Laurita Boni

Complex yessotoxins profile in Protoceratium reticulatum from north-western Adriatic sea revealed by LC-MS analysis.

While the occurrence of yessotoxin (YTX) has been reported worldwide from Protoceratium reticulatum, the biogenetic origin of some YTX analogues is still unknown, thus raising an issue whether they are metabolites of YTX formed in mussels or true products of different dinoflagellate species. Findings reported herein suggest that P. reticulatum from the north-western Adriatic sea is responsible for production, together with YTX (1), of homoYTX (2), 45-OHYTX (3), carboxyYTX (5), and noroxoYTX (7). YTX and its analogues have been determined by high performance liquid chromatography coupled with electrospray ion trap mass spectrometry (HPLC-MS and HPLC-MSn experiments). The result is the first to confirm production of these YTX analogues from this species and indicate it as a producing organism of homoYTX, 45-OHYTX, carboxyYTX, and noroxoYTX found in shellfish.

METABOLIC RESPONSES OF THE DIATOM ACHNANTHES BREVIPES (BACILLARIOPHYCEAE) TO NUTRIENT LIMITATION

The diatom Achnanthes brevipes C.A. Ag. was cultured in the presence of limiting concentrations of nitrogen (N) or inorganic phosphate (Pi). Growth, in terms of final yield, was more affected by N limitation than Pi limitation; N limitation had a greater effect also on protein and chlorophyll content. Carbohydrate concentrations increased under both nutrient starvation treatments, but N or Pi limitation had different effects. Total (intracellular plus extracellular) sugar content increased when cells were exposed to both types of nutrient limitation, but the extracellular polysaccharide fraction increased only in the presence of Pi starvation. Analyses were performed to identify the metabolic changes occurring in cells exposed to low phosphate because this was the main condition that affected carbohydrate extrusion. Activities of several enzymes involved in carbohydrate metabolism showed that under Pi limitation there was no activation of alternative reactions that were found to result in Pi liberation, instead of its consumption, in some higher plants and in the green alga Selenastrum minutum Naeg. Collins. Results showed that activities of pyruvate kinase, phosphorylating NAD‐dependent 3‐phosphate‐glyceraldehyde dehydrogenase, and 3‐phospho‐glycerate kinase were inhibited under Pi‐limited conditions compared with control cells, indicating limited glucose catabolism. Activity of uridine diphosphate glucose pyrophosphorylase, a key enzyme for the biosynthesis of the storage compound crysolaminarin, was also partly inhibited in Pi‐stressed cells. Our findings suggest that carbohydrate catabolism in A. brevipes is limited under Pi deficiency, whereas extracellular extrusion of carbohydrate is favored.

Increased production of extra- and intracellular metal-ligands in phytoplankton exposed to copper and cadmium

Several diatom and dinoflagellate species isolatedfrom the Adriatic Sea, grown in batch cultures in f/2medium, were exposed to two different concentrationsof Cu and Cd. The concentrations chosen caused areduction of growth, but were not lethal. Thespecies able to tolerate the highest Cu concentrations(in the order of 0.2–0.5 mg L-1) were diatoms,while some diatoms and all the dinoflagellates wereinhibited by concentrations between 0.01 and 0.05 mgCu L-1. Cadmium was less toxic in that it did notaffect diatom or dinoflagellate growth up to aconcentration of 0.2 mg L-1. Increasedproduction of extracellular polysaccharides was foundto be a general response to the presence of thepollutant. Cylindrotheca fusiformis, Achnanthesbrevipes and Prorocentrum micans wereinvestigated for their production of intracellularmetal-ligands. Molecules with Cd-binding propertiesand having a molecular weight comparable with that ofphytochelatins were found only in C.fusiformis, and their presence showed both adose- and time-dependent response. The presence orabsence of intracellular ligands in these algae isconsistent with their ability to exclude the metalextracellularly.

INVESTIGATION OF 4‐METHYL STEROLS FROM CULTURED DINOFLAGELLATE ALGAL STRAINS

The marine dinoflagellates Prorocentrum micans, Gonyaulax polyedra, Gymnodinium sp., and Alexandrium tamarense, collected from the Adriatic Sea during red‐tide blooms, were cultured to investigate the 4‐methyl sterol constituents. To ascertain a possible influence of cell age on the 4‐methyl sterol content, for one strain (Gymnodinium sp.)we investigated the composition of these constituents at exponential and stationary growing phases. The lipid material extracted with acetone from the lyophilized algal samples was fractionated by thin‐layer chromatography. The 4‐methyl sterols recovered from the layer were converted into the corresponding OTMS derivatives. Nine of 11 constituents were identified by gas chromatography and gas chromatography‐mass spectrometry; only two minor constituents were characterized by their gas chromatographic parameters. All free methyl sterols identified in the algal samples had been detected previously in various dinoflagellates. The 4‐methyl sterol fractions generally contained very few constituents. Except for the Gymnodinium sp. sample, collected at the exponential growing phase (GyD2 exp), which contains 4,24‐dimethylcholestan‐3‐ol as a unique constituent, dinosterol was the major component. Moreover, 4,24‐ethylcholestan‐3‐ol was also an important constituent of both Prorocentrum and Gonyaulax strains, whereas considerable amounts of dinostanol characterized all the Gymnodinium sp. strains. In addition, the latter contained several minor constituents such as 4‐methylcholestan‐3‐ol, 4,24‐dimethylcholesta‐22‐en‐3‐ol, and 4‐methyl‐24‐ethylcholestan‐3‐ol. 4‐Methyl‐24‐methylene‐cholestan‐3‐ol was a constituent of the Gymnodinium sp. sample, collected at the stationary growing phase (GyD2 stat)only, whereas 4‐methylgorgostanol was identified only in the Alexandrium tamarense Gt4 strain. Except for 4‐methyl‐24‐ethylcholesta‐8(14)‐en‐3‐ol, all the methyl sterol constituents from our algae show a saturated polynuclear system. The pathways by which side‐chain modifications occur in dinoflagellate 4‐methyl sterols are considered, and a map of the fragmentation pattern of the trimethylsilyl‐4‐methyl sterols under electronic impact is also reported.

Copper toxicity and carbohydrate production in the microalgae Cylindrotheca fusiformis and Gymnodinium sp.

The effect of copper on carbohydrate production in the diatom Cylindrotheca fusiformis and in the dinoflagellate Gymnodinium sp. was examined in batch cultures. Copper was shown to inhibit the growth of the two species to different extents : in particular the dinoflagellate was highly sensitive to the metal when metal chelators were absent. Analysis of the carbohydrate content showed that C. fusiformis produced a much higher total amount than Gymnodinium sp. and cells of the diatom which were exposed to toxic copper concentrations displayed a higher carbohydrate production which paralleled the increase in copper concentrations. The increased production could be attributed to extracellular rather than to intracellular carbohydrate and it occurred after 12 and 16 days of growth, respectively, for cells exposed to 0.2 and 0.5 mg l−1 Cu. The response of carbohydrate production by Gymnodinium sp. to the presence of copper was much smaller and occurred only in late stationary phase of growth (21st day). An anal...

High sensitivity bioassay of paralytic (PSP) and amnesic (ASP) algal toxins based on the fluorimetric detection of [Ca2+]i in rat cortical primary cultures

A high sensitivity bioassay able to recognise small amounts of paralytic and amnesic toxins in algal acetic extracts is described. The method is based on the measure of intracellular [Ca(2+)](i) in primary cultures of rat cortical neurones preloaded with Fura-2 and submitted to electrical field stimulation. Under normal conditions the basal [Ca(2+)](i) level was about 50-100 nM and was nearly doubled during the peaks induced by trains of electrical pulses at 10 Hz for 10 s. Saxitoxin (STX) 3.5 nM and tetrodotoxin (TTX) 24 nM halved the peaks height without affecting basal [Ca(2+)](i). Conversely, domoic acid increased the basal [Ca(2+)](i) (EC(50)=3. 7 microM) and decreased the calcium peaks (EC(50)=7.3 microM). CNQX (a competitive antagonist of AMPA/KA receptors) at 10 microM shifted to the right by a factor of 3 the concentration-response curves of domoic acid. The extracts of non-toxic algae were well tolerated by up to 10 microg protein/ml, whereas extracts of Alexandrium lusitanicum at 1-4 microg protein/ml reduced [Ca(2+)](i) peaks and increased basal calcium levels. This toxic effect of A. lusitanicum was unexpected since parallel HPLC analysis showed only the presence of gonyautoxins, known to act like saxitoxin. Therefore, the bioassay on rat cortical neurones revealed a complex composition of the toxins present in A. lusitanicum. The relevance of fluorimetric detection of [Ca(2+)](i) in primary neuronal cultures in the evaluation of algal risk is stressed.

Toxic or potentially toxic dinoflagellates from the Northern Adriatic Sea

The first occurrence of DSP (diarrhoetic shellfish poisoning) in the Adriatic Sea in 1989 and the risk of future toxic blooms prompted a survey of the toxic and potentially toxic dinoflagellates that have been found so far in the Adriatic Sea. A list of potential producers of DSP toxins (Dinophysis species), PSP toxins (Alexandrium species) and other toxins is given. The Alexandrium species are described by light and scanning electron microscopy. Information is given about their distribution and previous blooms in the Adriatic Sea

First cases of diarrhoetic shellfish poisoning in the Northern Adriatic Sea

Shellfish poisoning occurs when toxic phytoplankton species are present in their environment. Because shellfish such as mussels are filter feeders they concentrate toxins. It is also well known that sometimes also small amounts of toxic algae can produce serious shellfish poisoning. In the Adriatic Sea, dinoflagellate red tides are a recurring phenomenon, but no shellfish poisoning has been described despite the presence of potentially toxic species such as Alexandrium spp. (Protogonyaulax) and Dinophysis spp. (...)

Correlation between the presence of Gonyaulax fragilis (Dinophyceae) and the mucilage phenomena of the Emilia-Romagna coast (northern Adriatic Sea)

Abstract The formation of massive amounts of suspended mucilaginous organic matter which periodically affects the Adriatic Sea, has been regarded as a complex physico-chemical phenomenon resulting from the production of extracellular material by phytoplankton. Although the exact cause has remained obscure, the mechanism of its formation has usually been considered to be a long-term process, starting after the late winter-early spring blooms, and involving the participation of various algal species, mainly within the diatom group. In this paper we report on the results of a phytoplankton monitoring programme in northern Adriatic seawaters off the Emilia-Romagna coast of Italy which revealed the constant concomitant presence of the dinoflagellate Gonyaulax fragilis (Schutt) Kofoid and mucilaginous formations. In the early stages of the phenomenon the dinoflagellate was clearly observable by microscopic examination in the mucilage, but as the mucilage aged this alga almost completely decomposed and diatom cells increased in number and became predominant. Although characterized by a slow growth rate in culture, in natural seawater G. fragilis was observed to reach cell densities of up to 7.0×10 6 cells l −1 . The results of this study lead us to propose the hypothesis that the appearance of mucilage in the water column of the Adriatic Sea is the consequence of a seasonal growth of this dinoflagellate favoured by specific environmental circumstances.

The occurrence of Gonyaulax tamarensis Lebour bloom in the Adriatic Sea along the coast of Emilia-Romagna

Abstract The authors describe the occurrence of Gonyaulax tamarensis Lebour bloom in the Adriatic Sea between 200 and 1000 m off the coast of Emilia-Romagna during the period of August 14-20 1982.