Quantification of the Toxic Dinoflagellate Ostreopsis spp. by qPCR

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Quantification of the toxic dinoflagellate Ostreopsis spp. by qPCR assay in marine aerosol Silvia Casabianca, Anna Casabianca, Pilar Riobó, Jose Franco, Magda Vila, and Antonella Penna Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es305018s • Publication Date (Web): 12 Mar 2013 Downloaded from http://pubs.acs.org on March 18, 2013

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Environmental Science & Technology

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Quantification of the toxic dinoflagellate Ostreopsis spp. by qPCR assay in marine aerosol

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Silvia Casabianca†, Anna Casabianca†, Pilar Riobó‡, José Maria Franco‡, Magda Vila§,

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Antonella Penna†*

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†

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Pesaro, Italy

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‡

Unidad Asociada CSIC-IEO, Instituto de Investigaciones Marinas (IIM-CSIC), Vigo, Spain

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§

Institut de Ciències del Mar (ICM-CSIC), Barcelona, Spain

Department of Biomolecular Sciences, Section of Environmental Biology, University of Urbino,

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*Corresponding author: tel: 0039 0722 304908; fax: 0039 0722 304902;

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e-mail: [email protected].

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ABSTRACT

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We report the development and validation of a qPCR based method for estimation of the toxic

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benthic dinoflagellate Ostreopsis cf. ovata in the complex matrix of marine aerosol at Llavaneres

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beach (northwestern Mediterranean Sea). Toxic events in humans after inhalation or cutaneous

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contact have been reported during O. cf. ovata blooms and were attributed to palytoxin (PLTX) -

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like compounds produced by this microalga. Similar PCR efficiencies of plasmid and cellular

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environmental standard curves (98 and 100%, respectively) allowed obtaining the rDNA copy

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number per cell. The analytical sensitivity was set at 2 x 100 rDNA copy number and 8 x 10-4 cell

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per reaction. Based on spiking experiments, we evaluated the aerosol filter inhibitory activity and

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recovery rate of cells from filters, then normalized the abundance data of toxic O. cf. ovata. The

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abundance in marine aerosol during the bloom varied in the range of 1-102 cells per filter.

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Analytical determinations were also applied to detect palytoxin in field samples. No palytoxin was

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detected in the aerosol filters, and the estimation of PLTX like-compound concentrations in

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microepiphytic assemblages varied between 0.1 and 1.2 pg/cell.

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INTRODUCTION

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Marine aerosols are airborne particles of biological origin containing algal cells, bacteria, spores

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and viruses, which are carried out by bubbles to the sea surface microlayer and to the atmosphere.1

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Since the summer of 2005, in the Mediterranean Sea, growing concerns regarding exposure to

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harmful marine aerosols associated with toxic benthic dinoflagellate Ostreopsis spp. blooms have

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been reported, as they represent major health and economic risks to human populations.2 The genus

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Ostreopsis is comprised of toxic microorganisms that produce non-protein palytoxin (PLTX) and

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PLTX analogs,3,4,5 such as ovatoxins (OVTXs).6,7 Among the OVTXs, ovatoxin-a (OVTX-a) is the

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major toxin produced by O. cf. ovata.8 It was isolated from O. cf. ovata cultures and structurally

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identified as a palytoxin analogue by means of NMR techniques. In comparison with its PLTX

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compounds, OVTX-a possesses an extra hydroxyl group at the 42-position and lacks three hydroxyl

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groups at the 17-, 44-, and 64-positions.9

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During Ostreopsis spp. blooms, their abundance is variable among soft/hard substrata and surface

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seawater; the water cell concentration ranges from 1 x 104 to 1 x 106 cells/L, coinciding with ACS Paragon Plus Environment

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respiratory syndromes in people exposed to marine aerosols and skin irritation during recreational

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bathing along the Spanish, French and Italian coasts.10,11

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It has been hypothesized that aerosolized PLTX -like compounds, cells or cellular fragments of

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Ostreopsis spp. may be responsible for or are likely associated with respiratory symptoms in people

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frequenting beaches or the seaside.12 However, no in vitro studies have provided a clear explanation

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of the direct causes of human respiratory illness by Ostreopsis blooms.13 This is partially due to the

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fact that the complex aerosol matrix has never been analyzed during Ostreopsis blooms, either by

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chemical-analytical or molecular assays. However, it is also evident that harmful Ostreopsis

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occurrences and its high dispersal rate in the Mediterranean basin are very recent and although, a

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considerable amount of important data (based on ecology, chemical composition of toxins, as well

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as molecular and phylogenetic studies) has accumulated in recent years, they still represent only

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partial knowledge regarding this phenomenon.12,14

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In the Mediterranean Sea, two species have been identified, i.e. O. cf. ovata and O. cf. siamensis,

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which are differentially active producers of PLTX-like compounds based on strains, growth

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conditions and age of culture. It seems that Mediterranean/Atlantic O. cf. siamensis does not

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produce any appreciable amounts of PLTX,15,16 while on the contrary O. cf. ovata both field and

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cultured strains retain high level of PLTX compounds.5,6. These two species, O. cf. ovata and O. cf.

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siamensis, cannot be differentiated when co-occurring in a bloom by traditional optical microscopy

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due to the high morphometric variability and overlapping of the two morphotypes.17 Therefore, the

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taxonomic identification has been established based on coupled molecular and morphological SEM

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analyses.18,19 Nevertheless, the toxic O. cf. ovata genotype is predominant and it has been found

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with a greater frequency and abundance along the Mediterranean coasts20 with a highly variable

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range of PLTX-like compound productivity.21 Recently, molecular methods based on PCR and

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quantitative real-time PCR (qPCR) technologies have greatly improved the identification and

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estimation of different Ostreopsis species on macrophytes and surface seawater samples,22,23 thus

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providing a more accurate and specific alternative detection assay. ACS Paragon Plus Environment

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In this study, we developed a qPCR assay for the estimation of Ostreopsis cf. ovata cells in marine

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aerosol during a bloom together with a determination of PLTX-like compounds using hemolytic

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assay and liquid chromatography. Marine aerosol samples represent a difficult matrix containing an

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elevated concentration of microrganisms24 and chemical compounds (metals, inorganic and organic

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substances). In the air, the presence of a high concentration of biotic and abiotic particles could lead

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to cross-reactivity and bias in PCR assays. The new element in this molecular assay, based on a

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method previously developed by Perini et al.,23 is the species-specific detection and quantification

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of toxic Ostreopsis cells in the complex matrix of marine aerosol potentially contaminated with

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PLTX -like compounds. Moreover, we were able to provide normalized results of O. cf. ovata

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abundance in aerosol filters by taking into account the potential filter inhibitory activity and

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recovery rate.

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The qPCR method developed in this study was specific, sensitive and reproducible for the first quantification of toxic O. cf. ovata in marine aerosol.

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MATERIALS AND METHODS

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Study area. The present study was carried out at Llavaneres (41°33.13’N; 2°29.54’E) on the

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Catalan coast (Spain) from August 17-19, 2010 during an Ostreopsis spp. bloom event. Aerosol,

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seawater and macroalgal samples were collected intensively during a 36 h period.

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Sampling and sample treatment. A total of 12 marine aerosol samples were collected on 15 cm

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diameter QM-A quartz filters (Whatman, Maidstone, UK) using two High Volume Air Pump

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samplers (CAV-A/mb, 30 m3/h MCV, Barcelona, Spain). The air samplers worked continuously for

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3 days and filters were replaced every 6 or 7 h. In particular, six aerosol samples were collected

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with the sampling Air Pump located on the beach 0.5 m above sea level (Station S). The other six

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samples were collected with a second sampling Air Pump located 3 m above sea level, by a

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restaurant close to a large tank containing water pumped directly from the sea (Station A). The air

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samplers had a flow rate of 30 m3/h. Final samples of the quartz filters were split into two portions: ACS Paragon Plus Environment

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one half was directly placed in a 50 mL tube containing pure ethanol, and stored at +4°C until

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molecular analysis; the remaining half of the quartz filter was placed in a 50 mL tube containing

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pure methanol and stored at +4°C until toxin analysis.

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A total of 16 other field samples were collected: eight samples were wash seawater of the

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macroalga Corallina elongata and the other eight samples were surface seawater. Samples of C.

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elongata were harvested at a depth of 20-40 cm. Macroalgae and seawater samples were processed

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as described in Vila et al.25 and Battocchi et al.20 for microscopy and molecular analyses,

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respectively.

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Subsamples of sieved macroalgal washing seawater (20 mL), collected using a 140 or 200 µm net,

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were passed through GF/C filters (Whatman, Maidstone, UK). The filters were dried and stored at -

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20°C until toxin analyses.

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Microalgal cultures. A total of 51 Ostreopsis spp. isolates were obtained from surface seawater

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samples. Monoclonal cultures of Ostreopsis spp. were established as described by Penna et al.18 The

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isolates were maintained in F/4 medium at 23 ± 1°C. Light was provided by cool-white fluorescent

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bulbs (photon flux of 100 µE m-2 s-1) on a standard 14:10 h light/dark cycle. Other isolates of O. cf.

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ovata CBA1346 from the northwestern Adriatic Sea and O. cf. siamensis CSIC-D7 and VGO-

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OS5V from the Catalan Sea were also used in the molecular analyses of this study.

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Microscopy analyses. Counting was carried out on subsamples of the macroalgal microepiphytic

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assemblages (1-5 mL) and surface seawater (10-50 mL) as described by Battocchi et al.20

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Abundance in macroalga and surface seawater samples was expressed as the number of cells per

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gram of fresh weight (cells/g fw) and the number of cells per liter (cells/L), respectively. Ostreopsis

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spp. were identified under an inverted light microscope (Axiovert 40 CFL and Axiovert 135H, Zeiss

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or DM-IL, Leitz) at 200 or 400x magnification according to Fukuyo26 and Steidinger and Tangen27.

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The Utermöhl method was also used for the enumeration of O. cf. ovata CBA1681 for molecular

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filter spiking experiments.


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Quantitative PCR analysis of Ostreopsis cf. ovata. Marine aerosol sample processing. The

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preparation of samples (standard stringency washes) was performed as follows: the marine aerosol

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filter was placed in a tube containing 50 mL of pure ethanol and was shaken overnight at 60

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osc/min in order to wash the filter and recover the marine aerosol particulate. Next, the filter was

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discharged and the re-suspension was centrifuged at 4000 rpm for 10 min. The pellet was

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transferred to a 1.5 mL tube, centrifuged at 12000 rpm for 10 min and allowed to dry completely at

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room temperature for subsequent DNA extraction. Three new quartz filters were also processed and

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considered as blank samples.

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DNA extraction of field samples. Marine aerosol samples were lysed using a sonication procedure,

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while macroalgal microepiphytic assemblages, seawater and all Ostreopsis spp. cultured isolates

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were lysed using a freeze/thaw cycle protocol (three cycles of freeze/thaw at -80°C/+65°C for 15

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min). The lysis solution for all samples was prepared as described by Perini et al.23 The supernatants

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containing total genomic DNA (crude extract) were assayed by qPCR as described below. All

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samples analysed were processed in three determinations: 2 µl of undiluted, 1:10 and 1:100 diluted

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sub-samples of crude extracts. Results from 10 fold dilutions with a Ct difference between 3.3 and

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3.4 (∆Ct of 3.3 corresponds to an optimal efficiency of 100%) were accepted.

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Ostreopsis cf. ovata quantification. The field samples (marine aerosol, macroalgae and seawater)

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were analyzed by qPCR following the protocol of Perini et al.23 using designed primers for 204 bp

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specific amplified fragment. A plasmid (pLSUO) standard curve was constructed by amplifying 10-

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fold scalar dilutions with the copy number ranging from 1 x 106 to 1 x 102 (two replicates), and

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from 1 x 101 to 2 x 100 (four replicates). A cellular standard curve of four mixed crude extracts of 5

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x 102 or 2 x 103 cells from surface seawater samples taken at Llavaneres was generated with

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dilutions from 8 to 8 x 10-4 lysed cells. The qPCR assay was carried out in a final volume of 25 µL

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using the Hot-Rescue Real-Time PCR Kit-SG (Diatheva, Fano, Italy) in a StepOne Real-Time PCR

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System (Applied Biosystems, CA, USA). The thermal cycling conditions consisted of 10 min at

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95°C, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. No amplification of field ACS Paragon Plus Environment

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samples was evaluated by spiking amounts of 1 x 101 and 1 x 102 copies of pLSUO. The samples

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that were not inhibited are indicated as not detected.

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Quartz filter inhibitors and cell recovery rate. The inhibitory activity of the quartz filter was

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evaluated in spiking experiments. Amounts of 1 x 101 and 1 x 102 plasmid (pLSUO) copy number

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were added to ten-fold serial dilutions of crude extracts (1:1, 1:10, 1:100) and assayed by qPCR (n

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= 3 experiments).

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In order to determine the recovery rate of O. cf. ovata cells from marine aerosol samples, new

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quartz filters were contaminated by filtration under gentle vacuum (9.75 mmHg) with 2 mL of

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seawater solution containing different amounts (0.5 x 104, 1 x 103, 1 x 102, 5 x 101 or 1 x 101) of O.

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cf. ovata CBA1681 cells and assayed by qPCR. An amount of pure ethanol fixed 1 x 103 O. cf.

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ovata CBA1681 cells was harvested by centrifugation at 4000 rpm for 15 min. These cells were

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harvested, without the filtration step, and processed as the bioaerosol filters to simulate the same

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experimental conditions. This amount was established as 100% recovery rate of rDNA copy number

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per cell (n = 3 experiments).

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Quantitative PCR analysis of Ostreopsis cf. siamensis. O. cf. siamensis species-specific primer

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design was carried out using OLIGO ver. 6.65 and based on all Ostreopsis and other related

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dinoflagellates with the consensus LSU rRNA gene sequence alignment obtained by

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CLUSTALX2.28 Primers of Siamensis rt forward (5’-CACCACTGAGTGTGCGTACTG-3’) and

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Siamensis rt reverse (5’-GTTGGTGCGTACATTACTTCA-3’) were designed for the amplification

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of a 100 bp segment (Tm = 80.8°C) and synthesised by Eurofins MWG operon (Ebersberg,

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Germany). The primers were tested for species-specificity in silico by BLAST and in a qPCR assay

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on the genomic DNA of O. cf. ovata and O. cf. siamensis isolates and crude extracts from several

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macrophyte samples.

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To generate the plasmid standard curve (pLSUOs), the 645 bp partial LSU rDNA region was

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amplified with LSU D1R and LSU D2C primers29 from O. cf. siamensis VGO-OS5V genomic

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DNA and the fragment was cloned into the pCR 2.1 vector (Invitrogen, Carlsbad, CA, USA) ACS Paragon Plus Environment

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following the manufacturer’s instructions. The qPCR and thermal cycling conditions for

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quantitative O. cf. siamensis were the same as those used for O. cf. ovata with the only exception of

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the final primer concentration (200 nM). The method was applied to field samples and cultured

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isolates for O. cf. siamensis-specific identification and estimation.

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Quantitative PCR data analysis. Acquisition and data analyses were performed using StepOne

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Software ver. 2.1. Values were accepted when the PCR efficiency was between 95 and 100%

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(slope: -3.44 and -3.32). The total O. cf. ovata rDNA copy number of field (marine aerosol,

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macroalgal microepiphytic assemblages and seawater) and cultured isolate samples was obtained by

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interpolating the Ct on the plasmid standard. The cell number was calculated by dividing the total

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copy number by the normalized copy number per cell of O. cf. ovata collected at Llavaneres.

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Ostreopsis abundance in marine aerosol, macroalgae and surface seawater samples was expressed

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as cells/filter, cells/g fw and cells/L, respectively.

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Toxin analyses. A standard solution of palytoxin was obtained from Palythoa tuberculosa and

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provided by Wako Chemicals (Wako Chemical Industries, Ltd., Japan) as 100 µg of dry extract

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suspended in methanol/water (1:1 v/v) at final concentration of 25 ng/µL. The toxin content was

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determined in Ostreopsis spp. epiphytic assemblages from C. elongata samples. Subsamples of

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filtered macroalgal washing seawater (20 mL) were passed through GF/C filters; the filters were

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dried and stored frozen at -20°C until toxin analyses. Then, the filters were re-suspended in

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methanol and sonicated to homogenize the suspension using the 4710 sonication probe of an

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Ultrasonic Homogenizer (Cole-Parmer, Chicago, IL, USA). Finally, the suspension was centrifuged

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for 10 min at 7500 rpm at 10ºC. The supernatants were recovered and the filters were extracted

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again with methanol. Toxin extraction from aerosol filters (500 mL) was performed with boiling

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methanol at 70ºC in a Soxhlet extractor (Afora, Spain) for 10-12 hours. Distilled water was

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evaporated to dryness and then dissolved in 30 mL of methanol.


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Palytoxin was analyzed by a hemolytic assay30 and by liquid chromatography with fluorescence

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detection (LC-FLD).5 The limit of detection for palytoxin by these methods was 750 and 0.5 pg

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total for LC-FLD and the haemolytic assay, respectively.

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Environmental determinations. Meteorological data were obtained from the weather station

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situated in Port Balís (situated 1 km north of the Llavaneres sampling station).

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Statistical analyses. Data analyses were performed with parametric t-test, as well as non-

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parametric Mann-Whitney and Spearman correlation tests using GraphPad InStat (GraphPad

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Software Inc., La Jolla, CA, USA) and PAST ver. 2.0931 with a p-value < 0.05 determining

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significance.

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RESULTS

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Optimization of the lysis procedure. Two methods were tested to physically break cells: (i)

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sonication and (ii) freeze/thaw cycle protocols. The lysis efficiency was checked by microscopy by

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counting the entire cell number. Ethanol-fixed marine aerosol samples showed 35.5 ± 4.4% and

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88.3 ± 3.6% efficiency of the freeze/thaw cycles and sonication, respectively, while macroalgal

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microepiphytic assemblages, formalin-fixed seawater and cultures showed 99.6 ± 0.3% and 98.5 ±

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0.5% of efficiency, respectively. Therefore, in this study, sonication was used in marine aerosol

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samples and freeze/thaw cycles were applied in macroalgal microepiphytic assemblages, seawater

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and cultures.

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As macroalgal samples contain high amounts of debris, we checked for the presence of the

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inhibitors in equal amounts of Ostreopsis cells characterized by low, medium and high debris

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backgrounds (samples 2, 6 and 8, respectively) using 0.5 mL or 1 mL of lysis buffer. The results

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demonstrated no inhibitory effect on qPCR efficiency (see Supporting Information, Table S1).

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Subsequently, 0.5 mL of lysis buffer was used to process all kinds of environmental samples.

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Standard curve efficiency, reproducibility and cell copy number quantification. The typical

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characteristics of method validation were again confirmed as described by Perini et al.23 The use of ACS Paragon Plus Environment

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the pLSUO plasmid as the standard was validated. The PCR mean efficiency was 98-100% (mean

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standard curve: y = -3.34x + 34.48) with a linear correlation of a 6-log linear range (R2 = 0.99) and

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a quantification limit of 2 x 100 copies/reaction (Ct mean = 32.85 ± 0.66). The method

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reproducibility was assayed using the pLSUO standard curve by calculating the percentage of

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coefficient variation. The CVCt mean value of pLSUO was 1.5% and the CVCn mean value was 24%

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(see Supporting Information, Table S2).

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As the comparison of the pLSUO and cellular standard curves showed the same efficiency (98-

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100% and ∆s < 0.1, data not shown), it was possible to calculate the rDNA copy number per cell of

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O. cf. ovata by plotting the Ct mean value (23.42 ± 0.5) obtained from amplification of crude

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extract dilution corresponding to a single cell on the plasmid standard curve. The normalized copy

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number per cell of O. cf. ovata from Llavaneres was 2137 ± 190 (n = 5 experiments). This mean

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copy number per cell obtained from both 5 x 102 and 2 x 103 cells, 2126 ± 120 and 2170 ± 169,

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respectively, was not significantly different (t = 0.5963, P = 0.5599), showing that different cell

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concentrations do not affect the copy number per cell and quantification.

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Quantification of O. cf. ovata in marine aerosol samples. The potential presence of inhibitors in

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the quartz filters was verified by PCR spiking experiments. The Ct values of 30.9 ± 0.42 and 28.0 ±

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0.3 for 1 x 101 and 1 x 102 copy numbers, respectively, showed no significant difference (t = 1.481,

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P = 0.1769 and t = 1.439, P = 0.1880, respectively) (see also Table S2). The results showed that

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there was no inhibitory quartz filter effect.

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Due to the stringency of washes of the aerosol filters, Ostreopsis cellular loss needed to be

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evaluated by spiking experiments and, thus, by a recovery rate determination. The rDNA copy

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number per cell from the quartz filter was compared with that obtained from cultured O. cf. ovata

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CBA1681 ethanol-fixed cells (135471 ± 10714). The results showed a DNA recovery rate ranging

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from 65 to 20%. Quartz filters used as the blank revealed no amplification and no cross-activity

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(Table 1).


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The total rDNA content of O. cf. ovata in marine aerosol samples was estimated on the pLSUO

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curve and the abundance per quartz filter was calculated by dividing the total copy number by 2137

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± 190 (copy number per cell of O. cf. ovata). Samples collected at two Llavaneres stations were

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quantified by taking into account the percentage of total DNA recovery. In particular, the final

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abundance per filter in samples containing a cellular range from 0.2 to 7.9 was obtained by applying

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a recovery rate of 20%, while a recovery rate of 36% was applied for samples containing a cell

7

number higher than 27 cells per filter (samples 1A and 5A). Because marine aerosol samples were

8

derived from different volumes of filtered air, the cell number per filter was also normalized to the

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smaller volume (166 m3). The two sampling stations showed variable cell numbers ranging from 1

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to 102 with no amplification for sample 3A tested by a spiking experiment (Table 2). In particular,

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the mean Ct (31.11 ± 0.3 and 27.84 ± 0.2, for 1 x 101 and 1 x 102 spiked copies, respectively, n = 3

12

experiments) was not significantly different to the Ct of the plasmid standard curve (31.12 ± 0.3 and

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27.7 ± 0.25, for 1 x 101 and 1 x 102 copies, respectively, n = 3 experiments) (t = 0.0379, P = 0.9707,

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and t = 0.3846, P = 0.7105, respectively), indicating that no amplification of the marine aerosol

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sample was actually due to the absence of the LSU rDNA target sequence of O. cf. ovata and not to

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the presence of inhibitors.

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Abundance of O. cf. ovata and toxin content in environmental samples. In order to obtain the

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correct quantification of O. cf. ovata cells, the macroalgal microepiphytic assemblage and surface

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seawater samples were tested by qPCR to exclude the presence of O. cf. siamensis. No PCR

20

amplifications were obtained (data not shown). Furthermore, this evidence was also confirmed by

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no amplifications of 51 algal isolates positive only for O. cf. ovata. Therefore, all Ostreopsis spp.

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cells observed by microscopy were considered as belonging to O. cf. ovata.

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The macroalgal microepiphytic assemblage and surface seawater samples were analyzed by both

24

microscopy and qPCR assay. The temporal trend in O. cf. ovata mean abundance during the study

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period in the macroalgae, water column and marine aerosol is shown in Figure 1. During the three

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sampling days, the benthic macrophyte communities were covered by brown mucilage, which was ACS Paragon Plus Environment

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clearly dominated by O. cf. ovata. Abundance ranged between 7 x 104 and 7.6 x 105 cells/g fw in

2

macroalgae and between 3.5 x 103 and 1.1 x 105 cells/L in seawater. There was a significant

3

positive correlation between cell densities on the macroalga C. elongata and in the water column (n

4

= 8, Spearman r = 0.8095, P = 0.0218). Furthermore, a significant correlation was found between O.

5

cf. ovata abundance recovered by optical microscopy and qPCR determinations (n = 8, Spearman r

6

= 0.9286, P = 0.0022 and n = 8, Spearman r = 1, P < 0.0001 for macroalgae and surface seawater

7

samples, respectively).

8

A significant positive correlation (n = 6, Spearman r = 0.9276, P = 0.0167) was found between

9

O. cf. ovata abundance in the surface seawater and marine aerosol samples collected at Station S on

10

the beach, showing that higher abundance in the seawater is directly linked to an increased

11

abundance of airborne O. cf. ovata cells. Instead, no significant correlation was found between the

12

abundance in surface water and marine aerosol samples collected at Station A near the restaurant

13

and the tank (n = 5, Spearman r = - 0.9, P = 0.0833). The abundance of airborne O. cf. ovata was

14

significantly different between the two sampling stations (U = 44.5, P = 0.0011).

15

Then, all environmental samples including the quartz aerosol filters, macroalgae and seawater

16

samples were analyzed for the presence of palytoxin-like compounds by analytical and bio-

17

analytical determinations. The palytoxin content ranged from 0.1 to 1.2 pg/cell in macroalgal

18

microepiphytic assemblages during the bloom. The estimation of the palytoxin-like content was

19

calculated by taking into account the O. cf. ovata abundance both by qPCR and microscopy, which

20

showed no significant differences (t = 0.1052, P = 0.9177) (Table 3).

21

However, it was not possible to define the palytoxin content in aerosol filters because of the LOD of

22

the analytical and hemolytic assays employed in this study.

23

The Ostreopsis cf. siamensis qPCR assay. The designed primers were found to be species-specific

24

by BLAST. The primers were also tested in a qPCR assay using crude extracts of O. cf. ovata and

25

O. cf. siamensis isolates. Moreover, the primers were checked with crude extracts from several

26

macroalgal wash water samples collected from a coastal area of the central Adriatic Sea, where ACS Paragon Plus Environment

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1

Ostreopsis spp. has not been detected by microscopy analysis. All analyses demonstrated the high

2

species-specificity of the primers (data not shown). The O. cf. siamensis (pLSUOs) plasmid mean

3

standard curve (y = -3.36x + 36.66) showed an efficiency of 98 - 100%, a linear correlation of R2 =

4

0.99 and a quantification limit of 2 x 100 copies/reaction (Ct mean = 34.2 ± 0.8). The

5

reproducibility assayed by CVCn of the pLSUOs standard curve was 19% in the 1 x 106 to 2 x 100

6

copy number range (see Supporting Information, Table S3).

7

Environmental conditions. During the three consecutive sampling days, highly stable weather

8

conditions were observed, characterized by breezy conditions with a prevalent direction of 157-

9

202° (SSE, S and SSW) during the day. During the afternoon and evening the wind intensity ranged

10

between 10-16 m/s.

11 12

DISCUSSION

13

In the Mediterranean Sea, since 2004-2005, severe blooms of the toxic benthic dinoflagellate O.

14

cf. ovata have occurred in association with harmful marine aerosol formation.32 O. cf. ovata has

15

been found to produce PLTX and ovatoxins.6 During these intense bloom events, people

16

frequenting beaches or coastal littorals have been affected by respiratory and febrile syndromes.13 In

17

some cases, such as the Genoa bloom of O. cf. ovata in 2005, people exposed to sea-spray aerosol

18

during recreational bathing activities were hospitalized.2 In fact, it has been hypothesized that

19

respiratory intoxication seemed to be due to inhalation of seawater spray containing Ostreopsis cells

20

or cell fragments and aerosolized palytoxin-like compounds.12 Therefore, Ostreopsis blooms

21

represent emergent health and economic risks to human populations in the Mediterranean Sea that

22

must be urgently faced.

23

The presence of toxic microalgal species, such as O. cf. ovata, or aerosolized toxic compounds

24

in marine aerosol is probably related to algal species abundance and palytoxin-like compound

25

concentrations in the upper layer of surface seawater and physical mechanisms of aerosol formation

26

that are at play (e.g. wind).33 Therefore, in relation to that the current study was designed to develop ACS Paragon Plus Environment

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1

a qPCR assay for the quantification of toxic O. cf. ovata in marine aerosol. In particular, in order to

2

obtain DNA free from inhibitors and ready to use for qPCR from this complex matrix, standard

3

stringency washes and the DNA extraction method were optimized. The potential inhibitory activity

4

of the quartz filter and crude extracts from marine aerosol samples was checked by spiking PCR

5

experiments and by assaying efficiency in the qPCR assay. The results show no inhibitor effect in

6

either matrix. Moreover, the DNA recovery rate percentages from filters were efficient in relation to

7

the spiked cell number; marine aerosol sample quantification took these values into account. The

8

high sensitivity (2 x 100 copies of rDNA quantified) and specificity was demonstrated and the

9

reproducibility was realistic over the checked dynamic range displaying the reliability and accuracy

10

of the technical set-up over time and over a low range of quantification.

11

Given the potential variation in the rRNA gene copy number in O. cf. ovata,23 the strategy of

12

pooling different genomic DNAs from crude extracts of seawater samples was used to generate a

13

cellular standard curve to calculate the rDNA copy number/cell at the Llavaneres site. Then, we

14

applied the method for the estimation of O. cf. ovata abundance in marine aerosol samples collected

15

from the two sampling stations at Llavaneres beach during the bloom event of August 2010. It was

16

found that the O. cf. ovata abundance in marine aerosol samples collected at two stations was

17

significantly different. A significant positive correlation was found between O. cf. ovata abundance

18

in surface seawater and marine aerosol samples collected at Station S, on the beach, showing that

19

greater abundance in seawater is directly linked to increased abundance of O. cf. ovata cells in

20

marine aerosol. In contrast, no significant correlation was found between the abundance in surface

21

seawater and marine aerosol samples collected at the Station A, near the tank. However, these latter

22

marine aerosol samples from Station A contained the highest abundance of O. cf. ovata (from 12 to

23

102 cells per filter). This result may be related to the large tank near sampling Station A containing

24

seawater with O. cf. ovata cells pumped directly from the coast. The tank likely acted as a

25

mesocosm, allowing the proliferation of Ostreopsis that reached a higher concentration inside (1.4 x

26

106 cells/L) with respect to open seawater. The particular location of the aerosol pump sampler ACS Paragon Plus Environment

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1

behind the large tank was affected by south-easterly winds, which probably directed aerosol

2

droplets from the tank to the pump sampler.

3

The high-potential of the molecular method was again shown by the significant correlation

4

between qPCR and light microscopy of O. cf. ovata abundance determinations in natural samples.

5

Environmental samples of macroalgae and marine aerosol were also analyzed for the content of

6

PLTX-like compounds. Samples of epiphytic O. cf. ovata on C. elongata were positive for the

7

presence of PLTX compounds. The PLTX-like compounds, either aerosolized or contained in cells,

8

were not detected in aerosol samples, in contrast with the qPCR estimation of O. cf. ovata cells.

9

Although presence of O. cf. ovata in the aerosol filters was demonstrated by the molecular

10

determination it was not possible detect PLTX-like compounds by hemolytic assay neither LC-FLD

11

due to the sensitivity limit of detection of both methods. We assumed that in the sample with the

12

highest Ostreopsis abundance recorded in the aerosol filters by qPCR, which was 102 ± 3.8 cells

13

after 340 min of exposure and a filtered air volume of 166 m3, the palytoxin estimations in pg/cell

14

and the total toxin amount in the sample analyzed by the hemolytic assay should range between

15

0.01 and 0.1 pg total, which was five times lower than the LOD for this analysis, corresponding to

16

0.5 pg total. Further, considering the LC-FLD technique, the difference should be increased, since

17

estimated amounts ranged between 0.2 - 2 pg of palytoxin injected, with a LOD of 750 pg.

18

Moreover, the analysis of cultured extracts of O. cf. ovata isolates from Llavaneres by MALDI-

19

TOF15 revealed that the main ion detected in positive mode in O. cf. ovata extracts was m/z 2670

20

[M+Na+]+ which matched up with ovatoxin-a described by Ciminiello et al.6 in O. cf. ovata from

21

the Mediterranean areas.

22

Furthermore, we assumed that aerosolized palytoxin compounds could have passed through

23

the quartz filters, thereby negatively affecting the analytical determination of toxin levels. To test

24

this hypothesis, a control experiment was performed by bubbling filtered air through the quartz

25

filters in a distilled water tank in order to dissolve and collect potential PLTX-like compounds. No


15


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1

toxin compounds were present in the bubbled filtered water according to the analytical and

2

hemolytic determinations (data not reported).

3

Nevertheless, although the analytical determinations of PLTX in both aerosol and seawater

4

(data not shown) samples failed, some people frequenting the beach and restaurant during the O. cf.

5

ovata bloom reportedly experienced symptoms, such as rhinitis and fever. Only a few people

6

experienced irritation of the inner arms, strong irritation of the lips, scratchy throat and eye irritation

7

after snorkelling in the area. In particular, some people experienced a metallic taste in the mouth,

8

the typical symptom of palytoxin intoxication or irritation.34,35 In fact, recent evidence has

9

highlighted that palytoxin, and most likely, its congener Ostreopsis cf. ovata toxins, have the

10

potential to exert a pro-inflammatory response.36 Unfortunately, toxicological data referring to

11

palytoxin intoxication by inhalation are still scarce.37 In this study, based on our qPCR

12

quantification of O. cf. ovata cells in marine aerosol filters and the highest PLTX content per cell

13

recorded (1.2 pg), and based on human respiratory capacity in 24 h (10 m3),38 we speculated that a

14

person could inhale only a very small amount of PLTX, i.e. about 7.3 pg. It is likely that the

15

symptoms in sensitive people and/or those exposed to a level of aerosol could be caused by

16

palytoxin irritation together with other potentially allergenic substances derived from Ostreopsis

17

cells and associated microbial flora.35,39

18

In conclusion, the developed qPCR assay is a highly sensitive and specific quantification

19

method for toxic O. cf. ovata in a complex matrix, such as marine aerosol. In the future, the

20

application of the developed molecular assay together with the analytical technique and

21

epidemiological studies in areas affected by toxic blooms will allow us to relate the Ostreopsis

22

abundance in marine aerosol with respiratory syndromes in humans.

23 24

Moreover, efficient monitoring through accurate and adequate sampling will be fundamental to manage and prevent health and economic risks related to O. cf. ovata blooms in coastal areas.

25 26 ACS Paragon Plus Environment

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1

Acknowledgments

2

Thanks to Dr. Andrés Alastuey and collaborators from Institut Jaume Almera (CSIC) for lending us

3

High Volume Air Pumps; Cecilia Battocchi, Isabel Bravo, Mercedes Masó and Francisco Rodriguez

4

for sampling Ostreopsis at Llavaneres; the Aceña family of Restaurant Pins Mar (Sant Andreu) for

5

kindly offering us the use of their facilities; Isabel Manzano from CZ Veterinaria for supplying the

6

sheep blood for hemolytic assays. Meteorological data were kindly provided by the weather station

7

situated in Port Balís. Financial support was provided to A. P. by an Italian PRIN 2009 Project and

8

PNRA 2010 from MIUR; a Spanish EBITOX national project (CTQ 2008-06754-C04-04) and from

9

CCVIEO- Microalgae Culture Collection of Instituto Español de Oceanografía.

10 11

Supporting Information Available

12

Table S1: Ostreopsis cf. ovata LSU rDNA copy number per cell in Corallina elongata wash

13

seawater using different lysis volumes.

14

Table S2: Reproducibility of the qrt-PCR assay based on pLSUO plasmid standard curve.

15

Table S3: Reproducibility of the qrt-PCR assay of O. cf. siamensis based on pLSUOs plasmid

16

standard curve.

17

This information is available free of charge via the Internet at http://pubs.acs.org/.

18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

REFERENCES (1) O’Dowd, C. D.; de Leeuw, G. Marine Aerosol Production: a review of the current knowledge. Phil. Trans. R. Soc. A 2007, 365, 1753–1774. (2) Gallitelli, M.; Ungaro, N.; Addante, L. M.; Gentiloni Silver, N.; Sabbà, C. Respiratory illness as a reaction to tropical algal blooms occurring in a temperate climate. JAMA 2005, 293, 2599– 2600. (3) Ukena, T.; Satake, M.; Usami, M.; Oshima, Y.; Naoki, H.; Fujita, T.; Kan, Y; Yasumoto, T. Structure elucidation of ostreocin d, a palytoxin analog isolated from the dinoflagellate Ostreopsis siamensis. Biosci. Biotech. Biochem. 2001, 65, 2585–2588. (4) Lenoir, S.; Ten-Hage, L.; Turquet, J.; Quod, J. P.; Bernard, C.; Hennion, M. C. First evidence ACS Paragon Plus Environment

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of palytoxin analogues from an Ostreopsis mascarenensis (Dinophyceae) benthic bloom in the southwestern Indian Ocean. J. Phycol. 2004, 40, 1042–1051. (5) Riobó, P.; Paz, B.; Franco, J. M. Analysis of palytoxin-like in Ostreopsis cultures by liquid chromatography with precolumn derivatization and fluorescence detection. Anal. Chem. Acta 2006, 566, 217–223. (6) Ciminiello, P.; Dell’Aversano, C.; Dello Iacovo, E.; Fattorusso, E.; Forino, M.; Grauso, L.; Tartaglione, L.; Guerrini, F.; Pistocchi, R. Complex palytoxin-like profile of Ostreopsis ovata. Identification of four new ovatoxins by high-resolution liquid chromatography/mass spectrometry. Rapid Commun. Mass Spectrom. 2010, 24, 2735–2744. (7) Ciminiello P.; Dell’Aversano C.; Dello Iacovo E.; Fattorusso E.; Forino M.; Tartaglione L.; Crinelli R.; Carloni E.; Magnani M.; Battocchi C.; Penna A. The unique toxin profile of a Mediterranean Ostreopsis cf. ovata strain. HR LC-MSn characterization of ovatoxin-f, a new palytoxin congener. Chem. Res. Toxicol. 2012, 25, 1243–1252. (8) Ciminiello P.; Dell’Aversano C.; Dello Iacovo E.; Fattorusso E.; Forino M.; Grauso L.; Tartaglione L. Stereochemical Studies on Ovatoxin-a. Chem. Eur. J. 2012, 18, 16836–16843. (9) Ciminiello, P.; Dell’Aversano, C.; Dello Iacovo, E.; Fattorusso, E.; Forino, M.; Grauso, L.; Tartaglione, L.; Guerrini, F.; Pezzolesi, L.; Pistocchi, R.; Vanucci, S. Isolation and structure elucidation of ovatoxin-a, the major toxin produced by Ostreopsis ovata. J. American Chem. Soc. 2012, 134, 1869–1875. (10) Barroso García, P.; Rueda de la Puerta, P.; Parron Carreño, T.; Marín Martínez, P.; Guillen Enríquez, J. Brote con síntomas respiratorios en la provincia de Almería por una posible exposicion a microalgas toxicas. Gac. Sanit. 2008, 22, 578–584. (11) Tichadou, L.; Glaizal, M.; Armengaud, A.; Grossel, H.; Lemée, R.; Kantin, R.; Lasalle, J. L.; Drouet, G.; Rambaud, L.; Malfait, P.; Haro, L. D. Health impact of unicellular algae of the Ostreopsis genus blooms in the Mediterranean Sea: experience of the French Mediterranean coast surveillance network from 2006 to 2009. Clin. Toxicol. 2010, 48, 839–844. (12) Mangialajo, L.; Ganzin, N.; Accoroni, S.; Asnaghi, V.; Blanfuné, A.; Cabrini, M.; CattaneoVietti, R.; Chavanon, F.; Chiantore, M.; Cohu, S.; Costa, E.; Fornasaro, D.; Grossel, H.; MarcoMiralles, F.; Masó, M.; Reñé, A.; Rossi, A. M.; Montserrat Sala, M.; Thibaut, T.; Totti, C.; Vila, M.; Lemée, R. Trends in Ostreopsis proliferation along the Northern Mediterranean coasts. Toxicon 2011, 57, 408–420. (13) Tubaro, A.; Durando, P.; Del Favero, G.; Ansaldi, F.; Icardi, G.; Deeds, J. R.; Sosa, S. Case definitions for human poisonings postulated to palytoxins exposure. Toxicon 2011, 57, 478–495. (14) Parsons, M. L.; Aligizaki, K.; Bottein, M-Y. D; Fraga, S.; Morton, S. L.; Penna, A.; Rhodes, L. Gambierdiscus and Ostreopsis: reassessment of the state of knowledge of their taxonomy, geography, ecophysiology, and toxicology. Harmful Algae 2012, 14, 107–129. (15) Paz, B.; Riobó, P.; Franco, J. M. Preliminary study for rapid determination of phycotoxins in microalgae whole cells using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Comm. Mass Spectrom. 2011, 25, 3627–3639.


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(16) Ciminiello, P.; Dell’Aversano, C.; Dello Iacovo, E.; Fattorusso, E.; Forino, M.; Tartaglione, L.; Yasumoto, T.; Battocchi, C; Giacobbe, M.G.; Amorim, A.; Penna, A. Investigation of toxin profile of Mediterranean and Atlantic strains of Ostreopsis cf. siamensis (Dinophyceae) by liquid chromatography–high resolution mass spectrometry. Harmful Algae 2013, 23, 19–27. (17) Penna, A.; Vila, M.; Fraga, S.; Giacobbe, M.G.; Andreoni, F.; Riobó, P.; Vernesi, C. Characterization of Ostreopsis and Coolia (Dinophyceae) isolates in the Western Mediterranean Sea based on morphology, toxicity and internal transcribed spacer 5.8S rDNA sequences. J. Phycol. 2005, 41, 212–225. (18) Penna, A.; Fraga, S.; Battocchi, C.; Casabianca, S.; Giacobbe, M. G.; Riobo, P.; Vernesi, C. A phylogeographical study of the toxic benthic dinoflagellate genus Ostreopsis Schmidt. J. Biogeogr. 2010, 37, 380–841. (19) Bravo, I.; Vila, M.; Casabianca, S.; Rodriguez, F.; Rial, P.; Riobò, P.; Penna, A. Life cycle stages of the benthic palytoxin-producing dinoflagellate Ostreopsis cf. ovata (Dinophyceae). Harmful Algae 2012, 18, 24–34. (20) Battocchi, C.; Totti, C.; Vila, M.; Masó, M.; Capellacci, S.; Accoroni, S.; Reñé, A.; Scardi, M.; Penna, A. Monitoring toxic microalgae Ostreopsis (dinoflagellate) species in coastal waters of the Mediterranean Sea using molecular PCR-based assay combined with light microscopy. Mar. Poll. Bull. 2010, 60, 1074–1084. (21) Pezzolesi, L.; Guerrini, F.; Ciminiello, P.; Dell’Aversano, C.; Iacovo, E. D.; Fattorusso, E.; Forino, M.; Tartaglione, L.; Pistocchi, R. Influence of temperature and salinity on Ostreopsis cf. ovata growth and evaluation of toxin content through HR LC-MS and biological assays. Wat. Res. 2012, 46, 82–92. (22) 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. Ostreopsis cf. ovata bloom in the northern Adriatic Sea during summer 2009: ecology, molecular characterization and toxin profile. Mar. Poll. Bull. 2011, 62, 2512–2519. (23) Perini, F.; Casabianca, A.; Battocchi, C.; Accoroni, S.; Totti, C.; Penna, A. New approach using the real-time PCR method for estimation of the toxic marine dinoflagellate Ostreopsis cf. ovata in marine environment. PLoS ONE 2011, 6, e17699. (24) Pyankov, O. V.; Agranovski, I. E.; Pyankov, O.; Mokhonova, E.; Mokhonov, V.; Safatov, A. S.; Khromykh, A. A. Using a marine aerosol personal sampler in combination with real-time PCR analysis for rapid detection of airborne viruses. Environ. Microbiol. 2007, 9, 992–1000. (25) Vila, M.; Garcés, E.; Masó, M. Potentially toxic epiphytic dinoflagellates assemblages on macroalgae in the NW Mediterranean. Aquat. Microb. Ecol. 2001, 26, 51–60. (26) Fukuyo, Y., Taxonomical study on benthic dinoflagellates collected in coral reefs. Bull. Jap. Soc. Sci. Fish. 1981, 47, 967–978. (27) Steidinger, K.A.; Tangen, K. Dinoflagellates. In Identifying Marine Phytoplankton; Tomas, C. R., Ed.; Academic Press: San Diego, 1997; pp 387–584.


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(28) Larkin, M. A.; Blackshields, G.; Brown, N. P.; Chenna, R.; McGettigan, P. A.; McWilliam, H.; Valentin, F.; Wallace, I. M.; Wilm, A.; Lopez, R.; Thompson, J. D.; Gibson, T. J.; Higgins, D. G. Clustal W and Clustal X version 2.0. Bioinformatics 2007, 23, 2947–2948. (29) Scholin, C. A.; Herzog, M.; Sogin, M.; Anderson, D. M. Identification of group and strainspecific genetic markers from globally distributed Alexandrium (Dinophyceae). II. Sequence analysis of fragments of the LSU rRNA gene. J. Phycol. 1994, 30, 999–1011. (30) Riobó, P.; Paz, B.; Franco, J. M.; Vázquez, J. A.; Murado, M. A. Proposal for a simple and sensitive haemolytic assay for palytoxin. Toxicological dynamics, kinetics, ouabain inhibition and thermal stability. Harmful Algae 2008, 7, 415–429. (31) Hammer, Ø.; Harper, D. A. T.; Ryan P. D. PAST: Paleontological Statistics Software Package for Education and Data Analysis. Palaeont. Electr. 2001, 4, 1–9. (32) Ciminiello, P.; Dell’Aversano, C.; Fattorusso, E.; Forino, M.; Magno, G. S.; Tartaglione, L.; Grillo, C.; Melchiorre, N. The Genoa 2005 outbreak. Determination of putative palytoxin in Mediterranean Ostreopsis ovata by a new Liquid Chromatography Tandem Mass Spectrometry method. Anal. Chem. 2006, 78, 6153–6159. (33) Urbano, R.; Palenik, B.; Gaston, C. J.; Prather, K. A. Detection and phylogenetic analysis of coastal marine aerosols using culture dependent and independent techniques. Biogeosciences 2011, 8, 301–309. (34) Ramos, V.; Vasconcelos, V. Palytoxin and Analogs: Biological and Ecological Effects. Mar. Drugs. 2010, 8, 2021–2037. (35) Vila, M.; Arin, L.; Battocchi, C.; Bravo, I.; Fraga, S.; Penna, A.; Reñé, A.; Riobó, P.; Rodriguez, F.; Montserrat Sala, M.; Camp, J.; de Torres, M.; Franco, J. M. Management of Ostreopsis blooms in recreational waters along the Catalan coast (NW Mediterranean Sea): cooperation between a research project and a monitoring program. Cryptogamie 2012, 33, 143–152. (36) Crinelli, R.; Carloni, E.; Giacomini, E.; Penna, A.; Dominici, S.; Battocchi, C.; Ciminiello, P.; Dell'Aversano, C.; Fattorusso, E.; Forino, M.; Tartaglione, L.; Magnani, M. Palytoxin and an Ostreopsis Toxin Extract Increase the Levels of mRNAs Encoding Inflammation-Related Proteins in Human Macrophages via p38 MAPK and NF-kB. PLoS ONE 2012, 7, e38139. (37) Ito E.; Yasumoto, T. Toxicological studies on palytoxin and ostreocin-d administered to mice by three different routes. Toxicon 2009, 54, 244–251. (38) Grönkvist, M. J.; Emery M. J.; Gustafsson, P. M. Mechanisms of ventilation inhomogeneity during vital capacity breaths standing and supine. Respir. Physiol. 2002, 129, 345–355. (39) Delort, A.M.; Vaïtilingom, M.; Amato, P.; Sancelme, M.; Parazols, M.; Mailhot, G.; Laj, P.; Deguillaume, L. A short overview of the microbial population in clouds: Potential roles in atmospheric chemistry and nucleation processes. Atm. Res. 2010, 98, 249–260.


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1 2 3

4 5 6 7 8 9 10

Table 1. Spiking experiments and DNA recovery rate of Ostreopsis cf. ovata CBA1681 on quartz filters. Spiked cell number

LSU rDNA copy number/ cell ± SDa

CVcn (%)b

DNA recovery rate (%)c

0.5 x 104

88056 ± 8189

11

65

1 x 103

67576 ± 7842

12

50

1 x 102

51417 ± 8462

11

38

5 x 101

48555 ± 5409

16

36

1 x 101

26451± 3641

14

20

a

Mean LSU rDNA copy number per cell measured in triplicates ± standard deviation (SD). Coefficient of variation of copy number. c This data was obtained considering 135471 ± 10714 of O. cf. ovata CBA1681 copy number per cell representing 100% recovery. b


21


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1 2 3

4 5 6 7 8 9 10 11

Table 2. Quantitative PCR assay of Ostreopsis cf. ovata abundance in marine aerosol samples collected at Stations S and A at Llavaneres beach. Abundance/filter Abundance/filter Abundance/filter (166 m3)d with recovery ratec

Sample n°

Sampling day on August

Time interval (h)

Filtering duration (min)

Filtered air volume (m3)

Total LSU rDNA copy number/filtera

1S

17th

11:00 - 18:00

420

209

447 ± 161

0.2 ± 0.1

1 ± 0.4

1 ± 0.3

2S

17th

18:00 - 02:15

495

243

520 ± 260

0.2 ± 0.1

1 ± 0.6

1 ± 0.4

3S

18th

02:20 - 08:35

375

184

788 ± 394

0.4 ± 0.2

2 ± 0.9

2 ± 0.8

4S

18th

08:38 - 17:36

451

271

15174 ± 349

7.1 ± 0.2

36 ± 0.8

22 ± 0.5

5S

18th

17:42 - 01:20

458

225

6245 ± 1271

2.9 ± 0.6

15 ± 3

11 ± 2.2

6S

19th

01:30 - 07:54

394

194

12845 ± 1494

6 ± 0.7

30 ± 3.5

26 ± 3

1A

17th

12:20 - 18:00

340

166

78091 ± 2934

36.5 ± 1.4

101 ± 3.8

102 ± 3.8

2A

17th

18:00 - 02:23

503

255

16906 ± 3036

7.9 ± 1.4

40 ± 7.1

26 ± 4.6

3A

18th

02:38 - 08:26

348

170

n.d.b

n.d.b

n.d.b

n.d.b

4A

18th

08:31 - 17:25

534

269

9211 ± 576

4.3 ± 0.3

22 ± 1.3

13 ± 0.8

5A

18th

17:30 - 01:00

450

221

57429 ± 1795

26.9 ± 0.8

75 ± 2.3

56 ± 1.8

6A

19th

01:00 - 07:50

410

200

6398 ± 1538

3 ± 0.7

15 ± 3.6

12 ± 3

a

Mean values measured in triplicates ± standard deviation (SD). Not detected. c Abundance per filter in samples from 1S to 6S and samples 2A, 4A, 6A were obtained considering a recovery rate of 20%; abundance per filter in samples 1A and 5A were obtained considering a recovery rate of 38%. d Abundance per filter normalized to smaller volume of filtered air (166 m3). b


22

Page 23 of 25


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1 2 3 4

Table 3. Toxin content determined by LC-FLD of epiphytic Ostreopsis cf. ovata collected from macroalga Corallina elongata at Llavaneres beach. Sample no.

Sampling day on August

Palytoxin content (pg/cell)a ______________________________ qPCR

27 28 29

a

Microscopy

1

17th

0.12

0.11

2

17th

0.83

1.11

3

17th

0.47

0.38

4

17th

1.12

1.23

5

18th

0.26

0.27

6

18th

0.42

0.41

7

18th

0.82

0.68

8

19th

0.42

0.42

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Toxin content expressed as pg/cell determined by both qPCR assay and microscopy.


23


Page 24 of 25

24

5

10

4

10

3

10

2

10

1

10

0

20 Aug

10

19 Aug

6

17 Aug

10

18 Aug

qPCR seawater qPCR macroalgae qPCR aerosol

Ostreopsis cf.ovata abundance by q-PCR

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Sampling period

1 2

Figure 1. Quantitative PCR abundance of Ostreopsis cf. ovata on aerosol filters, surface water and

3

macroalgal microephiphytic assemblages at Station S at Llavaneres beach during the sampling

4

period. O. cf. ovata abundance determined by optical microscopy was omitted due to significant


24

Page 25 of 25


25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

correlation with qPCR abundance for both macroalgae and seawater samples (n = 8, Spearman r =

2

0.9286, P = 0.0022 and n = 8, Spearman r = 1.000, P < 0.0001 for macroalgae and surface seawater

3

samples, respectively).


25

Quantification of the Toxic Dinoflagellate Ostreopsis spp. by qPCR

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