blooms of the diatom genus pseudo-nitzschia h. peragallo in bizerte...

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This article was downloaded by: [Nipissing University] On: 18 October 2014, At: 13:34 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Diatom Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tdia20 BLOOMS OF THE DIATOM GENUS PSEUDO- NITZSCHIA H. PERAGALLO IN BIZERTE LAGOON (TUNISIA, SW MEDITERRANEAN) Inès Sahraoui a b , Asma Sakka Hlaili a , Hassine Hadj Mabrouk a , Claude Léger b & Stephen S. Bates b a Laboratoire de Cytologie Végétale et Phytoplanctonologie, Département des Sciences de la Vie, Faculté des Sciences de Bizerte , Université 7 novembre à Carthage , 7021, Zarzouna, Bizerte, Tunisie b Fisheries and Oceans Canada, Gulf Fisheries Centre , P.O. Box 5030, Moncton, NB, E1C 9B6, Canada Published online: 31 Oct 2011. To cite this article: Inès Sahraoui , Asma Sakka Hlaili , Hassine Hadj Mabrouk , Claude Léger & Stephen S. Bates (2009) BLOOMS OF THE DIATOM GENUS PSEUDO-NITZSCHIA H. PERAGALLO IN BIZERTE LAGOON (TUNISIA, SW MEDITERRANEAN), Diatom Research, 24:1, 175-190, DOI: 10.1080/0269249X.2009.9705789 To link to this article: http://dx.doi.org/10.1080/0269249X.2009.9705789 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

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Page 1: BLOOMS OF THE DIATOM GENUS               PSEUDO-NITZSCHIA               H. PERAGALLO IN BIZERTE LAGOON (TUNISIA, SW MEDITERRANEAN)

This article was downloaded by: [Nipissing University]On: 18 October 2014, At: 13:34Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Diatom ResearchPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/tdia20

BLOOMS OF THE DIATOM GENUS PSEUDO-NITZSCHIA H. PERAGALLO IN BIZERTELAGOON (TUNISIA, SW MEDITERRANEAN)Inès Sahraoui a b , Asma Sakka Hlaili a , Hassine Hadj Mabrouk a ,Claude Léger b & Stephen S. Bates ba Laboratoire de Cytologie Végétale et Phytoplanctonologie,Département des Sciences de la Vie, Faculté des Sciences deBizerte , Université 7 novembre à Carthage , 7021, Zarzouna,Bizerte, Tunisieb Fisheries and Oceans Canada, Gulf Fisheries Centre , P.O. Box5030, Moncton, NB, E1C 9B6, CanadaPublished online: 31 Oct 2011.

To cite this article: Inès Sahraoui , Asma Sakka Hlaili , Hassine Hadj Mabrouk , Claude Léger& Stephen S. Bates (2009) BLOOMS OF THE DIATOM GENUS PSEUDO-NITZSCHIA H. PERAGALLOIN BIZERTE LAGOON (TUNISIA, SW MEDITERRANEAN), Diatom Research, 24:1, 175-190, DOI:10.1080/0269249X.2009.9705789

To link to this article: http://dx.doi.org/10.1080/0269249X.2009.9705789

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever orhowsoever caused arising directly or indirectly in connection with, in relation to or arisingout of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

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Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

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Diatom Research (2009), Volume 24 (l), 175-190

BLOOMS OF THE DIATOM GENUS PSEUDO-NZTZSCHZA H. PERAGALLO IN BIZERTE LAGOON

(TUNISIA, SW MEDITERRANEAN)

I d s Sahraoui 19 ’?*, Asma Sakka Hlaili Hassine Hadj Mabrouk ’, Claude LCger ’ & Stephen S. Bates ’

Laboratoire de Cytologie Vkgktale et Phytoplanctonologie, Dkpartement des Sciences de la Vie, Facultk des Sciences de Bizerte, Universitk 7 novembre a Carthage,

7021 Zarzouna, Bizerte, Tunisie

Fisheries and Oceans Canada, GulfFisheries Centre, P.O. Box 5030, Moncton, NB, Canada El C 9B6

1

The phytoplankton composition, including the potentially toxic diatom genus Pseudo-nitzschiu, and related physico-chemical water properties were determined at four stations in Bizerte Lagoon (SW Mediterranean Sea) monthly, from March 2004 to March 2005. Total phytoplankton abundance was generally low (1.50-7.12 x lo5 cells L-’), but peaked in April 2004 (19.4 f 2.9 x lo5 cells L-I), July 2004 (19.9 f 11.1 x lo5 cells L-I) and March 2005 (12.2 f 4.8 x lo5 cells L-I), and was dominated by Plugioselmis spp., Pseudo-nitzschiu spp. and Thulussiosiru spp., respectively. The contribution of Pseudo-nitzschiu spp. to the total algal community was generally moderate (0.&8.9%, when detected). The genus Pseudo-nitzschiu was detected in 70% of the samples and thus appeared as a regular component of the phytoplankton that develops in the surface waters of Bizerte Lagoon. Distribution patterns of Pseudo-nitzschiu species in the “delicutissima” group (< 3 pm in width) showed a strong seasonality and were correlated with summer conditions (when temperature, salinity and silicate concentration increased), although they were present during at least seven months of the year, at all stations. Pseudo-nitzschiu species in the “seriutu” group (> 3 pm in width), conversely, revealed a narrower spatio-temporal distribution and appeared uncorrelated with the environmental factors measured. The causative species of the July peak was identified by scanning electron microscopy as Pseudo-nitzschia culliantha. Two out of four isolates of P. culliunthu from Bizerte Lagoon produced the neurotoxin domoic acid (causative agent of Amnesic Shellfish Poisoning) in batch culture. Our findings suggest the possibility of domoic acid contamination of bivalve molluscs in Bizert Lagoon, one of the main shellfish aquaculture areas in Tunisia, and therefore the need for continued vigilance.

INTRODUCTION

Reports of harmful algal blooms (HABs) have become more frequent in recent years (Smayda 1992, Fehling et al. 2005). The toxins produced by some algae are among the most potent naturally occurring compounds (GranCli & Legrand 200 1). Toxin accumulation in filter-feeding organisms can have devastating effects on higher level predators such as fishes, sea birds and marine mammals (Maneiro et al. 2005, Busse et al. 2006, Smith et al. 2006). HAB events may seriously impact public safety, fisheries, wildlife and aquaculture, resulting in considerable economic losses.

* Corresponding author: Inks Sahraoui ; tel.: + 216-71-889-330; fax: + 216-71-889-330. e-mail address: [email protected]

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176 I. SAHRAOUI., A. SAKKA HLAILI, H. HADJ MABROUK, C. LEGER & S.S. BATES

Domoic acid (DA), which was responsible for three deaths and the poisoning of over 100 people in November and December 1987 in Canada (Bates et al. 1998), has now been discovered in many parts of the world (Trainer et al. 2008). DA, responsible for a neurotoxic illness called Amnesic Shellfish Poisoning (ASP), is naturally produced by some species of the diatom genus Pseudo-nitzschia H. Peragallo. Among the -30 known morpho-species of the Pseudo-nitzschia genus, 11 to 12 are known to be toxic, although not always (Bates & Trainer 2006, Quiroga 2006, Trainer et al. 2008). Most of the toxigenic Pseudo-nitzschia species are cosmopolites (Hasle 2002), although an increasing number of cryptic species are being discovered (Orsini et al. 2004, Amato et al. 2007). In field work, Pseudo-nitzschia species have conventionally been divided into morphological categories, based on their cell widths as observed using light microscopy (e.g. Hasle & Syvertsen 1997): the “delicatissima group” (short and narrow cells) and the “seriata group” (long and wide cells). It is difficult to generalize about the ecology of the genus Pseudo-nitzschia because of the species’ diversity and differences among strains of presumably the same species. For example, different clades of P. delicatissima were found in the cold eastern Atlantic (Kaczmarska et al. 2008) and in the warm waters of the Mediterranean Sea and Gulf of Mexico (Orsini et al. 2004).

The occurrence of the Pseudo-nitzschia genus is well documented in the Mediterranean Sea (Orsini et al. 2002, 2004, Kaninou-Grigoriadou et al. 2005, Quiroga 2006, Amato et al. 2007, Quijano-Scheggia et al. 2008). In Tunisian (SW Mediterranean Sea) coastal waters and lagoons, Pseudo-nitzschia spp. were first noted and described by Turki & El Abed (2001). In spite of that, the history of toxic Pseudo-nitzschia blooms is poorly known; in fact, no ASP event has yet been recorded in Tunisian waters. This may be do in part to deficiencies in the harmful algae monitoring program, especially concerning DA-producing Pseudo- nitzschia spp., which are not monitored on a regular basis (DG (SANCO)/8622/2002, http://ec.europa.eu/food/fs/inspections/vi/reports/tun~s~~vi~rep~~ni~8622-2002sum~en.pdf).

Bizerte Lagoon is considered among the most important aquaculture areas in Tunisia, where aquaculture has existed for 40 years (Medhioub 1993). The lagoon is used almost exclusively for the culture of Pacific oysters (Crassostrea gigas Thunberg) and Mediterranean mussels (Mytilus galloprovincialis Lamarck). Clams (Tapes decussatus LinnC) are also very abundant and are commercialized within the region and for export (Dellali et al. 2001, Trigui El-Mnif et al. 2005). Recently, mussel production reached >100,000 tons per year (Khessiba et al. 2001). Therefore, the incidence of harmful microalgae may be an obstacle to the further development of shellfish farming in the area, especially since several potentially toxic algae (such as Pseudo-nitzschia spp.) have been identified several times in the Bizerte Lagoon (Turki & El Abed 2001, Sakka Hlaili et al. 2006,2007).

The establishment of monitoring programs for harmful phytoplankton species and their phycotoxins is considered a useful tool in the early warning of HAB events (Rhodes 1998, Grantli & Legrand 2001). In this context, we investigated the spatial and temporal distribution of the potentially toxic genus Pseudo-nitzschia in Bizerte Lagoon. Data is provided here concerning the main Pseudo-nitzschia groups (“delicatissima and “seriata”), including their seasonality and bloom dynamics. Also documented was the toxicity of P. calliantha Lundholm, Moestrup et Hasle isolated during one of the blooms.

MATERIALS AND METHODS Study site and sampling

Bizerte Lagoon (37” 8‘37” 14’ N, 9” 48’-9” 56’ E) is located on the northern coast of Tunisia. (Fig. 1). The lagoon is connected to the Mediterranean Sea through a 7-km long, 12-m deep channel. The ecosystem encompasses an area of 150 krn2 and has a mean depth of 8 m. Large volumes of water are exchanged between Bizerte Lagoon and the Mediterranean

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PSEUDO-NIESCHIA BLOOMS IN BIZERTE LAGOON 177

Sea. Water transport is strongly modulated by tidal currents. A semidiurnal tide (0.024.13 m of amplitude) requires >I year to renew the total water volume of the lagoon (Sakka Hlaili et al. 2008).

Fig. 1. Map of Bizerte Lagoon (Tunisia, SW Mediterranean Sea), showing the four sampling locations (*).

The system receives, in addition, a freshwater flux from Lake Ichkeul via the narrow Tinja Channel and other small surrounding rivers. The exchange with the sea is about one thousand times more important than the fresh water input, which may have a strong influence on the system’s water renewal (Harzallah 2002).

The sampling was carried out monthly, from March 2004 to March 2005, at four stations (Fig. 1). Station 1 is mainly influenced by marine water and reflects the status of Mediterranean coastal inshore waters. Stations 2 and 3 are both situated in the Channel of Bizerte. The former is subjected to intensive marine traffic as well as to various intensive industrial influences: cement fabrication, iron and steel works, and oil refineries. Station 3 is a well-known site for collecting wild mussels (Mytilus galloprovincialis). Station 4 is located in

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178 I. SAHRAOUI, A. SAKKA HLAILI, H. HADJ MABROUK, C. LEGER & S.S. BATES

the lagoon and is the most distant from the sea. At each station, temperature and salinity were recorded in situ, using a microprocessor conductivity meter (LF 196). Water was collected at 2 m (depth of the chlorophyll maximum), using a Hydrobios water sampler.

Water analyses Samples for nutrients (1000 mL) were collected in acid-washed vials, after filtering

through a Whatman GF/F filter; they were then stored frozen (-20°C) until analysis. Nitrite and nitrate were determined according to Wood et al. (1 967), and ammonia was analyzed following the procedure of Aminot & Chaussepied (1 983). Phosphate levels were determined as described in Murphy & Riley (1 962). Reactive dissolved silicate was analyzed according to Strickland & Parsons (1968). Detection limits of the analytic methods are 0.01, 0.02 and 0.1 pM, for nitrite, phosphate and silicate, respectively, and 0.05 pM for nitrate and ammonia.

For chlorophyll a (Chl a), seawater samples (500 mL) were filtered through Whatman GF/F filters. Pigment concentrations were determined using the standard spectrophotometric method (Parsons et al. 1984), following extraction with 10 mL 90% acetone overnight at 4°C in the dark. For phytoplankton identification and counting, water samples (100 mL) were fixed in acid Lugol’s solution (3% final concentration). Samples were allowed to settle in 50 mL chambers for 24 h and the cells were enumerated with an inverted microscope (1 OOx objective) (Utermohl 1958). At least 100 cells were counted in each sample.

Identijkation, culture, and toxicity of Pseudo-nitzschia Pseudo-nitzschia cells cannot be consistently distinguished at the species level by light

microscopy (Trainer et al. 2008). Depending on their valve width, cells were therefore separated into the “delicutissirnu” group (<3 pm in width) and the “seriata” group (>3 pm in width) (Hasle & Syvertsen 1997).

Clonal nonaxenic isolates of Pseudo-nitzschia were established from the July 2004 bloom. Cultures were grown in 125 mL flasks containing 75 mL of f72 medium (Guillard & Ryther 1962) plus 107 pM Si, at room temperature (- 20°C) and at a light intensity of - 100 pmol photons m-2 s-’ (12:12 h 1ight:dark cycle). For identification to the species level, aliquots of Pseudo-nitzschia cultures were prepared then examined by scanning electron microscopy (SEM) at the Digital Microscopy Facility (Sackville, New Brunswick, Canada), using the method of Kaczmarska et al. (2005). SEM was performed using a JOEL JSM-5600 SEM operating at 10 kV. Cell widths were measured from the scanning electron micrographs.

For toxicity examination, 20-mL aliquots from the stationary phase (day 33) of cultures growing under the above conditions were sonicated using a Vibra-Cell high-intensity ultrasonic processor, followed by filtration through a disposable polycarbonate filter (0.45 pm pore size) to remove the debris. DA in the whole culture (cells plus medium) was analyzed (detection limit - 1 .O ng mL-’) on a high performance liquid chromatograph using the FMOC fluorescence derivatization technique of Pocklington et al. (1 990).

Statistical analysis Analyses were done using SPSS 11.0 statistical software for Windows. Two-way

ANOVA was used to test the temporal and spatial variations of abiotic (temperature, salinity, NO;, NO3-, HPO?-, and biotic (Chl a, diatoms, total phytoplankton abundance) variables. The conditions of normality of distribution (Kolmogorov-Smirnov test) and homogeneity of variance (Bartlett-Box test) were respected. The Spearman’s correlation analysis was performed to assess whether the biotic and abiotic conditions affect the Pseudo- nitzschia species distribution. Generalized Linear Model Analysis of Variance (GLM ANOVA) was applied to assess whether time of year (month) or location (station) had a significant effect on the density distribution of species in the delicatissima and seriata groups.

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PSEUDO-NITZSCHIA BLOOMS IN BIZERTE LAGOON 179

RESULTS Water properties

The surface water temperature, which was similar among stations, went through a seasonal cycle characterized by a maximum of 24.7"C in August 2004 and a minimum of 11.8OC in January 2005 (Fig. 2a). Annual trends of salinity were similar for all locations sampled (Fig. 2b). Salinity was highest in July (38.3-39.8 psu) due to high evaporation. The lowest values were in November 2004 (28.9-34.5 psu), as a result of heavy rains.

Throughout the sampling period, nutrient concentrations were similar at all stations (Fig. 2c-f). The highest NOz- concentrations were detected in March 2005 (mean value: 0.66 pM) and the lowest in June 2004 (mean value: 0.08 pM) (Fig. 2c). N03- levels were minimal in June 2004 (mean value: 1.1 1 pM) and maximal in October 2004 (mean value: 3.56 pM) (Fig. 2d). Si(OH)4 concentrations exhibited a large variation throughout the year, and increased significantly in August 2004 (mean value: 2.25 pM) and September 2004 (mean value: 2.29 pM) (Fig. 2e). Lowest Si(OH)4 concentrations were recorded in December 2004 (mean value: 0.53 pM). PO:- levels remained relatively invariable throughout the year (constantly < 0.1 pM), except for July 2004 when they peaked (mean value 0.5 pM) (Fig. 20.

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Fig. 2. Monthly profiles of physicochemical variables at the four sampling stations. (a) temperature; (b) salinity; (c) nitrite; (d) nitrate; (e) silicate; (0 phosphate.

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180 I. SAHRAOUI, A. SAKKA HLAILI, H. HADJ MABROUK, C. LEGER & S.S. BATES

Phytoplankton dynamics

Highest levels of Chl a were found in March 2005 (8.0 yg L-I, at Station 2) (Fig. 3a). The lowest values (- 0.5 pg L-I) were recorded at Station 1 during June and November 2004 and January 2005, and at Station 4 during April and May 2004. Chl a also varied significantly among stations (P < 0.05). Higher Chl a levels were measured at Station 2 and lower levels at Station 1. Total phytoplankton abundance remained low (1.5CL7.1 x lo5 cells L-I) and relatively constant during late autumn and winter (Fig. 3b). It peaked during April 2004 (19.4 f 2.9 x lo5 cells L-I), July 2004 (19.9 f 11.1 x lo5 cells L-I) and March 2005 (12.2 f 4.8 x lo5 cells L-'), with pronounced concentrations at Station 4. Phytoplankton cell numbers were continually higher at Station 4 compared to those at Station 1 (P < 0.05). Over the year, changes in both total phytoplankton and diatom abundances paralleled each other in a significant fashion (P < 0.01) (Fig. 3b, c). However, diatoms did not account for the April peak of phytoplankton abundance, which was composed of flagellate species (Plagioselmis Butcher spp.). Diatom abundances were generally moderate (- 3 x lo5 cells L-') and increased considerably during summer 2004 and in March 2005 (Fig. 3c). The highest values were attained in July 2004 (17 x lo5 cells L-I, at Station 4). Diatom cell numbers were consistently higher at Station 4 compared to those at Station 1 (P < 0.05).

The taxonomic composition of algae at the four stations is illustrated in Fig. 4. Diatoms were diverse, with the presence of 13 genera. The highest contribution of diatoms occurred in March 2005 (54.1-84.7%), when large-celled Thalassiosira Cleve species bloomed. Diatoms also composed a considerable proportion of the phytoplankton in June (61.2-68.0%) and July 2004 (47.7-58.2%), when chain-forming species (Chaetoceros Ehrenberg spp., Leptocylindrus Cleve spp. and Pseudo-nitzschia spp.) were dominant. Some of the diatom species were found year round (mostly Nitzschia Hassall spp., Pseudo-nitzschia spp. and Chaetoceros spp.), but most were seasonal and some were rare (Fragilariopsis Hustedt spp., Pleurosigma W. Smith spp., Gyrosigma Hassall spp., Skeletonema Grev spp. and Thalassionema Grunow ex Mereschkowsky spp.). Dinoflagellates (mainly Gyrodinium Kfoid & Swezy spp., Gymnodinium Stein spp. and Prorocentrum Ehrenberg spp.) were present in most of the samples, but contributed little (maximum 13.5%) to the entire phytoplankton assemblage, with eight representative taxa. Flagellates, mainly represented by Cryptophyceae and Prasinophyceae, often contributed greatly to the phytoplankton community. Their relative abundances ranged between 14.1% in March 2005 and 98.7% in April 2004, when the cryptophyte Plagioselmis Butcher spp. dominated the phytoplankton.

Pseudo-nitzschia incidence and abundance

The contribution of Pseudo-nitzschia spp. to the algae did not vary significantly among the studied locations (P > 0.05). However, their presence was highly variable over time (Fig. 4). Allocation to the total planktonic community was significant in July 2004 (about 14.1%), moderate in May, June, August, September 2004 and in January 2005 (from 4.6 to 8.9%) and null in March, April and October 2004. Pseudo-nitzschia spp. abundance also varied over the year (Fig. 5) . The delicatissima group species were more abundant (between 0.03 and 3.0 x lo5 cells L-I) than the seriata group species (when detected, between 0.03 and 0.44 x lo5 cells L-I). The presence of cells in the delicatissima group was noted during at least seven months of the year, at all stations. The July peak of Pseudo-nitzschia spp. was exclusively composed of species in the delicatissirnu group. Conversely, the seriata group had a narrow spatio-temporal distribution, occurring for only three months at Station 3, four months at Stations 1 and 2, and five months at Station 4.

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PSEUDO-NITZSCHIA BLOOMS IN BIZERTE LAGOON 18 1 - Station 1 - -0 - Station 2

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Fig. 3. Monthly variation in (a) Chl a; (b) total phytoplankton abundance; and (c) diatom abundance at the four sampling stations. (Mean f SD, n = 2).

ANOVA analyses revealed that abundance variations of both the seriata and delicatissima groups were significantly modulated by time (month) (Table 1). Conversely, no significant effect of space (station) on cell number of these algae was detected. Abundances of the delicatissima group species were positively correlated with temperature, salinity and Si(OH), concentration (Table 2). Moreover, significant positive correlations were found with Chl a level and diatom cell number.

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182 I. SAHRAOUI, A. SAKKA HLAILI, H. HADJ MABROUK, C. LEGER & S.S. BATES

Fig. 4. Monthly variation in phytoplankton taxonomic composition at the four sampling stations.

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PSEUDO-NITZSCHIA BLOOMS IN BIZERTE LAGOON 183

Fig. 5. Monthly variation in concentration of the delicutissirnu group (<3 pm in cell width) and the seriutu group (>3 pm in cell width). (Mean f SD, n = 2).

Pseudo-nitzschia identijkation and toxicity Four monospecific cultures of Pseudo-nitzschia were obtained from the July bloom.

Strains IS-5 and IS-13 were isolated from Station 4, and IS-6 and IS-1 1 from Station 3 . SEM observation of these isolates revealed two striae ( 3 7 4 0 per 10 pm) per fibula (17-20 per 10 pm), one row of large poroids (4-5 per 1 pm) in each stria, and the presence of a central interspace with a central nodule (Fig. 6). The hymen of the valve poroids exhibited a flower pattern. These morphometrics were consistent with the recently described P. calliantha (Lundholm et al. 2003).

The concentration of DA was below the level of detection in strains IS-11 and IS-13. However, this toxin was present in strains IS-5 and IS-6, giving whole-culture (cells plus medium) DA concentrations of 149.1 ng mL-l and 13.4 ng mL-l, respectively.

Fig. 6. Scanning electron micrograph of Pseudo-nitzschiu cullianthu (strain IS-5), showing detail of central part of the cell.

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184 I. SAHRAOUI, A. SAKKA HLAILI, H. HADJ MABROUK, C. LEGER & S.S. BATES

DISCUSSION

Bizerte Lagoon has been investigated with gradually increasing effort in several disciplines for over 40 years. However, knowledge of the phytoplankton is based on relatively few studies. Azzouz (1966) was the first to study the phytoplankton community, in order to evaluate the potential for oyster culture in the lagoon. Since then, only a few reports have continued describing the phytoplankton. Following various mass mortality events in Tunisian aquaculture areas (Rhomdane et al. 1998), interest in potentially toxic microalgae started in 1995, with the establishment of a national monitoring program that operated in several regions of bivalve mollusc production, including Bizerte Lagoon (Turki & El Abed 2001). However, there was little or no attention focused on single species taxonomy, ecotoxicology, or bloom frequency and intensity. Our research was thus the first to explore the distribution pattern of the potentially toxic marine diatom genus Pseudo-nitzschia and associated phytoplankton in relation to environmental factors in Bizerte Lagoon.

Table 1. Statistical values for comparing Factors (A: month; B: station) against the delicatissima group species (<3 pm in cell width) and the seriata group species (>3 pm in cell width). Power values are calculated with a confidence level of 0.001.

delicatissima group seriata group

Factor df Fratio P Power df Fratio P Power

A 12 16.64 0.00 1 .oo 12 13.02 0.00 1 .oo B 3 1.25 3.76 0.35 3 0.80 0.49 0.16

Table 2. Spearman’s correlation matrix between Pseudo-nitzschia species in the delicatissima group (<3 pm in cell width) and in the seriata group (>3 pm in cell width), and the environmental data collected during the sampling period in Bizerte Lagoon. Significant r values are indicated in bold face for P < 0.05.

Parameter

delicatissima group seriata group

r P r P

Temperature

Salinity

NOz-

N03-

HPOZ-

Si(OH)4

Chl a

Diatoms

Total phytoplankton

0.570

0.626

- 0.167

- 0.204

0.059

0.337

0.258

0.488

0.186

0.000

0.000

0.238

0.147

0.677

0.015

0.008

0.000

0.059

-0.133

0.003

-0.158

- 0.004

- 0.206

0.107

- 0.209

0.013

-0.151

0.349

0.492

0.265

0.979

0.143

0.449

0.033

0.896

0.125

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PSEUDO-NIlZSCHIA BLOOMS IN BIZERTE LAGOON 185

Environmental conditions during this study seemed relatively comparable (although with some variation with season) with those measured during the last few years (Dellali et al. 2001, Harzallah 2002). The waters were euhaline (30-40 psu) and the yearly average temperature was 17.5”C. The NO; and PO:- concentrations remained below 1 pM, while the N03- and Si(OH)4 concentrations oscillated below 5 pM. Nutrients have often been considered as one of the major factors controlling the composition and abundance of members of the phytoplankton community, as well as the timing of blooms. In the lagoon, it has been demonstrated that nitrogen was the main limiting nutrient during winter, whereas during summer the phytoplankton seemed to be limited by nitrogen and phosphorus, which have shown an increasingly important anthropogenic loading (Sakka Hlaili et al. 2006).

Throughout the study period, the phytoplankton population was frequently dominated by flagellates (Fig. 4), which are recognized as being characteristic of oligotrophic systems (Carillo et al. 1995, Caroppo et al. 2005). Flagellates also composed over 50% of the algal biomass during winter 2001, and were highly linked to the nitrogen deficiency in the area (Sakka Hlaili et al. 2006). Similar results were also found in other Tunisian ecosystems (Guetari 2002). Sakka Hlaili et al. (2006) suggested that predominance of these small algae in Bizerte Lagoon may be due to their competitive ability to use low amounts of nutrients under the nitrate-deficient conditions. Unlike flagellates, diatoms contributed considerably to the phytoplankton community during spring and summer (Fig. 4), when nutrients were more available (Fig. 2). Diatom blooms are often a major food source for grazers during the spring in coastal regions. They can, however, be hazardous to marine biota and humans through excretion of oils and mucilage, and, in some cases, production of toxic compounds (McQuoid & Godhe 2004).

Our study revealed that potentially toxic diatoms of the genus Pseudo-nitzschia appeared commonly (in 70% of the samples) as a minor constituent of the phytoplankton in the surface waters of Bizerte Lagoon. Cell densities were usually low and often imperceptible in quantitative samples, but peak abundances reached > lo5 cells L-’ (in July 2004). Sakka Hlaili et al. (2006) have also reported the highest densities of Pseudo-nitzschia in summer surface waters (> lo5 cells L-’, July 2001), compared with winter, when the genus was undetectable. Blooms of Pseudo-nitzschia were often found during the spring and summer in other Mediterranean coastal areas. In French coastal waters, Pseudo-nitzschia spp. are known to bloom during the spring (Gailhard et al. 2002, Quiroga 2006). These blooms were also recorded in the spring in Italian coastal waters, although at times during the autumn (Orsini et al. 2004). Nowadays, the increase in Pseudo-nitzschia abundance at many locations is evidenced by long-term studies. This may be linked in part to heightened awareness and increased monitoring. However, it was demonstrated that the main cause was eutrophication in other areas of the world (e.g. Parsons et al. 2002). This could likewise be expected in Mediterranean waters, where eutrophication has been confirmed (e.g. Duarte et al. 2000, Karydis & Chatzichristofas 2003).

During our routine monitoring, species in the genus Pseudo-nitzschia were separated into the two groups, “delicatissima” and “seriata”, because of difficulties in species determination by light microscopy. The spatial patterns of both groups were similar among the four stations. This is in accordance with the environmental conditions that were also relatively unchangeable over these stations (Fig. 2). The comparable conditions can be the result of the semidiurnal tidal current, which results in a significant exchange of water into and out of the lagoon via the channel.

Temporal distributions showed that delicatissima group species were present during almost all of the sampling periods. Cell densities generally ranged between 3 x lo3 and 3 x lo5 cells L-’, with the highest values in July 2004 (Fig. 5). The seriata group species had a narrower spatio- temporal distribution than the delicatissima group species, and cell densities varied between

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186 I. SAHRAOUI, A. SAKKA HLAILI, H. HADJ MABROUK, C. LEGER & S.S. BATES

3 x lo3 and 4 x lo3 cells L-’. In other Mediterranean areas, members of the delicatissima group are also often the most important species in terms of abundance. For example, Pseudo-nitzschia delicatissima sensu Hasle is reported as a regular potential source of ASP toxins in the Gulf of Naples (Orsini et al. 2004). Pseudo-nitzschia delicatissima and P. calliantha are described as the most abundant taxa along the southern Adriatic coast (Caroppo et al. 2005) and the NE coast of the Spanish province of Catalonia (Quijano-Scheggia et al. 2008). Furthermore, P. calliantha composed the blooms during the summers of 2002 and 2003 in the Bay of Banuyls-sur-Mer in France (Quiroga 2006).

The statistical analysis demonstrated significant positive correlations between delicatissima group species abundance, temperature and salinity (Table 2). A higher abundance of Pseudo-nitzschia species commonly occurred during summer periods, coinciding with increases in temperature and salinity. The delicatissirnu group species were mainly responsible for the July 2004 bloom. This can be also demonstrated by the significant positive correlations between delicatissima group species occurrence, Chl a, and diatom concentration, resulting from their high cell density within the bloom. There was no significant relationship between delicatissima group species distribution and nitrate or phosphate. However, a previous study has demonstrated that the supply of phosphate stimulated the biomass and growth of P. delicatissirna and of P. pseudodelicatissirnu Hasle in Bizerte Lagoon (Sakka Hlaili et al. 2006). In the eastern Adriatic Sea, it was also shown that Pseudo-nitzschia blooms were significantly correlated with phosphorus concentration and temperature (BuriC et al. 2008). The apparent increase in phosphate concentration during July 2004 (Fig. 2f) may have explained the incidence of the delicatissima group species bloom. The tight relationship between silicate and delicatissirna group species abundance (Table 2) suggests that silicon may be a limiting nutrient for the growth and abundance of these species in Bizerte Lagoon waters. The relatively weak correlations obtained between the delicatissima group species and the environmental variables are suggestive of different species with different nutrient requirements present within the delicatissima group.

Our study revealed that Pseudo-nitzschia calliantha was the diatom species that bloomed in July 2004. Lundholm et al. (2003) recently described P. calliantha as a new species. Before then, it had been recorded as P. pseudodelicatissima in Canadian (Martin et al. 1990) and Danish (Lundholm et al. 1997, Skov et al. 1997) waters. The geographically widespread observations of this species indicate a more or less cosmopolitan distribution (Lundholm et al. 2003, Fehling 2004). In the Mediterranean basin, P. calliantha was reported in various areas along the French (Quiroga 2006), Italian (Lundholm et al. 2003, Caroppo et al. 2005, Amato et al. 2007, Congestri et al. 2008) and Croatian (Lundholm et ul. 2003) coasts.

The species in our study was identified as P. calliantha, based on morphometry and the distinctive “rosette” type of poroids (Lundholm et al. 2003). However, we found a wider range in the cell width (2.684.04 pm) compared to that reported by Lundholm et al. (2003) (1.3-1.8 pm). This may be due in part to the distortion of the cell valves in our culture at the time of measurement, a phenomenon commonly reported in actively growing cultures (Bates et al. 1998, Pan et al. 2001). The deformities could be a sign of the impending inability for hrther cell division; our cultures did, in fact, die out within a few months. However, the wider cell widths may also add support to recent observations of differences among other P. calliantha isolates. Amato et al. (2007) recently showed that P. calliantha may be composed of several “cryptic” species, with slight differences in morphological and genetic characteristics, and mating ability. Two of those morphotypes (clades pse3 and pse4) had cell widths of 1.7-2.6 and 1.7-2.4 pm, respectively. Likewise, Caroppo et al. (2005) reported P. calliantha cell widths of 1.5-2.2 pm. Three isolates of P. calliantha from the Chesapeake Bay, eastern USA, had cell widths of 2.80, 2.41 and 2.57 pm (cultures of Anne Thessen, University of Maryland; measurements by James Ehrman, Digital Microscopy Facility). These

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PSEUDO-NITZSCHIA BLOOMS IN BIZERTE LAGOON 187

studies indicate a wider upper range in cell width than originally reported by Lundholm et al. (2003). The P. calliantha strains studied by Caroppo et al. (2005) were from the southern Adriatic Sea, and those reported by Amato et al. (2007) were all from the Gulf of Naples (Albert0 Amato, personal communication), each part of the Mediterranean Sea. These differences in cell width, in addition to the other genetic and mating characteristics, may provide support for the presence of new species within the existing P. calliantha.

In 1988, a bloom of what was then called Pseudo-nitzschia pseudodelicatissima resulted in the closure of shellfish harvesting areas in the Bay of Fundy, eastern Canada, when blue mussels and soft-shell clams became contaminated with high levels of DA (Martin et al. 1990). The species was later reported to be P. calliantha (Lundholm et al. 2003), although there is still some discussion as to its actual identity (Kaczmarska et al. 2005, Bates & Trainer 2006). Lundholm et al. (1997) reported that an isolate of P. calliantha (then called P. pseudodelicatissima; Lundholm et al. 2003) produced DA, although other isolates were non-toxic (Skov et al. 1997). High numbers of non-toxic P. calliantha were found in inlets of Prince Edward Island, eastern Canada, in 2001 and 2002 (Bates & Trainer 2006), and did not result in any harvesting closures. As in the present study, there are thus toxic and apparently non-toxic strains of P. calliantha. Detecting apparently non-toxic and DA-producing strains within the same species is suggestive of a considerable intraspecific diversity occurring at a narrow spatial scale. It may also indicate either that the conditions tested were not conducive to toxin production, or that there are “cryptic”, non-toxic species within what is currently called P. calliantha.

In this study, P. calliantha of northern Tunisian waters was shown to bloom in the summer. At least two of the four P. calliantha isolates from the July bloom were able to produce DA in culture. Genetic studies would help to determine if the toxic and apparently non-toxic strains belong to different clades, even though they were isolated from the same stations. Our findings suggest that the incidence of toxic events in Bizerte Lagoon waters is a probability not to be excluded. A rigorous monitoring for ASP toxins, as well as an examination of Pseudo-nitzschia species distribution and related environment factors that affect their abundance, are therefore essential for predicting toxic events that can damage the aquaculture industry in the area.

ACKNOWLEDGEMENTS

We gratefully thank James Ehrman (Digital Microscopy Facility, Mount Allison University, Sackville, New Brunswick, Canada) for producing the scanning electron micrograph.

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Manuscript received February 2008; accepted for publication October 2008

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