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Since the 1990s red-tide blooms of Fibrocapsa japonica have frequently been observed in European waters; despite their recurrence also in the Adriatic coastal areas, they have never been conclusively linked to ichthyotoxic events. Since the toxicity of F. japonica is still under debate and its effects differ among the strains, in this study all the compounds previously postulated as involved in the toxic mechanism, such as brevetoxins, fatty acids and ROS, were screened for Adriatic strains. Whole algal extracts were analyzed for brevetoxin PbTx-2, which was not found, and for a qualitative and quantitative analysis of fatty acids. The fatty acid profile evidenced the presence of PUFAs, with considerable amounts of 18:4n-3, 20:4n-6, and 20:5n-3, as already found in different strains, but with a lower amount of arachidonic acid (20:4n-6). None of the PUFAs was released in the extracellular medium. Different toxicological assays (Vibrio fischeri, Artemia sp., haemolysis of fish erythrocytes) were performed using algal cellular, extracellular or subcellular samples as well as fractions of the algal extract, in order to identify the toxic compounds. The tested cultures were found to inhibit V. fischeri bioluminescence, to affect Artemia nauplii viability and to have haemolytic effects comparable to those previously observed in different strains. The solid phase extraction (SPE) of the Adriatic F. japonica extracts resulted in a high concentration of PUFAs in two main fractions, which caused mortality in Artemia nauplii and inhibition of V. fischeri bioluminescence. Fish (Dicentrarchus labrax) assays were also performed, reporting mortality after long exposure times and the cause of fish death was investigated. A significant increase of H2O2 in the tanks where sea basses were exposed to F. japonica was obtained, indicating that the presence of fish stimulates H2O2 production by algal cells and leading to the hypothesis of H2O2 involvement in F. japonica toxicity. The presence of oxidative stress in the exposed fish was confirmed by the increased malondialdehyde concentration in their gills. Overall these results indicate that since F. japonica cells can easily cling to fish gills, due to their high polysaccharide production, it is presumable that high PUFA amounts are released in loco, as a result of cell breakage, and that exert their toxicity together with ROS. This study shows that the Adriatic strains of F. japonica can be harmful to higher level organisms. Nevertheless a high cell density and a long lasting bloom are necessary to cause severe damage to fish gills or death for crustaceans.

The spatial and temporal distribution of the toxic benthic dinoflagellate Ostreopsis ovata was investigated along the Conero Riviera (northern Adriatic Sea) during summer 2007 to evaluate the role of substratum, depth and hydrodynamic conditions on bloom dynamics. Sampling was carried out from April to November on different substrata (seaweeds, mollusc shells and rocks) and in the water column. O. ovata bloom started in September, reaching maximum values in the first week of October (1.7 × 106 cells g−1 fw corresponding to 21.0 × 106 cells g−1 dw and 6.2 × 104 cells cm−2) and completely disappearing at the beginning of November. On average, the abundances of O. ovata on rocks were significantly higher than those on seaweeds. Seaweeds with branched thalli supported higher values of epiphytes than non-branched ones. The abundances of O. ovata were significantly higher in sheltered than in exposed sites indicating a major role of hydrodynamism in regulating bloom dynamics. O. ovata abundances showed a marked decrease with depth with significantly lower values at depths >3 m. In the northern Adriatic, bloom development did not show any relationship with temperature, as the peak occurred when temperature values were decreasing.

The effects of temperature, salinity, inorganic and organic nutrients on the growth and physiological performance of Fibrocapsa japonica have been investigated in strains isolated from the northern Adriatic Sea, where intense and regular blooms have occurred since 1997 in coastal waters during summer. Strains isolated at different times and from different locations appeared homogeneous in terms of both physiological responses and molecular (ITS-5.8S rDNA) characteristics. Growth rates were higher at temperatures between 20 and 26 °C and in a salinity range of 30–35 (0.7 div day −1 ). The temperature of 16 °C inhibited growth, more markedly at the lowest and highest salinity values, a result also confirmed by a lower photosynthetic efficiency and by an increase in cell volume due to impaired division. Higher cell concentrations were obtained with macronutrients at f/2 levels than in a fivefold diluted medium. Comparing the utilization of 200 μM nitrate to that of different N sources (inorganic and organic), F. japonica showed an efficient growth with equivalent amounts of ammonia, urea and amino acids, such as glycine and tryptophan; glutamate was less effective, while methionine had toxic effects. Organic phosphate, administered as glycerophosphate, could also sustain F. japonica growth, probably on account of an alkaline phosphatase whose activity was enhanced in the presence of the organic form. Vitamins were necessary for growth, though no further stimulation was observed when a surplus of vitamin B12 (3 nM) was added. The addition of 11.7 μM iron instead of 2.3 μM, as well as that of humic acid, with or without macronutrients, did not enhance algal growth either. These results led us to hypothesise that F. japonica blooms became more frequent due to a general seawater temperature increase and to the availability of organic forms which, in coastal anthropized areas, are especially abundant in summer periods.

The colonization of epilithic diatoms on artificial hard substrates (marble, quartzite and slate) was investigated on a seasonal basis in a subtidal site of the northern Adriatic Sea to determine if substrate-dependent differences in colonization occurs and to define the seasonal variations of microepilithic communities in terms of abundance, biomass and community structure. Artificial substrates (smoothed small discs mounted on a Plexiglas sheet) were placed at a depth of 8 m in April 2003, July 2003, January 2004 and February 2004. The discs were collected after 6–7 weeks for counting and taxonomic identification of the diatoms. The community structure of epilithic diatoms showed a dominance of motile species over the entire study period, followed by erect, adnate and tube-dwelling diatoms. Diatom density showed a marked seasonal variability, ranging from 365±407 (winter 2004) to 557156±82602 cells cm-2 (spring 2004). Biomass ranged from 0.02±0.01 to 17.53±3.20 μg C cm-2. Abundance and biomass values did not present any significant differences for the three substrates examined.

The diatom assemblage associated with the Antarctic sponge Mycale acerata was studied through an analysis of the diatom frustule and pigment concentrations in both the sponge ectosome and choanosome. Sponges were sampled weekly from November 2001 to February 2002 at Terra Nova Bay, Antarctica, at a depth of 25–35 m. The most abundant diatoms were Porannulus contentus, Fragilariopsis curta, Thalassiosira cf. gracilis, T. perpusilla and Plagiotropis sp. High abundances of P. contentus were found on the sponge ectosome up to the beginning of November, before the ice melted, while later frustules were incorporated inside, indicating that P. contentus lives epibiontically on M. acerata and represents a potential food source for the sponge. The presence of other diatom species was mainly related to the summer phytoplankton bloom. The sponge incorporates diatoms from the water column and utilises them as a food source, accumulating frustules inside the choanosome. The lack of planktonic diatom frustules at the beginning of the summer indicates that they are expelled or dissolved during the cold season.

Special issue Phytoplankton Life-Cycles and Their Impacts on the Ecology of Harmful Algal Bloom.-- 13 pages, 4 figures, 6 tables, A PCR-based assay was developed and applied to sediment and sediment trap samples for the detection of different cysts belonging to dinoflagellates and raphidophytes in European coastal areas. Oligonucleotide primers were designed based on the ITS-5.8S and LSU ribosomal gene sequences. The specificity and sensitivity of the PCR assay were assessed using genomic DNA from clonal cultures, plasmid copy number of cloned target sequences, as well as from sediment samples. Qualitative PCR determinations of different cysts in sediment and sediment trap samples were compared to taxonomic examinations by light microscopy. This molecular methodology permitted a fast and specific detection of target cysts in sediment samples. We also detected dinoflagellate and raphidophyte cysts at concentrations not detectable by microscopic methods or that are difficult to identify. The results given by molecular and microscopic methods were comparable. However, higher values of positive detection for target cysts were obtained by PCR than with microscopy. Some taxa were detected in 100% of the samples using PCR, while others were only found in 10% of the samples. The data obtained in this study showed that the PCR-based method is a valid tool for cyst identification in marine sediments, This study was financed by the EU funded Research Project SEED (GOCE-CT-2005-003875). E. Garcés was sustained by a Ramon y Cajal contract from the Spanish Ministry of Science and Education

Fibrocapsa japonica resting cysts were detected in coastal sediments of the Marche region (northern Adriatic Sea, Italy), where summer blooms regularly occur. Sampling was carried out along 18 transects (2 stations each) during May–June 2007. The identification and counting of raphidophyte cysts were carried out under the inverted microscope and molecular analyses were applied to sediment samples to confirm species-specific identification. Raphidophyte cysts were recorded in almost all stations with maximum abundance of 9 ± 5 and 12 ± 3 cysts g−1 dw for cysts of F. japonica and undetermined Raphidophyceae, respectively. Cyst formation was induced in F. japonica monoclonal cultures under different conditions: shadow-optimal temperature and dark-low temperature treatment; both experiments were performed at two salinity values (30 and 35). Cyst formation (preliminary cysts) was observed in both experiments, although with different encystment rates. However, only a further permanence in the dark at 15 °C in microplates led to the formation of mature cysts, probably supported by the possibility to adhere to solid surfaces. Before pre-cyst formation, vegetative cells showed a cytoplasmatic “brown body”.

The microalgal community associated with Eudendrium racemosum, a marine hydroid widely distributed in the Mediterranean Sea, was studied during an annual cycle, at monthly frequency, in a coastal station of the Ligurian Sea. Microalgae were represented mainly by diatoms, which exhibited higher abundance and biomass values between autumn and spring (max 46,752 cells mm-2 and 1.94 μg C mm-2, respectively), while during summer a significant decrease was observed (min 917 cells mm-2 and 0.013 μg C mm-2). High levels of abundance of filamentous cyanobacteria were observed in summer. Spatial distribution of epibiontic microalgae showed a markedly increasing gradient from the basal to the apical part of hydroid colonies. Considering the growth forms of diatom communities, motile diatoms (mainly small naviculoid taxa) were the most abundant in all the periods. Adnate (Amphora and Cocconeis spp.) were distributed mainly in the basal and central part of hydroid colonies and showed two peaks (autumn and summer). Erect forms (mainly Tabularia tabulata, Licmophora spp., Cyclophora tenuis) were mainly distributed in the apical part of the colonies and showed their maximum densities in spring-summer. Tube-dwelling (Berkeleya rutilans, Parlibellus sp.) were observed at low densities throughout the study period, without any significant temporal or spatial variability. Comparing the microalgal communities on marine hydroid to those grown on mimic substrata placed in the sampling station during summer, significantly higher values of abundance were observed in the hydroid, suggesting that microalgae may benefit from the polyp catabolites. This fact was particularly evident for the adnate diatoms, whose temporal trend paralleled the cycle of hydroid host. © 2006 Springer-Verlag.

Living diatoms are commonly found within Antarctic Porifera, and generally interpreted as additional food source, or as mutualists or parasites of sponge tissues. However, no data are available about temporal variations of the abundance of diatoms inside sponges especially during the winter period. In this paper we analysed the amount of diatom frustules and chlorophaeopigment concentration in six species of Antarctic sponges (Dendrilla antarctica, Homaxinella flagelliformis, Kirkpatrickia variolosa, Suberites montiniger, Haliclona dancoi, Haliclona penicillata) sampled weekly from November 2001 - before the ice melting - to February 2002. Frustule concentration in the sponge tissues was very low at the beginning of November in all the sponge species, and increased between 9 and 22 January, to reach maximum values between 29 January and 7 February. Diatom abundances were significantly higher in H. dancoi tissues, comparing to the other sponge species, reaching values up to 1217′178 cells *10 g - 1 dw at the end of January. Chlorophaeopigments, very low at the beginning of November, increased between 5 and 19 December, before the peak of diatoms. Maximum chlphaeo values (650.5 ′ 5.9 μg g - 1 dw) were observed in D. antarctica. The planktonic Fragilariopsis curta was the most common diatom species recorded inside sponges. These data clearly indicate that diatom concentration inside the sponge tissues is related to the summer phytoplankton bloom. The shift between the pigment and frustule peaks strongly suggests that diatoms are used as a food source by sponges and that their frustules are accumulated inside the sponge body. The lack of frustules at the beginning of summer indicates that diatom frustules are expelled or dissolved during the cold season.