Diatom ResearchVol. 26, No. 3, September 2011, 293–303

Toxicity of Pseudo-nitzschia populations from Bizerte Lagoon, Tunisia, southwestMediterranean, and first report of domoic acid production by P. brasiliana

INÈS SAHRAOUI1∗, STEPHEN S. BATES2, DONIA BOUCHOUICHA1, HASSINE HADJ MABROUK1 &ASMA SAKKA HLAILI1

1Laboratoire de Cytologie Végétale et Phytoplanctonologie, Université 7 novembre à Carthage, Bizerte, Tunisia2Fisheries and Oceans Canada, Gulf Fisheries Centre, Moncton, New Brunswick, NB, Canada E1C 9B6

Identification of species within the diatom genus Pseudo-nitzschia H. Peragallo, some of which produce the neurotoxin domoic acid(DA), causative agent of amnesic shellfish poisoning (ASP), is crucial for biotoxin monitoring and decision making concerning shellfishharvesting. The occurrence of this genus is reported here at species-complex resolution, for samples collected during one year from March2006 to February 2007 in Bizerte Lagoon (northern Tunisia) and at one coastal station located in the southwest Mediterranean Sea. Sixmorphospecies (P. brasiliana Lundholm, Hasle & Fryxell, P. calliantha Lundholm, Moestrup & Hasle, P. delicatissima (Cleve) Heidencomplex species, P. pseudodelicatissima (Hasle) Hasle, P. multistriata (Takano) Takano and P. seriata (Cleve) H. Peragallo group species)were observed in the field samples and their periodicity was studied. Five taxa (P. brasiliana, P. cf. delicatissima, P. pseudodelicatissima,P. multistriata and P. cf. seriata) were described for the first time for Bizerte Lagoon. Species of the P. delicatissima complex werethe most common, represented in 61% of the samples, followed by P. multistriata, P. calliantha and P. brasiliana, which were found in20–33% of the samples. The rarest species were P. pseudodelicatissima and species of the P. seriata group, which were present in only 7and 5% of the samples, respectively. Densest Pseudo-nitzschia blooms (>106cells L−1) were caused by P. delicatissima-complex species(March 2006) and P. brasiliana (October 2006). One P. brasiliana isolate and two strains of P. cf. delicatissima produced DA in culture(8.9 and 7.5–9.5 fg cell−1, respectively). This study gives the first report of DA production in P. brasiliana, based on the retention time ofa DA standard and its position relative to an internal standard, using high-performance liquid chromatography (HPLC). Our study callsattention to the probable occurrence of an ASP event in the region, where important shellfish aquaculture sites are located, and thereforethe requirement for a more intensive monitoring.

Keywords: Pseudo-nitzschia, ultrastructure, domoic acid, Pseudo-nitzschia brasiliana, toxicity, Bizerte Lagoon, aquaculture

IntroductionSince its implication in the first amnesic shellfish poisoning(ASP) event of 1987 in Canada, the genus Pseudo-nitzschiaH. Peragallo has rapidly become one of the best-studiedof all marine diatoms (Casteleyn 2009). High densitiesof domoic acid (DA)-producing Pseudo-nitzschia speciesand shellfish intoxications were subsequently registeredworldwide (Trainer et al. 2008), causing the suspensionof molluscan shellfish sales and heavy economic lossesto aquaculture. The economic significance of Pseudo-nitzschia species has accelerated the study of variousaspects of their biology and ecology (Bates 1998, Bateset al. 1998, Bates & Trainer 2006), which forms thebasis for monitoring, understanding and predicting Pseudo-nitzschia blooms. Information on the geographic distribu-tion of the genus Pseudo-nitzschia has also developed inrecent years (Hasle 2002, Casteleyn 2009). In particular,systematic studies of Pseudo-nitzschia have received con-siderable attention, emphasized by the need to distinguish

∗Corresponding author. Email: ineskalif@yahoo.fr

(Received 29 July 2010; accepted 13 May 2011)

toxic species from their non-toxic congeners. The genuscomprises more than 30 taxa of which 11 or 12 are potentialtoxin producers (Lundholm 2011). Pioneering taxonomicstudies have been based on morphometric characteristics ofthe frustule (Hasle 1965, 1993, Lundholm et al. 2002, 2003),which are still employed jointly with molecular tools in cur-rent systematic studies (e.g., Amato et al. 2007, Casteleyn2009, Moschandreou et al. 2010, Orive et al. 2010).

Bizerte Lagoon (SW Mediterranean) is an importantaquaculture area that supports an intensive fishery and hasrecently developed a considerable shellfish farming indus-try, namely mussels, oysters and clams (Tlili 2008). Foroyster and mussel production, ∼15 fixed structures (800 m2

each) are operating. Estimated annual production is ∼30tons of oysters and 100 tons of mussels. The most importantfraction of those seafood products is intended for the Euro-pean market. Water and shellfish quality are thus monitoredin Bizerte Lagoon, starting in 1995, within the frameworkof the Tunisian Harmful Algae Monitoring Program. This

ISSN 0269-249X print/ISSN 2159-8347 online© 2011 The International Society for Diatom Researchhttp://dx.doi.org/10.1080/0269249X.2011.597990http://www.tandfonline.com

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includes light microscopy identification and enumerationof toxic phytoplankton and measurement of toxins in shell-fish tissues using high-performance liquid chromatography(HPLC). Existing records of Pseudo-nitzschia based onelectron microscopy investigation are missing in the regionand data are usually limited to identification at the genericsize-group level, a distinction based on the width of thecell valve (Hasle 1965). Reported occurrences have beenreferred to as P. seriata (Cleve) H. Peragallo and P. del-icatissima (Cleve) Heiden (Turki & El Abed 2001, DalyYahia-Kéfi et al. 2005, Sahraoui et al. 2006). Data on thetoxicity of local Pseudo-nitzschia species are limited to thefindings of Sahraoui et al. (2009), who reported that a P. cal-liantha Lundholm, Moestrup & Hasle isolate from BizerteLagoon was a producer of DA.

This study documents the seasonal occurrence ofPseudo-nitzschia species populations over one year, bygathering light and electron microscopy data from sam-ples collected in Bizerte Lagoon from March 2006 toFebruary 2007. Data on the toxicity of local isolates ofPseudo-nitzschia species are also provided.

Materials and methodsStudy areaBizerte Lagoon (Fig. 1) is a shallow, restricted water body,located in northeast Tunisia, southwest Mediterranean Sea.It is connected to the Mediterranean through a narrow(300 m wide), shallow (12 m deep) and long (7 km length)channel. The mean depth of the lagoon is ∼8 m and the sur-face area is 120 km2 (Mzoughi et al. 2002). The climate istypical Mediterranean, i.e., hot–dry summer from May toSeptember and mild–rainy winter for the rest of the year.The lagoon is an enclosed water body with a high residence

1

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4 MJ

Tj

Mediterranean Sea

Tunisia

Bizerte Mediterranean Sea

Bizerte Lagoon

Ichkeul Lake

Tinja Channel

Musselaquaculture

Oysteraquaculture

Iron and steel metallurgy

Plastic and textile industries

Oil refinery

Harbor

Cement industry

37°10'

37°14'

37°12'

37°8'

37°16'

N

9°46' 9°48' 9°50' 9°52' 9°54' 9°56' E

Bizerte city

Menzel Abderrahmene city

Menzel Jemil city

Menzel Bourguiba city

1 km

N

Fig. 1. Map of Bizerte Lagoon, Tunisia, showing the sixsampling stations.

time (7 months) (Harzallah 2003), and nutrient loading fromseveral surrounding rivers and from urban, agricultural andindustrial activities (Sakka Hlaili et al. 2008).

Water samples were collected monthly at five stationslocated in Bizerte Lagoon and one (Sta 1) on the coastalMediterranean Sea (Fig. 1). Station Tj is at the interfacebetween the Tinja channel (connecting to Lake Ichkeul) andthe lagoon, and Sta MJ is at an aquaculture area. Samplingtook place over one year (from March 2006 to February2007), but was interrupted during three months (April andSeptember 2006, and January 2007).

SamplingSubsurface samples were obtained at 2 m (depth of thechlorophyll maximum, Sakka Hlaili et al. 2006) with aHYDRO-BIOS (Kiel-Holtenau, Germany) water samplerand fixed in Lugol’s iodine solution (3% final concentration)for microscopic analysis. Live samples were also obtainedto establish laboratory cultures of Pseudo-nitzschia species.Isolates were mainly obtained during bloom periods, butalso occasionally when Pseudo-nitzschia was at mod-erate concentrations, during the months indicated inTable 1. Monoclonal cultures were established by single-cell micropipette isolation. All cultures were grown inf/2 medium (Guillard & Ryther 1962) in 125 mL flasksincubated at 20C and an irradiance of 100 μmol photonsm−2 s−1, provided by cool-white fluorescent tubes, with a12:12 h light:dark cycle.

Species examinationPseudo-nitzschia cells in field samples were examinedmicroscopically, after settlement of a 50 mL sample (Uter-möhl 1958). The following taxonomic references wereused to identify the Pseudo-nitzschia species: Hasle (1965),Hasle (1993), Takano (1995), Lundholm et al. (2002),Lundholm et al. (2003) and Lundholm et al. (2006). Fivemorphotypes of Pseudo-nitzschia (see Results) were rec-ognized by evaluating cell shape and measuring frustuledimensions and colony overlap, using a 10× eyepiece and100× objective, under oil immersion. Counts were made ona total of 162 samples (6 stations × 3 replicates × 9 months).Later, the first 30 samples counted, from the March andMay 2006 samplings, were re-counted to verify the num-bers and morphotypes viewed. The relative abundance ofPseudo-nitzschia species was also verified by transmissionelectron microscopy (TEM) (see below) for one field sam-ple every month by counting the first 30 valves found in thesample. This operation was repeated three times and thenthe average of the relative abundance of Pseudo-nitzschiaspecies as calculated under TEM was compared with thatobtained using light microscopy (LM). The reliability ofour LM counting method for each morphospecies was con-firmed from the P-value (Student’s t-test) obtained, whichwas always between 0.01 < P < 0.05.

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Pseudo-nitzschia

inB

izerteL

agoon,2006–2007295

Table 1. Morphometric summary (transmission and scanning electron microscopy examination) of Pseudo-nitzschia species from Bizerte Lagoon during March–October 2006 andFebruary 2007.

Valve Striae Fibulae PoroidsSpecies Isolate Central Rows in(sampling period) designation Length (μm) Width (μm) Number in 10 μm Number in 10 μm Number in 1 μm interspace stria

P. brasiliana S-9 23.7–24.1 (23.9 ± 1.2) 2.8–2.9 (2.9 ± 0.3) 27 [10] 27 [10] 6 [10] Absent 2(October) [10] [10]

P. cf. delicatissima Is-1, Is-4, 45.1–57.9 (52.6 ± 3.1) 1.4–1.7 (1.6 ± 0.6) 40–41 (40.3 ± 0.6) 22–26 (23.6 ± 2.4) 9–11 (10.8 ± 0.9) Present 2(March) Is-9, Is-10, [30] [30] [30] [30] [30]

Is-19, Is-38,P. cf. delicatissima Is-6, Is-8 40.0–40.7 (40.2 ± 0.3) 2.0–2.3 (2.2 ± 0.4) 41–42 (41 ± 0.4) 25–27 (26.3 ± 0.6) 9–12 (10.5 ± 1.4) Present 2

(October) [10] [10] [10] [10] [10]P. multistriata (July) – – 3.3 [1] 46 [1] 27 [1] 11 [1] Absent 2P. pseudodelicatissima S-2 49.7–52.6 (51.1 ± 1.8) 1.2–1.3 (1.2 ± 0.5) 42 [10] 22 [10] 6 [10] Present 1

(May) [10] [10]P. cf. seriata

(February)– – 5.3 [1] 14 [1] 14 [1] 8 [1] Absent 2 + 1

Note: Designations are given only for isolates. The data are shown as: range of measurements (mean ± SD) [number of measurements]. The data for species isolated into culture do notinclude morphometrics from field samples.

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Electron microscopy was used to identify Pseudo-nitzschia cells in culture, as well as in two additionalconcentrated field samples collected in July 2006 and Febru-ary 2007. For this, 20 mL subsamples of cultures and fieldmaterial (concentrated 10-fold using a vacuum filtrationapparatus) were filtered then washed with 250 mL of dis-tilled water through a 3 μm pore size polytetrafluoroethy-lene (PTFE) membrane. The material (Pseudo-nitzschiafrustules) was then re-suspended in 5 mL distilled water andcleaned by adding 10 mL each of concentrated sulfuric andnitric acid in a boiling water bath for 60 min. Frustules werethen washed once more with 250 mL of distilled water inthe filtration apparatus and re-suspended in a final volumeof 5 mL.

For scanning electron microscope (SEM) examination,0.5–1.0 mL of a cleaned sample was dispersed on a 3 μmPTFE filter. Filters were mounted on aluminum stubs usingdouble-sided tape, rimmed with colloidal carbon and coatedwith ∼10 nm of gold, and examined using a JEOL JSM-5600 SEM operating at 10 kV, at the Digital MicroscopyFacility (Mount Allison University, Canada). For TEM, onedrop of cleaned sample was diluted 20:1 (for cultures) or5:1 (for field samples) and single drops were dried onto400-mesh copper grids bearing pure carbon support films,and examined using a Tecnai G2 TEM operating at 60 kV,at the Electron Microscopy Unit (Faculté des Sciences deBizerte, Tunisia).

Domoic acid analysisDomoic acid (DA) analyses were carried out on 20-mLaliquots taken from the stationary phase (day 29) of each ofthe Pseudo-nitzschia spp. established in culture. Aliquotswere sonicated using a Vibra-Cell high-intensity ultrasonicprocessor, followed by filtration through a disposable poly-carbonate filter (0.45 μm pore size) to remove the debris.DA in the whole culture (cells plus medium) was ana-lyzed (detection limit ∼1.0 ng mL−1) on a HPLC usingthe fluorenylmethylchloroformate (FMOC) fluorescencederivatization technique of Pocklington et al. (1990).

ResultsDuring our sampling period, water temperature and salinity(determined in situ using a Microprocessor Conductiv-ity Meter (WTW-LF-197, Brives B.V., Germany)), rangedfrom 15.0 to 28.5C and 33.2 to 37.7, respectively. More-over, the chlorophyll a concentration (0.5–7.7 μg L−1)and nutrient availability (determined as in Sahraoui et al.2009) (0.75–6.3 μM nitrate, 0.02–0.84 μM nitrite, 0.6–1.3 μM phosphate, 1.2–8.5 μM silicate) were relativelyhigh (Sahraoui et al. unpub.).

Light microscopy identificationGross morphological features viewed by LM allowedcolonies of linear cells with rounded apices in valve view

and sigmoid valve ends in girdle view, of ∼60 μm inlength and 2.5–3.0 μm in width, to be attributed to P. mul-tistriata (Takano) Takano. Chains of linear, narrow cellsof ∼2 μm in width (transapical axis) and 40–70 mm inlength (apical axis), with a very small overlap (1/9) of thetruncate-rounded apices, were attributed to the P. delicatis-sima complex. The P. pseudodelicatissima (Hasle) Haslecells exemplified by pointed apices in valve and girdle viewswere recognizable by the 2.0–2.5 μm width and 80–100 μmlength, and the relatively large overlap (1/6) of the celllength. Chains of linear, narrow cells of ∼2 μm in widthand 100–110 μm in length with pointed frustule apices wereattributed to P. calliantha. Colonies of four to six linearcells (1.8–2.0 μm wide, 25–30 μm long), with truncatedapices in girdle view, overlapping by 1/8 of the cell length,were assigned to P. brasiliana Lundholm, Hasle & Fryxell.Fusiform cells of 5–6 μm in width and 75–100 μm longwere assigned to the P. seriata group.

Ultrastructural examinationExamination by SEM and TEM revealed the followingcultured isolates: one as P. brasiliana, eight as P. cf. deli-catissima and one as P. pseudodelicatissima (Table 1). Inthe two field samples (July 2006 and February 2007), twoadditional species (P. multistriata and P. cf. seriata) weretentatively identified based on morphometric data obtainedby TEM observation on frustule fragments (Table 1).

For P. brasiliana (Fig. 2A), the transapical axis (width)is 2.8–2.9 μm and the apical axis (length) 23.7–24.1 μm.The fibulae and striae are arranged equally (Fig. 2B),with 27 in 10 μm. The striae contain two rows of poroidsarranged by six in each 1 μm. The most advalvar cop-ula (valvocopula) contains one row of large (∼0.2 μm indiameter) poroids (40 in 10 μm).

Pseudo-nitzschia cf. delicatissima cells (Fig. 3A) are1.4–2.3 μm wide and 40.0–57.9 μm long. Fibulae (22–27in 10 μm) are coarser than the striae (40–42 in 10 μm).

Fig. 2. Pseudo-nitzschia brasiliana, strain S-9 (SEM). A) Lin-ear valve showing broadly rounded ends; B) central part ofthe valve with no central interspace. Scale bars = 5 μm (A);1 μm (B).

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Fig. 3. Pseudo-nitzschia cf. delicatissima, strain Is-1 (SEM). A)symmetrical valve; B) structure of the striae with two rows ofsmall poroids. Scale bars = 5 μm (A); 1 μm (B).

Fig. 4. Pseudo-nitzschia pseudodelicatissima, strain S-2 (SEM).A) narrow symmetrical valve; B) structure of the striae with onerow of square shaped poroids. Scale bars = 5 μm (A); 1 μm (B).

Striae are perforated by two rows of poroids (Fig. 3B)positioned close to the interstriae, with 9–12 alternatingporoids in 1 μm.

Pseudo-nitzschia pseudodelicatissima cells (Fig. 4A)are 1.2–1.3 μm wide and 49.7–52.6 μm long. The numberof striae is 42 in 10 μm and the number of fibulae is 22 in10 μm. There is one row of square poroids, arranged trans-versely by six in 1 μm (Fig. 4B). The velum morphologyconsists of two large hymenate sectors separated by a thinsiliceous band.

In the July 2006 field sample, the P. multistriata frustulefragment examined (Fig. 5A) is 3.3 μm wide. The numberof fibulae and striae in 10 μm is 27 and 46, respectively. Thestriae on the valve surface consist of two rows of poroidsarranged by 11 in 1 μm.

The P. cf. seriata frustule fragment (Fig. 5B), foundin the February 2007 sample, is 5.3 μm wide. The numberof fibulae and striae is 14 in 10 μm. Striae are perforatedby eight poroids in 1 μm. The specimen has two rows ofporoids plus one incomplete third row per stria. That third

A B

Fig. 5. A): Pseudo-nitzschia multistriata, field material (Sta-tion 4, July 2006) (TEM); B): Pseudo-nitzschia cf. seriata, fieldmaterial (Sta 1, February 2007) (TEM). Scale bars = 1 μm.

row is composed of just a few single poroids visible betweenthe two solid rows of poroids. Because of this, and the lack ofan entire frustule, the species was only tentatively identifiedas P. cf. seriata.

Occurrence of Pseudo-nitzschia species in BizerteLagoon 2006–2007During the study period, the genus Pseudo-nitzschia wasa large component of the phytoplankton community, rep-resenting up to 68% (at Sta MJ, October 2006) of thetotal phytoplankton. The annual average concentration,of ∼ 2 × 105 cells L−1 (average of all stations), was alsonotable.

Distribution patterns of Pseudo-nitzschia species for thesamples taken during the nine months of this study (March2006 to February 2007) are presented in Fig. 6. The primaryPseudo-nitzschia species belonged to the P. delicatissimacomplex, detected in 61% of the samples, by number (aver-age of all stations and months). They were observed duringmost of the study period at Sta 1 (the Mediterranean Sea)and at Sta 2 (closest to the sea); P. delicatissima-complexspecies were found less frequently in the inner lagoonstations. The highest abundances of these species werefound in March 2006, over the entire study area, attainingmaximum concentrations at Sta 2 (1.3 × 106 cells L−1), Tj(2.3 × 106 cells L−1) and MJ (2.1 × 106 cells L−1). At thetwo latter stations, the spring bloom of P. delicatissima com-plex species declined in May, whereas it took longer (untilJuly) at Sta 1. Blooms of these species recurred at Sta Tj(1.7 × 105 cells L−1, October) and MJ (1.2 × 105 cells L−1,July and October), but declined more rapidly.

The second most important taxon was P. multistriata,present in 33% of the samples (average of all stations andmonths). The best occurrences of this species was at Sta 3and Tj, where it was detected five times during the survey.At Sta 1, 2 and MJ, however, the species was present

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37°16¢ P. delicatissima P. multistriata

P. calliantha P. brasiliana

P. pseudodelicatissima P. seriata

no cells

<104 cells L–1

>104 cells L–1

>105 cells L–1

>106 cells L–1

37°14¢

37°12¢

37°10¢

37°8¢

37°16¢

37°14¢

37°12¢

37°10¢

37°8¢

37°16¢

37°14¢

37°12¢

37°10¢

37°8¢

37°16¢

37°14¢

37°12¢

37°10¢

37°8¢

9°48¢ 9°50¢ 9°52¢ 9°54¢ 9°48¢ 9°50¢ 9°52¢ 9°54¢

Fig. 6. Cell abundance of Pseudo-nitzschia species in Bizerte Lagoon (March 2006 to February 2007). Each circle represents a monthof the sampling period (starting from the top: March, May, June, July, August, October, November, December, February).

only sporadically. The species seemed to have no particularseasonal pattern and reached maximum concentrations(> 1.5 × 105 cells L−1) in July at Sta 2 and Tj.

Pseudo-nitzschia calliantha was also a significantrepresentative of the genus. It was found in 25% of thesamples (average of all stations and months) and did notfollow a seasonal pattern. The species was more abundant

at Sta MJ, where it was found from August 2006 to February2007. The incidence of P. calliantha was often discrete, as itwas generally detected at only two stations simultaneously.In July, however, the species was noted at four differentstations (1, 3, 4 and Tj) at the same time. Highest con-centrations (> 105 cells L−1) were found at Sta 1 (February2007) and MJ (October 2006).

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Pseudo-nitzschia brasiliana was present in 20% ofthe samples (average of all stations and months) and itoccurred mostly in late summer/early fall of 2006, atall stations except Sta 4, where it appeared only sporad-ically. Pseudo-nitzschia brasiliana appeared first in May,in samples collected at Sta 1, 3 and Tj, at relatively lowconcentrations, never exceeding 6.0 × 104 cells L−1. Later,in August, the species exhibited two localized bloomsat Sta 2 and MJ, where it reached high concentrations(5.9 × 105 and 2.5 × 106 cells L−1, respectively). Bloomsof this species were present in the entire study area (exceptSta 1) in October, when it reached maximum concentra-tions at Sta Tj and MJ (1.4 × 106 and 4.7 × 106 cells L−1,respectively).

Pseudo-nitzschia pseudodelicatissima was present onlysporadically in our study area and was found in only 7%of the samples (average of all stations and months). Thespecies first appeared in May 2006, at Sta 3, Tj and MJ.Concentrations were relatively low (< 6.5 × 104 cells L−1)at Sta 3 and MJ, but were much higher at Sta Tj (1.4 ×105 cells L−1), where it continued to increase until Junereaching 3.4 × 105 cells L−1.

Species of the P. seriata group were also uncommonin Bizerte Lagoon waters and were present sporadically;only in February 2007, at Sta 1, 3 and Tj, at low concen-trations (19.2 × 103, 3.6 × 103 and 57.8 × 103 cells L−1,respectively).

Toxin analysisDomoic acid was detected only in P. brasiliana (strain S-9)and P. cf. delicatissima species (strains Is-6 and Is-8), allfrom isolates obtained at Sta MJ in October 2006 (Table 2).Concentrations on day 29 of the stationary phase were all

Table 2. Domoic acid (DA) content of Pseudo-nitzschiaisolates after 29 days (stationary phase) in batch culture.

Species(2006 sampling Isolate DA DAperiod, Station) designation (ng mL−1) (fg cell−1)

P. brasiliana S-9 11.4 8.9(October,

Sta MJ)P. cf.

delicatissimaIs-1, Is-4, nd –

Is-9, Is-10,(March, Sta 1, 3,

4, MJ)Is-19, Is-38

P. cf.delicatissima

Is-6 9.8 7.5

(October,Sta MJ)

Is-8 11.6 9.5

P. pseudodeli-catissima

S-2 nd –

(May, Sta 2)

Note: nd: not detected

of a similar magnitude, with 11.4 ng DA mL−1 in strainIs-8 of P. brasiliana and 9.8–11.6 ng DA mL−1 in strainsIs-6 and Is-8 of P. cf. delicatissima, respectively. CellularDA concentrations were also equivalent for the two species,with 8.9 fg cell−1 and 7.5–9.5 fg cell−1 for P. brasiliana andP. cf. delicatissima species, respectively.

DiscussionBefore this study was conducted, there was a paucityof data on Pseudo-nitzschia species in Tunisian waters,because of difficulties in species determination during rou-tine monitoring programs. This is the first assessment ofPseudo-nitzschia species distribution in northern Tunisianwaters. Our study was prompted by the fact that BizerteLagoon constitutes one of the most important sites foroyster, clam and mussel production in Tunisia. Moreover,the lagoon is connected to Lake Ichkeul, which was classi-fied as a National Park in 1980, and is a world heritage siteand a Biosphere Reserve.

Here, the potentially toxic diatoms of the genusPseudo-nitzschia were revealed as substantial members ofthe phytoplankton community of the Bizerte Lagoon sur-face waters. The genus appears to have acquired a moresignificant presence when compared with data obtained byprevious studies conducted in the area (Chikhaoui et al.2008, Grami 2009, Sahraoui et al. 2009). For example,the genus was present at a higher frequency (in > 90% ofthe samples) and was more abundant, ranging from 1.9 ×104 cells L−1 to 4.9 × 106 cells L−1 when compared withvalues obtained during a 2004–2005 survey (Sahraoui et al.2009), when it occurred in 70% of the samples and oscillatedbetween 3.0 × 103 and 2.3 × 105 cells L−1. Furthermore,our investigation confirmed previous observations on thepreference of Pseudo-nitzschia species for enclosed waterbodies with a high residence time and nutrient loading fromrun-off (Phlips et al. 2004, Spatharis et al. 2007). More-over, our results are in agreement with recent observationsthat blooms of Pseudo-nitzschia have been increasinglyrecorded in the Mediterranean Sea (Congestri et al. 2008).This is possibly as a result of continuing eutrophica-tion, which can be exacerbated in enclosed Mediterraneancoastal bays by elevated nutrient loading from rivers,together with direct discharges of untreated or poorlytreated domestic and industrial wastewater (Puigserver et al.2010).

Concentrations > 105 Pseudo-nitzschia spp. cells L−1

were found during more than two thirds of the studyperiod, and exceeded 106 cells L−1 in two instances (Marchand October 2006). Bizerte Lagoon therefore qualifiesas an important area for producing Pseudo-nitzschia spp.Blooms were frequently seen in the lagoon sites, but alsoat the coastal sea site (Sta 1), where Pseudo-nitzschia spp.concentrations > 105 cells L−1 were documented on threeoccasions. It is concluded that Bizerte Lagoon is a pro-lific area where potentially toxic cells of Pseudo-nitzschia

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are frequently observed and is therefore a potential sourceof cells that can be transported to the sea under someenvironmental conditions.

Our taxonomic investigation provides the first detailedrecord of the diatom Pseudo-nitzschia down to the specieslevel in the Bizerte Lagoon water system. Six Pseudo-nitzschia morphotypes were distinguished by lightmicroscopy and supported by electron microscopy obser-vations: P. brasiliana, P. delicatissima complex (whichincludes P. cf. decipiens Lundholm & Moestrup and P. cf.delicatissima; see below), P. calliantha, P. pseudodelicatis-sima, P. multistriata and the P. seriata group. Each of thesespecies is a potential producer of DA (Lundholm 2011). Thestudy did not, however, find any evidence of P. galaxiaeLundholm & Moestrup, P. caciantha Lundholm, Moestrup& Hasle, P. inflatula (Hasle) Hasle, P. fraudulenta (Cleve)Hasle or P. mannii Amato & Montresor, despite the factthat they have been confirmed from the Mediterranean Sea(Congestri et al. 2008, Moschandreou & Nikolaidis 2010).The bottle sampling method used here led to an accuratequantification of the more abundant species present in thedepth sampled, but may not be the most appropriate way togather qualitative presence/absence information about therarer species, which may be better achieved by net tows.Thus, although P. multistriata and P. brasiliana are indi-cated here as new records, they were perhaps present butnot detectable during the March 2004 to March 2005 studyin the same area (Sahraoui et al. 2009). For the same reason,the additional species may have escaped observation duringthis study.

Pseudo-nitzschia delicatissima-complex species werethe best represented species in our waters, presentin 61% of the samples and reaching high concentrations(>106 cells L−1) in the spring (March 2006). Examina-tion of six strains of P. delicatissima-complex speciesblooms from the Bizerte Lagoon revealed fine morpholog-ical features similar, but not identical, to P. delicatissima(Lundholm et al. 2006). Isolates Is-1, Is-4, Is-9, Is-10, Is-19and Is-38 of this species complex exhibited a higher densityof striae in 10 μm (40–41; 40.3 ± 0.6) compared with 35–40 (36.8 ± 1.5) in P. delicatissima (Lundholm et al. 2006),30–36 (34.5 ± 1.4) in P. dolorosa Lundholm & Moestrup(Lundholm et al. 2006) or 36–40 (37.8 ± 1.2) in P. areny-sensis Quijano-Scheggia, Garcés & Lundholm (Orive et al.2010). It does, however, resemble P. decipiens, althoughthis species has a slightly higher striae density (41–46;43.2 ± 1.2). The latter was erected by Lundholm et al.(2006), who found that this species was morphologicallysimilar to P. delicatissima but with a higher density of striaeand band striae.

Likewise, the ultrastructural morphology of strains Is-6 and Is-8 obtained in October 2006 (accompanying theP. brasiliana bloom) resembled P. delicatissima (Lund-holm et al. 2006). However, the two specimens had awider valve transapical axis (2.0–2.4 μm; 2.21 ± 0.46 μm)

compared with P. delicatissima (1.5–2.0 μm; 1.8 ± 0.8 μm)(Lundholm et al. 2006).

The variability between strains of the P. delicatis-sima complex in Bizerte Lagoon may be related to theco-existence of distinct genotypes sharing very similarmorphological features, suggesting the existence of cryp-tic taxa. In the Gulf of Naples, for example, phylogeniesshowed the presence of five distinct lineages within whatwas once considered to be a single morphospecies of P. del-icatissima (Orsini et al. 2004). Moreover, Lundholm et al.(2006) reported that P. delicatissima must be considered asa species whose genetic variation is slightly larger than inother Pseudo-nitzschia species.

Strains Is-6 and Is-8 of P. cf. delicatissima were shownto produce relatively low levels of cellular DA (7.5 and9.5 fg DA cell−1, respectively), on day 29 of the station-ary phase (Table 2). Conversely, the six other isolates ofthe P. delicatissima complex species tested below the limitof detection for DA. This again suggests the existence ofgenetic variability among strains of P. delicatissima speciesof Bizerte Lagoon. Differences in environmental factorscontrolling DA production (e.g., nutrients, bacteria) canbe also suspected here, as the toxic P. delicatissima-likeisolates Is-6 and Is-8 were obtained together with the toxicS-9 isolate of P. brasiliana (October 2006 water samples) incontrast to the non-toxic P. delicatissima isolates obtainedfrom water samples, March 2006.

Our investigation revealed P. multistriata as the sec-ond major Pseudo-nitzschia taxon, and a new record forBizerte Lagoon. Even though the species identity wasbased on TEM investigation of a single frustule fragment(most of the diatom material July sample was broken,perhaps due to the high pressure engendered by the vac-uum filtration method), this species was found in fixedwater samples, where it was easily distinguishable bylight microscopy. The seasonality of P. multistriata inBizerte Lagoon is in agreement with findings of Quijano-Scheggia et al. (2008), who noted that the species wasfound mainly in summer along the NE Spanish coast(NW Mediterranean Sea), with maximum abundances of105 cells L−1.

Pseudo-nitzschia brasiliana has not been reported pre-viously from Bizerte Lagoon or Tunisian coastal waters.The occurrence of P. brasiliana in our study was limitedto the late summer–early fall period, with maximum celldensities of ∼ 106 cells L−1, in agreement with Quijano-Scheggia et al. (2008) and Loureiro et al. (2009), for theNW Mediterranean. This is the second report of this speciesin the Mediterranean Sea, first from the southwest part ofthat sea.

The delineation of P. brasiliana was unclear untilLundholm et al. (2002) resolved much of the taxonomicuncertainty resulting from the morphological similaritywith P. linea Lundholm, Hasle & Fryxell and P. americana(Hasle) Fryxell. The morphometrics of P. brasiliana in

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this study (Table 1) are similar, but not identical, to thosedescribed for P. brasiliana by Lundholm et al. (2002); theyfound 1.8–3.0 μm width (compared with our 2.9–3.0 μm);20–26 fibulae and striae in 10 μm (compared with our27); and 7–10 poroids in 1 μm (compared with our 6).The P. brasiliana from Bizerte Lagoon was found in sur-face waters (planktonic mode of living) and was usuallyobserved in stepped colonies. In addition, the speciesshowed a single row of large poroids in the girdle band. Allof these features separated P. brasiliana from P. americana(Lundholm et al. 2002). Moreover, the species exhibited anequal number of fibulae and striae, which differentiates itfrom P. linea (Lundholm et al. 2002).

Importantly, this is the first study that reveals the toxi-city of P. brasiliana in culture (Table 2), as determined bythe identical retention times of the presumed DA peak andthat of the DA standard in the HPLC chromatogram. Theretention times were found to be very reproducible with theHPLC gradient system, the relative standard deviation usu-ally being < 0.4% (Pocklington et al. 1990). An internalstandard (dihydrokainic acid; DHKA) also serves as a ref-erence peak for the HPLC-FMOC data system to recognizeany unusual retention time variations, and the time differ-ence between the DA peak and the DHKA peak must bewithin a delta time of 2.2 ± 0.1 min to accept the peak asDA. This strain is no longer available for further testing, butfuture isolates of P. brasiliana from Bizerte Lagoon shouldbe analyzed by liquid chromatography coupled with tandemmass spectrometery (LC-MS/MS) to verify absolutely theidentity of DA.

Lundholm et al. (2002) previously tested the toxi-genicity of P. brasiliana on four strains obtained fromone locality (Bay of Sepetiba, Brazil). All were negativefor DA, using the ultraviolet photodiode array detectionHPLC method, which is less sensitive (detection limit of4–80 ng DA mL−1) than the HPLC-FMOC method usedin this study (∼1 ng DA mL−1). The authors, however,underlined the possibility that there could be toxic iso-lates of P. brasiliana, given the low number of isolates theytested from one location. Also, three strains from Spanishwaters (NW Mediterranean Sea) tested negative for DA,using LC-MS/MS (limit of detection of 1 ng DA mL−1)(Quijano-Scheggia et al. 2010). Their cultures, however,were harvested during the exponential phase; the stationaryphase was not sampled. The toxin content of our P. brasil-iana isolate (∼9 fg DA cell−1, day 29 of the stationaryphase) is on the low end of the spectrum in comparisonto other toxic Pseudo-nitzschia species, which may pro-duce up to 67 pg DA cell−1 (Trainer et al. 2008). Giventhe low toxicity and sporadic presence of P. brasiliana inBizerte Lagoon, it may be at first considered as a low threatfor the region. However, DA was also detected in seawatersamples of Bizerte Lagoon in October 2006 (ranging from0.5 to 2.0 μg DA L−1; I. Sahraoui, B. Grami, S.S. Bates, D.Bouchouicha, M.A. Chikhaoui, H. Hadj Mabrouk, A. Sakka

Hlaili, unpub. data). Moreover, because P. brasiliana wasable to reach high cell concentrations in August at Sta 2and MJ (5.9 × 105 and 2.5 × 106 cells L−1, respectively), itshould be considered as potential serious menace for BizerteLagoon.

Pseudo-nitzschia pseudodelicatissima was observedonly as a minor component of the Pseudo-nitzschia pop-ulation in Bizerte Lagoon. Zingone et al. (2006) reportedan irregular occurrence of the species, in the TyrrhenianSea (Mediterranean). This species is well known fromthe French Mediterranean coast (Quiroga 2006). Taxo-nomic investigations (Lundholm et al. 2003) have begun toresolve some of the ambiguities raised by the morphologi-cal similarity among P. pseudodelicatissima, P. callianthaand P. caciantha. However, it would not be surprising tofind other cryptic or pseudo-cryptic species within thisspecies. Poroids in our P. pseudodelicatissima specimenwere divided into two sectors, which is fewer than the four tofive ascribed to P. caciantha and the seven to ten attributedto P. calliantha (Lundholm et al. 2003). Our specimen wasalso more delicately structured (42 striae and 22 fibulae in10 μm) when compared with P. caciantha (28–31 inters-triae and 15–19 fibulae in 10 μm) or P. calliantha (15–22fibulae and 34–39 striae in 10 μm). The number of poroidsin 1 μm, however, was typical of P. pseudodelicatissima(5–6 poroids per 1 μm).

Unlike the morphological variability demonstrated byP. pseudodelicatissima in the Bay of Fundy (Kaczmarskaet al. 2005), our specimens from the Bizerte Lagoon closelycoincide with the emended circumscription (Lundholmet al. 2003). Strain S-2 of P. pseudodelicatissima obtainedfrom Bizerte Lagoon waters had negative results for DAproduction under our conditions. Although previous studieshave reported low toxicity in this species (Martin et al. 1990,Lundholm et al. 1997, Adams et al. 2000, Pan et al. 2001),these studies were carried out prior to the re-examination ofthe P. pseudodelicatissima complex (Lundholm et al. 2003,Amato & Montresor 2008). Indeed, molecular studies arerequired to determine unambiguously the possible presenceof cryptic or pseudo-cryptic species within the P. pseu-dodelicatissima and P. delicatissima complex in BizerteLagoon.

Species of the P. seriata group were uncommon inthe waters, confirming the narrow spatio-temporal speciescomplex distribution found previously in Bizerte Lagoon(Sahraoui et al. 2009). The ultrastructural data, althoughlimited, suggest that our specimens from the Bizerte Lagoonbelong to the P. seriata group. The identity of this specieswas difficult to verify unambiguously, based on the obser-vations of a single fragment of frustule.

Almost all of the diatom material of the Februaryfield sample was, in fact, damaged, likely by the highpressure during this particular filtration. Moreover, P. cf.seriata is usually associated with cold waters of higherlatitudes in the northern hemisphere (Hasle 2002, Fehling

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et al. 2004). However, new findings may be challengingthis notion. Pseudo-nitzschia seriata group species wererecently reported for the Balearic Archipelago in the north-west Mediterranean Sea (Puigserver et al. 2010). A recentinvestigation suggested that P. seriata is found in thesouthern hemisphere, based on the poroid and striae density,although there was uncertainty because their specimens hadonly two rows of poroids (Almandoz et al. 2009). Likewise,the specimen was similar to P. seriata, except it had onlyan incomplete third row of poroids, which is similar to thecultured cells described by Fehling et al. (2004). For now,the existence of this species in Tunisian waters remainsquestionable and requires confirmation.

Given the occurrence of toxigenic P. brasiliana andP. delicatissima complex species at high cell densities inBizerte Lagoon and the presence of DA in those waters, itappears that toxic events may occur in the area. Neverthe-less, to our knowledge, no human-related health problemshave ever been reported in Bizerte Lagoon. The lack ofrecords for cases of ASP may be due to the misidentifica-tion of gastrointestinal syndromes. Whatever the reasons,the occurrence of toxic and potentially toxic species callsfor an intense and regular monitoring of the Bizerte Lagoonwaters, especially at Sta MJ, where commercial shellfishaquaculture sites are located.

AcknowledgementsWe gratefully thank Claude Léger (Fisheries and Oceans Canada,Moncton, New Brunswick, Canada) for the DA analyses andJames Ehrman (Digital Microscopy Facility, Mount AllisonUniversity, Sackville, New Brunswick, Canada) for producingscanning electron micrographs used in this study.

ReferencesAdams N.G., Lesoing M. & Trainer V.L. 2000. Environmental

conditions associated with domoic acid in razor clams on theWashington coast. Journal of Shellfish Research 19: 1007–1015.

Almandoz G.O., Hernando M. & Ferrario M.E. 2009. SEMobservations of Pseudo-nitzschia from Beagle Channel:P. seriata in the southern hemisphere? Harmful Algae News39: 6–7.

Amato A. & Montresor M. 2008. Morphology, phylogeny, andsexual cycle of Pseudo-nitzschia mannii sp. nov. (Bacillario-phyceae): a pseudo-cryptic species within the P. pseudodeli-catissima complex. Phycologia 47: 487–497.

Amato A., Kooistra W.H.C.F., Levialdi Ghiron J.H., Mann

D.G., Pröshold T. & Montresor M. 2007. Reproductiveisolation among sympatric cryptic species in marine diatoms.Protist 158: 193–207.

Bates S.S. 1998. Ecophysiology and metabolism of ASP toxinproduction. In: Physiological ecology of harmful algalblooms (Ed. by D.M. Anderson, A.D. Cembella & G.M.Hallegraeff), pp. 405–426. Springer-Verlag, Heidelberg.

Bates S.S. & Trainer V.L. 2006. Ecology of harmful algae.In: Ecology of harmful algae (Ecological Studies Vol. 189)(Ed. by E. Granéli & J. Turner), pp. 81–93. Springer-Verlag,Heidelberg.

Bates S.S., Garrison D.L. & Horner R.A. 1998. Bloomdynamics and physiology of domoic-acid-producing Pseudo-nitzschia species. In: Physiological ecology of harmful algalblooms (Ed. by D.M. Anderson, A.D. Cembella & G.M.Hallegraeff), pp. 267–292. Springer-Verlag, Heidelberg.

Casteleyn G. 2009. Species structure and biogeography ofPseudo-nitzschia pungens. Ph.D. Thesis, Ghent University,Belgium. 201 pp.

Chikhaoui M.A., Sakka Hlaili A. & Hadj Mabrouk H.

2008. Réponses saisonnières du phytoplancton aux rapportsd’enrichissements N:Si:P dans la lagune de Bizerte (sud-ouest de la Méditerranée). Comptes Rendus de Biologie 331:389–408.

Congestri R., Plizzano S. & Albertano P. 2008. ToxicPseudo-nitzschia populations from the middle TyrrhenianSea (Mediterranean Sea, Italy). In: Algal toxins: nature,occurrence, effect and detection (Ed. by V. Evangelista,L. Barsanti, A.M. Frassanito, V. Passarelli & P. Gualtieri),pp. 197–210. NATO Science for Peace and Security. SeriesA. Chemistry and Biology. Springer, Dordrecht.

Daly Yahia-Kéfi O., Soussi S., Gómez F. & Daly Yahia

M.N. 2005. Spatiotemporal distribution of the dominantdiatom and dinoflagellate species in the Bay of Tunis(SW Mediterranean Sea). Mediterranean Marine Science 6:17–34.

Fehling J., Green D.H., Davidson K., Bolch C.J. & Bates S.S.

2004. Domoic acid production by Pseudo-nitzschia seriata(Bacillariophyceae) in Scottish waters. Journal of Phycology40: 622–630.

Grami B. 2009. Dynamique et fonctionnement des réseauxtrophiques planctoniques dans la lagune de Bizerte: Mod-élisation par la méthode inverse. Ph.D. Thesis, Université deCathage, Tunisia. 375 pp.

Guillard R.R.L. & Ryther J.H. 1962. Studies of marine plank-tonic diatoms. I. Cyclotella nana Hustedt, and Detonulaconfervacea (Cleve) Gran. Canadian Journal of Microbiol-ogy 8: 229–239.

Harzallah A. 2003. Transport de polluants dans la lagune deBizerte simulé par un modèle de circulation de l’eau. Bulletinde l’Institut National des Sciences et Technologies de la Merde Salammbô 30: 121–133.

Hasle G.R. 1965. Nitzschia and Fragilariopsis species studiedin the light and electron microscopes II. The group Pseudo-nitzschia. Skrifter utgitt av Det Norske Videnskaps-Akademii Oslo I Matematisk-Naturvidenskapelige Klasse. Ny Serie.18: 1–45.

Hasle G.R. 1993. Nomenclatural notes on marine planktonicdiatoms. The family Bacillariaceae. Nova Hedwigia, Beiheft106: 315–321.

Hasle G.R. 2002. Are most of the domoic acid-producing speciesof the diatom genus Pseudo-nitzschia cosmopolites? HarmfulAlgae 1: 137–146.

Kaczmarska I., LeGresley M.M., Martin J.L. & Ehrman J.

2005. Diversity of the diatom genus Pseudo-nitzschia Per-agallo in the Quoddy Region of the Bay of Fundy, Canada.Harmful Algae 4: 1–19.

Loureiro S., Garcés E., Fernández-Tejedor M., Vaqué D. &

Camp J. 2009. Pseudo-nitzschia spp. (Bacillariophyceae) anddissolved organic matter (DOM) dynamics in the Ebro Delta(Alfacs Bay, NW Mediterranean Sea). Estuarine, Coastaland Shelf Science 83: 539–549.

Dow

nloa

ded

by [

Fish

erie

s an

d O

cean

s C

anad

a], [

Step

hen

Bat

es]

at 1

2:49

18

Febr

uary

201

3

Pseudo-nitzschia in Bizerte Lagoon, 2006–2007 303

Lundholm N. 2011. Bacillariophyta. In: IOC-UNESCO Taxo-nomic reference list of harmful micro algae (Ed. by N. Lund-holm). Available from: http://www.marinespecies.org/HAB/aphia.php?p=taxlist&pid=148899&tRank=220&rComp=%3D&context_in=30&vOnly=1 [Accessed 18 July 2010].

Lundholm N., Skov J., Pocklington R. & Moestrup Ø. 1997.Studies on the marine planktonic diatom Pseudo-nitzschia.2. Autecology of P. pseudodelicatissima based on isolatesfrom Danish coastal waters. Phycologia 36: 381–388.

Lundholm N., Hasle G.R., Fryxell G.A. & Hargraves P.E.

2002. Morphology, phylogeny and taxonomy of specieswithin the Pseudo-nitzschia americana complex (Bacillar-iophyceae) with descriptions of two new species, Pseudo-nitzschia brasiliana and Pseudo-nitzschia linea. Phycologia41: 480–497.

Lundholm N., Moestrup Ø., Hasle G.R. & Hoef-Emden K. 2003.A study of the Pseudo-nitzschia pseudodelicatissima/cuspidatacomplex (Bacillariophyceae): what is P. pseudodelicatis-sima? Journal of Phycology 39: 797–813.

Lundholm N., Moestrup Ø., Kotaki Y., Hoef-Emden K.,

Scholin C. & Miller P. 2006. Inter- and intraspecific varia-tion of the Pseudo-nitzschia delicatissima complex (Bacil-lariophyceae) illustrated by rRNA probes, morphologicaldata and phylogenetic analyses. Journal of Phycology 42:464–481.

Martin J.L., Haya K., Burridge L.E. & Wildish D.J. 1990.Nitzschia pseudodelicatissima – a source of domoic acid inthe Bay of Fundy, eastern Canada. Marine Ecology ProgressSeries 67: 177–182.

Moschandreou K.K. & Nikolaidis G. 2010. The genus Pseudo-nitzschia (Bacillariophyceae) in Greek coastal waters. Botan-ica Marina 53: 159–172.

Moschandreou K.K., Papaefthimiou D., Katikou P., Kalopesa

E., Panou A. & Nikolaidis G. 2010. Morphology, phy-logeny and toxin analysis of Pseudo-nitzschia pseudodeli-catissima (Bacillariophyceae) isolated from the ThermaikosGulf, Greece. Phycologia 49: 260–273.

Mzoughi N., Hellal F., Dachraoui M., Villeneuve J.P.,

Cattini C., De Mora S.J. & El Abed A. 2002. Méthodologiede l’extraction des hydrocarbures aromatiques polycycliques.Application à des sédiments de la lagune de Bizerte (Tunisie).Comptes Rendus Geoscience 334: 893–901.

Orive E., Laza-Martfnez A., Seoane S. & Alonso A. 2010.Diversity of Pseudo-nitzschia in the southeastern Bay ofBiscay. Diatom Research 25: 125–145.

Orsini L., Procaccini G., Sarno D. & Montresor M. 2004. Mul-tiple rDNA ITS-types within the diatom Pseudo-nitzschiadelicatissima (Bacillariophyceae) and their relative abun-dances across a spring bloom in the Gulf of Naples. MarineEcology Progress Series 271: 87–98.

Pan Y., Parsons M.L., Busman M., Moeller P.D.R., Dortch Q.,

Powell C.L. & Doucette G.J. 2001. Pseudo-nitzschia sp. cf.pseudodelicatissima – a confirmed producer of domoic acidfrom the northern Gulf of Mexico. Marine Ecology ProgressSeries 220: 83–92.

Phlips E.J., Badylak S., Youn S. & Kelly K. 2004. The occur-rence of potentially toxic dinoflagellates and diatoms in asubtropical lagoon, the Indian River Lagoon, Florida, USA.Harmful Algae 3: 39–49.

Pocklington R., Milley J.E., Bates S.S., Bird C.J., De

Freitas A.S.W. & Quilliam M.A. 1990. Trace determi-nation of domoic acid in seawater and phytoplankton byhigh performance liquid chromatography of the fluorenyl-methoxycarbonyl (FMOC) derivative. International Journalof Environmental Analytic Chemistry 38: 351–368.

Puigserver M., Monerris N., Pablo J., Alos J. & Moya G. 2010.Abundance patterns of the toxic phytoplankton in coastalwaters of the Balearic Archipelago (NW Mediterranean Sea):a multivariate approach. Hydrobiologia 644: 145–157.

Quijano-Scheggia S., Garcés E., Flo E., Fernández-Tejedor

M., Diogène J. & Camp J. 2008. Bloom dynamics of thegenus Pseudo-nitzschia (Bacillariophyceae) in two coastalbays (NW Mediterranean Sea). Scientia Marina 72: 577–590.

Quijano-Scheggia S., Garcés E., Andree K.B., De La Iglesia

P., Diogène J., Fortuño J.M. & Camp J. 2010. Pseudo-nitzschia species on the Catalan coast: characterization andcontribution to the current knowledge of the distribution ofthis genus in the Mediterranean Sea. Scientia Marina 74:395–410.

Quiroga I. 2006. Pseudo-nitzschia blooms in the Bay of Banyuls-sur-Mer, northwestern Mediterranean Sea. Diatom Research21: 91–104.

Sahraoui I., Sakka Hlaili A. & Bates S.S. 2006. First detec-tion of toxic Pseudo-nitzschia calliantha in Bizerte Lagoon,Tunisia. Harmful Algae News 30: 8–9.

Sahraoui I., Sakka Hlaili A., Hadj Mabrouk H., Léger C.

& Bates S.S. 2009. Blooms of the diatom genus Pseudo-nitzschia H. Peragallo in Bizerte Lagoon (Tunisia, SWMediterranean). Diatom Research 24: 175–190.

Sakka Hlaili A., Grami B., Niquil N., Gosselin M., Hamel

D., Troussellier M. & Hadj Mabrouk H. 2008. Theplanktonic food web of the Bizerte lagoon (south-westernMediterranean) during summer: I. Spatial distribution underdifferent anthropogenic pressures. Estuarine, Coastal andShelf Science 78: 61–77.

Spatharis S., Danielidis D.B. & Tsirtsis G. 2007. RecurrentPseudo-nitzschia calliantha (Bacillariophyceae) and Alexan-drium insuetum (Dinophyceae) winter blooms induced byagricultural runoff. Harmful Algae 6: 811–822.

Takano H. 1995. Pseudo-nitzschia multistriata (Takano) Takano,a new combination for the pennate diatom Nitzschia multis-triata. Diatom 10: 73–75.

Tlili H. 2008. The CIHEAM watch letter. Winter, No. 4.Trainer V.L., Hickey B.M. & Bates S.S. 2008. Toxic diatoms.

In: Oceans and human health: risks and remedies from theseas (Ed. by P.J. Walsh, S.L. Smith, L.E. Fleming, H.M. Solo-Gabriele & W.H. Gerwick), pp. 219–237. Elsevier Science,New York.

Turki S. & El Abed A. 2001. On the presence of potentially toxicalgae in the lagoons of Tunisia. Harmful Algae News 22: 12.

Utermöhl H. 1958. Zur vervollkommung der quantitativenPhytoplankton Methodik. Mitteilungen der InternationaleVereinigung für Theoretische und Angewandte Limnologie9: 1–38.

Zingone A., Siano R., D’Alelio D. & Sarno D. 2006. Poten-tially toxic and harmful microalgae from coastal waters ofthe Campania region (Tyrrhenian Sea, Mediterranean Sea).Harmful Algae 5: 321–337.

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