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Variability in toxin profiles of the Mediterranean Ostreopsis cf. ovata and in structural features of the produced ovatoxins Luciana Tartaglione, Emma Dello Iacovo, Antonia Mazzeo, Silvia Casabianca, Patrizia Ciminiello, Antonella Penna, and Carmela Dell'Aversano Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03827 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 15, 2017

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Environmental Science & Technology

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Variability in toxin profiles of the Mediterranean Ostreopsis cf. ovata and in

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structural features of the produced ovatoxins

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Luciana Tartaglione,† Emma Dello Iacovo,† Antonia Mazzeo,† Silvia Casabianca,§‡ Patrizia Ciminiello, †

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Antonella Penna,§‡ Carmela Dell'Aversano†*

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Department of Pharmacy, School of Medicine and Surgery, University of Napoli Federico II, via D.

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Montesano 49, 80131, Naples, Italy, [email protected], §

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Department of Biomolecular Sciences, University of Urbino, Viale Trieste 296, 61121, Pesaro, Italy

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CoNISMa, Italian Interuniversity Consortium on Marine Sciences, Piazzale Flaminio 9, 00196, Rome, Italy

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TOC/Abstract graphic image:

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ABSTRACT:

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Fifty-five strains of Ostreopsis were collected in the Mediterranean Sea and analysed to characterize their

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toxin profiles. All the strains were grown in culture under the same experimental conditions and identified

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by molecular PCR assay based on the ITS-5.8S rDNA. A liquid chromatography-high resolution multiple

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stage mass spectrometry (LC-HRMSn) approach was used to analyze toxin profiles and to structurally

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characterize the detected toxins. Despite morphological and molecular characterization being consistent

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within the species O. cf. ovata, a certain degree of toxin variability was observed. All the strains produced

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ovatoxins (OVTXs), with the exception of only one strain. Toxin profiles were quite different from both

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qualitative and quantitative standpoints: 67% of the strains contained OVTX-a to –e, OVTX-g and isobaric

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PLTX, in 25% of them only OVTX-a, -d, -e and isobaric PLTX were present, while 4% produced only OVTX-b

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and –c. None of the strains showed a previously identified profile, featuring OVTX-f as dominant toxin,

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whereas OVTX-f was a minor component of very few strains. Toxin content was mostly in the range 4-70

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pg/cell with higher levels (up to 238 pg/cell) being found in strains from the Ligurian and South Adriatic Sea.

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Structural insights into OVTX-b, -c, -d and -e were gained and the new OVTX-l was detected in 36 strains.

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INTRODUCTION

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Over the last decade, massive blooms of the benthic dinoflagellate Ostreopsis cf. ovata have occurred in

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the Mediterranean Sea with consequent negative impacts on human health mainly by inhalation of toxic

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aerosols and/or skin contact [1,2]. Liquid chromatography-high resolution multiple stage mass

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spectrometry (LC-HRMSn) studies revealed O. cf. ovata as the producer of ovatoxins (OVTXs) [3,4],

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structural congeners of palytoxin (PLTX), one of the most potent non-protein marine toxins so far known.

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Interestingly, full scan HRMS spectra of PLTX and OVTXs represent a kind of fingerprint of this class of 2 ACS Paragon Plus Environment

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molecules that facilitate their identification [5]. Indeed they contain a number of ions singly, -doubly and –

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triply charged ions, including protonated ions, adducts with monovalent and divalent cations and some in-

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source formed fragment ions. This behavior, although makes quantitation quite challenging, is highly

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helpful for the straight-forward identification of palytoxin congeners. Even more informative are HRMSn

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spectra of these compounds that contain several diagnostic fragments and thus, have proven instrumental

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to the structural characterization of a number of unknown analogues, such as OVTX-a, -f, -g, –h, -i, -j1, -j2, –

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k and isobaric PLTX [3,4,6-9]; to date MS-based structural assignments are lacking only for OVTX-b, -c, -d,

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and -e.

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Toxin profiles of Mediterranean strains of O. cf. ovata reported so far appear to be dominated by OVTX-a

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followed by OVTX-b, the isomers OVTX-d and -e, OVTX-c, and isobaric PLTX [6,7, 10-12]. Consistently, only

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the major component, OVTX-a has been isolated and stereo-structurally elucidated [13,14]. A different

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toxin profile dominated by OVTX-f was reported for one Adriatic strain [6] and it is still considered to be

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unique, although OVTX-f was found as minor component in some French Mediterranean strains [12]. Some

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minor components of the toxin profile, such as OVTX-g and -h have been recently detected in Spanish and

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French strains, respectively [7,8]. Interestingly, OVTX-a, -d, and –e and isobaric PLTX have been detected

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also in other Mediterranean Ostreopsis species, such as in Lebanese and Cypriot strains of the recently

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identified O. fattorussoi [9,15], proving to be non-species-specific compounds. Some strains of O.

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fattorussoi from Cyprus were also demonstrated to produce only OVTX-i, j1, j2, and –k [9].

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From a quantitative standpoint, toxin content of Mediterranean O. cf. ovata strains apparently varies over

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an order of magnitude, from 30 to 300 pg/cell. However, the reported data refer to strains which were

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collected by several authors at very few sites of the Mediterranean basin in various periods, which were

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grown under different conditions, and, in some cases, analyzed by different analytical methods [6-8, 10-12].

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On the other hand, very low toxin contents have been recorded in Mediterranean O. fattorussoi (0.06-2.8

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pg/cell) [9,15] and O. cf. siamensis (0–0.8 fg/cell) [16] suggesting that, unlike O. cf. ovata, these two species

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do not represent a real threat to humans in the Mediterranean Sea. However, Ostreopsis blooms are not

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just a European problem. In fact, toxin-producing species of O. cf. ovata have been detected also in coastal

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waters of Japan [17,18], New Zealand [19], and Brazil [20], although only along the Brazilian coastlines were

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poisonings recorded concurrently to algal blooms [21]. Accordingly, high levels of OVTXs (60-468 pg/cell)

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have been reported for Brazilian O. cf. ovata strains, while very low toxin contents were reported for the

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New Zealand (0.013 pg/cell) and the Japanese (0-16 pg/cell) strains, the latter producing isomers of the

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Mediterranean ovatoxins [17-21].

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Variability in toxin profiles and contents and the fact that O. cf. ovata, although widely distributed, causes

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toxic outbreaks in humans only at some specific sites [2,20], suggest that toxic potential of O. cf. ovata

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could be different depending on geographical distribution. In more detail, in the Mediterranean basin and 3 ACS Paragon Plus Environment


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in Brazil O. cf. ovata is likely more toxic than in Japan and in South Pacific area; this parallels observations

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done for another species of Ostreopsis, namely O. cf. siamensis, which proved to have different toxicity

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depending on geographical origin, with strains from Japan being very toxic and Mediterranean/Atlantic

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strains being practically devoid of any toxicity [16].

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A systematic study on toxin profiles and content of Mediterranean O. cf. ovata has not been performed so

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far and the aim of this study was to ascertain the toxic potential of O. cf. ovata in the Mediterranean Sea by

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investigating a significant number of strains collected at 10 different coastal sites and cultured under the

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same conditions. A recently developed LC-HRMSn approach combining high mass accuracy with complete

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chromatographic separation of potentially interfering compounds [22] was used to characterize toxin

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profiles of the strains after their taxonomic identification by molecular analyses and to provide structural

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insight of the detected toxins.

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EXPERIMENTAL SECTION

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Collection of O. cf. ovata and batch culture conditions. Fifty-five strains of O. cf. ovata were collected

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at 10 sites of the Mediterranean basin, 4 sites from the Adriatic Sea (16 strains), 2 sites from the Ligurian

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Sea (16 strains), 3 sites from the Tyrrhenian Sea (17 strains), and 1 site from the Ionian Sea (6 strains)

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(Figure 1). In general, sampling sites were characterized by rocky bottom and shallow depths or artificial

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reefs. Samples of benthic macroalgae were collected at different depths (0.1 – 2 m). The strains were

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isolated by the capillary pipette method from macroalgal washing seawater (Table S1) and a total of 55

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clonal cultures were established, maintained and grown in 1 L glass bottles containing 400 mL of sterilized

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F/4 -Si medium [23] at a temperature of 23 ± 1 °C. Light was provided by cool-white fluorescent bulbs

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(photon flux of 100 µE m-2 s-1) with a standard 14:10 light–dark cycle. During the stationary phase (day 18-

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20 after culture inoculum) culture sub-samples were fixed with Lugol’s iodine and counted using the

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Utermöhl [24] or the Sedgewick-Rafter [25] methods. O. cf. ovata cells were harvested by centrifugation at

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4000 x g for 15 min at room temperature and pellets were stored at -80°C until chemical analyses.

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Microscopy Analyses. Ostreopsis spp. were analysed under an inverted light microscope (Axiovert 40 CFL, Zeiss) at 200 x or 400 x magnification according to Fukuyo [26] and Steidinger and Tangen [27].

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Molecular Identification by PCR assay. Genomic DNA was extracted from a 10 mL culture of all the

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strains, collected in exponential growth phase, using DNeasy Plant Kit (Qiagen, Valencia, CA, USA),

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according to the manufacturer’s instructions. PCR amplification using genus- and species-specific primers

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was performed as described in Penna et al. [28] and Battocchi et al. [29]. 4 ACS Paragon Plus Environment

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Extraction. Cell pellets were extracted with 3 mL of methanol/water (1:1, v/v), 0.2% acetic acid

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(Laboratory grade, Sigma Aldrich, USA). Each mixture was sonicated for 10 min in pulse mode, while cooling

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in ice bath and then centrifuged at 2000 x g for 10 min. The supernatant was decanted and each pellet was

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washed twice with 3 ml of the same extracting solvent. The extracts were combined and the volume

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adjusted to 9 ml. The obtained mixtures were analyzed directly by LC-HRMS after preparation.

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Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS). LC-HRMSn analyses (n= 1,2) were

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carried out on the crude extracts in order to characterize toxin profiles and measure toxin contents. A

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recently developed LC method which allowed chromatographic separation of most of the ovatoxins so far

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known was used in order to facilitate toxin identification and avoid interference in the quantification of the

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structural isomers (OVTX-d/OVTX-e) and of those toxin groups having quantification ions with very close

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mass differences (4-6 mDa) such as OVTX-a/OVTX-d/OVTX-e, OVTX-f/OVTX-b/PLTX, OVTX-b/OVTX-c,

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PLTX/OVTX-d/OVTX-e [22]. LC-HRMS experiments were performed on a hybrid linear ion trap LTQ Orbitrap

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XL™ Fourier Transform MS (FTMS) equipped with an ESI ION MAXTM source (Thermo-Fisher; San Jose, CA,

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USA) coupled to an Agilent 1100 LC binary system (Palo Alto, CA, USA). A 2.7 µm Poroshell 120 EC-C18, 100

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x 2.10 mm column (Agilent) was eluted at 0.2 mL/min with water (eluent A) and 95% acetonitrile/water

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(eluent B), both containing 30 mM acetic acid (HPLC grade, Sigma Aldrich, USA). Gradient elution was 28-

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29% B in 5 min, 29-30% B in 10 min, 30-100% B in 1 min, and hold for 5 min. Re-equilibration time was 14

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min. Injection volume was 5 µL. Under such conditions all the ovatoxins were chromatographically

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separated [22]. HR full MS experiments (positive ions) were acquired in the range m/z 800-1400 at a

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resolving power of 60,000. The following source settings were used: a spray voltage of 4.8 kV, a capillary

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temperature of 290°C, a capillary voltage of 50 V, a sheath gas and an auxiliary gas flow of 38 and 2

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(arbitrary units), respectively. The tube lens voltage was set at 120 V. HRMS2 data were acquired in collision

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induced dissociation (CID) mode at a resolving power of 30,000 by selecting the [M+H+Ca]3+ ions of OVTX-a

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to -g and isobaricPLTX as precursors [7] (Table S2). A collision energy of 25%, an activation Q of 0.250, and

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an activation time of 30 ms were used. Quantitative determination of OVTXs in the extracts was carried out

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by using extracted ion chromatograms (XIC) of [M+H+Ca]3+ ions of each congener at a 5 ppm mass

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tolerance. A reference extract of O. cf. ovata (OOAN0601) previously characterized [4] was injected under

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the same experimental conditions and used to identify individual toxins based on retention times, relative

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ratio of triply and doubly charged ions contained in Full scan HRMS spectra, exact masses (error 1-2 ppm)

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and isotopic patterns. In the absence of standards for ovatoxins, a calibration curve (triplicate injection) of

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PLTX standard (Wako Chemicals GmbH (Neuss, Germany) at five levels of concentration (1000, 100, 50, 25,

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and 12.5 ng/mL) was used, assuming for all the molecules a comparable molar response. Measured limit of

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quantitation (LOQ) for PLTX on the day of analysis was 6.25 ng/mL, as experimentally determined injecting

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multiple PLTX standards at decreasing concentrations till signal disappearance. Considering the extraction

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volume (9 mL) and the number of cells contained in each pellet sample (in the range 8.5 x 105 - 4.7 x 106 5 ACS Paragon Plus Environment


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cells), each PLTX congener could be detected in the extracts at minimum levels in the range 1.3-66 fg/cell,

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according to the following equation [(LOQ/cell number) x extract volume] x 106.

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RESULTS

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Molecular PCR Analysis. It had been previously demonstrated that O. cf. ovata is an

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Atlantic/Mediterranean/Pacific ribotype and that all the Mediterranean isolates have identical sequences

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of ITS-5.8S rDNA [30-33, 15]. Based on several ribosomal data sequences, species-specific primers were

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designed and applied on field samples and cultured strains using PCR based assay [28] for the species-

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specific taxonomical identification. PCR amplification based assay of the Ostreopsis sp. strains using genus-

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and species-specific primers confirmed the O. cf. ovata ribotype identification for all the analyzed strains

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(Figure S1). The ITS-5.8S rDNA sequences totally aligned without any mismatch [30, 31, 34, 15]. This proved

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that, in this study, Ostreopsis isolates could be assigned to the Mediterranean O. cf. ovata ribotype.

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Variability in toxin profiles. LC-HRMS analyses showed that, although the species did not show any

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variability based on the ITS-5.8S rDNA, 98% of the analysed strains produced toxins according to four

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recurring toxin profiles (Figure 2). Only one of the Adriatic strains (CBA 1703 from Portonovo) was

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practically devoid of any ovatoxin. Considering the extraction volume (9 mL), the number of cells contained

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in this sample (1.16 x 106 ) and the LOQ of the method (6.25 ng/mL), the presence of ovatoxins at levels ≥

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48 fg/cell could be excluded.

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Profile #1 was typical of 67% of the strains and it was found at all the 10 collection sites of the

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Mediterranean Sea (Figure 1). This profile contained most of the ovatoxins so far known (Figure 3a), namely

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OVTX-a as the dominant toxin (56.0%±10.1), followed by OVTX-b (26.1%±7.0), OVTX-d (6.9%±2.3), OVTX-e

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(4.1%±2.0), OVTX-c (3.8%±1.4), OVTX-g (0.6%±0.3), and isobaric PLTX (0.34%±0.18), listed in decreasing

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order of relative abundance. In two strains, OVTX-a was by far the major toxin produced, accounting for

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98% (CBA 1483 from site T2) and 77% (CBA 3112 from site A4) of the total toxin content (Table S3). OVTX-f

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was present as minor component of the toxin profile in only 4 strains (CBA 1586 from site T1, CBA 1427

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from site L1, CBA 1649 from site T2, and CBA 1704 from site T2) where it accounted for 3.0%±1.68 of the

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total toxin content. In 27 out of the 37 strains featuring Profile #1, the presence of a new ovatoxin emerged

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at m/z 900.4917 (z=3+) (C130H226O52N3Ca, RDB=19.5, ∆= 0.339) which represented 2.4%±1.7 of the total

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toxin content. We named it ovatoxin-l (OVTX-l). Besides the above mentioned base peak [M+H+Ca]3+, other

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ions were present in the full scan MS spectrum of OVTX-l, including [M+H+Mg]3+ at m/z 895.1650,

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[M+H+Na]2+at m/z 1342.2506, [M+H+K]2+ at m/z 1350.2334, all being consistent with a molecular formula


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of C130H225O52N3 for the molecule. At least for the major components, Profile #1 was in good agreement

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with all the previous reports on O. cf. ovata that revealed OVTX-a as the dominant toxin [7,11-13].

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Profile #2 was typical of 25% of the strains and it was found at all the Ligurian and at most of the Adriatic

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sites, and only at one Tyrrhenian site (T3, Alghero); none of the Ionian strains showed such profile (Figure

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1). Profile #2 contained only OVTX-a, -d, -e, -g, and isobaric PLTX (Fig. 3b). Ovatoxin-a was the major

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component accounting for 76.8%±4.2, followed by OVTX-d (12.4%±3.9), OVTX-e (6.3%±3.1), OVTX-g

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(0.9%±0.5), and isobaric PLTX (0.8%±0.3). In 8 out of the 14 samples featuring this profile, the new OVTX-l

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was also detected contributing significantly to the total toxin content (4.3%±2.6). A similar profile

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containing just OVTX-a, -d, -e, and isobaric PLTX has been already reported for one Adriatic strain

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(OOAN0816) [6,13]. A comparison between profile #1 and #2 indicates that the latter lacks those toxins

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(OVTX-b, -c, and -f) with two additional carbon atoms in their skeleton.

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Profile #3 was observed in two O. cf. ovata strains from the Adriatic site (A3, Portonovo) and it contained

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only OVTX-b (87.8%) and OVTX-c (11.3%) in one strain (CBA-3007) (Figure 3c) and non-quantifiable amounts

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of both toxins in another strain (CBA-3008). Both ovatoxin-b and –c present a molecular formula featuring

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C131 atoms, and thus 2 C atoms more than in PLTX itself and in most ovatoxins (OVTX-a, -d, -e, -g, -h)

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produced by O. cf. ovata.

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Profile #4 was found in only one strain from an Adriatic site (A3, Portonovo) that had been previously

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described [6]. It contained OVTX-f as the dominant toxin (50.1%) followed by OVTX-a (23.6%),-b (17.7%), -d

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(2.9%), -e (2.9%), -c (2.4%) and isobaric PLTX (0.29%).

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Geographically, the highest variability of toxin profiles was observed at one Adriatic site (A3 Portonovo)

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where all profiles #1 to #4 were found together with the only non-toxic strain. Both Profile #1 and #2 were

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present at most of the collection sites, with the only exception of sites T1, T2, and I1 where the only Profile

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#1 was detected, despite the significant number of strains collected (6 strains per site).

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For some strains (CBA 1410 and CBA 1416 from site L1; CBA 1550, CBA 1554 and CBA 1634 from site L2;

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CBA 1617 from site T3), cultures were grown for a long period of time (12 months) and biological replicates

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were obtained and analyzed under the same experimental conditions. The results were similar to those

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obtained initially with regard to the observed toxin profiles and to overall relative abundance of individual

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toxins (Table S3) while the absolute toxin contents decreased (Table S4) according to the age of the

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cultures (7-12 months).

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Since culturing conditions might affect toxin productivity, in our study all the O. cf. ovata strains were

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cultured under the same nutrient, temperature and salinity conditions, harvested on the same growth

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phase, and counted according to the same methodology. In addition, LC-HRMS analyses were performed 7 ACS Paragon Plus Environment


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under the same conditions using the same lot of PLTX standard. This allowed us to reliably estimate toxin

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content on a per cell basis. As a result, total toxin content of the strains was in the range 4 to 238 pg/cells

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(Figure 4, Table S4). Most of the strains (75%) produced OVTXs at levels of 4-70 pg/cell, while higher toxin

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contents (70-238 pg/cell) were observed in strains collected at sites L1 and L2 (Ligurian Sea) and at site A4

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(South Adriatic Sea), suggesting that strains from the Ligurian and the French-Mediterranean coasts as well

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as those from the south Adriatic Sea are likely to produce more toxins in the Mediterranean basin.

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Structural variability of ovatoxins by LC-HRMSn. Assorted O. cf. ovata extracts representative of each toxin

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profile were analysed by LC-HRMSn (n= 2,3) according to the approach developed by Ciminiello et al. [3] for

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structural investigation of unknown palytoxin congeners directly in the crude extract. The fragmentation

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patterns of individual toxins were interpreted in comparison with those of OVTX-a to -e contained in a

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reference extract of O. cf. ovata [10]. Notably, only molecular formula of OVTX-b to –e and elemental

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composition of the fragments originating from cleavage #4 (Figure 5) have been reported so far [4]. In this

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study, the whole fragmentation pattern of OVTX-b to -e has been interpreted thus gaining many insights

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into their structures (Table S5 and S6). The resulting structural hypotheses, which add to those of other

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ovatoxins already reported from Mediterranean O. cf. ovata and O. fattorussoi [3, 6-9], are summarized in

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Figure 5 versus planar structures of palytoxin [35] and ovatoxin-a [13] which have been fully elucidated by

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NMR.

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Ovatoxin-b (C131H227N3O53) compared to OVTX-a (C129H223N3O52) presented an additional C2H4O part

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structure in the region N-C8’ as suggested by the A-side fragment of cleavages #4, #11 to #13, #15 to #19,

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and #21 to #27 - all containing C2H4O more than the relevant fragments in OVTX-a - as well as by the

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internal fragments due to cleavage #2 and #1+4 observed in HRMS3 spectra. All the other fragments of

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OVTX-b (Table S5; Figure S2) were consistent with such structural hypothesis and confidently ruled out the

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presence of further structural difference between OVTX-a and OVTX-b. The C2H4O part of the structure

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could be likely due to two additional methylene and one hydroxyl functionalities. The presence of

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additional methylene groups in the region N-C8’ has been already reported for homo and bishomo

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palytoxin congeners isolated from Palythoa spp. [35].

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Ovatoxin-c (C131H227N3O54) compared to OVTX-a, presents an additional C2H4O part structure in the region

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N-C8’ similarly to OVTX-b - as inferred by the A-side fragments due to cleavage #4, #12, and #13 all

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containing C2H4O more than the relevant fragments in OVTX-a - and an additional O atom at C44,

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reasonably an hydroxyl group, as inferred by the A-side fragments due to cleavages #15 to #17, #19, #23,

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#25 and by the internal fragments #4+15 and #4+16 (Table S6).

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Ovatoxin-d (C129H223N3O53), compared to OVTX-a, presents just an additional hydroxyl group at C44 as

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inferred by the A-side fragments due to cleavage #16 onward (Table S6), all containing 1 oxygen more than 8 ACS Paragon Plus Environment

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the relevant fragments in OVTX-a. This structural feature is typical also of palytoxin [35], isobaric PLTX [7],

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OVTX-c as well as of OVTX-j1 and –k from O. fattorussoi [9].

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OVTX-e (C129H223N3O53) is a structural isomer of OVTX-d and, compared to OVTX-a, presents an additional

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hydroxyl group in the region stretching from N to C8’ as inferred by the A-side fragments due to cleavage

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#4 onward all containing 1 more O atom than relevant fragments of OVTX-a (Table S6).

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The fragmentation pattern of OVTX-a, -b, -d, and –e was confirmed to be the same for all the samples

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analysed in this study. Due to the low levels of OVTX-c, OVTX-g and isobaric PLTX contained in most

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samples, no HRMS2 confirmation could be obtained for such toxins. Work is on-going on structural

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characterization of OVTX-l.

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DISCUSSION

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Molecular and chemical investigation of 55 strains of O. cf. ovata from 10 sites located along the Italian and

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French-Mediterranean coasts allowed to ascertain that, although the species did not show any variability

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based on biomolecular results, a significant degree of variation in toxin profiles existed. In more detail, 37

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of the analyzed strains showed Profile #1, featuring OVTX-a>OVTX-b>>OVTX-d>OVTX-e>OVTX-c>OVTX-

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g≥isobPLTX, 14 strains showed Profile #2, characterized by OVTX-a>>OVTX-d>OVTX-e>OVTX-g≥isobPLTX,

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and 2 strains showed Profile #3, featuring only OVTX-b>>OVTX-c. Profile #4, dominated by OVTX-f [6],

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confirmed to be one of a kind. Only one strain was found not to contain any toxin. Variability in toxin

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profiles is not related to molecular features of the strains, since all the strains were found to belong to O.

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cf. ovata species. Further molecular methods, targeting strain genotypes, might help to understand

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whether the toxin profile could be related to the genotype variability even if it is highly challenging

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considering that the huge genome size of the dinoflagellate O. cf. ovata [36,37]. Chemical profiles are not

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intended to be chemotaxonomic markers. Indeed, Profile #4 was typical of just one O. cf. ovata strain [6]

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while Profile #2 was common to different Ostreopsis species, namely O. cf. ovata and O. fattorussoi [9,15].

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To date the only OVTX-a, as major component of the O. cf. ovata toxin profile, could be isolated in

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sufficient amount for full structure elucidation [13]. In the course of the isolation procedure, heavy toxin

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losses occurred as a result of the evaporation and re-dissolution steps leading to a final overall recovery of

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about 12% [13]. Thus, the HRMSn-based approach [3] employed herein for structural elucidation of the

260

minor congeners is currently the only tool that allows to gain structural insights into these very complex

261

compounds contained at sub-microgram levels in crude extracts. Structural hypotheses for OVTX-b to -e

262

add to those of ovatoxins and isobaric palytoxin previously reported [3, 6-9] and lay the foundations for

263

structure activity relationship studies. Overall, all these toxins, compared to the parent compound 9 ACS Paragon Plus Environment


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264

palytoxin, present an additional hydroxyl group at C42 and lack 2 hydroxyl groups, one at C17 and the other

265

one at C64. Actually, the lack of an OH at the latter position in OVTX-b to -k can only be hypothesized based

266

on the analogy with OVTX-a [13], because the region stretching from C53 to C78 does not fragment in MSn

267

experiments likely due to formation of a conjugated polyene [3,18]. Most of the ovatoxins (OVTX-a, -b, -e to

268

-i, and –j2), compared to PLTX, lack also the hydroxyl group at C44 while only a few (OVTX-b, -c and –e)

269

differ for substitution in the small region N-C8’, including an additional methylene and/or hydroxyl groups.

270

To date, only OVTX-f presents two additional methylenes in the region between C95-C102 and only OVTX-h

271

presents an open ring in the region C42-C49. An in depth analysis by T. Harwood et al. of the strain CBA2-

272

122 (personal communication) according to the micro-scale oxidation and LC-MS/MS analysis [38]

273

suggested that the OVTX-b formerly reported by Ciminiello et al. [6] in this strain was actually an isobaric

274

OVTX-b; this finding confirms the need for MSn structural confirmation when this class of molecules is

275

detected in an algal extract. Although differences among individual ovatoxins may appear small in the

276

frame of the complex structures (Figure 5), relative molar response of these compounds could be different

277

both in full scan MS spectra, due to a different ionization behavior, and in MSn spectra. In the latter a

278

difference in ion ratios of some fragment ions clearly emerges among various analogues (Table S2 and

279

Table S5 and S6). This make even more urgent the need for preparation of reference material for individual

280

ovatoxins that is currently lacking.

281

In 75% of the analyzed strains, toxin content was in the range 4-70 pg/cell with higher levels (up to 238

282

pg/cell) being found in strains from the Ligurian Sea (Genoa and Villefranche sur Mer) and from the South

283

Adriatic Sea (Giovinazzo, BA). Interestingly, sites L1, L2 and A4 (Figure 1) are the sites where the most

284

alarming toxic Ostreopsis-related outbreaks for number of people involved and severity of symptoms have

285

occurred so far [2,39-42]. These findings are in good agreement with the high toxin productivity (50 to 250

286

pg/cell) reported for the Spanish strains collected at Ebro Delta [7,43] and for the French strains collected

287

at site L2 (50 to 300 pg/cell) [12]. However, toxicity data on toxins produced by Ostreopis species are

288

currently limited to the only ostreocin-D [44] and ovatoxin-a [45]. All the other ovatoxins have not been

289

isolated yet and an effort in such a direction is desirable. Individual analogues could present different toxic

290

potency and thus even some of the less represented toxin profiles (such as Profile #3 and #4) could pose a

291

higher risk to humans. As long as toxicity studies will be performed, data reported herein will be useful to

292

correlate toxic potency with chemical structures and algal toxin profiles with potential risk to humans.

293

The geographical distribution of the most toxic strains provides a good foundation for policy makers and

294

managers with respect to monitoring and forecasting benthic harmful algal blooms and relevant

295

information for monitoring of toxins in environmental and seafood samples. We cannot exclude that

296

difference in toxin content may be explained with the physiological adaptations of different strains to

297

different environmental conditions (temperature, nutrients, salinity, allelopathic interactions, etc.). The 10 ACS Paragon Plus Environment

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Adriatic and the Tyrrhenian strains investigated by Guerrini et al. [10] presented significant differences in

299

cell volumes (the Adriatic strain being nearly twice the size of the Tyrrhenian one) although in a recent

300

biogeographical analysis, all the Atlantic/Mediterranean Ostreopsis samples have been assessed [46].

301

Results obtained on the biological replicates were in good agreement with previous observations by

302

Pistocchi et al. [10,47] showing that relative abundance of individual toxins does not depend on culturing

303

conditions while toxin productivity decreases with aging of the cultures. The seawater temperature,

304

investigated in the range 25-30° C [48,49], does not seem to be a primary driver and the maximal

305

abundance periods were site and year specific. The role of temperature requires further investigation and

306

the impact of other drivers has to be elucidated to better explain the bloom dynamics in field samples. Such

307

drivers may be inorganic nutrients concentration and/or their ratio, substrate characteristics (e.g.

308

composition of macroalgal communities), competitive and trophic interactions, or regulation of

309

dinoflagellates by parasites. Noteworthy, this study reports results obtained on cultured strains of O. cf.

310

ovata under laboratory controlled conditions. Since environmental variability could have (or not have) a

311

significant impact on toxin production, the obtained results should be further integrated with data obtained

312

on field samples in order to compare the production of toxins in a natural environment with that obtained

313

under controlled laboratory conditions.

314 315

ASSOCIATED CONTENT

316

Supporting Information

317

The Supporting information is available free of charge on the ACS Publication website at DOI:

318

Table S1: List of the O. cf. ovata strains with indication of the strain code, collection site and date, and cell

319

number.

320

Table S2: Precursor and characteristic product ions with relative ion ratios of OVTX-a to -g.

321

Table S3: Relative abundance of toxins contained in the Mediterranean O. cf. ovata strains.

322

Table S4: Total and individual toxin content of the Mediterranean O. cf. ovata strains.

323

Table S5: Ion assignments of fragment ions of OVTX-a and OVTX-b contained in their LC-HRMSn spectra.

324

Table S6: Ion assignments of fragment ions of OVTX-c, OVTX-d and OVTX-e contained in their LC-HRMSn

325

spectra.

326

Figure S1: Ostreopsis cf. ovata PCR species-specific assay. 11 ACS Paragon Plus Environment


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Page 12 of 23

Figure S2: HRMS2 spectrum of OVTX-b.

328 329

AUTHOR INFORMATION

330

Corresponding Author

331

* C. Dell’Aversano. Address: Department of Pharmacy, School of Medicine and Surgery, University of Napoli

332

Federico II, Via D. Montesano 49, 80131 Napoli, Italy; email: [email protected]; tel.: +39-081-678502; +39-

333

081-678552.

334

Notes

335

The authors declare there are no competing financial interest

336 337

ACKNOWLEDGEMENTS

338

This research was carried out in the frame of Programme STAR Linea 1 2013 (VALTOX, Napoli_call2013_08;

339

PI C. Dell’Aversano), financially supported by University of Napoli Federico II and Compagnia di San Paolo.

340

PRIN 2009. The author are grateful to Dr Tim Harwood for fruitful discussion about isobaric OVTX-b from

341

strain CBA2-122 which led to revise its assignment as a structural isomer of OVTX-b.

342 343

REFERENCES

344

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2011, 10, 165-172.

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504

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FIGURE CAPTIONS

505 506

Figure 1. Distribution of toxin profiles of the 55 strains of O. cf. ovata at 10 sites of the Mediterranean Sea.

507

Figure 2. Relative abundance of ovatoxins (OVTX) and isobaric palytoxin (isob PLTX) in the toxin profiles of

508

the Mediterranean O. cf. ovata.

509

Figure 3. Extracted ion chromatograms (XIC) of 3 representative extracts of O. cf. ovata featuring Profile #1

510

(a), Profile #2 (b), and Profile #3 (c).

511 512

Figure 4. Total toxin content (pg cell-1) of the 55 strains of O. cf. ovata collected in the Adriatic Sea at Trieste

513

(A1), Passetto (A2), Portonovo (A3), Giovinazzo (A4), in the Ligurian Sea at Genova (L1) and Villefranche sur

514

Mer (L2), in the Tyrrhenian Sea at Pisa (T1), Porto Romano (T2), and Alghero (T3), and in the Ionian Sea at

515

Taormina (I1).

516 517

Figure 5. Planar structure of palytoxin (PLTX) [35] and ovatoxin-a (OVTX-a) [13] and structural hypotheses

518

for ovatoxin-b to –k and isobaric palytoxin.

519 520 521 522


Page 19 of 23

523


FIGURE 1 Collection sites

A1 A2 A3 A4

Adriatic Sea (16 strains) Trieste Passetto (AN) Portonovo (AN) Giovinazzo (BA)

# O. cf. ovata strains Profile #1 Profile #2 Profile #3 Profile #4 Not toxic 1 1 2 4

2 1 1

2

1

1

2

1

1

Ligurian Sea (16 strains) L1

Genova

5

1

L2

Ville Franche sur Mer

3

7

Tyrrhenian Sea (17strains)

Profile #1 Profile #2 Profile #3 Profile #4

T1

Pisa

T2

Porto Romano (LT)

6

T3

Alghero

3

6 2

Ionian Sea (6 strains) I1

Taormina (CT)

6

Total

37

14

524 525



526

Page 20 of 23

FIGURE 2

Profile #1

Profile #2

Profile #3

Profile #4

OVTX-a

56% ± 10.1

76.8% ± 4.2

OVTX-b

26.1% ± 7.0

87.8%

17.7%

OVTX-c

3.8% ± 1.4

11.3%

2.4%

OVTX-d

6.9% ± 2.3

12.4% ± 3.9

2.9%

OVTX-e

4.1% ± 2.0

6.3% ± 3.1

2.9%

23.6%

OVTX-f

50.1%

OVTX-g

0.6% ± 0.29

0.9% ± 0.5

isob PLTX

0.34% ± 0.18

0.8% ± 0.3

0.29%

527 528


Page 21 of 23

529


FIGURE 3 OVTX-e 9.86 OVTX-a 11.35 OVTX-d 9.24 isobPLTX OVTX-b 7.94 10.47 OVTX-c 8.49 OVTX-g 12.57

a) 5

10

15 OVTX-a 11.26

b)

OVTX-d 9.18 OVTX-e 9.78 isobPLTX 7.93

5

10

OVTX-g 12.58 15

OVTX-b 10.39

OVTX-c 8.40 c) 5

530

10 Time, min

15

531 532


C5 CBA 3039 CBA 3041 CBA 3056 CBA 1703 CBA 1704 CBA 1706 CBA2-122 CBA 3007 CBA 3008 CBA 3010 CBA 3100 CBA 3106 CBA 3109 CBA 3111 CBA 3112 CBA 1409 CBA1410 CBA 1416 CBA 1425 CBA 1427 CBA 1428 CBA 1550 CBA 1554 CBA 1556 CBA 1566 CBA 1574 CBA 1576 CBA 1581 CBA 1634 CBA 1640 CBA 1632 CBA 1586 CBA 1588 CBA 1590 CBA 1597 CBA 1598 CBA 1601 CBA 1434 CBA 1437 CBA 1441 CBA 1477 CBA 1483 CBA 1649 CBA 1615 CBA 1617 CBA 1620 CBA 1621 CBA 1622 CBA 1291 CBA 1844 CBA 1845 CBA 1847 CBA 1851 CBA 1852

OVTXs pg/cell


533

180

534

535 160

140

A1 A2 A3

Adriatic A4 L1 L2

Ligurian T1 T2

Tyrrhenian

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Page 22 of 23

FIGURE 4 200

Profile #1 Profile #2 Profile #3 Profile #4

120

100

80

60

40

20

0

T3 I1

Ionian

536

537

538

22

Page 23 of 23


FIGURE 5

#26 105 O

OH

B side H2N

#24

O 103

110

O

#25

#27

#20

OH

OH HO

100 OH

115 #28

O

90

95

Me OH #21 #22

OH #23 HO

OH #5 OH

#4 #1 O HO 8'

N H

O 1' HN 1 #2

A side

Me

OH

3 OH #11

Me

HO OH 11 8 9

#3 OH Me 26

Me 31

O O

Me 37

OH OH 73

OH 70

OH #6

13

OH 79 OH 80 #19 78 OH

81

O OH 65 OH

16 OH O #7

17 #8 64 OH R5 18 19 #9 O OH 20 #18 #10 60 #16 OH HO Me #12 R 2 OH 52 56 O 49 50 53 41 42 OH #17 46 44 OH OH HO 45 R3 R4 R1

OH

OH

OH

#13 #14

#15

23

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Variability in Toxin Profiles of the Mediterranean ... - ACS Publications

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