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Investigation of toxin profile of Mediterranean and Atlantic strains ofOstreopsis cf. siamensis (Dinophyceae) by liquid chromatography–highresolution mass spectrometry

Patrizia Ciminiello a, Carmela Dell’Aversano a,*, Emma Dello Iacovo a, Ernesto Fattorusso a,Martino Forino a, Luciana Tartaglione a, Takeshi Yasumoto b, Cecilia Battocchi c, Mariagrazia Giacobbe d,Ana Amorim e, Antonella Penna c

a Dipartimento di Chimica delle Sostanze Naturali, Universita degli Studi di Napoli ‘‘Federico II’’, Via D. Montesano 49, 80131 Napoli, Italyb Japan Food Research Laboratories, Tama Laboratory, 6-11-10 Nagayama, Tama-shi, Tokyo 206-0025 Japanc Dipartimento di Scienze Biomolecolari, Sez. Biologia Ambientale, Universita di Urbino, Viale Trieste 296, 61121 Pesaro, Italyd Istituto per l’Ambiente Marino Costiero, Consiglio Nazionale delle Ricerche, Via San Raineri 86, 98122 Messina, Italye Centro de Oceanografia and Departamento de Biologia Vegetal, Faculdade Ciencias da Universidade de Lisboa, 1749-016 Lisboa, Portugal

1. Introduction

The benthic dinoflagellate Ostreopsis spp. is worldwide distrib-uted at circumtropical and temperate areas (Fukuyo, 1981; Faust,1999; Vila et al., 2001; Rhodes et al., 2010; Parsons et al., 2012). Inthe temperate region of the Mediterranean Sea, Ostreopsis spp.occur since the late ‘70s (Taylor, 1979; Tognetto et al., 1995), but inthe last decade O. confronta (cf.) ovata has become increasinglyfrequent with massive blooms (Mangialajo et al., 2011; Honsellet al., 2011; Pfannkuchen et al., 2012; Accoroni et al., 2011) whichcaused relevant negative impacts on benthic communities (Simoniet al., 2003; Totti et al., 2010) and on human health through skincontact (Tichadou et al., 2010), toxic aerosols (Durando et al., 2007)

and contaminated seafood (Aligizaki et al., 2008; ARPAC, 2008;Amzil et al., 2012).

Mediterranean Ostreopsis spp. isolates have been characterizedby morphological and molecular analyses (Penna et al., 2005;Battocchi et al., 2010; Aligizaki and Nikolaidis, 2006). However, todate, resolving the taxonomy of Ostreopsis spp. based only onmorphology has been difficult due to the morphological variabilityof both field material and cultured specimens. Further, none of theoriginal isolates from tropical areas from which Ostreopsis specieswere described has yet been sequenced for the genotypeassignment. At present, in the absence of molecular informationon the type material of Ostreopsis spp. the taxonomic status of thespecies remains uncertain and species-specific designationsapplied to strains of different genetic lineages have to be treatedwith caution. The use of designation O. cf. ovata or O. cf. siamensis

when referring to isolates from the Mediterranean Sea is advisable.Genus Ostreopsis was first described with the holotype Ostreopsis

Harmful Algae 23 (2013) 19–27

A R T I C L E I N F O

Article history:

Received 1 August 2012

Received in revised form 18 December 2012

Accepted 18 December 2012

Available online 22 January 2013

Dedicated to the memory of Prof. Ernesto

Fattorusso.

Keywords:

ITS-5.8S ribosomal genes

Mediterranean Sea

Ostreocin-b

Ostreocin-d

Ostreopsis cf. siamensis

Palytoxin

A B S T R A C T

Blooms of Ostreopsis spp. once confined to tropical and subtropical areas have recently spread to more

temperate regions such as the Mediterranean and the Southern-Atlantic coasts of Europe. However,

while O. confronta (cf.) ovata has caused several toxic outbreaks, the presence of O. cf. siamensis has been

reported rather occasionally and in very few regions; as a consequence, O. cf. ovata toxin profile has been

in-depth studied while poor information exists on toxicity of the Mediterranean and Atlantic O. cf.

siamensis. In the present study toxin profile of Mediterranean and Atlantic O. cf. siamensis isolates also

phylogenetically related has been studied through liquid chromatography–high resolution mass

spectrometry (LC–HRMS) versus a palytoxin standard, a crude extract of O. cf. ovata containing all the

ovatoxins so far known (ovatoxin-a to -f), and a Japanese O. siamensis extract which contained ostreocin-

d and ostreocin-b. The Mediterranean and Atlantic O. cf. siamensis strains were shown not to produce

either ostreocins, which are produced by the Japanese O. siamensis strain, or ovatoxins, which are

produced by the Mediterranean O. cf. ovata. Only sub-fg levels of palytoxin on a per cell basis were

detected in the Mediterranean strain. This study demonstrates that the Mediterranean and the Atlantic

O. cf. siamensis strains are devoid of any appreciable toxicity. Thus, at least in the European area, O. cf.

siamensis seems to present a much lower risk to human health than O. cf. ovata.

� 2013 Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: +39 081 678502; fax: +39 081 678552.

E-mail address: dellaver@unina.it (C. Dell’Aversano).

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siamensis from the Gulf of Siam (Thailand) by Schmidt (1901). Thetype species O. siamensis shows a worldwide distribution includingPacific areas (Asia, Australia, New Zealand and Hawaii), CaribbeanSea and Indian Ocean (Madagascar and Reunion Islands) (Rhodes,2011). But recently, morphological and phylogenetic or molecularanalyses of isolates identified the presence of O. cf. siamensis also inEuropean seawaters of the Mediterranean Sea and eastern Atlanticcoasts (Penna et al., 2010, 2012; Amorim et al., 2010; Laza-Martinez et al., 2011) confirming the previous morphological and/or phylogenetic finding of O. cf. siamensis in the Mediterranean Sea(Vila et al., 2001; Penna et al., 2005).

Ostreopsis spp. present some concern to human health due totheir ability to produce congeners of palytoxin (Fig. 1), one of themost potent marine toxins so far known. In the middle ‘90s, apalytoxin-like compound, named ostreocin-d, was isolated from aJapanese strain of O. siamensis (Usami et al., 1995). A few yearslater, its structure was fully elucidated as 42-hydroxy-3,26-didemethyl-19,44-dideoxypalytoxin (Fig. 1) basing on extensiveNMR evidence and confirmed by negative ion fast atombombardment (FAB) collision induced dissociation (CID) MS(Ukena et al., 2001, 2002). Some other palytoxin-like moleculeswere also detected in a Japanese O. siamensis culture but they werepresent in the algal extract in amounts too low for a completeNMR-based structure elucidation. Tentative structure of ostreocin-b was reported by Ukena (2001) (Fig. 1). Compared to ostreocin-d,ostreocin-b presents an additional hydroxyl at C-44, thus being the42-hydroxy-3,26-didemethyl-19-deoxypalytoxin.

Recently, liquid chromatography–high resolution mass spec-trometry (LC–HRMS) studies revealed also O. cf. ovata as aproducer of palytoxin-like compounds: putative palytoxin andovatoxins were identified in both field and cultured Mediterranean

strains of O. cf. ovata (Ciminiello et al., 2006, 2008, 2010, 2012c).Ovatoxin-a, the major component of O. cf. ovata toxin profile, wasrecently isolated and structurally elucidated as 42-hydroxy-17,44,64-trideoxypalytoxin (Fig. 1) basing on NMR and HRMSn

evidence (Ciminiello et al., 2012a,b). Ovatoxin-b, -c, -d, -e, and -fhave not been isolated yet and only their LC–HRMS and MSn datahave been reported so far: they present elemental compositionsimilar to that of palytoxin, differing for just a few carbon,hydrogen and/or oxygen atoms. Table 1 reports elementalformulae (M) of palytoxin, ovatoxins and ostreocins together withelemental composition of their relevant A and B moieties due tofragmentation between C-8 and C-9 of the molecules (Fig. 1) whichrepresent a key spectral feature of all palytoxin-like molecules(Ciminiello et al., 2011a).

In the Mediterranean area, O. cf. ovata has caused several toxicoutbreaks as recently reviewed by Mangialajo et al. (2011) while

Fig. 1. Structures of palytoxin, ovatoxin-a, ostreocin-d, and ostreocin-b.

Table 1Elemental formulae of palytoxin, ovatoxins and ostreocins so far known and

elemental composition of A- and B- moieties of each compound resulting from the

favored fragmentation between C-8 and C-9 of the molecule.

Toxin M A moiety B moiety

Palytoxin C129H223N3O54 C16H28N2O6 C113H195NO48

Ovatoxin-a C129H223N3O52 C16H28N2O6 C113H195NO46

Ovatoxin-b C131H227N3O53 C18H32N2O7 C113H195NO46

Ovatoxin-c C131H227N3O54 C18H32N2O7 C113H195NO47

Ovatoxin-d C129H223N3O53 C16H28N2O6 C113H195NO47

Ovatoxin-e C129H223N3O53 C16H28N2O7 C113H195NO46

Ovatoxin-f C131H227N3O52 C16H28N2O6 C115H199NO46

Ostreocin-d C127H219N3O53 C15H26N2O6 C112H193NO47

Ostreocin-b C127H219N3O54 C15H26N2O6 C112H193NO48

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the presence of O. cf. siamensis has been reported ratheroccasionally and in very few regions (Vila M. and Penna A. pers.comm.). As a consequence, Mediterranean O. cf. ovata toxin profilehas been in-depth studied and the presence of ovatoxins iscarefully monitored in both plankton and seafood while poorinformation exists on toxicity of the Mediterranean O. cf. siamensis.

In 2005, Penna and coworkers reported on characterization ofMediterranean Ostreopsis isolates based on morphology, phyloge-ny and toxicity. A delayed hemolytic activity neutralized byouabain was observed in various O. cf. siamensis strains from sitesof the Mediterranean Sea. Since the inhibition of hemolysis byouabain, a palytoxin antagonist, together with the delayed effect, isa characteristic of palytoxin (Habermann and Chatwal, 1982), theobtained results suggested the presence of palytoxin-like com-pounds in the analyzed O. cf. siamensis strains; however, nochemical analysis of palytoxin-like compounds was performed onthese strains.

More recently, Cagide et al. (2009) reported on the functionalactivity of Mediterranean O. cf. siamensis strains collected along thecoasts of Spain. The effects of the O. cf. siamensis strains on viability,membrane potential and intracellular calcium of neuroblastomacells were investigated and they resulted to be quite similar tothose induced by palytoxin standard and by an O. cf. ovata extract;thus, the analyzed O. cf. siamensis strains were supposed toproduce functionally active and highly toxic palytoxin-likecompounds, although neither palytoxin nor ovatoxin-a weredetected by LC–MS analyses.

In a recent study on qualitative determination of variousphycotoxins in dinoflagellate whole cells basing on matrix-assistedlaser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS), Paz et al. (2011) did not detect any ostreocinin a Spanish O. cf. siamensis cultured strain.

So, currently, poor chemical information exists on the toxinprofile of the Atlantic/Mediterranean O. cf. siamensis and itsrelation with the Japanese O. siamensis which indeed has beenthoroughly studied. The aim of the present study was to fill the gapby defining chemical composition of Mediterranean and EasternAtlantic O. cf. siamensis isolates also phylogenetically related. LC–HRMS experiments were carried out on one Italian and fourPortuguese O. cf. siamensis algal extracts and the obtained resultswere analyzed in comparison both to a palytoxin standard and acrude extract of O. cf. ovata containing all the ovatoxins so farknown (Ciminiello et al., 2012c) as well as to a sample obtainedfrom a Japanese O. siamensis strain which contained high levels ofostreocin-d together with small amounts of ostreocin-b. Theobtained results indicated that all the analyzed strains did notproduce ostreocins nor ovatoxins. Trace levels of palytoxin weredetected in the only Mediterranean strain. In addition, no toxicitywas detected in all the analyzed strains by mouse bioassay.

2. Materials and methods

2.1. Mediterranean and Atlantic Ostreopsis spp. strain isolation,

morphological and molecular identification

Mediterranean Ostreopsis spp. isolates were collected fromseawater samples at Taormina, Italy (Lat. 3785100.4300N, Long.1581802.8100E) where Ostreopsis spp. toxic blooms recur everysummer. This site is a semiclosed, shallow bay carpeted byseaweeds. Ostreopsis assemblages can be found both in watercolumn and macroalgae, as planktonic and epiphytic microflora.Atlantic Ostreopsis spp. isolates were obtained from seawatersamples and epiphytic seaweeds mats collected in two recreationalmarinas located on the West coast of Portugal, Sines (Lat.378570200N, Long. 885105300W) and Cascais (Lat. 3884103600N, Long.982405300W).

Ostreopsis strains were isolated as described by Penna et al.(2005). The Mediterranean O. cf. siamensis strain (CNR-T5) wasisolated in 2005 and kept in culture at temperature of 21 � 1 8C in f/2 medium, irradiance 100 mE m�2 s�1 with light:dark (L:D) cycle of14:10 h. The Atlantic O. cf. siamensis strains were isolated at Sines inJune and October 2008 and September 2009 (IO96-01, IO96-02 andIO96-03, respectively) and at Cascais in September 2010 (IO96-04);they were kept in culture at 19 � 1 8C in f/20 medium with irradianceof 40 mmol m�2 s�1 and a L:D cycle of 14:10.

In order to clarify the species-specific Ostreopsis taxonomicidentification, cell morphology and plate tabulation were studiedby light microscopy either under phase contrast, or staining withthe Fluorescent Brightener 28, according to Fritz and Triemer(1985). Alternatively, cultured specimens were analyzed byscanning electron microscopy following the protocol developedby Penna et al. (2005). Cell measures of a number of O. cf. siamensis

specimens were taken. The dorso-ventral length, the width,defined as the widest transdiameter, and the ratio between thetwo were used to describe cell size and morphology. Cell sizes ofthe Mediterranean CNR T5 clone were compared with those ofclones previously isolated from a Tyrrhenian site (Penna et al.,2005). The nomenclature used for the tabulation system followedthe modified Kofoid system proposed by Besada et al. (1982),following the recommendations by Fraga et al. (2011).

2.2. Taxonomic molecular identification by PCR assay and sequence

analysis

Genomic DNA was extracted and purified from 50 mL culture ofO. cf. siamensis IO 96-01, IO 96-02 and IO 96-03 in logarithmicgrowth phase using DNeasy Plant Kit (Qiagen, CA, USA) accordingto the manufacturer’s instructions. PCR amplifications using genusand species-specific primers was performed as described in Pennaet al. (2007) and Battocchi et al. (2010). Further, the strain IO 96-01was analyzed by nucleotide sequencing of ITS-5.8S rDNA. The PCRamplification and sequencing of ITS-5.8S rDNA protocols weredescribed in Penna et al. (2010). The sequence (JX065587) of ITS-5.8S rDNA was deposited in EMBL.

2.3. Ostreopsis cf. siamensis cultures

Cultures were grown in 1 L glass flasks containing 500 mLsterilized f/4 medium, a derivative of f/2 Guillard (1975) growthmedium, with an initial cell concentration of 1.0 � 103. Thetemperature was set at 23 � 1 8C. Light was provided by cool whitefluorescent bulbs (photon flux of 100 mE m�2 s�1) on a standard14:10 h L:D cycle. Culture samples were fixed with Lugol’s iodine andcounted using Utermhol method. A total volume of 6 L culture washarvested on 14th day of growth by centrifugation at 4000 rpm for15 min at room temperature or, alternatively, by filtration onto TSTPfilter type with 3 mm size pores (Millipore, MA, USA) under gentlevacuum. Then, filters were carefully washed with sterile artificialseawater and discharged; cells were collected by centrifugation at4000 rpm for 15 min at room temperature. Pellets containing from1.0 � 106 to 1.0 � 107 cells together with collected growth mediumwere stored at �80 8C and �20 8C, respectively, until chemicalanalyses.

2.4. Extraction

Cell pellet and growth medium of Mediterranean and AtlanticO. cf. siamensis cultures were separately extracted. Cell pellet wasextracted by adding 5 mL of a methanol/water (1:1, v/v) with 0.2%acetic acid solution and sonicating for 3 min in pulse mode, whilecooling in ice bath. The mixture was centrifuged at 5000 rpm for10 min, the supernatant was decanted and the pellet was washed

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twice with 5 mL of the above extraction solution. The extracts werecombined (volume = 15 mL) and divided into two 7.5 mL aliquots:one was used for mouse bioassay, the other was analyzed directlyby LC–HRMS (5 mL injected). Successively, the latter aliquot wasevaporated to dryness dissolved in 175 mL of methanol/water (1:1,v/v) with 0.2% acetic acid and re-analyzed by LC–HRMS (5 mLinjected). Recovery of the extraction procedure was 98% (Cimi-niello et al., 2008).

The growth medium was extracted five times with an equalvolume of butanol. The butanol layer was evaporated to dryness,dissolved in 35 mL of methanol/water (1:1, v/v) and analyzed byLC–HRMS (5 mL injected). Recovery of the extraction procedurewas 75% (Ciminiello et al., 2008).

2.5. Mouse bio-assay

Four dose levels corresponding to 1/100, 1/50, 1/10 and 1/2 ofthe Mediterranean and the Atlantic O. cf. siamensis pellet extractswere prepared. For each dose level, three replicates wereevaporated to dryness, dissolved each in 1 mL of 1% Tween 60solution, and intra-peritoneally injected into three CD1 male mice(18–20 g). Observation time was 24 h.

2.6. Liquid chromatography–high resolution mass spectrometry (LC–

HRMS)

LC–HRMS experiments were carried out on an Agilent 1100 LCbinary system (Palo Alto, CA, USA) coupled to a hybrid linear iontrap LTQ Orbitrap XLTM Fourier Transform MS (FTMS) equippedwith an ESI ION MAXTM source (Thermo-Fisher, San Jose, CA, USA).Chromatographic separation was accomplished by using a 3 mmgemini C18 (150 mm � 2.00 mm) column (Phenomenex, Torrance,CA, USA) maintained at room temperature and eluted at0.2 mL min�1 with water (eluent A) and 95% acetonitrile/water(eluent B), both containing 30 mM acetic acid. A slow gradientelution was used: 20–50% B over 20 min, 50–80% B over 10 min,80–100% B in 1 min, and hold 5 min.

HR full MS experiments (positive ions) were carried out in themass ranges m/z 800–1400 and m/z 2000–3000 at a resolving powerof 100,000. Actual resolution of the spectrum varies over the wholemass range starting from 80,000 at m/z 800 to 30,000 at m/z 3000.Calibration was performed just before the analyses by using amixture of caffeine, MRFA (L-methionyl-arginyl-phenylalanyl-ala-nine acetate � H2O) and Ultramark 1621 for the mass range m/z800–1400 and PPG 2007 for the mass range m/z 2000–3000. Thefollowing source settings were used: a spray voltage of 4 kV, acapillary temperature of 290 8C, a capillary voltage of 22 V, a sheathgas and an auxiliary gas flow of 35 and 1 (arbitrary units). The tubelens voltage was set at 110 V and 250 V in the experiments acquiredin mass range m/z 800–1400 and m/z 2000–3000 respectively.

HRMS2 data were acquired in collision induced dissociation(CID) mode at a 60,000 resolving power by selecting as precursorthe [M+H+Ca]3+ ion of each compound (Ciminiello et al., 2012a,c).A relative collision energy of 25%, an activation Q of 0.250, and anactivation time of 30 ms were used. Q refers to the q coordinate ofthe Mathieu stability diagram depicting ion trajectories in aquadrupole ion trap MS analyzer (March, 1997). Activation Q refersto the trapping field used when colliding ions.

Calculation of elemental formulae of ions contained in HRMSspectra was performed by using the mono-isotopic ion peak ofeach ion cluster. A mass tolerance of 5 ppm was used and theisotopic pattern of each ion cluster was considered in assigningmolecular formulae. Palytoxin standard (Wako Chemicals GmbH,Neuss, Germany) at five levels of concentration (25, 12.5, 6.25,3.13, and 1.6 ng mL�1) was used to generate a calibration curve andto measure limits of detection (LOD) and quantitation (LOQ) of the

method. Calibration points were the result of triplicate injection.Extracted ion chromatograms (XIC) for palytoxin, ovatoxins andostreocins were obtained by selecting the most abundant ion peaksof both [M+2H�H2O]2+ and [M+H+Ca]3+ ion clusters. A masstolerance of 5 ppm was used. Limit of detection (LOD) and ofquantitation (LOQ) for palytoxin standard in solvent under theused instrumental conditions were measured and resulted to be1.6 ng mL�1 and 3.13 ng mL�1.

3. Results and discussion

3.1. Morphological analyses

Ostreopsis cf. siamensis from Taormina (Isolabella, Sicily), as wellas cells from cultured material (CNR T5), appeared rather ovoid inshape, apical-antapically compressed, and pointed to the ventralside when observed in microscopy (Fig. 2a). The thecal plates werevery thin and cells contained golden-brown chloroplasts. Cellmeasures (field specimens and CNR T5, n = 20) were in the range35–55 mm in width, 50–70 mm in dorso-ventral length, and gave a1.35 DV/W mean ratio. Most of the Mediterranean O. cf. siamensis

cell sizes were within the measure range already reported for otherisolates from the Mediterranean, including Tyrrhenian specimens(CNR B5 clone, n = 70; Penna et al., 2005). Cell size sometimesoverlapped with cell measures of other species, such as O. ovata –the smallest species of the Ostreopsis genus – from the Mediterra-nean Sea, all the more because ‘‘small cells’’, possible gametes,were produced in culture.

The specific identification of Ostreopsis based only on morpho-metric criteria was, thus, quite uncertain, despite the carefulanalyses of thecal plates both under fluorescence and with thesupport of scanning electron microscopy (Fig. 2b, c and g).

No evident differences were found between the morphology ofthe Mediterranean and Atlantic strains. Cells of O. cf. siamensis

isolated from the Atlantic coast were markedly apical-antapicallycompressed with golden-brown chloroplasts occupying most ofthe cell, except for the ventral tip. In apical and antapical view cellswere tear-shaped (Fig. 2e). Live cells showed a characteristicspinning motion around the dorsal–ventral axis while attached bythe ventral side to particles and other surfaces. The dorsal–ventrallength of the wild cells (n = 15) ranged from 50 to 62 mm(mean = 58.49�4.11 mm) and the width from 41 to 50 mm(46.53 � 2.15 mm). The ratio DV/W ranged between 1.10 and 1.32,with a mean value of 1.26. In three week old cultures (IO96-01, 02, 03and 04) cell size showed a wider range (n = 83). The dorsal–ventrallength ranged from 35 to 73 mm (mean = 47.6 � 7.2 mm) and widthfrom 28 to 58 mm (mean = 38.1 � 6.7 mm) encompassing the rangefound for the Mediterranean strains and probably also reflecting thedifferentiation of different life-cycle stages in culture. The DV/W ratioranged from 1 to 1.46 (mean = 1.26 � 0.09).

The surface of thecal plates was smooth, covered with scatteredtrichocyst pores of only one type (Fig. 2f–h) as observed for theMediterranean strains (Fig. 2b). The margins of plates along thecingular groove had a row of aligned pores (Fig. 2g). In ventral view aventral opening could be recognized at the cingulum level (Fig. 2g).Given its position, the function of this opening may be related to cellattachment. A similar opening was described for O. labens by Faustand Morton (1995) who proposed it could be related to a feedingapparatus. As described for other species of Ostreopsis, O. cf. siamensis

from both the Atlantic and Mediterranean areas was observed toproduce an abundant filamentous network in culture.

3.2. Molecular analyses

The Mediterranean O. cf. siamensis CNR-T5 had been alreadycharacterized by phylogenetic analyses based on the alignment of

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the 5.8S gene and ITS nucleotide sequence, and then it wasincluded into O. cf. siamensis Mediterranean clade as reported byPenna et al. (2010).

PCR amplification based assay of the Atlantic Ostreopsis

spp. IO96-01, IO96-02 and IO96-03 strains using genus- and

species-specific primers confirmed the O. cf. siamensis genotypeidentification.

Further, the sequence alignment analysis of ribosomal ITS-5.8Sgene of Ostreopsis sp. IO96-03 in BLAST silico platform gave the100% identity with other O. cf. siamensis ribosomal sequences.

Fig. 2. Ostreopsis cf. siamensis: (a–d) Mediterranean specimens from culture CNR-T5 (Isolabella, Taormina), (e–h) Atlantic specimens from culture (strains IO96-01 and 04); (a)

Epithecal view in phase contrast with visible 40-Po plates; (b) scanning electron micrograph (SEM) of hypotheca with 2000 0 plate and scattered thecal pores; (c) SEM of apical

detail showing Po-20 plates; (d) cell in hypothecal view as seen in fluorescence light microscopy; (e) live cell in apical/antapical view in bright field; (f) epithecal view in phase

contrast; (g) ventral view of hypothecal surface in SEM evidencing the ventral opening (arrow); (h) hypothecal view in phase contrast. Scale bars: 20 mm, except (c, g) 5 mm.

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3.3. Chemical analyses

3.3.1. LC–HRMS of ostreocin-d and -b

In order to study toxin profiles of Mediterranean/Atlantic O. cf.siamensis isolates, a preliminary investigation of LC–HRMSbehavior of ostreocins under the same conditions developed forthe analysis of palytoxin and ovatoxins (Ciminiello et al., 2008,2010, 2011a, 2012a,c) was performed. So, a reference sample of aJapanese O. siamensis strain containing ostreocin-d, as majorcomponent, together with small amounts of ostreocin-b wasanalyzed by LC–HRMS in positive full MS mode. The obtained massspectra were interpreted in the light of those of palytoxin andovatoxin-a (Ciminiello et al., 2011a, 2012a).

Under the used LC conditions, ostreocin-d eluted at 10.24 min(0.54 and 1.21 min before palytoxin and ovatoxin-a, respectively)and ostreocin-b at 9.86 min. HR full MS spectra were acquiredin the mass ranges in which palytoxin-like compounds are knownto form several characteristic ions (Ciminiello et al., 2011a),namely:

- m/z 2500–3000, where the singly charged [M+H]+ ions appeartogether with ions due to subsequent water losses;

- m/z 800–1400, where the doubly-charged ions [M+H+K]2+,[M+H+Na]2+, and [M+2H�nH2O]2+ (n = 0–4) occur togetherwith the triply-charged ions [M+H+Ca]3+ and [M+H+Mg]3+ dueto adduct formation with divalent cations (Ciminiello et al.,2012a).

Relative ratios of all the above ions vary strongly with theionization conditions used: source temperature strongly influ-ences water molecule losses from singly, doubly, and triplycharged ions, while tube lens offset basically affects formation ofmultiply charged ions (110 V) versus singly charged ions (250 V).

Fig. 3 shows HR full MS spectra of ostreocin-d and -b containedin the Japanese O. siamensis reference sample in the mass ranges m/z 800–1400 and m/z 2500–3000. The relative abundance of ions foreach ostreocin was similar to that observed in HR full MS spectra ofpalytoxin standard. Assignment of the most intense ions ofostreocins is reported in Table 2 in comparison to palytoxin.

The fragmentation between C-8 and C-9, which is typical of allpalytoxin-like compounds so far known, as recently reviewed byCiminiello et al. (2011a,b), divides the molecules in the two

moieties A and B (Fig. 1); the following ions diagnostic of bothmoieties appear in LC–HRMS2 spectra:

- [A moiety+H�H2O]+ ion. This ion occurs at m/z 327.1908 forpalytoxin and at m/z 313.1754 for ostreocin-d and -b, respec-tively;

- [B moiety+Ca�nH2O]2+ (n = 0–5). [B moiety+Ca]2+ ion (mono-isotopic ion peak) occurs at m/z 1187.1210 in palytoxin, m/z1172.1188 in ostreocin-d, and m/z 1180.1155 in ostreocin-b.

Fig. 4 shows HRMS2 spectra of ostreocin-d and -b contained inthe Japanese O. siamensis reference sample in the mass regionswhere ions relevant to A- and B- moieties occur; a number of ionswere contained in other regions of the spectra whose assignment isnot reported herein as it was beyond the aim of this work.

3.3.2. LC–HRMS and toxicity of Mediterranean and Atlantic O. cf.

siamensisCrude extracts of O. cf. siamensis cultures were analyzed by LC–

HRMS to define toxin profile and by mouse bioassay to ascertaintotal toxicity.

HR full MS spectra of the extracts were acquired and analyzed inparallel to palytoxin standard at five levels of concentration, aMediterranean O. cf. ovata extract containing ovatoxin-a to -f(Ciminiello et al., 2012c) and the Japanese O. siamensis referencesample described above. Total ion chromatograms of the Atlanticand Mediterranean O. cf. siamensis extracts did not contain anypeak clearly indicating the presence of ovatoxins and ostreocins

m/z1280 129 0 130 0 131 0 132 0 133 0 134 0 135 0 2560 258 0 260 0 262 0 264 0 266 0870 88 0 89 0 90 0 91 0 92 0

Ostreo cin-d

Ostreo cin-b

m/z1280 129 0 130 0 131 0 132 0 133 0 134 0 135 0870 88 0 89 0 90 0 91 0 92 0 2560 258 0 260 0 262 0 264 0 266 0

Fig. 3. Expansion of HR full MS spectra of ostreocin-d and -b acquired in the mass range m/z 800–1400 and m/z 2500–3000.

Table 2Principal triply, doubly and singly charged ions (m/z of monoisotopic ion peaks) of

palytoxin, ostreocin-d and -b contained in their LC–HRMS spectra.

Toxin Palytoxin Ostreocin-d Ostreocin-b

[M+H]+ 2679.4900 2635.4707 2651.4453

[M+H+K]2+ 1359.2228 1337.2085 1345.2067

[M+H+Na]2+ 1351.2399 1329.2254 1337.2235

[M+2H]2+ 1340.2494 1318.2350 1326.2325

[M+2H�H2O]2+ 1331.2443 1309.2299 1317.2275

[M+2H�2H2O]2+ 1322.2389 1300.2246 1308.2226

[M+H+Ca]3+ 906.4844 891.8084 897.1403

[M+H+Mg]3+ 901.1586 886.4816 NA

NA = not assigned as signal had too low intensity or was overlapped to other signals.

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and even when extracted ion chromatograms (XICs) were obtainedfor each compound no peak relevant to the above toxins wasdetected; however, in the only Mediterranean strain a peak at10.78 min with an associated spectra containing [M+H+Ca]3+ ion ofpalytoxin at m/z 906.8160 (mono-isotopic ion peak at m/z906.4844 for C129H224N3O54Ca, D = 1.779 ppm, RDB = 19.5)revealed the presence of traces of palytoxin in pellet extract.Considering both the LOD and LOQ of palytoxin (1.6 ng mL�1 and3.13 ng mL�1, respectively) and the volume of the O. cf. siamensis

extract samples, the presence of palytoxin in the Mediterraneansample could be estimated at levels in the range 0.4–0.8 fg cell�1.

Since recent studies reported on a quite high structuralvariability of palytoxins produced by Ostreopsis spp. (Ciminielloet al., 2011b), it could not be excluded that palytoxin-likecompounds different from those reported so far were producedby the Mediterranean/Atlantic O. cf. siamensis strains. The presenceof unknown palytoxin congeners in the extracts was investigatedby analyzing HR full MS spectra of the chromatographic regionsrelatively close to palytoxin retention time (10.78 � 3 min).Although some bi- and tri-charged ions were observed, none ofthem could be assigned to a palytoxin-like compound on the basis ofthe reported MS behavior of palytoxins (Ciminiello et al., 2011a).

LC–HRMS results were further supported by mouse bioassayscarried out on all the Mediterranean and Atlantic O. cf. siamensis

extracts. No mice died even after injection of 1/2 of the extracts(corresponding approximately to 3 � 104 to 3 � 106 cells equiva-lent injected). It is to be noted that toxicity of palytoxin by intra-peritoneal injection is quite high with LD50 values of 0.31–1.5 mg kg�1 being reported (Munday, 2011).

In the light of the obtained results, the Mediterranean andAtlantic O. cf. siamensis strains do not produce either ostreocins,which are produced by the Japanese O. siamensis strain at levels ofabout 5 mg per liter of culture (Ukena et al., 2001), or ovatoxins,which are produced by the Mediterranean O. cf. ovata at levels of4–75 pg cell�1 (Ciminiello et al., 2008; Guerrini et al., 2010;Pistocchi et al., 2011; Honsell et al., 2011; Pfannkuchen et al., 2012;Accoroni et al., 2011). Only sub-fg levels of palytoxin on a per cellbasis were detected in one strain.

The obtained results are in agreement with Paz et al. (2011)who did not detect any ostreocin by MALDI-TOF MS in crudeextract and cells of a Mediterranean O. cf. siamensis collected atGirona (Spain).

A reliable comparison among the results presented herein andthose reported by Cagide et al. (2009) and Penna et al. (2005) forother Mediterranean O. cf. siamensis strains cannot be actuallydone as very different approaches were used. In both previousstudies, biomolecular methods were used for qualitative purposesand the presence of palytoxin-like compounds in O. cf. siamensis

extracts was deduced on the basis of functional effects onneuroblastoma cells (Cagide et al., 2009) and delayed hemolyticactivity (Penna et al., 2005). Biomolecular methods are known tobe highly sensitive and it cannot be excluded that even traceamounts of palytoxin could result into a functional effect beingdetected.

On account of all of the above results, the observation thatOstreopsis species are not necessarily toxin producers could bedrawn, as is the case of Mediterranean/Atlantic and Japanese O.cf. siamensis. This could be due to genetic variability amongstrains. However, we cannot rule out the possibility that O. cf.siamensis strains are all capable of producing the palytoxincongeners, but the gene cluster encoding for them may onlybe switched off or some genes of the toxin pathway are missing,as supposed for other dinoflagellates (Stuken et al., 2011). Inparticular, Murray et al. (2012) demonstrated that an Alexan-

drium tamarense strain Group V from Tasmania considered asno-toxic clade, is able to produce saxitoxins (STXs) and possessesthe sxtA gene.

However, at the present, only the saxitoxin gene clustersequences are known for Alexandrium species and Gymnodimnium

catenatum. No polyketide-like gene sequences, likely encoding forpalytoxin, are currently known.

Consequently, it is not possible to apply a molecular geneexpression analysis in different Ostreopsis species, that could be atool to demonstrate if the absence of toxicity in certain species orstrains observed by analytical determinations is due to the absenceof polyketide genes or to low levels of gene expression.

m/z1110 116 0 121 0260 31 0

Ostreoci n-d

m/z1110 116 0 121 0260 31 0

Ostreoci n-b

Fig. 4. Expansion of HRMS2 spectra of ostreocin-d and -b. Ions due to the favored cleavage between C-8 and C-9 which divides the molecule in the two moieties A- and B- are

labeled.

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4. Conclusions

This study demonstrates that the Mediterranean and theAtlantic O. cf. siamensis strains are devoid of any appreciabletoxicity. Thus, at least in the European area, O. cf. siamensis seemsto present a much lower risk to human health than O. cf. ovata thatwas shown to produce relatively high levels of palytoxin-likecompounds.

This is highly relevant under both a toxicological and anecological point of view, indicating that not all the Ostreopsis

species produce palytoxin-like compounds and, thus, can getinvolved in human intoxications or damage to marine benthiccommunities. A high variability in toxin content can occur amongdifferent Ostreopsis spp. and even within the same speciesdepending on the geographical origin of the strain. Therefore, acorrect species-specific identification of Ostreopsis spp. in environ-mental samples combined to a chemical analysis appear to behighly important, since toxic outbreaks can be likely related only toblooms of those Ostreopsis species able to produce high levels ofpalitoxin-like compounds.

Acknowledgments

This research was supported by PRIN 2009 granted to P. C. andA. P., Rome, Italy. A. Amorim acknowledges financial support byProject PTDC/MAR/100348/2008 and V. Veloso for technicalassistance with the cultures.[SS]

References

Accoroni, S., Romagnoli, T., Colombo, F., Pennesi, C., Di Camillo, C.G., Marini, M.,Battocchi, C., Ciminiello, P., Dell’Aversano, C., Dello Iacovo, E., Fattorusso, E.,Tartaglione, L., Penna, A., Totti, C., 2011. Ostreopsis cf. ovata bloom in thenorthern Adriatic Sea during summer 2009: ecology, molecular characteriza-tion and toxin profile. Marine Pollution Bulletin 62 (11), 2512–2519.

Aligizaki, K., Nikolaidis, G., 2006. The presence of the potentially toxic generaOstreopsis and Coolia (Dinophyceae) in the North Aegean Sea, Greece. HarmfulAlgae 5, 717–730.

Aligizaki, K., Katikou, P., Nikolaidis, G., Panou, A., 2008. First episode of shellfishcontamination by palytoxin-like compounds from Ostreopsis species (AegeanSea Greece). Toxicon 51, 418–427.

Amorim, A., Veloso, V., Penna, A., 2010. First detection of Ostreopsis cf. siamensis inPortuguese coastal waters. Harmful Algal News 42, 6–7.

Amzil, Z., Sibat, M., Chomerat, N., Grossel, H., Marco-Miralles, F., Lemee, R., Nezan, E.,Sechet, V., 2012. Ovatoxin-a and palytoxin accumulation in seafood in relationto Ostreopsis cf. ovata blooms on the French Mediterranean coast. Marine Drugs10, 477–496.

ARPAC, 2008. Il monitoraggio dell’Ostreopsis ovata lungo il litorale della Campania(giugno-agosto 2007), http://www.arpacampania.it/pubblicazioni.asp.

Battocchi, C., Totti, C., Vila, M., Maso, M., Cappellacci, S., Accoroni, S., Rene, A.,Scardi, M., Penna, A., 2010. Monitoring toxic microalgae Ostreopsis (dinofla-gellate) species in coastal waters of the Mediterranean Sea using molecularPCR-based assay combined with light microscopy. Marine Pollution Bulletin60, 1074–1084.

Besada, E.G., Loeblich, L.A., Loeblich III, A.R., 1982. Observations on tropical, benthicdinoflagellates form ciguatera-endemic areas: Coolia, Gambierdiscus, andOstreopsis. Bulletin of Marine Science 32, 723–735.

Cagide, E., Louzao, M.C., Espina, B., Vieytes, M.R., Jaen, D., Maman, L., Yasumoto, T.,Botana, L.M., 2009. Production of functionally active palytoxin-like compoundsby Mediterranean Ostreopsis cf. siamensis. Cellular Physiology and Biochemistry23, 431–440.

Ciminiello, P., Dell’Aversano, C., Fattorusso, E., Forino, M., Magno, G.S., Tarta-glione, L., Grillo, C., Melchiorre, N., 2006. The Genoa 2005 outbreak. Deter-mination of putative palytoxin in Mediterranean Ostreopsis ovata by a newliquid chromatography tandem mass spectrometry method. AnalyticalChemistry 78, 6153–6159.

Ciminiello, P., Dell’Aversano, C., Fattorusso, E., Forino, M., Tartaglione, L., Grillo, C.,Melchiorre, N., 2008. Putative palytoxin and its new analogue, ovatoxin-a, inOstreopsis ovata collected along the Ligurian coasts during the 2006 toxicoutbreak. Journal of the American Society for Mass Spectrometry 19, 111–120.

Ciminiello, P., Dell’Aversano, C., Dello Iacovo, E., Fattorusso, E., Forino, M., Grauso, L.,Tartaglione, L., Guerrini, F., Pistocchi, R., 2010. Complex palytoxin-like profile ofOstreopsis ovata. Identification of four new ovatoxins by high-resolution liquidchromatography/mass spectrometry. Rapid Communications in Mass Spec-trometry 24, 2735–2744.

Ciminiello, P., Dell’Aversano, C., Dello Iacovo, E., Fattorusso, E., Forino, M., Tarta-glione, L., 2011a. LC–MS of palytoxin and its analogues: state of the art andfuture perspectives. Toxicon 57, 376–389.

Ciminiello, P., Dell’Aversano, C., Fattorusso, E., Forino, M., Grauso, L., Tartaglione, L.,2011b. A 4-decade-long (and still ongoing) hunt for palytoxins chemicalarchitecture. Toxicon 57, 362–367.

Ciminiello, P., Dell’Aversano, C., Dello Iacovo, E., Fattorusso, E., Forino, M., Grauso, L.,Tartaglione, L., 2012a. High resolution LC–MSn fragmentation pattern of paly-toxin as template to gain new insights into ovatoxin-a structure. The key role ofcalcium in MS behavior of palytoxins. Journal of the American Society for MassSpectrometry 23 (5), 952–963.

Ciminiello, P., Dell’Aversano, C., Dello Iacovo, E., Fattorusso, E., Forino, M., Grauso, L.,Tartaglione, L., Guerrini, F., Pezzolesi, L., Pistocchi, R., Vanucci, S., 2012b.Isolation and structure elucidation of ovatoxin-a, the major toxin producedby Ostreopsis ovata. Journal of the American Chemical Society 134, 1869–1875.

Ciminiello, P., Dell’Aversano, C., Dello Iacovo, E., Fattorusso, E., Forino, M., Tarta-glione, L., Crinelli, R., Carloni, E., Magnani, M., Battocchi, C., Penna, A., 2012c. Theunique toxin profile of a Mediterranean Ostreopsis cf. ovata strain HR LC–MSn

characterization of ovatoxin-f, a new palytoxin congener. Chemical Research inToxicology 25, 1243–1252.

Durando, P., Ansaldi, F., Oreste, P., Moscatelli, P., Gasparini, L.M., Icardi, G., 2007.Ostreopsis ovata and human health: epidemiological and clinical features ofrespiratory syndrome outbreaks from a two-year syndromic surveillance,2005–06, in north-west Italy. Euro Surveillance 12 (23), pii¼3212.

Faust, M.A., 1999. Three new Ostreopsis species (Dinophyceae): O. marinus sp. nov.,O. belizeanus sp. nov., and O. caribbeanus sp. nov. Phycologia 38, 92–99.

Faust, M.A., Morton, S.L., 1995. Morphology and ecology of the marine dinoflagellateOstreopsis labens sp. nov. (Dinophyceae). Journal of Phycology 31, 456–463.

Fraga, S., Rodriguez, F., Caillaud, A., Diogene, J., Raho, N., Zapata, M., 2011. Gam-bierdiscus excentricus sp. nov (Dinophyceae), a benthic toxic dinoflagellate fromthe Canary Islands (NE Atlantic Ocean). Harmful Algae 11, 10–22.

Fritz, L., Triemer, R.E., 1985. A rapid and simple technique utilizing Calcofluor WhiteM2R for the visualization of dinoflagellate thecal plates. Journal of Phycology21, 662–664.

Fukuyo, Y., 1981. Taxonomical study on benthic dinoflagellates collected in coralreefs. Bulletin of the Japanese Society for the Science of Fish 47, 967–978.

Guerrini, F., Pezzolesi, L., Feller, A., Riccardi, M., Ciminiello, P., Dell’Aversano, C.,Tartaglione, L., Dello Iacovo, E., Fattorusso, E., Forino, M., Pistocchi, R., 2010.Comparative growth and toxin profile of cultured Ostreopsis ovata from theTyrrhenian and Adriatic Seas. Toxicon 55, 211–220.

Guillard, R.R.L., 1975. Culture of phytoplankton for feeding marine invertebrates. In:Smith, W.L., Chanley, M.H. (Eds.), Culture of Marine Invertebrate Animals.Plenum Press, New York, NY, pp. 29–60.

Habermann, E., Chatwal, G.S., 1982. Ouabain inhibits the increase due to palytoxinof cation permeability of erythrocytes. Naunyn-Schmiedeberg’s Archives ofPharmacology 319, 101–107.

Honsell, G., De Bortoli, M., Boscolo, S., Dell’Aversano, C., Battocchi, C., Fontanive, G.,Penna, A., Berti, F., Sosa, S., Yasumoto, T., Ciminiello, P., Poli, M., Tubaro, A., 2011.Harmful Dinoflagellate Ostreopsis cf. ovata Fukuyo: detection of ovatoxins infield samples and cell immunolocalization using antipalytoxin antibodies.Environmental Science & Technology 45 (16), 7051–7059.

Laza-Martinez, A., Orive, E., Miguel, I., 2011. Morphological and genetic characteri-zation of benthic dinoflagellates of the genera Coolia, Ostreopsis and Prorocentrumfrom the south-eastern Bay of Biscay. European Journal of Phycology 46, 45–65.

Mangialajo, L., Ganzin, N., Accoroni, S., Asnaghi, V., Blanfune, A., Cabrini, M.,Cattaneo-Vietti, R., Chavanon, F., Chiantore, M., Cohu, S., Costa, E., Fornasaro,D., Grossel, H., Marco-Miralles, F., Maso, M., Rene, A., Rossi, A.M., Sala, M.M.,Thibaut, T., Totti, C., Vila, M., Lemee, R., 2011. Trends in Ostreopsis proliferationalong the Northern Mediterranean coasts. Toxicon 57, 408–420.

March, R.E., 1997. An introduction to quadrupole ion trap mass spectrometry.Journal of Mass Spectrometry 32, 352–369.

Munday, R., 2011. Palytoxin toxicology: animal studies. Toxicon 57, 470–477.Murray, S.A., Wiese, M., Neilan, B.A., Orr, R.J.S., de Salas, M., Brett, S., Hallegraeff, G.,

2012. A reinvestigation of saxitoxin production and sxtA in the non-toxicAlexandrium tamarense Group V clade. Harmful Algae 18 (0), 96–104.

Parsons, M.L., Aligizaki, K., Dechraoui Bottein, M.-Y., Fraga, S., Morton, S.L., Penna, A.,Rhodes, L., 2012. Gambierdiscus and Ostreopsis: reassessment of the state ofknowledge of their taxonomy, geography, ecophysiology, and toxicology.Harmful Algae 14, 107–129.

Paz, B., Riobo, P., Franco, J.M., 2011. Preliminary study for rapid determination ofphycotoxins in microalgae whole cells using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Communications in MassSpectrometry 25, 3627–3639.

Penna, A., Vila, M., Fraga, S., Giacobbe, M.G., Andreoni, F., Riobo, P., Vernesi, C., 2005.Characterization of Ostreopsis and Coolia (Dinophyceae) isolates in the westernMediterranean Sea based on morphology, toxicity and internal transcribedspacer 5.8S rDNA sequences. Journal of Phycology 41, 212–225.

Penna, A., Bertozzini, E., Battocchi, C., Giacobbe, M.G., Galluzzi, L., Garces, E., Vila, M.,Luglie, A., Magnani, M., 2007. Monitoring of HAB species in the MediterraneanSea through molecular techniques. Journal of Plankton Research 29, 19–38.

Penna, A., Fraga, S., Battocchi, C., Casabianca, C., Giacobbe, M.G., Riobo, P., Vernesi, C.,2010. A phylogeographical study of the toxic benthic dinoflagellate genusOstreopsis Schmidt. Journal of Biogeography 37, 830–841.

Penna, A., Fraga, S., Battocchi, C., Casabianca, S., Perini, F., Capellacci, S., Casabianca,A., Riobo, P., Giacobbe, M.G., Totti, C., Accoroni, S., Vila, M., Rene, A., Scardi, M.,Aligizaki, K., Nguyen-Ngoc, L., Vernesi, C., 2012. Genetic diversity of the genus

P. Ciminiello et al. / Harmful Algae 23 (2013) 19–2726

Author's personal copy

Ostreopsis Schmidt: phylogeographical considerations and molecular method-ology applications for field detection in the Mediterranean Sea. Cryptogamie 33,153–163.

Pfannkuchen, M., Godrijan, J., Pfannkuchen, D.M., Ivesa, L., Kruzic, P., Ciminiello,P., Dell’Aversano, C., Dello Iacovo, E., Fattorusso, E., Forino, M., Tartaglione, L.,Godrijan, M., 2012. Toxin-producing Ostreopsis cf. ovata are likely to bloomundetected along coastal areas. Environmental Science & Technology 46,5574–5582.

Pistocchi, R., Pezzolesi, L., Guerrini, F., Vanucci, S., Dell’Aversano, C., Fattorusso, E.,2011. A review on the effects of environmental conditions on growth and toxinproduction of Ostreopsis ovata. Toxicon 57, 421–428.

Rhodes, L.L., Smith, K.F., Munday, R., Selwood, A.I., McNabb, P.S., Holland, P.T.,Bottein, M.Y., 2010. Toxic dinoflagellates (Dinophyceae) from Rarotonga, CookIslands. Toxicon 56, 751–758.

Rhodes, L.L., 2011. World-wide occurrence of the toxic dinoflagellate genusOstreopsis Schmidt. Toxicon 57, 400–407.

Schmidt, J., 1901. Peridiniales. Preliminary report of the botanical resultsof the Danish expedition to Siam (1899–1900). Botanische Pidsslerift212–221.

Simoni, F., Gaddi, A., Di Paolo, C., Lepri, L., 2003. Harmful epiphytic dinoflagellate onTyrrhenian Sea reefs. Harmful Algal News 24, 13–14.

Stuken, A., Orr, R.J.S., Kellmann, R., Murray, S.A., Neilan, B.A., Jakobsen, K.S., 2011.Discovery of nuclear-encoded genes for the neurotoxin saxitoxin in dinofla-gellates. PLoS One 6 (5), e20096.

Taylor, F.J.R., 1979. A description of the benthic dinoflagellate associated withmaitotoxin and ciguatoxin including observations on Hawaiian material. In:Taylor, D.L., Seliger, H.H. (Eds.), Toxic Dinoflagellate Blooms. Elsevier, NorthHolland, New York, pp. 71–76.

Tichadou, L., Glaizal, M., Armengaud, A., Grossel, H., Lemee, R., Kantin, R., Lasalle, J.L.,Drouet, G., Rambaud, L., Malfait, P., de Haro, L., 2010. Health impact of unicel-lular algae of the Ostreopsis genus blooms in the Mediterranean Sea: experienceof the French Mediterranean coast surveillance network from 2006 to 2009.Clinical Toxicology 48, 839–844.

Tognetto, L., Bellato, S., Moro, I., Andreoli, C., 1995. Occurrence of Ostreopsis ovata(Dinophyceae) in the Tyrrhenian Sea during summer 1994. Botanica Marina 38,291–295.

Totti, C., Accoroni, S., Cerino, F., Cucchiari, E., Romagnoli, T., 2010. Ostreopsis ovataalong the Conero Riviera (northern Adriatic Sea): relationships with environ-mental conditions and substrata. Harmful Algae 9, 233–239.

Ukena, T., Satake, M., Usami, M., Oshima, Y., Naoki, H., Fujita, T., Kan, Y., Yasumoto,T., 2001. Structure elucidation of ostreocin D, a palytoxin analog isolated fromthe dinoflagellate Ostreopsis siamensis. Bioscience Biotechnology and Biochem-istry 65, 2585–2588.

Ukena, T., 2001. Chemical studies on ostreocins, palytoxin analogs, isolated from thedinoflagellate Ostreopsis siamensis. Ph.D. Thesis, Graduate School of LifeSciences, Tohoku University.

Ukena, T., Satake, M., Usami, M., Oshima, Y., Fujita, T., Naoki, H., Yasumoto, T., 2002.Structural confirmation of ostreocin-D by application of negative-ion fast-atombombardment collision-induced dissociation tandem mass spectrometricmethods. Rapid Communications in Mass Spectrometry 16, 2387–2393.

Usami, M., Satake, M., Ishida, S., Inoue, A., Kan, Y., Yasumoto, T., 1995. Palytoxinanalogs from the dinoflagellate Ostreopsis siamensis. Journal of the AmericanChemical Society 117, 5389–5390.

Vila, M., Garces, E., Maso, M., 2001. Potentially toxic epiphytic dinoflagellatesassemblages on macroalgae in the NW Mediterranean. Aquatic MicrobialEcology 26, 51–60.

P. Ciminiello et al. / Harmful Algae 23 (2013) 19–27 27