Natural Astaxanthin Is a Green Antioxidant Able to Counteract Lipid Peroxidation and Ferroptotic Cell Death.

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.

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.

Extraction of astaxanthin from Haematococcus pluvialis with hydrophobic deep eutectic solvents based on oleic acid.

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.

Allelopathic interactions between phytobenthos and meiofaunal community in an Adriatic benthic ecosystem: Understanding the role of aldehydes and macroalgal structural complexity.

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).

Production of polyhydroxybutyrate by the cyanobacterium cf. Anabaena sp.

Polyhydroxybutyrate (PHB) production by the cyanobacterium cf. Anabaena sp. was here studied by varying the medium composition and the carbon source used to induce mixotrophic growth conditions. The highest PHB productivity (0.06 gPHB gbiomass-1 d-1) was observed when cultivating cf. Anabaena sp. in phosphorus-free medium and in the presence of sodium acetate (5.0 g L-1 concentration), after an incubation period of 7 days. A content of 40% of PHB on biomass, a dry weight of 0.1 g L-1, and a photosynthetic efficiency equal to the control were obtained. The cyanobacterium was then grown on a larger scale (10 L) to evaluate the characteristics of the produced PHB in relation to the main composition of the biomass (the content of proteins, polysaccharides, and lipids): after an incubation period of 7 days, a content of 6% of lipids (52% of which as unsaturated fatty acids with 18 carbon atoms), 12% of polysaccharides, 28% of proteins, and 46% of PHB was reached. The extracted PHB had a molecular weight of 3 MDa and a PDI of 1.7. These promising results demonstrated that cf. Anabaena sp. can be included among the Cyanobacteria species able to produce polyhydroxyalkanoates (PHAs) either in photoautotrophic or mixotrophic conditions, especially when it is grown under phosphorus-free conditions.

Survey of the allelopathic potential of Mediterranean macroalgae: production of long-chain polyunsaturated aldehydes (PUAs).

Chemical interactions between macroalgae and other organisms play an important role in determining species compositions and dominance patterns, and can explain the widespread success of some species in establishing their predominant populations in a specific coastal area. Allelopathy could act as a self-regulatory strategy of the algal community, being not only a succession regulator but also an active mechanism maintaining the species diversity especially in a delimited environment, such as the benthic ecosystem. Polyunsaturated aldehydes (PUAs) are among the most studied allelopathic compounds and are commonly released into the aquatic environment by different phytoplankton species in response to environmental stressors (e.g. wounding, grazing, or competition for nutrients). Diatom-released PUAs were observed to affect phytoplankton community dynamics and structure, and showed inhibitory effects on the reproduction and development of marine invertebrates. As for macroalgae, there are only a few reports that attest to the production of PUAs, and mostly refer to Ulva spp. In this study, the production of PUAs by several Mediterranean macroalgae was investigated at different sampling times, aiming at providing the first evidence of potential allelochemical activity. Results highlighted the potential production by macroalgae of a variety of aldehydes, among which some have never reported so far. Some species (i.e. D. polypodioides and U. cf. rigida) were found to produce higher PUAs amounts than others, and even a wider variety of structures (e.g. length of the carbon chain); these species might exert strong effects on epiphytic species or other organisms of the benthic community, especially considering the differential sensitivities of the various taxa. A high dPUA concentration (order of µM) potentially due to the release of PUAs by algal species was found, and might affect the population dynamics of the epiphytic organisms (e.g. microalgae, meiofauna), of grazers, as well as of the microbial community.

Comparative life cycle assessment of microalgae cultivation for non-energy purposes using different carbon dioxide sources.

The ability of microalgae to sequester carbon and at the same time synthesise valuable compounds with potential applications in nutraceutical, pharmaceutical and cosmetic industries makes them attractive for commercial deployment, especially in view of a blue bioeconomy. Among microalgae, the diatom Phaeodactylum tricornutum is considered as an important potential source of omega-3 polyunsaturated fatty acids, such as eicosapentanoic acid, an essential polyunsaturated fatty acid with anti-inflammatory and antimicrobial properties. The aim of this study was to perform the Life Cycle Assessment of the cultivation of P. tricornutum - at semi-industrial scale in photobioreactor - for the production of high-quality bioactive compounds comparing synthetic carbon dioxide supply to a supply with waste carbon dioxide from a biogas upgrading process hypothesizing industrial symbiosis network. The effect of renewable energy use instead of the European electricity mix was also examined. Primary data on the production process, including the stages of cleaning and sterilisation, cultivation, harvesting and freeze-drying, were used. The midpoint impact categories recommended in the ILCD Handbook were used for performing the impact assessment. A sensitivity analysis was also performed on algal productivity, culture medium recirculation factor and amount of solvents per cleaning cycle. Firstly, results indicate in general cultivation and freeze-drying as the most contributing stages to the impacts. Secondly, they demonstrate in the comparative assessment that the use of carbon dioxide from the biogas upgrading is a feasible and attractive alternative to the synthetic one, as it allows for the improvement of the environmental performance of the production process in all the analysed impact categories. Finally, sensitivity analysis suggests that the environmental performance could be further improved by acting on other key factors, such as electricity source, nutrients culture medium and cleaning solutions.

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.

Dinoflagellate resting cysts from surface sediments of the Adriatic Ports: Distribution and potential spreading patterns.

The ability of microalgae to preserve viable in coastal sediments as resting forms provides a reservoir of biodiversity and a useful tool to determine species spreadings. This study represents the first port baseline survey on dinoflagellate cysts, investigated in nine Adriatic ports during a cross border project. 40 dinoflagellate taxa were detected. The assemblages resulted in all ports dominated by Lingulodinium polyedra and Alexandrium minutum/affine/tamutum group. General separation to the western and eastern side of the Adriatic regarding cysts assemblage composition, partially abundance, was observed. Six taxa were detected as non-indigenous species for the Adriatic. Two taxa are included in the list of harmful aquatic organisms, indicating the potential threat of ballast waters in the Adriatic. Potential spreading of taxa by general circulation and ballast waters, intra- and extra-Adriatic was investigated. The entering in to force of the ballast waters management regulations should enhance prospects to minimize future harmful impacts.

Mediterranean Lithophyllum stictiforme (Corallinales, Rhodophyta) is a genetically diverse species complex: implications for species circumscription, biogeography and conservation of coralligenous habitats.

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.

Mediterranean Bioconstructions Along the Italian Coast.

Marine bioconstructions are biodiversity-rich, three-dimensional biogenic structures, regulating key ecological functions of benthic ecosystems worldwide. Tropical coral reefs are outstanding for their beauty, diversity and complexity, but analogous types of bioconstructions are also present in temperate seas. The main bioconstructions in the Mediterranean Sea are represented by coralligenous formations, vermetid reefs, deep-sea cold-water corals, Lithophyllum byssoides trottoirs, coral banks formed by the shallow-water corals Cladocora caespitosa or Astroides calycularis, and sabellariid or serpulid worm reefs. Bioconstructions change the morphological and chemicophysical features of primary substrates and create new habitats for a large variety of organisms, playing pivotal roles in ecosystem functioning. In spite of their importance, Mediterranean bioconstructions have not received the same attention that tropical coral reefs have, and the knowledge of their biology, ecology and distribution is still fragmentary. All existing data about the spatial distribution of Italian bioconstructions have been collected, together with information about their growth patterns, dynamics and connectivity. The degradation of these habitats as a consequence of anthropogenic pressures (pollution, organic enrichment, fishery, coastal development, direct physical disturbance), climate change and the spread of invasive species was also investigated. The study of bioconstructions requires a holistic approach leading to a better understanding of their ecology and the application of more insightful management and conservation measures at basin scale, within ecologically coherent units based on connectivity: the cells of ecosystem functioning.

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.

Allelopathic effects of diatom filtrates on the toxic benthic dinoflagellate Ostreopsis cf. ovata.

Ostreopsis blooms regularly occur in many Mediterranean coastal areas in late summer-autumn. In the northern Adriatic Sea, Ostreopsis blooms affect diatom-dominated microphytobenthic communities. In this study, the effects of the filtrates of some diatom species, both benthic (Tabularia affinis, Proschkinia complanatoides and Navicula sp.) and planktonic (Thalassiosira sp. and Skeletonema marinoi) on cell morphology, cytological features and growth of O. cf. ovata were investigated. Our results showed a marked decrease of O. cf. ovata growth when cells were exposed to all diatom filtrates tested. The highest inhibitions were observed for exposures to P. complanatoides and Navicula sp. filtrates (92.5% and 80%, respectively) and increased with the age of diatom culture. Moreover, a clear DNA degradation and abnormal forms of O. cf. ovata cells (83.8% of the total) were found at the highest concentrations using Navicula sp. filtrate after 10 days of the inoculum.

PUFAs and PUAs production in three benthic diatoms from the northern Adriatic Sea.

The production of polyunsaturated aldehydes (PUAs) has been reported by many planktonic diatoms, where they have been implicated in deleterious effects on copepod reproduction and growth of closeby microbes or suggested as infochemicals in shaping plankton interactions. This study investigates the production of PUAs by diatoms commonly occurring in the microphytobenthic communities in temperate regions: Tabularia affinis, Proschkinia complanatoides and Navicula sp. Results highlight the production of PUAs by the three benthic diatoms during stationary and decline phases, with intracellular concentrations from 1.8 to 154.4 fmol cell-1, which are within the range observed for planktonic species. The existence of a large family of PUAs, including some with four unsaturations, such as decatetraenal, undecatetraenal and tridecatetraenal, was observed. Since particulate and dissolved PUAs were positively correlated, together with cell lysis, equivalent concentrations may be released during late growth stages, which may affect benthic invertebrates grazing on them and other microalgae.

Genetic and morphological variation in an ecosystem engineer, Lithophyllum byssoides (Corallinales, Rhodophyta).

Lithophyllum byssoides is a common 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 about 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 pair-wise 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.

Photobioreactor cultivation and catalytic pyrolysis of the microalga Desmodesmus communis (Chlorophyceae) for hydrocarbons production by HZSM-5 zeolite cracking.

The study evaluated the growth of Desmodesmus communis on column photobioreactor and its thermochemical treatment by catalytic pyrolysis using HZSM-5 zeolite. D. communis showed good results in terms of growth (0.05gL-1d-1). Analytical pyrolysis of original algae and derived bio-oil mixed with zeolite was used as a screening method in order to gather information on the cracking process. Preparative pyrolysis on bench scale reactor was performed on algae biomass over a zeolite bed at 1:10 ratio (wt/wt). Py-GC-MS of biomass/catalyst mixture showed that the denitrogenation/deoxygenation increased with increasing zeolite load from 1:5 to 1:20 ratio and became significant at 1:10 ratio. The composition observed by analytical pyrolysis was featured by the predominance of alkylated monoaromatic hydrocarbons. The scaling-up to bench scale confirmed the results obtained with analytical pyrolysis in terms of monoaromatic hydrocarbons. However, low yield of catalytic oil (8% by weight) was observed.

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.

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.

Subcellular localization of dinoflagellate polyketide synthases and fatty acid synthase activity.

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 & Ø. 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 <10(-10) ) to PKSs, no transcripts for either Type I (cytosolic) or Type II (chloroplast) FAS are present. Further characterization of the FAS complexes may help to elucidate the functions of the PKS enzymes identified in dinoflagellates.