Improvement of In Vivo Fluorescence Tools for Fast Monitoring of Freshwater Phytoplankton and Potentially Harmful Cyanobacteria.

The use of multi-wavelength spectrofluorometers for the fast detection of algal taxa, based on chlorophyll a (Chl-a) emission spectra, has become a common practice in freshwater water management, although concerns about their accuracy have been raised. Here, inter-laboratory comparisons using monoalgal cultures have been performed to assess the reliability of different spectrofluorometer models, alongside Chl-a extraction methods. Higher Chl-a concentrations were obtained when using the spectrofluorometers than extraction methods, likely due to the poor extraction efficiencies of solvents, highlighting that traditional extraction methods could underestimate algal or cyanobacterial biomass. Spectrofluorometers correctly assigned species to the respective taxonomic group, with low and constant percent attribution errors (Chlorophyta and Euglenophyceae 6-8%, Cyanobacteria 0-3%, and Bacillariophyta 10-16%), suggesting that functioning limitations can be overcome by spectrofluorometer re-calibration with fresh cultures. The monitoring of a natural phytoplankton assemblage dominated by Chlorophyta and Cyanobacteria gave consistent results among spectrofluorometers and with microscopic observations, especially when cell biovolume rather than cell density was considered. In conclusion, multi-wavelength spectrofluorometers were confirmed as valid tools for freshwater monitoring, whereas a major focus on intercalibration procedures is encouraged to improve their reliability and broaden their use as fast monitoring tools to prevent environmental and public health issues related to the presence of harmful cyanobacteria.

Use of waste carbon dioxide and pre-treated liquid digestate from biogas process for Phaeodactylum tricornutum cultivation in photobioreactors and open ponds.

Phaeodactylum tricornutum is considered a promising source of polyunsaturated fatty acids (PUFAs), in particular eicosapentaenoic acid (EPA). In this study, P. tricornutum cultivation using waste products from anaerobic digestion (i.e. liquid digestate and CO2) was tested and scaled-up in closed and open prototype systems. The chemical composition of algal biomass was evaluated to optimize the lipid content. Algal productivity and composition, especially in terms of PUFAs, were not modified by the use of waste CO2. Digestate led to a lower protein (24%) content than medium (36-37%), without affecting lipid amount (about 37%). Algal and EPA productivity were nearly two-fold higher by using photobioreactors (0.075 g biomass L-1 day-1 and 1.62 mg EPA g-1 day-1) than open ponds, which are more influenced by environmental conditions. This study highlights that economic and environmental benefits could be achieved by using waste CO2 and liquid digestate from anaerobic digestion for microalgae cultivation.

Microbial dynamics during harmful dinoflagellate Ostreopsis cf. ovata growth: Bacterial succession and viral abundance pattern.

Algal-bacterial interactions play a major role in shaping diversity of algal associated bacterial communities. Temporal variation in bacterial phylogenetic composition reflects changes of these complex interactions which occur during the algal growth cycle as well as throughout the lifetime of algal blooms. Viruses are also known to cause shifts in bacterial community diversity which could affect algal bloom phases. This study investigated on changes of bacterial and viral abundances, bacterial physiological status, and on bacterial successional pattern associated with the harmful benthic dinoflagellate Ostreopsis cf. ovata in batch cultures over the algal growth cycle. Bacterial community phylogenetic structure was assessed by 16S rRNA gene ION torrent sequencing. A comparison between bacterial community retrieved in cultures and that one co-occurring in situ during the development of the O. cf. ovata bloom from where the algal strain was isolated was also reported. Bacterial community growth was characterized by a biphasic pattern with the highest contributions (~60%) of highly active bacteria found at the two bacterial exponential growth steps. An alphaproteobacterial consortium composed by the Rhodobacteraceae Dinoroseobacter (22.2%-35.4%) and Roseovarius (5.7%-18.3%), together with Oceanicaulis (14.2-40.3%), was strongly associated with O. cf. ovata over the algal growth. The Rhodobacteraceae members encompassed phylotypes with an assessed mutualistic-pathogenic bimodal behavior. Fabibacter (0.7%-25.2%), Labrenzia (5.6%-24.3%), and Dietzia (0.04%-1.7%) were relevant at the stationary phase. Overall, the successional pattern and the metabolic and functional traits of the bacterial community retrieved in culture mirror those ones underpinning O. cf. ovata bloom dynamics in field. Viral abundances increased synoptically with bacterial abundances during the first bacterial exponential growth step while being stationary during the second step. Microbial trends also suggest that viruses induced some shifts in bacterial community composition.

Inhibitory effect of polyunsaturated aldehydes (PUAs) on the growth of the toxic benthic dinoflagellate Ostreopsis cf. ovata.

Diatoms have been shown to produce and release a wide range of secondary metabolites that mediate interactions between individuals of different species. Among these compounds, different types of fatty acid derived long-chained polyunsaturated aldehydes (PUAs) have been related to multiple functions such as intra- or interspecific signals and adverse effect on the reproduction of marine organisms. Several studies have reported changes on growth, cell membrane permeability, flow cytometric properties and cell morphology in phytoplankton organisms exposed to PUAs, but little information is available on the effect of these compounds on benthic microalgae. Ostreopsis cf. ovata is a toxic benthic dinoflagellate which causes massive blooms along the Mediterranean coasts typically during the late summer period. In this study the effects of three toxic PUAs known to be produced by several algae (2E,4E-decadienal, 2E,4E-octadienal and 2E,4E-heptadienal) on the growth, cytological features and cell morphology of O. cf. ovata were investigated. Our results show a clear decrease of O. cf. ovata growth with longer-chain molecules than with shorter-chain ones, confirmed also by EC50 values calculated at 48h for 2E,4E-decadienal and 2E,4E-octadienal (6.6±1.5, 17.9±2.6µmolL(-1) respectively) and at 72h for 2E,4E-heptadienal (18.4±0.7µmolL(-1)). Moreover, morphological analysis highlighted up to 79% of abnormal forms of O. cf. ovata at the highest concentrations of 2E,4E-decadienal tested (9, 18 and 36µmolL(-1)), a gradual DNA degradation and an increase of lipid droplets with all tested PUAs. Further studies are needed to better clarify the interactions between diatoms and O. cf. ovata, especially on bloom-forming dynamics.

Effects of N and P availability on carbon allocation in the toxic dinoflagellate Ostreopsis cf. ovata.

Blooms of the toxic dinoflagellate Ostreopsis cf. ovata are usually associated with shallow and calm coastal waters, characterized by low nutrient concentrations. The algal cells typically cover the benthic substrates, such as the macroalgal and invertebrate communities and rocks, forming a mucilaginous film. Data reported on O. cf. ovata toxin production observed under both field and culture conditions show high variability in terms of toxic profile and cellular content; little is known about the environmental and physiological aspects which regulate the toxin dynamics. In this study, O. cf. ovata physiology was investigated using batch cultures supplied with nutrient concentrations similar to those found in the Adriatic Sea during the recurrent blooms and the observed cellular dynamics were compared with those found in a culture grown under optimal conditions, used as a reference. Data on the cellular C, N and P content during the growth highlighted a possible important role of the cellular nutritional status in regulating the toxin production that resulted to be promoted under specific intervals of the C:N and C:P ratios. The variable toxicity found for O. cf. ovata in various geographic areas could be related to the different in situ prevalent environmental conditions (e.g., nutrient concentrations) which affect the cellular elemental composition and carbon allocation. The obtained results strongly suggest that in the environment toxin production is steadily sustained by a low and constant nutrient supply, able to maintain appropriate cellular C:N (>12) or C:P (>170) ratios for a long period. These results explain to some extent the variability in toxicity and growth dynamics observed in blooms occurring in the different coastal areas.

Phylogenetic structure of bacterial assemblages co-occurring with Ostreopsis cf. ovata bloom.

Extensive blooms of the toxic epiphytic/benthic dinoflagellate Ostreopsis cf. ovata are being reported with increasing frequency and spatial distribution in temperate coastal regions including the Mediterranean. These blooms are of human and environmental health concern due to the production of isobaric palytoxin and a wide range of ovatoxins by Ostreopsis cf. ovata. Bacterial-microalgal interactions are important regulators in algal bloom dynamics and potentially toxin dynamics. This study investigated the bacterial assemblages co-occurring with O. cf. ovata (OA) and from ambient seawaters (SW) during the early and peak phases of bloom development in NW Adriatic Sea. Fractions of the bacterial assemblages co-occurring with O. cf. ovata (OA) and more closely associated to the mucilage layer (LA) embedding O. cf. ovata cells were also reported. In total, 14 bacterial phyla were detected by targeted 454 pyrosequencing of the 16S rRNA gene. The dominant bacterial phyla in the OA assemblages were Proteobacteria and Bacteroidetes; while at the class level, Alphaproteobacteria were the most abundant (83 and 66%, relative abundance, early and peak bloom phases), followed by Flavobacteria (7 and 19%, early and peak phases). Actinobacteria and Cyanobacteria were of minor importance (<5% of the relative bacterial abundance each). Gammaproteobacteria showed a notably presence in OA assemblage only at the early phase of the bloom (genus Haliea, 13%). The Alphaproteobacteria were predominately composed by the genera Ruegeria, Jannaschia and Erythrobacter which represented about half of the total phylotypes' contribution of OA at both early and peak phases of the O. cf. ovata bloom, suggesting interactions between this consortium and the microalga. Moreover, the highest contribution of Ruegeria (30% of the total phylotypes) was observed at the early phase of the bloom in LA assemblage. Microbial assemblages associated with the ambient seawaters while being also dominated by Alphaproteobacteria and Flavobacteria were partially distinct from those associated with O. cf. ovata due to the presence of genera almost not retrieved in the latter assemblages.

Modelling the Stoichiometric Regulation of C-Rich Toxins in Marine Dinoflagellates.

Toxin production in marine microalgae was previously shown to be tightly coupled with cellular stoichiometry. The highest values of cellular toxin are in fact mainly associated with a high carbon to nutrient cellular ratio. In particular, the cellular accumulation of C-rich toxins (i.e., with C:N > 6.6) can be stimulated by both N and P deficiency. Dinoflagellates are the main producers of C-rich toxins and may represent a serious threat for human health and the marine ecosystem. As such, the development of a numerical model able to predict how toxin production is stimulated by nutrient supply/deficiency is of primary utility for both scientific and management purposes. In this work we have developed a mechanistic model describing the stoichiometric regulation of C-rich toxins in marine dinoflagellates. To this purpose, a new formulation describing toxin production and fate was embedded in the European Regional Seas Ecosystem Model (ERSEM), here simplified to describe a monospecific batch culture. Toxin production was assumed to be composed by two distinct additive terms; the first is a constant fraction of algal production and is assumed to take place at any physiological conditions. The second term is assumed to be dependent on algal biomass and to be stimulated by internal nutrient deficiency. By using these assumptions, the model reproduced the concentrations and temporal evolution of toxins observed in cultures of Ostreopsis cf. ovata, a benthic/epiphytic dinoflagellate producing C-rich toxins named ovatoxins. The analysis of simulations and their comparison with experimental data provided a conceptual model linking toxin production and nutritional status in this species. The model was also qualitatively validated by using independent literature data, and the results indicate that our formulation can be also used to simulate toxin dynamics in other dinoflagellates. Our model represents an important step towards the simulation and prediction of marine algal toxicity.

Toxin levels and profiles in microalgae from the north-Western Adriatic Sea--15 years of studies on cultured species.

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.

Isolation and structure elucidation of ovatoxin-a, the major toxin produced by Ostreopsis ovata.

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.

Phytoplankton size-distribution and community structure in relation to mucilage occurrence in the northern Adriatic Sea.

The spatial and temporal variations of pico-, nano- and microphytoplankton abundance and composition were investigated over a 37 month period, focusing on the ecological role of different size classes of phytoplankton, and on the changes of the community structure that might occur during periods when large mucilage macroaggregates appear. Samples were collected monthly from June 1999 to July 2002 at 11 stations, along three transects covering the northern Adriatic basin. Highest abundances were observed in late-winter/spring for microphytoplankton (mainly diatoms), in spring-summer for nanophytoplankton, and in summer for picophytoplankton. The autotrophic component was more abundant in the summers of 2000 and 2002 (when large mucilage aggregates occurred) than in the summers of 1999 and 2001 (when a massive phenomenon was not observed). This increase was statistically significant for pico-, nano- and, among microphytoplankton, only for dinoflagellates. Blooms of picophytoplankton were often observed at the bottom layer during mucilage summers. The microphytoplankton community during mucilage phenomena was characterized by a species composition (Chaetoceros spp., Cerataulina pelagica, Pseudo-nitzschia delicatissima, P. pseudodelicatissima, Cylindrotheca closterium, Dactyliosolen fragilissimus) comparable to that observed in summers without extensive mucilage occurrence. However, some species appeared with significantly higher densities in the summers of 2000 and 2002: Ceratium furca, C. closterium, Oxytoxum spp., Hemiaulus hauckii and Gonyaulax fragilis. Microscopic observation of aggregates revealed that the microphytoplankton species composition inside the aggregates was comparable to that observed in the water column, with an enrichment of opportunistic species such as C. closterium and P. delicatissima. The presence of mucilage aggregates affects the phytoplankton populations in the water column, even when aggregates are at early stages. It seems that there is a mutual relationship between phytoplankton and aggregates, i.e., several diatom and dinoflagellate species may contribute to the aggregate formation and enlargement, but mucilage aggregates themselves may also affect the phytoplankton populations, allowing the development of a rich diatom community and in general enhancing nanophytoplankton growth.

Putative midkine family protein up-regulation in Patella caerulea (Mollusca, Gastropoda) exposed to sublethal concentrations of cadmium.

A cDNA sequence of a putative midkine (MK) family protein was identified and characterised in the mollusc Patella caerulea. The midkine family consists of two members, midkine and pleiotrophin (PTN), and it is one of the recently discovered cytokines. Our results show that this putative midkine protein is up-regulated in specimens of P. caerulea exposed to sublethal cadmium concentrations (i.e. 0.5 and 1 mg l(-1) Cd) over a 10-day exposure period. Semiquantitative RT-PCR and quantitative Real time RT-PCR estimations indicate elevated expression of midkine mRNA in exposed specimens compared to controls. Moreover, RT-PCR Real time values were higher in the viscera (here defined as the part of the soft tissue including digestive gland plus gills) than in the foot (i.e. foot plus head plus heart) of the limpets. At present, information on the functional signalling significance of the midkine family proteins suggests that the up-regulation of P. caerulea putative midkine family protein is a distress signal likely with informative value on health status of the organism and with potential prognostic capability.