Basin scale variability of Ostreopsis spp. blooms provides evidence of effectiveness of an integrated sampling approach.

Ostreopsis spp. blooms have been occurring in the last two decades in the Mediterranean Sea in association with a variety of biotic and abiotic substrata (macroalgae, seagrasses, benthic invertebrates, sand, pebbles and rocks). Cells proliferate attached to the surfaces through mucilaginous trichocysts, which lump together microalgal cells, and can also be found in the plankton and on floating aggregates: such tychoplanktonic behavior makes the quantitative assessment of blooms more difficult than planktonic or benthic ones. Different techniques have been so far applied for quantifying cell abundances of benthic microalgae for research, monitoring and risk assessment purposes. In this context, the Benthic Dinoflagellates Integrator (BEDI), a non-destructive quantification method for benthic dinoflagellate abundances, was developed and tested within the EU ENPI-CBCMED project M3-HABs. This device allows mechanical detachment of cells without collecting the benthic substrate, providing an integrated assessment of both epiphytic and planktonic cells, i.e. of the number of cells potentially made available in the water volume from "resuspension" which could have harmful effects on other organisms (including humans). The present study confirms the effectiveness of the BEDI sampling device across different environments across the Mediterranean Sea and constitutes the first large-scale study of Ostreopsis spp. blooms magnitude in function of different macro- and meso­habitat features across the basin.

Harmful epiphytic dinoflagellate assemblages on macrophytes in the Gulf of Tunis.

The spatio-temporal distribution of epiphytic and planktonic microalgae coupled with environmental factors was investigated for a one-year period in the Gulf of Tunis (northeastern Tunisia). Harmful microalgae assemblages were dominated by three toxic epiphytic dinoflagellates: Ostreopsis sp., Prorocentrum lima and Coolia monotis. They were observed, both on macrophytes (1.03 × 105 cells g-1 FW ; 1.3 × 104 cells g-1 FW and 865 cells g-1 FW, respectively) and in the water column (2.35 × 104 cells L-1; 3.72 × 103 cells L-1; 1.04 × 103 cells L-1, respectively). Species abundances decreased with depth and maximum concentrations were found in shallow waters (0.5-1 m). The highest species abundance was registered both on macroalgae and seagrass with no special preference observed for either of these substrates. Redundancy analyses (RDA) show significant changes in these species abundances according to sites and seasons. The proliferation of Ostreopsis sp. is widespread in summer, when water temperature is warm, and especially in bay zones. The occurrence of P. lima and C. monotis blooms was mainly correlated to nutrients. In this study, macrophyte beds in the Gulf of Tunis were a reservoir of potentially toxic species that could pose a real threat, both to ecosystems and to public health.

Assessing ultraphytoplankton and heterotrophic prokaryote composition by flow cytometry in a Mediterranean lagoon.

In the eutrophic Ghar El Melh Lagoon (GML, Tunisia), the distribution of heterotrophic prokaryotes, pico- and nanophytoplankton was studied at five stations in November 2012 at the single cell level, along with environmental factors. Flow cytometry analysis of ultraplankton (<10 µm) resolved (i) two heterotrophic prokaryote groups, low and high nucleic acid contents (LNA and HNA, respectively), and (ii) eight to nine ultraphytoplankton groups (cryptophyte-like cells, two nanoeukaryote subgroups, two picoeukaryote subgroups and three Synechococcus-like cells subgroups). Prochlorococcus was not detected. According to redundancy analysis (RDA), a significant difference was found in the distribution of the ultraplankton between stations (F = 2.61, p < 0.05); maximum proliferations of heterotrophic prokaryotes were observed in the inner parts of the lagoon at stations 3, 4 and 5 affected by urban, agricultural and industrial discharges. Ultraphytoplankton concentrations were the highest near the outlet of the lagoon at stations 1 and 2 influenced by freshwater outflow and oligotrophic Mediterranean water inflow, respectively. At station 1, the large ultraphytoplankton concentration derives from the high abundance of cryptophyte-like cells favoured by the freshwater outflow whereas at station 2, the input of oligotrophic Mediterranean water enhanced the abundance of Synechococcus and picoeukaryotes at the expense of nanoeukaryotes. Two trophic regimes were thus differentiated in GML.

Driving factors of dinoflagellate cyst distribution in surface sediments of a Mediterranean lagoon with limited access to the sea.

Seasonal distribution of dinoflagellate cysts were studied at five surface sediment study stations in Ghar El Melh Lagoon (GML) (Tunisia) in relation to physicochemical parameters and phytoplankton abundance in the water column. At least sixteen dinocyst types were identified, dominated mainly by Protoperidinium spp., Scrippsiella trochoidea complex, Lingulodinum machaerophorum, Alexandrium spp. and Gymnodinium spp., along with many round brown cysts. Cyst abundance ranged from 0 to 229g-1 dry sediment. No significant differences in cyst distribution were found among stations, though a significant variation was observed among seasons with cyst dominance in autumn. No significant variation was found between cyst abundance and the different abiotic factors monitored, neither in the water column (physicochemical parameters) nor in the sediment (% H2O). Low dinocyst abundance was consistent with the dominance of non-cyst-forming dinoflagellates in the GML water column.

Factors driving the seasonal distribution of zooplankton in a eutrophicated Mediterranean Lagoon.

The distribution of the zooplankton community was studied along with environmental factors at five sampling stations in Ghar El Melh Lagoon (GML) (Mediterranean Sea, northern Tunisia). GML is characterized by specific following properties: broad and shallow, freshwater supply (Station 1); connection to the sea (S2); stagnation (S3 especially), and eutrophic conditions with enhanced nutrient concentrations (S4 and S5). Samples were taken twice monthly from February 2011 to January 2012. Twenty-three zooplankton groups comprising 10 larval stages were identified. Zooplankton assemblages were largely dominated by copepods (37.25%), followed respectively by ciliates (21.09%), bivalve larvae (14.88%) and gastropod veligers (12.53%). Redundancy analysis indicated that while no significant difference was found in the distribution of zooplankton at any station, a strong difference was observed according to season. Both temporal and physicochemical fluctuations explain more than 50% of changes in zooplankton abundances.

Alexandrium pacificum Litaker sp. nov (Group IV): Resting cyst distribution and toxin profile of vegetative cells in Bizerte Lagoon (Tunisia, Southern Mediterranean Sea).

A high spatial resolution sampling of Alexandrium pacificum cysts, along with sediment characteristics (% H2O, % organic matter (OM), granulometry), vegetative cell abundance and environmental factors were investigated at 123 study stations in Bizerte Lagoon (Tunisia). Morphological examination and ribotyping of cells obtained from a culture called ABZ1 obtained from a cyst isolated in lagoon sediment confirmed that the species was A. pacificum. The toxin profile from the ABZ1 culture harvested during exponential growth phase was simple and composed of the N-sulfocarbamoyl toxins C1 (9.82pgtoxincell-1), the GTX6 (3.26pgtoxincell-1) and the carbamoyl toxin Neo-STX (0.38pgtoxincell-1). The latter represented only 2.8% of the total toxins in this strain. High abundance of A. pacificum cysts correlated with enhanced percentages of water and organic matter in the sediment. In addition, sediment fractions of less than 63µm were examined as a favorable potential seedbed for initiation of future blooms and outbreaks of A. pacificum in the lagoon. A significant difference in the cyst distribution pattern was recorded among the lagoon's different zones, with the higher cyst abundance occurring in the inner waters. Also, no correlation due to the specific hydrodynamics of the lagoon was observed in the spatial distribution of A. pacificum cysts and vegetative cells.

Bivalve and barnacle larvae distribution driven by water temperature in a Mediterranean lagoon.

The objective of this study was to explore the relationships between the distribution of some meroplanktonic species and water temperature. Meroplankton larvae abundance of bivalves, and barnacles and water temperature fluctuations were studied from February 2011 to January 2012 at five stations in Ghar El Melh lagoon (GML) Mediterranean Sea, northern Tunisia). According to redundancy analysis (RDA), a significant difference was found in the distribution of larvae among the seasons (F = 10.28, p < 0.001); summer and autumn appear to be the period of bivalve larvae development, whereas the arrival of barnacle larvae coincided with winter and spring. The generalized additive models (GAMs) show strong correlation of bivalve larvae with high temperature (F = 23.2; p < 0.001) and the affinity of barnacle larvae to low temperature values (F = 8.41; p = 0.004). This environmental parameter accounted for 26 % of the deviance in variability in larvae abundance. The development process of many generations of larvae may therefore have been predetermined by temperature.

Driving factors behind the distribution of dinocyst composition and abundance in surface sediments in a western Mediterranean coastal lagoon: report from a high resolution mapping study.

Species composition and abundance of dinocysts in relation to environmental factors were studied at 123 stations of surface sediment in Bizerte Lagoon. Forty-eight dinocyst types were identified, mainly dominated by Brigantidinium simplex, Votadinum spinosum, Alexandrium pseudogonyaulax, Alexandrium catenella, and Lingulodinum machaerophorum along with many round brown cysts and spiny round brown cysts. Cysts ranged from 1276 to 20126 cysts g(-1)dry weight sediment. Significant differences in cyst distribution pattern were recorded among the zones, with a higher cyst abundance occurring in the lagoon's inner areas. Redundancy analyses showed two distinct associations of dinocysts according to location and environmental variables. Ballast water discharges are potential introducers of non-indigenous species, especially harmful ones such as A. catenella and Polysphaeridium zoharyi, with currents playing a pivotal role in cyst distribution. Findings concerning harmful cyst species indicate potential seedbeds for initiation of future blooms and outbreaks of potentially toxic species in the lagoon.

Assessment of nitrogen and phosphate balance and the roles of bacteria and viruses at the water-sediment interface in the Allal El Fassi reservoir (Morocco).

Balances of nitrogen and phosphate were studied in the Allal El Fassi reservoir (Morocco); the results showing that nitrogen input (296 mg m(-2) d(-1)) was 161% higher than output (183 mg m(-2) d(-1)). Phosphate input (35.65 mg m(-2) d(-1)) was 865% higher than output (4.12 mg m(-2) d(-1)), causing a progressive increase in the internal phosphate stock. Sedimentation flux was equally high (53.80 and 18 mg m(-2) d(-1)) for both nitrogen and phosphate input, mainly from the Sebou River and in particulate form which immediately settles upon arrival in the reservoir. The release of nitrogen and phosphate from the sediment (5.40 and 1.15 mg m(-2) d(-1), respectively) depended on physicochemical and biological (bacteria and viruses) variability and the calcareous nature of the catchment basin. Calcium-bound phosphate prevailed in the reservoir. Drastic control of phosphate input is suggested to avoid accumulation of calcium-bound phosphate which may dissociate and thereby contribute to eutrophication.

Contrasting key roles of Ruppia cirrhosa in a southern Mediterranean lagoon: reservoir for both biodiversity and harmful species and indicator of lagoon health status.

The distribution of Ruppia cirrhosa meadow density and its epiphytic organisms in relation with environmental factors were studied in summer 2011 at five stations in the Ghar El Melh lagoon (GML; southern Mediterranean Sea). Eleven epiphytic groups were recognised among which diatoms and dinoflagellates were the dominant groups and greatest contributors to temporal dissimilarity. An overwhelming concentrations of harmful microalgae was recorded, mainly represented by the toxic dinoflagellate Prorocentrum lima with maximal concentrations attaining 6 × 10(5)cells 100g(-1) of Ruppia fresh weight. The epifauna community accounted for only 1.4% of total epiphyte abundance and was comprised predominantly of nematodes (47.51%), ciliates (32.59%), fish eggs (7.2%) and larvae (4.95%). PERMANOVA analyses revealed a significant spatio-temporal variation of all epiphytic groups (p<0.01). In this study, R. cirrhosa and its epiphytes were studied as potential early warning indicators of the health status of GML waters.

Factors contributing to heavy metal accumulation in sediments and in the intertidal mussel Perna perna in the Gulf of Annaba (Algeria).

This paper presents the results of a seasonal survey of heavy metals accumulated in sediments and in the soft parts of the body of the mussel Perna perna at four stations in the Gulf of Annaba (Algeria). Pooled soft tissues from 10 mussels representing the entire range of sizes were digested in nitric acid. Statistical analysis reveals a significant seasonal effect on all the measured metals, the highest values being recorded in winter. With the exception of Cr, the levels for all metals were significantly higher in the east, at the outlet of the Seybouse River, than at all other monitoring stations. The study also shows that north-western waters are subject to a significantly lower degree of heavy metal pollution than elsewhere in the gulf. Levels were nevertheless within the limits of public health standards. The results confirm the usefulness of P. perna as a bioindicator for heavy metal pollution.

Factors driving the seasonal distribution of planktonic and epiphytic ciliates in a eutrophicated Mediterranean lagoon.

We studied the distribution of planktonic and epiphytic ciliates coupled with environmental factors and microalgae abundance at five stations in Ghar El Melh Lagoon (Tunisia). Planktonic ciliates were monitored for a year and epiphytic ciliates were sampled during summer 2011 in concordance with the proliferation of the seagrass Ruppia cirrhosa. Ciliate assemblage was largely dominated by Spirotrichea followed respectively by Tintinnida of and Strombidiida. No significant difference was found in the distribution of ciliate species among the stations. Redundancy analysis indicates that abiotic factors (temperature and nutriments) have a significant effect on the dynamics of certain ciliates. For epiphytic ciliates, 4 species were identified: Tintinnopsis campanula, Aspidisca sp., Strombidium acutum and Amphorides amphora. Based on PERMANOVA analyses, ciliates exhibit significant correlations among months and stations. According to ACP, epiphyte distribution follows roughly those of R. cirrhosa and pH. Significant correlations were found between harmful dinoflagellates and both planktonic and epiphytic ciliates.

Seasonal dynamic and in situ division rates of the dominant Dinophysis species in Punic harbors of Carthage (Gulf of Tunis, South Mediterranean).

This study investigates spatio-temporal variations of in situ division rate (µ) of two species of the genus Dinophysis. Based on a postmitotic index approach, estimates of µ from net-haul samples were compared during five daily cell cycles studies in three stations of the Punic harbors of Carthage (Gulf of Tunis). To study the annual cycle of Dinophysis spp., weekly sampling were carried out from March 2008 to June 2010. In addition, sampling over 24 h was conducted on three-cycle studies in late spring and two-cycle studies in autumn, seasons characterized by Dinophysis proliferations. We recorded important abundances of Dinophysis sacculus stein (2.25 × 10(4) cells/l, June 2008) and Dinophysis cf. acuminata (3 × 10(4) cells/l, June 2009). Cellular division was phased in D. sacculus stein and D. cf. acuminata, but the division time, required for the population cohorts to pass from one phase to another, and values of estimated division rates varied between seasons and stations for the same species. D. cf. acuminata exhibit moderate (0.22 day(-1)) to high (0.68 day(-1)) value of µ. In contrast, D. sacculus shows very low µ values (0.02-0.17 day(-1)) in autumn season to moderate values (0.21-0.35 day(-1)) in late spring. The results presented here confirm that the postmitotic index approach allowed estimated of µ at low field depths and concentrations (10(2)-10(3) cells/l).

Dynamics of harmful dinoflagellates driven by temperature and salinity in a northeastern Mediterranean lagoon.

To attempt to determine the effects of temperature and salinity on the dynamics of the dinoflagellate community, a monthly sampling was carried out from October 2008 to March 2009 at eight sampling stations in Ghar El Melh Lagoon (GML; Mediterranean Sea, Northern Tunisia). Dinoflagellates were dominant among plankton, accounting for 73.9 % of the lagoon's overall plankton community, and were comprised of 25 different species among which 17 were reported in the literature as harmful. While no significant difference was found in the distribution of dinoflagellates among the stations, a strong monthly difference was observed. This temporal variability was due to an increase in the abundance of Prorocentrum micans from December to February, leading to a strong decrease in the Shannon diversity index from station to station. At the onset of P. micans development, dinoflagellate abundances reached 1.26.10(5) cells l(-1). A redundance analysis indicates that both temperature and salinity have a significant effect on the dynamics of the dinoflagellate community. Using a generalized additive model, both temperature and salinity appear to have significant nonlinear relationships with P. micans abundances. Model predictions indicate that outbreaks of P. micans may occur at a temperature below 22.5 °C and with salinity above 32.5. We discuss our results against a backdrop of climate change which, by affecting temperature and salinity, is likely to have an antagonistic impact on P. micans development and subsequently on the dinoflagellate dynamics in GML.

Occurrence of diarrhetic shellfish poisoning (DSP) toxins in clams (Ruditapes decussatus) from Tunis north lagoon.

The main diarrhetic shellfish toxins, okadaic acid (OA) and dinophysistoxin-1, 2 (DTX-2, 2) were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as pyrenacyl esters in clams (Ruditapes decussatus) collected in Tunis north lagoon from January 2007 to June 2008. Sample analyses by LC-MS/MS displayed OA and related congeners (DTX-2, 2) with a highest detected level of 21 µg OA eq/kg shellfish meat for the samples of January 2007. Nevertheless, all samples were MBA negative. During the study period, potentially toxic dinoflagellate Dinophysis sacculus was recorded all year, blooming at different times. Highest concentrations were recorded during January 2007 with 4.6 × 10(4) cells per liter and 4.10(4) cells per liter in the northern and southern districts, respectively. Results show that there is no significant correlation between D. sacculus densities in water column and diarrhetic shellfish poisoning (DSP) toxins concentrations unregistered in clams. These data reveal that DSP toxicity in clams of Tunis north lagoon is low according to European regulatory limit (160 µg OA eq/kg shellfish meat). However, a potential threat, in this area, is represented by DSP toxic species as D. sacculus and provides grounds for widen and reinforcing sanitary control of the phycotoxin measures in the region.

First recorded proliferation of Coolia monotis (Meunier, 1919) in the North Lake of Tunis (Tunisia) correlation with environmental factors.

Coolia monotis is a potentially toxic epiphytic dinoflagellate widespread along the Mediterranean coasts, where it is frequently detected year round at low concentrations. However, it only proliferates recurrently in some localities. The North Lake of Tunis is one of the affected areas in the southwestern part of the Mediterranean Sea. This site is one of the most productive aquatic Tunisian areas (Recreational Fisheries and shellfish collecting). In the south part of this area of study, recurrent C. monotis proliferation (5 x10(5) cells per liter) took place in late spring and early summer of 2006. During this proliferation, the spatial distribution of C. monotis species, phytoplankton community, and abiotic factors were studied. The composition of the phytoplankton community exhibited a clear dominance of dinoflagellates over other genera. We suggest that proliferation development of C. monotis was linked to climatic conditions, water temperature (r = 0.24, p < 0.05) and high concentrations of nitrogenous nutrients, essentially NH(4)(+) (r = 0.18, p < 0.05) and NO(3)(-) (r = 0.21, p < 0.05).