coding genes (Avise, 1994; Saccone et al., 2000). Some mitochondrial genes are better molecular clocks than the rDNA because they have a relatively constant mutation rate, what makes them ideal for cross-taxa phylogenetic studies (Saccone et al., 2000). For example, the Cytochrome b (cob) is one of the mitochondrial genes that are most widely used for phylogenetic population analyses (Taylor and Hellberg, 2003). The combination of nuclear and mitochondrial genes has been shown to provide robust phylogenetic trees among different organisms, including protists of the group Alveolata (Serizawa et al., 2000; Rathore et al., 2001; Zhang et al., 2005). Furthermore, the Cytochrome c oxydase 1 (cox1) gene proved to be useful to separate different clades of the dinoflagellate genus Symbiodinium (Takabayashi et al., 2004). In late May 2006, a sudden bloom of Dinophysis spp. was detected during a routine weekly monitoring sampling at one fixed station in the Galician Rı´as Baixas. A species of Dinophysis, smaller and with a more regular oval contour than the typical D. acuminata from the area, was the overwhelmingly dominant species in the size-fractioned (20–77 mm) plankton concentrates. This small Dinophysis was identified as D. ovum Schu¨tt. This work undertakes a morphometric analysis of this species along with its toxinological characterization by LC/MS analyses in plankton concentrates and in single cell isolates. For the first time, sequences of the mitochondrial Cytochrome c oxidase 1 (cox1), combined with ITS rRNA are applied in phylogenetic studies of Dinophysis spp. 2. Material and methods 2.1. Plankton samples Sampling was carried out on board RV J. M. Navaz at a fixed station (Bueu, P2, 42821.400 N, 8846.420 W) in Rı´a de Pontevedra

Fig. 1. Map of the Galician Rı´as Baixas (NW Spain), and location of the sampling point (P2) in Ria de Pontevedra.

(Fig. 1) on 12 June 2006. Seawater from 3 to 5 m depth was pumped with a submersible pump during 10 min, and passed through a set of superimposed meshes. The 20–77 mm size-fractioned concentrate was selected for Dinophysis spp. studies, and diluted with seawater into 5 l bottles so the plankton material was kept fresh and alive during transportation (1 h) to the laboratory. 2.2. Cells measurements and imaging Maximum length (L) and dorso-ventral depth (W) of the large hypothecal plates of Dinophysis cells were measured, following Balech (2002), with a micrometer inserted in the ocular of the microscope. Measurements were obtained of 44 normal-sized cells of D. ovum. Differential Interference Contrast (DIC) digitized micrographs of live cells were obtained under a NIKON ECLIPSE TE2000-S inverted microscope with a NIKON D70 camera. For the Scanning Electron Microscopy (SEM) examination, a simplified method was used that skips the critical point-drying steps from the conventional methods. This technique is not appropriate for naked dinoflagellates, but has proved to work well with hardy thecate dinoflagellates, including Dinophysis spp. Lugol-fixed samples were diluted with distilled water (1 ml sample in 2 ml of water). Cells of D. ovum and D. acuminata cooccurring in the same sample were isolated one by one with a microcapillary pipette under the inverted microscope, placed in 5 mm polycarbonate membrane filters (Isopore, Millipore Corp., Bedford, MA), and rinsed again with distilled water. Filters were dried inside Petri dishes, to avoid dust contamination, at room temperature and further sealed over aluminium stubs. Specimens, sputtered-coated with gold (30 nm) with a K550 X sputter coater (Emitech Ltd., Ashford, Kent, UK), were examined and photographed under a PHILIPS XL30 scanning electron microscope (FEI Company, Hillsboro, OR USA) with an acceleration voltage of 10 kV. 2.3. Phytoplankton counts in samples for toxin analyses Two subsamples were taken from the plankton concentrates collected for toxins analyses: (1) a 50-ml subsample was taken and preserved with Lugol’s iodine acidic solution (Lovegrove, 1960) for cell counts; (2) another 15-ml subsample was taken for toxin extraction and analyses. For identification and quantification of microplankton, 3 ml of the Lugol-fixed subsample were placed in a sedimentation chamber over 1 h; all specimen in the whole bottom-surface of the chamber were scanned and counted under a

N. Raho et al. / Harmful Algae 7 (2008) 839–848

NIKON ECLIPSE TE2000-S inverted microscope. Differential Interference Contrast (DIC) digitized micrographs were taken with a NIKON D70 camera coupled to the microscope. To estimate toxin content per cell in plankton concentrates, the total amount of toxins estimated from the 15-ml extract (next section) was divided between the number of cells of Dinophysis contained in that volume. 2.4. Toxin extraction from phytoplankton concentrates and from single cell isolates Back in the laboratory, a raw 15 ml sample from the 5 l bottles was centrifuged (1000 rpm, 20 min), and the supernatant eliminated; 500 ml of MeOH were added to the pellet and the mixture sonicated for 1 min; remains in the sonication probe were rinsed with 250 mL of methanol and the final sample of 750 ml was kept at 20 8C until analysis. To prepare for the LC/MS analysis, samples were sonicated again for 1 min and the remains in the sonication probe rinsed with 250 ml of methanol, so the final extract sample had a volume of 1 ml. Extracts were centrifuged at 5000 rpm for 20 min, the supernatant collected, and the pellet re-suspended in 500 ml for a second extraction repeating the same procedure. Finally the two supernatants were combined, mixed in a vortex and methanol added to a final volume of 2 ml. A 200 ml aliquot of these extracts was filtered through 0.45 mm filters (Gelman Nylon Acrodisc 13 mm) prior to injection of 35 ml subsamples into the LC/ MS system. Dinophysis ovum-like cells were isolated one by one, from the plankton concentrates with a microcapillary pipette under the inverted microscope, at 100 and 400 magnification; transferred 2–3 times through sterile seawater and finally placed (with as little seawater as possible) in a 1.5 ml eppendorf tube with 500 ml methanol, and then treated and kept until analysis in the same way as the plankton concentrate extracts. To prepare for the LC/MS analysis, the samples were transferred to a glass vial of 1.8 ml, the remains in the eppendorf tube washed twice with 200 ml of methanol, and the supernatants combined and mixed in a vortex prior to be dried at 40 8C under reduced pressure on a Speed Vac (Savant, Instruments Inc., Holbrook, NY). The dried toxin extract was re-suspended in 200 ml of methanol, mixed in a vortex and filtered through 0.45-mm filters (Gelman Nylon Acrodisc 13 mm) prior to injection of 10 and 20 ml subsamples into the LC/MS system. 2.5. LC/MS analyses For toxins identification a LC/MS system was used. The chromatographic separation was performed on a Thermo Finnigan Surveyor with a Waters XTerra C18 column (2.1 mm  150 mm) packed with 5 mm particles at 35 8C. The mobile phase consisted of (A) 2 mM ammonium acetate at pH 5.8 and (B) 100% methanol, with a flow rate of 0.2 ml min1. A linear gradient elution from 60% to 100% B was run 20 min and held at 100% B for 2 min, then decreased to 60% B over 3 min and held at 60% B for 5 min. The spectral measurements were performed using an ion-trap mass spectrometer (Thermo Finnigan LCQ-Advantage) equipped with a micro-electrospray ionization interface (mESI). The mass spectrometer was operated at a temperature of 250 8C and a spray voltage of 3.0 and 4.5 kV for positive and negative analyses, respectively. A flow of 20 ml min1 for the sheath gas, and of 10 ml min1 for the auxiliary gas were used. Full scan data were acquired from m/z 200 to 2000, in both negative and positive ionization modes. Negative mode was performed to confirm the toxin spectrum. The MS spectrum shifted prominent ions at m/z 805 [M+H]+, 822 [M+NH4]+, 827 [M+Na]+

841

and 803 [MH] for okadaic acid (OA) and dinophysistoxin-2 (DTX2); 876 [M+NH4]+, 881 [M+Na]+, 857 [MH] and 918 [M+CH3COO] for pectenotoxin-2 (PTX2). In the case of extracts from single cell isolates, 20 ml were injected into the LC/MS. To obtain a discernible signal, a single ion monitoring (SIM) analysis and only one ionization mode were used according to the possibilities of the mass spectrometer, and so, positive ionization mode was used to record signals of the [M+NH4]+ and [M+Na]+ ions at m/z 822 and 827 for both OA and DTX2, and at m/z 876 and 881 for PTX2. The same column and particle-packing were used for toxin analyses of single cell isolates. OA and PTX2 certified reference standards (NRC, Canada) and a secondary reference material of DTX2 (1 mg ml1) were used for the calibration of the LC/MS and quantification of toxins. For the phytoplankton concentrates analyses, different volumes of a working solution (0.7 ng ml1) of OA, DTX2 and PTX2, were used to generate a calibration curve (minimum of 5 points). The linearity of each point was tested according to van Trijp and Roos (1991). Calibration lines were obtained for each toxin. The amounts of toxins injected ranged from 0.8 to 6.6 ng for OA and DTX2 (ions at m/z 805 plus 822 and 827) and from 1 to 6.0 ng for PTX2 (ions at m/z 876 plus 881). For the analyses of single cell isolates, calibration curves were obtained just for OA, the only toxin detected in the plankton concentrates. A working solution (3 pg ml1) of OA was used to generate a five-point calibration curve, from 5 to 40 pg. 2.6. PCR amplification and sequencing of single-cell isolates D. ovum-like cells were isolated, one by one, as explained in Section 2.4, but they had an additional final rinse in phosphate buffer solution (PBS 1). The selected cells were then transferred to 200 ml Eppendorf tubes containing 5 ml of lysis buffer (0.005% SDS with 400 ng ml1 Proteinase K) and treated following the procedure of Kai et al. (2006). Amplifications were carried out in a thermocycler Thermal Cycler 2720 (Applied Biosystems). All PCR reaction mixtures (50 ml) contained 3 single cells of Dinophysis and 1 mM of dNTP, 3 mM MgCl2, 1 mM of each primer, 5 ml of 10 PCR buffer and 0.025 u ml1 DNA Polimerase (AmpliTaq DNA Polymerase, Roche Molecular Systems). Amplification was examined for correct length, purity and shield on 1% agarose gels stained with 0.5 mg ml1 ethidium bromide and visualizated by UV-transilumination. Amplification of the mitochondrial citocrome c oxidase 1 (cox1) was performed with the primers Dinocox1F (50 -AAAAATTGTAATCATAAACGCTTAGG-30 ) and Dinocox1R (50 -TGTTGAGCCACCTATAGTAAACATTA-30 ) (Lin et al., 2002), with an initial denaturation at 948 C for 10 min, 40 cycles at 94 8C for 1 min, 55 8C for 1 min and 72 8C for 3 min, followed by a 10-min extension step at 72 8C. The ITS1-5,8S-ITS2 regions were amplified with primers ITSB2 (5 0 -CCTGCAGTCGACA(AG)AATGCTTAA(AG)TTCAGC(AG)GG-3 0 ) and ITSA2 (50 -CCAAGCTTCTAGATCGTAACAAGG(ACT)TCCGTAGGT30 ) (Adachi et al., 1994) with an initial denaturation step of 94 8C for 10 min followed by 30 cycles of 94 8C for 1 min, 40 8C for 1 min, 72 8C for 1.5 min, and a final extension of 72 8C for 10 min. 2.7. Sequence analysis PCR products were sequenced using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (ABI) and an Applied Biosystem ABI 310 (PE Applied Biosystems, Foster City, CA, USA) automated sequencer. The obtained sequences were compared to those available in GenBank using the Basic Local Aligment Search Tool (BLAST) algorithm to identify known sequences with

842

N. Raho et al. / Harmful Algae 7 (2008) 839–848

high similarity. The selected sequences were aligned with CLUSTAL X (Thompson et al., 1997) and Neighbor Joining (NJ) (Saitou and Nei, 1987) phylogenetic constructions were performed with PAUP* 4.0 (ver. 4.0b10) (Swofford, 1998) using the Kimura 3 parameter (Kimura, 1981). Finally, support for the NJ branches was tested by Bootstrap (Felsenstein, 1985) analysis of 1000 repetitions. 3. Results 3.1. Description of D. ovum Schu¨tt Cells in side view somewhat irregularly egg-shaped, rather asymmetrical with an apex narrower than the broad rounded antapex; ventral contour until R3 (third rib of the left sulcal list) slightly convex, and from there onwards, more or less broad and rounded; dorsal contour always more strongly convex than the ventral one. The broader depth of the cell is at the height of R3, or slightly lower. Its plastids have a beautiful golden colour. The ratio between the maximum length and dorso-ventral depth (L:D) of the large hypothecal plates (LHP) ranges from 1.3 to 1.4. These LHP exhibit a rough areolation, and most of the areolae have a central pore (Schu¨tt, 1865; Schiller, 1933). The original description (Schu¨tt, 1895, Pl.1 Fig. 61–3) does not provide a size range for the species, but illustrates three specimens (Fig. 2a–c) that, according to the author, are drawn with a magnification of 640. Therefore, these specimens (45 mm 

31 mm in the original drawings) are 70 mm long and 48 mm deep. The description of Schiller (1933) provides a size range of L = 44– 62 mm; D = 38–45 mm. This last author states that D. ovum is a warm-subtropical water species present in the Mediterranean and Adriatic seas, and in the Atlantic up to the English Channel. Redrawn figures corresponding to the original description of D. ovum by Schu¨tt (1865) and several morphotypes of the same species from Abe´ (1967) are shown in Fig. 2. The specimens from the Galician Rı´as have their large hypothecal plates with a very regular and rotund outline, almost symmetric in relation to the longitudinal axis (Fig. 3). Measurements from a 44–cells sample were: L (length) = 36.6  2.4 mm (range 32–42 mm), D (dorso-ventral depth) = 26.4  2.1 mm (range 22.5–32 mm), and C (cingular width) = 8.5  1.2 mm (range 7.5– 12 mm). The epicone is not visible in lateral view. The cingular lists have no ribs. The left sulcal list has a smooth contour and not very prominent ribs. The thecae are ornamented with areolae, and there are plastids present in the cytoplasm. The main differences between D. ovum and D. acuminata are that D. acuminata from Galician coastal waters is larger (L = 44.1–58.7 mm; D = 24.3–43.4 mm), dorso-ventrally inclined, has a more straight ventral margin, a slightly tapered antapical contour and larger left sulcal lists with more prominent ribs than D. ovum (Fig. 3). The latter is widest below the middle region of the lage hypothecal plate (LHP) and has a lower L:D ratio than D. acuminata, that is widest around the middle region

Fig. 2. Dinophysis ovum Schu¨tt. (a–c) Redrawn from the original description by Schu¨tt (1895), Fig. 61–3. (d–k) Redrawn from Abe´ (1967), Fig. 10a–e, g, f, and j, respectively.

N. Raho et al. / Harmful Algae 7 (2008) 839–848

843

Fig. 3. Differential interference contrast (DIC) micrographs of (a–b) normal-sized, (c–d) intermediate and (e–f) small cells of D. ovum (left column) and D. acuminata (right column) from Rı´a de Pontevedra (NW Spain). Scale bars 20 mm.

of the LHP. The right sulcal list of D. ovum ends close to the base of the second rib (R2) of the left sulcal list, whereas in D. acuminata is longer and ends near the middle point between the bases of R2 and R3 (Fig. 4). Differences between D. ovum and D. acuminata are more notorious when their small cells are considered. Those of D. ovum retain almost the same rotund

contour of the normal-sized cells, with a convex dorsal margin, whereas the small cells of D. acuminata have a pointed antapical region and an almost straight dorsal margin (Fig. 3). Table 1 summarizes morphometric measurements on the Galician specimens and the original and other descriptions of D. ovum and D. acuminata.

N. Raho et al. / Harmful Algae 7 (2008) 839–848

844

Fig. 4. Scanning electron microscopy micrographs of specimens from Rı´a de Pontevedra (NW Spain): (a) D. acuminata, (b) D. ovum and (c) Detail of the right sulcal list of D. ovum. Scale bars 20 mm.

Table 1 Morphometry of D. ovum from the original description, and from specimens of Galician and other parts of the world and comparison with measurements (Galician and others) of D. acuminata. L and D are maximum length and dorso-ventral depth D. ovum

D. acuminata

Author

L (mm)

D (mm)

Author

L (mm)

D (mm)

Schu¨tt (1895) Schiller (1933) Burns and Mitchell (1982) This paper

62.5 44–62 30 32–42

48.4 38–45 22 22.5–32

Larsen and Moestrup (1992) Zingone et al. (1998) Reguera (2003)

38–58 42–58 44.1–58.7

30–38 25–38 24.3–43.4

3.3. LC/MS analyses of the phytoplankton-concentrate and of single cell isolates

3.2. Composition of plankton concentrates Analysis of the lugol-fixed size-fractioned plankton concentrates revealed a population dominated by dinoflagellates and with undetectable levels of other microalgal groups. On 12 June, D. ovum was the dominant species and represented 47% of the phytoplankton assemblage and 97% of the total Dinophysis spp. (Table 2). It was a very ephemeral bloom, as one week later, on 19 June, dinoflagellates were still the dominant group, but species of the genus Ceratium furca (Ehrenberg) Clapare`de et Lachmann and C. fusus (Ehrenberg) Dujardin became dominant, and D. acuminata (3.5%) and D. ovum (4%) represented a very small part of the microplankton assemblage. Table 2 Relative abundance and composition of the 20-77 mm size fraction in a plankton concentrate from 12 June 2006 collected in Rı´a de Pontevedra (NW Spain) Species

Cell (ml1)

%

Ceratium furca C. fusus C. horridum Dinophysis acuminata D. caudate D. rotundata D. ovum Diplopsalis lenticula Protoperidinium diabolus P. divergens P. cf. leonis P. steinii P. cf. ovatum Scrippsiella sp.

280 27 7 34 13 27 2221 387 500 47 13 434 700 33

5.9 0.6 0.1 0.7 0.3 0.6 47.0 8.2 10.6 1.0 0.3 9.2 14.8 0.7

Total Dinoflagellates

4729

Total

4729

Fig. 5a and b shows chromatograms and mass spectra of the standards extracted from the full-scan (m/z 200–2000) positive ion LC/MS analysis. Peaks corresponding to OA, DTX2 and PTX2 (at 8.0, 8.9 and 12.1 min, respectively), presented spectra with [M+H]+, [M+NH4]+ and [M+Na]+ ions at m/z 805, 822 and 827 for OA and DTX2; [M+NH4]+ and [M+Na]+ ions at m/z 876 and 881 for PTX2. Fig. 5c and d shows chromatograms and the corresponding spectra obtained from the full-scan (m/z 200–2000) positive ion LC/MS analysis of the phytoplankton-concentrate. The peaks corresponded to OA (Fig. 5c) and traces of PTX2 (Fig. 5d) (at 7.9 and 12.0 min, respectively). The estimated cell toxin quota of OA was 8.5 pg cell1 of Dinophysis. Fig. 6a shows the LC/MS chromatograms and mass spectra in positive mode of the predominant ions of the OA, [M+NH4]+ and [M+Na]+ at m/z 822 and 827 in the standard solution prepared for analysis of single cell isolates. A peak occurred at a retention time of 7.6 min that presented the typical spectra of OA with [M+NH4]+ and [M+Na]+ ions at m/z 822 and 827. Fig. 6b shows the LC/MS chromatograms and mass spectra in positive mode of the OA present in the single cell isolates of D. ovum. The estimated cell toxin quota of OA was 7.0 + 0.3 pg cell1. 3.4. Molecular analyses of D. ovum 3.4.1. Analyses of internal transcribed spacers ITS1 and ITS2 The ITS rDNA region of D. ovum (GenBank accession no. AM931581) and D. acuminata (GenBank accession no. AM931580) isolated from Galician waters was sequenced. Phylogenetic analyses of D. ovum were performed and compared with the

N. Raho et al. / Harmful Algae 7 (2008) 839–848

845

Fig. 5. LC/MS chromatograms, in positive ionization mode, with their retention time (left) corresponding to the OA, DTX2 and PTX2 standards and the plankton concentrate, and mass spectra for [M+NH4]+ and [M+Na]+ (right): (a) OA and DTX2 standards; (b) PTX2 standard; (c) and (d) OA and PTX2, respectively, found in the plankton concentrate rich in Dinophysis ovum.

corresponding sequences from 16 species of Dinophysis available at the GenBank and with the sequence from D. acuminata obtained in this study. The Neighbour Joining (NJ) tree (Fig. 7) shows that larger-sized species of Dinophysis, D. caudata Kent and D. tripos Gourret, were the most divergent. The new D. acuminata AM931580 was grouped with other D. acuminata strains from France, Vigo (Spain), Australia, Japan and UK. This clade contained the smaller-sized Dinophysis species, such as D. acuminata, D. norvegica, D. dens Pavillard, D. sacculus, D. ovum and D. acuta. Curiously, specimens labeled as D. dens, a life-history stage of D. acuta (Reguera et al., 2004) that may be confused with other smaller-sized species of Dinophysis co-occurring in the samples, did not group together with the D. acuta group. D. ovum grouped with all the D. sacculus strains, and with D. pavillardii, a taxon considered a synonym of D. sacculus (Zingone et al., 1998). 3.4.2. Analyses of cytochrome oxydase 1 The PCR amplification of mitochondrial genes of D. ovum AM931583 and D. acuminata AM931587 yielded a DNA fragment of 1037 bp (Fig. 8). Alignment of the sequences from both species showed 25 nucleotide mismatches distributed in three sites: 7 mismatches between positions 58 and 118; 5 between 197 and

240, and 13 from 913 to 955. Thus, the last region showed to be the most variable, with 13 different bases and with two extra nucleotides on the sequence from D. acuminata. 4. Discussion and conclusions 4.1. Morphology The species of Dinophysis, D. ovum, described here, can be separated from D. acuminata on morphological bases, at the light microscope, by experts acquainted with the genus in their locality. Nevertheless, D. ovum has probably been misidentified as D. acuminata in different parts of the world where either D. ovum is the only predominant morphospecies of the ‘‘D. acuminata complex’’ or scattered cells of this species appear embedded in a population of D. acuminata. The Galician specimens observed during this event are much smaller than those described by Schu¨tt (1895) and Schiller (1933), but the lowest ranges of the L and D measurements are slightly higher than the D. ovum from New Zealand (Burns and Mitchell, 1982). Cells of Dinophysis very similar to the Galician D. ovum were labeled as D. cf acuminata, and formed dense blooms in Thermaikos Gulf (Greece) (Koukaras and Nikolaidis, 2004; Figs. 3, 5, 7 and 8); similar morphotypes are

846

N. Raho et al. / Harmful Algae 7 (2008) 839–848

Fig. 6. LC/MS chromatograms in positive ionization mode, for selected ion monitoring (SIM) of the predominant ions in the OA standard solution: [M+H4]+ and [M+Na]+ at m/z 822 and 827, with their retention time (left) and mass spectra for [M+NH4]+ and [M+Na]+ (right). (a) OA standard, (b) OA in single cell isolates of D. ovum.

Fig. 7. Phylogenetic tree based on ITS1-5,8S-ITS2 sequences, inferred by Neighbour Joining, that shows the relationship of D. ovum (in bold) from Galician coastal waters with other species of Dinophysis. The tree is rooted on Prorocentrum minimum (EF579797). The Bootstrap values (1000 replicates) are shown at the internal branches. GenBank accession numbers of the genes are shown after the organism’s name. Bar indicates substitutions per site.

N. Raho et al. / Harmful Algae 7 (2008) 839–848

847

Fig. 8. Aligment of the cox1 nucleotide (nt) positions where substitutions occurred. D. ovum is shown on the first line, with the numbers of nt positions shown on the top. Dots indicate regions where nucleotides of D. acuminata AM931587 were identical to those of D. ovum AM931583. Dash lines indicate gaps.

labeled as ‘‘D. acuminata from the South’’ by Portuguese phytoplanktologists (T. Moita, personal communication) to distinguish the D. ovum-like specimens from the Algarve region (Southern Portugal) from the larger D. acuminata from Northern Portugal. The D. ovum described here is also very similar to Dinophysis specimens. labeled as D. acuminata in the monitoring programme on the Atlantic coast of Andalucia (Southern Spain) (L. Velo-Sua´rez, personal communication). Other Dinophysis spp. from the D. acuminata complex with morphology close to that of D. ovum are D. sphaerica Stein and D. similis Kofoid and Skogsberg. These two species could be very easily confused with D. ovum if they coincided in the same sample; a closer look would reveal that they are larger than the latter and have ribs in their cingular lists. 4.2. Toxin composition LC–MS analyses of plankton concentrates where D. ovum was the dominant species and of single cell isolates of D. ovum showed that OA was the only lipophilic toxin present in this species at detectable levels with the analytical methods described for this study. D. ovum did not contain DTX2 or PTX2, toxins that together with OA are major components of D. acuta from Galician waters and the main cause of shellfish harvesting closures in the autumn (Pizarro et al., 2008). OA has been reported as the only toxin present in single cell isolates of D. acuminata (Ferna´ndez et al., 2001) and in mussels exposed to blooms of this species in Galicia and Portugal (Ferna´ndez et al., 2003; Vale et al., in press). Therefore, D. acuminata and D. ovum from Galician coastal waters only have OA acid and are undistinguishable on the basis of their toxin profile. Nevertheless, while D. acuminata is a frequent and persistent species in the Galician Rı´as, and the main cause of prolonged shellfish harvesting closures (due to okadaic acid contamination) from spring to autumn, D. ovum, up until the event described here, was a rare species in the area. Values of cell toxin quota for OA estimated from plankton concentrates (8.5 pg cell1) and from single cell isolates (7.1 pg cell1) were very close in samples from 12 June. This was probably due to the fact that the plankton concentrate was very rich in D. ovum, the dominant species in the microplankton assemblage. 4.3. Molecular analyses Previous molecular studies with Dinophysis spp. (Edvardsen et al., 2003; Marı´n et al., 2001b) have shown that the ITS rRNA genes within the species of this genus exhibit low levels of sequence variability and yield poor results for phylogenetic analyses. Results from the present work on analyses of the ITS rDNA of D. ovum and D. acuminata are in agreement with these observations.

The need to explore other gene sequences led us to try the analyses of mitochondrial genes, because they have been claimed to show a greater variability than nuclear genes (Avise, 1994; Saccone et al., 2000). Results from this work, where the sequence of mitochondrial genes have been applied for the first time on molecular studies of Dinophysis spp, are very promising. A mismatch of 25 bp between the alignments of mitochondrial genes of D. ovum and D. acuminata is, by far, the largest difference ever observed between DNA sequence alignments of smaller-sized species of Dinophysis – including D. acuminata – that usually group in the same clade when the sequence of their ITS rDNA region is compared. We can conclude that the mitochondrial genes are, so far, the most appropriate to tag different sequences of DNA if the objective is to achieve a species-specific separation of Dinophysis spp. Acknowledgements We express our gratitude to the crew of RV J.M. Navaz for their helpful attitude, to Dr. M. Elbra¨chter for help with the original description of D. ovum and to Dr. P. Hess for providing us with DTX2 secondary reference material. This work was funded by two subprojects, DINOPHYSIS Galicia and DINOGENES, of the coordinated project PHYCODISIS (CTM2004-04078-C03) and by an institutional Grant from Fundacio´n Areces to the CBMSO. N. Raho was funded by an MAE-AECI grant, G. Pizarro by a Chilean CEQUA-IFOP pre-doctoral fellowship and L. Escalera by an FPI grant from the Spanish MEC. This is a contribution to the SCOR-IOC GEOHAB Programme, Core Research Project of ‘‘HABs in Upwelling Systems’’.[SS] References Abe´, T.H., 1967. The armoured Dinoflagellata: II. Prorocentridae and Dinophysidae (B): Dinophysis and its allied genera. Publ. Seto Mar. Biol. Lab. XV, 37–78. Adachi, M., Sako, Y., Ishida, Y., 1994. Restriction fragment length polymorphism of ribosomal DNA internal transcribed spacer and 5.8S regions in Japanese Alexandrium species (Dinophyceae). J. Phycol. 30, 857–863. Avise, J.C., 1994. Molecular Markers, Natural History and Evolution. Chapman and Hall, New York, p. 511. Balech, E., 2002. Dinoflagelados tecados to´xicos del Cono Sur Americano. In: Sar, E.A., Ferrario, M.E., Reguera, B. (Eds.), Floraciones Algales Nocivas en el Cono Sur Americano. Vigo, pp. 123–144. Bhattacharya, D., Medlin, L.K., 1995. The phylogeny of plastid: a review based on comparisons of small-subunit ribosomal RNA coding regions. J. Phycol. 31, 489– 498. Bravo, I., Delgado, M., Fraga, S., Honsell, G., Montresor, M., Sampayo, M.A.M., 1995. The Dinophysis genus: toxicity and species definition in Europe. In: Lassus, P., Arzul, G., Erard-Le Denn, E., Gentien, P., Marcaillou Le Baut, C. (Eds.), Harmful Marine Algal Blooms. Lavoisier, Parı´s, pp. 843–845. Burns, D.A., Mitchell, J.S., 1982. Dinoflagellates of the genus Dinophysis Ehrenberg from New Zealand coastal waters. N. Z. J. Mar. Fresh. Ecol. 16, 289–298. Edvardsen, B., Shalchian-Tabrizi, K., Jakobsen, K.S., Medlin, L.K., Dahl, E., Brubak, S., Paasche, E., 2003. Genetic variability and molecular phylogeny of Dinophysis

848

N. Raho et al. / Harmful Algae 7 (2008) 839–848

species (Dinophyceae) from Norwegian waters inferred from single cell analyses of rDNA. J. Phycol. 39, 395–408. Felsenstein, J., 1985. Confidence limits on phylogenetic: on approach using the bootstrap. Evolution 39, 783–791. Ferna´ndez, M.L., Reguera, B., Ramilo, I., Martı´nez, A., 2001. Toxin content of Dinophysis acuminata acuta and D. caudata from the Galician Rı´as Baixas. In: Hallegraeff, G.M., Blackburn, S.I., Bolch, C.J., Lewis, R.J. (Eds.), Harmful Algal Blooms. Intergovernmental Oceanographic Commission of UNESCO, Paris, pp. 360–363. Ferna´ndez, M.L., Mı´guez, A., Cacho, E., Martı´nez, A., 2003. European approaches to marine toxin control, Towards harmonization. In: Villalba, A., Reguera, B., Romalde, J.L., Beiras, R. (Eds.), Molluscan Shellfish Safety. Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, Santiago de Compostela, pp. 159–167. Guillou, L., Ne´zan, E., Cueff, V., Erard-Le Denn, E., Cambon-Bonavita, M.A., Gentien, P., Barbier, G., 2002. Genetic diversity and molecular detection of three toxic dinoflagellate genera (Alexandrium, Dinophysis, and Karenia) from French coasts. Protist 153, 223–238. Hart, M.C., Green, D.H., Bresnan, E., Bolch, C.J., 2007. Large subunit ribosomal RNA gene variation and sequency heterogeneity of Dinophysis (Dinophyceae) species from Scottish coastal waters. Harmful Algae 6, 271–287. Kai, K.L., Cheung, Y.K., Yeung, P.K.K., Wong, J.T.Y., 2006. Development of single-cell PCR methods for the Raphidophyceae. Harmful Algae 5, 649–657. Kimura, M., 1981. Estimation of evolutionary distances between homologous nucleotide sequences. Proc. Natl. Acad. Sci. U.S.A. 78, 454–458. Koukaras, K., Nikolaidis, G., 2004. Dinophysis blooms in Greek coastal waters (Thermaikos Gulf NW Aegean Sea). J. Plankton Res. 26, 445–457. Larsen, J., Moestrup, Ø., 1992. Potentially toxic phytoplankton. 2. Genus Dinophysis (Dinophyceae). In: Lindley, J.A. (Ed.), ICES Identification Leaflets for Plankton, vol. 180. pp. 1–12. Lassus, P., Bardouil, M., 1991. Le Complexe Dinophysis acuminata: identification des espe`ces le long des coˆtes Franc¸aises. Cryptogamie. Algol 12, 1–9. Lee, J.S., Igarashi, T., Fraga, S., Dahl, E., Hovgaard, P., Yasumoto, T., 1989. Determination of diarrhetic shellfish toxins in various dinoflagellate species. J. Appl. Phycol. 1, 147–152. Lin, S., Zhang, H., Spencer, D.F., Norman, J.E., Gray, M.W., 2002. Widespread and extensive editing of mitochondrial mRNAS in Dinoflagellates. J. Mol. Biol. 320, 727–739. Lovegrove, T., 1960. An improved form of sedimentation apparatus for use with an inverted microscope. J. Cons. CIEM 25, 279–284. Marı´n, I., Aguilera, A., Gonza´lez-Gil, S., Reguera, B., Abad, J.P., 2001a. Genetic analysis of several species of Dinophysis causing diarrhetic shellfish outbreaks in Galicia (NW Spain). In: Hallegraeff, G.M., Blackburn, S.L., Bolch, C.J., Lewis, R.J. (Eds.), Harmful Algal Blooms 2000. Intergovernmental Oceanographic Commission of UNESCO, Tasmania, pp. 222–225. Marı´n, I., Aguilera, A., Reguera, B., Abad, J.P., 2001b. Preparation of DNA suitable for PCR amplification from fresh or fixed single dinoflagellate cells. Biotechniques 30, 88–93. Moestrup, Ø., 2004. Taxonomic reference list of toxic algae. Intergovernmental Oceanographic Commission of UNESCO, http://ioc.unesco.org/hab/data.htm. Pizarro, G., Escalera, L., Gonza´lez-Gil, S., Franco, J.M., Reguera, B., 2008. Growth, behaviour and cell toxin quota of Dinophysis acuta Ehrenberg during a daily cycle. Mar. Ecol. Prog. Ser. 353, 89–105. Puel, O., Galgani, F., Dalet, C., Lassus, P., 1998. Partial sequence of the 24S rRNA and PCR assay of the toxic dinoflagellate Dinophysis acuminata. Can. J. Fish. Aquat. Sci. 55, 597–604. Rathore, D., Wahl, A.M., Sullivan, M., McCutchan, T.F., 2001. A phylogenetic comparison of gene trees constructed from plastid mitochondrial and genomic DNA of Plasmodium species. Mol. Biochem. Parasitol. 114, 89–94. Reguera, B. 2003. Biologı´a, autoecologı´a y toxinologı´a de las principales especies del ge´nero Dinophysis associadas con envenenamiento diarreoge´nico por bivalvos

(DSP). Ph D Thesis, University of Barcelona, http://www.tesisenxarxa.net/TESIS_UB/AVAILABLE/TDX-0628104-112836//TESISREGUERA.pdf. Reguera, B., Gonza´lez-Gil, S., 2001. Small cell and interminate cell formation in species of Dinophysis (Dinophyceae Dinophysiales). J. Phycol. 37, 318–333. Reguera, B., Gonza´lez-Gil, S., Delgado, M., 2004. Formation of Dinophysis dens Pavillard and D. diegensis Kofoid from laboratory incubations of Dinophysis acuta Ehrenberg and D. caudata Saville-Kent. In: Steidinger, K.A., Landsberg, J.H., Tomas, C.R., Vargo, G.A. (Eds.), Harmful Algae 2002. Florida Fish and Wildlife Conservation Commission, and Intergovernmental Oceanographic Commission of UNESCO, St. Petersburg, FL, USA, pp. 440–442. Reguera, B., Pizarro, G., 2007. Planktonic dinoflagellates which produce polyether toxins of the old ‘‘DSP complex’’. In: Botana, L.M. (Ed.), Seafood and Freshwater Toxins: Pharmacology, Physiology, and Detection. second edn. Taylor & Francis, London, pp. 257–284. Rehnstam-Holm, A.S., Godhe, A., Anderson, D.M., 2002. Molecular studies of Dinophysis (Dinophyceae) species from Sweden and North America. Phycologia 41, 348–357. Saccone, C., Gissi, C., Lanave, C., Larizza, A., Pesole, G., Reyes, A., 2000. Evolution of the mitocohondrial genetic system: an overview. Gene 261, 153–159. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Saldarriaga, J.F., Taylor, F.J.R., Keeling, P.J., Cavalier-Smith, T., 2001. Dinoflagellate nuclear SSU rRNA phylogeny suggests multiple plastid losses and replacements. J. Mol. Evol. 53, 204–213. Saunders, G.W., Hill, D.R.A., Sexton, J.P., Andersen, R.A., 1997. Small-subunit ribosomal RNA sequences from selected dinoflagellates: testing classical evolutionary hypotheses with molecular systematic methods. Plant. Syst. Evol. 11, 237–259. Schiller, J., 1933. Dinoflagellatae. In: Rabenhorst, L. (Ed.), Kryptogamen-Flora, vol. 10, no. 3. I Teil. Akademische Verlags-gesellschaft, Leipzig, p. 617. Schu¨tt, F., 1895. Die Peridineen der Planktonexpedition I. Theil. Studien u¨ber die Zellen der Peridineen. Ergebnisse der Plankton Expedition der HumboldtStiftung. IV. M. a. A. Lipsius and Tischler, Kiel, Leipzig, pp. 1–170. Serizawa, K., Suzuki, H., Tsuchiya, K., 2000. A phylogenetic view on species radiation in Apedemus inferred from variation of nuclear and mitochondrial genes. Biochem. Genet. 38, 27–40. Swofford, D.L., 1998. PAUP* Phylogenetic Analysis using Parsimony (* and other methods). Version 4. Sinauer Associates Inc., Sunderland, Massachussets. Takabayashi, M., Santos, S.R., Cook, C.B., 2004. Mitochondrial DNA phylogeny of the symbiotic dinoflagellates (Symbiodinium Dinophyta). J. Phycol. 40, 160–164. Taylor, M.S., Hellberg, M.E., 2003. Genetic evidence for local retention of pelagic larvae in a Caribbean reef fish. Science 299, 107–108. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 24, 4876–4882. Vale, P., Botelho, M.J., Rodrigues, S.M., Gomes, S.S., Sampayo, M.A.M., in press. Two decades of marine biotoxin monitoring in bivalves from Portugal (1986–2006): a review of exposure assessment. Harmful Algae 7, 11–25. van Egmond, H.P., Aune, T., Lassus, P., Speijers, G.J.A., Waldock, M., 1993. Paralytic and diarrhoeic shellfish poisons: occurrence in Europe, toxicity, analysis and regulation. J. Nat. Toxins 2, 41–82. van Trijp, J.M.P., Roos, A.H., 1991. Model for the calculation of calibration curves. RIKILT Report 91, 02. Zhang, H., Bhattacharya, D., Lin, S., 2005. Phylogeny of dinoflagellates based on mitochondrial cytochrome b and nuclear small subunit rDNA sequence comparisons. J. Phycol. 41, 411–420. Zingone, A., Montresor, M., Marino, D., 1998. Morphological variability of the potentially toxic dinoflagellate Dinophysis sacculus (Dinophyceae) and its taxonomic relationship with D. pavillardii and D. acuminata. Eur. J. Phycol. 33, 259–273.