Franca Guerrini

Complex palytoxin-like profile of Ostreopsis ovata. Identification of four new ovatoxins by high-resolution liquid chromatography/mass spectrometry.

Over the past decades, Italian coastlines have been plagued by recurring presence of the benthic dinoflagellate Ostreopsis ovata. Such an alga has caused severe sanitary emergencies and economic losses due to its production of palytoxin-like compounds. Previous studies have confirmed the presence of ovatoxin-a (OVTX-a) as the major toxin of the algal toxin profile together with small amounts of putative palytoxin (PLTX). In our ongoing research on O. ovata toxins we report herein on in-depth investigation of an O. ovata culture carried out by high-resolution (HR) liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS(2)). Particularly, the presence of putative PLTX and OVTX-a was confirmed and the occurrence in the extract of four new palytoxin-like compounds, OVTX-b, -c, -d, and -e, was highlighted. Elemental formulae have been assigned to the new ovatoxins and information has been gained about their structural features. A quantitative study of the O. ovata culture extract indicated that the whole of the new ovatoxins represents about 46% of the total toxin content and, thus, their presence has to be taken into account when LC/MS-based monitoring programs of either plankton or contaminated seafood are carried out.

Extraction of hydrocarbons from microalga Botryococcus braunii with switchable solvents

Lipid extraction is a critical step in the development of biofuels from microalgae. Here a new procedure was proposed to extract hydrocarbons from dried and water-suspended samples of the microalga Botryococcus braunii by using switchable-polarity solvents (SPS) based on 1,8-diazabicyclo-[5.4.0]-undec-7-ene (DBU) and an alcohol. The high affinity of the non-ionic form of DBU/alcohol SPS towards non-polar compounds was exploited to extract hydrocarbons from algae, while the ionic character of the DBU-alkyl carbonate form, obtained by the addition of CO(2), was used to recover hydrocarbons from the SPS. DBU/octanol and DBU/ethanol SPS were tested for the extraction efficiency of lipids from freeze-dried B. braunii samples and compared with n-hexane and chloroform/methanol. The DBU/octanol system was further evaluated for the extraction of hydrocarbons directly from algal culture samples. DBU/octanol exhibited the highest yields of extracted hydrocarbons from both freeze-dried and liquid algal samples (16% and 8.2% respectively against 7.8% and 5.6% with n-hexane).

Comparative growth and toxin profile of cultured Ostreopsis ovata from the Tyrrhenian and Adriatic Seas.

Massive blooms of the benthic dinoflagellate Ostreopsis ovata Fukuyo have recently occurred along the whole Italian coastlines, both Tyrrhenian and Adriatic, resulting sometimes in benthonic biocenosis sufferings and, occasionally, in human health problems. In this work, two strains of O. ovata collected in 2006 along the Adriatic and Tyrrhenian coastlines and grown in culture were studied to characterize their growth and toxin profile. The two strains showed different cell volumes, the Adriatic strain being nearly twice bigger than the Tyrrhenian, but they had similar slow growth rates. Liquid chromatography-mass spectrometry (LC-MS) analyses indicated that both strains produce putative palytoxin (pPLTX) and ovatoxin-a (OVTX-a), a palytoxin-like compound presenting 2 oxygen atoms less than palytoxin. Toxin content was determined at the end of the stationary and exponential growth phases and reached the highest value in the Adriatic strain at the end of the stationary phase, with concentrations of 353.3 microg l(-1) for OVTX-a and 30.4 microg l(-1) for pPLTX. Toxin released in the growth medium was also measured and resulted to be the highest at the end of the stationary phase, suggesting that a long lasting bloom could enhance the toxin content in the water and cause toxic effects in people inhaling the aerosol.

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.

A review on the effects of environmental conditions on growth and toxin production of Ostreopsis ovata.

Since the end of the 1990s the occurrence of blooms of the benthic dinoflagellates Ostreopsis spp. is spreading in many tropical and temperate regions worldwide, sometimes causing benthonic biocenosis suffering and occasional human distress. Ostreopsis ovata has been found to produce palytoxin-like compounds, a class of highly potent toxins. As general, the highest abundances of Ostreopsis spp. are recorded during warmer periods characterized by high temperature, salinity, and water column stability. Moreover, as these cells are easily resuspended in the water column, the role of hydrodynamism in the blooms development and decline has been highlighted. The environmental conditions appear, therefore, to be one of the main factors determining the proliferation of these species as testified by several field surveys. Laboratory studies on the effect of environmental parameters on growth and toxicity of O. ovata are rather scarce. With regard to the effects of temperature, culture results indicate that different strains blooming along Italian coasts displayed different optima, in accordance to blooming periods, and that higher toxin levels correlated with best growth conditions. Additionally, in relation to an Adriatic strain, cell growth positively correlated with the increase in salinity, while toxicity was lowest at the highest salinity value (i.e. 40). For the same strain, both nitrogen and phosphorus limitation determined a decrease in cell toxicity showing different behaviour with respect to many other toxic dinoflagellates.

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.

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...

Characterization of 27-hydroxy-13-desmethyl spirolide C and 27-oxo-13,19-didesmethyl spirolide C. Further insights into the complex adriatic Alexandrium ostenfeldii toxin profile.

Alexandrium ostenfeldii is a widespread toxic dinoflagellate that has recently bloomed across the Adriatic Sea, seriously threatening both shellfish consumers and aquacultures. In 2007 we reported on preliminary studies carried out on field samples and cultures of A. ostenfeldii. At the time, along with three major spirolides - among which 27-hydroxy-13,19-didesmethyl spirolide C (3) proved to be a novel compound - a number of new minor spirolides were detected. Unfortunately, for all of them only Mass Spectrometry-based structural hypotheses could be ventured due to their very small amount. In the present paper we report on isolation and High Resolution Mass Spectrometry- and NMR-based structural elucidation of two of those minor spirolides detected in our previous study.

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.