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In the Mediterranean Sea coralline red algae play a particularly important role, as their calcified thalli provide hard substratum in several benthic biocenoses covering large portions of rocky bottom (i.e. coralligenous concretions, rims of Lithophyllum byssoides, rims of articulated corallines). Since its opening 250 Myr ago, the Mediterranean has undergone a complex oceanographic history, due to geological and climatic events such as the Atlantic opening, the isolation from the Indian Ocean, and the Messinian Salinity Crisis, which determined phases of connection and separation from the adjacent oceans. These events profoundly affected the evolutionary history of Mediterranean marine organisms, fueling a high biological diversity. Paleontological evidence indicates that corallines have been major bioconstructors in the Mediterranean for a long time and that some common species have occurred in the basin for at least 10-11 Myr (Lithophyllum dentatum, L. incrustans, L. pustulatum). The limited amount of molecular data available has hindered our understanding of the evolution and diversity of this group, but recent studies provide major insights in these topics. The recent discovery of Pneophyllum cetinaensis shows that the Mediterranean is the only geographical region in which coralline algae have successfully crossed the border between sea and freshwater, a transition that probably took place during the last glaciation. Lithophyllum byssoides, one of the few genuinely intertidal Mediterranean seaweeds, has a high haplotypic diversity and Mediterranean populations probably represent different species from Atlantic populations, a pattern observed for many animal species. Similarly sharp haplotypic differentiation between Mediterranean and Atlantic populations has been documented for species of Mesophyllum and Corallina. Lithophyllum cabiochiae, the main bioconstructor of coralligenous concretions, represents a highly diverse complex of species, rather than a single species. These studies suggest that Mediterranean corallines represent a more evolutionarily complex group than believed so far, worthy of further detailed investigation.

In the Mediterranean biogenic habitat built by coralline algae (orders Corallinales, Hapalidiales and Sporolithales) show a great diversity and a large bathymetric extent. The genus Lithophyllum is the most species-diverse coralline genus. and plays a key role in the formation of extensive bioconstructions, as the coralligenous concretions in the circalittoral and the “trottoirs” in the mid littoral. As part of a large-scale investigation of the marine bioconstructions of the Italian shores, we account here some results on the distribution, diversity and ecology for L. byssoides, L. cabiochae and L. stictaeforme.

International audience; Lithophyllum species in the Mediterranean Sea function as algal bioconstructors, contributing to the formation of biogenic habitats such as coralligenous concretions. In such habitats, thalli of Lithophyllum, consisting of crusts or lamellae with entire or lobed margins, have been variously referred to as either one species, L. stictiforme, or two species, L. stictiforme and L. cabiochiae, in the recent literature. We investigated species diversity and phylogenetic relationships in these algae by sequencing three markers (psbA and rbcL genes, cox2,3 spacer), in conjunction with methods for algorithmic delimitation of species (ABGD and GMYC). Mediterranean subtidal Lithophyllum belong to a well‐supported lineage, hereby called the L. stictiforme complex, which also includes two species described from the Atlantic, L. lobatum and L. searlesii. Our results indicate that the L. stictiforme complex consists of at least 13 species. Among the Mediterranean species, some are widely distributed and span most of the western and central Mediterranean, whereas others appear to be restricted to specific localities. These patterns are interpreted as possibly resulting from allopatric speciation events that took place during the Messinian Salinity Crisis and subsequent glacial periods. A partial rbcL sequence from the lectotype of L. stictiforme unambiguously indicates that this name applies to the most common subtidal Lithophyllum in the central Mediterranean. We agree with recent treatments that considered L. cabiochiae and L. stictiforme conspecific. The diversity of Lithophyllum in Mediterranean coralligenous habitats has been substantially underestimated, and future work on these and other Mediterranean corallines should use identifications based on DNA sequences.

Astaxanthin is a red orange xanthophyll carotenoid produced mainly by microalgae but which can also be chemically synthesized. As demonstrated by several studies, this lipophilic molecule is endowed with potent antioxidant properties and is able to modulate biological functions. Unlike synthetic astaxanthin, natural astaxanthin (NAst) is considered safe for human nutrition, and its production is considered eco-friendly. The antioxidant activity of astaxanthin depends on its bioavailability, which, in turn, is related to its hydrophobicity. In this study, we analyzed the water-solubility of NAst and assessed its protective effect against oxidative stress by means of different approaches using a neuroblastoma cell model. Moreover, due to its highly lipophilic nature, astaxanthin is particularly protective against lipid peroxidation; therefore, the role of NAst in counteracting ferroptosis was investigated. This recently discovered process of programmed cell death is indeed characterized by iron-dependent lipid peroxidation and seems to be linked to the onset and development of oxidative-stress-related diseases. The promising results of this study, together with the “green sources” from which astaxanthin could derive, suggest a potential role for NAst in the prevention and co-treatment of chronic degenerative diseases by means of a sustainable approach.

Macroalgae produce several allelopathic substances, including polyunsaturated aldehydes (PUAs), which may inhibit photosynthesis and growth rates of other algal species, and grazing. Additionally, macroalgal structural complexity is an important factor in determining abundance patterns and size structure of epiphytic organisms. In this study the PUAs production of two Mediterranean macroalgae, Dictyopteris polypodioides, (DP, Phaeophyceae, Dictyotales) and Cystoseira compressa (CC, Phaeophyceae, Fucales), was characterized to clarify the relationships between the meiobenthic and microphytobenthic communities. Results showed a higher PUAs production and a diverse qualitative profile for DP, which reported long-chain compounds (i.e. C14-C16) as main aldehydes, than CC, with the short-chain C6:2 as the main compound, as well as variability among sampling times. A clear separation of the meiofauna and microphytobenthos assemblages was found for the macroalgae, but with different temporal trends. Dissimilarities were due to five microalgal orders, namely Naviculales, Lyrellales, Gonyaulacales (i.e. Ostreopsis), Bacillariales, and Licmophorales, and to the meiofaunal groups nematodes, copepods, and copepod nauplii, which were more abundant on DP than on CC. Results indicate that macroalgal complexity is a major determinant of the meiofaunal community structure (accounting for 26% of the variation), rather than PUAs production itself (17%). PUAs effects seem species-specific, thus affecting some grazers instead of the entire community. Conversely, microphytobenthos affected the meiofauna assemblages, particularly harpacticoids, confirming the role of these organisms as the primary food source of all marine food chain producers. Since PUAs are produced also by several epiphytic diatoms, the understanding of their effects on the community structure and on the relationships among taxa in the field is complicated and requires further in-depth investigations in simplified systems (i.e. microcosms).

Three novel hydrophobic deep eutectic solvents (DESs) based on oleic acid and terpenes (thymol, DL-menthol, and geraniol) were prepared, characterized, and used to extract astaxanthin from the microalga Haematococcus pluvialis without any pre-treatment of the cells. The three DES were composed of Generally Recognized As Safe (GRAS) and edible ingredients. All the tested DESs gave astaxanthin recovery values of about 60 and 30% in 6 h if applied on freeze-dried biomass or directly on algae culture, respectively. The carotenoid profile was qualitatively identical to what was obtained by using traditional organic solvents, regardless of the DES used; the monoesters of astaxanthin with C18-fatty acids were the main compounds found in all the carotenoid extracts. The thymol:oleic acid DES (TAO) could preserve astaxanthin content after prolonged oxidative stress (40% of the astaxanthin initially extracted was still present after 13.5 h of light exposure), thanks to the superior antioxidant properties of thymol. The capacity of improving astaxanthin stability combined with the intrinsic safety and edibility of the DES components makes the formulation astaxanthin-TAO appealing for the food ingredients/additives industry.

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.

We thank the University of Bologna (RFO program) for funding. The microalga Haematococcus pluvialis is capable of accumulating natural astaxanthin when subjected to external stress and shifted towards a red-cyst phase, characterized by a robust and multilayered cell wall. In the present study, still-unexplored solvents were applied for extracting astaxanthin from H. pluvialis, directly from algae culture and without any pre-treatment of the cells. Among the tested solvents, some of them (e.g. ethyl acetate and 2-methyltehydrofuran) gave excellent astaxanthin recovery (>80%) in a short time (30 min) and others (e.g. isoamyl acetate, well known as a human-compatible solvent and already in use as a food additive) gave an astaxanthin recovery close to 90% in 1 hour and thus they are exploitable in the natural astaxanthin market. Almond oil is proved to be able to extract astaxanthin and keep H. pluvialis alive, without affecting the algal photosynthetic activity, providing the possibility to milk and regeneratively cultivate H. pluvialis and avoid an uneconomical loss of biomass.

The feasibility of the green alga Desmodesmus communis for biomass production was investigated, firstly testing different nitrogen forms in the growth medium and the effect of CO2-enriched air supply, secondarily scaling up the cultivation system in 70 L photobioreactors (PBRs). Maximum nitrogen uptake rate obtained in the performed kinetic experiment was higher for ammonium than for nitrate (188.0 vs 11.7 μmol g−1 h−1); however, D. communis cultured in PBRs with only aeration grew faster with nitrate reaching a biomass yield (1.23 g L−1) and a productivity (0.036 g L−1 day−1) about twofold higher than with ammonium, which caused a pH decrease in the medium affecting the algal growth. CO2 supply allowed algal growth optimization, maintaining a high productivity with both nitrogen sources, slightly higher with nitrate (0.050 vs 0.038 g L−1 day−1). Additionally, nitrate-supplied cells showed higher lipids (19.0 vs 9.4%) and proteins (33.0 vs 27.2%) values than those grown with ammonium. The semi-continuous scaled-up cultivation performed for 5 months attests the potential utilization of this species for valuable algal biomass production exploitable in various industrial applications.

Plastid-encoded rbcL and psbA sequences from branched, Caribbean Sea Lithophyllum specimens indicate that four species are present, not one. Short (263 base pairs) rbcL sequences from an isolectotype of L. kaiseri (Gulf of Suez) and the holotypes of L. congestum, L. daedaleum and L. platyphyllum (Caribbean Sea) show that L. congestum and L. daedaleum are conspecific with L. kaiseri, the last having nomenclatural priority. Lithophyllum platyphyllum, currently considered a synonym of L. congestum, is recognised as a valid species. Lithophyllum stictaeforme, originally described from the Mediterranean Sea, is not conspecific with L. kaiseri (as L. congestum) as previously suggested. Lithophyllum neocongestum sp. nov. and L. pseudoplatyphyllum sp. nov. are proposed. Together with L. platyphyllum, these three branched species are so far endemic to the Caribbean Sea. This is the first report, documented by DNA sequence data, of a coralline species (L. kaiseri) widespread through the tropical Indo-West Pacific Oceans, Red Sea and Caribbean Sea.

Flocculation induced by pH decrease is here presented as a solution to pre-concentrate algal cultures of species belonging to Scenedesmaceae and preserve the integrity of the biomass, while maintaining the photosynthetic capacity of non-harvested cells. The low-pH flocculation method gives highly effective results exclusively by the strength of the acid (hydrochloric or formic) used for the treatment, and is applicable to cultures having high cell densities (> 2 g L− 1). High flocculation efficiencies (> 90%) are obtained also by using heterogeneous acids, such as solid acid carbons from naturally available compounds, which are proposed as cheap recyclable materials to be exploited for this purpose. Additionally, the potential reuse of the filtered medium (sterilized or not) and of the supernatant (containing non-harvested cells) is investigated to avoid great water losses and to re-inoculate new cultures.

In the last decade Ostreopsis cf. ovata blooms have been among the most intense along the entire Mediterranean coast, leading to ecological and human health problems, that are associated with the toxins (palytoxin-like compounds) produced by these algal cells. These compounds are secondary metabolites, whose rates of synthesis depend on the metabolism of their precursors. In general, growth dynamics and toxicity of dinoflagellates reflect the physiological status of the organism. The aim of the present study was to investigate the cellular production of the main biochemical compounds likely involved in the growth and toxicity dynamics of O. cf. ovata during exponential to the late stationary phase in batch cultures of an Adriatic strain. Removal of major nutrients from the medium was monitored along with concentration, biovolume and production of the main cellular components (e.g. polysaccharides, proteins, lipids and toxins). Nutrient uptake, as well as toxin production rates were calculated in the different growth periods. Nutrients (N and P) were completely depleted when cells entered stationary phase and the greatest net toxin production rate (RTOX) occurred during the first days of growth. The various palytoxins reported a relative abundance quite stable during the different growth phases, while the total toxin cellular amount increased along the growth curve. Total and extracellular released polysaccharides, as well as the lipid content increased greatly during the stationary phase, while proteins were mainly produced by cells during the exponential phase. The continuous release of polysaccharides could facilitate cell aggregation and the formation of the benthic community during algal blooms. The trend of production of the main cellular compounds in O. cf. ovata and the growth dynamics of this species lead us to hypothesize that the fast growth of this dinoflagellate, associated with the rapid use of environmental resources (nutrients, and phosphates in particular), may be an ecological/ adaptive strategy which could favor this organism in competition with other species.

Dinoflagellates are prolific producers of polyketide secondary metabolites. Dinoflagellate polyketide synthases (PKSs) have sequence similarity to Type I PKSs, megasynthases that encode all catalytic domains on a single polypeptide. However, in dinoflagellate PKSs identified to date, each catalytic domain resides on a separate transcript, suggesting multiprotein complexes similar to Type II PKSs. Here, we provide evidence through coimmunoprecipitation that single-domain ketosynthase and ketoreductase proteins interact, suggesting a predicted multiprotein complex. In Karenia brevis (C.C. Davis) Gert Hansen & O. Moestrup, previously observed chloroplast localization of PKSs suggested that brevetoxin biosynthesis may take place in the chloroplast. Here, we report that PKSs are present in both cytosol and chloroplast. Furthermore, brevetoxin is not present in isolated chloroplasts, raising the question of what chloroplast-localized PKS enzymes might be doing. Antibodies to K. brevis PKSs recognize cytosolic and chloroplast proteins in Ostreopsis cf. ovata Fukuyo, and Coolia monotis Meunier, which produce different suites of polyketide toxins, suggesting that these PKSs may share common pathways. Since PKSs are closely related to fatty acid synthases (FAS), we sought to determine if fatty acid biosynthesis colocalizes with either chloroplast or cytosolic PKSs. [(3) H]acetate labeling showed fatty acids are synthesized in the cytosol, with little incorporation in chloroplasts, consistent with a Type I FAS system. However, although 29 sequences in a K. brevis expressed sequence tag database have similarity (BLASTx e-value

Lithophyllum byssoides is a comm on coralline alga in the intertidal zone of Mediterranean coasts, where it produces biogenic concretions housing a high algal and invertebrate biodiversity. This species is an ecosystem engineer and is considered a target for conservation efforts, but designing effective conservation strategies currently is impossible due to lack of information abo ut its population structure. The morphological and molecular variation of L. byssoides was investigated using morphoanatomy and DNA sequences (psbA and cox2,3) obtained from populations at 15 localities on the Italian and Croatian coasts. Lithophyllum byssoides exhibited a high number of haplotypes (31 psbA haplotypes and 24 cox2,3 haplotypes) in the central Mediterranean. The psbA and cox2,3 phylogenies were congruent and showed seven lineages. For most of these clades, the distribution was limited to one or a few localities, but one of them (clade 7) was widespread across the central Mediterranean, spanning the main biogeographic boundaries recognized in this area. The central Mediterranean populations formed a lineage separate from Atlantic samples; psbA pairwise divergences suggested that recognition of Atlantic and Mediterranean L. byssoides as different species may be appropriate. The central Mediterranean haplotype patterns of L. byssoides were interpreted as resulting from past climatic events in the hydrogeological history of the Mediterranean Sea. The high haplotype diversity and the restricted spatial distribution of the seven lineages suggest that individual populations should be managed as independent units.

An increase in the frequency of extensive blooms of the toxic benthic dinoflagellate Ostreopsis cf. ovata has been reported in temperate coastal regions including the Mediterranean. Dimethylsulfoniopropionate (DMSP) can mediate a mutualist interaction between dinoflagellates and their associated bacteria that could have an effect on the development of blooms. In this study the production of DMSP by O. cf. ovata is reported for the first time.

This article describes the toxic effects of imidazolium ionic liquids bearing alkyl (BMIM), monoethoxy (MOEMIM), and diethoxy (M(OE)(2)MIM) side chains toward two marine diatoms, Skeletonema marinoi and Phaeodactylum tricornutum. MOEMIM and M(OE)(2)MIM cations showed a lower inhibition of growth and photosynthetic efficiency with respect to their alkyl counter part, with both algal Species. However, a large difference in sensitivity was found between S. marinoi and P. tricornutum, the first being much more Sensitive to the action of ionic liquids than the second one. The effects of salinity on BMIM Cl toxicity toward S. marinoi revealed that a decrease from salinity 35 to salinity 15 does not influence the biological effects toward the alga. Finally, Fourier transform infrared (FT-IR) microscopy of alga cells after ionic liquids exposure allowed us to detect an alteration of the organic cellular components related. to silica uptake and organization. On the basis of these results, the different behavior of the two diatom species can be tentatively ascribed to different silica uptake and organization in outer cell walls

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.

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. ovate 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 0. 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. (C) 2016 Elsevier B.V. All rights reserved.

Fioriture di Ostreopsis spp. sono state osservate nell'ultimo ventennio nel Mediterraneo e la loro diffusione aumenta nel mondo ogni anno, rappresentando una potenziale minaccia per la salute umana e per l'ecosistema marino (Rhodes et al., 2011). In particolare la specie O. cf. ovata negli ultimi anni è presente in varie aree costiere europee, tra cui l'Italia. Nonostante in queste zone non siano stati registrati casi di intossicazione umana per consumo di prodotti ittici, la problematica è di grande interesse a seguito dell'elevata tossicità della palitossina e dei congeneri, denominati ovatossine, prodotti da questa dinoflagellata. Le fioriture hanno spesso mostrato differenti caratteristiche in termini di periodo di sviluppo e relative condizioni abiotiche, di densità algale e di tossicità (Cohu et al., 2013). Allo scopo di individuare i fattori abiotici (e.g. temperatura, salinità, luce) e biotici (e.g. presenza di specie algali competitrici o batteri) che maggiormente influenzano le fioriture ed il loro livello di tossicità, sono stati effettuati studi in coltura utilizzando ceppi di O. cf. ovata isolati da coste italiane, saggiando differenti condizioni. In primo luogo è stato evidenziato che le diverse ovatossine analizzate negli estratti di O. cf. ovata aumentano quantitativamente lungo la crescita (Pezzolesi et al., 2014) con un rilascio nel mezzo extracellulare maggiore in fase stazionaria. Inoltre il profilo tossinologico ha evidenziato un'alta variabilità sia in termini qualitativi (i.e. assenza di alcune ovatossine) che quantitativi (i.e. contenuto di tossina per cellula o litro di coltura) in funzione dei differenti ceppi esaminati. In generale è emerso che temperatura, salinità e concentrazione dei nutrienti sono fattori abiotici che influiscono nella produzione cellulare di tossine (Pistocchi et al., 2011; Pezzolesi et al., 2012; Vanucci et al., 2012b). Nello specifico la disponibilità di nutrienti è importante per la biosintesi di questi composti che contengono azoto e hanno alto peso molecolare. I risultati hanno evidenziato che in carenza di azoto (N), in particolare, e di fosforo (P) i quantitativi di tossine prodotte sono inferiori. Tra i fattori che maggiormente influenzano la crescita e la tossicità di O. cf. ovata è emersa anche la competizione con altre microalghe (e.g. diatomee o dinoflagellate bentoniche), con effetti sulla crescita; sembrano inoltre essere esclusi possibili effetti allelopatici delle ovatossine verso le altre microalghe indagate. La comunità batterica associata ad O. cf. ovata in coltura sembra anch'essa interferire sulla produzione di tossine tramite re-mineralizzazione di sostanza organica, influenzando i quantitativi di N e P disponibile, e tramite interazioni di tipo mutualistico (Vanucci et al., 2012a). I risultati ottenuti evidenziano che da un lato lo stato fisiologico delle cellule è strettamente legato alle condizioni chimicofisiche e allo stato trofico del sistema e dall'altro questo si riflette sulla produzione e sul rilascio delle tossine. Cohu S., Mangialajo L., Thibaut T., Blanfuné A., Marro S., Lemée R., 2013. Harmful Algae 24, 32-44. Pezzolesi L., Guerrini F., Ciminiello P., Dell'Aversano C., Dello Iacovo E., Fattorusso E., Forino M., Tartaglione L., Pistocchi R., 2012. Water Research 46, 82-92. Pezzolesi L., Pistocchi R., Fratangeli F., Dell'Aversano C., Dello Iacovo E., Tartaglione L., 2014. Harmful Algae 36, 1-10. Pistocchi R., Pezzolesi L., Guerrini F., Vanucci S., Dell'Aversano C., Fattorusso E., 2011. Toxicon 57, 421-428. Rhodes L., 2011. Toxicon 57 (3), 400–407. Vanucci S., Guerrini F., Pezzolesi L., Dell'Aversano C., Ciminiello P., Pistocchi R., 2012a. Cryptogamie, Algologie 33, 105-112. Vanucci S., Pezzolesi L., Pistocchi R., Ciminiello P., Dell'Aversano C., Dello Iacovo E., Fattorusso E., Tartaglione L., Guerrini F., 2012b. Harmful Algae 15, 78-90.

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.

During the last decades, massive blooms of the toxic benthic dinoflagellate Ostreopsis cf. ovata have occurred in different coastal areas of the Mediterranean Sea typically during the late summer period. O. cf. ovata blooms have been related to human intoxication and to mass mortalities of benthic invertebrates due to the production of palytoxin-like compounds, including isobaric palytoxin, ovatoxins and mascarenotoxins. In the last decades, several microalgae have been shown to release a wide range of secondary metabolites that mediate interactions between individuals of different species. A variety of diatoms genera produce and release different types of fatty acid derived long-chained polyunsaturated aldehydes (PUAs). These compounds have been related to multiple functions such as adverse effect on the reproduction of various invertebrates, intra- or interspecific signals, and have effects on a variety of marine organims as well, including co-occurring microalgae. Moreover, several studies have highlighted changes on growth, cell membrane permeability, flow cytometric properties and cell morphology due the exposition of microalgae to PUAs. The relationships between O. cf. ovata and co-occurring microalgae has been previously investigated (Pichierri et al., 2014), showing that some benthic diatoms exherted a negative effect on the growth rate of O. cf. ovata. The aim of this study was to investigate the effects of three toxic algal-derived PUAs, namely the 2E,4E-decadienal, 2E,4E-octadienal and the 2E,4E heptadienal, on the growth and cell morphology of O. cf. ovata. O. cf. ovata was cultured under controlled laboratory conditions at different PUAs concentrations (from 0.1 to 36 µmol l−1) and using two different exposition times (24 and 72 h). Our results showed a clear decrease of O. cf. ovata growth when cultures were exposed to the various PUAs compared with the control. After 72 h of exposition at the maximum concentration tested, the growth decrease respect to the control was 92.4%, 81.5% and 48.6% for 2E,4E-decadienal, 2E,4E-octadienal and 2E,4E heptadienal, respectively. Although a concentration-dependent decrease of the growth rate was observed for all three aldehyde compounds, the effect was stronger with longer-chained molecules than with shorter-chained ones. Moreover, at the highest concentrations of 2E,4E-decadienal (from 6 to 36 µmol l−1), aberrant forms of O. cf. ovata cells were observed, showing a marked decrease of cell dimensions, a contraction of cytoplasm and the formation of abnormal vesicle-like structures. These data pointed out for the first time the negative effect of PUAs, which are compounds produced by some diatoms species, on the growth and morphology of O. cf ovata cells and confirm the possible allelopathic role of these molecules in the relationships among phytoplankton species. Further studies are needed to better clarify the interactions between diatoms and O. cf. ovata, especially on bloom-forming dynamics.

Fioriture di Ostreopsis spp. sono state osservate nell'ultimo ventennio nel Mediterraneo e la loro diffusione aumenta nel mondo ogni anno, rappresentando una potenziale minaccia per la salute umana e per l'ecosistema marino (Rhodes et al., 2011). In particolare la specie O. cf. ovata negli ultimi anni è presente in varie aree costiere europee, tra cui l'Italia. Nonostante in queste zone non siano stati registrati casi di intossicazione umana per consumo di prodotti ittici, la problematica è di grande interesse a seguito dell'elevata tossicità della palitossina e dei congeneri, denominati ovatossine, prodotti da questa dinoflagellata. Le fioriture hanno spesso mostrato differenti caratteristiche in termini di periodo di sviluppo e relative condizioni abiotiche, di densità algale e di tossicità (Cohu et al., 2013). Allo scopo di individuare i fattori abiotici (e.g. temperatura, salinità, luce) e biotici (e.g. presenza di specie algali competitrici o batteri) che maggiormente influenzano le fioriture ed il loro livello di tossicità, sono stati effettuati studi in coltura utilizzando ceppi di O. cf. ovata isolati da coste italiane, saggiando differenti condizioni. In primo luogo è stato evidenziato che le diverse ovatossine analizzate negli estratti di O. cf. ovata aumentano quantitativamente lungo la crescita (Pezzolesi et al., 2014) con un rilascio nel mezzo extracellulare maggiore in fase stazionaria. Inoltre il profilo tossinologico ha evidenziato un'alta variabilità sia in termini qualitativi (i.e. assenza di alcune ovatossine) che quantitativi (i.e. contenuto di tossina per cellula o litro di coltura) in funzione dei differenti ceppi esaminati. In generale è emerso che temperatura, salinità e concentrazione dei nutrienti sono fattori abiotici che influiscono nella produzione cellulare di tossine (Pistocchi et al., 2011; Pezzolesi et al., 2012; Vanucci et al., 2012b). Nello specifico la disponibilità di nutrienti è importante per la biosintesi di questi composti che contengono azoto e hanno alto peso molecolare. I risultati hanno evidenziato che in carenza di azoto (N), in particolare, e di fosforo (P) i quantitativi di tossine prodotte sono inferiori. Tra i fattori che maggiormente influenzano la crescita e la tossicità di O. cf. ovata è emersa anche la competizione con altre microalghe (e.g. diatomee o dinoflagellate bentoniche), con effetti sulla crescita; sembrano inoltre essere esclusi possibili effetti allelopatici delle ovatossine verso le altre microalghe indagate. La comunità batterica associata ad O. cf. ovata in coltura sembra anch'essa interferire sulla produzione di tossine tramite re-mineralizzazione di sostanza organica, influenzando i quantitativi di N e P disponibile, e tramite interazioni di tipo mutualistico (Vanucci et al., 2012a). I risultati ottenuti evidenziano che da un lato lo stato fisiologico delle cellule è strettamente legato alle condizioni chimicofisiche e allo stato trofico del sistema e dall'altro questo si riflette sulla produzione e sul rilascio delle tossine. Cohu S., Mangialajo L., Thibaut T., Blanfuné A., Marro S., Lemée R., 2013. Harmful Algae 24, 32-44. Pezzolesi L., Guerrini F., Ciminiello P., Dell'Aversano C., Dello Iacovo E., Fattorusso E., Forino M., Tartaglione L., Pistocchi R., 2012. Water Research 46, 82-92. Pezzolesi L., Pistocchi R., Fratangeli F., Dell'Aversano C., Dello Iacovo E., Tartaglione L., 2014. Harmful Algae 36, 1-10. Pistocchi R., Pezzolesi L., Guerrini F., Vanucci S., Dell'Aversano C., Fattorusso E., 2011. Toxicon 57, 421-428. Rhodes L., 2011. Toxicon 57 (3), 400–407. Vanucci S., Guerrini F., Pezzolesi L., Dell'Aversano C., Ciminiello P., Pistocchi R., 2012a. Cryptogamie, Algologie 33, 105-112. Vanucci S., Pezzolesi L., Pistocchi R., Ciminiello P., Dell'Aversano C., Dello Iacovo E., Fattorusso E., Tartaglione L., Guerrini F., 2012b. Harmful Algae 15, 78.

Although nutrient implication in bloom dynamics has not yet been clarified, in Ostreopsis cf. ovata cultures the importance of nutrient concentrations (N and P) for cell growth and toxicity has been remarked. Nutrient uptake pattern in cultured strains also evidenced the need for balanced N/P ratios for optimal growth of this species; moreover it appears that the intracellular nutrient ratios tend towards balanced conditions independently by concentrations (N/P ratio) in the external medium. The present study was therefore performed in order to get better insights into nutrient utilization by O. cf. ovata. The characterization of the nutrient uptake kinetics showed that this species has higher Vmax and higher affinity values for P uptake than for N. In addition, a study performed with cultures grown at different N/P ratios, showed that alkaline phosphatase activity was induced when external P was depleted or present in low amounts. These findings attest to the high efficiency of O. cf. ovata in both inorganic and organic P acquisition, an aspect that could confer advantages towards competing species.

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.

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.

In the Mediterranean Sea, blooms of Ostreopsis cf. ovata and Ostreopsis siamensis have become increasingly frequent in the last decade and O. cf. ovata was found to produce palytoxin-like compounds (putative palytoxin, ovatoxin-a, -b, -c, -d and -e), a class of highly potent toxins. The environmental conditions seem to play a key role in influencing the abundance of Ostreopsis spp. High cell densities are generally recorded in concomitance with relatively high temperature and salinity and low hydrodynamics conditions. In this study the effects of temperature and salinity on the growth and toxicity of an Adriatic O. cf. ovata isolate were investigated. The highest growth rates of the Adriatic strain were recorded for cultures grown at 20 °C and at salinity values of 36 and 40, in accordance with natural bloom surveys. Toxicity was affected by growth conditions, with the highest toxin content on a per cell basis being measured at 25 °C and salinity 32. However, the highest total toxin content on a per litre basis was recorded at 20 °C and salinity 36, since under such conditions the growth yield was the highest. O. cf. ovata had lethal effects on Artemia nauplii and juvenile sea basses, and produced haemolysis of sheep erythrocytes. A comparison between haemolysis neutralization assay and HR LC-MS results showed a good correlation between haemolytic effect and total toxin content measured through HR LC-MS. Considering the increasing need for rapid and sensitive methods to detect palytoxin in natural samples, the haemolytic assay appears a useful method for preliminary quantification of the whole of palytoxin-like compounds in algal extracts.

High resolution (HR) LC-MS investigation of an Adriatic Ostreopsis ovata culture is reported herein. It highlighted the presence of putative palytoxin and ovatoxin-a in combination with four new ovatoxins. Elemental formulae and information about their structural features were gained.