In this study, we optimized procedures to enumerate viruses from marine sediments by epifluorescence microscopy using SYBR Green I as a stain. The highest virus yields from the bulk of the sediments were obtained by utilizing pyrophosphate and 3 min of sonication. The efficiency of extraction benthic viruses by pyrophosphate-ultrasound treatment was about 60% of the extractable virus particles. Samples treated with nucleases had increased virus counts, suggesting a masking effect of extracellular DNA. No significant differences were observed between virus counts obtained by epifluorescence microscopy and transmission electron microscopy. Both formaldehyde and glutaraldehyde gave significant reductions of virus counts after only 24 h of sediment storage, but no further loss occurred after 7 days.Viruses are now considered to be an important component of all aquatic microbial communities. The reevaluation of the role of viruses in marine ecosystems is due to the discovery of very high virus abundance (see reference 13 for a review). Recent studies have stressed the ecological implication of viruses in the release of dissolved organic matter, nutrient recycling (18), and the pathways of organic carbon utilization, with cascade effects on marine microbial food webs and organicmatter cycling (12). The available methods for the determination of virus abundance in aquatic environments include counting by transmission electron microscopy (TEM) (1, 2, 16, 21), by flow cytometry (17), and by epifluorescence microscopy (EFM) (9,14,20,26,29). The last technique allows an accurate and easily performed enumeration, avoiding the use of expensive and bulky equipment (13). In addition, EFM is reported to be up to seven times more efficient than TEM for counting viruses (15,28).Available information dealing with benthic virus ecology is scant. This is due to the lack of adequate protocols for easily determining their abundance and distribution in marine sediments (6). The main objective of this work was the optimization of procedures to enumerate viruses in different marine sediments. We focused our attention on EFM counting using SYBR Green I as a stain (20) in order to address the following issues: (i) virus dislodgment from sediment particles (using surfactant and ultrasound treatments), (ii) the efficiency of virus extraction from bulk sediment (by the number of postsonication washings), and (iii) stain-counting accuracy and efficiency (by removing possible interferences due to extracellular DNA in virus counting and by comparison with TEM counts). In addition, we tested the effects of preservatives on virus abundance in long-time-course experiments carried out on fixed sediment samples.Sediment sampling and selection of sediment. In order to make the protocol for virus counting suitable for the widest variety of sediment samples, two different sediment types were selected in this study: shallow sands and deep-sea muds. As deep-sea samples are not generally analyzed immediately, the effects of long-term storage with preservat...

Since 2005, the benthic dinoflagellate Ostreopsis cf. ovata has bloomed across the Mediterranean basin, provoking serious toxic outbreaks. LC/MS studies have identified a number of palytoxin-like compounds, termed ovatoxins, along with trace amounts of putative palytoxin as the causative agents of the O. cf. ovata -related human sufferings. So far, any risk assessment for ovatoxins as well as establishment of their allowance levels in seafood has been prevented by the lack of pure toxins. The present paper reports on the isolation, NMR-based structural determination, and preliminary mouse lethality evaluation of ovatoxin-a, the major toxic compound contained in O. cf. ovata extracts. Availability of pure ovatoxin-a will open the double prospect of fully evaluating its toxicity and preparing reference standards to be employed in LC/MS quantitative analyses. Elucidation of ovatoxin-a's complex structure will ultimately herald the understanding of the molecular bases of ovatoxins bioactivity.

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The Northern Adriatic Sea is the area of the Mediterranean Sea where eutrophication and episodes related to harmful algae have occurred most frequently since the 1970s. In this area, which is highly exploited for mollusk farming, the first occurrence of human intoxication due to shellfish consumption occurred in 1989, nearly 10 years later than other countries in Europe and worldwide that had faced similar problems. Until 1997, Adriatic mollusks had been found to be contaminated mostly by diarrhetic shellfish poisoning toxins (i.e., okadaic acid and dinophysistoxins) that, along with paralytic shellfish poisoning toxins (i.e., saxitoxins), constitute the most common marine biotoxins. Only once, in 1994, a toxic outbreak was related to the occurrence of paralytic shellfish poisoning toxins in the Adriatic coastal waters. Moreover, in the past 15 years, the Adriatic Sea has been characterized by the presence of toxic or potentially toxic algae, not highly widespread outside Europe, such as species producing yessotoxins (i.e., Protoceratium reticulatum, Gonyaulax spinifera and Lingulodinium polyedrum), recurrent blooms of the potentially ichthyotoxic species Fibrocapsa japonica and, recently, by blooms of palytoxin-like producing species of the Ostreopsis genus. This review is aimed at integrating monitoring data on toxin spectra and levels in mussels farmed along the coast of the Emilia-Romagna region with laboratory studies performed on the species involved in the production of those toxins; toxicity studies on toxic or potentially toxic species that have recently appeared in this area are also reviewed. Overall, reviewed data are related to: (i) the yessotoxins producing species P. reticulatum, G. spinifera and L. polyedrum, highlighting genetic and toxic characteristics; (ii) Adriatic strains of Alexandrium minutum, Alexandrium ostenfeldii and Prorocentrum lima whose toxic profiles are compared with those of strains of different geographic origins; (iii) F. japonica and Ostreopsis cf. ovata toxicity. Moreover, new data concerning domoic acid production by a Pseudo-nitzschia multistriata strain, toxicity investigations on a Prorocentrum cf. levis, and on presumably ichthyotoxic species, Heterosigma akashiwo and Chattonella cf. subsalsa, are also reported.

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