Yessotoxins profile in strains of Protoceratium reticulatum from Spain and USA

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0041-0101/$ - see

doi:10.1016/j.tox

�CorrespondiOceanografıa, C

Vigo, Espana, S

E-mail addre

Toxicon 50 (2007) 1–17

www.elsevier.com/locate/toxicon

Yessotoxins profile in strains of Protoceratium reticulatumfrom Spain and USA

Beatriz Paza,�, Pilar Rioboa, Isabel Ramilob, Jose M. Francoa

aFitoplancton Toxico, Instituto Investigaciones Marinas (CSIC), Eduardo Cabello 6, 36208 Vigo, SpainbFitoplancton Toxico, Instituto Espanol de Oceanografıa, Centro Oceanografico de Vigo (IEO), Cabo Estay, Apdo 1552,

36200 Vigo, Espana, Spain

Received 27 October 2006; received in revised form 8 February 2007; accepted 8 February 2007

Available online 23 February 2007

Abstract

Seven strains of Protoceratium reticulatum isolated from Spain and the USA were cultured in the laboratory.

Yessotoxins (YTXs) quantification and toxin profile determination were performed by LC–FLD and LC–MS/MS.

The four Spanish strains were found to produce YTX and known YTX analogs, however, YTX was not detected in any of

the three USA strains. Among the strains that produced YTXs, toxin production ranged between 2.9 and 28.6 pg/cell. The

YTX profile was substantially different between strains, in three out of the four Spanish strains YTX was the main toxin

and in the fourth homoYTX was the prominent toxin. This work demonstrates that YTX is not always the main toxin in

P. reticulatum and a high variability in YTX amounts and profile found in other locations is confirmed.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Protoceratium reticulatum; Strains; YTXs; HomoYTXs; LC–FLD; LC–MS/MS; Toxin profile

1. Introduction

Yessotoxins (YTXs), a group of disulphatedpolyether toxins, are mainly produced by thedinoflagellate Protoceratium reticulatum (Claparedeet Lachmann) Butschli ( ¼ Gonyaulax grindleyi

Reinecke) and have been reported in strains fromNew Zealand (Satake et al., 1997), Japan (Satakeet al., 1999), Norway (Ramstad et al., 2001; Samdalet al., 2004), Italy (Ciminiello et al., 2003b), UK,Canada (Stobo et al., 2003) and Spain (Paz et al.,2004). YTXs are accumulated in shellfish and are

front matter r 2007 Elsevier Ltd. All rights reserved

icon.2007.02.005

ng author. Current Address: Instituto Espanol de

entro Oceanografico de Vigo, Apdo. 1552, 36280

pain. Tel.: +34 986 492111; fax: +34 986 498626.

ss: [email protected] (B. Paz).

toxic to mice when injected intraperioneally (Auneet al., 2002; Tubaro et al., 2003), causing falsepositives in the mouse bioassay for diarrheticshellfish poisoning (DSP). YTXs have been foundto be nondiarrhoeic toxins to humans (Tubaroet al., 1998; De la Rosa et al., 2001; Alfonso et al.,2003) but have been found to be potent cytotoxins(Bianchi et al., 2004; Konishi et al., 2004; Perez-Gomez et al., 2006). The European Union estab-lished a maximum level permitted in shellfish of1mg/Kg (CEE, 2002). YTXs have been detected inshellfish in Japan (Murata et al., 1987), Norway(Aesen et al., 2005), Chile, New Zealand (Yasumotoand Takizawa, 1997), Italy (Yasumoto and Takiza-wa, 1997; Ciminiello et al., 2003a) and Spain(Arevalo et al., 2004; Mallat et al., 2006).

.

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ARTICLE IN PRESSB. Paz et al. / Toxicon 50 (2007) 1–172

P. reticulatum produces a rich array of YTXanalogs, which have recently been discovered andisolated. The existence of about 100 analogs inP. reticulatum have been reported, although onlythe structure of about 22 of them have beenidentified (Miles et al., 2006a). The YTX productionand toxin profile in different P. reticulatum strainshave been found to be dependent on the origin ofthe strain (Ciminiello et al., 2003b; Eiki et al., 2005;Samdal et al., 2006). For instance trinorYTX, thefirst analog discovered in algae (Satake et al., 1999),was detected in Japanese (Satake et al., 1999) andNorwegian (Samdal et al., 2006) strains, but not inNew Zealand strains (Miles et al., 2005a). OtherYTX analogs, 1a-homoYTX, noroxoYTX, 45-hydroxyYTX and carboxyYTX, have been foundin one Italian strain (Ciminiello et al., 2003b). Butthere is some disagreement about the occurrence of45-hydroxyYTX and carboxyYTX in algae, becauseevidence suggests that they are produced mainly bythe metabolism in shellfish (Aesen et al., 2005). In aNew Zealand strain, which was studied in depth, acomplex YTX profile has been reported with a widerange of analogs not detected until now in any otherstrains such as 40-epi-YTX and YTX-enone isomersof noroxoYTX (Miles et al., 2004), 41a-homoYTXs,9-methyl-41a-homoYTXs, nor-ring-A-YTXs (Mileset al., 2005a), 44,55-dihydroxyYTXs derived from41a-homoYTX and 9-methyl-41a-homoYTX(Finch et al., 2005), hidroxy-amideYTXs derivedfrom the 41a-homoYTXs and 9-methyl-41a-homo-YTX (Miles et al., 2005b), 45-OHdinorYTX,oxotrinorYTXs (Miles et al., 2006a). The 32-O-mono- and di-glycosylYTXs were detected in strainsfrom Japan, Spain and New Zealand (Cooney et al.,2003; Souto et al., 2005; Miles et al., 2006b) and the32-O-mono, -di- and -tri-arabinosides of 1a-homo-YTX, until now, were only detected in two strainsisolated in Japan (Konishi et al., 2004). In otherJapanese strains the trinorhomoYTX was alsodetected (Satake et al., 2006). Nevertheless, in spiteof the high variability in the reported YTX profile, itseems that the major toxin in P. reticulatum isusually YTX, and only homoYTX, together with32-O-mono, -di- and -tri-arabinosides of 1a-homo-YTX, which was found to be the main toxin in twoJapanese strains (Konishi et al., 2004). Moreover,the amount of YTXs produced by each strain wasdifferent, ranging from 0 to 34 pg/cell. This varia-bility in production could be due to the strain, butalso due to the different culture conditions, methodof extraction or method of analysis used (Stobo

et al., 2003; Eiki et al., 2005; Samdal et al., 2006). Itis also assumed that YTX amounts and profile inshellfish are dependent on the profile of eachdinoflagellate strain.

Therefore investigation of different strains, re-garding the YTXs profile, the amounts of toxinproduced and its possible release into the medium, isessential in order to determine the potential toxicityof each P. reticulatum strain. Taking this intoaccount, the present study has focused on thedetermination of YTX production and YTX profilein several Spanish and USA strains of P. reticulatum.

2. Material and methods

2.1. Cultures

The seven strains of P. reticulatum, used in thisstudy, were obtained from the collection of phyto-plankton cultures at the Centro Oceanografico inVigo. Strains VGO757, VGO758 and VGO764 wereisolated from cysts in the Lagoon El Alfacs (Deltadel Ebro, Spain). The other four strains wereisolated from cells, GG1AM in La Atunara, (Cadiz,Spain), CCMP404 in Salton Sea (California, USA),CCMP1720 and CCMP1721 in Biscaney Bay(Florida, USA) (Table 1).

The species of the USA strains were identified inthe CCMP and the species of the Spanish strainswere identified in the CCVIEO: GG1AM strain bySantiago Fraga and VGO 757, 758 and 764 byIsabel Bravo. In order to calculate the biovolume,cells were assumed to be spheres and their diameterswere measured with the aid of a micrometereyepiece using the average value between lengthand width, n ¼ 30. ANOVA (po0.05) was used tocompare the cell volume of the different strains.

All of the cultures were grown in 3L Erlenmeyerflasks containing 2L of L1 medium without silicates(Guillard and Hargraves, 1993). Cultures wereinoculated with 35–65mL (500 cells/mL) in theexponentially growing phase of each P. reticulatum

strain, maintained at 1971 1C, at a salinity of 30and under an irradiance of 100–125 mmol photons/m2� s on a 12:12 h light:darkness regime. Cultures

were gently shaken once a day. To determine cellyield, 5mL aliquots of samples were collected whencultures reached the stationary phase at 31–33 days(Table 1). Cells were fixed with Lugol’s solution andwere counted by optical microscopy in a Sedge-wick–Rafter chamber.

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Table 1

Location, yield and YTX contents in Protoceratium reticulatum strains by LC–FLD analyses

Strain Location Yiel Culture YTXs cells YTXs medium Total YTXs % YTXs

(Cell/mL) Days (pg/cell) (ng/mL) (pg/cell) (pg/cell) (cells)

VGO758 Els Alfacs (Delta Ebro, Spain) 9700 31 18.7 93.01 10.0 28.6 65

VGO764a Els Alfacs (Delta Ebro, Spain) 24180 31 18.2a 54.73a 2.3a 20.5a 89

VGO757 Els Alfacs (Delta Ebro, Spain) 18633 31 14.3 19.41 1.0 15.3 93

GG1AM La Atunara (Cadiz, Spain) 16275 31 1.8 18.34 1.1 2.9 62

CCMP404 Salton Sea (California, USA) 5727 33 — — — — —

CCMP1720 Biscayne Bay (Florida, USA) 4396 33 — — — — —

CCMP1721 Biscayne Bay (Florida, USA) 5940 33 — — — — —

aIt was determined by LC–MS analyses that the main toxin in VGO764 strain is homoYTX. HomoYTX amount was calculated

assuming that homoYTX gave the same molar response than YTX.

B. Paz et al. / Toxicon 50 (2007) 1–17 3

2.2. YTXs extraction

YTXs were extracted from cells and culturemedium, separately. For this purpose, 50mLaliquots of each culture were harvested in thestationary phase, which was reached at 31–33 daysdepending on the strain (Table 1) and were filteredthrough 1.4 mmGF/C glass fiber filters (25mmdiameter) (Whatman, Maidstone, England). Thecells in the filter were extracted twice with MeOH.The MeOH cell extract and the filtered culturemedium were respectively loaded onto two differentSep-Pak C18 light cartridges (Waters USA) for thepurification of YTXs in solid phase (SPE). Car-tridges were washed with 4mL of 20% MeOH andYTXs were eluted using 4mL of 70% MeOH. Thisfraction was dried under a N2 stream and re-suspended in 0.5mL of MeOH for LC–MS analysisor derivatized with DMEQ-TAD, re-suspended inMeOH and finally quantified by LC–FLD (Pazet al., 2006).

In previous studies it has been found that toxin isreleased into the medium. The proportion of toxinbetween medium and cells varies depending on theculture phase (Paz et al., 2004, 2006). Therefore allthe toxin produced by cells in terms of pg/cell, wasdetermined as the sum of YTXs in both culturemedium and cells.

2.3. Toxin analysis by LC– FLD

YTX determination and quantification wereperformed by LC–FLD. Analysis was carried outby a system equipped with a Hitachi L-6200 Apump, a Jasco FP-920 fluorescence detector, aHitachi AS-4000 autosampler and a Lichrospher100 RP18 5 mm (4.6� 125mm) cartridge column. A

mobile phase of 100mM ammonium acetate, pH5.8:MeOH (3:7) at a flow rate of 0.75mL/min and35 1C column temperature, was used. The excitationand emission wavelengths were 370 and 440 nm,respectively (Yasumoto and Takizawa, 1997; Pazet al., 2004).

2.4. Toxin analysis by LC– MS

For YTXs identification a LC–MS system wasused. The separation column was an Xterra MS C185 mm (2.1� 150mm) cartridge at 35 1C. As mobilephase 2mM ammonium acetate (pH 5.8) (A) andMeOH (B), in a gradient elution (40–30% A in5min, 30–20% A in 5min, followed by 5min with20% A, then 20–0% A in 5min and 0% A for2min), were used. As flow rate and injectionvolume, 0.20mL/min and 10 mL were used, respec-tively. Mass spectral measurements were performedusing an ion trap mass spectrometer, ThermoFinnigan LCQ-Advantage, equipped with a micro-electrospray ionization (mESI), in negative ionmode. ESI was performed with a 4.5 kV sprayvoltage and 200 1C capillary temperature, flow15mL/min for sheath gas and 5mL/min forauxiliary gas. Full scan data were acquired fromm/z 500 to 2000. MS spectrum shifted prominentions at m/z [M-H]�, [M-2H+Na]� and [M-2H]�2,in different proportions depending on the YTXanalog. Therefore for MS quantification the threeprominent ions for each analog were selected. Thecontribution percentages of each analog to the totalYTXs production for medium and cells werecalculated, separately. Subsequent LC–MS3 forYTXs were performed by applying a supplementaryvoltage (Collision Energy, CE) of 35% and 40% onthe [M-H]� ion.

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Strain

VG

O76

4

VG

O75

8

VG

O75

7

GG

1AM

CC

MP4

04

CC

MP1

721

CC

MP1

720

Mea

n ±

2 SD

Vol

µm

3 x103

50

40

30

20

10

0

Fig. 1. ANOVA (o0.05) analyses for cell biovolume � 103 mm3

of the Protoceratium reticulatum strains used in this study.

B. Paz et al. / Toxicon 50 (2007) 1–174

For YTXs identification by LC–FLD andLC–MS a YTX standard purchased from theInstitute of Environmental Science and ResearchLimited (New Zealand), a 45-OHYTX and carbox-yYTX standards provided by Professor T. Yasu-moto and a G-YTXA extract purified from cells ofP. reticulatum (Souto et al., 2005) were used. Thesame YTXs standards were used for homoYTXsidentification because homoYTXs eluted in thesame retention times as YTX homologues and thefragmentation pattern is superimposable on that ofYTXs, but shifted in 14 mass units (Ciminiello et al.,2003b). However quantification was performedusing just the YTX standard, due to the lack ofappropriate standards for all the YTX and homo-YTX analogs detected. The assumption was madethat all the analogs would give the same molarresponse as YTX.

3. Results and discussion

3.1. Cultures of P. reticulatum

Under the culture conditions used in the currentstudy, the seven strains of P. reticulatum achievedthe stationary phase in about 31–33 days dependingon the strain. The cell yield ranged among strainsbetween 4396 and 24180 cell/mL. The lowest celldensity was achieved by CCMP404 and the highestby VGO764 (Table 1).

The cell biovolume ranged from 3.7 to12.4� 103 mm3. The strain with smallest volumewas VGO764 and the highest was CCMP1720(Fig. 1). No significant differences (po0.05) in cellbiovolume were found among the strainsCCMP1720, CCMP1721 and CCMP404, the sameoccurred among the strains GG1AM, VGO757 andVGO758. However, significant differences (po0.05)in biovolume were found between the strainsCCMP1720, CCMP1721 and CCMP404 with re-gard to the strains GG1AM, VGO757, VGO758and VGO764 (po0.05). Furthermore there weresignificant differences (po0.05) between the strainsGG1AM, VGO757 and VGO758 with regard toVGO764. (Fig. 1).

3.2. LC– FLD determination and quantification of

YTX

Chromatographic analysis of the SPE 70%MeOH fraction of both, medium and cells showedthe characteristic double peak for derivatized YTX

only in the four Spanish strains. Comparison withthe retention time of the YTX standard (Fig. 2),demonstrated YTX production by the four Spanishstrains: GG1AM, VGO758, VGO757 and VGO764(Fig. 2). The three USA strains: CCMP404,CCMP1720 and CCMP1721, were confirmed asnot producing YTXs (Fig. 2). Owing to the factsome reported YTX analogs are more polar thanothers (Miles et al., 2005a), the 20%MeOH fractionof the SPE purification was analysed, but deriva-tized YTXs were not detected in any of the strains.

Total toxin production differed from strain tostrain, between 2.9 and 28.6 pg/cell. The leastproductive one was GG1AM and the most wasVGO758, whereas YTX was not detected in strainsCCMP404, CCMP1720 and CCMP1721 (Table 1).Under the current culture conditions YTX wasfound both, inside cells and in the culture medium,at the end of the culture. In all the strains most ofthe toxin remains inside the cells, in differringpercentages, depending on the strain (Table 1). InVGO758 and GG1AM strains the quantity of toxinreleased into the culture medium represents 35%and 38% of total toxin, respectively (Table 1), thisbeing a considerable amount. These percentages arenot a permanent characteristic of the strains,because cellular content of toxin is subject to cultureconditions and varies substantially depending onthe culture phase (Paz et al., 2004, 2006).

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-5

0

5

10

15YTX standard

-5

0

5

10

15

GG1AM

-5

5

15

25

35

45 VGO758

-5

5

15

25

35

45 VGO757

-5

15

35

55

75

95

VGO764

-5

0

5

10

15

CCPM404

-5

0

5

10

15

0 5 10 15 20

Minutes

CCPM1720

-5

0

5

10

15

0 5 10 15 20

Minutes

CCPM1721

Flu

ore

scence

Flu

ore

scence

Fig. 2. LC–FLD chromatograms of cells from Protoceratium reticulatum strains: 6 ng of YTX standard, GG1AM, VGO758, VGO757,

VGO464, CCPM404, CCMP1720 and CCMP1721. Similar chromatograms were obtained for culture medium.


The Spanish strains contained similar levels ofYTX to those found in isolates from Japan, Italy orNew Zealand, determined by LC–FLD or LC–MS,in which the YTX content ranged from 0.3 to15.7 pg/cell (Satake et al., 1999; Boni et al., 2000;Stobo et al., 2003; Ciminiello et al., 2003b; Eikiet al., 2005; Mitrovic et al., 2005; Rhodes et al.,2006) (Table 2). Nevertheless, amounts of YTXdetected were lower to that obtained in some NewZealand and Norwegian strains, 30–34 pg/cell,determined by ELISA (Samdal et al., 2004)(Table 2). It has been reported that differences intoxin concentration, were due to different analytical

methods employed, because some YTX analogswere quantified by ELISA but not by LC–MS orLC–FLD (Samdal et al., 2005). The antibodies usedin the ELISA analysis have a broad cross-reactivitywith most of the YTX analogs, therefore ELISAdetects all the YTX analogs jointly. Thereforesamples with a mixture of analogs will give a higherresult when analysed by ELISA than with chemicalmethods (LC–MS or LC–FLD) in which some ofthe YTX analogs may not be quantified, becausethey are determined separately. Taking this intoaccount, it is likely that the productivity of thosestrains analysed by different analytical methods

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Table 2

Some of the reported YTX concentration in different Protoceratium reticulatum strains

P. reticulatum strain Location Analysis technique YTXs Reference

(pg/cell)

Yamada Bay Japan LC–FLD 14 Satake et al. (1999)

Harina Nada Japan LC–FLD n.d. Satake et al. (1999)

New Zealand New Zealand LC–FLD 3.0 Satake et al. (1999)

Mutsu Bay Japan LC–FLD 0.9–11 Eiki et al. (2005)

Emilia-Romagna Italy LC–FLD 15.7 Boni et al. (2000)

UW351 UK LC–MS 0.3 Stobo et al. (2003)

UW409 Canada LC–MS 5 Stobo et al. (2003)

Adriatic Italy LC–MS 11.4 Ciminiello et al. (2003b)

CAWD40 New Zealand LC–MS 10–15 Mitrovic et al. (2005)

CAWD40 New Zealand ELISA 30 Samdal et al. (2004a)

AP2 Norway ELISA 19–22 Samdal et al. (2004a)

Sognfj03 Norway ELISA 19–34 Samdal et al. (2004a)

CAWD40 New Zealand ELISA 8.3 Rhodes et al. (2006)

CAWD127 New Zealand ELISA n.d. Rhodes et al. (2006)


were in similar ranges. Consequently, to compareresults it is essential that they be obtained with thesame technique. The absence of YTX in someP. reticulatum strains has also been reported inNew Zealand in the strain CAWD127 (Rhodeset al., 2006) and in Harina Nada, Japan (Satakeet al., 1999) (Table 2).

Despite the LC–FLD method being effective forquantification and detection of YTX and someYTX analogs, it is not able to detect analogswithout a conjugated diene in the side chain, as incarboxyYTX, carboxyhomoYTX, adriatoxin ordiOHYTXs. Another disadvantage is that homo-YTXs peaks overlap with YTXs peaks and areindistinguishable. To elucidate the presence ofYTXs analogs in the samples it was necessary toapply the LC–MS/MS analysis.

3.3. LC– MS/MS identification of YTXs

Identification of YTX analogs were performed byLC–MS and MS3. The two fractions harvested inthe SPE cleanup from both, culture medium andcells were analysed. YTXs were only detected in the70% MeOH fraction. The Spanish strains VGO757,VGO758 and GG1AM showed a similar toxinprofile, in which YTX was the main toxin, togetherwith small quantity of YTX analogs (Fig. 3). Incontrast, in the VGO764 strain the homoYTX wasthe main toxin and the minor components deter-mined were analogs of the homoYTX (Fig. 4). Aswas previously determined by LC–FLD, YTX orany of the analogs were not found in the USA

strains. Retention time and spectrum of injectedstandards used for LC–MS assignment are shown inFig. 5.

3.3.1. Profile of toxin in strains VGO757, VGO758

and GG1AM

LC–MS and MS3 analysis of VGO757, VGO758and GG1AM strains revealed the presence of YTXwith m/z 1141 (11.18min) as the main toxin,together with noroxoYTX-enone at m/z 1047(8.12min), 32-O-monoglycosylYTX at m/z 1273(10.45min), two unknown YTX analogs with m/z

1173 (8.08min) and with m/z 1157 (9.15min),respectively. Also trace amounts of 44,55-diOH-41a-homoYTX at m/z 1189 ( 8.61min), 32-O-diglycosylYTX at m/z 702 (9.72min) and homo-YTX with m/z 1155 (11.12min) (Tables 3 and 4)were detected. Without ruling out the possibilitythat these strains also contain other known andunknown YTX analogs. The assignment of theseanalogs is described below.

Ion m/z 1173 [M-H]� (8.08min): This ion was thefirst detected by LC–MS analyses (Fig. 3a). Theextracted ion chromatogram also showed promi-nent ions at m/z 587 [M-2H]�2 and m/z 1195[M-2H+Na]�. MS2 fragmentation of the m/z 1173[M-H]� ion gave an ion at m/z 1093 [M-SO3H]�.Additional MS3 fragmentation originated ions atm/z 924 and 855 (Fig. 6a). This daughter ionsscheme was consistent with the characteristicfragmentation of the YTX side chain (Fig. 7). Theretention time was different to that of carboxyYTX(4.69min) (Fig. 5a). The mass and fragmentation

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Fig. 3. Selected ion chromatograms and mass spectra obtained from negative LC–MS analyses of culture medium extract from

Protoceratium reticulatum VGO758 strain. (a) at m/z 1173 [M-H]� for a YTX analog, (b) at m/z 1047 [M-H]� for noroxoYTX-enone, (c)

at m/z 1157 [M-H]� for a YTX analog, (d) at m/z 636 [M-2H]�2 plus 1273 [M-H]� for 32-O-monoglycosilYTX, (e) at m/z 1141 [M-H]�

for YTX. Similar YTX pattern was obtained for GG1AM and VGO757 strains.


pattern indicates that this compound with m/z 1173[M-H]� should be the carboxiYTX, but the differentretention time to that of carboxyYTX standardsuggests that there is an unknown toxin belongingto the YTX group. A similar YTX analog waspreviously detected in algal samples, but itsstructure remains unidentified (Miles et al., 2005a).More structural studies will be necessary in order tofully identify this YTX analog.

Ion m/z 1047 [M-H]� (8.12min): This was thesecond compound detected by LC–MS (Fig. 3b) andshowed the same mass as that of the threeketoYTXs ( ¼ noroxoYTX) (Ciminiello et al.,

2003b; Miles et al., 2004). A common fragmentat m/z 967 [M-SO3H]� for ketoYTXs wasobtained in the MS2 analyses of the m/z 1047[M-H]� ion. The MS3 fragmentation of 967[M-SO3H]� ion was difficult and only a very weakion at m/z 883 was obtained (Fig. 7b), even whenapplying a high CE of 50. This fragment indicatesthat the m/z 1047 ion was the 1,3-enone isomer ofheptanor-41-oxoYTX ( ¼ noroxoYTX-enone) (Mileset al., 2004) and not any of the other ketoYTXanalogs (heptanor-41-oxoYTX or 40-epi-heptanor-41-oxoYTX) (Ciminiello et al., 2002a; Miles et al.,2004).

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Fig. 4. Selected ion chromatograms and mass spectra obtained from negative LC–MS analyses of culture medium extract from

Protoceratium reticulatum VGO764 strain. (a) at 1187 [M-H]� for a homoYTX analog, (b) at m/z 1061 [M-H]� for noroxohomoYTX-

enone, (c) at m/z 1203 [M-H]� for 44,55-diOH-9Me-41a-homoYTX? in the cell extract, (d) at m/z 1171 [M-H]� for a homoYTX analog,

(e) at m/z 709 [M-2H]�2 plus 1441 [M-2H+Na]� for 32-O-diglycosilhomoYTX, (f) at m/z 643 [M-2H]�2 plus 1287 [M-H]� for 32-O-

monoglycosilhomoYTX and (g) at m/z 1155 [M-H]� for homoYTX.


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Fig. 5. Selected ion chromatograms and mass spectra obtained from negative LC–MS analyses of YTXs standards. (a) at m/z 1173 [M-M-

H]� for carYTX, (b) at m/z 1157 [M-H]� for 45-OHYTX, (c) at m/z 636 [M-2H]�2 plus 1273 [M-H]� for 32-O-monoglycosilYTX and (d)

at m/z 1141 [M-H]� for YTX.

Table 3

Contribution percentage of each analog detected by LC–MS3 analyses in culture medium of the Protoceratium reticulatum strains

Rt m/z YTXs Culture medium Abundance (%)

Analog GG1AM VGO758 VGO757 VGO764

8.08 1173, 586 carboxiYTXanalog? 1.30 1.03 0.47 —

7.97 1187, 594 carboxihomoYTXanlog? — — — 0.75

8.12 1047 noroxoYTX-enone 0.98 1.04 0.97 —

8.03 1061 noroxohomoYTX-enone — — — 1.44

8.61 1189, 594 44,55-diOH-41a-homoYTX tr — tr —

8.68 1203, 601 44,55-diOH-9Me-41a-homoYTX — — — tr

9.15 1157, 578 45-OHYTXanalog? 1.05 1.18 0.58 —

9.18 1171, 585 45-OHhomoYTXanalog? — — — 1.00

9.72 1427, 702 32-O-diglycosylYTX tr tr — —

9.78 1441a, 709 32-O-diglycosylhomoYTX — — — 0.39

10.45 1273, 636 32-O-monoglycosylYTX 3.95 0.22 0.38 —

10.34 1287, 643 32-O-monoglycosylhomoYTX — — — 0.51

11.18 1141, 570 YTX 92.72 96.54 97.60 —

11.12 1155, 577 homoYTX tr tr tr 95.92

For each analog is given the retention time (Rt), m/z values for [M-H]� and [M-2H]�2 ions, respectively, the name for the analog and the

relative abundance observed in the LC–MS analyses. In bold, predominant pseudomolecular ion, tr ¼ traces.aFor this case the m/z is for the [M-2H+Na]� ion.


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Table 4

Contribution percentage of each analog detected by LC–MS3 analyses in cells in the analysed Protoceratium reticulatum strains

Rt m/z YTXs cells Abundance (%)

Analog GG1AM VGO758 VGO757 VGO764

8.12 1047 noroxoYTX-enone 0.51 0.13 0.11 —

8.08 1061 noroxohomoYTX-enone — — — 0.10

8.61 1189, 594 44,55-diOH-41a-homoYTX — — tr —

8.68 1203, 601 44,55-diOH-9Me-41a-homoYTX — — — 0.44

9.20 1157, 578 45-OHYTXanalog? 0.89 0.18 0.31 —

9.16 1171, 585 45-OHhomoYTXanalog? — — — 0.19

10.38 1273, 636 32-O-monoglycosylYTX 1.42 0.42 0.23 —

10.36 1287, 643 32-O-monoglycosylhomoYTX — — — 0.09

11.18 1141, 570 YTX 97.18 99.28 99.34 —

11.10 1155, 577 homoYTX — — — 99.17

For each analog is given the retention time (Rt), m/z values for [M-H]� and [M-2H]�2 ions, respectively, the name for the analog and the

relative abundance observed in the LC–MS analyses. In bold, predominant pseudomolecular ion, tr ¼ traces.


Ion m/z 1157 [M-H]� (9.15min): The extractedion peak also showed prominent ions at m/z 578[M-2H]�2 and m/z 1179 [M-2H+Na]�. The se-lected-ion plot at m/z 1157 [M-H]� gave a short,wide peak, as if there were several compounds withthe same mass eluting at different times (Fig. 3c).An inspection of the MS/MS spectrum of the peakshowed the typical fragmentation pattern of the45-OHYTX: MS2 of m/z 1157 [M-H]� ion dis-played a daughter ion at m/z 1077 [M-SO3H]�, andMS3 of this gave the m/z ions at m/z 924, 855 and713 (Fig. 6c). Retention time did not showcoincidence with 45-OHYTX standard (8.32min)(Fig. 6). This suggests the presence of a YTX analogdifferent to 45-OHYTX. This is supported bythe fact that most studies have indicated that45-OHYTX is produced only by metabolism inshellfish. There was one study in which 45-OHYTXwas found in algae (Ciminiello et al., 2003b), butchromatographic peaks for YTX analogs showedclosed retention times, therefore 45-OHYTX stan-dard and the m/z 1157 [M-H]� ion are practicallyindistinguishable. A series of four YTX analogswith m/z 1157 [M-H]�, and different to 45-OHYTX, have also been found by Miles et al.(2004), however structures have not been deter-mined yet. It is possible that our unknown YTXs atm/z 1157 [M-H]� might be the same as thatmentioned by Miles et al. (2004) because theirfragmentation pattern and chromatographic beha-viour were similar.

Ion m/z 1273 [M-H]� (10.40min): For thiscompound the prominent ion was di-charged atm/z 636 [M-2H]�2 (Fig. 3d) and the m/z 1295

[M-2H+Na]� ion appeared at very low intensity.The MS2 of m/z 1273 [M-H]� gave the ion at m/z

1193 [M-SO3H]� and MS3 displayed daughter ionsat m/z 1061, 1056 and 987 (Fig. 6d). The m/z 1061ion was generated due to the loss of a pentose unit.The m/z 1056 and 987 ions maintain the pentoseunit. The retention time and fragmentation patternmatches with the G-YTX A standard ( ¼ 32-O-monoglycosylYTX) (Fig. 5c) (Souto et al., 2005; Pazet al., 2006).

Ion m/z 1141 [M-H]� (11.18min): This was themost relevant peak in these Spanish strains (Fig. 3e).MS3 analyses showed the m/z 924, 855 and 713 ions,which is characteristic of YTX (Fig. 6e). Theirassignment as YTX was confirmed using a YTXstandard, which resulted in perfect coincidencein both retention time (Fig. 5d) and MS3 fragmen-tation.

Other minor compounds detected (data notshown): (i) The m/z 1189 [M-H]� ion (8.61min)was found in trace amounts in strains GG1AM andVGO 757. MS2 and MS3 fragmentation of m/z 1189[M-H]� gave two peaks at m/z 1109 and 925,respectively. This ion scheme indicated that itbelonged to the YTXs series. It is possible that thiscompound will be the 44,55-diOH-41a-homoYTX,whose structure was recently elucidated in a NewZealand P. reticulatum strain (Finch et al., 2005). (ii)In strains GG1AM and VGO 758, the 32-O-diglycosylYTX (Miles et al., 2006b), was detectedin trace amounts. This glycosyl derivative showedup as a prominent ion di-charged at m/z 702 [M-2H]�2 together with 1427 [M-2H+Na]� eluting at9.72min, the nono-charged ion (m/z 1403 [M-H]�)

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VGO758 strain. Applying a CE of 40% in the ion: (a) m/z

1093 [M-SO3H]�, (b) m/z 967 [M-SO3H]� in this case applying a

CE of 50%, (c) m/z 1077 [M-SO3H]�, (d) m/z 1193 [M-SO3H]�,

(e) m/z 1061 [M-SO3H]�. Similar MS3 spectra pattern was

obtained for GG1AM and VGO757 strains.


was not detected. Fragmentation was not performeddue to the small signal obtained. (iii) The m/z 1155[M-H]� ion (11.15min), was also found in smallquantities, eluting in the same retention time asYTX. MS3 fragmentation displayed the m/z 938fragment characteristic of 1a-homoYTX. This wasthe only compound from the homoYTXs seriesfound in these three strains.

3.3.2. Profile of toxin in strain VGO764

LC–MS and MS3 analysis of the VGO764 strainshowed a very different YTX profile to that of theabove mentioned strains. The main toxin was the

1a-homoYTX at m/z 1155 (11.12min), togetherwith noroxohomoYTX-enone at m/z 1061(8.03min), a possible 44,55-diOH-9-Me-1a-homo-YTX with m/z 1203 (8.55min), 32-O-diglycosylho-moYTX at m/z 709 (9.78min), 32-O-monoglycosylhomoYTX at m/z 643 (10.38min),and two unknown homoYTX analogs with m/z

1187 (7.97min) and with m/z 1171 (9.18min)(Tables 3 and 4). Also trace amounts of 32-O-triglycosylhomoYTX at m/z 775 (9.34min) and anunknown analog with m/z 1189 (7.41min) weredetected. This strain might also contain otherhomoYTX analogs. The assignment of these ana-logs is described below.

Ion m/z 1187 [M-H]� (7.97min): This was thefirst compound eluted which also showed pseudo-molecular ions at m/z 594 [M-2H]�2 and m/z 1209[M-2H+Na]� (Fig. 4a). The MS2 fragmentation ofthe m/z 1187 [M-H]� ion generates an ion at m/z

1107 [M-SO3H]�, which gave daughter ions in theMS3 spectrum at m/z 938, 869 and 727 (Fig. 8a).This was the characteristic fragmentation of thepolycyclic backbone skeleton of homoYTXs (Cimi-niello et al., 2003b) (Fig. 7). The mass andfragmentation of this peak was the same as that ofcarboxyhomoYTX (Ciminiello et al., 2000), butretention time was different to that of the carbox-yYTX standard (4.69min). Moreover, carboxyho-moYTX appears to be produced by metabolism inshellfish and not in algae (Ciminiello et al., 2000).These facts suggest that this compound is ahomoYTX different to carboxyhomoYTX andwhich has not been previously detected. This analogshould be the homologue of the ion at m/z 1173 [M-H]� in the YTX series, found in strains GG1AM,VGO757 and VGO 758.

Ion m/z 1061 [M-H]� (8.03min): This iondisplayed a peak with the same mass as that of thenoroxohomoYTX (Fig. 4b). In the MS2 analysis afragment ion with m/z 981 [M-SO3H]� wasobtained. The MS3 fragmentation of this ion gavea very weak peak at m/z 898 (Fig. 8b) even whenapplying a high CE. This fragmentation pattern isdifferent to that of the noroxohomoYTX (Cimi-niello et al., 2001), but is similar to that ofnoroxoYTX-enone (m/z 1047 [M-H]�) shifted in14 mass units. Therefore the m/z 1061 [M-H]� ionshould be the 1,3-enone isomer of noroxoho-moYTX, the homologue of noroxoYTX-enone(Ciminiello et al., 2001; Miles et al., 2005a) in theYTXs series. The noroxohomoYTX was previouslyidentified in mussels (Ciminiello et al., 2001), but, to

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Fig. 7. Structures and characteristic ion product ions observed in the LC–MS3 analyses of [M-SO3H]� ions of common yessotoxin

skeletons (n ¼ 1) and above of homoyessotoxin skeletons (n ¼ 2).


our knowledge the noroxohomoYTX-enone, hasnot been previously mentioned either in dinoflagel-late or in shellfish. Therefore it is possible thatseveral ketohomoYTXs exist, as happens withketoYTXs, but this would need to be confirmed infuture studies.

Ion at m/z 1203 [M-H]� (8.55min): was foundmainly in cells (Fig. 4c). MS2 of m/z 1203 [M-H]�

showed a fragment ion at m/z 1123 [M-SO3H]�, andthe MS3 gave a daughter ion at m/z 938 (Fig. 8c).This is the same LC–MS3 spectrum as that of the44,55-diOH-9-Me-41a-homoYTX, a YTX analogrecently identified in a New Zealand strain ofP. reticulatum (Finch et al., 2005). Taking intoaccount that this strain produces mainly 1a-homoYTX analogs we think that the m/z 1203 [M-H]�

ion could be 44,55-diOH-9-Me-1a-homoYTX,which should therefore generate the same fragmen-tation pattern. Further work will be required todetermine its assignment.

Ion m/z 1171 [M-H]� (9.18min): The next peakfound was for m/z 1171 (Fig. 4d). Fragmentation byMS2 of the m/z 1171 [M-H]� ion showed an m/z

1091 [M-SO3H]� daughter fragment, MS3 of thisfragment generated the ions at m/z 938, 869 and 727(Fig. 8d). Fragmentation scheme was consistentwith the characteristic fragmentation of the homo-YTXs side chain (Fig. 7). This pattern was

coincident with the 45-OHhomoYTX, previouslydetected in shellfish but not in algae (Ciminielloet al., 2002a), however, the retention time differedsubstantially to the 45-OHYTX standard(8.32min). These facts indicated that this ion is ahomoYTX analog different to 45-OHhomoYTXand had not been previously detected.

Ion m/z 709 [M-2H]�2 (9.70min): As wasdetermined in the glycosyl analogs, the generatedpeak showed a prominent di-charged ion, where asmall signal at m/z 1441 [M-2H+Na]� was detectedand the mono-charged ion at m/z 1419 [M-H]� wasnot detected at all (Fig. 4e). Fragmentation of themain ion at m/z 709 [M-2H]�2 was not possible.MS2 fragmentation of the m/z 1441 [M-2H+Na]�

ion showed as the prominent daughter ion the oneat m/z 1305 [M-C3H13O+H]� and also the ion atm/z 1177 due to the loss of the glycosyl units. MS3

fragmentation of m/z 1305 [M-C9H13O+H]� iongave the ions at m/z 1235 and 829 (Fig. 8e). ThisLC–MS3 fragmentation matches with that of pro-toceratin II determined by Konishi et al (2004) in aJapanese strain. Thus indicating the presence of 32-O-triglycosylhomoYTX ( ¼ protoceratin II) in theVGO764 strain.

Ion m/z 1287 [M-H]� (10.34min): The generatedpeak showed as prominent ion the m/z 643[M-2H]�2 (Fig. 4f). The same retention time was

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Fig. 8. MS3 spectra for selected peaks observed during LC–MS3

analyses of culture medium from Protoceratium reticulatum

VGO764 strain. Applying a CE of 40% in the ion: (a) m/z

1107 [M-SO3H]�, (b) m/z 981 [M-SO3H]� in this case applying a

CE of 50%, (c) m/z 1123 [M-SO3H]�, (d) m/z 1091 [M-SO3H]�,

(e) m/z 1304 [M-C9H13O]�, (f) m/z 1207 [M-SO3H], (g) m/z 1075

[M-SO3H]�.


recorded for G-YTX standard. MS2 fragmentationof the ion m/z 1287 [M-H]� gave one daughter ionat m/z 1207 [M-SO3H]� and by MS3 ions at m/z

1075 and 1071 (Fig. 8f). LC–MS3 fragmentationmatches with the G-YTX standard (Souto et al.,2005; Paz et al., 2006) differing in 14 units. Thusindicating the presence of 32-O-monoglycosylho-moYTX which has already been identified andcharacterised by Konishi et al (2004) in a Japanesestrain, together with the di and tri-32-O-glycosyl-homoYTX (Konishi et al., 2004). These being theonly homoYTX derivates identified up till now inalgae.

Ion m/z 1155 [M-H]� (11.12min): This was theprominent ion (Fig. 4g) detected. MS2 gave afragment ion at m/z 1075 [M-SO3H]�, and theMS3 generated daughter ions at m/z 938, 869 and727 (Fig. 8g). This MS/MS spectrum is character-istic of the 1a-homoYTX side chain. The assign-ment of 1a-homoYTX was confirmed bycomparison with the retention time and fragmenta-tion of the YTX standard (11.15min) (Fig. 6d). MS3

of homoYTXs is superimposable on the fragmenta-tion of YTXs, but shifted in 14 mass units(Ciminiello et al., 2002b) (Fig. 8).

Other minor compounds detected (data notshown): (i) An unknown analog with m/z 1189[M-H]� (7.41min), which overlapped with the signalof the m/z ion 1187 at 7.97 was detected. Thiscompound also displayed pseudomolecular ions atm/z 595 [M-2H]�2 and m/z 1211 [M-2H+Na]�

(Fig. 4a). The MS2 fragmentation of the m/z 1189[M-H]� ion generates an ion at m/z 1109 [M-SO3H]�, which gave daughter ions in the MS3

spectrum at m/z 938, 869 and 727 characteristic ofhomoYTX analogs. (ii) Also trace amounts of 32-O-triglycosylhomoYTX at m/z 775 [M-2H]�2

(9.34min) were detected in the extracts.

3.3.3. General toxin profile of strains

For VGO757, VGO758 and GG1AM strains(Fig. 4) YTX was the main toxin, but these strainsalso produced noroxoYTX-enone, 32-O-monoglyco-sylYTX, 32-O-diglycosylYTX, 44,55-diOH-41a-homoYTX, homoYTX, two unknown YTX analogs(Tables 3 and 4) and possible traces of other YTXanalogs. The VGO764 strain (Fig. 5) producedas main toxin the 1a-homoYTX, together withnoroxohomoYTX-enone, 32-O-triglycosylhomoYTX,32-O-diglycosylhomoYTX, 32-O-monoglycosylho-moYTX, 44,55-diOH-9-Me-41a-homoYTX and threeunknown homoYTX analogs (Tables 3 and 4). It is


possible that it could also contain other homoYTXanalogs.

It was observed that the analogs found in thethree Spanish strains where YTX prevailed, weresimilar to those found in the strain in which thehomoYTX prevailed, but as homoYTX analogs(Tables 3 and 4). Only trace amounts of homoYTXwere found in the VGO757, VGO758 and GG1AMstrains. But neither YTX nor YTX analogs werefound in VGO764 strain. These findings indicatethat there were at least two kinds of P. reticulatum,the YTXs producers and the homoYTXs producers.

The calculated contribution percentage of ana-logs to the total toxin production was very lowranging between 0.1% and 3.9% depending on theanalog and the strain. The presence of analogs wasmore important in the culture medium than in cells.Depending on the strain either YTX or homoYTX,was practically the only toxin detected. In theculture medium of the strains VGO757, VGO758and GG1AM the YTX represented between 92.7%and 97.6% of total toxin; and in strain VGO 764 thehomoYTX corresponded to a 95.9% (Table 3). TheYTX which remained inside the cells in strainsVGO757, VGO758 and GG1AM ranged from97.2% to 99.3% of total toxin; and homoYTX inVGO764 strain was a 99.2% (Table 4).

The low contribution percentage of YTX analogswas also found in other strains in different studies.In the Norwegian strain AP2 the percentage oftrinorYTX, the most abundant analog, representeda 3.8% of total toxin production, and for the otheranalogs (32-O-mono- and -di-glycosideYTX, ke-toYTXs, dihydroxyYTXs, etc.) corresponded to1–2% (Samdal et al., 2006). In one Italian strainall the analogs were about 5% of total YTXs(Ciminiello et al., 2003b).

The YTXs profile determined for the Spanishstrains differed from that of other recently studiedstrains, such as the New Zealand (100 YTXs) (Mileset al., 2006a), Norwegian (5 YTXs) (Samdal et al.,2006), Italian (5 YTXs) (Ciminiello et al., 2003b) andJapan (3 YTXs) (Satake et al., 2006) strains, whichmainly produce YTX. To our knowledge, only twoother Japanese strains (4 homoYTXs) (Konishi et al.,2004), showed a different profile, and they producedmainly homoYTX, as did the VGO 764 strainstudied here. Unlike the VGO 764 strain in theJapanese strain 32-O-mono, -di- and -tri-arabino-sides of 2-homoYTX were also detected, however,YTX analogs were not detected in any of the MutsuBay strains (Eiki et al., 2005). In the Yamada Bay

(Satake et al., 1999) and in the Norwegian (Samdalet al., 2006) strains 45,46,47-trinorYTX have beenfound, an analog not detected in any of the Spanishstrains studied here. In the New Zealand strainCAWD40, which was studied in depth, a complexYTX profile with 100 analogs was reported, but onlythe structure of about 20 of them have been identified(Miles et al., 2006a). In a Norwegian strain (AP2) theYTX together with 32-O-mono- and -di-glycosi-deYTX, trinorYTX, three ketoYTXs two dihydrox-yYTXs and a hydroxyYTX (not 45-OHYTX) werealso found (Samdal et al., 2006). Moreover, in anItalian strain, as in our strains, YTX, homoYTX andnoroxoYTX, were detected, but also 45-OHYTXand carboxyYTX (Ciminiello et al., 2003b) were notfound in any of the other studied strains. Finally inthe Japanese strain, only YTX, trinorYTX andthinorhomoYTX were found (Satake et al, 2006),but neither trinorYTX nor thinorhomoYTX weredetected in the Spanish strains studied here. Thesedifferences in YTX profile have been found amongstrains from different locations and evaluated bydifferent analytical methods, therefore differencescould be due to the different method of extractionor analysis employed. In the current study thestrains were isolated in the same location, grownunder the same conditions and analysed by the samemethod, therefore differences obtained can only bedue to strains with a different YTX profile andproduction.

3.4. LC– MS analysis vs. LC– FLD analysis

LC–MS analysis revealed that the strain VGO764produces mainly homoYTX instead of YTX,whereas LC–FLD analysis showed that all the fourSpanish strains produce YTX. As was expected,homoYTX and YTX were indistinguishable byLC–FLD. Therefore the analysis of new strainsmust first be approached by LC–MS. Then, once themain toxin produced by each strain is identified, itcan be determined and quantified by LC–FLDanalysis, assuming that homoYTX gives the samemolar response as YTX and taking into accountthat the amount of other analogs is low.

3.5. Cell biovolume vs. toxin profile

The significant differences (po0.05) found be-tween the strains in cell biovolume (Fig. 1) wereconsistent with differences found in YTXs profile.Similarly, CCMP1720, CCMP1721 and CCMP404

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showed the highest cell biovolume and were notYTX producers, whereas strains GG1AM,VGO757, VGO758 with medium biovolume wereYTX producers. Finally, the strain with the smallestbiovolume produces homoYTXs. These findingssuggest that the studied strains could be differentspecies. In this sense YTX was also found inGonyaulax spinifera (Rhodes et al., 2006) a dino-flagellate species different from P. reticulatum.Previous studies suggested two different organismsbeing responsible for production of these homo-logue series. A genetic study of the strains isnecessary to confirm this hypothesis.

In summary, the YTX profile was substantiallydifferent between strains. In three out of the fouranalysed strains YTX was the main toxin and inthe fourth homoYTX was the prominent toxin.P. reticulatum is able to produce compounds belongingto both YTX and homoYTX series. Toxin profile ofP. reticulatum determined only by LC–MS appearedto be, definitively, more complex than that previouslydetermined. Differences found among strains in YTXamounts, analogs produced and percentage of eachanalog produced could act as the ‘‘molecular finger-print’’ (Cembella, 2003), to distinguish and identifystrains from different locations.

Small amounts of sample were used to detect byLC–MS the presence of different YTX analogs inthe strains studied. Further work, culturing on largescale these dinoflagellates including isolation ofthese derivatives and NMR spectral analysis willbe required to fully identify and characterise thestructure of the unknown YTX, and above all, thehomoYTX analogs detected for the first time in thisstudy.

Acknowledgments

We thank Dr. C. O. Miles for his valuablecomments on MS analysis. We thank S. Fraga andIsabel Bravo for the morphological identification ofthe strains. This study was supported by projectAGL2005-07924-CO4-01/02 with the collaborationof the project ACU-02-005 INIA and CCVIEO.Beatriz Paz Pino is a pre-doctoral fellow of CSIC-Xunta de Galicia.

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Yessotoxins profile in strains of Protoceratium reticulatum from Spain and USA

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