Harmful Algae 36 (2014) 1–10

Growth dynamics in relation to the production of the main cellularcomponents in the toxic dinoflagellate Ostreopsis cf. ovata

Laura Pezzolesi a,*, Rossella Pistocchi a,**, Francesca Fratangeli a, Carmela Dell’Aversano b,Emma Dello Iacovo b, Luciana Tartaglione b

a Department of Biological, Geological and Environmental Sciences (BiGeA), University of Bologna, Via S’Alberto 163, 48123 Ravenna, Italyb Department of Pharmacy, University of Naples ‘‘Federico II’’, Via D. Montesano 49, 80131 Naples, Italy

A R T I C L E I N F O

Article history:

Received 2 August 2013

Received in revised form 25 March 2014

Accepted 25 March 2014

Keywords:

Ostreopsis

Palytoxin

Ovatoxin

Proteins

Polysaccharides

Lipids

A B S T R A C T

In the last decade Ostreopsis cf. ovata blooms have been among the most intense along the entire

Mediterranean coast, leading to ecological and human health problems, that are associated with the

toxins (palytoxin-like compounds) produced by these algal cells. These compounds are secondary

metabolites, whose rates of synthesis depend on the metabolism of their precursors. In general, growth

dynamics and toxicity of dinoflagellates reflect the physiological status of the organism. The aim of the

present study was to investigate the cellular production of the main biochemical compounds likely

involved in the growth and toxicity dynamics of O. cf. ovata during exponential to the late stationary

phase in batch cultures of an Adriatic strain. Removal of major nutrients from the medium was

monitored along with concentration, biovolume and production of the main cellular components (e.g.

polysaccharides, proteins, lipids and toxins). Nutrient uptake, as well as toxin production rates were

calculated in the different growth periods. Nutrients (N and P) were completely depleted when cells

entered stationary phase and the greatest net toxin production rate (RTOX) occurred during the first days

of growth. The various palytoxins reported a relative abundance quite stable during the different growth

phases, while the total toxin cellular amount increased along the growth curve. Total and extracellular

released polysaccharides, as well as the lipid content increased greatly during the stationary phase, while

proteins were mainly produced by cells during the exponential phase. The continuous release of

polysaccharides could facilitate cell aggregation and the formation of the benthic community during

algal blooms. The trend of production of the main cellular compounds in O. cf. ovata and the growth

dynamics of this species lead us to hypothesize that the fast growth of this dinoflagellate, associated with

the rapid use of environmental resources (nutrients, and phosphates in particular), may be an ecological/

adaptive strategy which could favor this organism in competition with other species.

� 2014 Elsevier B.V. All rights reserved.

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Harmful Algae

jo u rn al h om epag e: ww w.els evier .c o m/lo cat e/ha l

1. Introduction

Several dinoflagellate species are known to form dense bloomsposing severe health problems and ecosystem threats through theproduction of toxins or other noxious substances.

As most of these toxins are secondary metabolites, their rate ofsynthesis depends on the metabolism of their precursors (i.e.primary metabolites). Due to the complex regulation of primarymetabolic processes, it is not easy to predict the conditions thatpromote or depress the synthesis of toxins (Pistocchi, 2014).

* Corresponding author. Tel.: +39 0544 937372; fax: +39 0544 937411.** Corresponding author. Tel.: +39 0544 937376; fax: +39 0544 937411.

E-mail addresses: laura.pezzolesi@unibo.it (L. Pezzolesi),

rossella.pistocchi@unibo.it (R. Pistocchi).

http://dx.doi.org/10.1016/j.hal.2014.03.006

1568-9883/� 2014 Elsevier B.V. All rights reserved.

Moreover, secondary metabolites may be more likely affected byperturbations as they are not fully integrated into the homeostasisof the cell.

In general, growth dynamics and toxicity of dinoflagellatesreflect the physiological status of the organism (Flynn and Flynn,1995). In order to better investigate the physiological mechanismsof population dynamics and bloom formation in dinoflagellates, itis necessary to improve the knowledge of growth regulation,nutrient uptake and starvation responses. At the same time otherfactors, such as cellular biosynthesis, regulation of toxins andallelochemicals, may be likely implicated in the bloom dynamics.Presently there are few studies concerning some of these aspects indinoflagellates and they are mainly focused on paralytic shellfishpoisoning (PSP)- toxins producing algae, such as Alexandrium spp.and Gymnodinium catenatum (Anderson et al., 1990; Flynn et al.,1994, 1996; Flynn and Flynn, 1995; Cembella, 1998; John and

L. Pezzolesi et al. / Harmful Algae 36 (2014) 1–102

Flynn, 2000; Lippemeier et al., 2003; Etheridge and Roesler, 2005;Touzet et al., 2007; Li et al., 2011; Yang et al., 2011; Xu et al., 2012).

Among the dinoflagellates, species of Ostreopsis are importantcomponents of subtropical and tropical marine environments. Intemperate regions their presence has increased greatly in the lastseveral years (Mangialajo et al., 2011; Rhodes, 2011). For theMediterranean Sea, the toxic bloom-forming species O. ovata, byfar the most abundant (Penna et al., 2010; Totti et al., 2010;Accoroni et al., 2011; Mangialajo et al., 2011), and O. siamensis

regularly occur, and are very similar in shape, dimensions and platearrangement. Human health effects ascribed to aerosols orcutaneous exposure to seawater were reported during Ostreopsis

blooms in different areas of the Mediterranean Sea (Gallitelli et al.,2005; Tichadou et al., 2010; Del Favero et al., 2012). Symptomsassociated with these events mainly involved the upper respirato-ry tract; however the cause-effect correlation between illness andthe involvement of algal toxins has not been clarified yet. O. cf.ovata blooms in the Adriatic Sea are among the most intense of theentire Mediterranean coasts (Mangialajo et al., 2011) and have ledto ecological problems, such as benthic invertebrate deaths(Accoroni et al., 2011), associated with the high ovatoxins (OVTXs)levels produced by these algal cells. These compounds arechemically related to the palytoxins, which are high molecularweight polyketides that act as potent marine toxins (Ciminielloet al., 2011). The HR LC–MS analysis of several strains (Accoroniet al., 2012; Ciminiello et al., 2012a; Pezzolesi et al., 2012; Honsellet al., 2013) reported that ovatoxin-a is the predominant toxin,together with decreasing amounts of ovatoxin-b, -d/e, -c andputative palytoxin. However, recently a new ovatoxin, designatedOVTX-f, was detected in an isolate from Portonovo (Adriatic Sea,Italy), accounting for 50% of the total toxin content (Ciminielloet al., 2012b). Toxins concentration on a per cell basis waspreviously found to increase from exponential to senescent phase,independently of the growth conditions (Guerrini et al., 2010;Pistocchi et al., 2011; Vanucci et al., 2012a,b) however, it was notdetermined in which phase their synthesis mostly occur.Understanding the regulation of toxin biosynthesis in toxicdinoflagellates is an area of key interest, and little is presentlyknown about the role these compounds play in the biology of thealgal cell. Some toxins (e.g. brevetoxins) appear to be putativelysynthesized by complex enzymes called polyketide synthases(PKS); however the involvement of these proteins remains to beconfirmed (Monroe et al., 2010). Polyketides are synthesized in amanner analogous to fatty acid biosynthesis through the sequen-tial addition of carboxylic acid building blocks by polyketidesynthases (PKS) (Shimizu, 2003). Recently, antibodies to K. brevis

PKSs recognized similar proteins from an Adriatic strain of O. cf.ovata (Van Dolah et al., 2013) demonstrating for the first time thepresence of PKS proteins in this species, opening the possibilitythat these proteins could have an alternative or additional role andbe involved in fatty acid synthesis.

Blooms of O. cf. ovata are often associated with thickmucilaginous mats which help the algal cells to colonize asubstrate (Graneli et al., 2002; Guerrini et al., 2010; Totti et al.,2010). The mucilaginous mats adhere loosely to the substrata sothat it can easily be resuspended in the water column by wavemechanical action (Totti et al., 2010). A new investigation oncytological and metabolic features of O. cf. ovata (Honsell et al.,2013) showed unique features of the mucilage network abundant-ly produced by this species to colonize benthic substrates, with anew role of trichocysts, never described before.

The ecology of O. cf. ovata blooms relative to environmentalparameters have been investigated by several authors (Vila et al.,2001; Penna et al., 2005; Totti et al., 2010; Accoroni et al., 2011,2012) however, the bloom mechanism for this species is complexand far from understood. Toxins produced by these species are

relatively new and only the ovatoxin-a structure has been recentlycharacterized (Ciminiello et al., 2012a). Relatively little is knownabout the biosynthetic linkage among the various ovatoxins asthey are supposed to be all very similar in structure and may beprecursors for the others.

The assimilation of nutrients such as NO3-N and PO4-P togetherwith environmental factors (e.g. temperature, salinity, irradiance)have all been shown to affect toxin content (Pezzolesi et al., 2012;Scalco et al., 2012; Vanucci et al., 2012b). Nutrient depletion wasreported to affect both growth and toxin dynamics. In fact, althoughpalytoxin-like compounds (e.g. ovatoxins) do not contain phospho-rous, P-nutrition has been shown to influence toxin production: withP-depletion there is up to 40% decrease in toxicity in O. cf. ovata cells(day 22), in comparison to 53% decrease once N-limited (Vanucciet al., 2012b). This last result was less surprising as ovatoxins are N-containing toxins. Results apparently differed from those reportedfor several harmful flagellates which increased their cell toxincontent mainly under P-limitation (e.g. Prymnesium parvum,Johansson and Graneli, 1999; Chrysochromulina polylepis, Edvardsenet al., 1990; Dahl et al., 2005; the planktonic dinoflagellateProtoceratium reticulatum, Guerrini et al., 2007; the benthicdinoflagellates Gambierdiscus toxicus, Tindall and Morton, 1998;and Prorocentrum lima, Tomas and Baden, 1993; Vanucci et al., 2010).However, different behaviors may occur with different nutrienttreatments depending on elemental composition of the toxins.

The present study was undertaken to investigate the cellularproduction of the main biochemical compounds likely to beinvolved in the growth and toxicity dynamics of O. cf. ovata, fromthe first days of growth through the exponential to the latestationary phase. Using batch cultures of an Adriatic O. cf. ovata

strain (OOAB0801), the profiles of cellular palytoxins weremonitored along with the change in cell concentration and size,the chlorophyll-a content, and nutrient-uptake (NO3-N and PO4-P).Moreover the cellular protein amount, as well as the totalpolysaccharides and lipid content were measured. Toxin produc-tion rates were calculated in order to evaluate the relationshipbetween these metabolites and the other main compoundssynthetized in the cells and to better understand their productiondynamics throughout the growth cycle.

2. Materials and methods

2.1. Experimental setup and culture conditions

A strain of O. cf. ovata Fukuyo (OOAB0801) isolated in 2008using capillary pipette method (Hoshaw and Rosowski, 1973) inthe Western Adriatic Sea during a bloom nearby Bari (Pugliaregion, Italy) was used in this study. After initial growth inmicroplates, cells were grown in batch cultures in autoclaved,1.2 mm filtered natural seawater at salinity 36, adding macro-nutrients at a five-fold diluted f/2 concentration (Guillard, 1975)and selenium. Cultures were maintained under illumination fromcool white light at a photon flux density of 110–120 mmol m�2 s�1,at 20 � 1 8C on a 16:8 h light/dark cycle in a growth chamber.

Preliminary experiments optimized sample volumes requiredfor the various analyses and compared several quantificationmethods of each compound. Due to the large volume of culturesrequired for the entire experiment and the slow growth of thesecells (Pezzolesi et al., 2012), three replicate flasks were establishedand sampled in triplicate (A1 + A2, B1 + B2, C1 + C2). Experimentalcultures consisted therefore in 3 L Erlenmeyer flasks, inoculatedwith cells collected from a culture at early stationary phase andfresh medium to a final volume of 2500 mL, in order to have aconcentration of about 200 cell mL�1 at the beginning of theexperiment (day 0). Most of the analyses were performed at day 1,2, 5, 7, 9, 12, 15, 21, 27, 35. Since these cells tend to form mucous

L. Pezzolesi et al. / Harmful Algae 36 (2014) 1–10 3

aggregates which could prevent a correct subsampling, everysampling day cells were gently mixed and an aliquot (120–280 mLdepending on the total volume required for the analyses) wascollected from each of the paired flasks and combined (e.g. A1 + A2)in order to obtain only one ‘‘representative subculture’’ (i.e. A, B, C)to use for further samplings. Aliquots for the analyses were thencollected from this last flask. After 15 days as a consequence of thedecreased volume, the remaining culture in the flasks correspond-ing to the same replicate were combined.

After gentle mixing aliquots of 10–50 mL were fixed (1% Lugolsolution; Throndsen, 1978) before cell counting and measurement.Cells were allowed to settle for about 5 min, then cell counts wereperformed using the Utermohl method (Hasle, 1978) and a ZeissAxioplan inverted microscope at 320� magnification under brightfield and phase contrast illumination. For each sample cellconcentration was estimated by averaging the cells enumeratedin 4 transects of the chamber, at least in triplicates. Specific growthrate (m, day�1) was calculated using the following equation:

m ¼ ln N1 � ln N0

t1 � t0

where N0 and N1 were cell density values at time t0 and t1.Cells were measured using a digital monitor system (Nis

Elements BR 2.20 software) with a Nikon Digital Sight DS-U1camera connected to the inverted microscope. Calculations of cellvolume were performed with the assumption of ellipsoid shapeusing the following equation (Sun and Liu, 2003):

V ¼ p6� a � b � c

where a = dorsoventral diameter (DV), b = width (W), c = meananteriorposterior diameter (AP).

2.2. Nutrient determination

Nitrate and phosphate analyses were performed on filteredculture medium aliquots (Whatman GF/F filters, pore size 0.7 mm)and analyzed spectrophotometrically (UV/VIS, JASCO 7800, Tokyo,Japan) according to Strickland and Parsons (1972).

The nutrient (NO3-N and PO4-P) uptake (U) was calculatedaccording to Lim et al. (2006) from the residual nutrientconcentrations in the medium (C) and the difference in celldensities (g) between days, when the depletion of nutrients waslinear. The following equations were used:

U ¼ �C1 � C0

gDt

g ¼ N1 � N0

ln N1 � ln N0

where C0 and C1 were the nutrient concentrations (mM) at time t0

and t1, and N0 and N1 were the corresponding cell densities(cell mL�1).

2.3. Chlorophyll-a determination

A volume of 10 mL culture sample was withdrawn. Cells werecentrifuged at 2550 � g for 15 min at 4 8C. The supernatant wasremoved and cells were then resuspended in 3 mL of acetone (90%,v/v), homogenized with strong vortex mixing and stored overnightat 4 8C to allow the pigments extraction. At the end of theincubation period (20–24 h), samples were centrifuged at 2550 � g

for 10 min, the absorbance of extracts was measured at 665 and750 nm (UV/VIS, JASCO 7800, Tokyo, Japan) and the concentration

of chlorophyll-a was determined according to the followingequation (Ritchie, 2006):

Chlorophyll-a ðmg L�1Þ ¼ 11:4062 � ½Að665sÞ � Að750sÞ� � v

� 103=ðco � VÞ

where A(665s) = the blank corrected absorbance at 665 nm,A(750s) = the blank corrected absorbance at 750 nm, v = volumeof acetone solution used for the extraction (mL), co = cell pathlength (cm), V = volume of filtered sample (mL).

2.4. Determination of protein content

The protein determination was carried out using the algalpellets obtained from 25 mL culture aliquots by centrifugation at2550 � g at 4 8C for 15 min, and preserved at �80 8C. Before theprotein determination, the algal pellet was resuspended in 1 mLNaOH 0.1 M, and was sonicated for 15 s (4 cycles). The cell proteinconcentration was estimated with the Folin Phenol reagent (Lowryet al., 1951) using bovine serum albumin (BSA) as the standard.

2.5. Determination of carbohydrate content

Total and extracellular polysaccharides were extracted follow-ing the Myklestad and Haug protocol (1972) from culture aliquotsand from the supernatants obtained by the centrifugationperformed for protein determination (see ‘‘Determination ofprotein content’’), respectively. Samples were stored at �20 8Cprior to analysis. Two volumes (30 mL) of absolute ethanol wereadded to one volume (15 mL) of culture and stored at �20 8C for24 h. The solution was centrifuged for 15 min at 14,700 � g at 4 8C,and the pellet was used to measure the polysaccharides content.The carbohydrate digestion was carried out using 80% sulfuric acidsolution at room temperature for 20 h, and the total carbohydratecontent of microalgae was determined by the Phenol SulfuricMethod (Dubois et al., 1956; Hellebust and Craigie, 1973) usingglucose as the standard.

2.6. Determination of lipid content

Culture aliquots (100 mL) for lipids quantification werecollected at day 7, 12, 21, 35 and stored at �20 8C prior toanalysis. The total lipids were extracted by mixing 60 mLchloroform/methanol (2:1, v/v) with the samples using a slightlymodified version of Bligh and Dyer’s method (1959). The mixturewas treated with ultrasound for 5 min, then lipids were extractedunder reflux at 55–60 8C for 2 h for three cycles. After phaseseparation and filtration over celite, total lipids were determined inthe chloroform phase by gravimetric analysis following evapora-tion of the solvent at room temperature.

2.7. Toxin analysis

2.7.1. Sample extraction

Cell samples were obtained by filtering a variable volume ofculture (150–250 mL) using glass fiber filters (Whatman GF/F, poresize 0.7 mm), and the obtained filters was stored at �80 8C until theextraction. All organic solvents used for the toxin extraction andanalysis were of distilled-in-glass grade (Carlo Erba, Milan, Italy).Water was distilled and passed through a MilliQ water purificationsystem (Millipore Ltd., Bedford, MA, USA). For the toxin extraction,1–3 mL of a methanol/water (1:1, v/v) solution was added to eachfilter and then sonicated for 2 min in pulse mode, while cooling inan ice bath. The mixture was centrifuged at 3000 � g for 15 min,the supernatant was decanted and the pellet was washed twice

0 3 6 9 12 15 18 21 24 27 30 33 36100

1000

10000

conc

(cel

l mL-1

)

Time (day)

Fig. 1. Growth pattern and corresponding growth rates (inset) of O. cf. ovata cells.

Bars indicate standard deviation.

L. Pezzolesi et al. / Harmful Algae 36 (2014) 1–104

with 0.5–1 mL of methanol/water (1:1, v/v). The extracts werecombined and the volume adjusted to 2–5 mL with extractingsolvent. The obtained mixture was analyzed directly by LC–HRMS(5 mL injected). Recovery percentages of the above extractionprocedures were estimated to be 98% (Ciminiello et al., 2006).

2.7.2. Liquid chromatography–high resolution mass spectrometry

(LC–HRMS)

LC–HRMS experiments were carried out on an Agilent 1100 LCbinary system (Palo Alto, CA, USA) coupled to a hybrid linear iontrap LTQ Orbitrap XLTM Fourier Transform MS (FTMS) equippedwith an ESI ION MAXTM source (Thermo-Fisher, San Jose, CA, USA).Chromatographic separation was accomplished by using a 3 mmGemini C18 (150 � 2.00 mm) column (Phenomenex, Torrance, CA,USA) maintained at room temperature and eluted at 0.2 mL min�1

with water (eluent A) and 95% acetonitrile/water (eluent B), bothcontaining 30 mM acetic acid (Laboratory grade, Carlo Erba). Aslow gradient elution was used: 20–50% B over 20 min, 50–80% Bover 10 min, 80–100% B in 1 min, and hold 5 min. This gradientsystem allowed a partial chromatographic separation of mostpalytoxin-like compounds.

HR full MS experiments (positive ions) were acquired in therange m/z 800–1400 at a resolving power of 60,000. The followingsource settings were used in all LC–HRMS experiments: a sprayvoltage of 4 kV, a capillary temperature of 290 8C, a capillaryvoltage of 22 V, a sheath gas and an auxiliary gas flow of 35 and 1(arbitrary units). The tube lens voltage was set at 110 V.

Quantitative determinations of putative palytoxin, ovatoxin-a,-b,-c,-d, and -e in the extracts were carried out using a calibrationcurve (triplicate injection) of palytoxin standards (Wako Chemi-cals GmbH, Neuss, Germany) at four levels of concentration (25,12.5, 6.25, and 3.13 ng mL�1) and assuming that their molarresponses were similar to that of palytoxin. Calibration curveequation was y = 8565.2106x � 228102.4309 and its linearity wasexpressed by R2 = 0.999. Extracted ion chromatograms (XIC) forpalytoxin and each ovatoxins were obtained by selecting the mostabundant ion peaks of both [M+2H-H2O]2+ and [M+H+Ca]3+ ionclusters. A mass tolerance of 5 ppm was used.

The total toxin production rate mTOX (pg PLTXs mL�1 day�1) inthe cultures throughout the growth phase was calculated using thefollowing equation:

mTOX ¼lnðC1T1=C0T0Þ

t1 � t0

where CtTt was the toxin concentration (pg PLTXs mL�1) calculatedby multiplying the cell concentration Ct (cells mL�1) by the cellulartoxin content Tt (pg cell�1) at time t.

To account for the effect of cell growth rates on toxin production,the net toxin production rate RTOX (pg PLTXs cell�1 day�1) wasdetermined over each growth phase according to Anderson et al.(1990):

TTOX ¼C1T1 � C0T0

Cðt1 � t0Þ

where C is the ln average of the cell concentration:

C ¼ C1 � C0

lnðC1=C0Þ

2.8. Statistical analysis

Differences in cell abundance, cell size, biovolume, and intra-cellular and extra-cellular concentrations of the various com-pounds during the growth were tested by the analysis of variance

(ANOVA), using Statistica (StatSoft) software. Whenever a signifi-cant difference for the main effect was observed (p < 0.05) aTukey’s pairwise comparison test was also performed.

3. Results

3.1. Cell growth and dimensions

The O. cf. ovata (strain OOAB0801) growth curve is shown inFig. 1. Cultures had initial cell densities of 150–200 cells mL�1 anda 7 day exponential growth phase with a mean growth rate of0.52 day�1. Cells were actively dividing from the beginning of thegrowth, as reported by the growth rate calculated between days 1and 2 which was 1.00 day�1 (Fig. 1), followed by the samplesbetween days 2 and 5 and between days 5 and 7 which were 0.41and 0.40 day�1, respectively.

Cell yields in the stationary phase were on average5462 � 698 cells mL�1, with a maximum of 6692 � 227 cells mL�1

at day 35. Over the growth cycle O. cf. ovata cells varied significantly(ANOVA, p < 0.05) in size and biovolume (Fig. 2). The dorsoventral towidth (DV/W) ratio was not significantly different over the growth withvalues around 1.4, while the dorsoventral to anteroposterior (DV/AP)ratio were around 1.6 at the beginning of the growth and significantly(ANOVA, p < 0.05) increased to 1.8–1.9 during active cell division,reaching a maximum value of 2.11 in the early stationary phasefollowed by a decreased to 1.7–1.8 in the late stationary phase. The DVdiameter was significantly different (ANOVA, p < 0.01) among thegrowth phases, starting from a value of 54 mm at the beginning of thegrowth and ranging from 45 to 54–55 mm from the exponential to thestationary phase, respectively. Biovolume values were 33,000 mm3 atday 0, corresponding to the biovolume of the cells of the inocula,withdrawn from acclimatized cultures at the beginning of thestationary phase. Cell volumes in the exponential phase showed adecreasing trend, presumably due to cell division reaching a minimum(19,000 mm3) at day 5. In the stationary phase cell volumes increasedsignificantly (ANOVA, p < 0.01) and were in the range 30,000–35,000 mm3.

Chlorophyll-a reached the highest concentration while cellswere growing exponentially (183.6 mg L�1 corresponding to47.5 pg cell�1, day 7), and then decreased to a value of 70–76 mg L�1, which corresponds to a cellular concentration of 12–13 pg cell�1 in the stationary phase (days 21–27, Fig. 3). Nutrients(N and P) were totally depleted by day 9 (Fig. 4), as they wererapidly taken up by the cells during the first days. Nitrate nitrogenin particular was consumed at a rate of 29.0 pmol day�1 cell�1

during the first 2 days (Table 1), while the value for PO4-P was2.5 pmol day�1 cell�1, then uptake rates decreased to 12.2 and5.4 pmol day�1 cell�1 for N, and 0.5 and 0.1 pmol day�1 cell�1 for P,between days 5–7, and days 7–9, respectively. P was taken up bycells more rapidly than N, as attested by the increase of the N:Pratio in the culture medium (which was 24 at day 0) to values of 26,31, and 50 at days 2, 5, and 7, respectively.

1.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.0

0 1 2 5 7 9 12 15 21 27 350

5000

10000

15000

20000

25000

30000

35000

40000

45000 biovolume

DV

/ AP

DV

/ W

Biov

olum

e ( µ

m3 )

Time (da y)

DV/AP DV/W

Fig. 2. Mean cellular volume, dorsoventral to anteroposterior (DV/AP) ratio and

dorsoventral to width (DV/W) ratio of O. cf. ovata cells. Bars indicate standard

deviation.

020406080100120140160180200

0 3 6 9 12 15 18 21 24 27 30 33 360

306090

120150180210240270300

µg L-1

Chl

-a (p

g ce

ll-1)

Chl

-a (µ

g L-1

)

Time (day)

pg cell-1

Fig. 3. Content of chlorophyll-a (chl-a) in O. cf. ovata cells during the growth curve.

Bars indicate standard deviation.

0123456789101112

0 3 6 9 12 15 18 21 24 27 30 33 360

20406080

100120140160180200220

NO3-N

PO4 -

P (µ

M)

NO

3 -N

(µM

)

Time ( day)

PO4-P

Fig. 4. Nitrogen (NO3-N) and phosphorus (PO4-P) concentrations measured in O. cf.

ovata culture medium. Bars indicate standard deviation.

02468101214161820

0 3 6 9 12 15 18 21 24 27 30 33 3606

121824303642485460

% ex tracell po lysaccharide s µg mL n g cell

Poly

sacc

harid

es (n

g ce

ll-1)

Poly

sacc

harid

es ( µ

g m

L-1)

Time (day)

Fig. 5. Total polysaccharides content, expressed either on a culture volume

(mg mL�1) and on a cell (ng cell�1) basis, and corresponding extracellular release (%)

calculated throughout the growth. Bars indicate standard deviation.

L. Pezzolesi et al. / Harmful Algae 36 (2014) 1–10 5

3.2. Cellular components

3.2.1. Proteins, polysaccharides and lipids

Total polysaccharides content either expressed per cell or perculture volume (ng cell�1 or mg mL�1) increased significantly(ANOVA, p < 0.01) immediately after O. cf. ovata cells entered thestationary phase (Fig. 5). The cellular amount in the exponentialphase decreased as a consequence of the cell division whichoccurred during the first days and divided the internal poly-saccharides content among daughter cells. The polysaccharidesamount was in fact 4.27 ng cell�1 at day 2, then significantlydecreased (ANOVA, p < 0.01) to the minimum value of0.73 ng cell�1 at day 7, while the maximum concentration foundduring the stationary phase was 6.35 ng cell�1 (day 27). During thegrowth curve, cells were continuously producing polysaccharides,reaching a total maximum value of 36.0 mg mL�1 at day 35,concurrently with an extracellular amount of 4.1 mg mL�1. Despite

Table 1Growth rates (m) and corresponding cell densities, total (mTOX) and net (RTOX) toxin p

between different days during the growth curve.

Period (day) m (day�1) Cell density (cell mL�1) mTOX (pg mL�1 day�1) RT

1–2 1.00 174–475

2–5 0.41 475–1715

5–7 0.40 1715–3841

1–7 0.52 174–3841 0.40 2.

7–9 0.11 3841–4733 0.48 3.

9–27 4733–5630 0.05 0.

that the extracellular release increased significantly during theprogression of the growth curve and resulted significantly higher(ANOVA, p < 0.01) during the stationary phase, the percentage ofextracellular release calculated throughout the growth reported amaximum value, accounting for 54% of the total, at day 5 whichwas significantly higher than those measured in the stationaryphase (11–12%).

Analyses of the cellular proteins, expressed as ng cell�1,reported a decreasing trend during the exponential phase as aconsequence of cell division. Conversely, the protein content in thealgal culture, expressed as mg mL�1, increased and was propor-tional to the algal biomass during the growth (Fig. 6). The proteincontent was significantly different (ANOVA, p < 0.01) between theexponential and the stationary phase, while it remained almostconstant and around a value of 44–45 mg mL�1 in this latter period.

Total lipids were extracted from the culture at specific days,corresponding to different growth stages, in particular the

roduction rates, nutrient (N-NO3 and P-PO4) uptakes (U) of O. cf. ovata calculated

OX (pg cell�1 day�1) UN-NO3(pmol cell�1 day�1) UP-PO4

(pmol cell�1 day�1)

29.0 2.5

16.9 0.9

12.2 0.5

2

3 5.4 0.1

8

02468101214161820

0 3 6 9 12 15 18 21 24 27 30 33 3605

101520253035404550

µg mL-1

Prot

eins

(ng

cell-1

)

Prot

eins

(µg

mL-1

)

Time (day)

ng cell-1

Fig. 6. Protein content expressed either on a culture volume (mg mL�1) and on a cell

(ng cell�1) basis. Bars indicate standard deviation.

0 3 6 9 12 15 18 21 24 27 30 33 360123456789

10111213

OVTX-a OVTX-b OVTX-c OVTX-d,e pPLTX

conc

(pg

cell-1

)

Time (day)

Fig. 7. Individual toxin content of putative palytoxin (pPLTX), ovatoxin (OVTX)-a,b,c

and -d plus -e of O. cf. ovata cells during the growth, expressed on a cell basis

(pg cell�1). Bars indicate standard deviation.

5060708090

100

OVTX-a OVTX-b OVTX-c OVTX-d,e pPLTXon

c (n

g m

L-1)

con

c (%

)

708090100110120130140150

tot (ng mL-1)

L. Pezzolesi et al. / Harmful Algae 36 (2014) 1–106

exponential phase (day 7), beginning (day 12) mid (day 21) andlate (day 35) stationary phase. Lipid content increased significantly(ANOVA, p < 0.01) from the beginning to the end of the growthperiod (Table 2), reporting a value of 8.7 mg mL�1 which increasedby 3.5 times at day 35 (30.7 mg mL�1). Although still statisticallydifferent, the cellular lipid content at the end of the stationaryphase was only two-fold higher than that at day 7 (4.6 vs.2.3 ng cell�1).

3.2.2. Toxins

The LC–HRMS analysis of the algal extracts showed thepresence of the main algal toxins produced by this species (pPLTX,ovatoxin-a,-b,-c,-d,-e) (Figs. 7 and 8). The concentration of all theindividual toxins decreased in a parallel manner at the beginning ofthe growth, as a result of the cell division, which occurred at a highrate (0.52 day�1, Fig. 7) until the end of the exponential phase.From day 7, cells started to accumulate toxins and the cellulartoxin content increased with the progression of the growth period,and was significantly higher (ANOVA, p < 0.01) in the stationaryphase where a maximum total PLTXs value of 21.5 pg cell�1 wasobserved (Fig. 7). Quantitative analyses (Fig. 8) showed that themost abundant toxin was ovatoxin-a (52–55%), followed byovatoxin-b (25–29%), d + e (11–16%), c (4–7%), and putativepalytoxin (1–2%). The relative percentage of the single toxinsremained constant throughout the growth curve as values werenot significantly different among days. Total toxin contentexpressed on a culture volume basis (Fig. 8) had an increasingtrend from the beginning of the growth with an almost constantrate during the exponential phase which increased rapidly whilecells were entering the stationary phase (days 7–12), reaching avalue of 65.3 ng mL�1 at day 12. During the stationary phase, cellscontinued to produce toxins and at day 35 a maximum amount of134.1 ng mL�1 was detected.

Despite the high cellular toxin content during the stationarygrowth phase, toxin production rate (mTOX) was the highest during

Table 2Lipids content along the growth curve, expressed either on a

culture volume (mg mL�1) and on a cell (ng cell�1) basis.

Day Lipids

(mg mL�1) (ng cell�1)

7 8.67 2.26

12 20.67 3.97

21 22.33 4.00

35 30.67 4.58

the exponential phase, ranging from maximum values of about0.40 and 0.48 pg PLTXs mL�1 day�1, between days 1–7 and days 7–9, to a minimum of 0.05 pg PLTXs mL�1 day�1 during the stationaryphase (days 9–27, Table 1). When the effect of cell growth rates ontoxin production was considered and the net toxin production rate(RTOX) was calculated, the trend was not affected, althoughproduction rates reported higher values, especially in theexponential phase when cells were dividing; in fact RTOX valuesvaried from 2.2 and 3.3 pg PLTXs cell�1 day�1 between days 1–7and days 7–9 to 0.8 pg PLTXs cell�1 day�1 in the stationary phase(days 9–27).

3.2.3. Relative abundances of the main compounds

Fig. 9 summarizes the relative composition (%) of the maincompounds in the algal cell at specific days of growth (exponentialphase, early-mid-late stationary phase). The results of this studysuggest that proteins are mainly produced during the exponentialphase, when nutrients are available, then their cellular concen-tration slightly decreased (from 68% at day 7 to 41% at day 35,Fig. 9) while cells start to accumulate polysaccharides (from 4% to30% at day 7 and 35, respectively). Lipid content is relativelystable, while toxins, although at very low levels, increase theirconcentration, and their relative abundance within the cell isdouble at the end of the growth (from 0.06% to 0.13% at day 7 and35, respectively).

0 3 6 9 12 15 18 21 24 27 30 33 360

10203040

tota

l c

rela

tive

Time (day)

0102030405060

Fig. 8. Total toxin content of O. cf. ovata cells, expressed on a culture volume basis

(ng mL�1), and relative concentration (%) of the single toxins during the growth.

Bars indicate standard deviation.

Fig. 9. Relative abundance (%) of the main cellular components (lipids,

polysaccharides, proteins, toxins) within O. cf. ovata cells during exponential

phase and early, mid, late stationary phase (days 7 and 12, 21, 35, respectively).

L. Pezzolesi et al. / Harmful Algae 36 (2014) 1–10 7

4. Discussion

Ostreopsis cf. ovata cells of different strains grown undercomparable conditions, in terms of nutrients (NO3-N and PO4-P asfive-fold diluted f/2), salinity (36), light (about 100 mmol m�2 s�1)and temperature (20 8C), reported similar growth patterns(Pezzolesi et al., 2012; Vanucci et al., 2012b; Vidyarathna et al.,2012) to those observed in this study, as evidenced by the growth

rates (0.4–0.5 day�1) and the entrance into the stationary phasearound days 7–9. However, differences in the final cell yields wereobserved among the various experiments and algal strains.Nutrient uptakes’ patterns were also comparable, with N and Ptotally consumed when cells entered the stationary growth phase(day 9). The depletion of P in the culture medium, compared to thatof N, was more rapid, which is similar with a previous study(Vanucci et al., 2012b) where a different Adriatic strain was used.This result was not surprising as growth and carbon fixation maycontinue for several generations in P-depletion and be limitedunder high N:P ratio (John and Flynn, 2000). A rapid P-uptakewithin few days of growth was found also for other benthicdinoflagellates (e.g. Prorocentrum hoffmannianum, P. lima), forwhich P was the major nutrient affecting the growth (Aikman et al.,1993; Vanucci et al., 2010). Interestingly, P was found to beimplicated in the regulation of toxin metabolism in Alexandrium

minutum (Flynn et al., 1994), as phosphorylation of enzymes is acommon mode of metabolic regulation; in addition P-depletionaffected toxin metabolism, as observed in O. cf. ovata (Vanucciet al., 2012b).

During rapid growth, cells exhibited a wide size range aspreviously observed both in natural samples from the Adriatic(Accoroni et al., 2012) and other areas (Aligizaki and Nikolaidis,2006; Honsell et al., 2011) and from cultured strains (Pin et al.,2001; Rossi et al., 2010; Vanucci et al., 2012a,b). Although it shouldbe taken into account that morphological differences existbetween cells from cultures and field samples (Aligizaki andNikolaidis, 2006), our results are in accordance with those found byAccoroni et al. (2012) during an O. cf. ovata bloom in the Adriaticregion. We also observed a decreasing trend (from about 54–55 to45 mm) in the dorsoventral (DV) diameter size, reported also forother dinoflagellates (e.g. Alexandrium minutum, Touzet et al.,2007; A. ostenfeldii, Jensen and Moestrup, 1997; A. catenella, Liet al., 2011), while cells undergo active cell division in theexponential phase. As for the DV/AP ratio, data are in accordancewith O. cf. ovata values (2.07–2.33) found in other studies(Aligizaki and Nikolaidis, 2006; Guerrini et al., 2010; Selina andOrlova, 2010; Accoroni et al., 2012; Pfannkuchen et al., 2012) indifferent areas worldwide. The relatively constant DV/W ratio, incontrast to the variable DV/AP ratio, along the growth curve leadsto the hypothesis that while growing, cells decreased or increasedtheir size maintaining proportional DV and W dimensions;conversely, a relative decrease in the AP diameter occurred duringcell division. This Southern Adriatic strain had cellular volumesslightly higher than those observed in previous studies with otherstrains (Pezzolesi et al., 2012; Vanucci et al., 2012b; Vidyarathnaand Graneli, 2013). Moreover, it is important to point out as thatthroughout the growth stages, biovolumes decrease during theexponential phase as a consequence of the active cell division,while they increase in stationary phase cells. This trend waspreviously observed under different nutrient conditions (Vanucciet al., 2012b), but results were clarified in this study through alonger period of sampling along the growth curve.

Results showed that the amount of chlorophyll-a (either asmg L�1 and as pg cell�1) varied among the growth phases, reachingthe maximum value while cells were entering the stationary phaseand falling rapidly once nutrients were totally exhausted from thealgal medium. The chl-a profile was not representative of the algalbiomass (e.g. cell density) during the growth. The decreasing trendwas also observed for the dinoflagellate Alexandrium minutum,which illustrated a decrease in chl-a content more rapidly onnitrate than phosphate exhaustion (Flynn et al., 1994; Grzebyket al., 2003) and for N-depleted O. cf. ovata cultures (Vidyarathnaand Graneli, 2013), as chl-a represents one of the most importantN-rich compounds in the algal cells. However, in this last studythey found an increasing chl-a content till the end of the growth in

L. Pezzolesi et al. / Harmful Algae 36 (2014) 1–108

N, P-sufficient cultures, and the maximum value was around day22 (about 90 mg L�1) when cells were entered the decaying phase:this different result can be explained considering the relatively lowgrowth rate (m, 0.21 day�1) found in the study and the fact thatnutrients were not totally uptaken even at the end of theexperiment (decaying phase), being 9.9 and 2.8 mM for NO3

�

and PO43�, respectively.

The polysaccharide profile found in this study was inaccordance with previous results on O. cf. ovata (Vidyarathnaand Graneli, 2012, 2013), where the lowest levels were recordedduring mid-exponential phase (0.4–0.5 ng cell�1 vs 0.73 ng cell�1

in this study) and then increased in the stationary phase(9.72 ng cell�1 vs 6.35 ng cell�1 in this study), despite experimen-tal cultures were grown at slightly different growth conditions. Ourresults indicate that the cell division during the exponential phaseclearly influenced the polysaccharide content, and that cellsstarted to accumulate these compounds especially in the station-ary phase, likely due to nutrient limitation, as reported for otheralgal species (Guerrini et al., 1998; Liu and Buskey, 2000). It isnotable as polysaccharides were mainly extruded from the cellsduring the first days of growth (exponential phase), reporting highpercentages of release (27–54%). The extrusion can be seen as acolonization strategy, in fact while cells are dividing and the celldensity is increasing, the release of polysaccharides could help inthe creation of the algal population, as cells may more easilyaggregate in the formation of a mucous mat that more stronglyattach to the substrate (Graneli et al., 2002; Honsell et al., 2013).The presence of mucilage offers many adaptive benefits toindividual algae, such as increased settling rates, aid in thesequestration of vital nutrients, reduction of the palatability ofalgae and defence against grazing (Reynolds, 2007). As observed byTotti et al. (2010), during the period of O. cf. ovata proliferation, thecharacteristic brownish, spotty network-shaped mat covers all thebenthic substrata and adheres loosely, unlike benthic diatoms thatpossess structures ensuring a strong attachment to the substrata(Round et al., 1990). These extracellular compounds may protectthe cells against grazing (Liu and Buskey, 2000), once morefacilitating the development of the algal community and stimu-lating bacterial nutrient re-mineralization (Guerrini et al., 1998).Recently, Honsell et al. (2013) found that Ostreopsis mucilage isformed by acidic polysaccharides and by a very high number oftrichocysts sticking together to form a complex network offilaments. Additionally, authors revealed the presence of neutrallipid droplets within cells in all growth phases, which seem toincrease from exponential to stationary phase. Our results on thelipid content compliment previous data on O. cf. ovata (Honsellet al., 2013) as we report an increasing production of total lipidsduring the growth, with a maximum value at the end of thestationary phase (day 35).

In our study no variation in toxin composition was observedduring the growth stages. O. cf. ovata toxin profile was relativelystable and independent of culture conditions (Pistocchi et al.,2011), unlike the cellular toxin content, which may be apopulation-specific marker. O. cf. ovata toxins are in fact producedproportionally during the different growth phases, leading tohypothesize that none can probably act as precursor for the others.As for the toxin content, values found for this Southern Adriaticstrain are similar to previous results (Pezzolesi et al., 2012) where aNorthern Adriatic strain was used (toxin content: 16 pg cell�1 and155 mg L�1 in the stationary phase on a per cell and per culturevolume basis, respectively). However, it is important to point outthat the toxin quota in these algal cells is strongly influenced by thegrowth conditions (e.g., temperature, salinity, nutrients) and bythe characteristic of the strains (e.g. isolation site, but also age ofthe strain), as variation in the toxin content has been reported byseveral authors (Guerrini et al., 2010; Ciminiello et al., 2012a,b;

Pezzolesi et al., 2012; Pistocchi et al., 2012; Vanucci et al., 2012b).These results demonstrated decreasing toxin quotas duringexponential growth despite high net toxin production rates (RTOX),thus changes in toxin content per cell may simply reflect changesin cell size or the fact that exponentially growing cells may dilutetheir toxin content through cellular division (Graneli and Flynn,2006). Intracellular toxin quota cannot be interpreted as a directmeasure for the rate of toxin production (Cembella, 1998). Thegreatest net toxin production rate (RTOX) occurs in the exponentialphase during the first days and when nutrients were totallyexhausted, then it decreased during the stationary phase,suggesting that palytoxins, like brevetoxins (Johnson et al.,2012), could not be considered ‘‘classical secondary metabolites’’,which are by definition produced by aged or stressed cells. Thisresult demonstrated an uncoupling of toxin production fromgrowth at the stationary phase, as observed also for Dinophysis

acuminata (Tong et al., 2011). We cannot exclude the involvementof nutrient-depletion, as previously suggested for A. minutum byLippemeier et al. (2003). In Alexandrium fundyense and otherAlexandrium spp. toxin synthesis appears to be somehowuncoupled from cell division (Anderson et al., 1990; Etheridgeand Roesler, 2005; Yang et al., 2011); however the toxicity is notdirectly related to other physiological responses (e.g. photosyn-thesis and/or growth rate, Touzet et al., 2007), thus it cannot bemodeled solely as a function of growth rate. As for the saxitoxinproducing species A. minutum, Lippermeier et al. (2003) attributedthe elevated toxin production during P-limitation to a potentialarrest of the cell cycle in G1, the cell cycle stage when toxinsynthesis occurs, implying a continuous expression of G1-specificgenes among which are included at least some of the genes codingfor PSP toxin biosynthetic enzymes. Generally, changes in toxincontent are associated with disturbed/unbalanced physiology, andN-rich (e.g. PSP) toxins are synthesized during N-upshock and P-stress, not during N-downshock (Flynn et al., 1994), as A. fundyense

and P. bahamense var. compressum had the highest toxin contentsduring exponential growth in nutrient replete medium (Andersonet al., 1990; Usup et al., 1994). On the other hand, O. cf. ovata toxinproduction may be more comparable to Pseudo-nitzschia multi-

series’s, which accumulated the majority of domoic acid (DA)during the stationary phase (Bates et al., 1998).

To our knowledge, no previous data on the Ostreopsis proteincontent are available. The trend of growth and production of themain cellular compounds in O. cf. ovata leads to the hypothesis thatthe fast growth of this dinoflagellate, associated with the rapid useof environmental resources (nutrients, and phosphates in particu-lar), may be an ecological/adaptive strategy which could favor thisorganism in competition with other species. O. cf. ovata is a benthicmicroalgae that adheres to the substrate creating a mucilaginousmatrix, as evidenced by the high release of extracellularpolysaccharides. This microalga extrudes trichocysts (Honsellet al., 2011) and the presence of a network of trichocyst filamentsembedding O. cf. ovata cells could contribute to provide moremechanical resistance to the mucilage, copiously produced by thisspecies: this could represent an advantage for Ostreopsis species inthe colonization of different surfaces and could explain, at least inpart, its rapid proliferation without being dispersed by hydro-dynamism (Honsell et al., 2013). Another advantage is theproduction of toxins that could be a defense against other speciesand a strategy against grazing.

The enhancement of toxin, lipid, protein and polysaccharidecontent requires energy and an intra-cellular pools of N and P forthe metabolic activities, as phosphorus is involved in the energeticmetabolism for ATP production and in the regulation ofintracellular functions, while nitrogen is involved in the photo-synthetic activities and in the maintenance of basic and essentialcellular functions. This study improves the knowledge on O. cf.

L. Pezzolesi et al. / Harmful Algae 36 (2014) 1–10 9

ovata toxin and growth dynamics under nutrient replete condi-tions, showing results that could be of great interest for furtherinvestigations. Additional experiments are underway to investi-gate the leakage of these toxins into the surrounding mediumduring the growth, to better understand the total toxin productiondynamic, together with the elementary composition of the algalcells, either in nutrient replete and deplete conditions.

Conflict of interest

The authors declare no competing financial interest.

Acknowledgements

This research was supported by Italian PRIN2009 project fromMIUR. We are grateful to Dr. Mackenzie Zippay for English revisionof the manuscript [SS].

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