Harmful Algae 9 (2010) 590–599

Effects of different levels of N- and P-deficiency on cell yield, okadaic acid,DTX-1, protein and carbohydrate dynamics in the benthic dinoflagellateProrocentrum lima

Silvana Vanucci a, Franca Guerrini b, Anna Milandri c, Rossella Pistocchi b,*a Dipartimento di Biologia Animale ed Ecologia Marina, Universita di Messina, Salita Sperone 31, 98166 S. Agata, Messina, Italyb Centro Interdipartimentale di Ricerca per le Scienze Ambientali, Universita di Bologna, Via Sant’Alberto 163, 48100 Ravenna, Italyc Centro di Ricerche Marine, Viale A. Vespucci 2, 47042 Cesenatico (FC), Italy

A R T I C L E I N F O

Article history:

Received 28 July 2009

Received in revised form 10 March 2010

Accepted 29 April 2010

Keywords:

Sacca di Goro lagoon

Prorocentrum lima

Nutrient limitation

Okadaic acid

Benthic dinoflagellates

EPS

A B S T R A C T

Prorocentrum lima (Ehrenberg) Dodge is a cosmopolitan epiphytic dinoflagellate that produces biotoxins

which are causative of diarrhetic shellfish poisoning (DPS). Here we report on effects of several nitrogen (N)

and phosphorous (P) limited conditions on cell yield, okadaic acid (OA) and dinophysistoxin-1 (DTX-1)

contents synoptically with cell carbohydrate, exopolysaccharide (EPS) and cell protein concentrations in a

P. lima strain isolated from the Sacca di Goro lagoon (Northern Adriatic Sea). Batch culture experiments

were set to assess changes induced by four nitrogen-limited levels (1/3-N, 1/10-N, 1/20-N, and 1/50-N) and

four phosphorus-limited levels (1/3-P, 1/10-P, 1/20-P, and 1/50-P) with respect to control nutrient

conditions (f/2 medium; NO3� and PO4

3� concentrations: 883 and 36.3 mM, respectively; N/P ratio: 24).

Low nutrients availability determined lower cell yields starting from 1/10-N and 1/3-P levels and the

pattern observed was dependent on nutrient dynamics, as shown by N and P analyses performed in culture

media during growth. Final cell yield decreased significantly up to 4.7- and 5.6-fold under 1/50-N and 1/50-

P-limited levels with respect to control values, while cell volume increased with respect to control (up to

30% and 35% for N- and P-experiment, respectively). On overall, OA concentration ranged from 6.69 to

15.80 pg cell�1, while DTX-1 ranged from 0.12 and 0.39 pg cell�1 resulting in unusual high OA/DTX-1

ratios. The study indicates that protein, carbohydrate, EPS, and toxin concentrations displayed remarkable

different patterns under the two kinds of nutrient deficiencies. The main differences can be summarised as:

(i) significant decrease of cell protein concentration (up to 2-fold) under N-limitation, conversely no

significant changes in protein concentration under P-limitation; (ii) significant increase of cell

carbohydrate (up to 2.8-fold and 3.4-fold for N- and P-limitation, respectively) and cell OA amount (up

to 1.9-fold and 2.3-fold, N- and P-limitation, respectively) under both N- and P-limitations, however

different level-deficiency dependent patterns were displayed under the two nutrient conditions; (iii)

significant increase of EPS concentration (up to 6.50-fold) under P-limitation, conversely no significant

changes in EPS concentration under N-limitation. Data presented here indicate that P. lima adopts different

eco-physiological strategies to face N-limitation or P-limitation. This study provides the first evidence for

an increase in EPS production by benthic dinoflagellates under P-limited conditions; the ecological

significance of this increase is discussed.

� 2010 Elsevier B.V. All rights reserved.

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1. Introduction

Prorocentrum lima (Ehrenberg) Dodge is an epiphytic-benthicdinoflagellate widely distributed in both tropical and temperatecoastal waters and lagoons (e.g. Faust, 1991; Levasseur et al., 2003;Nascimento et al., 2005). It occurs in sand attached to the surface ofmacroalgae, seagrass, benthic debris, associated with coral reefs or

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

E-mail address: rossella.pistocchi@unibo.it (R. Pistocchi).

1568-9883/$ – see front matter � 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.hal.2010.04.009

attached to floating detritus in mangrove habitats (Faust, 1991). Itis also found in association with seaweeds fouling aquaculturefacilities or growing on rocks of the intertidal zone (e.g. Mortonet al., 1999; Lawrence et al., 2000; Maranda et al., 2007a; Levasseuret al., 2003). P. lima is also encountered in several Mediterraneanbenthic ecosystems (e.g. Vila et al., 2001; Simoni et al., 2004;Nagahama and Fukuyo, 2005; Turki et al., 2006; Aligizaki et al.,2009; Ingarao et al., 2009).

In the past, P. lima had also been linked to the ciguatera tropicalsyndrome as it is a frequent component of the epiphyticcommunity of ciguatera-endemic areas (Tindall and Morton,

S. Vanucci et al. / Harmful Algae 9 (2010) 590–599 591

1998). To date, all cultured isolates of P. lima, regardless of theirorigin or season of isolation, produce among others the lipophylicpolyether compounds okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 DTX-2 (McLachlan et al., 1997; Mortonet al., 1999), which are the principal toxins responsible fordiarrhetic shellfish poisoning (DSP) in humans (Murakami et al.,1982). OA, DTX-1 and DTX-2 are potent inhibitors of the essentialeukaryotic protein serine/threonine phosphatases 1, 2A, and 2B(e.g. Takai et al., 1992; Mackintosh and Mackintosh, 1994). Theidentification of several other toxins from P. lima cultures have alsobeen reported, although their relative importance in P. lima toxicityis still under debate (Torigoe et al., 1988; Hu et al., 1995; Holmeset al., 2001; Suarez-Gomez et al., 2001; Bravo et al., 2001;Fernandez et al., 2003; Lu et al., 2005; Vale et al., 2009).

OA and DTX-1 have been considered as the major causativeagents of DSP outbreaks also in several coastal areas of the AdriaticSea (Fattorusso et al., 1992; Draisci et al., 1995; Pavela-Vrancicet al., 2002); however, so far, no DSP events have been reported inItalian waters associated to the presence of a relative highabundance of P. lima, except in the Sacca di Goro lagoon (Lido diSpina, Pompei et al., 2006).

The eco-physiological role of OA and of its precursors andderivatives are still partially to be clarified; allelopathic activityhas been clearly proposed for OA produced by P. lima (Paul et al.,2007), in fact it has a growth inhibitory effect on several speciesof microalgae (Windust et al., 1996) and on co-occurringdinoflagellates (Sugg and VanDolah, 1999). Recent studies havealso shown that P. lima toxins have several biological effectsincluding grazing deterrence, ichthyotoxicity and hepatotoxicitytowards a range of marine organisms (Ajuzie, 2007, 2008).Moreover, in marine mammals and turtles it may be suspected tohave long-term effects due to low levels of chronic exposure(Takahashi et al., 2008) since OA has been identified as a tumourpromoter (e.g. Fujiki, 1992; Creppy et al., 2002; Vilarino et al.,2008).

Eco-physiological factors involved in cell biotoxin productionare numerous. Generally, it is largely accepted that nutrientimbalance is one of the main driving forces involved in toxinproduction. Both the availability and composition of nutrients havea significant impact on the algal community structure and biomass,and also on phytoplankton biochemistry, changes in nutrientsupply may ultimately affect toxin production (Graneli and Flynn,2006). Moreover, it is also well documented that alteredpolysaccharide metabolism is a prompt distress signal of nutrientstarvation or nutrient imbalance in diatoms (Myklestad, 1974;Passow et al., 1994). Changes in polysaccharide production and/oraccumulation and/or release may satisfy a cell strategy for copingnutrient deficiency that prevents temporarily protein and nucleicacid syntheses (Fraga, 2001 and references therein; Flynn et al.,2008). Many other functions have been proposed for exopoly-saccharides: epiphytic diatoms typically produce extra-cellularpolysaccharides for substrate adhesion, self-regulation of positionand for competitive advantage (e.g. Underwood and Paterson,2003; Underwood et al., 2004). Recently, it has also been reportedthe production of conspicuous amount of mucilage by planktonicdinoflagellates, both in the field and in laboratory conditions(Alldredge et al., 1998; Mackenzie et al., 2002; Pompei et al., 2003;Pistocchi et al., 2005; Nikolaidis et al., 2008). The environmentalfactors inducing this production are still not elucidated as well asthe ecological role of extra-cellular polysaccharide production inbenthic dinoflagellates which has only been proposed as an openquestion (Faust, 1995). So far, it is well known that this ecologicallyrelevant group of organisms among which there are the highlytoxic Ostreopsis spp. Gambierdiscus toxicus, and Prorocentrum spp.,release copious amount of mucilage both in situ and in culture (Heilet al., 1993). Nevertheless, no information is available on

polysaccharide dynamics synoptically with toxin production inbenthic biotoxin producer dinoflagellates.

To date, most of the studies regarding P. lima report on itspronounced variable toxicity and toxin profile within and betweensites (Bouaıcha et al., 2001; Bravo et al., 2001); the development oftoxic events seems also quite unpredictable even under monitor-ing survey (Maranda et al., 2007a,b; Okolodkov et al., 2007;Aligizaki et al., 2009). Whereas, studies on effects of some nutrientconditions and nutrient sources on cell growth rate and toxiccontent are scant (e.g. Morlaix and Lassus, 1992; Tomas and Baden,1993; McLachlan et al., 1994; Sohet et al., 1995; Varkitzi et al.,2010). These studies show that growth limitation in P. lima induceshigher toxin contents per cell; as a general feature, the mediagiving the least production of cells gave the most OA content percell, whichever nutrient is limiting. Additionally, P. lima cultures instationary phase are more toxic than nutrient-replete or exponen-tial phase cultures (Haecky, 1991; McLachlan et al., 1994; Quilliamet al., 1996; Nascimento et al., 2005).

To our knowledge, there is no information on relationshipsamong cell toxin content, polysaccharide and protein productionand/or accumulation in P. lima under nutrient imbalancedconditions. As mentioned, changes in carbohydrate and proteincell pools as well as changes in cell toxin production are indicativeof cell response to environmental constraints. The aim of thepresent study was to investigate on effects of nutrient deficiencyon cell growth, toxin (OA and DTX-1) content, and on protein andpolysaccharide production/accumulation in P. lima. To do this a P.

lima strain, isolated from the Sacca di Goro lagoon, was grownunder four nitrogen-limited levels (1/3-N, 1/10-N, 1/20-N, and 1/50-N) and four phosphorus-limited levels (1/3-P, 1/10-P, 1/20-P,and 1/50-P) with respect to control nutrient conditions (f/2medium; NO3

� and PO43� concentrations: 883 and 36.3 mM,

respectively; N/P ratio: 24).

2. Materials and methods

2.1. Sampling site and laboratory cultures

The Sacca di Goro is an estuarine environment of the Po RiverDelta, Northern Italy (448470–448500N and 128150–128200E). It is alarge lagoon (26 km2) with an average depth of 1.5 m and it isconnected to the sea by a 2 km wide mouth. It is subjected toanthropogenic eutrophication since the last two decades and itexperiences abnormal proliferation of macroalgae (Ulva rigida andGracilaria sp.), oxygen depletion and anoxic crises. The catchmentarea is heavily exploited for agriculture, whilst the lagoon is one ofthe most important aquaculture systems in Italy (Viaroli et al.,1995).

Several strains of P. lima were isolated by the capillarypipette method (Hoshaw and Rosowski, 1973) in the summer2002, from the coastal lagoon of Goro located in the north-western coastal area of Adriatic Sea (Italy). After an initialgrowth in microplates, the cells were cultured in sterileErlenmeyer flasks sealed with cotton plugs at 20 8C and undera 16:8 h L:D cycle (ca. 90 mmol m�2 s�1 from cool white lamp);cultures were established in natural seawater, at salinity of 25,where the nutrients were added at the f/2 concentration(Guillard, 1975).

Cell counts were made following Utermohl method and specificgrowth rate (m, day�1) was calculated using the followingequation:

m ¼ ln N1 � ln N0

t1 � t0

where N0 and N1 are cell density values at time t0 and t1.

Table 1Nitrate and phosphate concentrations (mM) added in the different growth media of P. lima bath cultures and related N:P ratios.

Control 1/3-N 1/10-N 1/20-N 1/50-N 1/3-P 1/10-P 1/20-P 1/50-P

NO3� (mM) 883 294 88.3 44.2 17.7 883 883 883 883

PO43� (mM) 36.3 36.3 36.3 36.3 36.3 12.1 3.63 1.81 0.73

N/P 24 8.2 2.4 1.2 0.49 73 243 488 1210

S. Vanucci et al. / Harmful Algae 9 (2010) 590–599592

Calculation of cell volume was made with the assumption ofellipsoid shape using the following equation (Sun and Liu, 2003):

V ¼ p6

� �� a� b� c

where a = dorsoventral diameter (length), b = width, c = cross-section (height).

In order to evaluate nutrient limitation effects, experiments onP. lima were performed under nine different N/P ratios (Table 1);cells were grown in duplicate Erlenmeyer flasks containing 800 mLmedium. All cultures were grown under same temperature, light,and salinity values described above. Cell counts were performedevery 3–5 days in settling chambers (Utermohl, 1958); cells werecollected at late stationary phase of growth (day 33).

2.2. Nutrient analysis

Nitrate and phosphate analyses were performed on filteredculture medium aliquots (Millipore cellulose filters, pore size0.45 mm) and analysed spectrophotometrically according toStrickland and Parsons (1972).

2.3. Carbohydrate analysis

Aliquots of 40 mL culture were centrifuged at 10,200 � g for15 min in a Beckman J2-HS centrifuge (rotor JS-13.1) to separatecells from supernatant; 40 mL of ethylic alcohol were added to20 mL supernatant (in duplicate) and kept at �20 8C for 24 h, thenthe polysaccharides were pelleted by centrifuging at 10,200 � g for15 min at 4 8C and the supernatant discarded. The two fractions,cells and pelleted polysaccharides, were analyzed according toMyklestad and Haug (1972). Briefly, 1 mL 80% H2SO4 was added tothe pellets and incubated 20 h at 20 8C; the suspension was then 7-fold diluted with distilled water and 1–2 mL were used forpolysaccharides analysis following the phenol–sulfuric acidmethod of Dubois et al. (1956) using glucose as a standard;carbohydrates were measured by adding 50 mL 80% phenol and5 mL 96% sulfuric acid to 1 mL culture extracts supplemented with1 mL filtered seawater (salinity 25); for each culture conditionthere were two replicate flasks and every experiment was repeated2 or 3 times. Values presented in figures are the means � standarddeviations (SD) of different experiments.

2.4. Protein analysis

Ten-milliliter aliquots of algal cultures (in triplicate) werecollected by centrifugation at 10,200 � g for 15 min; proteins wereanalyzed in pellets according to the method of Lowry et al. (1951)using albumin as standard, after resuspending the cells in 0.1NNaOH and sonicating for 1 min.

2.5. Sample extraction and clean-up

For toxicity studies, 700 mL culture of P. lima in duplicate werecollected by centrifugation at 4600 � g for 25 min at 10 8C toseparate cell pellets from the culture media. Each pellet was addedto 8 mL methanol 80% and sonicated for 15 min in pulse mode,while cooling in ice bath. The mixture was centrifuged at 2000 � g

for 15 min, the supernatant was decanted and the pellet waswashed twice with 8 mL of methanol 80%. The three supernatantswere combined, the volume was adjusted to 25 mL with methanol80% and analyzed directly by LC–MS (5 mL injected).

2.6. Liquid chromatography–mass spectrometry analyses

LC–MS–MS measurements were carried out on a liquidchromatograph coupled to a triple quadruple mass spectrometerVarian 1200L with an electrospray ionization source (Varian Inc.,Walnut Creek, CA, USA). LC separation was conducted on a 5 mmPolaris C18, 150 mm � 2.0 mm column (Varian Inc., Walnut Creek,CA, USA). The mobile phase consisted of methanol:water (13:87, v/v) (eluent A) and methanol:water (90:10, v/v) (eluent B), bothcontaining 50 mM formic acid and 4 mM ammonium hydrate.Flow rate was 200 mL min�1. A gradient elution was programmed.Multiple reaction monitoring (MRM) experiments were carried outin negative ion mode in order to investigate the presence of OA (m/z 803 > 255) and DTX1 (m/z 817 > 255). For identification andquantification purposes okadaic acid standard solution and MUS-bcertified reference material were purchased by NRC (Halifax,Canada).

2.7. SEM observations

Culture aliquots were collected by centrifugation, dehydratedby immersion in alcohol at increasing gradations, and treated in aCritical Point Dryer. The cells were placed on a stub and sputteredwith gold–palladium for observation under the SEM (Philips EM515, 25 kV).

2.8. Statistical analysis

Differences in cell abundance, cell biovolume, intra-cellular andextra-cellular carbohydrate concentrations, OA and DTX1 con-centrations among cultures maintained under different inorganicnutrient conditions 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. Nutrient concentrations

Nutrient concentration changes during cell growth underdifferent N-nitrogen and P-phosphorus levels are reported inFig. 1. Under control conditions, initial nitrogen concentration(883 mM) remained high (341 mM) over stationary growth phase(see next section), while initial phosphorus concentration (36 mM)decreased to 1.35 mM by late exponential phase and declined to0.22 mM at late stationary phase.

Under N-limited conditions at stationary phase, N concentra-tions decreased to 1.8 and 1.4 mM in 1/3-N and 1/10-N treatments,respectively, whereas N was exhausted (<0.1 mM) in 1/20-N and 1/50-N treatments. Initial phosphate concentration (36 mM for all Ntreatments) decreased to <2 mM within 13 days in all treatments;at late stationary phase residual P concentrations were <0.15 mM

Fig. 1. Nitrogen (NO3�) and phosphorus (PO4

3�) concentrations measured in P. lima cultures grown under different nutrient conditions. Bars indicate standard deviations.

S. Vanucci et al. / Harmful Algae 9 (2010) 590–599 593

in 1/3-N and 1/10-N treatments and <0.1 mM in 1/20-N, and 1/50-N treatments.

Under all P-limited conditions, initial nitrate concentration(883 mM for all P levels) remained high in all P treatments from478 to 735 mM (from 1/3-P to 1/50-P level) over stationary phase.Whereas, phosphorus concentrations rapidly decreased within thefirst 7 days of growth, and at late stationary phase residualP concentrations were dropped down to <0.1 mM.

3.2. Cell growth and biovolume

In Fig. 2 are reported scanning electron and light micrographs ofthe P. lima strain isolated in the Sacca di Goro lagoon.

Fig. 2. Scanning electron micrographs of the P. lima strain isolated from the Sacca di Goro

and (d).

Growth curves of P. lima under different N and P levels areshown in Fig. 3. Culture initial cell densities (i.e. control culture andnutrient limited cultures) were on average 300–400 cells mL�1.During the early exponential phase (i.e. from 1st to 7th day)growth rate was similar in all nutrient conditions and did notshow any significant difference between controls and treatmentsand among treatments (growth rate: 0.23 � 0.015 and0.22 � 0.014 div day�1, N-experiment and P-experiment, respective-ly; ANOVA, all p > 0.05). Under both nutrient limited conditions,stationary phase was attained after 23rd day in control (N/P ratio: 24,which is typical of f/2 medium) and in 1/3-nutrient depleted cultures(i.e. 1/3-N and 1/3-P-depleted levels); while stationary phase wasreached after 18th day under more severe limited nutrient levels.

lagoon (a) and (b); light microscopy micrographs of the isolated strain in culture (c)

Fig. 3. P. lima growth curves in control conditions (C) and in nutrient limited

conditions. (a) Different N-limited levels; (b) different P-limited levels. Bars

indicate standard deviations.

Fig. 4. Cell protein concentration in P. lima cultures grown under control (C) and

under different nutrient limited conditions. Bars indicate standard deviations.

S. Vanucci et al. / Harmful Algae 9 (2010) 590–599594

Under N-limited conditions, at the stationary phase, cell yieldshowed decreasing values with increasing N deficiency (range:3.31 � 104–7.04 � 103 cells mL�1, control and 1/50-N treatment,respectively; ANOVA, p < 0.001) attaining to a 4.7-fold lower celldensity under 1/50-N-limited level when compared to control.Particularly, cell yield was significantly reduced with respect tocontrol starting from 1/10-N level (53% lower cell density withrespect of control; ANOVA, p < 0.05). Cell yield decrease wasaccompanied by a cell volume increase (range: 5574–7244 mm3,control and 1/50-N treatment, respectively; ANOVA, p < 0.001;Table 2). As for cell yield, cell volume changed significantly withrespect to control starting from 1/10-N level (19% higher cellvolume with respect to control; ANOVA, p < 0.05), and the relativecell volume increase with respect to control ranged from 7% to 30%(1/3-N and 1/50-N treatments, respectively).

Under P-limited conditions, cell yield showed a decreasingtrend as P-limitation increased, attaining to a 5.6-fold lower valuein 1/50-P treatment with respect to control (range: 3.50 � 104–6.25 � 103 cells mL�1, control and 1/50-P treatment, respectively;ANOVA, p < 0.001). Significant yield decrease was readily observed

Table 2Biovolumes of P. lima cells grown under different nutrient conditions and protein, cell

Cell volume (mm3) Protein (fg/mm3) Cell carbohydra

Control 5573.5�54.4 212.1�20.8 252.7�55.1

1/3-N 5988.5�133.6 167.7�12.7 308.3�21.8

1/10-N 6627.0�110.3 109.6�16.5 413.6�21.8

1/20-N 6629.0�338.0 88.4�4.9 437.8�44.2

1/50-N 7243.5�45.9 92.4�10.6 537.5�32.1

1/3-P 6293.0�169.7 251.6�18.5 233.5�45.8

1/10-P 6667.0�116.0 261.2�27.0 367.7�61.1

1/20-P 7091.0�172.5 282.1�46.0 450.3�39.3

1/50-P 7513.0�52.3 324.9�9.6 566.1�31.1

under 1/3-P-limited level when compared to control (39% withrespect to control; ANOVA, p < 0.01). Cell yield decrease wasaccompanied by a cell volume increase (range: 5574–7513 mm3,control and 1/50-P treatment, respectively; ANOVA, p < 0.001;Table 2). As for cell yield, cell volume changed significantly withrespect to control starting from 1/3-P level (ANOVA, p < 0.05), andthe relative volume increase ranged from 13% to 35% (for 1/3-P and1/50-P, respectively).

3.3. Cell protein concentration

Protein concentration on a cell basis decreased significantlyunder N-limited conditions (range: 1.18–0.59 ng cell�1 for controland 1/20-N level, respectively; ANOVA, p < 0.001; Fig. 4). Proteinconcentration was significantly responsive to N deficiency startingfrom the 1/10-N level when compared to control (ANOVA,p < 0.05), showing thereafter almost stable values with nosignificant differences among 1/10-N, 1/20-N, 1/50-N treatments(ANOVA, ns). Conversely, under P-limited conditions, proteinconcentration showed a slight increasing trend with increasing Pdeficient level although not significant (range: 1.37–1.93 ng cell�1,control and 1/50-P treatment, respectively; ANOVA, ns; Fig. 4).

Protein concentrations expressed on a cell volume basis rangedfrom 88 to 212 fg mm3 and from 212 to 325 fg mm3 for N-experiment and for P-experiment, respectively (Table 2), showingsame pattern and statistical analysis results (data not shown) ofthat ones found for protein expressed per cell.

3.4. Cell carbohydrate and EPS concentrations

Under N-limited conditions, cell carbohydrate concentration ona cell basis displayed opposite pattern than that one observed forprotein concentration showing significant increasing trend withincreasing N-limited level (Fig. 5; range: 1.41–3.89 ng cell�1,control and 1/50-N treatment, respectively; ANOVA, p < 0.001).

carbohydrate, EPS and toxin content on a cell volume basis.

te (fg/mm3) EPS (fg/mm3) OA (fg/mm3) DTX-1 (fg/mm3)

168.5�2.0 1.22�0.01 0.022� 0.009

120.7�7.2 1.71�0.12 0.023� 0.005

125.8�10.3 1.73�0.06 0.028� 0.008

115.2�15.2 1.69�0.08 0.037� 0.009

139.0�4.6 1.74�0.02 0.054� 0.004

190.8�1.8 1.56�0.02 0.027� 0.003

347.1�0.7 1.76�0.15 0.039� 0.001

541.7�46.8 2.23�0.18 0.045� 0.001

728.2�109.4 1.43�0.05 0.035� 0.004

Fig. 5. Cell carbohydrate and extra-cellular polysaccharide concentrations in P. lima

cultures grown under control (C) and under nitrogen limited conditions. Bars

indicate standard deviations.

Fig. 7. OA and DTX-1 cell concentrations in P. lima cultures grown under control and

under nitrogen limited conditions. Bars indicate standard deviations.

S. Vanucci et al. / Harmful Algae 9 (2010) 590–599 595

As for protein concentration, cell carbohydrate changed signifi-cantly at the 1/10-N threshold level when compared to control(ANOVA, p < 0.05). By contrast, exopolysaccharide (EPS) concen-tration on cell basis did not change significantly (Fig. 5; range:0.72–1.00 ng cell�1, 1/3-N treatment and 1/50-N treatment,respectively; ANOVA, ns). The cell carbohydrate to EPS concentra-tion ratio was significantly higher in all treatments with respect tocontrol (ratio range: 1.50–3.87, control and 1/50-N treatment,respectively, data not shown; ANOVA, all p < 0.05) attaining toalmost stable values (i.e. 3.75–3.87) in the 1/10–1/50-N range.

Under P-depleted conditions, cell carbohydrate concentrationshowed a significant increasing trend with the increasing P-depleted level (Fig. 6; range: 1.25–4.25 ng cell�1, for control and 1/50-P treatment, respectively; ANOVA, p < 0.001) and significantdifferences were found among 1/10-P, 1/20-P and 1/50-P treat-ments (ANOVA, p < 0.05 for all). EPS concentration followed thesame pattern of that one observed for cell carbohydrateconcentration (range: 0.84–5.46 ng cell�1, control and 1/50-Ptreatment, respectively; ANOVA, p < 0.001; Fig. 6) attaining to avalue 6.8 times higher than control value under 1/50-P level.Significant differences were found among 1/10-P, 1/20-P, and 1/50-P treatments (ANOVA, all p < 0.05). The cell carbohydrate toEPS ratio decreased with increasing P-depleted level due to arelative higher increase of EPS than cell carbohydrate concentra-tion in all treatments (ratio range: 1.49–0.78, control and 1/50-Ptreatment, respectively, data not shown), and the decrease wassignificantly lower with respect to control (about 1.8-fold lower) in1/20-P and P-1/50-P treatments (ANOVA, p < 0.05 for both) whenEPS concentration exceeded cell carbohydrate concentration.

Cell carbohydrate concentrations per biovolume quota rangedfrom 253 to 537 fg mm3 and from 253 to 566 fg mm3 for N-experiment and for P-experiment, respectively; whereas EPSconcentrations ranged from 139 to 169 fg mm3 and from 169 to728 fg mm3 for N-experiment and for P-experiment, respectively

Fig. 6. Cell carbohydrate and extra-cellular polysaccharide concentrations in P. lima

cultures grown under control (C) and under phosphorous limited conditions. Bars

indicate standard deviations.

(Table 2). The data per cell volume quota showed same trend andstatistical analysis results of that ones reported on a cell basis.

3.5. Toxin concentrations

The investigated P. lima strain contained okadaic acid (OA) asthe most abundant toxin, while DTX-1 was the second toxin inconcentration and it was almost two orders of magnitude lowerthan OA. These two toxins were then chosen for monitoringchanges due to nutrient deficiency.

OA concentration on cell basis increased significantly under alltested N levels with respect to control (range: 6.69–12.50 pg cell�1; control and 1/50-N treatment, respectively,ANOVA, p < 0.001; Fig. 7) attaining to the highest value in 1/50-N treatment (1.9-fold higher with respect to control); whereas nosignificant differences were found among 1/10-N, 1/20-N and 1/50-N treatments (ANOVA, ns for all). DTX-1 per cell concentrationwas more than one order of magnitude lower than OA concentra-tion and ranged from 0.12 to 0.39 pg cell�1 (control and 1/50-Ntreatment, respectively; Fig. 7). Although DTX-1 concentrationshowed an increasing trend with the increasing N-depletion level,DTX-1 value was significantly higher only in 1/50-N treatment(3.6-fold) when compared to control (ANOVA, p < 0.05). The OA toDTX-1 concentration ratio (OA/DTX-1 ratio range: 32.06–77.38; 1/50-N and 1/3-N, respectively; data not shown) showed a slightdecreasing trend with increasing N-depletion level due to a relativehigher increase of DTX-1 with respect to OA concentration,however no significant differences were found between controland treatments (ANOVA, ns).

Under P-limited conditions, OA per cell concentration increasedalong with increasing P-deficiency up to 1/20-P level attaining to a2.3-fold higher value than control (Fig. 8; range: 6.87–15.80 pg cell�1 control and 1/20-P treatment, respectively);whereas at 1/50-P level OA dropped at 10.75 pg cell�1. OAconcentration showed a prompt significant increase under 1/3-Plevel (ANOVA, p < 0.05). As for N-depleted treatments, DTX-1concentration (range: 0.13–0.32 pg cell�1, control and 1/20-Ptreatment, respectively) was more than one order of magnitudelower than OA concentrations while displaying similar OA pattern(Fig. 8). The OA/DTX-1 ratio (range: 40.7–57.5; 1/50-P and 1/3-P,respectively) did not show any significant difference amongtreatments except between 1/3-P and 1/50-P treatment (ANOVA,p < 0.05). OA and DTX-1 concentrations per biovolume quotaranged from 1.22 to 1.74 fg mm3 and from 0.022 to 0.054 fg mm3

for N-experiment; while OA and DTX-1 concentrations rangedfrom 1.22 to 2.23 fg mm3 and from 0.022 to 0.045 fg mm3 for P-experiment, respectively; Table 2). The data showed same patternand statistical analysis results of that ones reported per cell basis.

Fig. 8. OA and DTX-1 cell concentrations in P. lima cultures grown under control and

under phosphorous limited conditions. Bars indicate standard deviations.

S. Vanucci et al. / Harmful Algae 9 (2010) 590–599596

4. Discussion

4.1. Cell growth and nutrients

As general, nutrient limited conditions applied in this study didnot affect the early exponential growth phase of P. lima and themean growth rate (0.22 div day�1) was within the range reportedfor several worldwide distributed P. lima strains (Sohet et al., 1995;Nascimento et al., 2005 and references therein, Varkitzi et al.,2010). Starting from the late exponential phase, growth wasaffected by nutrient deprivation and this determined significantlower final cell yields with respect to control conditions. P. lima

growth pattern was dependent on nutrient dynamics as indicatedby N and P analyses performed during growth. Nitrogen levels aswell as its uptake rates (data not shown) became limiting startingfrom 1/10-N treatment, while P was more rapidly depleted andinfluenced cell yield already at the 1/3-P treatment. As alreadyshown for a different P. lima strain (Varkitzi et al., 2010) Prepresents the nutrient that mainly affects the growth of thisdinoflagellate; its uptake rates were high during the exponentialgrowth phase while nitrogen maintained high uptake rates alsoduring the stationary phase. In our case, by applying more severe Nand P-deficiencies, we could observe that N utilization decreasedin cells facing a strong P-limitation (1/20- and 1/50-P) so thatresidual N in these cultures was increasingly high.

4.2. Protein, carbohydrate and EPS

Cell protein and carbohydrate estimations reported here forcontrol cultures (mean values: 1.30 and 1.34 ng cell�1, for proteinand cellular carbohydrate concentration, respectively) are compa-rable with those ones reported for the Australian P. lima clones byMorton and Tindall (1995).

On the whole protein content, cell carbohydrate and EPSproduction/accumulation displayed crucial different patternchanges when comparing N-limited and P-limited conditions.

These differences can be summarised as follows: (i) significantdecrease of cell protein concentration (up to 2-fold) under N-limitation, conversely no significant changes in protein concentra-tion under P-limitation; (ii) significant increase of cell carbohydrateconcentration (up to 2.8-fold and 3.4-fold for N- and P-limitations,respectively) under both N- and P-limitations however differentlevel-deficiency dependent patterns were displayed under the twonutrient conditions; (iii) significant increase of EPS concentration(up to 6.50-fold) under P-limitation, conversely no significantchanges in EPS concentration under N-limitation.

These findings clearly indicate that the two nutrient imbal-anced conditions affect differently, at least in part, important cell

metabolic pathways, and that P. lima adopts different eco-physiological strategies to face N-limitation or P-limitation.

Nitrogen deprivation at the 1/3-N-limited level (i.e. N mediumconcentration: 294 mM; N/P ratio: 8.2) did not significantly affectcell yield together with protein and cell carbohydrate concentra-tions, indicating that cellular metabolic activities were notsubstantially injured. Actually, in this treatment medium nitrogenconcentrations remain high (�200 mM) by mid growth stationaryphase, and only at late stationary phase values drop down,although they still were not limiting (>2 mM). Protein synthesiswas significantly affected starting from 1/10-N level at whichcultures experienced low N concentrations (�11 mM) startingfrom late exponential phase and this likely triggered a cascade ofmetabolic impairments such as prevention of cell duplication. Thehighest increasing rate of cell carbohydrate accompanied with thesignificant decrease of cell yield with respect to balancedconditions observed at this N-deficient level corroborate thishypothesis. Cell carbohydrates are likely stored as carbonskeletons for further utilization when N would be available againfor amino-acid and protein synthesis in support of cell growth (e.g.Cullen, 1985; Fraga, 2001). Under more severe N-limited condi-tions N levels and uptake rates after the exponential phase greatlydecreased in 1/10-N treatment attaining exhaustion in the 1/20-and 1/50-N treatments; in these conditions all variables showedrather steady values indicating that in our N-limited experimentthe initial medium nitrogen concentration of 88.3 mM (N/P ratio:2.4) is the limiting value for coping induced imbalancedmetabolism.

Differently from nitrogen, phosphate deficiency significantlyaffected cell yield readily at the lowest imbalanced level (i.e. 1/3-Pthreshold level; phosphorus concentration: 12.1 mM; N/P ratio:73) at which most P was consumed within the first 7 days, thusindicating a rapid impairment of P-depending cell metabolismsuch as nucleic acid duplication, and more generally, P-dependingcell membrane and enzymatic activities.

Under increasing P-deprivation cell carbohydrate and EPSconcentrations also increased constantly and unlike for nitrogendeficiency, a clear P-limited level-dependent increasing trend wasfound for both of them.

As mentioned above, while the role of intra-cellular carbohy-drates may be clearly linked to cellular metabolic needs (e.g.carbon storage), the function of the polysaccharide release bymicroalgae is not univocal (Wood and Van Valen, 1990; Decho andLopez, 1993; Reynolds, 2007). The release of EPS and othercarbohydrate-rich exudates into the environment have beenproposed as an advantageous mechanism for regulating theaccumulation of excess photosynthates; under particular condi-tions the rate of excretion increases with depletion of essentialnutrients and the rate of bacterial nutrient re-mineralisation canbe sufficiently rapid to have a positive effect on the microalgaepopulation. This carbohydrate dynamics are reported for plank-tonic (Myklestad, 1974; Passow et al., 1994; Obernosterer &Herndl, 1995; Giroldo et al., 2007) and benthic diatoms (Under-wood and Paterson, 2003; Haynes et al., 2007) in both laboratoryand natural conditions. Yet, Guerrini et al. (2000) found that in thediatom Achnanthes brevipes extra-cellular carbohydrate concen-tration increased only under P-deficiency but not under Ndeficiency and this difference was mainly addressed to a reducedcarbohydrate catabolism and storage compound synthesis in theformer condition.

4.3. Toxins

OA and DTX1 were the main toxins produced by the studied P.

lima strain. The OA cell contents reported here under controlconditions (mean value: 6.78 pg cell�1) are well within the range of

S. Vanucci et al. / Harmful Algae 9 (2010) 590–599 597

values reported in literature for many strains isolated fromdifferent geographic regions (Murakami et al., 1982; Tomas andBaden, 1993; Morton and Tindall, 1995), including Europeancoastal areas (Bravo et al., 2001; Nascimento et al., 2005).Particularly, control OA values reported for our strain arecomparable with those reported for the strains isolated from thelagoon of Cies Islands (Bravo et al., 2001). By contrast, the DTX-1cell concentrations (mean value: 0.12 pg cell�1) were one order ofmagnitude lower than those reported for European lagoons, withthe exception of two strains (Bravo et al., 2001; Nascimento et al.,2005), and comparable to those reported for other geographicareas (Tomas and Baden, 1993). The isolation of P. lima strainswhich do not produce DTX-1 in association with relative low OAproduction have been reported from several regions (Murakamiet al., 1982; Sanriku coast Japan, Koike et al., 1998; Holmes et al.,2001). However, it must be emphasised that our strain showedrelative high OA concentrations associated with very low DTX-1concentrations even under control conditions with the result of aOA/DTX-1 ratio of about 60, quite higher than most of the OA/DTX-1 ratios reported in literature, including those for European strains(OA/DTX-1 ratio range: 2.4–4.5, Bravo et al., 2001; 0.14–3.20,Nascimento et al., 2005; 0.16–0.60, Foden et al., 2005).

In this study, toxin production was induced irrespective ofwhich nutrient was limiting; this finding is in accordance withprevious studies on P. lima (McLachlan et al., 1994; Sohet et al.,1995; Varkitzi et al., 2010) and it corroborates the knowledge thattoxin production is modulated by cell physiological conditionswhich are affected by a wide spectrum of factors, including severalenvironmental stressors such as nutrient availability (reviewed inLegrand et al., 2003; Ianora et al., 2006). Therefore, DSP toxin over-production is likely a non-specific cell response to imbalanced cellphysiology.

Overall, data reported here show that OA content increasedsignificantly up to 1.9- and 1.4-fold (1/50-N level, per cell andbiovolume, respectively) under N-limitation with respect to controlconditions. Whereas, under P-limitation OA increased significantlyup to 2.3- and 1.8-fold (1/20-P level, per cell and biovolume,respectively) with respect to control conditions. The increase in celltoxin content is generally related to cell toxin accumulation due toincreased cell volume as a consequence of inhibition of cell division(Graneli and Flynn, 2006); still, although we are not in a position todemonstrate it, the higher toxin values reported here respect tocontrol, when expressing concentration per biovolume, indicates aninduction of toxin production in unbalanced nutrient cultures.

Generally, DTX-1 increased at a slight higher rate withincreasing nutrient deficiency when compared with OA, neverthe-less the OA/DTX-1 ratio did not change significantly. A slightdecrease in the OA/DTX-1 ratio due to a relative higher increase ofDTX-1 with respect to OA was previously observed by McLachlanet al. (1994) under stressing nitrogen concentrations, and morerecently by Nascimento et al. (2005) towards stationary growthphase when nutrients are coming limiting.

In this study OA maximum increase was almost of the sameorder of magnitude under both N- and P-limited conditions,however toxin induction showed a remarkable different deficiencydepend trend under the two conditions, and this difference wasshared with other studied parameters. In fact, OA increasedsignificantly at the lowest deficiency level (1/3 threshold level) forboth nutrients, and this prompt change suggests a rapid enhancedallelopathic cell response to imbalanced nutrient conditions.However, while under more severe N-limited levels (i.e. in the1/10-1/50-N level range) OA showed rather steady values, under P-limitation toxin synthesis followed a level-dependent pattern withthe exception of the 1/50-P level when we can reasonably supposethat the ATP levels as well as the general cell metabolism wereseverely affected.

As general, a correlation between nutrient limitation andtoxicity increase has clearly been established for various algaeproducing N-containing toxins, such as Alexandrium and Pseudo-

nitzschia species, where P-limitation was mostly found to beaccompanied by high toxin levels (Boyer et al., 1987; Andersonet al., 1990; Pan et al., 1996; Bechemin et al., 1999; Graneli andFlynn, 2006).

The dynamics of OA toxins are not univocal; OA levels havebeen shown to enhance under both P and N stresses in all P. lima

strains but, for example, only under N-limitation in Dinophysis

acuminata (Johansson et al., 1996). The behaviour of our strain issimilar to that one of other flagellates producing no N- or little N-containing toxins such as Prymnesium parvum and Crysochromulina

polylepis which show enhanced toxin levels under either nitrogenor phosphorous deficiency (Graneli and Flynn, 2006).

5. Conclusions

In the present study we analysed cell yield, protein, toxin (DTX-1 and OA) contents, synoptically with cell carbohydrate and EPSproduction/accumulation in P. lima under different levels ofnitrogen and phosphate deficiencies. Both N-limited and P-limitedconditions applied in our experiments affected all variables takeninto consideration with greater effects found for EPS and toxinproduction under phosphate limitation.

Benthic dinoflagellates are since long time known to releasecopious amount of mucilage, nevertheless, this is the first time thatenhanced EPS production under P-limited conditions is reported inbenthic dinoflagellates. This finding deserves interest in the lightthat as along with P. lima other toxic species are characterised bylarge aggregate formations as those observed for culturedOstreopsis ovata (Heil et al., 1993; Guerrini et al., 2010). Theproduction of EPS will offer these benthic species competitiveadvantages related to the several functions proposed for mucilagewhich may all be different from an original purpose of a merehomeostatic mechanism to balance cell stoichiometry (Reynolds,2007). Considering that these benthic species are easily suspendedand found in the upper layers of the water column it can be arguedthat the increase of exopolysaccharide excretion would facilitatebuoyancy as well as nutrient or DOM acquisition. The documentedmixotrophic behaviour of most benthic dinoflagellates (Stoeckeret al., 2006) could be of help in DOM demolition. Finally, thecombined increase of toxin and polysaccharides observed in P. lima

under P-deprivation could also have a synergistic role inallelopathy by decreasing animal feeding rate and/or the growthof competing photosynthetic species.

Acknowledgements

Financial support was provided by PRIN funds from MURST(Rome, Italy). A special thanks to Dr. Tiziana Romagnoli (UniversitaPolitecnica delle Marche, Ancona) for realizing SEM pictures.[SS]

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