Mohamed Laabir

NITROGENOUS NUTRITION OF ALEXANDRIUM CATENELLA (DINOPHYCEAE) IN CULTURES AND IN THAU LAGOON, SOUTHERN FRANCE1

Alexandrium catenella (Whedon et Kofoid) Balech was isolated from Thau lagoon (northern Mediterranean) and its growth and uptake characteristics measured for nitrate, ammonium, and urea. Although affinity constants did not indicate a preference for ammonium over nitrate, there was a strong inhibition of nitrate uptake by ammonium when both nitrogen (N) sources were present. Nitrogen budgets during growth in cultures revealed major imbalances between decreases in dissolved N and increases in particulate N, indicating excretion of dissolved organic N during the early part of the growth phase and uptake during the later part. A quasi‐unialgal bloom in November 2001 (4×106 cells·L−1) allowed measurements of uptake of nitrate, nitrite, ammonium, and urea; net and gross growth rate of A. catenella; and grazing rates on this organism. The affinity constants indicate that it is not a strong competitor for the N nutrients tested when these are in low concentrations (<10 μgat N·L−1), compared with other members of the phytoplankton community. Indirect evidence from cultures indicate that dissolved organic N compounds could be important in triggering those blooms. Finally, the strongly unbalanced growth observed in the field indicates that A. catenella exhibits a storage rather than a growth response to a nutrient pulse and is adapted to low frequency events such as the passage of frontal disturbances. The disappearance of A. catenella was due to grazing that balanced growth at the peak of the bloom.

New evidence of the copepod maternal food effects on reproduction.

Failure of female reproductive capacity in the copepod Calanus helgolandicus was related to number and combination of the phytoplankton species in the diets. The maternal food effects were detectable at different levels: fecundity, oogenesis and hatching. Fecundity and hatching were normal with two single (ca. Isochrysis galbana and Prorocentrum minimum) and one mixed (Phaeodactylum tricornutum+Dunaliella tertiolecta+Pavlova lutherii+I. galbana+P. minimum) diets. With the single P. lutherii diet, fecundity decreased, but hatching remained optimal. The daily egg production and hatching rates decreased significantly in females fed the other single P. tricornutum, D. tertiolecta and mixed (P. tricornutum+D. tertiolecta+P. lutherii+I. galbana) diets, or starved. The fecundity decrease coincided with gonad atresia, which was reversible when P. tricornutum and P. lutherii diets were replaced by P. minimum diet. It was irreversible when D. tertiolecta was replaced by P. minimum, leading to female sterilization expressed by the deterioration of OS3 and OS2 oocytes, as a function of the feeding duration. We assume that atresia of female gonads was caused by the limitation of essential nutrients in food, such as fatty acids, which induced catabolism and recycling of yolk reserves and thus, maintenance of gonad integrity and low spawning rates. With the D. tertiolecta diet, abnormally high increase of ornithine concentrations in eggs showed that the ornithine metabolism and polyamine pathway were affected during oogenesis, leading atresia of oocytes to be deeply disturbed and followed up by necrosis of the gonads.

Unexpected Genetic Diversity among and within Populations of the Toxic Dinoflagellate Alexandrium catenella as Revealed by Nuclear Microsatellite Markers

ABSTRACT Since 1998, blooms of Alexandrium catenella associated with paralytic shellfish poisoning have been repeatedly reported for Thau Lagoon (French Mediterranean coast). Based on data obtained for rRNA gene markers, it has been suggested that the strains involved could be closely related to the Japanese temperate Asian ribotype of the temperate Asian clade. In order to gain more insight into the origin of these organisms, we carried out a genetic analysis of 61 Mediterranean and 23 Japanese strains using both ribosomal and microsatellite markers. Whereas the phylogeny based on ribosomal markers tended to confirm the previous findings, the analysis of microsatellite sequences revealed an unexpected distinction between the French and Japanese populations. This analysis also highlighted great intraspecific diversity that was not detected with the classical rRNA gene markers. The Japanese strains are divided into two differentiated A. catenella lineages: the Sea of Japan lineage and the east coast lineage, which includes populations from the Inland Sea and the Pacific Ocean. A. catenella strains isolated from Thau Lagoon belong to another lineage. These findings indicate that microsatellite markers are probably better suited to investigations of the population genetics of this species that is distributed worldwide. Finally, application of the population genetics concepts available for macroorganisms could support new paradigms for speciation and migration in phytoplankton assemblages.

Gene Expression in Proliferating Cells of the Dinoflagellate Alexandrium catenella (Dinophyceae)

ABSTRACT Understanding the conditions leading to harmful algal blooms, especially those produced by toxic dinoflagellate species, is important for environmental and health safety. In addition to investigations into the environmental conditions necessary for the formation of toxic blooms, we postulate that investigating gene expression in proliferating cells is essential for understanding bloom dynamics. Expressed sequence tags were produced from cultured cells of the toxic dinoflagellate Alexandrium catenella sampled during the initiation phase of growth using Sangers method and by 454 pyrosequencing. A significant proportion of identified genes (ca. 25%) represented enzymes and proteins that participate in a variety of cellular regulatory mechanisms that may characterize proliferating cells, e.g., control of the cell cycle and division, regulation of transcription, translation and posttranslational protein modifications, signaling, intracellular trafficking, and transport. All of the several genes selected for gene expression assays due to their involvement in metabolism and the cell cycle were overexpressed during exponential growth. These data will be useful for investigating the mechanisms underlying growth and toxin production in toxic Alexandrium species and for studying and monitoring the development of toxic blooms.

Influence of Environmental Factors on the Paralytic Shellfish Toxin Content and Profile of Alexandrium catenella (Dinophyceae) Isolated from the Mediterranean Sea

Laboratory experiments were designed to study the toxin content and profile of the Alexandrium catenella strain ACT03 (isolated from Thau Lagoon, French Mediterranean) in response to abiotic environmental factors under nutrient-replete conditions. This dinoflagellate can produce various paralytic shellfish toxins with concentrations ranging from 2.9 to 50.3 fmol/cell. The toxin profile was characterized by carbamate toxins (GTX3, GTX4 and GTX5) and N-sulfocarbamoyl toxins (C1, C2, C3 and C4). C2 dominated at 12–18 °C, but only for salinities ranging from 10 to 25 psu, whereas GTX5 became dominant at temperatures ranging from 21 to 30 °C at almost all salinities. There was no significant variation in the cellular toxin amount from 18 °C to 27 °C for salinities ranging between 30 and 40 psu. At salinities of 10 to 25 psu, the toxin concentrations always remained below 20 fmol/cell. Toxin content was stable for irradiance ranging from 10 to 70 μmol photons/m2/s then slightly increased. Overall, the toxin profile was more stable than the toxin content (fmol/cell), except for temperature and/or salinity values different from those recorded during Alexandrium blooms in Thau Lagoon.

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 lagoons 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.

Exposure to the Neurotoxic Dinoflagellate, Alexandrium catenella, Induces Apoptosis of the Hemocytes of the Oyster, Crassostrea gigas

This study assessed the apoptotic process occurring in the hemocytes of the Pacific oyster, Crassostrea gigas, exposed to Alexandrium catenella, a paralytic shellfish toxins (PSTs) producer. Oysters were experimentally exposed during 48 h to the toxic algae. PSTs accumulation, the expression of 12 key apoptotic-related genes, as well as the variation of the number of hemocytes in apoptosis was measured at time intervals during the experiment. Results show a significant increase of the number of hemocytes in apoptosis after 29 h of exposure. Two pro-apoptotic genes (Bax and Bax-like) implicated in the mitochondrial pathway were significantly upregulated at 21 h followed by the overexpression of two caspase executor genes (caspase-3 and caspase-7) at 29 h, suggesting that the intrinsic pathway was activated. No modulation of the expression of genes implicated in the cell signaling Fas-Associated protein with Death Domain (FADD) and initiation-phase (caspase-2) was observed, suggesting that only the extrinsic pathway was not activated. Moreover, the clear time-dependent upregulation of five (Bcl2, BI-1, IAP1, IAP7B and Hsp70) inhibitors of apoptosis-related genes associated with the return to the initial number of hemocytes in apoptosis at 48 h of exposure suggests the involvement of strong regulatory mechanisms of apoptosis occurring in the hemocytes of the Pacific oyster.

Toxicity and Growth Assessments of Three Thermophilic Benthic Dinoflagellates (Ostreopsis cf. ovata, Prorocentrum lima and Coolia monotis) Developing in the Southern Mediterranean Basin

Harmful benthic dinoflagellates, usually developing in tropical areas, are expanding to temperate ecosystems facing water warming. Reports on harmful benthic species are particularly scarce in the Southern Mediterranean Sea. For the first time, three thermophilic benthic dinoflagellates (Ostreopsis cf. ovata, Prorocentrum lima and Coolia monotis) were isolated from Bizerte Bay (Tunisia, Mediterranean) and monoclonal cultures established. The ribotyping confirmed the morphological identification of the three species. Maximum growth rates were 0.59 ± 0.08 d−1 for O. cf. ovata, 0.35 ± 0.01 d−1 for C. monotis and 0.33 ± 0.04 d−1 for P. lima. Toxin analyses revealed the presence of ovatoxin-a and ovatoxin-b in O. cf. ovata cells. Okadaic acid and dinophysistoxin-1 were detected in P. lima cultures. For C. monotis, a chromatographic peak at 5.6 min with a mass m/z = 1061.768 was observed, but did not correspond to a mono-sulfated analogue of the yessotoxin. A comparison of the toxicity and growth characteristics of these dinoflagellates, distributed worldwide, is proposed.

Distribution of resting cysts of the potentially toxic dinoflagellate Alexandrium pseudogonyaulax in recently-deposited sediment within Bizerte lagoon (Mediterranean coast, Tunisia).

This study investigated the spatial distribution of Alexandrium pseudogonyaulax resting cysts in recently-deposited sediment of Bizerte lagoon (South-Western Mediterranean, Tunisia). This lagoon is the subject of many anthropogenic impacts, such as holding important fishing and aquaculture activities. A. pseudogonyaulax has been shown to produce Goniodomin A, which is a biologically-active compound. We showed that this dinoflagellate produces two types of resting cysts, which could be distinguished by the presence or the absence of a paratabulate wall. The average cyst density across the whole lagoon was rather high, reaching 639 cysts g(-1) of dry sediment (DS). Cyst densities varied widely among the sampled stations, with the highest density of 1685 cyst g(-1)DS being recorded at station 51 near a mussel farm. With respect to sediment characteristics, the highest cyst densities were found within silty sediments with high water content values. The distribution of A. pseudongoyaulax cysts in Bizerte lagoon appears to be related to hydrodynamic factors.

Paralytic Toxins Accumulation and Tissue Expression of α-Amylase and Lipase Genes in the Pacific Oyster Crassostrea gigas Fed with the Neurotoxic Dinoflagellate Alexandrium catenella

The pacific oyster Crassostrea gigas was experimentally exposed to the neurotoxic Alexandrium catenella and a non-producer of PSTs, Alexandrium tamarense (control algae), at concentrations corresponding to those observed during the blooming period. At fixed time intervals, from 0 to 48 h, we determined the clearance rate, the total filtered cells, the composition of the fecal ribbons, the profile of the PSP toxins and the variation of the expression of two α-amylase and triacylglecerol lipase precursor (TLP) genes through semi-quantitative RT-PCR. The results showed a significant decrease of the clearance rate of C. gigas fed with both Alexandrium species. However, from 29 to 48 h, the clearance rate and cell filtration activity increased only in oysters fed with A. tamarense. The toxin concentrations in the digestive gland rose above the sanitary threshold in less than 48 h of exposure and GTX6, a compound absent in A. catenella cells, accumulated. The α-amylase B gene expression level increased significantly in the time interval from 6 to 48 h in the digestive gland of oysters fed with A. tamarense, whereas the TLP gene transcript was significantly up-regulated in the digestive gland of oysters fed with the neurotoxic A. catenella. All together, these results suggest that the digestion capacity could be affected by PSP toxins.